UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA

Transcription

1 UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA MOLECULAR MECHANISMS OF MICROGLIA REACTIVITY TO BILIRUBIN: EVALUATION OF POTENTIAL NEUROLOGICAL EFFECTS Sandra Isabel Leitão da Silva DOUTORAMENTO EM FARMÁCIA (Biologia Celular e Molecular) 2010

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3 UNIVERSIDADE DE LISBOA FACULDADE DE FARMÁCIA MOLECULAR MECHANISMS OF MICROGLIA REACTIVITY TO BILIRUBIN: EVALUATION OF POTENTIAL NEUROLOGICAL EFFECTS Sandra Isabel Leitão da Silva Research advisors: Dora Maria Tuna de Oliveira Brites, PhD. Rui Fernando Marques da Silva, PhD. DOUTORAMENTO EM FARMÁCIA (Biologia Celular e Molecular) 2010

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5 MOLECULAR MECHANISMS OF MICROGLIA REACTIVITY TO BILIRUBIN: EVALUATION OF POTENTIAL NEUROLOGICAL EFFECTS MECANISMOS MOLECULARES DE REACTIVIDADE DA MICROGLIA À BILIRRUBINA: AVALIAÇÃO DE POTENCIAIS EFEITOS NEUROLÓGICOS Dissertação apresentada à Faculdade de Farmácia da Universidade de Lisboa para obtenção do grau de Doutor em Farmácia (Biologia Celular e Molecular) Sandra Isabel Leitão da Silva 2010

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7 Para a elaboração da presente tese de doutoramento foram usados integralmente como capítulos, artigos científicos publicados, ou submetidos para publicação, em revistas científicas internacionais indexadas. Estes trabalhos foram realizados em colaboração com os seguintes autores: Ana Rita Vaz, Catarina Osório, Andreia Barateiro, Adelaide Fernandes, Ana Sofia Falcão, Maria José Diógenes, Maria Alexandra Brito, Nico van Rooijen, Ana Sebastião, Rui Silva e Dora Brites. De acordo com o disposto no ponto 1 do artigo nº41 do Regulamento de Estudos Pós- Graduados da Universidade de Lisboa, deliberação nº 93/2006, publicada em Diário da República II Série nº de Julho de 2003, a Autora desta dissertação declara que participou na concepção e execução do trabalho experimental, na interpretação dos resultados obtidos e na redacção dos manuscritos.

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9 Os estudos apresentados nesta dissertação foram realizados no grupo de investigação Neuron Glia Biology in Health & Disease, Research Institute for Medicines and Pharmaceutical Sciences (imed.ul), Faculdade de Farmácia da Universidade de Lisboa. Parte do trabalho foi também realizado no Departamento de Investigação Cellular Neuroscience do Max-Delbrück Centrum em Berlim, Alemanha, sob a orientação do Professor Doutor Helmut Kettenmann. O trabalho foi subsidiado pelos projectos FCT-POCTI/SAU/MMO/55955/2004 e FCT- PTDC/SAU-NEU/64385/2006 concedidos à Professora Doutora Dora Brites pela Fundação para a Ciência e Tecnologia (FCT), sendo que a Autora usufruiu de uma bolsa de Doutoramento (SFRH/BD/30326/2006) concedida pela FCT, Lisboa, Portugal.

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11 Para o meu Pai

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13 Acknowledgements/Agradecimentos As minhas primeiras palavras de agradecimento vão naturalmente para a Professora Doutora Dora Brites, orientadora deste doutoramento. Gostaria de lhe agradecer o facto de me ter aberto as portas do mundo da ciência e por tão bem me ter guiado nos seus trilhos. Gostaria de salientar as suas notáveis qualidades científicas e o seu enorme espírito crítico, bem como o seu sentido de justiça e igualdade. Os seus elevados padrões de rigor científico e o seu constante encorajamento ao longo deste caminho fizeram-me acreditar que podia chegar ao fim e, mais importante do que isso, que era sempre possível fazer mais e melhor. Como já lhe tenho dito muitas vezes, o seu olhar consegue sempre ver mais além! Os seus ensinamentos ser-me-ão úteis ao longo da vida académica e pessoal e não esquecerei o tempo que passei a trabalhar ao seu lado nem o facto de ter depositado em mim a confiança para desempenhar esta tarefa. Só espero ter estado à altura das suas expectativas! Ao Professor Doutor Rui Silva, co-orientador desta tese, quero agradecer a constante ajuda e encorajamento para superar as dificuldades que foram surgindo ao longo deste percurso académico. O seu olhar crítico sobre as questões essenciais foram extremamente valiosas para a concretização deste trabalho. Quero também agradecer a preocupação e carinho que sempre demonstrou para comigo e a forma como sempre me conseguia fazer sorrir mesmo quando tudo corria mal! À Professora Doutora Alexandra Brito gostaria de agradecer por, apesar de não ter uma responsabilidade directa sobre o meu trabalho, ter sempre demonstrado interesse e disponibilidade para me ajudar. Os seus conselhos científicos foram pertinentes, assertivos e de extrema validade. Gostaria também de lhe agradecer a amizade que desenvolvemos ao longo dos anos. I would also like to thank Professor Helmut Kettenmann and his lab, in the Research Department Cellular Neuroscience from the Max-Delbrück Centrum in Berlin, Germany for welcoming me in Berlin during my training period and for providing me with so much valuable knowledge. Gostaria também de agradecer às Professoras Doutoras Ana Sebastião e Maria José Diógenes pela imensa disponibilidade que demonstraram desde o primeiro dia da colaboração que encetámos e pelo seu contributo científico para este trabalho. À Liliana Bernardino quero endereçar um agradecimento sentido pela ajuda tão preciosa que me prestou na implementação das culturas organotípicas de cortes de hipocampo. A tua enorme disponibilidade e simpatia, bem como a fé que sempre tiveste que tudo ia correr bem, fizeram-me acreditar que seria possível completar esta tarefa hercúlea!

14 Ao Pedro Pereira quero agradecer a ajuda preciosa nos aspectos mais técnicos do trabalho com os cortes e pela paciência e disponibilidade que sempre demonstrou. À Adelaide tenho prometido um merecido agradecimento desde o primeiro dia em que entrei no CPM! Foste tu quem me encorajou a enveredar pelo mundo da ciência e foste também tu quem me deu a mão nas vezes em que me perdi pelo caminho Por todos os momentos em que dispensaste o teu tempo para me ajudares, pelo enorme contributo intelectual e científico que deste a esta tese e pela forma como me fizeste sentir acarinhada neste grupo, um enorme obrigado! Sem a tua ajuda tenho a certeza que este trabalho não seria o mesmo! À Sofia tenho que agradecer os ensinamentos que me transmitiu durante este período e a forma atenciosa e carinhosa como me tratou desde o início, fazendo-me sentir em casa e parte integrante desta equipa! As nossas conversas à hora do lanche foram uma fonte de motivação e de força para continuar, bem como o teu exemplo de perseverança apesar das dificuldades! Um agradecimento muito especial à minha companheira de jornada, Rita. Fizemos esta travessia quase de mãos dadas e estivemos presentes uma para a outra nos teus e nos meus momentos bons e maus. A tua amizade foi um factor determinante para conseguir chegar até aqui e só espero ter correspondido na mesma medida! Mereces toda a felicidade do mundo e eu tenho a certeza que a encontrarás e que alcançarás o sucesso tanto a nível profissional como pessoal. Quero agradecer também à Andreia, colega e amiga deste laboratório que se tornou a nossa casa ao longo destes anos. Muito obrigado pela tua boa-disposição e por toda a ajuda que me prestaste. Só tenho pena que não estejas aqui neste momento mas esperamos o teu regresso! Não posso deixar de agradecer às meninas do nosso laboratório e futuras doutoras: à Inês, com quem tanto me identifico quer a nível pessoal quer a nível profissional, muito obrigado por me ouvires tantas vezes e por me encorajares quando desanimei. Tenho a certeza que também tu alcançarás este momento e todo o sucesso que mereces! À pequena Ema que tanto cresceu desde que cá chegou! És um doce de pessoa, sempre pronta para ouvir e ajudar e tão interessada nos outros e no mundo que a rodeia. Tens crescido tanto nos últimos tempos que vamos ter que deixar de te chamar Pequena! À Filipa quero agradecer todo o apoio que me ofereceste, o que deste e o que não deste uma vez que é característica tua estar sempre pronta para ajudar em qualquer circunstância! Desejo-te as maiores felicidades no teu percurso académico e pessoal.

15 Não posso deixar de fora a Cibelle, a nossa Belli, que me ajudou tanto com o seu conhecimento infinito sobre cortes e imunohistoquímica! Gostei muito de partilhar contigo o mesmo espaço e de desenvolvermos esta amizade! Gostaria ainda de agradecer à Eduarda, que por cá passou e avançou para outros voos, por todas as conversas inspiradoras que tivemos e que me fizeram ter mais força para superar esta prova! Mesmo não estando cá, a tua ajuda foi preciosa. Agradeço também à Catarina Osório, que fez parte dos trabalhos aqui apresentados e que sempre se mostrou disponível para me acompanhar neste percurso. Um agradecimento especial para a Inês Milagre e para a Maria João Nunes que perderam tanto tempo com as minhas dúvidas e questões e que nunca hesitaram em me ajudar quando eu precisei! Gostaria também de agradecer à Professora Elsa, que sempre se preocupou tanto comigo e que ao longo deste caminho me foi dando alento e motivação para continuar! Cheguei ao fim e estou viva e de saúde! Quero agradecer a todos os inquilinos deste pequeno T0 que partilhamos aqui na cave! Os momentos de diversão e as conversas animadas contribuíram e muito para fazer deste sítio um local de trabalho muito agradável! Quero agradecer especialmente às minhas colegas de faculdade que também seguiram o caminho da ciência, Maria João e Cati, e que tanto me influenciaram nesta escolha! Quero agradecer também aos meus queridos amigos Rita, Cláudia e Badalo, João e Ju e a todos os restantes membros da nossa grande família! Muito obrigado por estarem sempre atentos e dispostos a ajudar e por me encorajarem constantemente! Continuo a achar o condomínio fechado uma ideia vencedora! Aos meus sogros Fernanda e Guedes e aos meus cunhados Carla e Zé obrigado por me fazerem sentir parte desta família e pelo interesse que demonstraram no meu trabalho. Aos meus manos Ana e Nuno quero agradecer do fundo do coração todo o amor e carinho que têm por mim e que me deu força neste caminho. Saber-vos por perto e saber que acreditam em mim e nas minhas capacidades tem sido o meu alento, a minha motivação e só espero que se possam orgulhar de mim neste momento! Aos meus sobrinhos Jaime e Vasco, Gui, Miguel e Francisco quero agradecer por trazerem tanta luz e sorrisos para a minha vida! São vocês que fazem a vida ter mais cor! À minha mãe, um agradecimento sentido a quem sempre acreditou em mim e me ensinou a acreditar em mim mesma. És a pessoa mais corajosa que conheço à face da terra e o teu exemplo de força e coragem para superar a prova mais difícil de todas

16 inspira-me a dar o meu melhor para superar as pequenas provas da vida. Obrigada por tudo! Um agradecimento do fundo do meu coração e da minha alma vai para uma das pessoas mais importantes da minha vida: o meu Pai. Dedico-te esta tese porque sempre me ensinaste a ser perseverante e a não desistir nunca e por isso prometi a mim mesma que completaria esta tarefa e que seria em tua honra. Espero que, estejas onde estiveres, possas ter orgulho em mim Por fim, um agradecimento muito especial ao homem da minha vida, o Tiago. Muito obrigado por todos os momentos que passámos juntos e também por entenderes as minhas ausências ao longo deste caminho. Nunca, nem por um segundo, duvidaste da minha capacidade de empreender esta tarefa e foi isso que me impulsionou a conclui-la! Adoro-te!

17 Contents Contents Abbreviations... xxi Abstract... xxv Resumo... xxvii Chapter I - General Introduction The brain an integrated network of interactive neurons and glia Neurons Oligodendrocytes Astrocytes Microglia Heterogeneity of microglial phenotypes Extracellular matrix Role of microglia in different brain development stages Embryonic period Neonatal period Microglia in the aging brain Functional roles of microglia activation and overactivation Surveillance functions and role in synaptic plasticity Role of microglia in innate and adaptive responses Phagocytosis Production of mediators in neuroprotection and neurodegeneration Microglial reactivity and modulation by cell interplay Models for the evaluation of microglial reactivity Reciprocal reactivity modulation by interplay of microglia with neighboring cells Involvement of microglia in the progression of neurological diseases Acute and chronic neurological diseases Neonatal hyperbilirubinemia Molecular mechanisms of bilirubin-induced CNS injury Global aims of the thesis References Chapter II - Features of bilirubin-induced reactive microglia: from phagocytosis to inflammation Introduction xvii

18 Contents 2. Material and Methods Chemicals Primary culture of microglia Cell treatment Measurement of cytokine release Western blot assay Detection of NF-κB activation Morphological Analysis Assessment of microglial phagocytic properties Gelatin zymography Evaluation of microglial cell death Statistical analysis Results UCB triggers IL-1β, TNF-α and IL-6 secretion following different temporal profiles p38 and ERK1/2 phosphorylation is elicited by UCB in microglia at an early time point NF-κB signalling pathway is triggered in UCB-activated microglia UCB-induced NF-κB translocation depends on both ERK1/2 and p Microglia depict morphological changes upon UCB stimulation UCB differently modulates microglial phagocytosis depending on exposure time Release of active MMPs is enhanced upon UCB stimulation of microglia UCB-stimulated microglia evidence enhanced COX-2 expression UCB reduces microglial viability leading to loss of membrane integrity and increased caspase activity Discussion References Chapter III - Dynamics of neuron-glia interplay upon exposure to unconjugated bilirubin Introduction Material and Methods Chemicals Primary culture of microglia Primary culture of astrocytes xviii

19 Contents 2.4. Primary neuronal cell cultures Neuron-microglia mixed cultures Cell treatment Measurement of cytokine release Gelatin zymography Assessment of microglial phagocytic properties Quantification of nitrite levels Evaluation of cell death Neurite Extension and Ramification Statistical Analysis Results Conditioned media from UCB-treated astrocytes and neurons modulate microglial secretion of cytokines, as compared to UCB-activated microglia Conditioned media from UCB-treated astrocytes and neurons have opposing effects on microglial MMP-9 activity, as compared to UCB-activated microglia Nitric oxide (NO) release by microglia is elicited by UCB and is higher by naïve microglia exposed to conditioned medium from UCB-treated neurons Loss of viability in UCB-stimulated microglia is less in naïve microglia exposed to conditioned medium from UCB-treated astrocytes (ACM), but higher if treated with medium from neurons incubated with UCB (NCM) Microglial phagocytic properties enhanced by UCB further increase by medium collected from neurons exposed to UCB UCB-induced neuronal network impairment is prevented by microglia environment Neuronal cell death triggered by UCB is diminished in the presence of microglia Discussion References Chapter IV - Unconjugated bilirubin neurotoxicity is modulated by microglia and prevented by glycoursodeoxycholic acid and interleukin Introduction Material and Methods Chemicals Organotypic-cultured hippocampal slices Preparation of microglia-depleted organotypic-cultured hippocampal slices Organotypic-cultured hippocampal slices treatment xix

20 Contents 2.5. Assessment of cell death in organotypic-cultured hippocampal slices Quantification of nitrite levels Primary neuronal cell cultures Cell treatment Neurite extension and ramification Measurement of glutamate Evaluation of cell death Western blot assay Statistical Analysis Results Microglia modulate UCB-induced glutamate release and NO production in organotypic-cultured hippocampal slices NMDA receptors and NO are implicated in UCB-induced neurite impairment and in cell death UCB-elicited accumulation of extracellular glutamate is reduced by both GUDCA and IL-10, but not abolished GUDCA or IL-10 pre-treatment counteracts impairment of neurite outgrowth and ramification, as well as cell death in UCB-treated neurons UCB decreases the expression of pre-synaptic proteins and this event is abrogated by GUDCA, but not by IL Hampering of UCB-induced NO and glutamate production, as well as cell death by GUDCA was reproduced in hippocampal slices Discussion References Chapter V - Final considerations Concluding remarks and perspectives References xx

21 Abbreviations ACM Astrocyte conditioned medium AD Alzheimer s disease AGUDC Ácido glicoursodesoxicólico ALS Amyotrophic lateral sclerosis AMPA α-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate AP-1 Activator protein-1 ATP Adenosine triphosphate Aβ Amyloid β BBB Blood-brain barrier BDNF Brain-derived neurotrophic factor BIND Bilirubin-induced neurological dysfunction BNC Bilirrubina não conjugada CNS Central nervous system COX-2 Cyclooxigenase-2 CR3 Complement receptor 3 CSF Colony stimulating factor DIV Days in vitro ECM Extracellular matrix ERK1/2 Extracellular signal regulated kinases 1 and 2 GABA γ-aminobutyric acid GDNF Glial cell-line derived neurotrophic factor GFAP Glial fibrillary acidic protein GLT-1 Glutamate transporter-1 GTP Guanosine triphosphate GUDCA Glycoursodeoxycholic acid HIV Human immunodeficiency virus HSA Human serum albumin Iba1 Ionized calcium-binding adaptor molecule 1 Ig Immunoglobulin IKK Inhibitor of nuclear factor-κb kinase complex IL Interleukin IL-1R Interleukin-1 receptor inos Inductible nitric oxide synthase xxi

22 IRAK 4 IL-1R-associated kinase 4 IκB Inhibitor of nuclear factor-κb JNK1/2 c-jun N-terminal kinases 1 and 2 LDH Lactate dehydrogenase L-NAME N-ω-nitro-L-arginine methyl ester LPS Lipopolysaccharide LTP Long term potentiation Mac 1 Macrophage receptor 1 MAP-2 Microtubule associated protein-2 MAPK Mitogen-activated protein kinase MCAO Middle cerebral artery occlusion MEKK MAPK kinase kinase mglur Metabotropic glutamate receptor MHC Major histocompatibility complex MK-801 [(+)-5-methyl-10,11-dihydro- 5Hdibenzo[a,d]cyclohepten-5,10-imine maleate)] MMP Matrix metalloproteinase MRI Magnetic resonance imaging Mrp1 Multidrug resistance associated protein 1 MS Multiple sclerosis MyD88 Myeloid differentiation factor 88 NCM Neuron conditioned medium NF-κB Nuclear factor-κb NGF Nerve growth factor NMDA N-methyl-D-aspartate nnos Neuronal nitric oxide synthase NO Nitric oxide NT Neurotrophin OPC Oligodendrocyte precursor cell OSC Organotypic slice culture OX Orexin PAMP Pathogen-associated molecular pattern PARP Poly-ADP ribose polymerase PD Parkinson s disease PGE2 Prostaglandin E2 RNS Reactive nitrosative species xxii

23 ROS SNAP-25 STAT TACM TF TGF-β TLR TNCM TNFR TNF-α TRAF TREM-2 UCB UDCA Reactive oxygen species Synaptosomal-associated protein-25 Signal transducer and activator of transcription Conditioned medium from UCB-treated astrocytes Transcription factors Transforming growth factor-β Toll-like receptor Conditioned medium from UCB-treated neurons Tumour necrosis factor receptor Tumour necrosis factor-α TNFR-associated factor Triggering receptor expressed on myeloid cells-2 Unconjugated bilirubin Ursodeoxycholic acid xxiii

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25 Abstract Abstract Microglia are active sensors in the brain that rapidly engage adequate functional activity states in response to injury to restore homeostasis. During the neonatal period, the brain is more vulnerable to several injury conditions such as the one induced by hyperbilirubinemia, a common situation observed in the newborn, where excessive levels of unconjugated bilirubin (UCB) can reach and damage the brain. Although UCB-induced neuronal and astrocytic toxicity have already been approached, the role of microglia in this condition remains unclear. Thus, this thesis intended to investigate microglial reactivity to UCB and to characterize the intervention of other brain cells in the modulation of their response. Isolated microglial cells showed to acquire a phagocytic phenotype upon UCB exposure that preceded the release of pro-inflammatory cytokines. This release showed to involve activation of upstream signalling pathways such as mitogen-activated protein kinases (MAPKs) and nuclear factor-κb (NF-κB). We next investigated whether and how the microenvironment influenced microglial response to UCB. Our findings revealed that soluble factors released by UCB-stimulated astrocytes refrained microglial activation while neuron-microglia interaction, evaluated using conditioned media and mixed culture models, signalled microglial clearance functions but also enhanced its inflammatory potential, ultimately leading to microglia demise. Finally, we evaluated microglial neuroprotective or neurotoxic effects in a cell-to-cell concerted action in response to UCB. Microglia revealed to participate in glutamate homeostasis, and to induce the release of this neurotransmitter and of nitric oxide (NO) in UCB-treated organotypiccultured hippocampal slices, molecules that showed to be key players in UCB-induced neurotoxicity. Moreover, our results point to interleukin (IL)-10 and glycoursodeoxycholic acid (GUDCA) as promising therapies in neonatal hyperbilirubinemia. In conclusion, microglia displays a dual activation profile in response to UCB stimulation which is tailored by the influence of neighbouring cells. Collectively, these data contribute for the understanding of microglia s role in hyperbilirubinemia and reinforce their remarkable functional plasticity. Keywords: Astrocytes; Hyperbilirubinemia; Inflammation; Intracellular signalling; Microglia; Neurons; Neuron-glia dynamics; Phagocytosis. xxv

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27 Resumo Resumo As células da micróglia são sensores activos de dano no cérebro e rapidamente adquirem estados funcionais de activação adequados à restauração da homeostase. Durante o período neonatal o cérebro está mais vulnerável a certas lesões tais como as induzidas pela hiperbilirrubinémia, uma condição comum no recém-nascido, na qual níveis excessivos de bilirrubina não conjugada (BNC) podem atingir e lesar o cérebro. A toxicidade desencadeada pela BNC em neurónios e astrócitos já foi alvo de estudos prévios, no entanto o papel da micróglia nesta condição ainda não está completamente esclarecido. Assim, foi objectivo desta tese investigar a reactividade da micróglia à BNC e caracterizar a intervenção das restantes células do cérebro na modulação da sua resposta. As células de micróglia isoladas reagem à exposição à BNC pela aquisição de um fenótipo fagocítico que precede a libertação de citocinas pró-inflamatórias. Esta libertação parece envolver a activação a montante de vias de sinalização tais como as cinases proteicas activadas por mitogénios (mitogen-activated protein kinases, MAPKs) e o factor nuclear-κb (nuclear factor-κb, NF-κB). Em seguida investigámos a influência do micro-ambiente celular na resposta da micróglia desencadeada pela BNC. Verificámos então que astrócitos expostos à BNC libertam factores solúveis que contêm a activação da micróglia. Por outro lado, a interacção entre neurónios e micróglia, avaliada através do modelo dos meios condicionados e do das culturas mistas, parece sinalizar a micróglia a empreender funções de eliminação de restos celulares, ao mesmo tempo que aumenta o potencial inflamatório desta célula levando, em última análise, à degeneração celular. Por fim, estudámos os efeitos neuroprotectores ou neurotóxicos da micróglia face à BNC num modelo onde as interacções celulares são preservadas. Os resultados obtidos em culturas organotípicas de fatias de hipocampo revelam a participação das células da micróglia na homeostase do glutamato para além de libertarem este neurotransmissor em conjunto com o óxido nítrico (nitric oxide, NO). Os dados obtidos demonstram ainda que o glutamato e o NO são peças chave na neurotoxicidade provocada pela BNC. Por fim, a interleucina-10 e o ácido glicoursodesoxicólico (AGUDC) salientam-se como promissores agentes terapêuticos na hiperbilirrubinémia neonatal. Em conclusão, a micróglia apresenta um duplo perfil de activação em resposta à BNC que é modulaado pela influência das células adjacentes. Em conjunto, estes dados contribuem para uma melhor clarificação do papel da micróglia na hiperbilirrubinémia e corroboram a sua notável plasticidade funcional. xxvii

28 Resumo Palavras chave: Astrócitos; Fagocitose; Hiperbilirrubinémia; Inflamação; Interacção neurónio-glia; Micróglia; Neurónios. xxviii

29 Chapter I GENERAL INTRODUCTION 1

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31 General Introduction 1. The brain an integrated network of interactive neurons and glia Neurons are the functioning unit of the central nervous system (CNS) and are responsible for information processing. Glia make up 90% of the cells in the human brain, and although their name derives from the greek word glue, research in the past 20 years has proven that these cells not only provide physical support to neurons but make important contributions in the formation and operation of the neural circuitry and play a key role in the brain s immune functions (Fig.I.1) (Allen and Barres, 2009; Miller, 2005). Neuron Oligodendrocyte Microglia Astrocyte Blood vessel Fig. I.1. The brain as an integrated network. Glial cells (i.e. astrocytes, microglia and oligodendrocytes) are intimately related to neurons and blood vessels, contributing for their support and participating in various key brain functions. Adapted from Allen and Barres (2009). The major difference between glia and neurons is that the latter ones fire action potentials that underlie sensation, movement and thought, while glial cells lack this capacity. Nevertheless, emerging research suggests that glial cells participate in information processing and interact with synapses (Wake et al., 2009). In fact, the 3

32 Chapter I acknowledgement of neuron-glia crosstalk during synaptic transmission has progressively challenged the classical view of the brain as a network of neuronal contacts but rather as an integrated circuit of interactive neurons and glia (Bezzi and Volterra, 2001). Hence glia is emerging as critical participants in every aspect of brain development, function, and disease (Barres, 2008) Neurons Neurons are highly specialized nerve cells responsible for communicating information in both chemical and electrical forms throughout the body (Bloom, 2003). Their structure comprises a cell body, or soma, from which an elaborate arborisation tree emerges. Neuronal processes give neurons the regionalization of their functions, their polarity and capacity to connect to other neurons, to sensory cells and to effector cells (Hammond, 2001). Information is received and processed through the dendritic branches, whose major structural components are the actin and microtubule cytoskeleton together with microtubule-associated proteins (MAPs), responsible for stabilization (Chen and Ghosh, 2005). The neuronal axon is a long cytoplasmic process that culminates in a highly specialized structure, the pre-synaptic terminal, which, together with the post-synaptic terminal of the adjacent neuron forms the synapse (Bloom, 2003). The high molecular weight MAP-2 protein is more common in dendrites than in axons and for this reason, MAP-2A and MAP-2B antibodies coupled to fluorescent molecules are useful tools for labelling dendrites, allowing morphometric analysis in cell cultures (Hammond, 2001). Electrical signalling in the nervous system involves the movement of ions across the neuronal plasma membrane through specific transmembrane proteins called ion channels. These ionic currents evoke transient changes of membrane potential (action potentials) which fire neuronal communication (Hammond, 2001). In the synapse, neurotransmitters are released and establish communication between adjacent neurons or between neurons and effector or sensory cells (Bloom, 2003). Several molecules serve as neurotransmitters, such as acetylcholine, amino acids like glutamate, γ- aminobutyric acid (GABA) and glycine and neuropeptides and their release at the presynaptic terminal is induced by calcium-regulated vesicle exocytosis. Neurotransmitters released into the synaptic cleft bind their specific receptors in the post-synaptic terminal of the adjacent neuron and propagate action potential (Hammond, 2001). Glutamate is the major excitatory neurotransmitter in the CNS and activates two main types of post-synaptic receptors: ionotropic glutamate receptors that are ligandgated ion channels and are divided into N-methyl-D-aspartate (NMDA), α-amino-3- hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) and kainate receptors, and 4

33 General Introduction metabotropic glutamate receptors (mglurs) that are receptors coupled to guanosine triphosphate (GTP)-binding proteins. Glutamate s concentration in the synaptic cleft must be kept low as high or sustained glutamate receptor activation may induce neuronal death via intracellular calcium and/or sodium deregulation, a mechanism called excitotoxicity (Gras et al., 2006) Oligodendrocytes Oligodendrocytes are the myelinating cells of the CNS and constitute about 5 to 10 % of the total glial population. Their processes enwrap neuron axons and form myelin, an insulating lipid-rich membrane sheath that speeds the conduction of electrical impulses. Therefore, saltatory propagation of action potentials occurs at the nodes of Ranvier between oligodendrocyte myelin sheaths (Bradl and Lassmann, 2010). Oligodendrocytes are vulnerable to cytotoxicity induced by dysfunctional astrocytes (Sharma et al., 2010) or activated microglia as oligodendroglial cell death can be initiated by increased levels of cytokines and reactive oxygen species (ROS) (Sherwin and Fern, 2005). Demyelination due to damage to oligodendrocytes leads to various diseases from neurodevelopment to neurodegeneration such as periventricular leukomalacia (Volpe, 2009) and multiple sclerosis (MS) (Allen and Barres, 2009), respectively. Moreover, the existence of direct chemical synapses between pyramidal neurons and oligodendrocyte precursor cells (OPCs) in the mammalian hippocampus has been demonstrated, underscoring the active role of glial cells in information processing (Bergles et al., 2000) Astrocytes Astrocytes comprise about 85% of the glial population and are star-shaped cells. They exert structural functions, are required for neuronal survival, neurite formation and angiogenesis and maintain CNS homeostasis by regulating ph, ionic concentrations and osmolarity (Montgomery, 1994). Astrocytes also provide metabolic support to neurons by fueling their activity with energy and substrates and remove excess neurotransmitter molecules from the extracellular space (Tsacopoulos and Magistretti, 1996). These cells take part in the neurovascular unit of the blood-brain barrier (BBB) since their end-feet ensheathe endothelial cells in micro-vessels (Cardoso et al., 2010). By signalling blood vessels to expand or narrow, astrocytes regulate local blood flow to provide oxygen and nutrients to neurons in need. Moreover, astrocytes were shown to induce tight junctions in endothelial cells, thus participating actively in the establishment of the CNS boundaries (Yamagata et al., 1997). More recently, bidirectional communication between neurons and astrocytes was demonstrated. This fact altered the long-lasting paradigm of the bipartite synapse, established between a pre- and a post-synaptic neuronal element, 5

34 Chapter I leading to the novel concept of the tripartite synapse, where astrocytic participation in the regulation of synaptic activity (Araque et al., 1999; Perea et al., 2009) and plasticity (Barker and Ullian, 2010) is acknowledged. Moreover, astrocytes can communicate to each other through the generation of calcium waves (Cornell-Bell et al., 1990) and to neurons by the activation of calcium-based signalling cascades triggered by neurotransmitters released by neurons which feedback the production of neuroactive substances (Araque et al., 1999). Furthermore, Alvarez-Maubecin et al. (2000) demonstrated gap-junctional electrical coupling between neurons and astrocytes and also that selective changes in the membrane potential of glia modulate neuronal excitability. Considering these interesting concepts, a new role has been established for glial cells, particularly astrocytes, in the so-called neuron-exclusive functions such as information processing Microglia Microglia are small glial cells that reside within the CNS parenchyma and constitute about 10 to 20 % of the total glial cell population (Chew et al., 2006; Vilhardt, 2005). They are ubiquitously distributed in the brain, being present in both grey and white matter and exhibiting a higher density within the hippocampus, basal ganglia and substantia nigra (Walter and Neumann, 2009). Microglial cells share many properties of macrophages as they belong to the mononuclear phagocyte lineage (Vilhardt, 2005). The notion that microglial cells are the resident immune cells of the CNS has altered the concept of the brain as an immunologically privileged organ supported by the existence of the BBB. Although microglial immunocompetent functions are not on the same scale as that of peripheral leucocytes, it is well established that they act as the brain s innate immune system (Aloisi, 2001; Streit, 2002) Heterogeneity of microglial phenotypes The traditional classification of microglial subtypes described a resting and an activated state. In the resting state microglia would display a ramified morphology, with small bipolar or rod-shaped cell bodies bearing multiple branching processes (Chew et al., 2006; Kim and de Vellis, 2005), while in the activated state, microglia would undergo morphological changes acquiring an amoeboid appearance, up-regulate several cell surface markers and produce a plethora of bioactive substances (Ladeby et al., 2005). 6

35 General Introduction However, emerging research has updated the concept of microglial activation redefining it as a shift in activity states rather than an activation process in itself (Fig. I.2). 1. Surveying microglia Activating signals 2. Alerted microglia 6. Post activated microglia Calming signals 5. Elimination 4.1. Reactive phenotype 3. Reactive phenotype Feedback signals 4. Reactive phenotype Fig. I.2. Microglial activation is a shift between activity states. Microglia in its resting state plays a very important role in surveying the brain parenchyma (1) and can be prompted into an activated state (2) due to the presence of activating signals (such as inflammatory mediators and neurotransmitters) or the absence of constitutive calming signals, such as chemokines or neurotrophins that trigger a transition to an alerted state. During the activation process, microglia commit to distinct response phenotypes which can be altered throughout the pathological process (3, 4 and 4.1) by the influence of other nerve and immune cells (illustrated as feedback signals). At the end of this process, microglia can either return to a resting state (1), be eliminated (5) or step into a post-activated state (6), in which the cells acquire a kind of memory (indicated in the figure by a floppy disk icon). Adapted from Hanisch (2008). Therefore, microglial activation should no longer be considered an all-or-none event but an adaptive response to microenvironmental changes. (Hanisch and Kettenmann, 2007). Indeed, Kreutzberg (1996) had already dubbed microglia as a sensor of pathology. The discoveries made by Nimmerjahn et al. (2005) and Davalos et al. (2005) demonstrated that resting microglia showed highly motile extension and retraction of processes which enabled them to constantly scan the neural parenchyma. These novel 7

36 Chapter I findings, that we will further explore below, produced a dramatic change in the concept of resting microglia since this phenotype could no longer be considered dormant or inactive but rather a surveying state with an important role in the normal and healthy brain (Hanisch and Kettenmann, 2007; Raivich, 2005). Hence, microglia function as local sentinels that can detect microdamages in the brain and rapidly repair such minute insults without even being noticed. However, stronger or more prolonged insults may trigger more drastic changes in microglial functional phenotype (Hanisch and Kettenmann, 2007). Some authors now define activated microglia as a cell working to restore CNS parenchymal homeostasis, in accordance with the new concept of microglial activation phenotypes (Streit and Xue, 2009). The traditional classification of microglial subtypes based on morphological criteria is no longer suitable, being replaced by the definition of functional activation states (Schwartz et al., 2006). Furthermore, microglia do not constitute a single and uniform cell population but comprise a family of different cell phenotypes with ultimate beneficial or destructive outcomes, being microglial responses tailored in regional and insult-specific manners (Carson et al., 2007) Extracellular matrix The extracellular matrix (ECM) accounts for about 20% of the adult brain volume and is composed of collagen, proteoglycans, hyaluronan, fibronectin, elastins, laminins, vitronectin, thrombospondins, tenascins, among other proteins and carbohydrates (Zimmermann and Dours-Zimmermann, 2008). Mounting evidence demonstrates that ECM is not merely mechanical scaffolding for nervous cells. ECM components are involved in neural migration, proliferation, axon growth and guidance, or synapse formation, processes that are essential during brain development. Furthermore, several cell matrix receptors, like integrins, and cadherins have been shown to affect long-term potentiation (LTP), revealing the implication of ECM in synaptic plasticity, as reviewed in Pavlov et al. (2004). Interactions with ECM can also regulate immune cell action in both a positive and negative fashion. For instance, ECM breakdown, from either microbe colonization or resulting from the action of matrix metalloproteinases (MMPs) released during inflammation, can produce fragments of specific ECM proteins that may act as pro-inflammatory agents. In contrast, ECM proteins can also contribute to the downregulation of inflammation since decorin and biglycan were reported to inhibit the classical pathway of complement activation (Morwood and Nicholson, 2006). Moreover, the interaction of microglial receptors with ECM components such as cell adhesion molecules contributes to microglial chemotaxis (Hristova et al., 2010; Raivich, 2005). 8

37 General Introduction 2. Role of microglia in different brain development stages The function of microglia depends on the age of the cell or organism (Walter and Neumann, 2009). Therefore, we will next address the involvement of microglia in different development stages of the brain (Fig. I.3). Neonatal period Aging brain CNS development and remodelling Phagocytosisof cellular debris Embryonic development Resident immune cell population Reactivityto brain injury Active pathology sensors Functional plasticity Adult stage Senescence and dystrophy Lossoffunction Fig. I.3. Microglia along brain development. Summary of the role of microglia in different brain development stages, from embryonic period to the aging brain Embryonic period The origin of microglia has been a very contentious issue over the years. However current view concerning microglial ontogenesis comprises a mesodermal origin for microglia corroborating the original observations made by Pio del Rio-Hortega that microglia arise from polyblasts or embryonic cells of the meninges (Cuadros and Navascues, 1998). In fact, microglia are descendants of mesodermal fetal macrophages which appear early in development and derive from primitive macrophages of the yolk sac. The entry of fetal macrophages into the neuroepithelium and invasion through the CNS occurs later in embryonic development, at about mid-gestation or earlier in rodents, and late during the first trimester in humans (Alliot et al., 1999; Chan et al., 2007; Streit, 2001). Additionally, it is now accepted that microglial cells can be acutely derived from blood-borne monocytes in the adult upon certain pathologic conditions (Graeber and Streit, 2009; Kaur et al., 2001). After their entry in the CNS, microglial precursors proliferate and spread through the CNS by tangential and radial migration. Finally, mesodermal precursors originate resident microglia that display an amoeboid-globoid appearance, with an enlarged cell body and retracted processes (Chew et al., 2006; Cuadros and Navascues, 1998). Microglia play an active role in CNS development and remodelling since they phagocyte dead cells, cellular debris and aberrant axons (Kim and de Vellis, 2005; Streit, 2001). Later in development microglia also participate in neuritogenesis, axonal growth and 9

38 Chapter I guidance, as well as on the subsequent synaptogenesis and even in vasculogenesis. The production of trophic factors by microglia also contributes for the development, proliferation and growth of neurons and glia (Cuadros and Navascues, 1998; Nakajima and Kohsaka, 2004) Neonatal period In the neonatal period microglial cells transform into a more ramified morphology constituting the resident immune cell population and presenting a slow turnover rate (Cuadros and Navascues, 1998). An interesting study suggests that resident microglia, in contrast to immune cells in other tissues, are not terminally differentiated along the myeloid lineage. This immature state of microglial cells could explain their graded response to activation as well as their enormous plasticity and capacity to assume different phenotypes upon brain injury (Santambrogio et al., 2001). Birth is not a deadline for the end of brain maturity since several key processes such as synaptogenesis and gliogenesis take place postnatally rendering the brain highly vulnerable to injury during the neonatal period. (Rice and Barone, 2000). Interestingly, microglia has been implicated in neonatal pathologic conditions such as hypoxic-ischemic (Vexler and Yenari, 2009) or excitotoxic brain injury (Dommergues et al., 2003). In these cases, activated microglial cells rapidly accumulate in the injured neonatal brain displaying up-regulation of cell surface markers such as major histocompatibility complex I and II (MHC I and II) or complement receptors and producing inflammatory mediators (Doverhag et al., 2010) and ROS. The inflammatory response, in conjunction with excitotoxic and oxidative responses, is the major contributor to ischemic injury in the immature brain (Vexler and Yenari, 2009) Microglia in the aging brain An additional phenotype has recently been added to the concept of microglial functional plasticity: a dystrophic or senescent phenotype (Streit et al., 2004). The prevalence of degenerating microglia increases dramatically in Alzheimer s disease (AD), co-localizing with neurofibrillary degeneration (Graeber and Streit, 2009). Moreover, fragmentation of cytoplasm (cytorrhexis) has been pointed to be indicative of widespread microglial degeneration in amyotrophic lateral sclerosis (ALS) models (Fendrick et al., 2007) and beading and clusters of fragmented twigs have also been demonstrated in the aged brain (Hasegawa-Ishii et al., 2010). Such degenerative changes may underlie a loss of microglial functionality and support, leading to pathological outcomes like neurodegenerative changes and synapse loss (Streit and Xue, 2009). This concept contributes for the growing notion that microglial activities are 10

39 General Introduction essentially beneficial, becoming destructive only when they escape from the strict control imposed on them (Schwartz et al., 2006). 3. Functional roles of microglia activation and overactivation The definition of microglia s function in the adult brain is not simple since it encompasses the ability to respond to environmental changes and toxic insults (Fig. I.4). Dystrophy loss of function hv Surveying functions Chemotaxis migration fg Debris clearance Neurotrophic functions fg Productionof inflammatory mediators Fig. I.4. Functional activation states of microglia. Resident microglia can become activated to adopt one of many diverse phenotypes depending on several circumstantial variables and these reactive phenotypes may be alternated along disease progression. Adapted from Perry et al. (2010). Perhaps the most comprehensive term is pathology sensor coined by Kreutzberg (1996). In fact, microglia assume an activated state and exhibit immunological functions in response to danger signals. Nevertheless, several evidences have demonstrated that overactivation might lead to microglial cell death, possibly as a safety mechanism to prevent further deleterious effects (Liu et al., 2001; Polazzi and Contestabile, 2006). In the following sections we will approach the several functions performed by microglia in the healthy and injured brain. 11

40 Chapter I 3.1. Surveillance functions and role in synaptic plasticity As stated above, resident and so-called resting microglia are not functionally silent cells. In vivo two-photon microscopy demonstrated that microglial processes are remarkably motile presenting high velocities of extension and retraction interspersed with brief static periods. By continuously sampling the environment with their highly motile protrusions, microglia exert an important surveillance function and completely scan the brain parenchyma every few hours (Nimmerjahn et al., 2005). Interestingly, adenosine triphosphate (ATP) released from damaged tissue regulates microglial branch dynamics in the intact brain and mediates a rapid microglial response towards injury (Davalos et al., 2005). Recently microglia have been shown to directly interact with synapses by establishing intimate but transient connections with pre-synaptic and post-synaptic elements. The frequency of these connections in the healthy brain corresponded with the static periods observed by Nimmerjahn et al. (2005), while this connectivity increased significantly during ischemic lesion (Wake et al., 2009) or after excitotoxic brain injury (Hasegawa et al., 2007). Such observations led to the assumption that microglia monitor the synapse functional status making brief contacts with the healthy ones and prolonged contacts with the injured or pathological synapses. Additionally, pre-synaptic buttons disappear after prolonged contact with microglia in ischemic conditions, suggesting that microglia might produce synaptic stripping in a similar fashion as the one observed after facial nerve axotomy (Wake et al., 2009). In this latter case, microglia become activated and promote the detachment of afferent synaptic terminals from the surface of motoneurons in an attempt to remove the excitatory input and limit further deleterious outcomes (Kreutzberg, 1996; Moran and Graeber, 2004). These findings underscore microglia s function as sensors of the integrity of the CNS circuitry and may render them a novel epithet as the CNS electrician (Graeber and Streit, 2009). Moreover, mounting evidence of microglial interaction with synaptic elements contributes for the concept that microglial cells also participate in synaptic plasticity, a process connoted as a hallmark of neurons (Ben Achour and Pascual, 2010). Synaptic plasticity is the basis of learning and memory and consists in a modification of synaptic strength (Ben Achour and Pascual, 2010). Evidence of communication between microglia, astrocytes and neurons relies on the expression of matching sets of transmitters and receptors. Indeed, microglia express a variety of neurotransmitter receptors such as AMPA, mglurs, GABA receptor, as well as purinergic, cholinergic, adrenergic, cannabinoid, dopaminergic and opioid receptors. Activation of those receptors may trigger the release of several neuroactive substances like glutamate, ATP, nitric oxide (NO), cytokines and chemokines (Pocock and Kettenmann, 2007). 12

41 General Introduction The high density of purinergic P2X7 receptors in microglia mediates microglial participation in the synapse and incited the emergence of quadpartite synapse concept (Fig. I.5) (Bennett, 2007). Astrocyte ATP P2X7 Microglia ATP P2X7 AMPA TNF α AMPA Pre synaptic neuron Glu NMDA Post synaptic neuron Fig. I.5. Microglia are vital members of the quadpartite synapse, which also includes pre-synaptic and post-synaptic neurons, and astrocytes. Input to the presynaptic terminal induces glutamate (Glu) release which binds to α-amino-3-hydroxyl-5- methyl-4-isoxazole-propionate (AMPA) and N-methyl-D-aspartate (NMDA) receptors on the post-synaptic neuron and also to AMPA receptors on the astrocytes end-feet, leading to the release of adenosine triphosphate (ATP) from the astrocyte. This acts as a chemoattractant driving microglia into intimate contact with synapses. ATP binds to P2X7 receptors on the pre-synaptic terminal, potentiating glutamate release from the terminal and acts on P2X7 receptors on microglia, leading to the release of tumour necrosis factor (TNF)-α which increases the effectiveness of the AMPA receptors on both the neuron and astrocytes. Adapted from Bennett et al. (2009). ATP released from astrocyte processes at the synapse acts as a chemoattractant on microglia driving them into intimate contact with synapses. Once microglia processes are interlaced at the synapse they can themselves release ATP due to nerve terminalderived glutamate. ATP may also act on synaptic microglia P2X7 receptors to evoke the release of cytokines such as tumour necrosis factor-α (TNF-α), which can then increase AMPA receptors in the post-synaptic membrane processes. Thus the P2X7 receptor 13

42 Chapter I mediates mechanisms that give rise to significant changes in the efficacy of synapses (Bennett et al., 2009) Role of microglia in innate and adaptive responses Microglia constitute the first line of defence against invading microorganisms (Town et al., 2005) and are intricately implicated in the initiation and propagation of the inflammatory response (Chew et al., 2006). Activation of microglia can result in different functional phenotypes entailing several characteristic features such as up-regulation of cell surface markers, production of pro-inflammatory mediators and phagocytosis (Hanisch and Kettenmann, 2007). Some authors refer to microglial activation as a continuum where the innate or phagocytic response is at one end and the adaptive response (comprising antigen presentation functions) is at the other (Town et al., 2005). Therefore, the duration and type of stimulation received by microglia will determine the activation features displayed by these cells reinforcing their remarkable plasticity in response to injury (Town et al., 2005) Phagocytosis Phagocytosis is defined as the process by which large particles (>0,5 µm) are recognized, internalized and digested by phagocytic cells involving actin-dependent mechanisms. This process is critical for the removal of infectious agents or senescent cells and is performed by immune cells of the body such as macrophages and microglia. Interaction of specific receptors expressed by phagocytic cells with ligands in the surface of the particle is the first step for particle internalization, followed by actin polymerization at the site of ingestion and internalization culminating in the formation of a phagolysosome. Recognition mechanisms can be cell-mediated by cellular receptors like integrins or mannose and scavenger receptors or humoral by the opsonisation of the invading pathogen by antibodies or complement proteins which are then recognized by specific receptors (Aderem and Underhill, 1999). Activation of microglial phagocytic receptors can be associated with the production of inflammatory products, as occurs in the case of Toll-like receptors (TLRs) or Fc- receptors interaction with microbes (Neumann et al., 2009). On the other hand, phagocytosis may occur in the absence of inflammation by the engagement of several receptors such as complement, scavenger, phosphatidylserine (Neumann et al., 2009) or purinergic (Koizumi et al., 2007) receptors as well as triggering receptor expressed on myeloid cells-2 (TREM-2) (Takahashi et al., 2005). In fact, removal of apoptotic cells by phagocytosis is described as a rather silent process which is essential during brain development as well as in some pathologic conditions. Studies in hippocampal slices 14

43 General Introduction show that microglia rapidly respond to neuronal death leading to phagocytic clearance (Petersen and Dailey, 2004). Several putative eat-me signals have been established for apoptotic cells such as phosphatidylserine externalization, interaction with vitronectin receptors and changes in the pattern of glycosilation of surface proteins allowing microglial recognition of apoptotic cells by lectin receptors (Ravichandran, 2003; Witting et al., 2000). As described previously, the phagocytic role of microglia in CNS development and remodelling is widely established as these cells promote a clearance of dead or dying cells and debris. In addition, microglial phagocytic activity is not limited to clearing the corpses but it is also implicated in promoting axon pruning and cell death (Mallat et al., 2005) as demonstrated by the studies of Marin-Teva et al. (2004) demonstrating that microglia provoke the death of developing Purkinje cells by producing high levels of superoxide. A putative hypothesis for the recognition of these Purkinje cells as prey for phagocytic microglia involves the activation of caspase-3 which can lead to the production of chemotactic agents like lysophosphatidylcholine (Lauber et al., 2003). A role for microglia in the removal of unwanted synapses tagged by complement has also been demonstrated (Stevens et al., 2007). In the context of brain pathology, phagocytosis performed by microglia also plays an essential role in the clearance of tissue debris and, therefore, in the generation of a pro-regenerative environment (Neumann et al., 2009). In fact, microglia is able to phagocyte myelin debris upon a demyelinating insult (Glezer et al., 2007; Takahashi et al., 2007) and amyloid-β (Aβ) from senile plaques (Kakimura et al., 2002), thus triggering repair. Nonetheless, an inadequate regenerative response may prevail if insufficient clearance by microglia occurs (leading to pathology) or when microglial phagocytic properties decline with aging (Zhao et al., 2006) Production of mediators in neuroprotection and neurodegeneration Activated microglia release a plethora of soluble factors, which may be proinflammatory and cytotoxic or, in contrast, exert neuroprotective or neurotrophic functions (Block and Hong, 2005; Kim and de Vellis, 2005). In fact, microglia are recognized producers of neurotrophic molecules such as neurotrophins (NT), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), basic fibroblast growth factor, hepatocyte growth factor, glial cell-line derived neurotrophic factor (GDNF), plasminogen, platelet-derived growth factor, among several others (Nakajima et al., 2001a; Nakajima and Kohsaka, 2004). This panoply of molecules have been reported to exert important roles in the development of the CNS given their implication in the survival, maturation and differentiation of neurons, 15

44 Chapter I astrocytes and oligodendrocytes as well as their involvement in neurite outgrowth and synapse homeostasis (Kim and de Vellis, 2005; Nakajima and Kohsaka, 2004). Furthermore, upon exposure to toxic insults, microglia also proved to secrete molecules with a neuroprotective potential. For instance, lipopolysaccharide (LPS) stimulation of microglia led to increased production of BDNF, NT-4/5 and NGF suggesting the participation of these cells in neuronal regeneration (Miwa et al., 1997; Nakajima et al., 2001a). After middle cerebral artery occlusion (MCAO), microglia secretes increased amounts of insulin-like growth factor-1 that can promote neural precursors proliferation, survival and differentiation, thus rendering microglia a pro-neurogenic role upon stroke (Thored et al., 2009). In addition, glutamate may signal microglia to release neurotrophic factors like BDNF, GDNF and NGF as an attempt to rescue neurons from excitotoxic damage (Liang et al., 2010). Microglia can also play additional neuroprotective actions as glutamate scavengers by the expression of glutamate transporter-1 (GLT-1) (Nakajima et al., 2001b). Moreover, GLT-1 expression by microglia is up-regulated upon motoneuron injury in axotomized rat facial nucleus (Lopez-Redondo et al., 2000) or in response to TNF-α production (Persson et al., 2005). Glutamine synthetase, the enzyme responsible for converting glutamate into the less toxic glutamine, is also expressed in activated microglia (Chretien et al., 2002; Rimaniol et al., 2000). Furthermore, the concept of protective autoimmunity, developed by Schwartz et al. (2003) illustrates an interaction between activated microglia and the adaptive immune system (namely T-cells) which ultimately contributes for the orchestration of glutamate homeostasis (Schwartz et al., 2003). On the other hand, activation of microglia is also associated with the release of a myriad of potentially cytotoxic substances such as ROS, proteases, pro-inflammatory cytokines, chemokines, glutamate, among others (Aloisi, 2001; Kim and de Vellis, 2005). In fact, microglia is one of the principal sources of cytokines in the brain, such as IL-1β, TNF-α and IL-6 (Hanisch, 2002). Cytokines are low molecular weight proteins which participate in a wide range of biological responses such as development and modulation of inflammation (Acarin et al., 2001; Gebicke-Haerter, 2001). Interleukin (IL)-1β and TNFα are two important cytokines with pleiotropic functions (Fig. I.6). Both cytokines are implicated in CNS inflammation given their ability to induce the expression of adhesion molecules and chemokines leading to BBB breakdown and facilitating leukocyte recruitment into the CNS (Allan and Rothwell, 2003; Hanisch, 2002; Lee and Benveniste, 1999). 16

45 General Introduction Fig. I.6. Overview of the signaling pathways following cytokine receptor activation. Tumour necrosis factor (TNF)-α interacts with its specific receptor TNFR-1 initiating intracellular cascades through the recruitment of TNFR-associated factor (TRAF) 2. This event may lead to TNF-α-induced phosphorylation of members of the mitogen-activated protein kinases (MAPKs) family such as MAPK kinase kinases (MEKKs) and subsequently of the MAPKs [namely p38, extracellular signal regulated kinases 1 and 2 (ERK1/2) and c-jun N-terminal kinases 1 and 2 (JNK1/2)]. MAPKs activation may then induce the activation of transcription factors (TF) such as nuclear factor-kb (NF-kB). Stimulation of the NF-kB pathway may be initiated by the phosphorylation of the inhibitor of NF-κB (IκB) kinase complex (IKK) which is crucial for the degradation of IκB allowing NF-κB to translocate into the nucleus and promote gene transcription. Additionally, Interleukin (IL)-1 may also trigger NF-κB and MAPKs activation by the engagement of its membrane receptor IL-1R and consequent recruitment of myeloid differentiation factor 88 (MyD88). This latter adaptor protein associates with IL-1R-associated kinase 4 (IRAK 4) and subsequently activates TRAF6. These pathways may consequently result in modification of the inflammatory response and even cell death. IL-1β has been broadly implicated in acute neuronal injuries such as the ones resulting from ischemic or excitotoxic challenges (Hagberg et al., 1996). In fact, studies where the action of IL-1β was inhibited culminated in marked reduction of the extent of cell death elicited by ischemic, traumatic or excitotoxic brain injury (Allan and Rothwell, 17

46 Chapter I 2003). TNF-α was reported to promote neuronal (Venters et al., 2000) and oligodendroglial cell death and is also involved in demyelination (Akassoglou et al., 1998). Nevertheless, the neuroprotective or neurotoxic outcomes induced by TNF-α seem to depend on the existence of two distinguishable signalling pathways mediated through the TNF receptor (TNFR) 1 and 2 (Arnett et al., 2001). Indeed, a recent report demonstrates that microglia up-regulates TNFR2 expression in response to an inflammatory stimulus and that this event leads to the induction of anti-inflammatory pathways (Veroni et al., 2010). In addition, IL-6 can have both anti- and pro-inflammatory actions since it can significantly reduce ischemic brain damage after MCAO (Loddick et al., 1998) or, in contrast, contribute to the pathophysiology of many diseases (Gadient and Otten, 1997). Microglial cells express surface receptors for this multitude of cytokines, thus propagating the inflammatory response (Aloisi, 2001). Among the upstream signalling pathways involved in the production of cytokines by microglia are mitogen-activated protein kinases (MAPKs), responsible for the phosphorylation of several transcription factors such as nuclear factor-κb (NF-κB) or activator protein-1 (AP- 1) (Koj, 1996; Roux and Blenis, 2004). The nuclear translocation of these factors may culminate in the production of the above described cytokines as well as chemokines, complement factors and others (Nakajima et al., 2006). A correlation between Notch-1 and NF-κB was recently confirmed in LPSstimulated microglia demonstrating that Notch signalling may amplify the proinflammatory response of microglia by enhancing NF-κB signalling (Cao et al., 2010). The signal transducers and activators of transcription (STAT) pathway might also be activated in microglia upon ischemic lesion and lead to enhanced production of inflammatory products (Justicia et al., 2000; Planas et al., 1997). Cytokine release can also raise the production of other inflammatory mediators such as NO (Chao et al., 1995). Microglial cells are reported to generate a burst of NO in response to injury, which may lead to neuronal dysfunction and cell death (Bal-Price and Brown, 2001; Cassina et al., 2002; Kawase et al., 1996; Sunico et al., 2010). Interestingly, oxidative stress may lead to excessive glutamate production in microglia, culminating in excitotoxic brain damage (Barger et al., 2007; Golde et al., 2002). The release of glutamate in microglia is conveyed by the Xc antiporter, an heterodimeric protein complex which exchanges extracellular cystine for intracellular glutamate (Barger and Basile, 2001; Domercq et al., 2007). Glutamate is an excitatory neurotransmitter but when present in the synaptic space above a certain threshold will lead to excitotoxicity, that is, to the excessive activation of its receptors (namely NMDA, AMPA or kainate 18

47 General Introduction receptors) with subsequent increase in intracellular calcium influx that culminates in neuronal death (Johnston, 2005). The extensive list of bioactive substances released by microglia in response to brain injury encompasses molecules with chemoattractant properties such as chemokines and MMPs (involved in the degradation of ECM components facilitating leukocyte recruitment); molecules implicated in the initiation and propagation of inflammation like prostanoids produced by cyclooxigenase-2 (COX-2) and complement factors; substances involved in endoplasmic reticulum stress such as cathepsins; antiinflammatory cytokines like IL-10 and transforming growth factor (TGF)-β; and others (Aloisi, 2001; Kim and de Vellis, 2005; Nakajima and Kohsaka, 2004). The inflammatory response engaged by microglia has also been implied in the inhibition of neurogenesis (Ekdahl et al., 2003; Monje et al., 2003) adding on the notion that inflammation equals toxicity. Nevertheless, inflammation is a normal response of the organism to infection, injury and trauma and some authors envisage this response as primarily beneficial (Amor et al., 2010; Harry and Kraft, 2008) and as an internal mechanism for repair (Schwartz et al., 2003). Yet, deregulation of the inflammatory response and chronic immune activation can lead to deleterious effects and ultimately cell death (Graeber and Streit, 2009; Harry and Kraft, 2008). It is, therefore, important to recognize the beneficial aspects of the immune response and harness them, in order to contribute to damage resolution instead of aggravating injury. 4. Microglial reactivity and modulation by cell interplay 4.1. Models for the evaluation of microglial reactivity Activation of microglia comprises the up-regulation of several surface markers, which in the resting or surveying state are expressed at low or moderate levels (Aloisi, 2001; Guillemin and Brew, 2004). Some of these markers have been used to identify microglia in tissue sections and cell cultures (Fig. I.7) but, the existence of a single specific molecular marker for microglia is still unknown (Graeber and Streit, 2009). This is explained by the fact that microglia share several antigens with macrophages, endothelial cells, lymphocytes, among other cells (Guillemin and Brew, 2004). Therefore, the known microglia markers are cell-specific in the sense that they do not label other glial cells or neurons (Graeber and Streit, 2009). Some of the most commonly used surface markers are lectins (carbohydrate binding proteins) such as wheat germ agglutinin, mistletoe lectin, Ricinus communis agglutinin-1, Griffonia simplicifolia B4 isolectin and Lycopersicon esculentum (tomato) lectin, which present an increased 19

48 Chapter I affinity for microglial cell but also bind neurons and myelin nodes and membranes (Acarin et al., 1994). Other very commonly used markers are β2-integrins (CD11a, CD11b and CD11c); the ligand for CD11a is intercellular adhesion molecule-1 whereas the ligands for CD11b and CD11c are complement proteins (Kim and de Vellis, 2005). CD11b is also commonly known as complement rerceptor-3 (CR3), and the antibodies aimed at its recognition are orexin (OX) 42 or macrophage receptor 1 (Mac 1) depending on the rat or mouse origin of the protein, respectively. Additional antibodies have been proposed for the detection of activated microglia such as ED-1, also called CD-68, which is expressed on the membranes of cytoplasmic granules such as phagolysosomes and has been shown to correlate with phagocytic activity (Damoiseaux et al., 1994); or OX-6 that recognizes MHC class II antigens (Guillemin and Brew, 2004). One of the most versatile immunocytochemical marker for microglia is ionized calcium-binding adaptor molecule 1 (Iba1), an EF-hand protein which is up-regulated in activated microglia (Imai et al., 1996). A B Fig. I.7. Surface markers used for microglial labelling. Rat primary cortical microglia are labelled with ionized calcium-binding adaptor molecule 1 (Iba1) (A) or with orexin (OX) 42, an antibody raised against rat complement receptor 3 (B). Scale bar represents 20 µm. However, the expression of these surface markers depends on the activation state of microglial cells, as demonstrated by Cristovão and colleagues (2010), and its presence is not always constant in all activation processes. For instance, activated microglia accumulated in the facial nucleus after axotomy do not express ED-1 (Graeber et al., 1998), and MHC class II can be also found in ramified microglia besides being present in the activated state (Streit et al., 2004). These observations further corroborate the functional plasticity exhibited by microglia. Additionally, different behaviors have been 20

49 General Introduction reported for microglial cells when applying in vitro or in vivo models which may be attributable to the fact that these cells are always activated to same extent when in culture somewhat limiting the ability to study microglial actual functions (Farber and Kettenmann, 2005) and also to the lack of interaction with neighboring cells, a determinant factor for the modulation of microglial reactivity (Hanisch and Kettenmann, 2007). The abundance of surface receptors expressed by microglia as well as the panoply of soluble factors produced by these cells certainly vouches for its ability to interact with other cells. The most striking example is the capacity of microglia to function as an antigen presenting cell. Microglia bind the processed antigens to MHC complex on its surface and engage interaction with T-cells. Optimal T-cell-microglia adhesion and reciprocal activation are achieved through additional interactions between adhesion and co-stimulatory molecules i.e. lymphocyte function-associated antigen 1, B7-1 and B7-2 molecules (CD80 and CD86), CD40 and their specific counterparts in the surface of T- cells (Aloisi, 2001; van Kooten and Banchereau, 2000). Therefore, when evaluating microglial response towards toxic or external stimulus several experimental approaches can be used in order to explore the cross-talk between microglia and nerve or immune cells (Fig. I.8). The conditioned medium model is used in this thesis and aims at the evaluation of the influence of soluble factors on cell reactivity. In fact, soluble factors released by either neurons or glial cells have proven to reciprocally influence the development and reactivity of these cells (Gomes et al., 2001). Mixed culture models, also applied in this thesis, rely on cell-to-cell interactions, such as ligand-receptor interaction and phagocytosis. Finally, organotypic slice cultures (OSCs) are heralded as the most approximate model to the in vivo conditions. In this model the three dimensional architecture of the brain is maintained, recapitulating the cellular contacts existent between neurons and glial cells. OSC are thick sections ( µm) usually obtained from the early post-natal period by the roller tube method (in which the cultured tissue is maintained attached to a coverslip rotating in the incubator) or by the interface method developed by Stoppini et al. (1991), where explanted tissues are layered onto a semiporous membrane and kept at an air liquid interface (Lossi et al., 2009). 21

50 Chapter I Isolated cultures Conditioned medium CM Mixed cultures Organotypic slice cultures Evaluation of reactivity at a cellular and molecular level Assessment of modulatory effects of released soluble factors Investigation of cell to cell proximity dependent interactions Evaluation of cellular interplay in a 3D brain structure Fig. I.8. Schematic representation of experimental models and respective main applications used for the evaluation of microglial reactivity Reciprocal reactivity modulation by interplay of microglia with neighboring cells Neurons have been mainly regarded as the victims of overactivated microglia, nevertheless, compelling evidence demonstrates the existence of reciprocal signalling between neurons and microglia (Biber et al., 2007). Some authors have listed several signals by which microglial function seems to be controlled and divided them in off or on signals. Off signals are constitutively found in the healthy brain and its absence may trigger microglial activation (Biber et al., 2007). The classical example of an off signal is CD200, a member of the immunoglobulin (Ig) superfamily constitutively expressed at the neuronal membrane, which is able to bind to its receptor CD200R, at the surface of microglia, leading to the inhibition of microglial inflammatory functions (Hoek et al., 2000). Among the off signals are also chemokines such as CX3CL1 or fractalkine that exert a similar microglial inhibition (Mizuno et al., 2003; Zujovic et al., 2000). Therefore, off signals can be envisaged as a way of neurons to maintain microglia in a resting state in the healthy brain. On signals, by the other hand, are described as substances produced by endangered or injured neurons and may lead to microglial activation. Some of the on signals described are purines like ATP (Koizumi et al., 2007), chemokines like CCL21 (Zhao et al., 2007) and MMPs (Kim et al., 2005), 22

51 General Introduction which may induce microglial chemotaxis towards the injury site and enhance its phagocytic activity. Whether the outcome resulting from neuron-microglia signalling is either beneficial or detrimental is the question many researchers try to answer employing different study models. Using the conditioned medium model (Polazzi and Contestabile, 2006) found that soluble factors released by healthy neurons lead to an overactivation of LPS-stimulated microglia culminating in the apoptotic elimination of microglia, probably as a safety mechanism. Reciprocally, activated microglia release factors such as NO that cause neuronal cell death and neurite destruction (Munch et al., 2003). In addition, the severity of neuronal injury appears to modulate the production of neurotrophic or neurotoxic molecules by microglia (Lai and Todd, 2008). In fact, some studies have reported an increased production of neurotrophins by microglia upon exposure to neuronal conditioned medium (Nakajima et al., 2007). However, not only soluble factors but also cellular interaction has to be accounted for when studying microglial response to activating factors. In fact, if unstimulated microglia promotes neuronal survival by the production of soluble factors, when in contact with neurons (in a mixed culture system) and upon LPS stimulation, they lead to neuronal death, a situation that failed to occur when conditioned media from LPS-stimulated microglia was applied to neurons (Zhang and Fedoroff, 1996). Similarly, a proximity dependent mechanism, probably mediated by NO, seems to govern microglial-induced neuronal death (Gibbons and Dragunow, 2006). In contrast, there are also examples of neuron-microglia mixed cultures where neurons seem to reduce microglial responsiveness to LPS (Chang et al., 2001). Microglia in OSCs have demonstrated to migrate specifically towards sites of excitotoxic injury (Heppner et al., 1998) and to participate in neuronal death in both excitotoxicity (Bernardino et al., 2008; Dehghani et al., 2004) and hypoxia-ischemia models (Leonardo et al., 2009) owing to the production of pro-inflammatory cytokines, NO and MMPs. However, the neurotoxic or neuroprotective effects of microglia upon excitotoxic injury may also depend on the concentration of inflammatory cytokines produced and on the signalling pathways engaged (Bernardino et al., 2005). Regarding microglial interplay with astrocytes, the latter cells produce substances like colony stimulating factors (CSFs) which act as mitogens promoting microglial proliferation (Suzumura et al., 1990). Moreover, microglia are able to sense astrocytic calcium waves responding to it by the activation of purinergic receptors (Farber and Kettenmann, 2005). Upon brain injury, nucleotides, chemokines and adhesion molecules rapidly activated at the astrocytes surface act as chemoatractants driving microglia towards the lesion site (Davalos et al., 2005; Raivich, 2005). 23

52 Chapter I Conversely, astrocytes have been reported to down-regulate microglial ROS production, probably as a mechanism of preventing excessive brain inflammation (Min et al., 2006). The same was verified by Eskes et al. (2003) in an astrocyte-microglia mixed culture where TNF-α release was decreased after exposure to a neurotoxicant. In accordance, astrocytes were shown to counteract microglial glutamate-induced neuronal death in a model using both conditioned media and mixed cultures (Liang et al., 2008). In contrast, in a study employing OSCs, astrocytes react to excitotoxicity by the production of the chemokines CXCL10 which interact with its specific microglial receptor culminating in neuronal death (van Weering et al., 2010). Another study regarding the role of IL-18 in neuropathic pain demonstrated that this cytokine mediates astrocyte-microglia interaction and further enhances pain (Miyoshi et al., 2008). Microglia has been long implicated in demyelinating diseases given its ability to phagocyte myelin and to produce potential harmful factors such as TNF-α, which may lead to oligodendroglial death (Selmaj and Raine, 1988a; Selmaj and Raine, 1988b). A recent report showed that conditioned medium from LPS-activated microglia attenuated primary OPC proliferation without inducing cell death (Taylor et al., 2010). In contrast, microglial activation was shown to enhance OPC recruitment and to promote debris removal, rendering the brain tissue more responsive to myelin repair (Glezer et al., 2007). In summary, interplay between CNS cells may have a strong and diverse influence on the cascade of microglial responses and, in exchange, microglial activation may also interfere with the reactivity of the remaining nerve cells leading to beneficial or detrimental outcomes depending on the environmental circumstances. 5. Involvement of microglia in the progression of neurological diseases Microglial activation is the consequence of virtually all conditions associated with neuronal injury. Therefore, the involvement of microglia in several neurological diseases is notorious. In fact, the presence of activated microglia in regions of brain injury led to the assumption that these cells where accountable for pathological effects (Harry and Kraft, 2008). Nevertheless, emerging research as abovementioned proved the essential roles of microglia in the brain s normal functions as well as their neuroprotective effects in some pathologic conditions Acute and chronic neurological diseases Among the most common acute neurological diseases are CNS infections by different strains of bacteria, viruses or parasites (Rock et al., 2004). Innate immune cells such as microglia interact with invading pathogens by pattern recognition receptors that 24

53 General Introduction bind to structures designated as pathogen-associated molecular patterns (PAMPs) (Janeway, 2001). Among the pattern recognition receptors are TLRs, CD14 receptor, mannose receptors and CR3 and their interaction with PAMPs leads to the expression of MHC class II and co-stimulatory molecules thus facilitating antigen presentation (Janeway, 2001; Town et al., 2005). In fact, some authors stated that microglial role in the defence of CNS against invading microorganisms relies mainly on their ability to call in the troops, meaning systemic lymphocytes, monocytes and neutrophils (Rock et al., 2004). Moreover, upon activation due to pathogen recognition, phagocytosis is engaged. Phagocytic engulfment of the pathogen is often followed by a respiratory burst involving the production of ROS (Block and Hong, 2005) aiming at eliminating the pathogen. Microglia also participate in the opsonization of pathogens aiding in their recognition and elimination (Hadas et al., 2010). In acute CNS injury like entorhinal cortex injury (Rappert et al., 2004) or facial nerve axotomy (Graeber et al., 1998) microglia rapidly migrates towards the lesion and proceeds with the removal of injured neurons, thus exerting a pro-regenerative function. In hypoxic-ischemic brain injury, a leading cause of mortality and morbidity, microglia and macrophages accumulate at the lesion site (McRae et al., 1995) and may cause injury through the production of pro-inflammatory mediators (Doverhag et al., 2010; Hagberg et al., 1996) and consequent neuronal death (Dean et al., 2010). Yet, contrasting data suggest that inhibition of microglial activation worsens hypoxic-ischemic brain injury in a neonatal mouse model (Tsuji et al., 2004). In addition, ablation of microglial cells results in significant increase in infarct size after MCAO in the adult stage (Lalancette-Hebert et al., 2007). In this context microglia have also been demonstrated to produce neurotrophic factors in response to stroke in the adult (Thored et al., 2009), and therefore to promote recovery. Compelling evidences have demonstrated that microglial activation is one of the hallmarks of chronic neurodegenerative diseases such as AD, Parkinson s disease (PD), MS, ALS, human immunodeficiency virus (HIV) associated dementia, among others (Gebicke-Haerter, 2001), displaying both causative and exacerbating roles. The role of microglia in neurodegenerative diseases is supported by the prevalence of progressive neuronal loss and pathological levels of cytotoxic products like ROS and proinflammatory cytokines, to the production of which microglia may be accounted for (Lull and Block, 2010). AD is a neurodegenerative disease characterized by the presence of insoluble plaques containing Aβ and intraneuronal neurofibrillary tangles in the cortical region of the brain (Lull and Block, 2010). The presence of MHC class II positive microglia was 25

54 Chapter I reported in clusters formed around Aβ containing plaques in postmortem AD tissue (McGeer et al., 1988; McGeer et al., 1987). Aβ recruits and activates microglia leading to the release of NO (Ii et al., 1996), TNF-α (Dheen et al., 2005) and superoxide (Qin et al., 2002), which may ultimately culminate in neuronal damage. Moreover, chemokine receptors such as CX3CR1, which are critical for microglia neuron communication seem to be required for neuron loss in a mouse model of AD (Fuhrmann et al., 2010). Although these evidences point to microglia as neurotoxic and disease aggravating in AD, microglia was also reported to phagocyte Aβ (Das et al., 2003; Koenigsknecht- Talboo and Landreth, 2005), thus playing a neuroprotective role. The clearance of perivascular Aβ deposits by microglia seems to be mediated by the chemokine CCR2 (El Khoury et al., 2007). In fact, immunization of animals with Aβ peptide proved to provide protection against AD progression since it prompted microglial clearance of Aβ (Schenk et al., 1999). However, clinical trials in humans were arrested due to the emergence of meningoencephalitis symptoms (Senior, 2002). A contrasting theory defends that microglia do not cause bystander damage but instead become senescent and dysfunctional with normal aging, becoming disabled and deprived of their normal neuronsupporting functions. Consistently, signs of microglial degeneration were found in AD tissue, where microglia presented structural abnormalities and cell death (Streit, 2002). PD is characterized by the degeneration of dopaminergic neurons in the substantia nigra of the brain (Kim and Joh, 2006). Activated microglia was observed in the striatum of postmortem PD brains (McGeer et al., 1988) and was associated with neuronal damage due to the production of oxidative stress (Dexter et al., 1994) and inflammatory mediators (Mogi et al., 1994). In addition, minocycline, a known blocker of microglia activation, prevents loss of dopaminergic neurons (Wu et al., 2002), further reinforcing the implication of inflammation and microglia in PD progression. However, minocycline has also direct anti-apoptotic effects which may modulate microglial response since this depends on the severity of cell death or injury (Kraft et al., 2009). In fact, microglia may be neuroprotective in the early stages of disease but become neurotoxic upon dopaminergic neuron degeneration (Sawada et al., 2006). MS is the most prevalent inflammation-mediated demyelinating disease in which the myelin sheath is damaged by an autoimmune response with a clear inflammatory component (Block and Hong, 2005). Increased microglial activity was demonstrated around MS lesions (Banati et al., 2000). Microglia may contribute for the disease progression by the production of inflammatory cytokines and ROS (Murphy et al., 2010), and also by their antigen-presentation function which sparks the autoimmune response targeting myelin (Mack et al., 2003). Nevertheless, phagocytic clearance of debris has been proven favorable for myelin repair (Glezer et al., 2007). In fact, a beneficial role has 26

55 General Introduction been described for the microglial phagocytic TREM-2 receptor in an experimental autoimmune encephalomyelitis (EAE) model (Piccio et al., 2007). ALS is a very incapacitating neurodegenerative disorder characterized by the degeneration of motor neurons (Henkel et al., 2009). Studies using a transgenic animal model of ALS demonstrated the presence of structural abnormalities like cytorrhexis in microglial cells (Fendrick et al., 2007), thus reinforcing the concept that microglial dysfunction may underlie the onset or progression of chronic diseases. HIV-associated dementia is a neurological syndrome that develops in the later stages of HIV infection (Block and Hong, 2005). Microglia share some surface receptors like CD4 and chemokine receptors with peripheral lymphocytes, which enables them to harbor HIV and consequently act as viral replication sites (Garden, 2002). Indeed, microglia are activated by HIV proteins such as Tat (D'Aversa et al., 2004) and gp120 (Kong et al., 1996) leading to the production of ROS, cytokines, chemokines and NO (Brabers and Nottet, 2006) which ultimately lead to neuronal death. Some authors claim even that persistent infection with HIV may disable or deplete microglia leading the way for opportunist CNS infections and dementia (Streit, 2002) Neonatal hyperbilirubinemia Hyperbilirubinemia is a frequent condition in the neonatal period, defined by an increase in serum bilirubin above 5 to 7 mg/dl and occurring in up to 60% of full term newborns and 80% of preterm infants (Porter and Dennis, 2002; Stevenson et al., 2001). Generally designated as neonatal jaundice, it is characterized by a yellowish discoloration of the skin and mucous membranes (Cohen et al., 2010; Porter and Dennis, 2002). Unconjugated bilirubin (UCB) is formed by the catabolism of heme (Gourley, 1997; Stevenson et al., 2001) and may reach excessive levels in newborn and preterm infants due to its overproduction and to the limited ability to excrete this molecule as a result of the of enzymatic machinery immaturity in these infants (Dennery et al., 2001). In most cases, unconjugated hyperbilirubinemia occurs in a transient and non-injurious manner and is called physiologic jaundice (Ostrow et al., 2002). When UCB exceeds physiological concentrations, multifocal deposition of UCB may take place in selected regions of the brain originating bilirubin encephalopathy (Gourley, 1997; Ostrow et al., 2004). However, the serum bilirubin levels associated to the term physiologic jaundice are under scrutiny since they may not accurately define a harmless hyperbilirubinemia condition (Maisels, 2006). In fact, some studies have reported a protective effect of UCB against oxidative stress injury, when present in low levels (Dore and Snyder, 1999; Dore et al., 1999). Contrastingly, the same UCB levels may reveal safe in normal newborn infants while become potentially hazardous in 27

56 Chapter I premature infants (Stevenson et al., 2001; Watchko and Maisels, 2003) or even in term neonates displaying additional pathologies, such as sepsis (Hansen et al., 1993; Kaplan and Hammerman, 2005) or hypoxia-ischemia (Ahdab-Barmada and Moossy, 1984; Falcão et al., 2007b), which may pose as risk factors for bilirubin encephalopathy. For instance, data from magnetic resonance imaging (MRI) showed that severe cerebral palsy occurred for relatively low serum bilirubin levels in preterm infants but only at high levels in full terms (Gkoltsiou et al., 2008). Regarding prematurity as a risk factor for bilirubin toxicity, data from in vitro (Falcão et al., 2005; Falcão et al., 2006) and in vivo studies (Conlee and Shapiro, 1997; Keino and Kashiwamata, 1989) point to the existence of windows of developmental susceptibility of the CNS to bilirubin toxicity (Shapiro, 2010). The term acute bilirubin encephalopathy is used to describe the sharp manifestations of bilirubin toxicity seen in the first weeks after birth (American Academy of Pediatrics, 2004). Such manifestations, that might be reversible, progress from lethargy and decreased feeding to hypotonia and hypertonia, high-pitched cry, impairment of upward gaze, fever and seizures (Shapiro, 2003). Recommended therapeutic approaches for acute bilirubin encephalopathy are phototerapy and in the more severe cases exchange transfusion (American Academy of Pediatrics, 2004). The classic form of chronic bilirubin encephalopathy is called kernicterus, originally a pathological term referring to the yellow staining (-icterus) of the deep nuclei of the brain (kern-, relating to the basal ganglia) (Shapiro, 2010). This condition is characterized by chronic and permanent sequelae like athetoid cerebral palsy, deafness or hearing loss, impairment of upward gaze and dental enamel hypoplasia, or even death. Brain regions more prone to neuropathological lesions produced by hyperbilirubinemia are the globus pallidus and subthalamic nucleus, auditory and oculomotor brainstem nuclei, cerebellum and hippocampus (Shapiro, 2003; Shapiro, 2005). The increasing prevalence of bilirubin encephalopathy (Ebbesen, 2000; Hansen, 2000) due to earlier hospital discharge and to the implementation of less aggressive clinical approaches in the management of neonatal jaundice (Maisels and Newman, 1998) has reawakened interest in understanding the pathophysiology of this condition. Moreover, the incidence of severe jaundice is at near epidemic proportions among some developing countries (Gordon et al., 2005; Katar, 2007; Owa and Ogunlesi, 2009). There is evidence that even moderate levels of unconjugated bilirubin can produce subtle encephalopathy, referred to as bilirubin-induced neurological dysfunction (BIND) (Shapiro, 2003; Shapiro, 2005). BIND is associated with cognitive disturbances, mild neurological abnormalities (Hyman et al., 1969; Odell et al., 1970; Rubin et al., 1979), isolated hearing loss (Bergman et al., 1985; Salamy et al., 1989) and auditory 28

57 General Introduction neuropathy (Rance et al., 1999; Simmons and Beauchaine, 2000). Recent data have demonstrated a high incidence of acute auditory neuropathy spectrum disorder among neonates with severe jaundice (Saluja et al., 2010). Nonetheless, auditory dysfunction may occur in children with or without other signs of classical kernicterus given the high sensitivity of the auditory system to bilirubin toxicity (Shapiro and Nakamura, 2001). The cellular mechanism underlying bilirubin-induced hearing dysfunction seems to involve protein kinases A and C mediated GABA/glycinergic synaptic transmission in postnatal rat ventral cochlear nucleus neurons (Li et al., 2010). In addition, hyperbilirubinemia has been shown to cause degeneration of excitatory synaptic terminals in the auditory brainstem and this is associated with activation of neuronal nitric oxide synthase (nnos) (Haustein et al., 2010). Regarding cognitive disturbances and neurological abnormalities, moderate hyperbilirubinemia has been associated with minor neurologic dysfunction throughout the first year of life (Soorani-Lunsing et al., 2001). Additional studies have demonstrated that neonatal hyperbilirubinemia can have an impact on learning and memory later in infancy (Zhang et al., 2003) as well as affect long-term cognitive ability (Seidman et al., 1991). An interesting association has also been made between hyperbilirubinemia and increased vulnerability for later development of mental disorders (Dalman and Cullberg, 1999). In fact, neonatal jaundice has been considered a perinatal risk factor for autism spectrum disorders and sensory processing disorder given its higher prevalence in children suffering from these disorders (May-Benson et al., 2009). An association between hyperbilirubinemia and developmental delay, attention-deficit disorder, and autism was also observed (Jangaard et al., 2008). Similarly, schizophrenic patients showed a significantly higher incidence of hyperbilirubinemia relative to patients suffering from other psychiatric disorders, and the psychiatric symptom score was significantly higher in schizophrenic patients with hyperbilirubinemia than in patients without hyperbilirubinemia (Miyaoka et al., 2000). Another study using Gunn rats, the animal model for bilirubin encephalopathy, demonstrated that these animals displayed stereotypical behaviors, considered as positive symptoms of schizophrenia, as well as more aggressive behaviors (Hayashida et al., 2009). These findings further strengthen the neurodevelopmental hypothesis which proposes that a proportion of schizophrenia is the result of an early brain insult, either pre or perinatal, which affects brain development leading to abnormalities expressed in the mature brain (Gupta and Kulhara, 2010). In fact, UCB has been proven to cause impairment of neurotransmitter release in synaptic vesicle membranes (Roseth et al., 1998), alteration of long-term synaptic plasticity (Chang et al., 2009) and decreased synaptic activity in hippocampal slices (Hansen, 1994). In addition, UCB leads to neurite outgrowth impairment in immature cortical 29

58 Chapter I neurons (Falcão et al., 2007c) and also interferes with axonal growth cone area and spine formation (Fernandes et al., 2009). These findings further confirm that UCB s effects in the developing brain may lead to decreased synaptic connectivity, which may underlie the emergence of neurodegenerative diseases Molecular mechanisms of bilirubin-induced CNS injury Bilirubin-induced neurotoxicity is initiated at the level of the membrane. Indeed, UCB interacts with whole nerve cell and mitochondrial membranes disrupting its redox status and increasing oxidative damage (Rodrigues et al., 2002b). Rough endoplasmic reticulum abnormalities were also observed in astrocytes exposed to UCB (Silva et al., 2001a). UCB-induced membrane permeabilization leads to mitochondrial swelling and release of cytochrome c (Rodrigues et al., 2000b), which is followed by caspase-3 processing, poly-adp ribose polymerase (PARP) cleavage and Bax translocation, resulting in apoptotic cell death (Rodrigues et al., 2002a). Studies have also showed the involvement of caspase-8 in cell death provoked by UCB (Seubert et al., 2002). Susceptibility to UCB-induced damage differs with age and cellular type. Younger cells are more susceptible to UCB-induced injury (Falcão et al., 2005; Falcão et al., 2006; Rodrigues et al., 2002c; Silva et al., 2002) and to this may account the reduced expression of multidrug resistance associated protein 1 (Mrp1) in immature cells (Falcão et al., 2007a), an efflux pump involved in UCB export. Moreover, neurons are more vulnerable to death mechanisms than astrocytes (Falcão et al., 2006; Silva et al., 2002), in which an inflammatory response is triggered by UCB (Fernandes et al., 2004). Neurons are also more exposed than astrocytes to oxidative injury by UCB, for which accounts the lower glutathione stores in neuronal cells (Brito et al., 2008b) (Fig. I.9). In addition, UCB causes a bioenergetic and oxidative crisis in immature neurons, inhibiting cytochrome c oxidase activity and increasing glycolitic activity as well as ROS production (Vaz et al., 2010). Interestingly, a recent report demonstrated that oxidative damage by UCB may be mediated by overstimulation of glutamate receptors (Brito et al., 2010). Glutamate and NMDA receptors are particularly important in the pathogenesis of neonatal neuronal injury (Johnston, 2005). In fact, UCB inhibits glutamate uptake by astrocytes (Silva et al., 1999) prolonging glutamate s presence in the synaptic cleft, which may engender NMDA receptors overstimulation and excitotoxicity in both in vitro (Grojean et al., 2000; Grojean et al., 2001) and in vivo studies (Hoffman et al., 1996; McDonald et al., 1998). Astrocytes and microglia react to UCB through the increased production of proinflammatory cytokines and glutamate (Fernandes et al., 2006; Fernandes et al., 2004; 30

59 General Introduction Gordo et al., 2006). Upstream signalling pathways involved in astrocytic cytokine production elicited by UCB are MAPKs and NF-κB, which also contribute to the observed cell death (Fernandes et al., 2007a; Fernandes et al., 2006). In addition, activation of p38 MAPK is involved in bilirubin-induced granule cerebellar neuronal death (Lin et al., 2003). Moreover, decreased viability and apoptotic and/or necrotic cell death is also observed in oligodendrocytes (Genc et al., 2003) and microglial cells exposed to UCB (Gordo et al., 2006). Neuron Astrocyte Microglia Cell death (necrosis and apoptosis) Oxidative stress Cytokine release Glutamate secretion Sensitivity along DIV Cell death (necrosis and apoptosis) Oxidative stress Cytokine release Glutamate secretion and reuptake inhibition Sensitivity along DIV Cell death (necrosis and apoptosis) Cytokine release Glutamate secretion Morphological changes Fig. I.9. Major findings regarding the molecular mechanisms involved in unconjugated bilirubin (UCB)-induced toxicity. UCB triggers several toxicity-related molecular mechanisms in neurons, astrocytes and microglia. Moreover, the intensity of the observed deleterious effects depends on cell type and also on maturation stage, given that both immature neurons and astrocytes are more vulnerable to UCB-induced toxicity. (DIV days in vitro). Concerning potential therapeutical strategies to prevent UCB-induced injury, glycoursodeoxycholic acid (GUDCA) and IL-10 have already been assayed with positive results. Indeed, ursodeoxycholic acid (UDCA) and its conjugates have demonstrated neuroprotective effects by the stabilization of mitochondrial membranes (Sola et al., 2002), inhibition of mitochondrial swelling (Rodrigues et al., 2000c), cytochrome c release (Rodrigues et al., 2000a) and, ultimately, by the decrease of apoptotic cell death (Silva et al., 2001b). UDCA oral administration induces a rapid and sustained decrease in plasma UCB concentrations in Gunn rats (Cuperus et al., 2009). Moreover, recent reports have described GUDCA to prevent UCB-induced alterations in protein oxidation, lipid peroxidation, impairment of glutathione homeostasis and neuron cell death (Brito et al., 2008a) as well as its ability to ameliorate UCB-induced mitochondrial respiratory chain dysfunction and to restore cellular antioxidant potential (Vaz et al., 2010). In 31

60 Chapter I addition, both GUDCA and IL-10 can modulate astrocytic reactivity to UCB decreasing the elicited inflammatory properties and reducing cell death (Fernandes et al., 2007b). 32

61 General Introduction 6. Global aims of the thesis The main goal of the present work is to explore the role of microglia in hyperbilirubinemia by addressing microglial reactivity, characterizing the dynamics of neuron-glia interplay and identifying potential therapeutic strategies. As a first step we sought to investigate the cellular and molecular mechanisms of isolated microglial reactivity towards UCB by the evaluation of cell survival and by the assessment of the different phenotypes acquired by microglia when facing UCB. Therefore we intended to evaluate microglial phagocytic abilities and to characterize the inflammatory response engaged by these cells by studying the temporal profile of proinflammatory cytokines secretion as well its underlying signalling pathways and by the analysis of indicators of the inflammatory response such as MMPs activation and COX-2 up-regulation. Since microglia exhibited different activation stages in the presence of UCB, displaying both phagocytic and inflammatory phenotypes in a sequenced fashion along exposure time, we decided to study how neuron-glia interplay could modulate the effect of UCB. For this matter we used a conditioned media model to evaluate the influence of soluble factors released by UCB-stimulated astrocytes and neurons in microglial reactivity. We also assessed the influence of neuron-microglia cell-to-cell interactions in UCB-induced deleterious effects on immature neurons in a neuron-microglia mixed culture. Finally, we advanced one step further and sought to investigate the role of microglia in the modulation of UCB-induced neurotoxicity in organotypic-cultured hippocampal slices where selective ablation of microglia was performed. Furthermore, we aimed at unravelling the molecular mechanisms triggering UCB-induced neurotoxicity which may underlie the association of hyperbilirubinemia with mental disorders. Ultimately we evaluated the ability of GUDCA and IL-10 to prevent UCB s deleterious effects in both neuronal cultures and organotypic-cultured hippocampal slices. 33

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67 General Introduction Haustein, M. D., Read, D. J., Steinert, J. R., Pilati, N., Dinsdale, D., Forsythe, I. D., Acute hyperbilirubinaemia induces presynaptic neurodegeneration at a central glutamatergic synapse. J Physiol. Hayashida, M., Miyaoka, T., Tsuchie, K., Yasuda, H., Wake, R., Nishida, A., Inagaki, T., Toga, T., Nagami, H., Oda, T., Horiguchi, J., Hyperbilirubinemia-related behavioral and neuropathological changes in rats: a possible schizophrenia animal model. Prog Neuropsychopharmacol Biol Psychiatry. 33, Henkel, J. S., Beers, D. R., Zhao, W., Appel, S. H., Microglia in ALS: the good, the bad, and the resting. J Neuroimmune Pharmacol. 4, Heppner, F. L., Skutella, T., Hailer, N. P., Haas, D., Nitsch, R., Activated microglial cells migrate towards sites of excitotoxic neuronal injury inside organotypic hippocampal slice cultures. Eur J Neurosci. 10, Hoek, R. M., Ruuls, S. R., Murphy, C. A., Wright, G. J., Goddard, R., Zurawski, S. M., Blom, B., Homola, M. E., Streit, W. J., Brown, M. H., Barclay, A. N., Sedgwick, J. D., Downregulation of the macrophage lineage through interaction with OX2 (CD200). Science. 290, Hoffman, D. J., Zanelli, S. A., Kubin, J., Mishra, O. P., Delivoria-Papadopoulos, M., The in vivo effect of bilirubin on the N-methyl-D-aspartate receptor/ion channel complex in the brains of newborn piglets. Pediatr Res. 40, Hristova, M., Cuthill, D., Zbarsky, V., Acosta-Saltos, A., Wallace, A., Blight, K., Buckley, S. M., Peebles, D., Heuer, H., Waddington, S. N., Raivich, G., Activation and deactivation of periventricular white matter phagocytes during postnatal mouse development. Glia. 58, Hyman, C. B., Keaster, J., Hanson, V., Harris, I., Sedgwick, R., Wursten, H., Wright, A. R., CNS abnormalities after neonatal hemolytic disease or hyperbilirubinemia. A prospective study of 405 patients. Am J Dis Child. 117, Ii, M., Sunamoto, M., Ohnishi, K., Ichimori, Y., beta-amyloid protein-dependent nitric oxide production from microglial cells and neurotoxicity. Brain Res. 720, Imai, Y., Ibata, I., Ito, D., Ohsawa, K., Kohsaka, S., A novel gene iba1 in the major histocompatibility complex class III region encoding an EF hand protein expressed in a monocytic lineage. Biochem Biophys Res Commun. 224, Janeway, C. A., Jr., How the immune system protects the host from infection. Microbes Infect. 3, Jangaard, K. A., Fell, D. B., Dodds, L., Allen, A. C., Outcomes in a population of healthy term and near-term infants with serum bilirubin levels of >or=325 micromol/l (>or=19 mg/dl) who were born in Nova Scotia, Canada, between 1994 and Pediatrics. 122, Johnston, M. V., Excitotoxicity in perinatal brain injury. Brain Pathol. 15, Justicia, C., Gabriel, C., Planas, A. M., Activation of the JAK/STAT pathway following transient focal cerebral ischemia: signaling through Jak1 and Stat3 in astrocytes. Glia. 30, Kakimura, J., Kitamura, Y., Takata, K., Umeki, M., Suzuki, S., Shibagaki, K., Taniguchi, T., Nomura, Y., Gebicke-Haerter, P. J., Smith, M. A., Perry, G., Shimohama, S., Microglial activation and amyloid-beta clearance induced by exogenous heat-shock proteins. FASEB J. 16, Kaplan, M., Hammerman, C., Understanding severe hyperbilirubinemia and preventing kernicterus: adjuncts in the interpretation of neonatal serum bilirubin. Clin Chim Acta. 356, Katar, S., Glucose-6-phosphate dehydrogenase deficiency and kernicterus of South-East anatolia. J Pediatr Hematol Oncol. 29, Kaur, C., Hao, A. J., Wu, C. H., Ling, E. A., Origin of microglia. Microsc Res Tech. 54, 2-9. Kawase, M., Kinouchi, H., Kato, I., Akabane, A., Kondo, T., Arai, S., Fujimura, M., Okamoto, H., Yoshimoto, T., Inducible nitric oxide synthase following hypoxia in rat cultured glial cells. Brain Res. 738, Keino, H., Kashiwamata, S., Critical period of bilirubin-induced cerebellar hypoplasia in a new Sprague-Dawley strain of jaundiced Gunn rats. Neurosci Res. 6, Kim, S. U., de Vellis, J., Microglia in health and disease. J Neurosci Res. 81, Kim, Y. S., Joh, T. H., Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease. Exp Mol Med. 38,

68 Chapter I Kim, Y. S., Kim, S. S., Cho, J. J., Choi, D. H., Hwang, O., Shin, D. H., Chun, H. S., Beal, M. F., Joh, T. H., Matrix metalloproteinase-3: a novel signaling proteinase from apoptotic neuronal cells that activates microglia. J Neurosci. 25, Koenigsknecht-Talboo, J., Landreth, G. E., Microglial phagocytosis induced by fibrillar betaamyloid and IgGs are differentially regulated by proinflammatory cytokines. J Neurosci. 25, Koizumi, S., Shigemoto-Mogami, Y., Nasu-Tada, K., Shinozaki, Y., Ohsawa, K., Tsuda, M., Joshi, B. V., Jacobson, K. A., Kohsaka, S., Inoue, K., UDP acting at P2Y6 receptors is a mediator of microglial phagocytosis. Nature. 446, Koj, A., Initiation of acute phase response and synthesis of cytokines. Biochim Biophys Acta. 1317, Kong, L. Y., Wilson, B. C., McMillian, M. K., Bing, G., Hudson, P. M., Hong, J. S., The effects of the HIV-1 envelope protein gp120 on the production of nitric oxide and proinflammatory cytokines in mixed glial cell cultures. Cell Immunol. 172, Kraft, A. D., McPherson, C. A., Harry, G. J., Heterogeneity of microglia and TNF signaling as determinants for neuronal death or survival. Neurotoxicology. 30, Kreutzberg, G. W., Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, Ladeby, R., Wirenfeldt, M., Garcia-Ovejero, D., Fenger, C., Dissing-Olesen, L., Dalmau, I., Finsen, B., Microglial cell population dynamics in the injured adult central nervous system. Brain Res Brain Res Rev. 48, Lai, A. Y., Todd, K. G., Differential regulation of trophic and proinflammatory microglial effectors is dependent on severity of neuronal injury. Glia. 56, Lalancette-Hebert, M., Gowing, G., Simard, A., Weng, Y. C., Kriz, J., Selective ablation of proliferating microglial cells exacerbates ischemic injury in the brain. J Neurosci. 27, Lauber, K., Bohn, E., Krober, S. M., Xiao, Y. J., Blumenthal, S. G., Lindemann, R. K., Marini, P., Wiedig, C., Zobywalski, A., Baksh, S., Xu, Y., Autenrieth, I. B., Schulze-Osthoff, K., Belka, C., Stuhler, G., Wesselborg, S., Apoptotic cells induce migration of phagocytes via caspase-3-mediated release of a lipid attraction signal. Cell. 113, Lee, S. J., Benveniste, E. N., Adhesion molecule expression and regulation on cells of the central nervous system. J Neuroimmunol. 98, Leonardo, C. C., Hall, A. A., Collier, L. A., Gottschall, P. E., Pennypacker, K. R., Inhibition of gelatinase activity reduces neural injury in an ex vivo model of hypoxia-ischemia. Neuroscience. 160, Li, C. Y., Shi, H. B., Chen, Z. N., Ye, H. B., Song, N. Y., Yin, S. K., Protein kinase A and C signaling induces bilirubin potentiation of GABA/glycinergic synaptic transmission in rat ventral cochlear nucleus neurons. Brain Res. 1348, Liang, J., Takeuchi, H., Doi, Y., Kawanokuchi, J., Sonobe, Y., Jin, S., Yawata, I., Li, H., Yasuoka, S., Mizuno, T., Suzumura, A., Excitatory amino acid transporter expression by astrocytes is neuroprotective against microglial excitotoxicity. Brain Res. 1210, Liang, J., Takeuchi, H., Jin, S., Noda, M., Li, H., Doi, Y., Kawanokuchi, J., Sonobe, Y., Mizuno, T., Suzumura, A., Glutamate induces neurotrophic factor production from microglia via protein kinase C pathway. Brain Res. 1322, Lin, S., Yan, C., Wei, X., Paul, S. M., Du, Y., p38 MAP kinase mediates bilirubin-induced neuronal death of cultured rat cerebellar granule neurons. Neurosci Lett. 353, Liu, B., Wang, K., Gao, H. M., Mandavilli, B., Wang, J. Y., Hong, J. S., Molecular consequences of activated microglia in the brain: overactivation induces apoptosis. J Neurochem. 77, Loddick, S. A., Turnbull, A. V., Rothwell, N. J., Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat. J Cereb Blood Flow Metab. 18, Lopez-Redondo, F., Nakajima, K., Honda, S., Kohsaka, S., Glutamate transporter GLT-1 is highly expressed in activated microglia following facial nerve axotomy. Brain Res Mol Brain Res. 76, Lossi, L., Alasia, S., Salio, C., Merighi, A., Cell death and proliferation in acute slices and organotypic cultures of mammalian CNS. Prog Neurobiol. 88, Lull, M. E., Block, M. L., Microglial activation and chronic neurodegeneration. Neurotherapeutics. 7,

69 General Introduction Mack, C. L., Vanderlugt-Castaneda, C. L., Neville, K. L., Miller, S. D., Microglia are activated to become competent antigen presenting and effector cells in the inflammatory environment of the Theiler's virus model of multiple sclerosis. J Neuroimmunol. 144, Maisels, M. J., What's in a name? Physiologic and pathologic jaundice: the conundrum of defining normal bilirubin levels in the newborn. Pediatrics. 118, Maisels, M. J., Newman, T. B., Jaundice in full-term and near-term babies who leave the hospital within 36 hours. The pediatrician's nemesis. Clin Perinatol. 25, Mallat, M., Marin-Teva, J. L., Cheret, C., Phagocytosis in the developing CNS: more than clearing the corpses. Curr Opin Neurobiol. 15, Marin-Teva, J. L., Dusart, I., Colin, C., Gervais, A., van Rooijen, N., Mallat, M., Microglia promote the death of developing Purkinje cells. Neuron. 41, May-Benson, T. A., Koomar, J. A., Teasdale, A., Incidence of pre-, peri-, and post-natal birth and developmental problems of children with sensory processing disorder and children with autism spectrum disorder. Front Integr Neurosci. 3, 31. McDonald, J. W., Shapiro, S. M., Silverstein, F. S., Johnston, M. V., Role of glutamate receptor-mediated excitotoxicity in bilirubin-induced brain injury in the Gunn rat model. Exp Neurol. 150, McGeer, P. L., Itagaki, S., Boyes, B. E., McGeer, E. G., Reactive microglia are positive for HLA-DR in the substantia nigra of Parkinson's and Alzheimer's disease brains. Neurology. 38, McGeer, P. L., Itagaki, S., Tago, H., McGeer, E. G., Reactive microglia in patients with senile dementia of the Alzheimer type are positive for the histocompatibility glycoprotein HLA-DR. Neurosci Lett. 79, McRae, A., Gilland, E., Bona, E., Hagberg, H., Microglia activation after neonatal hypoxicischemia. Brain Res Dev Brain Res. 84, Miller, G., Neuroscience. The dark side of glia. Science. 308, Min, K. J., Yang, M. S., Kim, S. U., Jou, I., Joe, E. H., Astrocytes induce hemeoxygenase-1 expression in microglia: a feasible mechanism for preventing excessive brain inflammation. J Neurosci. 26, Miwa, T., Furukawa, S., Nakajima, K., Furukawa, Y., Kohsaka, S., Lipopolysaccharide enhances synthesis of brain-derived neurotrophic factor in cultured rat microglia. J Neurosci Res. 50, Miyaoka, T., Seno, H., Itoga, M., Iijima, M., Inagaki, T., Horiguchi, J., Schizophreniaassociated idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome). J Clin Psychiatry. 61, Miyoshi, K., Obata, K., Kondo, T., Okamura, H., Noguchi, K., Interleukin-18-mediated microglia/astrocyte interaction in the spinal cord enhances neuropathic pain processing after nerve injury. J Neurosci. 28, Mizuno, T., Kawanokuchi, J., Numata, K., Suzumura, A., Production and neuroprotective functions of fractalkine in the central nervous system. Brain Res. 979, Mogi, M., Harada, M., Riederer, P., Narabayashi, H., Fujita, K., Nagatsu, T., Tumor necrosis factor-alpha (TNF-alpha) increases both in the brain and in the cerebrospinal fluid from parkinsonian patients. Neurosci Lett. 165, Monje, M. L., Toda, H., Palmer, T. D., Inflammatory blockade restores adult hippocampal neurogenesis. Science. 302, Montgomery, D. L., Astrocytes: form, functions, and roles in disease. Vet Pathol. 31, Moran, L. B., Graeber, M. B., The facial nerve axotomy model. Brain Res Brain Res Rev. 44, Morwood, S. R., Nicholson, L. B., Modulation of the immune response by extracellular matrix proteins. Arch Immunol Ther Exp (Warsz). 54, Munch, G., Gasic-Milenkovic, J., Dukic-Stefanovic, S., Kuhla, B., Heinrich, K., Riederer, P., Huttunen, H. J., Founds, H., Sajithlal, G., Microglial activation induces cell death, inhibits neurite outgrowth and causes neurite retraction of differentiated neuroblastoma cells. Exp Brain Res. 150, 1-8. Murphy, A. C., Lalor, S. J., Lynch, M. A., Mills, K. H., Infiltration of Th1 and Th17 cells and activation of microglia in the CNS during the course of experimental autoimmune encephalomyelitis. Brain Behav Immun. 24,

70 Chapter I Nakajima, K., Honda, S., Tohyama, Y., Imai, Y., Kohsaka, S., Kurihara, T., 2001a. Neurotrophin secretion from cultured microglia. J Neurosci Res. 65, Nakajima, K., Kohsaka, S., Microglia: neuroprotective and neurotrophic cells in the central nervous system. Curr Drug Targets Cardiovasc Haematol Disord. 4, Nakajima, K., Matsushita, Y., Tohyama, Y., Kohsaka, S., Kurihara, T., Differential suppression of endotoxin-inducible inflammatory cytokines by nuclear factor kappa B (NFkappaB) inhibitor in rat microglia. Neurosci Lett. 401, Nakajima, K., Tohyama, Y., Kohsaka, S., Kurihara, T., 2001b. Ability of rat microglia to uptake extracellular glutamate. Neurosci Lett. 307, Nakajima, K., Tohyama, Y., Maeda, S., Kohsaka, S., Kurihara, T., Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons. Neurochem Int. 50, Neumann, H., Kotter, M. R., Franklin, R. J., Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. 132, Nimmerjahn, A., Kirchhoff, F., Helmchen, F., Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science. 308, Odell, G. B., Storey, G. N., Rosenberg, L. A., Studies in kernicterus. 3. The saturation of serum proteins with bilirubin during neonatal life and its relationship to brain damage at five years. J Pediatr. 76, Ostrow, J. D., Pascolo, L., Brites, D., Tiribelli, C., Molecular basis of bilirubin-induced neurotoxicity. Trends Mol Med. 10, Ostrow, J. D., Pascolo, L., Tiribelli, C., Mechanisms of bilirubin neurotoxicity. Hepatology. 35, Owa, J. A., Ogunlesi, T. A., Why we are still doing so many exchange blood transfusion for neonatal jaundice in Nigeria. World J Pediatr. 5, Pavlov, I., Lauri, S., Taira, T., Rauvala, H., The role of ECM molecules in activity-dependent synaptic development and plasticity. Birth Defects Res C Embryo Today. 72, Perea, G., Navarrete, M., Araque, A., Tripartite synapses: astrocytes process and control synaptic information. Trends Neurosci. 32, Perry, V. H., Nicoll, J. A., Holmes, C., Microglia in neurodegenerative disease. Nat Rev Neurol. 6, Persson, M., Brantefjord, M., Hansson, E., Ronnback, L., Lipopolysaccharide increases microglial GLT-1 expression and glutamate uptake capacity in vitro by a mechanism dependent on TNF-alpha. Glia. 51, Petersen, M. A., Dailey, M. E., Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices. Glia. 46, Piccio, L., Buonsanti, C., Mariani, M., Cella, M., Gilfillan, S., Cross, A. H., Colonna, M., Panina- Bordignon, P., Blockade of TREM-2 exacerbates experimental autoimmune encephalomyelitis. Eur J Immunol. 37, Planas, A. M., Justicia, C., Ferrer, I., Stat1 in developing and adult rat brain. Induction after transient focal ischemia. Neuroreport. 8, Pocock, J. M., Kettenmann, H., Neurotransmitter receptors on microglia. Trends Neurosci. 30, Polazzi, E., Contestabile, A., Overactivation of LPS-stimulated microglial cells by cocultured neurons or neuron-conditioned medium. J Neuroimmunol. 172, Porter, M. L., Dennis, B. L., Hyperbilirubinemia in the term newborn. Am Fam Physician. 65, Qin, L., Liu, Y., Cooper, C., Liu, B., Wilson, B., Hong, J. S., Microglia enhance beta-amyloid peptide-induced toxicity in cortical and mesencephalic neurons by producing reactive oxygen species. J Neurochem. 83, Raivich, G., Like cops on the beat: the active role of resting microglia. Trends Neurosci. 28, Rance, G., Beer, D. E., Cone-Wesson, B., Shepherd, R. K., Dowell, R. C., King, A. M., Rickards, F. W., Clark, G. M., Clinical findings for a group of infants and young children with auditory neuropathy. Ear Hear. 20, Rappert, A., Bechmann, I., Pivneva, T., Mahlo, J., Biber, K., Nolte, C., Kovac, A. D., Gerard, C., Boddeke, H. W., Nitsch, R., Kettenmann, H., CXCR3-dependent microglial recruitment is essential for dendrite loss after brain lesion. J Neurosci. 24, Ravichandran, K. S., "Recruitment signals" from apoptotic cells: invitation to a quiet meal. Cell. 113,

71 General Introduction Rice, D., Barone, S., Jr., Critical periods of vulnerability for the developing nervous system: evidence from humans and animal models. Environ Health Perspect. 108 Suppl 3, Rimaniol, A. C., Haik, S., Martin, M., Le Grand, R., Boussin, F. D., Dereuddre-Bosquet, N., Gras, G., Dormont, D., Na+-dependent high-affinity glutamate transport in macrophages. J Immunol. 164, Rock, R. B., Gekker, G., Hu, S., Sheng, W. S., Cheeran, M., Lokensgard, J. R., Peterson, P. K., Role of microglia in central nervous system infections. Clin Microbiol Rev. 17, , table of contents. Rodrigues, C. M., Solá, S., Brites, D., 2002a. Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology. 35, Rodrigues, C. M., Solá, S., Castro, R. E., Laires, P. A., Brites, D., Moura, J. J., 2002b. Perturbation of membrane dynamics in nerve cells as an early event during bilirubininduced apoptosis. J Lipid Res. 43, Rodrigues, C. M., Sola, S., Silva, R. F. M., Brites, D., 2000a. Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization. Mol Med. 6, Rodrigues, C. M., Solá, S., Silva, R. F. M., Brites, D., 2000b. Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization. Mol Med. 6, Rodrigues, C. M., Solá, S., Silva, R. F. M., Brites, D., 2002c. Aging confers different sensitivity to the neurotoxic properties of unconjugated bilirubin. Pediatr Res. 51, Rodrigues, C. M., Stieers, C. L., Keene, C. D., Ma, X., Kren, B. T., Low, W. C., Steer, C. J., 2000c. Tauroursodeoxycholic acid partially prevents apoptosis induced by 3- nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition. J Neurochem. 75, Roseth, S., Hansen, T. W., Fonnum, F., Walaas, S. I., Bilirubin inhibits transport of neurotransmitters in synaptic vesicles. Pediatr Res. 44, Roux, P. P., Blenis, J., ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev. 68, Rubin, R. A., Balow, B., Fisch, R. O., Neonatal serum bilirubin levels related to cognitive development at ages 4 through 7 years. J Pediatr. 94, Salamy, A., Eldredge, L., Tooley, W. H., Neonatal status and hearing loss in high-risk infants. J Pediatr. 114, Saluja, S., Agarwal, A., Kler, N., Amin, S., Auditory neuropathy spectrum disorder in late preterm and term infants with severe jaundice. Int J Pediatr Otorhinolaryngol. Santambrogio, L., Belyanskaya, S. L., Fischer, F. R., Cipriani, B., Brosnan, C. F., Ricciardi- Castagnoli, P., Stern, L. J., Strominger, J. L., Riese, R., Developmental plasticity of CNS microglia. Proc Natl Acad Sci U S A. 98, Sawada, M., Imamura, K., Nagatsu, T., Role of cytokines in inflammatory process in Parkinson's disease. J Neural Transm Suppl Schenk, D., Barbour, R., Dunn, W., Gordon, G., Grajeda, H., Guido, T., Hu, K., Huang, J., Johnson-Wood, K., Khan, K., Kholodenko, D., Lee, M., Liao, Z., Lieberburg, I., Motter, R., Mutter, L., Soriano, F., Shopp, G., Vasquez, N., Vandevert, C., Walker, S., Wogulis, M., Yednock, T., Games, D., Seubert, P., Immunization with amyloid-beta attenuates Alzheimer-disease-like pathology in the PDAPP mouse. Nature. 400, Schwartz, M., Butovsky, O., Bruck, W., Hanisch, U. K., Microglial phenotype: is the commitment reversible? Trends Neurosci. 29, Schwartz, M., Shaked, I., Fisher, J., Mizrahi, T., Schori, H., Protective autoimmunity against the enemy within: fighting glutamate toxicity. Trends Neurosci. 26, Seidman, D. S., Paz, I., Stevenson, D. K., Laor, A., Danon, Y. L., Gale, R., Neonatal hyperbilirubinemia and physical and cognitive performance at 17 years of age. Pediatrics. 88, Selmaj, K., Raine, C. S., 1988a. Tumor necrosis factor mediates myelin damage in organotypic cultures of nervous tissue. Ann N Y Acad Sci. 540, Selmaj, K. W., Raine, C. S., 1988b. Tumor necrosis factor mediates myelin and oligodendrocyte damage in vitro. Ann Neurol. 23, Senior, K., Dosing in phase II trial of Alzheimer's vaccine suspended. Lancet Neurol. 1, 3. 43

72 Chapter I Seubert, J. M., Darmon, A. J., El-Kadi, A. O., D'Souza, S. J., Bend, J. R., Apoptosis in murine hepatoma hepa 1c1c7 wild-type, C12, and C4 cells mediated by bilirubin. Mol Pharmacol. 62, Shapiro, S. M., Bilirubin toxicity in the developing nervous system. Pediatr Neurol. 29, Shapiro, S. M., Definition of the clinical spectrum of kernicterus and bilirubin-induced neurologic dysfunction (BIND). J Perinatol. 25, Shapiro, S. M., Chronic bilirubin encephalopathy: diagnosis and outcome. Semin Fetal Neonatal Med. 15, Shapiro, S. M., Nakamura, H., Bilirubin and the auditory system. J Perinatol. 21 Suppl 1, S52-5; discussion S Sharma, R., Fischer, M. T., Bauer, J., Felts, P. A., Smith, K. J., Misu, T., Fujihara, K., Bradl, M., Lassmann, H., Inflammation induced by innate immunity in the central nervous system leads to primary astrocyte dysfunction followed by demyelination. Acta Neuropathol. 120, Sherwin, C., Fern, R., Acute lipopolysaccharide-mediated injury in neonatal white matter glia: role of TNF-alpha, IL-1beta, and calcium. J Immunol. 175, Silva, R., Mata, L. R., Gulbenkian, S., Brito, M. A., Tiribelli, C., Brites, D., Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and ph. Biochem Biophys Res Commun. 265, Silva, R. F. M., Mata, L. M., Gulbenkian, S., Brites, D., 2001a. Endocytosis in rat cultured astrocytes is inhibited by unconjugated bilirubin. Neurochem Res. 26, Silva, R. F. M., Rodrigues, C. M., Brites, D., 2001b. Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid. J Hepatol. 34, Silva, R. F. M., Rodrigues, C. M., Brites, D., Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin. Pediatr Res. 51, Simmons, J. L., Beauchaine, K. L., Auditory neuropathy: case study with hyperbilirubinemia. J Am Acad Audiol. 11, Solá, S., Brito, M. A., Brites, D., Moura, J. J., Rodrigues, C. M., Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes. Clin Sci (Lond). 103, Soorani-Lunsing, I., Woltil, H. A., Hadders-Algra, M., Are moderate degrees of hyperbilirubinemia in healthy term neonates really safe for the brain? Pediatr Res. 50, Stevens, B., Allen, N. J., Vazquez, L. E., Howell, G. R., Christopherson, K. S., Nouri, N., Micheva, K. D., Mehalow, A. K., Huberman, A. D., Stafford, B., Sher, A., Litke, A. M., Lambris, J. D., Smith, S. J., John, S. W., Barres, B. A., The classical complement cascade mediates CNS synapse elimination. Cell. 131, Stevenson, D. K., Dennery, P. A., Hintz, S. R., Understanding newborn jaundice. J Perinatol. 21 Suppl 1, S21-4; discussion S35-9. Stoppini, L., Buchs, P. A., Muller, D., A simple method for organotypic cultures of nervous tissue. J Neurosci Methods. 37, Streit, W. J., Microglia and macrophages in the developing CNS. Neurotoxicology. 22, Streit, W. J., Microglia as neuroprotective, immunocompetent cells of the CNS. Glia. 40, Streit, W. J., Sammons, N. W., Kuhns, A. J., Sparks, D. L., Dystrophic microglia in the aging human brain. Glia. 45, Streit, W. J., Xue, Q. S., Life and death of microglia. J Neuroimmune Pharmacol. 4, Sunico, C. R., Gonzalez-Forero, D., Dominguez, G., Garcia-Verdugo, J. M., Moreno-Lopez, B., Nitric oxide induces pathological synapse loss by a protein kinase G-, Rho kinasedependent mechanism preceded by myosin light chain phosphorylation. J Neurosci. 30, Suzumura, A., Sawada, M., Yamamoto, H., Marunouchi, T., Effects of colony stimulating factors on isolated microglia in vitro. J Neuroimmunol. 30, Takahashi, K., Prinz, M., Stagi, M., Chechneva, O., Neumann, H., TREM2-transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis. PLoS Med. 4, e

73 General Introduction Takahashi, K., Rochford, C. D., Neumann, H., Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2. J Exp Med. 201, Taylor, D. L., Pirianov, G., Holland, S., McGinnity, C. J., Norman, A. L., Reali, C., Diemel, L. T., Gveric, D., Yeung, D., Mehmet, H., Attenuation of proliferation in oligodendrocyte precursor cells by activated microglia. J Neurosci Res. 88, Thored, P., Heldmann, U., Gomes-Leal, W., Gisler, R., Darsalia, V., Taneera, J., Nygren, J. M., Jacobsen, S. E., Ekdahl, C. T., Kokaia, Z., Lindvall, O., Long-term accumulation of microglia with proneurogenic phenotype concomitant with persistent neurogenesis in adult subventricular zone after stroke. Glia. 57, Town, T., Nikolic, V., Tan, J., The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2, 24. Tsacopoulos, M., Magistretti, P. J., Metabolic coupling between glia and neurons. J Neurosci. 16, Tsuji, M., Wilson, M. A., Lange, M. S., Johnston, M. V., Minocycline worsens hypoxicischemic brain injury in a neonatal mouse model. Exp Neurol. 189, van Kooten, C., Banchereau, J., CD40-CD40 ligand. J Leukoc Biol. 67, van Weering, H. R., Boddeke, H. W., Vinet, J., Brouwer, N., de Haas, A. H., van Rooijen, N., Thomsen, A. R., Biber, K. P., CXCL10/CXCR3 signaling in glia cells differentially affects NMDA-induced cell death in CA and DG neurons of the mouse hippocampus. Hippocampus. Vaz, A. R., Delgado-Esteban, M., Brito, M. A., Bolanos, J. P., Brites, D., Almeida, A., Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical neurons: assessment of the protective effects of glycoursodeoxycholic acid. J Neurochem. 112, Venters, H. D., Dantzer, R., Kelley, K. W., Tumor necrosis factor-alpha induces neuronal death by silencing survival signals generated by the type I insulin-like growth factor receptor. Ann N Y Acad Sci. 917, Veroni, C., Gabriele, L., Canini, I., Castiello, L., Coccia, E., Remoli, M. E., Columba-Cabezas, S., Arico, E., Aloisi, F., Agresti, C., Activation of TNF receptor 2 in microglia promotes induction of anti-inflammatory pathways. Mol Cell Neurosci. 45, Vexler, Z. S., Yenari, M. A., Does inflammation after stroke affect the developing brain differently than adult brain? Dev Neurosci. 31, Vilhardt, F., Microglia: phagocyte and glia cell. Int J Biochem Cell Biol. 37, Volpe, J. J., Brain injury in premature infants: a complex amalgam of destructive and developmental disturbances. Lancet Neurol. 8, Wake, H., Moorhouse, A. J., Jinno, S., Kohsaka, S., Nabekura, J., Resting microglia directly monitor the functional state of synapses in vivo and determine the fate of ischemic terminals. J Neurosci. 29, Walter, L., Neumann, H., Role of microglia in neuronal degeneration and regeneration. Semin Immunopathol. 31, Watchko, J. F., Maisels, M. J., Jaundice in low birthweight infants: pathobiology and outcome. Arch Dis Child Fetal Neonatal Ed. 88, F Witting, A., Muller, P., Herrmann, A., Kettenmann, H., Nolte, C., Phagocytic clearance of apoptotic neurons by Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J Neurochem. 75, Wu, D. C., Jackson-Lewis, V., Vila, M., Tieu, K., Teismann, P., Vadseth, C., Choi, D. K., Ischiropoulos, H., Przedborski, S., Blockade of microglial activation is neuroprotective in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine mouse model of Parkinson disease. J Neurosci. 22, Yamagata, K., Tagami, M., Nara, Y., Mitani, M., Kubota, A., Fujino, H., Numano, F., Kato, T., Yamori, Y., Astrocyte-conditioned medium induces blood-brain barrier properties in endothelial cells. Clin Exp Pharmacol Physiol. 24, Zhang, L., Liu, W., Tanswell, A. K., Luo, X., The effects of bilirubin on evoked potentials and long-term potentiation in rat hippocampus in vivo. Pediatr Res. 53, Zhang, S. C., Fedoroff, S., Neuron-microglia interactions in vitro. Acta Neuropathol. 91, Zhao, C., Li, W. W., Franklin, R. J., Differences in the early inflammatory responses to toxin-induced demyelination are associated with the age-related decline in CNS remyelination. Neurobiol Aging. 27,

74 Chapter I Zhao, P., Waxman, S. G., Hains, B. C., Modulation of thalamic nociceptive processing after spinal cord injury through remote activation of thalamic microglia by cysteine cysteine chemokine ligand 21. J Neurosci. 27, Zimmermann, D. R., Dours-Zimmermann, M. T., Extracellular matrix of the central nervous system: from neglect to challenge. Histochem Cell Biol. 130, Zujovic, V., Benavides, J., Vige, X., Carter, C., Taupin, V., Fractalkine modulates TNFalpha secretion and neurotoxicity induced by microglial activation. Glia. 29,

75 Chapter II FEATURES OF BILIRUBIN-INDUCED REACTIVE MICROGLIA: FROM PHAGOCYTOSIS TO INFLAMMATION Sandra L. Silva, Ana R. Vaz, Andreia Barateiro, Ana S. Falcão, Adelaide Fernandes, Maria A. Brito, Rui F. M. Silva, Dora Brites Research Institute for Medicines and Pharmaceutical Sciences (imed.ul), Faculdade de Farmácia, University of Lisbon, Av. Professor Gama Pinto, Lisbon , Portugal. Neurobiology of Disease (2010) 40:

76 Acknowledgments The authors thank Elsa Rodrigues for her expertise with gene reporter assays. This work was supported by POCI/SAU-MMO/55955/2004 and PTDC/SAU-NEU/64385/2006 grants, from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal and FEDER (to D.B.). S.L.S. was recipient of a PhD fellowship (SFRH/BD/30326/2006) from FCT. 48

77 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia Abstract Microglia constitute the brain s immunocompetent cells and are intricately implicated in numerous inflammatory processes included in neonatal brain injury. In addition, clearance of tissue debris by microglia is essential for tissue homeostasis and may have a neuroprotective outcome. Since unconjugated bilirubin (UCB) has been proven to induce astroglial immunological activation and neuronal cell death, we addressed the question of whether microglia acquires a reactive phenotype when challenged by UCB and intended to characterize this response. In the present study we report that microglia primary cultures stimulated by UCB react by the acquisition of a phagocytic phenotype that shifted into an inflammatory response characterized by the secretion of the pro-inflammatory cytokines tumour necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6, up-regulation of cyclooxygenase (COX)-2 and increased matrix metalloproteinase (MMP)-2 and -9 activities. Further investigation upon upstream signalling pathways revealed that UCB led to the activation of mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB at an early time point, suggesting that these pathways might underlie both the phagocytic and the inflammatory phenotypes engaged by microglia. Curiously, the phagocytic and inflammatory phenotypes in UCB-activated microglia seem to alternate along time, indicating that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurred or there could be two distinct sub-populations of microglial cells, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. In conclusion, microglial cells are relevant partners to consider during bilirubin encephalopathy and the modulation of its activation might be a promising therapeutic target. Keywords: Microglial activation; Cyclooxygenase-2; Hyperbilirubinemia; Inflammatory signalling pathways; Matrix metalloproteinases; Mitogen activated protein kinases; Nuclear factor-κb; Phagocytic activity. 49

78 Chapter II 1. Introduction Hyperbilirubinemia is a common condition in the neonatal period and results from a limited ability of the newborns to excrete an over produced bilirubin (Dennery et al., 2001; Watson, 2009). Physiological to pathological transition is driven by the multifocal deposition of unconjugated bilirubin (UCB) in selected regions of the brain leading to encephalopathy and kernicterus (Hansen, 2002; Porter and Dennis, 2002). This event is directly correlated with death, as well as with impairments of neural development and hearing (Oh et al., 2003). Additionally, moderate hyperbilirubinemia has been proven to be associated with a significant increase in minor neurologic dysfunction throughout the first year of life (Soorani-Lunsing et al., 2001) and has also been related to the outcome of mental disorders such as schizophrenia (Miyaoka et al., 2000). The cytotoxic effects of UCB in the central nervous system (CNS) have been broadly studied and comprise several features such as: perturbation of nerve cell and mitochondria membranes (Rodrigues et al., 2002b; Rodrigues et al., 2002c); inhibition of glutamate uptake prolonging its presence in the synaptic cleft (Silva et al., 1999; Silva et al., 2002); N-methyl-D-aspartic acid (NMDA)-mediated excitotoxicity (Brito et al., 2010; Grojean et al., 2000; Grojean et al., 2001; McDonald et al., 1998); and increase in intracellular calcium (Brito et al., 2004). All these events may culminate in cell death by both necrosis and apoptosis (Rodrigues et al., 2002a; Silva et al., 2001) being neurons more susceptible to death mechanisms than astrocytes (Falcão et al., 2006; Silva et al., 2002). Some of the injurious effects of UCB on astrocytes are an elevated glutamate secretion and the activation of inflammatory pathways that lead to cytokine release (Falcão et al., 2006; Fernandes et al., 2006; Fernandes et al., 2004). Furthermore, our group was the first to demonstrate that UCB activates and damages microglial cells (Gordo et al., 2006). Indeed microglia showed to be the most reactive brain cells when compared to astrocytes and neurons, as they evidence increased UCB-induced cell death, release of glutamate and cytokine production (Brites et al., 2009). Microglial cells reside within the CNS parenchyma (Streit, 2002) and engage several important roles in the developing brain (Cuadros and Navascues, 1998; Kim and de Vellis, 2005) as well as in pathological conditions (Block and Hong, 2005; Nakajima and Kohsaka, 2004). In response to injury, microglia turn into an activated state and display a complexity of phenotypic alterations that illustrate what is called reactive microglia. This activation entails several features such as: dramatic morphologic changes by the acquisition of an amoeboid phenotype (Kreutzberg, 1996); up-regulation of intracellular enzymes and cell surface markers, release of pro-inflammatory mediators, oxygen radicals and proteases (Kim and de Vellis, 2005), antigen presentation (Aloisi, 2001) and phagocytosis (Chew et al., 2006). 50

79 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia Moreover, the implication of microglia to neonatal pathologic conditions has been acknowledged (McRae et al., 1995; Vexler and Yenari, 2009), since the production of inflammatory mediators by these cells is a major contributor to hypoxic- ischemic injury in the neonatal brain (Doverhag et al., 2010). Microglial activation must not be viewed as an on/off process, but rather as a shift between activity states, altering between a surveying and an effector status. In fact, microglia s activation process is an adaptive one but, depending on the circumstances in which it occurs, may have neuroprotective or neurotoxic outcomes (Hanisch and Kettenmann, 2007). Indeed, microglial involvement in various neurodegenerative disorders is notorious, namely in Parkinson s disease (Tansey et al., 2008), Alzheimer s disease (Kim and Joh, 2006), multiple sclerosis (Jack et al., 2005; Muzio et al., 2007), and human immunodeficiency virus (HIV)-associated dementia (Gonzalez-Scarano and Baltuch, 1999), mostly owing to its inflammatory character. Yet, microglial phagocytic role in numerous neurodegenerative diseases as well as in acute brain injury is essential for tissue debris removal and contributes for a pro-regenerative environment (Neumann et al., 2009). Moreover, phagocytic clearance of debris may be considered a protective measure as it constitutes an attempt to restrain further detrimental inflammatory responses (Napoli and Neumann, 2009). In this study we characterize the microglial response to UCB stimulation by evaluating both its phagocytic properties and the inflammatory mechanisms engaged upon activation. We observed, for the first time, that UCB induces an increase in the phagocytic properties of microglia, followed by a shift into a rather inflammatory response with prolonged exposure time. Moreover, this inflammatory response triggered by UCB follows different temporal profiles of interleukin (IL)-1β, tumour necrosis factor (TNF)-α and IL-6 secretion. Remarkably, an increase in TNF-α and IL-1β release is observed prior to the secretion of IL-6. Our findings also point to mitogen-activated protein kinases (MAPKs) and nuclear factor (NF)-κB as probable signalling pathways involved in microglial reactivity to UCB as they might be entailed either in inflammation (Hanisch et al., 2001) or phagocytosis (Sun et al., 2008; Tanaka et al., 2009). In fact, we demonstrate that MAPKs phosphorylation is an essential step for NF-κB nuclear translocation. Furthermore, our results reveal the induction of cyclooxygenase (COX)-2 expression and matrix metalloproteinase (MMP)-2 and -9 activity in a later phase of microglial response to UCB, implicating these events in the overall deleterious and inflammatory response. We provide evidence that UCB-induced cytokine secretion may participate in MMP activation and hypothesize that these events might be reciprocally regulated, further contributing to the complex network of microglia activation process. 51

80 Chapter II Taken together, these results strongly imply a multiple response of microglia to UCB, suggesting that those cells are relevant partners to consider during bilirubin encephalopathy. 2. Material and Methods 2.1. Chemicals Dulbecco s modified Eagle s medium-ham s F12 medium (DMEM-Ham s F-12), Opti-MEM medium, fetal bovine serum (FBS), L-glutamine, sodium pyruvate and nonessential aminoacids (NEA) were purchased from Biochrom AG (Berlin, Germany). Antibiotic antimycotic solution (20X), human serum albumin (HSA; fraction V, fatty acid free), bovine serum albumin (BSA), Hoechst dye, biotinylated tomato lectin (Lycopersicon esculentum), avidin-fluorescein isothiocyanate (FITC), avidintetramethylrhodamine isothiocyanate (TRITC), fluorescent latex beads 1μm (2.5%), mouse anti-β-actin, FITC-labelled goat anti-rabbit IgG, rabbit anti-glial fibrillary acidic protein (GFAP), TRITC-labelled goat anti-rabbit IgG, Coomassie Brilliant Blue R-250 and propidium iodide (PI) were from Sigma Chemical Co. (St. Louis, MO). UCB was also obtained from Sigma and purified according to the method of McDonagh and Assisi (1972). Trypsin/Ethylenediamine tetraacetic acid (EDTA) solution (0.25% trypsin, 1 mm EDTA in Hank s balanced salt solution) and Alexa Fluor 594 chicken anti-goat IgG were purchased from Invitrogen Corporation (Carlsbad, CA). FuGENE HD Transfection Reagent was acquired from Roche Molecular Biochemicals (Mannheim, Germany); Dual Luciferase reporter assay system was from Promega (Madison, WI, USA); and caspase-3, -8 and -9 substrates, Ac-DEVD-pNA, Ac- IETD-pNA and Ac-LEHD-pNA, respectively, were purchased from Calbiochem (San Diego, CA). Concentrated solutions (10 mm) of MAPK pathways inhibitors SB (p38 MAPK inhibitor; Calbiochem, La Jolla, CA, USA), and U0126 [Extracellular signal regulated kinase (ERK1/2)-upstream inhibitor; Promega, Madison, WI, USA], were prepared in dimethylsulfoxide. Recombinant rat IL-1β and DuoSet ELISA kits were from R&D Systems, Inc. (Minneapolis, MN, USA). Nitrocellulose membrane, Hyperfilm ECL and Horseradish peroxidase-labelled goat anti-mouse IgG were obtained from Amersham Biosciences (Piscataway, NJ, USA). LumiGLO, Cell lysis buffer, rabbit anti-phosphorylated-p38 (P-p38) and rabbit anti-phosphorylated-erk 1/2 (P-ERK1/2) were from Cell Signalling (Beverly, MA, USA). Mouse anti-phosphorylated-c-jun N-terminal kinase (P-JNK1/2), rabbit anti-p65 NF-κB subunit and horseradish peroxidase-labelled goat anti-rabbit IgG were from Santa 52

81 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia Cruz Biotechnology (Santa Cruz, CA, USA). Goat anti-ionized calcium-binding adaptor molecule 1 (Iba1) was from Abcam (Cambridge, UK). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany) Primary culture of microglia Animal care followed the recommendations of European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (Council Directive 86/609/EEC) and National Law 1005/92 (rules for protection of experimental animals). All animal procedures were approved by the Institutional Animal Care and Use Committee. Every effort was made to minimize the number of animals used and their suffering. Mixed glial cultures were prepared from 1-to-2 day-old Wistar rats as previously described (McCarthy and de Vellis, 1980), with minor modifications (Gordo et al., 2006). Cells (4 x 10 5 cells/cm 2 ) were plated on uncoated 12 or 6-well tissue culture plates (Corning Costar Corp., Cambridge, MA) in culture medium (DMEM-Ham s F-12 medium supplemented with 2 mm L-glutamine, 1 mm sodium pyruvate, nonessential amino acids 1X, 10% FBS, and 1% antibiotic-antimycotic solution) and maintained at 37ºC in a humidified atmosphere of 5% CO 2. Microglia were isolated as previously described by Saura et al. (2003). Briefly, after 21 days in culture, microglia were obtained by mild trypsinization with a trypsin- EDTA solution diluted 1:3 in DMEM-Ham s F12 for min. The trypsinization resulted in detachment of an upper layer of cells containing all the astrocytes, whereas the microglia remained attached to the bottom of the well. The medium containing detached cells was removed and replaced with initial mixed glial-conditioned medium. Twenty-four hours after trypsinization, the attached cells were subjected to the different treatments. The use of 21-days-in-vitro cells intents to achieve the maximal yield and purity of the cultures. In fact, astrocyte contamination was less than 2%, as assessed by immunocytochemical staining with a primary antibody against GFAP followed by a species-specific fluorescent-labelled secondary antibody. Microglia were counterstained with a biotinylated tomato lectin (Lycopersicon esculentum), using a 1:166 dilution in 1% Triton X-100 in phosphate-buffered saline (PBS) overnight at 4ºC followed by 1 h incubation at room temperature with avidin-tritc in a 1:100 dilution in PBS and the nuclei immunostained with Hoechst dye Thus, the high purity level of microglia cultures excludes interference of contaminating astroglial cells. 53

82 Chapter II 2.3. Cell treatment Microglial cells were incubated in the absence (control) or in the presence of 50 µm UCB plus 100 µm HSA, from 5 min to 48 h, at 37ºC. A UCB stock solution (10 mm) was prepared in 0.1 M NaOH immediately before use and the ph of the incubation medium was restored to 7.4 by addition of equal amounts of 0.1 M HCl. All the experiments with UCB were performed under light protection to avoid photodegradation. To study the role of MAPK pathways in microglial response to UCB, cells were pretreated for 20 min with 10 µm of the MAPK inhibitors prior to UCB stimulation: SB203580, a selective inhibitor of p38 MAPK and U0126, a selective inhibitor of the MAPK kinases (MEK)1/2, upstream kinases in the ERK1/2 pathway. The involvement of IL-1β in MMP activation was investigated by treating microglia with 2 ng/ml recombinant rat IL-1β or vehicle alone, in the presence of 100 µm HSA, for 30 min and 1 h at 37ºC. The selected concentration of cytokine was based on the maximal levels obtained in our culture model upon UCB stimulation Measurement of cytokine release Aliquots of the cell culture media were collected at the end of the incubations and, after removal of cellular debris by short centrifugation, placed in a 96-well microplate and assessed in triplicate for TNF-α, IL-1β and IL-6 with specific DuoSet ELISA Development kits from R&D Systems, according to the manufacturer s instructions. Results were expressed as pg/ml Western blot assay Western blot assay was carried out as usual in our lab (Fernandes et al., 2006). Briefly, total protein was extracted from primary microglia using Cell lysis buffer. Protein extracts were separated on a 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoretic transfer onto a nitrocellulose membrane and blocking with 5% milk solution, the blots were incubated with primary antibody overnight at 4ºC [anti-p-p38 MAPK (1:1000), anti-p-erk1/2 (1:1000), anti-p- JNK1/2 (1:200), rabbit anti-cox-2 (1:1000) or anti-β-actin (1:10000) in 5% (w/v) bovine serum albumin] and with horseradish peroxidase-labelled secondary antibody [antimouse (1:5000) or anti-rabbit (1:5000)] for 1 h at room temperature. Protein bands were detected by LumiGLO and visualized by autoradiography with Hyperfilm ECL. 54

83 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia 2.6. Detection of NF-κB activation To assay the transcriptional activity of NF-κB, reporter gene analysis was applied. A reporter plasmid under control of NF-κB binding sites was provided by Dr. Guy Haegeman (Flanders Interuniversity Institute for Biotechnology and University of Gent, Belgium). NF-κB-dependent reporter plasmids, p(il6κb)350hu.il6p-luc+, contain three NF-κB binding sites in the promoter region, while NF-κB-independent plasmids, p50hu.il6p-luc+, do not (Vanden Berghe et al., 1998). These reporter genes were introduced into microglial cells using FuGene HD Transfection Reagent. After 24 h of transfection, cells were treated with 50 µm UCB plus 100 µm HSA from 30 min to 4 h, at 37ºC. Luciferase assays were carried out using a Dual Luciferase Reporter Assay System (Promega), according to the instructions in manufacturer s manual. Firefly and renilla luciferase activities were measured using a luminometer (Berthold Technologies, Wildbad, Germany). Firefly luciferase activity value was normalized to renilla luciferase activity value from psv-sport-rluc plasmid. Readings of promoter activities of NF-κBindependent plasmids, p50hu.il6p-luc+ and p1168hil6m NF-κB-luc (plasmid presenting a mutation in NF-κB binding sites), were also performed. Results were presented as fold change of the relative luciferase activity compared to the respective control. For immunofluorescence detection of NF-κB nuclear translocation, cells were fixed for 20 min with freshly prepared 4% (w/v) paraformaldehyde in PBS and a standard immunocytochemical technique was performed using a polyclonal rabbit anti-p65 NF-κB subunit antibody (1:200) as the primary antibody and a FITC-labelled goat anti-rabbit antibody (1:160) as the secondary antibody. To identify the total number of cells, microglial nuclei were stained with Hoechst dye as previously described. Fluorescence was visualized using a Leica DFC490 camera adapted to an AxioSkope microscope (Zeiss). Pairs of U.V. and green-fluorescence images of ten random microscopic fields (original magnification: 400X) were acquired per sample. NF-κBpositive nuclei (identified by localization of the NF-κB p65 subunit staining exclusively at the nucleus) and total cells were counted (>500 cells per sample) to determine the percentage of NF-κB-positive nuclei. Results were expressed as fold change vs. respective control Morphological Analysis For morphological analysis, cells were fixed as described above and a standard indirect immunocytochemical technique was carried out using a primary antibody raised against Iba-1 (goat, 1:500) and a secondary Alexa Fluor 594 chicken anti-goat antibody 55

84 Chapter II (1:200). Fluorescent images were acquired using a Leica DFC490 camera attached to an AxioSkope microscope (Zeiss) Assessment of microglial phagocytic properties After treatment with UCB, cells were incubated with % (w/w) 1 μm fluorescent latex beads for 75 min at 37ºC and fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS. Labelling with tomato lectin was performed followed by avidin- TRITC and the nuclei counterstained with Hoechst dye. U.V., green and redfluorescence images of fifteen random microscopic fields (original magnification: 630X) were acquired per sample. The number of ingested beads per cell was counted in approximately 250 cells per sample Gelatin zymography Aliquots of culture supernatant were analyzed by SDS-PAGE zymography in 0.1% gelatine 10% acrylamide gels under non-reducing conditions. After electrophoresis, gels were washed for 1 h with 2.5% Triton X-100 (in 50 mm Tris ph7.4; 5 mm CaCl 2 ; 1μM ZnCl 2 ) to remove SDS and renature the MMP species in the gel. Then the gels were incubated in the developing buffer (50 mm Tris ph7.4; 5 mm CaCl 2 ; 1μM ZnCl 2 ) overnight to induce gelatine lysis. For enzyme activity analysis, the gels were stained with 0.5% Coomassie Brilliant Blue R-250 and destained in 30% ethanol/10% acetic acid/h 2 O. Gelatinase activity, detected as a white band on a blue background, was quantified by computerized image analysis and normalized with total cellular protein Evaluation of microglial cell death Necrotic-like cell death was assessed by monitoring the cellular uptake of the fluorescent dye propidium iodide [PI; 3,8-diamino-5-(3-(diethylmethylamino)propyl)-6- phenyl phenanthridinium diiodide]. PI readily enters and stains non-viable cells, but cannot cross the membrane of viable cells. This dye binds to double-stranded DNA and emits red fluorescence (630 nm; absorbance 493 nm). Unpermeabilized adherent cells cultured on coverslips were incubated with a 75 μm PI solution for 15 min in the absence of light. Subsequently, cells were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS and the nuclei immunostained with Hoechst dye. Red-fluorescence and U.V. images of ten random microscopic fields (original magnification: 400X) were acquired per sample and the percentage of PI positive cells was counted and expressed as fold vs. respective control. 56

85 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia Activities of caspase-3, -8 and -9 were measured by a commercial colorimetric method (Calbiochem, Darmstadt, Germany). Cells were harvested, washed with ice-cold PBS and lysed for 30 min on ice in the lysis buffer [50 mm HEPES (ph 7.4); 100 mm NaCl; 0.1% (w/v) CHAPS; 1 mm dithiothreitol (DTT); 0.1 mm EDTA]. The activities of caspase-3, -8 and -9 were determined in cell lysates by enzymatic cleavage of chromophore p-nitroanilide (pna) from the substrate Ac-DEVD-pNA for caspase-3, Ac- IETD-pNA for caspase-8 and Ac-LEHD-pNA for caspase-9, according to manufacturer s instructions. The proteolytic reaction was carried out in protease assay buffer [50 mm HEPES (ph 7.4); 100 mm NaCl; 0.1% (w/v) CHAPS; 10 mm DTT; 0.1 mm EDTA; 10% (v/v) glicerol], containing 2 mm specific substrate. Following incubation of the reaction mixtures for 1 to 2 h at 37ºC, the formation of pna was measured in a microplate reader (PR 2100, BioRad Laboratories, Inc.) at λ= 405 nm with a reference filter at 620 nm. Readings were normalized to total protein content determined using a protein assay kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer s specification, and expressed as fold change of respective control Statistical analysis Results of, at least, three different experiments were expressed as mean ± S.E.M. Significant differences between two groups were determined by the two-tailed t- test performed on the basis of equal and unequal variance as appropriate. Comparison of more than two groups was done by ANOVA using Instat 3.05 (GraphPad Software, San Diego, CA, USA) followed by multiple comparisons Bonferroni post-hoc correction. Statistical significance was considered for a p value less than Results 3.1. UCB triggers IL-1β, TNF-α and IL-6 secretion following different temporal profiles Supporting evidence reports reactive microglia as one of the main sources of proinflammatory cytokines in the brain (Hanisch, 2002), which are known to exert deleterious effects in nerve cells (Rothwell, 1999). Previous results suggested that UCB is able to induce an inflammatory response by microglia (Gordo et al., 2006). Thus, we intended to further characterize those inflammatory events by the evaluation of the temporal secretion profile of IL-1β, TNF-α and IL-6. In Figure II.1 it can be observed that UCB stimulates cytokine release in a time-dependent manner but following different temporal profiles. In fact, TNF-α and IL-1β seem to be the first to be up-regulated, as an increase in those cytokine levels in culture supernatants can be observed as early as 2 h 57

86 Chapter II after the addition of 50 μm UCB. However, while peak values for TNF-α are reached at 4 h of UCB exposure (changing from 365 pg/ml for control conditions to 520 pg/ml for UCB, p<0.05) and decline gradually along time of exposure (although an additional increase is noticed at 24 h), the maximum release of IL-1β was only achieved at 12 h, but in a much higher amount (ranging from 980 pg/ml for control conditions to 1700 pg/ml for UCB, p<0.05). On the other hand, IL-6 secretion was only noticed from 2 h on, reaching peak levels at 8 h of UCB exposure (shifting from 1650 pg/ml for control conditions to 2050 pg/ml for UCB, p<0.01) and decreasing thereafter. These results seem to indicate that microglia responds to UCB stimulus with a rather inflammatory profile which is manifested for prolonged incubation periods. As earlier results on astrocytes demonstrated that UCB-induced cytokine secretion involves MAPK and NFκB activations (Fernandes et al., 2007; Fernandes et al., 2006) we sought to verify if the same inflammatory signalling pathways are maintained by microglia. Fig. II.1. UCB induces the release of TNF-α, IL-1β and IL-6 by microglia following different temporal profiles. Rat cortical microglial cells were treated with 50 µm UCB in the presence of 100 µm HSA for the indicated time periods. TNF-α, IL-1β and IL-6 concentrations in the media were determined by ELISA and expressed as mean ± SEM cytokine release from four independent experiments performed in triplicate, after deduction of cytokine values in control assays. *p<0.05 and **p<0.01 vs. respective control p38 and ERK1/2 phosphorylation is elicited by UCB in microglia at an early time point MAPKs have been reported by several studies to be involved in the production of inflammatory mediators by microglia (Bhat et al., 1998; Lee et al., 2000; Waetzig et al., 2005), but their involvement on UCB microglial stimulation is still not known. So, we assessed the phosphorylated (activated) forms of all three MAPKs (p38, ERK1/2 and JNK1/2) in total cell lysates of UCB-exposed microglia, by western blot, using specific antibodies. 58

87 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia As shown in Figure II.2, upon UCB stimulation, P-p38 and P-ERK1/2 expression were significantly up-regulated in a rapid but transient manner. A 1.4-fold induction (p<0.05 vs. control) was observed for P-p38 as early as 15 min after UCB exposure, and this activation was sustained until 30 min of incubation (1.3-fold, p<0.05 vs. control), fading afterwards. A second activation peak was observed after 2 h of exposure (1.4- fold, p<0.05), declining to control levels at 6 h. In regard to P-ERK1/2, a 1.2-fold increase (p<0.05 vs. control) was observed at 15 min of exposure and, as for P-p38, a second peak was observed, but now smaller (1.12 -fold, p<0.05) and at 1 h of exposure, declining from this point on. A weaker increase in P-JNK1/2 is perceived at 15 and 30 min of UCB exposure; however, this effect is not significantly different from the respective controls. Figure II.2. MAPKs activation is elicited by UCB in microglia. Rat cortical microglia were exposed to 50 µm UCB in the presence of 100 µm HSA for the indicated time periods. Total cell lysates were analysed by western blotting with antibodies specific for the phosphorylated forms of the three MAPKs, P-p38, P-ERK1/2 and P-JNK1/2. (A) Representative results of one experiment are shown. Similar results were obtained in three independent experiments. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein expression and expressed as mean ± SEM fold change compared with control conditions. *p<0.05 vs. respective control. 59

88 Chapter II Prolonging UCB incubation to longer periods did not modify the pattern of MAPK activation (data not shown). These results indicate that MAPK activation is a rather early event on microglial activation, seeming to involve two activation peaks. Next we found interesting to check if this activation could be followed by the engagement of NF-κB nuclear translocation NF-κB signalling pathway is triggered in UCB-activated microglia NF-κB is described as a convergent point of signalling for microglial activation by cytokines and other stressors (Glezer et al., 2007) and its implication in the inflammatory response induced by UCB in astrocytes has already been established (Fernandes et al., 2006). Hence, we examined the effect of UCB on NF-κB transactivation in microglial cells by gene reporter assay (Figure II.3A). The results indicated that UCB markedly induced NF-κB activation at 15 and 30 min of exposure (1.4-fold, p<0.01 for both time points). It should be stated that readings of the promoter activities of p50hu.il6p-luc+ and p1168hil6m NF-κB-luc plasmids (empty and mutated vectors, respectively) showed no change in the presence or absence of UCB (data not shown), thus validating the assay. To further confirm the activation of this signalling pathway we investigated NF-κB activation in microglia exposed to UCB at various time points by the immunochemical assessment of the cytoplasmic or nuclear localization of p65 NF-κB subunit. Interestingly we found NF-κB translocation into the nucleus to be significantly increased from 15 min to 2 h of exposure when compared to the respective controls (Figure II.3B-C), which is in line with our previous observations regarding NF-κB transcriptional activation. Maximum levels of nuclear NF-κB were observed at 30 min (2.2-fold, p<0.01), while from 4 h onwards NF-κB was mostly localized in the cellular cytoplasm. These results follow the ones from MAPK activation, suggesting that, as previously verified in astrocytes, both events are connected. 60

89 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia Fig. II.3. UCB activates NF-κB signalling pathway in microglia. Rat cortical microglia were exposed to 50 µm UCB in the presence of 100 µm HSA for the indicated time periods. (A) Microglial cells were transiently transfected with NF-κB-dependent luciferase plasmids and control plasmids. Relative luciferase activities were plotted as fold change of respective controls. To further confirm NF-κB activation, cells were fixed and immunostained with an antibody against p65 NF-κB subunit. (B) Representative results of one experiment are shown. Scale bar, 20 μm. (C) The percentage of NF-κB-positive nuclei was calculated and expressed as fold change vs. respective control. Results are mean ± SEM from three independent experiments performed in triplicate. *p<0.05 and **p<0.01 vs. respective control UCB-induced NF-κB translocation depends on both ERK1/2 and p38 To assess whether MAPKs phosphorylation is an essential step for UCB-evoked NF-κB translocation, we investigated this event after exposure of microglia to UCB alone or in combination with the MAPK inhibitors SB (p38 selective inhibitor) and UO126 [(MEK)1/2 selective inhibitor, upstream kinases in the ERK1/2 pathway]. The use of 30 min and 1 h incubation periods was based on previous results showing that maximal translocation of NF-κB to the nucleus in UCB-challenged microglia occurs between 30 min and 4 h. 61

90 Chapter II As expected, pre-incubation with SB and U0126 completely abrogated UCB-stimulated NF-κB nuclear translocation after 30 min (p<0.01) and 1 h (p<0.05) of UCB stimulation (Figure II.4), thus providing proof of concept that p38 and ERK1/2 phosphorylation are required for the engagement of NF-κB pathway upon UCB exposure. Fig. II.4. Phosphorylation of p38 and ERK1/2 is essential for UCB-evoked NF-κB activation. Rat cortical microglia were exposed for 30 min and 1 h to 50 µm UCB alone or in combination with 10 µm of the p38 inhibitor SB or the ERK1/2 inhibitor U0126. Cells were fixed and immunostained with an antibody against p65 NF-κB subunit. (A) Representative results of one experiment are shown. Scale bar, 20 μm. (B) The percentage of NF-κB-positive nuclei was calculated and expressed as fold change vs. respective control. Results are mean ± SEM from three independent experiments performed in triplicate. **p<0.01 vs. respective control, p<0.05 and p<0.01 vs. UCB alone. 62

91 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia So far our results have described the engagement of early inflammatory signalling pathways that culminate in the acquisition of an inflammatory phenotype characterized by the release of proinflammatory mediators. We next intended to characterize UCB-stimulated microglia at a morphological level, in order to verify the acquisition of this activated state Microglia depict morphological changes upon UCB stimulation Modification of microglial morphology is one of the hallmarks of its activation profile and has been widely used to categorize different activation states (Chew et al., 2006; Kim and de Vellis, 2005; Lynch, 2009; Raivich et al., 1999). For that reason, we evaluated the morphology and reactivity of UCB-stimulated microglia by immunocytochemistry after 4 and 24 h incubation periods. Our results indicate that, after a short UCB exposure, microglia shifted from an elongated morphology to a large and amoeboid shape (Figure II.5). This phenotype is characteristic of activated or reactive microglia (Nakajima and Kohsaka, 2004). In contrast, for longer exposure periods, microglia display fragmented and condensed cytoplasm, a feature described by other authors (Fendrick et al., 2007; Hasegawa-Ishii et al., 2010) as cytorrhexis, indicative of microglia degeneration and senescence. Fig. II.5. UCB-stimulated microglia show reactive morphological changes. Rat cortical microglia were exposed to 50 µm UCB in the presence of 100 µm HSA for the indicated time periods. Cells were fixed and immunostained with an antibody against Iba1 reactivity marker. Representative results of one experiment are shown. Scale bar, 20 μm. Interestingly, the inflammatory phenotype previously described occurs after prolonged UCB incubation periods. However, activation features were also observed for shorter stimulations. For that matter, we sought to evaluate the phagocytic properties of UCB-challenged microglia and, more importantly, to verify whether this reactive phenotype occurred prior, simultaneously or after the inflammatory response triggered by UCB, in order to further characterize the chronologic events of UCB microglial activation. 63

92 Chapter II 3.6. UCB differently modulates microglial phagocytosis depending on exposure time Phagocytosis is one of the main features of microglial activation dumping cell debris prior to cell regeneration, and can be involved in the pathogenesis of several brain dysfunctions (Neumann et al., 2009). However, this microglial property was never investigated under UCB stimulation. As can be seen in Figure II.6, a sharp increase in the uptake of fluorescent latex beads by UCB-stimulated microglia occurs from 2 h on, reaching a maximum peak after 4 h of exposure, (~50%, p<0.05). This is a strong evidence that UCB is able to induce microglial phagocytic properties in a rather early time of exposure. Fig. II.6. Microglial phagocytosis is differently modulated by UCB. Rat cortical microglia were exposed to 50 µm UCB in the presence of 100 µm HSA for the indicated time periods and incubated with 1 μm fluorescent latex beads as described in Methods. (A) Representative results of one experiment are shown. Scale bar, 20 μm. (B) Results are expressed as number of ingested beads per cell (± SEM) from three independent experiments. *p<0.05 vs. respective control. 64

93 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia Unspecific entry of latex beads due to UCB-induced cell permeabilization was excluded by performing the phagocytosis assay in microglial cells incubated with UCB for 4 h and simultaneously determining PI uptake. Dead cells did not appear to engulf particles (data not shown). Interestingly, prolonging UCB incubation for 8 and 12 h slightly reverts this phagocytic ability although it approached control values for 24 h of exposure. This may indicate that, after an initial phagocytic reaction, microglial cells shifted to the previously observed inflammatory response to UCB stimulus. To add on the characterization of microglial behaviour we advanced to the evaluation of other markers of its inflammatory response such as MMPs activity and COX-2 expression Release of active MMPs is enhanced upon UCB stimulation of microglia MMPs are a family of proteases with many important roles in normal development although they may also participate in several neuronal diseases such as multiple sclerosis, ischemia and neuroinflammation given their ability to degrade the basal lamina surrounding the blood-brain barrier allowing infiltration of immune cells and, thus, aggravating inflammatory reactions in the CNS (Michaluk and Kaczmarek, 2007; Rosenberg, 2002). Since microglia have been reported to secrete active MMPs further contributing to neuronal injury (Kauppinen and Swanson, 2005; Woo et al., 2008), and given the fact that cytokines are reported to stimulate MMPs secretion and activity (Gottschall and Yu, 1995; Lin et al., 2009; Vincenti and Brinckerhoff, 2007), we intended to evaluate the levels of active MMPs secreted by these cells in response to UCB and to verify if this activation could be ascribed to UCB-induced IL-1β. Cell supernatants collected after UCB incubation were subjected to gelatin zymography for the assessment of MMP-2 and MMP-9 activity levels. As can be seen in Figure II.7 there is a slight but significant increase in the activity of MMP-2 and MMP-9 (1.07-fold and 1.08-fold, p<0.05, respectively) after a prolonged exposure time (24 h) to UCB. 65

94 Chapter II Fig. II.7. MMP-2 and MMP-9 activities are enhanced upon UCB stimulation. Culture supernatants from rat cortical microglial cells were harvested after incubation with 50 µm UCB in the presence of 100 µm HSA or with 2 ng/ml IL-1β for the indicated time periods and subjected to zymography as described in Methods. (A) Representative gels of one experiment are reported. MMP-2 and MMP-9 were identified by their apparent molecular mass of 67 and 92 kda, respectively. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to total protein content and expressed as mean ± SEM fold change compared with control conditions. *p<0.05 and **p<0.01 vs. respective control. Since this event occurs after the onset of cytokine secretion elicited by microglia, a possible regulation of this event by inflammatory mediators could be occurring. In fact, stimulation of microglia with 2 ng/ml of IL-1β (which correspond to maximal levels obtained in our culture model upon UCB stimulation) increases significantly MMP-2 and MMP-9 activity at both 12 (1.3-fold and 1.2-fold, p<0.05 and p<0.01, respectively) and 24 h of exposure (1.2-fold and 1.3-fold, p<0.01, respectively) UCB-stimulated microglia evidence enhanced COX-2 expression COX-2 is the enzyme responsible for the production of prostanoids such as prostaglandin E2 (PGE2), which is known to be involved in the initiation and propagation of the immune response (de Oliveira et al., 2008). The expression of this enzyme can be induced by lipopolysaccharide (LPS) and cytokines (Levi et al., 1998) and has been proven to be elevated in Alzheimer s disease (Yokota et al., 2003) and ischemia (Iadecola et al., 1999). As depicted in Figure II.8, a significant up-regulation of COX-2 expression was only noticed after 12 and 24 h of UCB exposure (1.13-fold, p<0.01 and 66

95 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia 1.18-fold, p<0.05, respectively), when compared to the respective controls, again suggesting a later response of microglia to UCB, in line with the previous results. Finally, we were interested in examining how UCB interaction affected microglial cell survival. Fig. II.8. COX-2 expression is up-regulated by UCB in microglia. Rat cortical microglia were exposed to 50 µm UCB in the presence of 100 µm HSA for the indicated time periods. Total cell lysates were analysed by western blotting. (A) Representative results of one experiment are shown. Similar results were obtained in three independent experiments. (B) The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein and expressed as mean ± SEM fold change compared with control conditions. *p<0.05 vs. respective control UCB reduces microglial viability leading to loss of membrane integrity and increased caspase activity To evaluate the necrotic-like cell death we used the uptake of the fluorescent dye PI as an indicator of membrane integrity and cell damage since this polar substance can only enter dead or dying cells. To address the possible involvement of the apoptotic pathways in microglial demise we determined the relative levels of caspases activity, since these proteases have been traditionally viewed as central regulators of apoptosis (Fink and Cookson, 2005). As depicted in Figure II.9, UCB stimulation only arouses increased PI uptake from 4 to 12 h of exposure, reaching maximum significance at 8 h. Accordingly, the activities of initiator caspases -8 and -9 were found to be significantly elevated in response to UCB from 2 to 12 h of exposure reaching maximum activity at 6 h (Figure II.10) while effector caspase-3 was significantly increased at a relatively later time point (from 4 to 12 h of exposure). It is interesting to notice that cell death seems to occur on the onset of inflammatory response and when phagocytic activity declines, again suggesting a possible double response from microglia to UCB. 67

96 Chapter II Fig. II.9. UCB induces microglial decreased viability and membrane disruption. Rat cortical microglia were exposed to 50 µm UCB in the presence of 100 µm HSA for the indicated time periods and incubated with 75 μm PI as described in Methods. The percentage of PI-positive cells was calculated and expressed as fold vs. respective control. Results are mean ± SEM from three independent experiments performed in triplicate *p<0.05 and **p<0.01 vs. respective control. Fig. II.10. Microglial apoptotic cell death is elicited by UCB. Rat cortical microglia were exposed to 50 µm UCB in the presence of 100 µm HSA for the indicated time periods. The activities of caspases -3, -8 and -9 were determined in cell lysates by enzymatic cleavage of chromophore p-nitroanilide (pna) from specific substrates. Results are expressed as fold of respective control at each time point. Data are means ± SEM from at least three independent experiments. *p<0.05 and **p<0.01 vs. respective control. 68

97 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia 4. Discussion In this paper we describe, for the first time, different activation stages of microglia in the presence of UCB, since these cells display both phagocytic and inflammatory phenotypes. Indeed, this study is original in depicting the increased phagocytic properties of microglia upon UCB stimulation. In addition, our group was also the first to implicate microglial cells in the inflammatory response elicited by UCB (Gordo et al., 2006). Thus, in this study we further investigated the activation profile of microglia under UCB stimulation, by the evaluation of some of its characteristical features, and the signalling events involved in cell response. In fact, previous studies performed by our group have proven that UCB induces immunological responses in astrocytes by the activation of inflammatory pathways and secretion of glutamate (Fernandes et al., 2006; Fernandes et al., 2004), and also that UCB is neurotoxic (Falcão et al., 2007; Silva et al., 2001). Accordingly, it is conceivable that increased microglia reactivity may further contribute to neuronal injury during hyperbilirubinemia. Microglia contribute to both innate and adaptive immune responses in the brain (Chew et al., 2006). As innate immune cells, they constitute the first line of defence against invading microorganisms. The hallmark indicators of such response are the production of pro-inflammatory cytokines, the up-regulation of cell surface antigens and phagocytosis (Town et al., 2005). In addition, phagocytosis of debris by microglia can be beneficial in several pathological conditions such as multiple sclerosis (Takahashi et al., 2007) and Alzheimer s disease (Simard et al., 2006) as it restricts lesion extension and facilitates tissue recovery. The fact that UCB may alter the function of various cells of the immune system (both in vivo and in vitro) seems to be firmly established and a wide range of immunosuppressive effects on peripheral immune cells are summarized by Vetvicka et al. (1991) such as alterations on antigen expression, chemotaxis, bactericidal activity, proliferative response of T lymphocytes, or antibody production. On the other hand, an increase in phagocytosis of both peripheral blood granulocytes and monocytes after UCB treatment was reported by Miler et al. (1985). Thus, a rather contradictory immunosuppressive immunostimulant status seems to be observed upon UCB challenge that might be explained by dose or time-dependent effect (Vetvicka et al., 1991). Our findings demonstrate that, in conditions that intend to mimic a mild hyperbilirubinemia, enhancing of microglial phagocytic properties by UCB is an early, but transient, event that seems to be lost with increased time of exposure. Thus, we may assume that phagocytosis is the first response towards UCB insult and may constitute a neuroprotective measure. 69

98 Chapter II Various conditions have been shown to greatly modify microglial phagocytic activity, such as cytokines (Koenigsknecht-Talboo and Landreth, 2005), and LPS (Sun et al., 2008), among others. Interestingly, the study performed by von Zahn et al. (1997) reports an induction of nearly two-fold increase in the uptake of uncoated latex particles by TNF-α-stimulated microglia, substantiating this cytokine as an autocrine activator of microglial immune functions. Indeed, UCB-activated microglia are reportedly one of the main sources of TNF-α, even when compared to astrocytes (Brites et al., 2009). Similarly to what we already observed for astrocytes (Fernandes et al., 2006), our results point to TNF-α as the first cytokine to be released by microglia upon UCB challenge, and, remarkably, its temporal profile of secretion is rather paralleled by the observed phagocytic alterations. TNF-α secretion reaches a maximum level at 4 h of UCB exposure, when microglial phagocytic properties are significantly increased and a decline in UCB-induced TNF-α release is observed from this point on, coinciding with decreased phagocytosis elicited by UCB. Besides its role as microglial phagocytosis inducer, several experiments have also implicated TNF-α in demyelination (Akassoglou et al., 1998) and neuronal degeneration (Allan and Rothwell, 2003; Silva et al., 2006), and this cytokine, along with IL-1β, participates in astrogliosis (Hanisch, 2002). IL-1β is involved in fever induction and edema, stimulation of COX-2, release of nitric oxide (NO) and free radicals (Rothwell, 1999), also participating in the recruitment of circulating leukocytes into the CNS due to its ability to up-regulate the expression of adhesion molecules and chemokine synthesis (Lee and Benveniste, 1999; Sedgwick et al., 2000). IL-6 can have both pro and antiinflammatory functions and is produced in the early phases of CNS insult (Raivich et al., 1999). Our results clearly imply microglia as an important player in the inflammatory response instigated by UCB, since the observed early release of TNF-α, previously discussed, is followed by a later but intense secretion of IL-6 and an even stronger induction of IL-1β. Strikingly, UCB seems to induce a major release of pro-inflammatory cytokines in a time period in which phagocytosis is already absent. Recent reports have, in fact, substantiated the existence of a non-phlogistic (non-inflammatory) phagocytic response from microglia (Neumann et al., 2009), triggered by apoptotic stimuli and potentially mediated by phosphatidylserine receptors (PRs) and triggering receptor expressed on myeloid cells-2 (TREM2) (Hsieh et al., 2009; Takahashi et al., 2005). Additionally, IL-1β and PGE2 were shown to suppress microglial ability to phagocytise insoluble fibrillar β- amyloid deposits, suggesting that a pro-inflammatory milieu inhibits this type of phagocytosis (Koenigsknecht-Talboo and Landreth, 2005). 70

99 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia Our findings also suggest a role for MMPs in UCB-induced microglia reactivity, since their activity is enhanced upon prolonged exposure periods to this molecule. MMP- 9 has been associated with glutamate dysfunction (Michaluk and Kaczmarek, 2007) and its release can be a cause of microglia-induced neuron death (Kauppinen and Swanson, 2005). In addition, inhibition of gelatinases (MMP-2 and -9) showed efficacy in reducing neural injury and dampening neuroinflammation (Leonardo et al., 2008). Thus, these proteases seem to actively participate in inflammatory events and their activity is very tightly regulated (Sternlicht and Werb, 2001). Cytokines are firmly established inducers of MMP expression and secretion (Gottschall and Yu, 1995; Ito et al., 1996), and the induction of MMPs has been shown to be mediated by MAPKs, NF-κB and activator protein-1 signalling pathways (Lin et al., 2009; Shakibaei et al., 2007; Vincenti and Brinckerhoff, 2007; Woo et al., 2008). Interestingly, in our study model, MMPs enhanced activity occurs at a later time of exposure, when MAPKs and NF-κB activation as well as cytokine secretion have already taken place, suggesting that these events might be involved in the activation of MMPs induced by UCB. Moreover, active MMPs may also participate in the regulation of cytokine activity by promoting the secretion and activation of these molecules (Chauvet et al., 2001; Kim et al., 2005; Nuttall et al., 2007; Woo et al., 2008) or, on the other hand, by negatively regulating their biological activities (Ito et al., 1996). As stated above, MMPs increased activity in UCB-stimulated microglia takes place after the peak of cytokine secretion, suggesting a possible reciprocal regulation between pro-inflammatory cytokines and MMPs, since the latter molecules could be involved in the termination of the inflammatory response by means of the degradation of IL-1β. Our results further address this issue since IL-1β demonstrated to intensely elevate MMP-2 and -9 activation providing proof of concept that IL-1β secretion produced upon UCB stimulation, is at least in part, responsible for MMP activation. Discrepancy between the activation levels observed for UCB or IL-1β alone may be a result of UCB-multiple cytokine activation which results in a pleiotropic regulatory loop, absent in the second condition. COX-2 expression can also be induced in microglia by several inflammatory conditions. As can be seen in our results, UCB is able to induce up-regulation of COX-2 in microglial cells in a profile very similar to the observed for IL-6 and IL-1β, thus contributing to the overall inflammatory environment described so far and again pointing to an inflammatory response secondary to phagocytosis. Therefore, our studies suggest a dual role for microglia upon UCB stimulation, shifting from a phagocytic and possibly neuroprotective phenotype towards an inflammatory and deleterious one. This is 71

100 Chapter II consistent with our findings demonstrating that microglia portray altered morphological features after a prolonged UCB exposure, typical of an activated state. As previously observed for astrocytes (Fernandes et al., 2006; Fernandes et al., 2004) we show here that UCB stimulation of microglial cells also involves the activation of MAPKs and NF-κB. MAPKs can be activated by a variety of different stimuli (Roux and Blenis, 2004), and the engagement of this signalling pathway can lead to the phosphorylation of several substrates, including transcription factors such as NF-κB, which may ultimately lead to the enhanced transcription of genes encoding for proinflammatory cytokines (Koj, 1996). Activation of p38 and ERK1/2 are regarded as essential steps for cytokine induction since their involvement in TNF-α, IL-1β, IL-6, COX- 2 and inductible nitric oxide synthase (inos) expression in microglia has been widely established (Bhat et al., 1998; Hanisch et al., 2001; Lee et al., 2000). Intriguingly, MAPKs activation, particularly p38, seems to be also involved in the induction of microglia phagocytosis (Sun et al., 2008; Tanaka et al., 2009). In this regard our data indicate a rapid activation of p38 and ERK1/2 by UCB in microglial cells, which occurs prior to the production of inflammatory mediators previously reported. In fact, MAPKs activation by UCB in microglia is triggered at a much earlier stage than in astrocytes (Fernandes et al., 2006), reinforcing the greater responsiveness of these glial cells during hyperbilirubinemia but also suggesting that the early phagocytic response of microglia cells to UCB may be under the control of p38 and ERK1/2 activation. In this case, the latter activation peak observed would engage the pro-inflammatory cascade that results in IL-1β and IL-6 enhanced secretion, as well as the induction of COX-2 and MMPs, a feature already observed for other immune cells (Gong et al., 2008; Hwang et al., 1997). Moving downstream on the intracellular signalling pathways is the original observation that, as in astrocytes (Fernandes et al. 2007; Fernandes et al. 2006), NF-κB activation is also present in microglia exposed to UCB. Interestingly, maximum activation of NF-κB takes place during and after the early MAPKs phosphorylation and again prior to the production of IL-1β, TNF-α and IL-6, postulating a possible involvement of NF-κB in both phagocytic and inflammatory responses elicited by UCB in microglia. The observations that NF-κB nuclear translocation in UCB-stimulated microglia is completely abrogated when microglia are pretreated with p38 and ERK1/2 inhibitors provided an unequivocal proof of MAPKs involvement in NF-κB engagement in UCB-challenged microglia which had already been previously established by other authors in different disease models (Wilms et al., 2003). 72

101 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia It isworthwhile to mention that cell viability and membrane integrity are compromised from 4 h onwards, indicating increased cell damage induced by UCB on microglial cells from this point onwards. In fact, UCB-induced apoptotic and necrotic microglial cell death have already been established (Gordo et al., 2006). Our results indicate that both the extrinsic and intrinsic apoptotic pathways are triggered culminating in the activation of effector caspase-3 and consequently causing cell death. However, cell death phenomena reach maximum peaks between 6 to 8 h but decreased for longer incubation periods. Together with the findings described above these data portray an interesting hypothesis for microglia response to UCB stimulus. So, it is conceivable that, either a shift on global microglia reaction occurs, or there are two distinct subpopulations of microglial cells displaying complementary activation features, one directed at eliminating the damaged cells by phagocytosis, that died after engulfment of beads, and another engaging a more delayed inflammatory response. In fact, fragmentation of cytoplasm (cytorrhexis) which is suggested in our 24 h morphological observations, has been pointed to be indicative of widespread microglial degeneration in amyotrophic lateral sclerosis models (Fendrick et al., 2007). Degenerative changes in microglia such as beading and clusters of fragmented twigs have also been demonstrated in the aged brain (Hasegawa-Ishii et al., 2010). Which of the above mentioned hypotheses is the more valid demands further elucidation and will clarify the multifaceted profile of microglia activation under UCB stimulation. The complex network of UCB-induced events in microglia, as well as the proposed interactions between them, is depicted in Figure II.11. In conclusion, our experiments evidence that phagocytosis is differently modulated by UCB depending on the time of exposure, prevailing at an early time point, which is followed by the release of inflammatory cytokines, and activation of MMP-2 and -9, as well as of COX-2 activation. Thus, microglial phagocytosis and inflammatory response stand out as important events prompted by UCB. To what extent the activation of microglia by UCB has a beneficial or detrimental outcome is yet to be determined in future studies, where the influence of other nerve cells will be evaluated. Nevertheless, modulation of microglial activation seems to be a promising target in neonatal bilirubin encephalopathy. 73

102 Chapter II UCB Cell death 0 h 15 min 30 min 2 h 4 h 8 h 12 h 24 h MAPKs NF κβ Phagocytosis Inflammation TNF α IL 1β IL 6 COX 2 MMP 2 MMP 9 Fig. II.11. Schematic representation of time-dependent microglial activation induced by UCB. Upon UCB stimulation of microglial cells, MAPK and NK-κB signalling pathways are engaged, culminating in the generation of a phagocytic response followed by an inflammatory profile. Both phenotypes might alternate due to a reciprocal regulatory effect or to the existence of two different sub-populations engaging both types of response, being the phagocytic sub-population firstly extinguished and replaced by a rather inflammatory sub-population. This inflammatory profile is characterized by the increased release of pro-inflammatory cytokines TNF-α, IL-1β and IL-6, the up-regulation of COX-2 and enhanced activities of MMP-2 and MMP-9. Regulatory interactions between the UCB-induced events are portrayed in the figure. 5. References Akassoglou, K., Bauer, J., Kassiotis, G., Pasparakis, M., Lassmann, H., Kollias, G., Probert, L., Oligodendrocyte apoptosis and primary demyelination induced by local TNF/p55TNF receptor signaling in the central nervous system of transgenic mice: models for multiple sclerosis with primary oligodendrogliopathy. Am J Pathol. 153, Allan, S. M., Rothwell, N. J., Inflammation in central nervous system injury. Philos Trans R Soc Lond B Biol Sci. 358, Aloisi, F., Immune function of microglia. Glia. 36, Bhat, N. R., Zhang, P., Lee, J. C., Hogan, E. L., Extracellular signal-regulated kinase and p38 subgroups of mitogen-activated protein kinases regulate inducible nitric oxide synthase and tumor necrosis factor-alpha gene expression in endotoxin-stimulated primary glial cultures. J Neurosci. 18, Block, M. L., Hong, J. S., Microglia and inflammation-mediated neurodegeneration: multiple triggers with a common mechanism. Prog Neurobiol. 76, Brites, D., Fernandes, A., Falcão, A. S., Gordo, A. C., Silva, R. F. M., Brito, M. A., Biological risks for neurological abnormalities associated with hyperbilirubinemia. J Perinatol. 29 Suppl 1, S8-13. Brito, M. A., Brites, D., Butterfield, D. A., A link between hyperbilirubinemia, oxidative stress and injury to neocortical synaptosomes. Brain Res. 1026,

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104 Chapter II Hansen, T. W., Mechanisms of bilirubin toxicity: clinical implications. Clin Perinatol. 29, , viii. Hasegawa-Ishii, S., Takei, S., Chiba, Y., Furukawa, A., Umegaki, H., Iguchi, A., Kawamura, N., Yoshikawa, K., Hosokawa, M., Shimada, A., Morphological impairments in microglia precede age-related neuronal degeneration in senescence-accelerated mice. Neuropathology. Hsieh, C. L., Koike, M., Spusta, S. C., Niemi, E. C., Yenari, M., Nakamura, M. C., Seaman, W. E., A role for TREM2 ligands in the phagocytosis of apoptotic neuronal cells by microglia. J Neurochem. 109, Hwang, D., Jang, B. C., Yu, G., Boudreau, M., Expression of mitogen-inducible cyclooxygenase induced by lipopolysaccharide: mediation through both mitogen-activated protein kinase and NF-kappaB signaling pathways in macrophages. Biochem Pharmacol. 54, Iadecola, C., Forster, C., Nogawa, S., Clark, H. B., Ross, M. E., Cyclooxygenase-2 immunoreactivity in the human brain following cerebral ischemia. Acta Neuropathol. 98, Ito, A., Mukaiyama, A., Itoh, Y., Nagase, H., Thogersen, I. B., Enghild, J. J., Sasaguri, Y., Mori, Y., Degradation of interleukin 1beta by matrix metalloproteinases. J Biol Chem. 271, Jack, C., Ruffini, F., Bar-Or, A., Antel, J. P., Microglia and multiple sclerosis. J Neurosci Res. 81, Kauppinen, T. M., Swanson, R. A., Poly(ADP-ribose) polymerase-1 promotes microglial activation, proliferation, and matrix metalloproteinase-9-mediated neuron death. J Immunol. 174, Kim, S. U., de Vellis, J., Microglia in health and disease. J Neurosci Res. 81, Kim, Y. S., Joh, T. H., Microglia, major player in the brain inflammation: their roles in the pathogenesis of Parkinson's disease. Exp Mol Med. 38, Kim, Y. S., Kim, S. S., Cho, J. J., Choi, D. H., Hwang, O., Shin, D. H., Chun, H. S., Beal, M. F., Joh, T. H., Matrix metalloproteinase-3: a novel signaling proteinase from apoptotic neuronal cells that activates microglia. J Neurosci. 25, Koenigsknecht-Talboo, J., Landreth, G. E., Microglial phagocytosis induced by fibrillar betaamyloid and IgGs are differentially regulated by proinflammatory cytokines. J Neurosci. 25, Koj, A., Initiation of acute phase response and synthesis of cytokines. Biochim Biophys Acta. 1317, Kreutzberg, G. W., Microglia: a sensor for pathological events in the CNS. Trends Neurosci. 19, Lee, S. J., Benveniste, E. N., Adhesion molecule expression and regulation on cells of the central nervous system. J Neuroimmunol. 98, Lee, Y. B., Schrader, J. W., Kim, S. U., p38 map kinase regulates TNF-alpha production in human astrocytes and microglia by multiple mechanisms. Cytokine. 12, Leonardo, C. C., Eakin, A. K., Ajmo, J. M., Collier, L. A., Pennypacker, K. R., Strongin, A. Y., Gottschall, P. E., Delayed administration of a matrix metalloproteinase inhibitor limits progressive brain injury after hypoxia-ischemia in the neonatal rat. J Neuroinflammation. 5, 34. Levi, G., Minghetti, L., Aloisi, F., Regulation of prostanoid synthesis in microglial cells and effects of prostaglandin E2 on microglial functions. Biochimie. 80, Lin, C. C., Kuo, C. T., Cheng, C. Y., Wu, C. Y., Lee, C. W., Hsieh, H. L., Lee, I. T., Yang, C. M., IL-1 beta promotes A549 cell migration via MAPKs/AP-1- and NF-kappaBdependent matrix metalloproteinase-9 expression. Cell Signal. 21, Lynch, M. A., The multifaceted profile of activated microglia. Mol Neurobiol. 40, McCarthy, K. D., de Vellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 85, McDonagh, A. F., Assisi, F., The ready isomerization of bilirubin IX- in aqueous solution. Biochem J. 129, McDonald, J. W., Shapiro, S. M., Silverstein, F. S., Johnston, M. V., Role of glutamate receptor-mediated excitotoxicity in bilirubin-induced brain injury in the Gunn rat model. Exp Neurol. 150, McRae, A., Gilland, E., Bona, E., Hagberg, H., Microglia activation after neonatal hypoxicischemia. Brain Res Dev Brain Res. 84,

105 Unconjugated bilirubin-induced phagocytic and inflammatory role of microglia Michaluk, P., Kaczmarek, L., Matrix metalloproteinase-9 in glutamate-dependent adult brain function and dysfunction. Cell Death Differ. 14, Miler, I., Vetvicka, V., Sima, P., Taborsky, L., The effect of bilirubin on the phagocytic activity of mouse peripheral granulocytes and monocytes in vivo. Folia Microbiol (Praha). 30, Miyaoka, T., Seno, H., Itoga, M., Iijima, M., Inagaki, T., Horiguchi, J., Schizophreniaassociated idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome). J Clin Psychiatry. 61, Muzio, L., Martino, G., Furlan, R., Multifaceted aspects of inflammation in multiple sclerosis: the role of microglia. J Neuroimmunol. 191, Nakajima, K., Kohsaka, S., Microglia: neuroprotective and neurotrophic cells in the central nervous system. Curr Drug Targets Cardiovasc Haematol Disord. 4, Napoli, I., Neumann, H., Microglial clearance function in health and disease. Neuroscience. 158, Neumann, H., Kotter, M. R., Franklin, R. J., Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. 132, Nuttall, R. K., Silva, C., Hader, W., Bar-Or, A., Patel, K. D., Edwards, D. R., Yong, V. W., Metalloproteinases are enriched in microglia compared with leukocytes and they regulate cytokine levels in activated microglia. Glia. 55, Oh, W., Tyson, J. E., Fanaroff, A. A., Vohr, B. R., Perritt, R., Stoll, B. J., Ehrenkranz, R. A., Carlo, W. A., Shankaran, S., Poole, K., Wright, L. L., Association between peak serum bilirubin and neurodevelopmental outcomes in extremely low birth weight infants. Pediatrics. 112, Porter, M. L., Dennis, B. L., Hyperbilirubinemia in the term newborn. Am Fam Physician. 65, Raivich, G., Bohatschek, M., Kloss, C. U., Werner, A., Jones, L. L., Kreutzberg, G. W., Neuroglial activation repertoire in the injured brain: graded response, molecular mechanisms and cues to physiological function. Brain Res Brain Res Rev. 30, Rodrigues, C. M., Solá, S., Brites, D., 2002a. Bilirubin induces apoptosis via the mitochondrial pathway in developing rat brain neurons. Hepatology. 35, Rodrigues, C. M., Solá, S., Brito, M. A., Brites, D., Moura, J. J., 2002b. Bilirubin directly disrupts membrane lipid polarity and fluidity, protein order, and redox status in rat mitochondria. J Hepatol. 36, Rodrigues, C. M., Solá, S., Castro, R. E., Laires, P. A., Brites, D., Moura, J. J., 2002c. Perturbation of membrane dynamics in nerve cells as an early event during bilirubininduced apoptosis. J Lipid Res. 43, Rosenberg, G. A., Matrix metalloproteinases in neuroinflammation. Glia. 39, Rothwell, N. J., Annual review prize lecture cytokines - killers in the brain? J Physiol. 514 ( Pt 1), Roux, P. P., Blenis, J., ERK and p38 MAPK-activated protein kinases: a family of protein kinases with diverse biological functions. Microbiol Mol Biol Rev. 68, Saura, J., Tusell, J. M., Serratosa, J., High-yield isolation of murine microglia by mild trypsinization. Glia. 44, Sedgwick, J. D., Riminton, D. S., Cyster, J. G., Korner, H., Tumor necrosis factor: a masterregulator of leukocyte movement. Immunol Today. 21, Shakibaei, M., John, T., Schulze-Tanzil, G., Lehmann, I., Mobasheri, A., Suppression of NF-kappaB activation by curcumin leads to inhibition of expression of cyclo-oxygenase-2 and matrix metalloproteinase-9 in human articular chondrocytes: Implications for the treatment of osteoarthritis. Biochem Pharmacol. 73, Silva, R. F. M., Falcão, A. S., Fernandes, A., Gordo, A. C., Brito, M. A., Brites, D., Dissociated primary nerve cell cultures as models for assessment of neurotoxicity. Toxicol Lett. 163, 1-9. Silva, R. F. M., Mata, L. R., Gulbenkian, S., Brito, M. A., Tiribelli, C., Brites, D., Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and ph. Biochem Biophys Res Commun. 265, Silva, R. F. M., Rodrigues, C. M. P., Brites, D., Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid. J Hepatol. 34, Silva, R. F. M., Rodrigues, C. M. P., Brites, D., Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin. Pediatr Res. 51,

106 Chapter II Simard, A. R., Soulet, D., Gowing, G., Julien, J. P., Rivest, S., Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron. 49, Soorani-Lunsing, I., Woltil, H. A., Hadders-Algra, M., Are moderate degrees of hyperbilirubinemia in healthy term neonates really safe for the brain? Pediatr Res. 50, Sternlicht, M. D., Werb, Z., How matrix metalloproteinases regulate cell behavior. Annu Rev Cell Dev Biol. 17, Streit, W. J., Microglia as neuroprotective, immunocompetent cells of the CNS. Glia. 40, Sun, H. N., Kim, S. U., Lee, M. S., Kim, S. K., Kim, J. M., Yim, M., Yu, D. Y., Lee, D. S., Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-dependent activation of phosphoinositide 3-kinase and p38 mitogen-activated protein kinase signal pathways is required for lipopolysaccharide-induced microglial phagocytosis. Biol Pharm Bull. 31, Takahashi, K., Prinz, M., Stagi, M., Chechneva, O., Neumann, H., TREM2-transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis. PLoS Med. 4, e124. Takahashi, K., Rochford, C. D., Neumann, H., Clearance of apoptotic neurons without inflammation by microglial triggering receptor expressed on myeloid cells-2. J Exp Med. 201, Tanaka, T., Ueno, M., Yamashita, T., Engulfment of axon debris by microglia requires p38 MAPK activity. J Biol Chem. 284, Tansey, M. G., Frank-Cannon, T. C., McCoy, M. K., Lee, J. K., Martinez, T. N., McAlpine, F. E., Ruhn, K. A., Tran, T. A., Neuroinflammation in Parkinson's disease: is there sufficient evidence for mechanism-based interventional therapy? Front Biosci. 13, Town, T., Nikolic, V., Tan, J., The microglial "activation" continuum: from innate to adaptive responses. J Neuroinflammation. 2, 24. Vanden Berghe, W., Plaisance, S., Boone, E., De Bosscher, K., Schmitz, M. L., Fiers, W., Haegeman, G., p38 and extracellular signal-regulated kinase mitogen-activated protein kinase pathways are required for nuclear factor-kappab p65 transactivation mediated by tumor necrosis factor. J Biol Chem. 273, Vetvicka, V., Sima, P., Miler, I., Bilej, M., The immunosuppressive effects of bilirubin. Folia Microbiol (Praha). 36, Vexler, Z. S., Yenari, M. A., Does inflammation after stroke affect the developing brain differently than adult brain? Dev Neurosci. 31, Vincenti, M. P., Brinckerhoff, C. E., Signal transduction and cell-type specific regulation of matrix metalloproteinase gene expression: can MMPs be good for you? J Cell Physiol. 213, von Zahn, J., Moller, T., Kettenmann, H., Nolte, C., Microglial phagocytosis is modulated by pro- and anti-inflammatory cytokines. Neuroreport. 8, Waetzig, V., Czeloth, K., Hidding, U., Mielke, K., Kanzow, M., Brecht, S., Goetz, M., Lucius, R., Herdegen, T., Hanisch, U. K., c-jun N-terminal kinases (JNKs) mediate proinflammatory actions of microglia. Glia. 50, Watson, R. L., Hyperbilirubinemia. Crit Care Nurs Clin North Am. 21, , vii. Wilms, H., Rosenstiel, P., Sievers, J., Deuschl, G., Zecca, L., Lucius, R., Activation of microglia by human neuromelanin is NF-kappaB dependent and involves p38 mitogenactivated protein kinase: implications for Parkinson's disease. FASEB J. 17, Woo, M. S., Park, J. S., Choi, I. Y., Kim, W. K., Kim, H. S., Inhibition of MMP-3 or -9 suppresses lipopolysaccharide-induced expression of proinflammatory cytokines and inos in microglia. J Neurochem. 106, Yokota, O., Terada, S., Ishizu, H., Ishihara, T., Ujike, H., Nakashima, H., Nakashima, Y., Kugo, A., Checler, F., Kuroda, S., Cyclooxygenase-2 in the hippocampus is up-regulated in Alzheimer's disease but not in variant Alzheimer's disease with cotton wool plaques in humans. Neurosci Lett. 343,

107 Chapter III DYNAMICS OF NEURON-GLIA INTERPLAY UPON EXPOSURE TO UNCONJUGATED BILIRUBIN Sandra L. Silva, Catarina Osório, Ana R. Vaz, Andreia Barateiro, Ana S. Falcão, Rui F. M. Silva, Dora Brites Research Institute for Medicines and Pharmaceutical Sciences (imed.ul), Faculdade de Farmácia, University of Lisbon, Av. Professor Gama Pinto, Lisbon , Portugal. Journal of Neurochemistry, 2010 (submitted) 79

108 Acknowledgments This work was supported by PTDC/SAU-NEU/64385/2006 grant, from Fundação para a Ciência e a Tecnologia (FCT), Lisbon, Portugal (to D.B.). S.L.S. was recipient of a PhD fellowship (SFRH/BD/30326/2006) from FCT. 80

109 Glial and neuronal pathological alterations by unconjugated bilirubin Abstract Microglia are the main players of the brain immune response acting as active sensors that rapidly respond to injurious insults by shifting into different activated states. Elevated levels of unconjugated bilirubin (UCB) induce cell death, immunostimulation and oxidative stress in both neurons and astrocytes. We recently reported that microglia phagocytosis phenotype precedes the release of pro-inflammatory cytokines upon UCB exposure. We investigated whether and how microglia microenvironment influences the response to UCB. Our findings, revealed that conditioned media derived from UCBtreated astrocytes reduce microglial inflammatory reaction and cell death, suggesting an attempt to refrain microglial overactivation. Conditioned medium from UCB-challenged neurons, although down-regulating TNF-α and IL-1β promoted the release of IL-6 and nitric oxide, the activation of matrix metalloproteinase-9, and cell death, as compared to UCB-direct effects on microglia. Moreover, soluble factors released by UCB-treated neurons showed to intensify the phagocytic properties manifested by microglia under direct exposure to UCB. Results from neuron-microglia mixed cultures incubated with UCB evidenced that sensitized microglia was able to prevent neurite outgrowth impairment and cell death. In conclusion, our data indicate that stressed neurons signal microglial clearance functions, but also overstimulate its inflammatory potential ultimately leading to microglia demise. Keywords: Astrocyte- and neuron-conditioned media; Inflammatory reaction; Nerve cell death; Neurite outgrowth and branching; Neuron-microglia mixed cultures; Nitrosative stress; Phagocytosis; Unconjugated bilirubin. 81

110 Chapter III 1. Introduction Hyperbilirubinemia is a common disorder of the brain that occurs in the neonatal period (Dennery et al., 2001; Porter and Dennis, 2002) and has been associated with minor neurologic dysfunction (Soorani-Lunsing et al., 2001), motor and auditory impairment (Oh et al., 2003; Shapiro and Nakamura, 2001). In fact, recent findings have strongly correlated auditory neuropathy disorder and acute bilirubin-induced neurotoxicity (Saluja et al., 2010). Moreover, neonatal hyperbilirubinemia might be a vulnerability factor for mental disorders (Dalman and Cullberg, 1999) such as schizophrenia (Hayashida et al., 2009; Miyaoka et al., 2000). Interestingly, elevated levels of unconjugated bilirubin (UCB) have also been associated with sepsis (Zhai et al., 2009) and fever (Kaplan and Hammerman, 2005). Indeed, previous reports from our work group have already demonstrated the immunostimulant properties of UCB in neurons (Falcão et al., 2006) and, in a more marked fashion, in astrocytes (Falcão et al., 2005; Fernandes et al., 2006; Fernandes et al., 2004) and microglia (Gordo et al., 2006; Silva et al., 2010), thus reinforcing the immunostimulant effects of UCB. Microglial cells have been acknowledged as the brain s immune system constituting a link between the central nervous system (CNS) a barrier protected organ and the general immune system (Aloisi, 2001; Streit, 2002). Microglia can perform a plethora of diverse functions that range from immune surveillance (Nimmerjahn et al., 2005) and phagocytic debris clearance (in both physiologic and pathologic conditions) (Cuadros and Navascues, 1998; Neumann et al., 2009; Streit, 2001), to the production of inflammatory mediators and reactive oxygen and nitrosative species (ROS and RNS, respectively) (Aloisi, 2001; Kim and de Vellis, 2005) which may contribute to nerve cell dysfunction and death (Gibbons and Dragunow, 2006). Our group has recently shown that microglia reacts to UCB by the acquisition of a phagocytic phenotype that shifts into a rather inflammatory profile (Silva et al., 2010). Accordingly, some authors have actually described microglia as active sensors that rapidly respond to microenvironmental changes with the engagement of different reactivity profiles (Hanisch and Kettenmann, 2007). Indeed, cellular interactions may be relevant for microglial reactivity. Neurons have proven its role as key immune modulators in the brain as they can influence the release of inflammatory or neurotrophic mediators form microglia (Biber et al., 2007; Lai and Todd, 2008; Polazzi and Contestabile, 2006). Furthermore, damaged neurons can signal microglia to engage phagocytic clearance, thus limiting injury extension (Petersen and Dailey, 2004; Witting et al., 2000). Astrocytes can also attenuate microglial toxicity (Giulian et al., 1993; Rozenfeld et al., 2003) and down-regulate its inflammatory reaction (Eskes et al., 2003; Min et al., 2006) or, in opposite, further activate microglia through 82

111 Glial and neuronal pathological alterations by unconjugated bilirubin increased ROS production (Yang et al., 1998). Finally, microglial reactivity is widely recognized as a cause of cell demise (Gibbons and Dragunow, 2006; Munch et al., 2003; Wang et al., 2005), that in some circumstances may also lead to a neuroprotective outcome (Minghetti et al., 2005; Zhang and Fedoroff, 1996). Considering that interplay between CNS cells may have such a strong and diverse influence on the cascade of microglial responses, this study was undertaken to ascertain if UCB-activated astrocytes or neurons could modulate the reactive response of microglia. Our findings indicate that soluble factors released by UCB-treated astrocytes dampen microglial inflammatory reaction upon UCB exposure. On the other hand, conditioned medium from neurons incubated with UCB, although exerting a similar down-regulation in microglial production of inflammatory cytokines, up-regulated matrix metalloproteinases (MMP) and nitric oxide (NO) secretion. Furthermore, neurons were able to augment microglial phagocytic abilities when facing UCB and ultimately increased UCB-induced microglial cell death. Microglia-neuron interplay in mixed cultures upon UCB stimulation revealed a neuroprotective role of microglia in preventing neurite impairment and necrotic-like cell death by UCB. We hypothesize that UCB-injured neurons might be signaling microglia to engage a phagocytic phenotype while hampering inflammatory reaction in an attempt to constrain lesion extent. 2. Material and Methods 2.1. Chemicals Neurobasal medium, B-27 Supplement (50x), Hanks balanced salt solution (HBSS), HBSS without Ca 2+ and Mg 2+, gentamicin (50 mg/ml), trypsin (0.5 g/l), Alexa Fluor 594 chicken anti-goat IgG and Trypsin/Ethylenediamine tetraacetic acid (EDTA) solution (0.25% trypsin, 1 mm EDTA in HBSS) were acquired from Invitrogen (Carlsbad, CA, USA). Dulbecco s modified Eagle s medium (DMEM), DMEM-Ham s F12 medium, fetal bovine serum (FBS), sodium pyruvate, non-essential aminoacids (NEA) and L- glutamine, were purchased from Biochrom AG (Berlin, Germany). Antibiotic antimycotic solution (20X), human serum albumin (HSA; Fraction V, fatty acid free), Hoechst dye, biotinylated tomato lectin (Lycopersicon esculentum), avidin-fluorescein isothiocyanate (FITC), avidin-tetramethylrhodamine isothiocyanate (TRITC), fluorescent latex beads 1 μm (2.5%), propidium iodide (PI), N-1-naphthylethylenediamine, and Coomassie Brilliant Blue R-250 were purchased from Sigma Chemical Co. (St. Louis, MO, USA). UCB was also obtained from Sigma and purified according to the method of McDonagh and Assisi, (1972). 83

112 Chapter III DuoSet ELISA kits were from R&D Systems, Inc. (Minneapolis, MN, USA). Goat anti-ionized calcium-binding adaptor molecule 1 (Iba1) was from Abcam (Cambridge, UK) and mouse anti-microtubule associated protein (MAP)-2 antibody was from Chemicon (Temecula, CA, USA). FITC-labeled horse antibody anti-mouse was acquired from Vector (Burlingame, CA, USA). Lactate dehydrogenase (LDH) cytotoxicity detection kit was purchased from Roche Molecular Biochemicals (Manheim, Germany). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany) Primary culture of microglia Animal care followed the recommendations of European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (Council Directive 86/609/EEC) and National Law 1005/92 (rules for protection of experimental animals). All animal procedures were approved by the Institutional Animal Care and Use Committee. Every effort was made to minimize the number of animals used and their suffering. Mixed glial cultures were prepared from 1 to 2 day-old Wistar rats as previously described (McCarthy and de Vellis, 1980), with minor modifications (Gordo et al., 2006). Cells (4 X 10 5 cells/cm 2 ) were plated on uncoated 12 or 6-well tissue culture plates (Corning Costar Corp., Cambridge, MA, USA) in culture medium (DMEM-Ham s F-12 medium supplemented with 2 mm L-glutamine, 1 mm sodium pyruvate, NEA 1X, 10% FBS, and 1% antibiotic-antimycotic solution) and maintained at 37ºC in a humidified atmosphere of 5% CO 2. Microglia were isolated as previously described by Saura et al., (2003). Briefly, after 21 days in vitro (DIV), microglia were obtained by mild trypsinization with a trypsin- EDTA solution diluted 1:3 in DMEM-F12 for min. The trypsinization resulted in detachment of an upper layer of cells containing all the astrocytes, whereas the microglia remained attached to the bottom of the well. The medium containing detached cells was removed and replaced with initial mixed glial-conditioned medium. Twenty-four hours after trypsinization, the attached cells were subjected to the different treatments Primary culture of astrocytes Astrocytes were isolated from 2-day-old rats as previously described (Blondeau et al., 1993), with minor modifications (Silva et al., 1999). Cells were resuspended in DMEM containing 11 mm sodium bicarbonate, 71 mm glucose and 1% antibiotic antimycotic solution supplemented with 10% FBS, plated (2.0 X 10 5 cells/cm2) on 75 cm 2 culture flasks and cultured for 10 DIV. 84

113 Glial and neuronal pathological alterations by unconjugated bilirubin 2.4. Primary neuronal cell cultures Neurons were isolated from foetuses of day pregnant Wistar rats, as described previously (Silva et al., 2002). Pregnant rats were anesthetized and decapitated. The foetuses were collected in HBSS and rapidly decapitated, the brain cortex was mechanically fragmented, and the fragments transferred to a 0.5 g/l trypsin in Ca 2+ and Mg 2+ free HBSS medium and incubated for 15 min at 37ºC. After trypsinization, cells were washed twice in calcium and magnesium free HBSS containing 10% FBS, and resuspended in Neurobasal medium supplemented with 0.5 mm L- glutamine, 25 µm L-glutamic acid, 2% B-27 Supplement, and 0.12 mg/ml gentamicin. Aliquots of 1 X 10 5 cells/cm 2 were plated on poly-d-lysine coated 75 cm 2 culture flasks and maintained at 37ºC in a humidified atmosphere of 5% CO 2. Every 3 days, 0.5 ml of old medium was removed by aspiration and replaced by the same volume of fresh medium without L-glutamic acid Neuron-microglia mixed cultures Primary cortical microglia were isolated from mixed glial cultures by mild trypsinization as described above. Remaining microglia was removed by trypsinization from the bottom of the plates and seeded on top of 2 DIV neurons at a density of 3.6 X 10 4 cells/cm 2. Mixed neuron-microglia cultures were kept for 24 h before treatment with UCB Cell treatment Microglial cells were incubated in the absence (control) or in the presence of 50 µm UCB plus 100 µm HSA, for 4 h or 24 h, at 37ºC. A UCB stock solution (10 mm) was prepared in 0.1 M NaOH immediately before use and the ph of the incubation medium was restored to 7.4 by addition of equal amounts of 0.1 M HCl. All the experiments with UCB were performed under light protection to avoid photodegradation. To study the role of soluble factors released by astrocytes or neurons upon UCB exposure, microglial cells were incubated for 4 or 24 h with conditioned medium from either 10 DIV astrocytes or 8 DIV neurons prior exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. Neuron-microglia mixed cultures at 3 DIV were incubated in the absence (control) or in the presence of 50 µm UCB plus 100 µm HSA, for 24 h, at 37ºC. Neuron-microglia mixed cultures at 3 DIV were incubated in the absence (control) or in the presence of 50 µm UCB plus 100 µm HSA, for 24 h, at 37ºC. 85

114 Chapter III 2.7. Measurement of cytokine release Aliquots of the cell culture media were collected at the end of the incubations and, after removal of cellular debris by short centrifugation, placed in a 96-well microplate and assessed in triplicate for TNF-α, IL-1β and IL-6 with specific DuoSet ELISA Development kits from R&D Systems, according to the manufacturer s instructions. Results were expressed as fold change vs. control Gelatin zymography Aliquots of culture supernatant were analyzed by sodium dodecyl sulphatepolyacrylamide gel electrophoresis (SDS-PAGE) zymography in 0.1% gelatine 10% acrylamide gels under non-reducing conditions. After electrophoresis, gels were washed for 1 h with 2.5% Triton X-100 (in 50 mm Tris ph7.4; 5 mm CaCl 2 ; 1μM ZnCl 2 ) to remove SDS and renature the MMP species in the gel. Then the gels were incubated in the developing buffer (50 mm Tris ph7.4; 5 mm CaCl 2 ; 1μM ZnCl 2 ) overnight to induce gelatine lysis. For enzyme activity analysis, the gels were stained with 0.5% Coomassie Brilliant Blue R-250 and destained in 30% ethanol/10% acetic acid/h 2 O. Gelatinase activity, detected as a white band on a blue background, was quantified by computerized image analysis and normalized for total cellular protein Assessment of microglial phagocytic properties After treatment with UCB, cells were incubated with % (w/w) 1 μm fluorescent latex beads for 75 min at 37ºC and fixed with freshly prepared 4% (w/v) paraformaldehyde in phosphate-buffered saline (PBS). Microglia were counterstained with a primary antibody raised against Iba-1 (goat, 1:500) followed by a secondary Alexa Fluor 594 chicken anti-goat antibody (1:200). Green- and red-fluorescence images of fifteen random microscopic fields (original magnification: 630X) were acquired per sample. The number of ingested beads per cell was counted in approximately 250 cells per sample Quantification of nitrite levels Nitric oxide levels were estimated by measuring the concentrations of nitrites, which are the resulting NO metabolites. Briefly, supernatants free from cellular debris were mixed with Griess reagent in 96-well tissue culture plates for 10 min at room temperature in the dark. The absorbance at 540 nm was determined using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA). 86

115 Glial and neuronal pathological alterations by unconjugated bilirubin Evaluation of cell death Necrotic-like cell death was estimated by evaluating the activity of LDH released by nonviable cells, as well as by monitoring the cellular uptake of the fluorescent dye PI [3,8-diamino-5-(3-(diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide]. LDH was determined in the incubation medium using a cytotoxicity detection kit as described previously (Silva et al., 2002). The reaction was performed in a 96-well microplate and the absorbance measured at 490 nm. All readings were corrected for the possible interference of UCB absorption. The maximum amount of releasable LDH was obtained by lysing non-incubated cells with 2.0% Triton X-100 in DMEM for 5 min and the results were expressed as fold change vs. control. PI readily enters and stains non-viable cells, but cannot cross the membrane of viable cells. This dye binds to double-stranded DNA and emits red fluorescence (630 nm; absorbance 493 nm). Unpermeabilized adherent cells cultured on coverslips were incubated with a 75 μm PI solution for 15 min in the absence of light. Subsequently, cells were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS and the nuclei stained with Hoechst dye. Red-fluorescence and U.V. images of ten random microscopic fields (original magnification: 400X) were acquired per sample and the percentage of PI positive cells was counted and expressed as fold vs. respective control Neurite Extension and Ramification Neurite extension and ramification were assessed by the immunofluorescence detection of the cytoskeletal protein MAP-2, as described previously (Falcão et al., 2007). Briefly, cells were fixed as described above and a standard indirect immunocytochemical technique was carried out using a mouse anti-map-2 antibody (1:100) as the primary antibody and a horse FITC-labeled anti-mouse antibody (1:227) as the secondary antibody. In neuron-microglia mixed cultures, microglia were counterstained with a primary antibody raised against Iba-1 (goat, 1:500) followed by a secondary Alexa Fluor 594 chicken anti-goat antibody (1:200) and the nuclei stained with Hoechst dye. Fluorescence was visualized using a Leica DFC490 camera adapted to an AxioSkope microscope. Green- and red-fluorescence images of ten random microscopic fields (original magnification: 400X) were acquired per sample. Evaluation of neurite extension and number of nodes from individual neurons, were traced manually using ImageJ software (National Institutes of Health). 87

116 Chapter III Statistical Analysis Results of at least three different experiments, carried out in duplicate, were expressed as mean ± SEM. Differences between groups were determined by one-way ANOVA with Dunnett s or Bonferroni s multiple comparisons post tests, using Instat 3.05 (GraphPad Software, San Diego, CA). Statistical significance was considered for a p value less than Results 3.1. Conditioned media from UCB-treated astrocytes and neurons modulate microglial secretion of cytokines, as compared to UCB-activated microglia Microglial cells are important sources of cytokines in the brain (Hanisch, 2002). In fact, a recent report from our group demonstrated that microglia, upon UCB stimulation, engages an inflammatory reaction which entails IL-1β IL-6 and TNF-α production and follows different temporal profiles of secretion (Silva et al., 2010). However, the influence of neighbouring cells must be considered when evaluating the response of microglia to toxic molecules like UCB. Indeed, several authors have described that neurons or astrocytes can modulate the inflammatory reaction undertaken by microglia (Giulian et al., 1993; Min et al., 2006; Mott et al., 2004; Polazzi and Contestabile, 2006). For that matter we compared cytokine secretion by microglia directly exposed to UCB or to astrocytes and neuron conditioned media (ACM and NCM, respectively) after incubation with UCB. Our results indicated that both UCB-treated ACM and NCM completely counteracted (p<0.01) the UCB-induced increase in IL-1β (1.5 fold, p<0.01), as depicted in Figure III.1A. Regarding TNF-α production (Figure III.1B), only NCM was able to significantly prevent (p<0.05) the elevation elicited by UCB (1.5 fold, p<0.05). Curiously, both ACM and NCM obtained after treatment with UCB led to an IL-6 overproduction by microglia (2.7 and 2.8-fold, respectively; p<0.01 vs. control) which proved to be significantly higher than the one triggered by the direct effect of UCB in microglia (p<0,01 for both UCB-treated ACM and NCM), as can be seen in Figure III.1C. Therefore, these findings indicate that UCB-stimulated astrocytes and neurons dampen microglial inflammatory response triggered by UCB in isolated cells, since the release of proinflammatory mediators such as IL-1β and TNF-α is down-regulated while the secretion of IL-6, which is considered to exert anti-inflammatory effects (Loddick et al., 1998), is greatly enhanced. 88

117 Glial and neuronal pathological alterations by unconjugated bilirubin Fig. III.1. Conditioned media derived from astrocytes (ACM) and neurons (NCM) incubated with UCB modulate the secretion of IL-1β, TNF-α and IL- 6 by microglia directly exposed to UCB. Rat cortical microglial cells were treated for 24 h with 50 µm UCB in the presence of 100 µm HSA, or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. IL-1β, TNF-α and IL-6 concentrations in the media were determined by ELISA and expressed as mean ± SEM fold change vs. control from three independent experiments performed in triplicate. *p<0.05 and **p<0.01 vs. control; p<0.05 and p<0.01 vs. UCB alone. 89

118 Chapter III 3.2. Conditioned media from UCB-treated astrocytes and neurons have opposing effects on microglial MMP-9 activity, as compared to UCB-activated microglia UCB has been previously shown to enhance the activity of MMP-9 and -2, proteases that actively participate in inflammatory events. Moreover, the same study also evidenced the existence of regulatory interactions between cytokine secretion and MMP activation upon UCB exposure (Silva et al., 2010). Since conditioned media from UCBexposed astrocytes and neurons showed to modulate the secretion of inflammatory mediators, we wondered what effect could ACM and NCM exert on MMP activation. The results show that UCB-treated NCM and direct UCB stimulation have similar activation levels of MMP-2 (p<0.05 vs. control) but ACM from astrocytes exposed to UCB did not produce any significant effect (Figure III.2A). Curiously, our findings evidenced opposing effects of ACM and NCM upon UCB stimulation on MMP-9 activity by microglia directly exposed to UCB (Figure III.2B). While ACM abrogated (p<0.05) the increase in MMP-9 activity elicited by UCB in microglia (1.1-fold, p<0.05), NCM further aggravated such effect (1.2-fold; p<0.01 vs. control and p<0.05 vs. UCB alone) Nitric oxide (NO) release by microglia is elicited by UCB and is higher by naïve microglia exposed to conditioned medium from UCB-treated neurons Microglia and astrocytes generate an NO burst in response to injury, which may be cytotoxic (Cassina et al., 2002; Kawase et al., 1996) and trigger the clearance of damaged cells from the CNS (Bal-Price and Brown, 2001; Golde et al., 2002). Although UCB has been shown to elicit NO release in neurons (Brito et al., 2010), no evidence was ever confirmed regarding microglial cells exposed to UCB. Therefore, this study is the first to demonstrate that UCB can induce the generation of NO by microglia (1.3-fold; p<0.05 vs. control). Moreover, the results displayed in Figure III.3 indicate that the evoked NO production is even more enhanced when non-treated (naïve) microglia are exposed to conditioned medium derived from neurons incubated with UCB, NCM, (1.5- fold; p<0.01 vs. control and p<0.05 vs. UCB alone). On the other hand, conditioned medium from astrocytes exposed to UCB, ACM, did not significantly alter the amount of NO release by UCB-stimulated microglia (1.3-fold, p<0.05 vs. control but N.S. vs. UCB alone). Data show that neighbouring neurons may further increase the nitrosative stress induced by UCB in microglia. 90

119 Glial and neuronal pathological alterations by unconjugated bilirubin Fig. III.2. MMP-9 activation in microglia directly exposed to UCB is prevented by conditioned medium derived from UCB-treated astrocytes (ACM), but enhanced by conditioned medium derived from UCB-treated neurons (NCM). Culture supernatants from rat cortical microglial cells were harvested after 24 h incubation with 50 µm UCB in the presence of 100 µm HSA or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. Samples were subjected to zymography as described in Methods. (A) Representative gels of one experiment are reported. MMP-2 and MMP-9 were identified by their apparent molecular mass of 67 and 92 kda, respectively. (B,C) Intensity of the bands was quantified by scanning densitometry, standardized with respect to total protein content and expressed as mean ± SEM fold change compared with those obtained in control conditions. *p<0.05 and **p<0.01 vs. respective control; p<0.05 vs. UCB alone. 91

120 Chapter III Fig. III.3. NO release by UCB-activated microglia is intensified by conditioned medium derived from UCB-treated neurons (NCM), but not changed by conditioned medium derived from UCB-treated astrocytes (ACM). Rat cortical microglial cells were treated for 24 h with 50 µm UCB in the presence of 100 µm HSA or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. NO production was estimated by the quantification of nitrite levels and expressed as mean ± SEM fold change from three independent experiments performed in duplicate. *p<0.05 and **p<0.01 vs. control and p<0.05 vs. UCB alone Loss of viability in UCB-stimulated microglia is less in naïve microglia exposed to conditioned medium from UCB-treated astrocytes (ACM), but higher if treated with medium from neurons incubated with UCB (NCM) To evaluate whether the loss of microglia viability upon exposure to ACM and NCM was different from the one produced by the direct action of UCB on microglial cell death, we assessed the extracellular accumulation of LDH, a characteristic feature of cell membrane integrity loss. Our results evidenced that ACM does not produce any change over the results observed in the absence of UCB (Figure III.4) and was significantly less (p<0.01) than the release of LDH obtained in UCB-stimulated microglia (1.5 fold, p<0.01). In contrast, the values achieved with NCM-treated microglia were clearly higher (1.8 fold, p<0.01 vs. control and p<0.05 vs. UCB alone). This finding strengthens again the notion that neurons may further damage reactive microglia increasing UCB injury in brain parenchyma. 92

121 Glial and neuronal pathological alterations by unconjugated bilirubin Fig. III.4. LDH leakage by UCB-activated microglia is completely abrogated by conditioned medium derived from UCB-treated astrocytes (ACM) but enhanced by conditioned medium derived from UCB-treated neurons (NCM). Culture supernatants from rat cortical microglial cells were harvested after 24 h incubation with 50 µm UCB in the presence of 100 µm HSA or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. Samples were used for determination of LDH activity. Results are mean ± SEM from three independent experiments performed in duplicate. **p<0.01 vs. control; p<0.05 and p<0.01 vs. UCB alone Microglial phagocytic properties enhanced by UCB further increase by medium collected from neurons exposed to UCB Recently we have described that microglia reacts to UCB by the acquisition of an early phagocytic phenotype that afterwards shifted to a rather inflammatory state (Silva et al., 2010). Since icteric NCM was shown to decrease the production of proinflammatory cytokines, but to promote microglia demise, we wondered how microglia would react if surrounded by neurons in an icteric parenchyma. Interestingly, as can be seen in Figure III.5, the uptake of fluorescent latex beads by microglia is stimulated in the presence of UCB, but even more by the icteric NCM (~40%, p<0.05). Therefore, UCB seems to induce the release of soluble factors from neurons that stimulate the phagocytic role of microglia. Whether deleterious consequences occur either by an increased time of exposure to UCB or by higher UCB levels, are to be seen in future studies. 93

122 Chapter III Fig. III.5. Microglia phagocytic ability increases upon exposure to UCB, and this property is enhanced by icteric NCM derived from UCB-treated neurons. Rat cortical microglial cells were treated for 24 h with 50 µm UCB in the presence of 100 µm HSA or with ACM or NCM from cells exposed to the same UCB and HSA levels for 12 h. Controls were performed in the absence of UCB. Cells were incubated with 1 μm green fluorescent latex beads as described in Methods. (A) Microglial cells were counterstained with an antibody raised against Iba-1 (red) Representative results of one experiment are shown. Scale bar 20 μm. (B) Results are expressed as number of ingested beads per cell (± SEM) from three independent experiments. *p<0.05 and **p<0.01 vs. control; p<0.05 and p<0.01 vs. UCB alone. 94

123 Glial and neuronal pathological alterations by unconjugated bilirubin 3.6. UCB-induced neuronal network impairment is prevented by microglia environment Considering that neurons showed to greatly influence microglial reactivity using a conditioned medium model and that the phagocytic profile seems to be promoted under these conditions, we decided to invert the picture and take a look at the effects of UCBreactive microglia on neurons. For that matter we used a neuron-microglia mixed culture system where cell-to-cell contact could take place. Since immature cortical neurons have previously been shown to be more vulnerable than the most differentiated ones to UCBinduced cell death, glutamate release and TNF-α secretion (Falcão et al., 2006), we chose to explore the effect of microglia on 3 DIV neurons, a differentiation stage where UCB seems to produce a more injurious outcome. As previously reported by Falcão et al. (2007) 24 h exposure of immature cortical neurons to UCB decreases neuronal survival and causes an impairment of neurite development. The involvement of microglia in neurite outgrowth during brain development has been acknowledged (Cuadros and Navascues, 1998), as well as in pathologic events (Munch et al., 2003; Rozenfeld et al., 2003). Activated microglia has been accounted for as either neurotoxic (Zhang and Fedoroff, 1996) or neuroprotective in several models (Nakajima et al., 2007) due to its inflammatory and phagocytic nature. Nonetheless, the impact of microglia on neuronal plasticity in an icteric milieu had not been previously evaluated. Hence, we decided to verify if the UCB-elicited network impairment in neurons is modulated by the presence of microglia when mixed cultures of both cell types are used. As represented in Figure III.6, microglia when cultivated with neurons are able to protect the injury caused by UCB at the level of neurite extension (p<0.01) and ramification (p<0.05). 95

124 Chapter III Fig. III.6. Reduction of neurite extension and ramification by UCB in immature neurons is counteracted by the presence of microglia. Cortical neurons and neuronmicroglia mixed cultures at 3 DIV were exposed for 24 h, to either no addition (control), or to 50 µm UCB as described in Methods. Neurite extension (A) and number of nodes (B) were identified by immunolabeling for MAP-2 (green), quantified by ImageJ and expressed as arbitrary units ± SEM. Microglial cells were counterstained with an antibody raised against Iba-1 (red). Representative results of one experiment are shown in (C). **p<0.01 vs. control; p<0.05 and p<0.01 vs. UCB alone. Scale bar 40 µm. Therefore, defensive properties on the neuronal injury by UCB seem to be a key role of microglia when UCB gets into the brain. We hypothesize that the additional presence of risk factors such as sepsis or prematurity may change the protective role of microglia. We also questioned if the effects observed were really a protection or resulted from the increased microglia phagocytosis previously observed. Thus we next investigated the influence of microglia in neuron viability upon UCB exposure. 96

125 Glial and neuronal pathological alterations by unconjugated bilirubin 3.7. Neuronal cell death triggered by UCB is diminished in the presence of microglia Interplay between neurons and microglia is widely recognized (Biber et al., 2007; Zhang and Fedoroff, 1996). In fact, several reports have demonstrated that activated microglia can decrease neuronal viability (Gibbons and Dragunow, 2006) or, by the other hand, exert a neuroprotective role (Figueiredo et al., 2008; Nakajima et al., 2007). Indeed, as demonstrated in Figure III.7, the propensity of microglia to decrease cellular necrosis is reinforced in the presence of UCB, with a completely abrogated UCB-induced death of immature neurons (p<0.01) in mixed cultures of neurons and microglia. Fig. III.7. Decreased neuronal survival by UCB disappears when microglia is nearby. Cortical neurons or neuron-microglia mixed cultures at 3 DIV were exposed for 24 h, to either no addition (control), or 50 µm UCB and further incubated with 75 μm PI as described in Methods. Results are percentage of PI-positive cells and are expressed as mean ± SEM from three independent experiments performed in triplicate **p<0.01 vs. respective control and p<0.01 vs. UCB alone. These results reinforce the neuroprotective role that microglia may exert in preventing UCB neurological damage. However, given the acknowledged phagocytic nature of microglia and the fact that icteric NCM further enhances that property, we believe that this apparent protection results from the phagocytic clearance of UCBinjured neurons, similarly to the events described in other disease models (Napoli and Neumann, 2009; Neumann et al., 2009; Tanaka et al., 2009). 97

126 Chapter III 4. Discussion Cell-to-cell interactions as well as the release of soluble mediators are essential regulators of many critical functions in brain health and disease. In this study we demonstrated, for the first time, that astrocytes and neurons stimulated by UCB can modulate microglial reactivity through different pathways. UCB-treated astrocytes lead to a decrease in the inflammatory response elicited by UCB in microglia and ultimately prevent UCB-induced cell demise. On the other hand, although UCB-exposed neurons release soluble factors with the capacity to down-regulate microglial production of proinflammatory cytokines, neurons also lead to increased NO production and further aggravate UCB-induced MMP-9 activity in microglia. Interestingly, neurons exacerbate microglial early phagocytic phenotype while also increase microglia demise by UCB. Our study is the first to evaluate if injurious effects of UCB on neurons are modified in the presence of microglial cells, thus taking into consideration probable cellular interactions. Curiously, our findings show that microglial cells counteract the deleterious effects produced by UCB on neuronal cell death and neurite extension and ramification. Altogether, these observations allow us to hypothesize that UCB-evoked injury in neurons is signaling microglia to engage phagocytic clearance in an attempt to limit injury extension, rather than exacerbating inflammation. The contribution of microglial immune response to either damage or repair is a broadly discussed subject (Amor et al., 2010). Additionally, the influence of neighboring cells on microglial reactivity is renowned, substantiating the observed immunomodulatory effects produced by icteric ACM and NCM. In fact, when evaluating the role of microglia in organometallic compounds toxicity, astrocytes were shown to dampen TNF-α production and, additionally, were not able to induce morphogical changes (Eskes et al., 2003). Furthermore, astrocytes can attenuate microglial activation in several disease models, thus preventing neurodegeneration (Giulian et al., 1993; Rozenfeld et al., 2003). It is also interesting to notice that, in our work model, UCB-treated astrocytes and neurons greatly enhanced IL-6 production by microglia. Similar effects were observed in studies regarding microglial reactivity to methylmercury where interaction between activated microglia and astrocytes was shown to increase local IL-6 release, which may cause astrocyte reactivity and ultimately culminate in neuroprotection (Eskes et al., 2002). We have recently demonstrated that UCB-induced cytokine release by microglia, particularly IL-1β, originate MMP-9 and -2 activation (Silva et al., 2010). Thus, the observed decrease in microglial MMP-9 activity after exposure to icteric ACM could be explained by the regulatory interactions between cytokines and MMPs, since the 98

127 Glial and neuronal pathological alterations by unconjugated bilirubin production of the formers is also hampered in this particular case. Collectively, our findings suggest an overall protective effect exerted by astrocytes on microglia, upon UCB stimulation. We theorize that microglia, when isolated, respond intensively to UCB stimulation, but previously activated astrocytes drive microglia to refrain its response. Other authors have postulated that this is a feasible mechanism to prevent excessive brain inflammation (Min et al., 2006). In this work we also describe the down-regulatory effect of icteric NCM upon UCB-stimulated microglial secretion of pro-inflammatory cytokines, particularly IL-1β and TNF-α. Accordingly, neurons reduce glial secretion of TNF-α after treatment with the bacterial endotoxin lipopolysaccharide (LPS) emphasizing their role as immune responsiveness regulators (Chang et al., 2001; Mott et al., 2004). Similarly, neurons constitutively release factors that maintain microglia in a quiescent state in the healthy brain (Biber et al., 2007), such as the protein CD200, a molecule shown to downregulate microglial inflammatory response (Broderick et al., 2002; Deckert et al., 2006). Nevertheless, under inflammatory or deleterious conditions, microglia s behavior might be modified, as suggested by the study of Hasegawa et al. (2007) where the contact rate between activated microglia and hippocampal pyramidal cells dendrites is increased. Additionally, the severity of neuronal injury has been reported to determine the release of trophic or pro-inflammatory mediators form microglia (Lai and Todd, 2008). For example, interaction of microglia with apoptotic neurons leads to a progressive down-regulation of pro-inflammatory microglial functions (Minghetti et al., 2005). Considering that neurons reveal higher susceptibility to cell death than astrocytes, in the presence of high UCB levels (Brites et al., 2009; Silva et al., 2002), we postulate that this event leads to the release of factors by neurons which give the impression to influence microglial response to UCB in a more marked fashion than those produced by astrocytes. In fact, our results reveal that conditioned medium derived from UCB-stimulated neurons dampens microglial production of pro-inflammatory cytokines, yet increases MMP-9 activity and NO secretion, culminating in microglial death. These observations led to the assumption that UCB-injured neurons might be signaling microglia to shift its activity state rather than exerting a down- or up-regulation of its functions. Indeed, a previous report regarding microglial reactivity to UCB has already identified two different phenotypes (phagocytic and inflammatory) which occurred in an alternate fashion possibly indicating the existence of two different microglial subpopulations reacting towards UCB (Silva et al., 2010). In the present study we observed that soluble factors released by UCB-damaged neurons are able to enhance the microglial phagocytic ability when compared to the one showed by UCB-stimulated microglia. These data reinforce the belief that neuronmicroglia interaction modifies the response phenotype of the latter cells, upon UCB 99

128 Chapter III exposure. Consistently, other authors have reported that microglia recognize and phagocyte dying neurons (Petersen and Dailey, 2004; Witting et al., 2000) providing a clearance of potentially harmful debris. Although MMP-9 activation and increase in NO production are normally included in the inflammatory reaction engaged by microglia, in this case we may consider that these events can be related to the acquisition of a phagocytic profile. In fact, NO has been proven to regulate phagocytosis (Kopec and Carroll, 2000) and to trigger the clearance of damaged cells (Cassina et al., 2002; Kawase et al., 1996). Furthermore, MMPs can be involved in chemoattraction of microglial cells to injured sites since they participate in the cleavage of fractalkine from neuronal membranes following toxic insults (Chapman et al., 2000). MMPs also have the ability to degrade the basal lamina surrounding the blood-brain barrier allowing infiltration of immune cells (Michaluk and Kaczmarek, 2007; Rosenberg, 2002) consequently participating in phagocytic events (Buss et al., 2007). Nevertheless, the engagement of such a dramatic phenotype such as microglial phagocytosis may culminate in cell death. Our observations regarding the influence of UCB-reactive neurons on microglial response seem to corroborate this concept since cell death is further aggravated under these conditions. Other studies have also reported that NCM produces an overactivation of LPS-stimulated microglia culminating in apoptotic elimination as a safety mechanism (Polazzi and Contestabile, 2006). Besides studying the influence of soluble factors released by astrocytes or neurons stimulated by UCB on microglial response, we intended to advance one step further in the understanding of UCB-induced neurotoxicity and add neuron-microglia cellto-cell interactions to the puzzle. For this matter we used neuron-microglia mixed cultures to study the effects of UCB on network dynamics and cell death, since isolated immature neurons proved to be more affected by UCB than more differentiated cells in these particular features (Falcão et al., 2007). Unexpectedly, microglia proved to completely counteract UCB-elicited neurite impairment and loss of viability. Other authors have proven opposite results concerning the effects of microglia on neurite outgrowth and cell viability. For instance, Munch et al. (2003) demonstrated that microglial activation induces cell death and inhibits neurite outgrowth in neuroblastoma cells. Microglia is also involved in neuron loss in Alzheimer s disease (Fuhrmann et al., 2010) and may cause neuronal cell death and degeneration (Gibbons and Dragunow, 2006; Rozenfeld et al., 2003). Taking into account all these confounding aspects, we hypothesize that this apparent microglial protection of UCB-induced neurotoxicity may indeed be regarded as a neuroprotective measure aiming at limiting injury extension by the phagocytic clearance of damaged neurons. Phagocytosis of amyloid deposits or 100

129 Glial and neuronal pathological alterations by unconjugated bilirubin cellular debris by microglia was also proven to be important in Alzheimer s disease (Simard et al., 2006) and multiple sclerosis (Takahashi et al., 2007). Thus, clearance of tissue debris performed by microglia following injury might constitute a regenerative measure (Napoli and Neumann, 2009; Neumann et al., 2009). In summary, our findings indicate that astrocytes activated by UCB release soluble factors that reduce microglial inflammatory reaction, probably in an attempt to refrain deleterious overactivation. On the other hand, UCB-stimulated neurons downregulate microglial production of inflammatory cytokines but further increase its phagocytic abilities and ultimately lead to microglial elimination. Furthermore, microglia counteract UCB-evoked impairment of neurite extension and ramification as well as neuronal cell death in a mixed culture model. Hence, we hypothesize that UCB-injured neurons might be signaling microglia to engage a phagocytic phenotype while hampering inflammatory reaction in an attempt to constrain lesion extent. In conclusion, this study introduces important knowledge to the understanding of the molecular basis of UCBinduced neurotoxicity as it adds, for the first time, an important piece to the puzzle, e.g. cellular interactions between microglia and other nerve cells in a model mimicking acute neonatal jaundice. 5. References Aloisi, F., Immune function of microglia. Glia. 36, Amor, S., Puentes, F., Baker, D., van der Valk, P., Inflammation in neurodegenerative diseases. Immunology. 129, Bal-Price, A., Brown, G. C., Inflammatory neurodegeneration mediated by nitric oxide from activated glia-inhibiting neuronal respiration, causing glutamate release and excitotoxicity. J Neurosci. 21, Biber, K., Neumann, H., Inoue, K., Boddeke, H. W., Neuronal 'On' and 'Off' signals control microglia. Trends Neurosci. 30, Blondeau, J. P., Beslin, A., Chantoux, F., Francon, J., Triiodothyronine is a high-affinity inhibitor of amino acid transport system L1 in cultured astrocytes. J Neurochem. 60, Brites, D., Fernandes, A., Falcão, A. S., Gordo, A. C., Silva, R. F. M., Brito, M. A., Biological risks for neurological abnormalities associated with hyperbilirubinemia. J Perinatol. 29 Suppl 1, S8-13. Brito, M. A., Vaz, A. R., Silva, S. L., Falcão, A. S., Fernandes, A., Silva, R. F. M., Brites, D., N-Methyl--Aspartate Receptor and Neuronal Nitric Oxide Synthase Activation Mediate Bilirubin-Induced Neurotoxicity. Mol Med. 16, Broderick, C., Hoek, R. M., Forrester, J. V., Liversidge, J., Sedgwick, J. D., Dick, A. D., Constitutive retinal CD200 expression regulates resident microglia and activation state of inflammatory cells during experimental autoimmune uveoretinitis. Am J Pathol. 161, Buss, A., Pech, K., Kakulas, B. A., Martin, D., Schoenen, J., Noth, J., Brook, G. A., Matrix metalloproteinases and their inhibitors in human traumatic spinal cord injury. BMC Neurol. 7, 17. Cassina, P., Peluffo, H., Pehar, M., Martinez-Palma, L., Ressia, A., Beckman, J. S., Estevez, A. G., Barbeito, L., Peroxynitrite triggers a phenotypic transformation in spinal cord astrocytes that induces motor neuron apoptosis. J Neurosci Res. 67,

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131 Glial and neuronal pathological alterations by unconjugated bilirubin Hayashida, M., Miyaoka, T., Tsuchie, K., Yasuda, H., Wake, R., Nishida, A., Inagaki, T., Toga, T., Nagami, H., Oda, T., Horiguchi, J., Hyperbilirubinemia-related behavioral and neuropathological changes in rats: a possible schizophrenia animal model. Prog Neuropsychopharmacol Biol Psychiatry. 33, Kaplan, M., Hammerman, C., Understanding severe hyperbilirubinemia and preventing kernicterus: adjuncts in the interpretation of neonatal serum bilirubin. Clin Chim Acta. 356, Kawase, M., Kinouchi, H., Kato, I., Akabane, A., Kondo, T., Arai, S., Fujimura, M., Okamoto, H., Yoshimoto, T., Inducible nitric oxide synthase following hypoxia in rat cultured glial cells. Brain Res. 738, Kim, S. U., de Vellis, J., Microglia in health and disease. J Neurosci Res. 81, Kopec, K. K., Carroll, R. T., Phagocytosis is regulated by nitric oxide in murine microglia. Nitric Oxide. 4, Lai, A. Y., Todd, K. G., Differential regulation of trophic and proinflammatory microglial effectors is dependent on severity of neuronal injury. Glia. 56, Loddick, S. A., Turnbull, A. V., Rothwell, N. J., Cerebral interleukin-6 is neuroprotective during permanent focal cerebral ischemia in the rat. J Cereb Blood Flow Metab. 18, McCarthy, K. D., de Vellis, J., Preparation of separate astroglial and oligodendroglial cell cultures from rat cerebral tissue. J Cell Biol. 85, McDonagh, A. F., Assisi, F., The ready isomerization of bilirubin IX- in aqueous solution. Biochem J. 129, Michaluk, P., Kaczmarek, L., Matrix metalloproteinase-9 in glutamate-dependent adult brain function and dysfunction. Cell Death Differ. 14, Min, K. J., Yang, M. S., Kim, S. U., Jou, I., Joe, E. H., Astrocytes induce hemeoxygenase-1 expression in microglia: a feasible mechanism for preventing excessive brain inflammation. J Neurosci. 26, Minghetti, L., Ajmone-Cat, M. A., De Berardinis, M. A., De Simone, R., Microglial activation in chronic neurodegenerative diseases: roles of apoptotic neurons and chronic stimulation. Brain Res Brain Res Rev. 48, Miyaoka, T., Seno, H., Itoga, M., Iijima, M., Inagaki, T., Horiguchi, J., Schizophreniaassociated idiopathic unconjugated hyperbilirubinemia (Gilbert's syndrome). J Clin Psychiatry. 61, Mott, R. T., Ait-Ghezala, G., Town, T., Mori, T., Vendrame, M., Zeng, J., Ehrhart, J., Mullan, M., Tan, J., Neuronal expression of CD22: novel mechanism for inhibiting microglial proinflammatory cytokine production. Glia. 46, Munch, G., Gasic-Milenkovic, J., Dukic-Stefanovic, S., Kuhla, B., Heinrich, K., Riederer, P., Huttunen, H. J., Founds, H., Sajithlal, G., Microglial activation induces cell death, inhibits neurite outgrowth and causes neurite retraction of differentiated neuroblastoma cells. Exp Brain Res. 150, 1-8. Nakajima, K., Tohyama, Y., Maeda, S., Kohsaka, S., Kurihara, T., Neuronal regulation by which microglia enhance the production of neurotrophic factors for GABAergic, catecholaminergic, and cholinergic neurons. Neurochem Int. 50, Napoli, I., Neumann, H., Microglial clearance function in health and disease. Neuroscience. 158, Neumann, H., Kotter, M. R., Franklin, R. J., Debris clearance by microglia: an essential link between degeneration and regeneration. Brain. 132, Nimmerjahn, A., Kirchhoff, F., Helmchen, F., Resting microglial cells are highly dynamic surveillants of brain parenchyma in vivo. Science. 308, Oh, W., Tyson, J. E., Fanaroff, A. A., Vohr, B. R., Perritt, R., Stoll, B. J., Ehrenkranz, R. A., Carlo, W. A., Shankaran, S., Poole, K., Wright, L. L., Association between peak serum bilirubin and neurodevelopmental outcomes in extremely low birth weight infants. Pediatrics. 112, Petersen, M. A., Dailey, M. E., Diverse microglial motility behaviors during clearance of dead cells in hippocampal slices. Glia. 46, Polazzi, E., Contestabile, A., Overactivation of LPS-stimulated microglial cells by cocultured neurons or neuron-conditioned medium. J Neuroimmunol. 172, Porter, M. L., Dennis, B. L., Hyperbilirubinemia in the term newborn. Am Fam Physician. 65, Rosenberg, G. A., Matrix metalloproteinases in neuroinflammation. Glia. 39,

132 Chapter III Rozenfeld, C., Martinez, R., Figueiredo, R. T., Bozza, M. T., Lima, F. R., Pires, A. L., Silva, P. M., Bonomo, A., Lannes-Vieira, J., De Souza, W., Moura-Neto, V., Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration. Infect Immun. 71, Saluja, S., Agarwal, A., Kler, N., Amin, S., Auditory neuropathy spectrum disorder in late preterm and term infants with severe jaundice. Int J Pediatr Otorhinolaryngol. Saura, J., Tusell, J. M., Serratosa, J., High-yield isolation of murine microglia by mild trypsinization. Glia. 44, Shapiro, S. M., Nakamura, H., Bilirubin and the auditory system. J Perinatol. 21 Suppl 1, S52-5; discussion S Silva, R., Mata, L. R., Gulbenkian, S., Brito, M. A., Tiribelli, C., Brites, D., Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and ph. Biochem Biophys Res Commun. 265, Silva, R. F. M., Rodrigues, C. M., Brites, D., Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin. Pediatr Res. 51, Silva, S. L., Vaz, A. R., Barateiro, A., Falcão, A. S., Fernandes, A., Brito, M. A., Silva, R. F. M., Brites, D., Features of bilirubin-induced reactive microglia: From phagocytosis to inflammation. Neurobiol Dis. 40, Simard, A. R., Soulet, D., Gowing, G., Julien, J. P., Rivest, S., Bone marrow-derived microglia play a critical role in restricting senile plaque formation in Alzheimer's disease. Neuron. 49, Soorani-Lunsing, I., Woltil, H. A., Hadders-Algra, M., Are moderate degrees of hyperbilirubinemia in healthy term neonates really safe for the brain? Pediatr Res. 50, Streit, W. J., Microglia and macrophages in the developing CNS. Neurotoxicology. 22, Streit, W. J., Microglia as neuroprotective, immunocompetent cells of the CNS. Glia. 40, Takahashi, K., Prinz, M., Stagi, M., Chechneva, O., Neumann, H., TREM2-transduced myeloid precursors mediate nervous tissue debris clearance and facilitate recovery in an animal model of multiple sclerosis. PLoS Med. 4, e124. Tanaka, T., Ueno, M., Yamashita, T., Engulfment of axon debris by microglia requires p38 MAPK activity. J Biol Chem. 284, Wang, X., Chen, S., Ma, G., Ye, M., Lu, G., Involvement of proinflammatory factors, apoptosis, caspase-3 activation and Ca2+ disturbance in microglia activation-mediated dopaminergic cell degeneration. Mech Ageing Dev. 126, Witting, A., Muller, P., Herrmann, A., Kettenmann, H., Nolte, C., Phagocytic clearance of apoptotic neurons by Microglia/Brain macrophages in vitro: involvement of lectin-, integrin-, and phosphatidylserine-mediated recognition. J Neurochem. 75, Yang, L., Tanaka, J., Zhang, B., Sakanaka, M., Maeda, N., Astrocytes modulate nitric oxide production by microglial cells through secretion of serine and glycine. Biochem Biophys Res Commun. 251, Zhai, R., Sheu, C. C., Su, L., Gong, M. N., Tejera, P., Chen, F., Wang, Z., Convery, M. P., Thompson, B. T., Christiani, D. C., Serum bilirubin levels on ICU admission are associated with ARDS development and mortality in sepsis. Thorax. 64, Zhang, S. C., Fedoroff, S., Neuron-microglia interactions in vitro. Acta Neuropathol. 91,

133 Chapter IV UNCONJUGATED BILIRUBIN NEUROTOXICITY IS MODULATED BY MICROGLIA AND PREVENTED BY GLYCOURSODEOXYCHOLIC ACID AND INTERLEUKIN-10 Sandra L. Silva, 1 Ana R. Vaz, 1 Maria J. Diógenes, 2,3 Nico van Rooijen, 4 Ana M. Sebastião, 2,3 Adelaide Fernandes, 1 Rui F. M. Silva, 1 Dora Brites 1 1 Research Institute for Medicines and Pharmaceutical Sciences (imed.ul), Faculdade de Farmácia, University of Lisbon, Av. Professor Gama Pinto, Lisbon , Portugal 2 Institute of Pharmacology and Neurosciences, Faculty of Medicine University of Lisbon, Lisbon, Portugal. Av. Professor Egas Moniz, Lisbon, Portugal 3 Unit of Neurosciences, Institute of Molecular Medicine, University of Lisbon, Portugal. Av. Professor Egas Moniz, Lisbon, Portugal 4 Department of Molecular Cell Biology, Faculty of Medicine, Amsterdam, The Netherlands. Van der Boechorststraat 7, 1081 BT, Amsterdam, The Netherlands Acta Neuropathologica, 2010 (submitted) 105

134 Acknowledgments The authors would like to thank Prof. Helmut Kettenmann s research group for sharing their expertise on microglia, Liliana Bernardino for her skill with organotypic-cultured hippocampal slices and Pedro Pereira and Ana Isabel Pereira for technical assistance. This work was supported by PTDC/SAU-NEU/64385/2006 grant, from Fundação para a Ciência e a Tecnologia, Lisbon, Portugal (to D.B.). S.L.S. was recipient of a PhD fellowship (SFRH/BD/30326/2006) from FCT. 106

135 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia Abstract Microglia has recently emerged as a crucial mediator of CNS inflammation since they are exquisitely sensitive to brain injury. The role of microglia during neonatal jaundice is unrecognized, although we evidenced that microglia reacts to unconjugated bilirubin (UCB) by engaging a phagocytic phenotype followed by the secretion of cytokines and glutamate. In addition, UCB injury to neurons involve short- and long-term alterations in neurite outgrowth and synaptic density, namely in hippocampal neurons, and is mediated by oxidative and nitrosative stress, raising the possibility of having an impact on infant s learning. This study investigated microglia neuroprotective or neurotoxic effects in a cell-tocell concerted action in response to UCB, namely in the production of glutamate and nitric oxide (NO), using organotypic cultured-hippocampal slices. Involvement of glutamate and NO in UCB-mediated toxicity to immature neurons was addressed using MK-801 (a NMDA glutamate-subtype receptor antagonist) and L-NAME (a non-specific NO synthase inhibitor). Therapeutic potential of glycoursodeoxycholic acid (GUDCA) and interleukin (IL)-10, with antioxidant and immunossupressive properties, in UCB-induced neurodegeneration was also evaluated. Microglia revealed to participate in glutamate homeostasis and to induce the release of this neurotransmitter and NO in UCB-treated hippocampal slices, which showed to be mediators in neuritic impairment, and cell death. Either GUDCA or IL-10 counteracted these insidious effects on immature neurons, but only GUDCA showed preventive effects on cell death, synaptic changes and release of glutamate and NO in UCB-treated cortical neurons and hippocampal slices. Collectively our data reveal microglia, glutamate and NO as key players in UCBinduced neurotoxicity and point to GUDCA as a promising therapy in infant s hyperbilirubinemia. Keywords: Glutamate-mediated neurotoxicity; Glycoursodeoxycholic acid; Interleukin-10; Long-term disabilities; Microglia-neuron interactions; Neurite outgrowth impairment; Nitric oxide; Organotypic-cultured hippocampal slices; Unconjugated bilirubin. 107

136 Chapter IV 1. Introduction Microglial cells are the first to respond to neural insults (Morioka et al., 1991) and its reactivity may be differentially modulated depending on microenvironmental signals (Zhang and Fedoroff, 1996). Activated microglia promote neuronal survival in ischemic lesion and facial nerve axotomy models (Moran and Graeber, 2004). Nevertheless, microglia can also release molecules, which may be harmful in acute brain insults and neurodegenerative diseases (Raivich et al., 1999). Hyperbilirubinemia, a very common neonatal condition, characterized by increased serum levels of unconjugated bilirubin (UCB) (Dennery et al., 2001), is associated with minor neurologic dysfunction (Soorani-Lunsing et al., 2001) and correlated with the emergence of long-term neurodevelopment disabilities (Dalman and Cullberg, 1999; Miyaoka et al., 2000). Several toxic effects have been accounted for UCB neurotoxicity such as cell death by both necrosis and apoptosis (Rodrigues et al., 2002a; Silva et al., 2001) and neuronal oxidative injury (Brito et al., 2008b; Vaz et al., 2010). UCB has also been shown to inhibit glutamate uptake prolonging its presence in the synaptic cleft (Silva et al., 1999; Silva et al., 2002) and to enhance its secretion in both astrocytes (Falcão et al., 2005; Fernandes et al., 2004) and microglia (Gordo et al., 2006) enabling N-methyl-D-aspartic acid (NMDA)-mediated excitotoxicity (Brito et al., 2010; Grojean et al., 2000; Grojean et al., 2001; McDonald et al., 1998). Microglia is one of the brain s major sources of reactive oxygen species (ROS) and reactive nitrosative species (RNS). In fact, microglial activation upon UCB exposure has been demonstrated to engender inflammation (Silva et al., 2010), glutamate secretion (Gordo et al., 2006) and nitric oxide (NO) increased production (unpublished results). The involvement of glutamate in cell death (Barger and Basile, 2001; Barger et al., 2007; Hahn et al., 1988; Lee et al., 2000; Liang et al., 2008; Takeuchi et al., 2008) and neuritic outgrowth regulation (Hoffman et al., 1996; Lee et al., 2005; Mattson et al., 1988; Monnerie et al., 2003) has been widely established. Intimately linked to excitotoxicity is the generation of destructive free radicals, especially RNS (Ha et al., 2010). These species are accountable for synapse and neuron injury, can exacerbate excitotoxicity (Sunico et al., 2010) and are broadly implicated in neurodegenerative diseases (Bishop and Anderson, 2005). In a very recent paper it was evidenced that excitotoxicity plays a key role in UCB-induced oxidative damage to rat cortical mature neurons (Brito et al., 2010). Finally, UCB seems to produce neurodevelopmental deficits since it has deleterious effects in neurogenesis, neuritogenesis and synaptogenesis (Falcão et al., 2007; Fernandes et al., 2009). Indeed, early exposure to UCB leads to impairment of 108

137 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia neuronal development by reducing dendrite extension and ramification, as well as dendritic spine formation and synapse establishment (Falcão et al., 2007). Moderate concentrations of UCB inhibit the induction of long term potentiation (LTP) in the hippocampus by short-term (Zhang et al., 2003) or prolonged exposure (Chang et al., 2009), indicating that neonatal jaundice may have deleterious consequences in learning and memory. In fact, it was observed that the more the history of jaundice was severe the highest difficulty for learning was revealed (Weir and Millar, 1997). Glycoursodeoxycholic acid (GUDCA) was proven to reduce cell death induced by UCB in astrocytes (Fernandes et al., 2007) and neurons (Brito et al., 2008a; Vaz et al., 2010), and to abrogate the UCB-induced oxidative damage in neurons (Brito et al., 2008a; Vaz et al., 2010). Moreover, immunosuppressive properties of GUDCA and interleukin (IL)-10 were demonstrated on the release of pro-inflammatory cytokines by UCB-treated astrocytes (Fernandes et al., 2007). IL-10 has also been shown to reduce neuronal degeneration after central nervous system (CNS) injury (Bachis et al., 2001; Park et al., 2007). However, to the best of our knowledge, no evidence was established regarding the effect of GUDCA and IL-10 in the prevention of UCB-induced neurite impairment along neuronal maturation. Therefore, this study aimed at evaluating whether the production of glutamate and NO could be elicited by UCB in an ex-vivo model of hippocampal slice cultures and, more importantly, if this effect could be ascribed to microglial cells. This culture model maintains the cytoarchitecture of the tissue, allowing interaction of multiple cell types in the brain, namely neurons, astrocytes and microglia (Cho et al., 2007). Furthermore, we intended to clarify the role of glutamate and NO in UCB-induced neurotoxicity by using MK-801 (a NMDA glutamate-subtype receptor antagonist) and L-NAME (a non-specific NO synthase inhibitor), and to elucidate the protective effects of GUDCA and IL-10 in both isolated immature neurons and hippocampal slice cultures. Our studies clearly demonstrate a key role of NO and overactivation of NMDA receptors in UCB-induced impairment of neuritic outgrowth and cell death. Microglia revealed to directly participate in glutamate homeostasis and NO production. Additionally, we show, for the first time, that GUDCA and IL-10 prevent UCB-induced loss of cell viability and extracellular glutamate accumulation. Moreover, they completely abrogate the deleterious effects produced by UCB in neuronal network dynamics in a very sustained manner along cellular differentiation, indicating that both short- and longterm UCB harmful consequences on CNS could be in this way counteracted. Furthermore, our findings report that UCB is able to down-regulate the expression of presynaptic proteins, adding on the detrimental effects of UCB on synaptic plasticity, and that only GUDCA prove efficacy in preventing this injurious outcome. 109

138 Chapter IV 2. Material and Methods 2.1. Chemicals Neurobasal medium, B-27 Supplement (50x), Hanks balanced salt solution (HBSS), HBSS without Ca 2+ and Mg 2+, gentamicin (50 mg/ml), trypsin (0.5 g/l) and Alexa Fluor 594 chicken anti-goat IgG were acquired from Invitrogen (Carlsbad, CA, USA). Dulbecco s modified Eagle s medium (DMEM), fetal bovine serum (FBS), and L- glutamine, were purchased from Biochrom AG (Berlin, Germany). Antibiotic antimycotic solution (20X), human serum albumin (HSA; fraction V, fatty acid free), bovine serum albumin (BSA), Hoechst dye, mouse anti-β-actin antibody, propidium iodide (PI), N-1-naphthylethylenediamine, N-ω-nitro-L-arginine methyl ester (NAME), [(+)-5-methyl- 10,11-dihydro-5Hdibenzo[a,d]cyclohepten-5,10-imine maleate)] (MK-801) and DAPI were purchased from Sigma Chemical Co. (St. Louis, MO). UCB was also obtained from Sigma and purified according to the method of McDonagh and Assisi, (1972). Mouse anti-microtubule associated protein (MAP)-2, mouse anti- synaptosomalassociated protein (SNAP)-25 and mouse anti-synaptophysin antibodies were from Chemicon (Temecula, CA) and fluorescein isothiocyanate (FITC)-labeled horse antibody anti-mouse was acquired from Vector (Burlingame, CA). GUDCA (minimum 96% pure) was obtained from Calbiochem, Darmstadt, Germany and recombinant rat IL-10 from R&D Systems, Minneapolis, MN. Nitrocellulose membrane and Hyperfilm ECL were from Amersham Biosciences (Piscataway, NJ, USA). Horseradish peroxidase-labeled goat anti-rabbit IgG and antimouse IgG antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Cell lysis buffer and LumiGLO were from Cell Signaling (Beverly, MA, USA). L-glutamic acid kit was purchased from Roche Molecular Biochemicals (Manheim, Germany). All other chemicals were of analytical grade and were purchased from Merck (Darmstadt, Germany) Organotypic-cultured hippocampal slices Animal care followed the recommendations of European Convention for the Protection of Vertebrate Animals Used for Experimental and other Scientific Purposes (Council Directive 86/609/EEC) and National Law 1005/92 (rules for protection of experimental animals). The Institutional Animal Care and Use Committee approved all animal procedures. Every effort was made to minimize the number of animals used and their suffering. Organotypic-cultured hippocampal slices were prepared from P8-P10 Wistar rat brains, according to the interface culture method (Stoppini et al., 1991), as previously 110

139 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia described (Markovic et al., 2005; Synowitz et al., 2006). Briefly, mice were killed by decapitation, their brains removed under sterile conditions and the two hippocampi isolated and cut in 400 µm coronal sections using a McIlwain tissue chopper. The hippocampal slices were then transferred onto the 0.4-μm polycarbonate membrane in the upper chamber of a Transwell tissue insert (Falcon model 3090, Becton Dickinson, Lincoln Park, NJ), which were placed into a six-well plate (Falcon model 3502, Becton Dickinson). Thereafter, slices were incubated in 1 ml culture medium per well containing DMEM supplemented with 10% heat-inactivated FBS (medium 1). After overnight equilibration of the organotypic-cultured hippocampal slices in medium 1, this was exchanged for cultivation medium (medium 2). Medium 2 (100 ml) contains 25 ml heatinactivated horse serum, 580 μl bicarbonate (7.5%), 2 ml of L-glutamine solution, 25 ml HBSS, 10 μl of insulin (10 mg/ml), 1.2% glucose, 80 μl vitamin C (1 mg/ml), 1 ml antibiotic-antimycotic solution, and 500 μl of 1 mol/l Tris in DMEM. Medium was changed every day Preparation of microglia-depleted organotypic-cultured hippocampal slices Microglia-depleted organotypic-cultured hippocampal slices were obtained by 24 h treatment with liposomes filled with clodronate as described in (Markovic et al., 2005). Liposomes were obtained from GOT Therapeutics (Berlin, Germany) and from the Department of Molecular Cell Biology of the Free University of Amsterdam. For the preparation of clodronate-liposomes, 86 mg of phosphatidylcholine and 8 mg of cholesterol were combined with 10 ml of a clodronate (0.7 M; a gift of Roche Diagnostics GmbH, Mannheim, Germany) solution and sonicated gently. The resulting liposomes were washed to eliminate free drug. All liposomes were passed through a 12 mm filter immediately prior to use in order to eliminate large lipid aggregates (Van Rooijen and Sanders, 1994) Organotypic-cultured hippocampal slices treatment Slices were maintained in vitro for a minimum of 3 days prior to use, a period over which tissues recover from experimental trauma caused by the isolation procedure (Huuskonen et al., 2005). A stock solution of purified UCB was prepared in 0.1 N NaOH immediately before use and the ph of the incubation medium was restored to 7.4 by addition of equivalent amounts of 0.1 N HCl. All the experiments with UCB were performed under light protection to avoid photodegradation. Organotypic-cultured hippocampal slices were incubated in the absence (control) or in the presence of 50 µm UCB plus 100 µm HSA, 111

140 Chapter IV for 24 h, at 37ºC. When appropriate, slices were pre-incubated with 50 µm GUDCA (from a 5 mm stock solution) 1 h prior to UCB addition Assessment of cell death in organotypic-cultured hippocampal slices Cell death in organotypic-cultured hippocampal slices was assessed by monitoring the cellular uptake of the fluorescent dye PI [3,8-diamino-5-(3- (diethylmethylamino)propyl)-6-phenyl phenanthridinium diiodide]. PI readily enters and stains non-viable cells, but cannot cross the membrane of viable cells. This dye binds to double-stranded DNA and emits red fluorescence (630 nm; absorbance 493 nm). After UCB treatment, slices were exposed to 2 µg/ml PI for 2 h and fixed by immersion in 4% paraformaldehyde for 30 min, cryoprotected in 30% sucrose and stored at -20ºC until use. Slices were cut in 15 µm-thick sections and the nuclei were stained with DAPI (2ug/mL). Cellular uptake of PI was recorded by fluorescence microscopy using a rhodamine filter and a Leica DFC490 camera adapted to an AxioSkope microscope. The percentage of PI-positive cells was quantified using ImageJ software (National Institutes of Health) Quantification of nitrite levels Nitric oxide levels were estimated by measuring the concentrations of nitrites (NO - 2 ), which are the resulting NO metabolites. Briefly, supernatants free from cellular debris were mixed with Griess reagent [1 part 1% (w/v) sulfanilamide in 5% H 3 PO 4, 1 part 0.1% (w/v) N-1-naphthylethylenediamine (v/v)] in 96-well tissue culture plates for 10 min at room temperature in the dark. The absorbance at 540 nm was determined using a microplate reader (Bio-Rad Laboratories, Hercules, CA, USA) Primary neuronal cell cultures Neurons were isolated from foetuses of day pregnant Wistar rats, as described previously (Silva et al., 2002). Pregnant rats were anesthetized and decapitated. The foetuses were collected in HBSS and rapidly decapitated, the brain cortex was mechanically fragmented, and the fragments transferred to a 0.5 g/l trypsin in Ca 2+ and Mg 2+ free HBSS medium and incubated for 15 min at 37ºC. After trypsinization, cells were washed twice in calcium and magnesium free HBSS containing 10% FBS, and resuspended in Neurobasal medium supplemented with 0.5 mm L- glutamine, 25 µm L-glutamic acid, 2% B-27 Supplement, and 0.12 mg/ml gentamicin. Aliquots of 1 x 10 5 cells/cm 2 were plated on poly-d-lysine coated 12-well tissue culture plates and maintained at 37ºC in a humidified atmosphere of 5% CO 2. Every 3 days,

141 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia ml of old medium was removed by aspiration and replaced by the same volume of fresh medium without L-glutamic acid Cell treatment A 100 mm stock solution of L-NAME, a competitive nitric oxide synthase (NOS) inhibitor, and a 1 mm stock solution of MK-801, a NMDA receptor antagonist, were prepared in phosphate-buffered saline (PBS), ph 7.4. A stock solution of purified UCB was prepared as described above. Neurons at 3 days in vitro (DIV) were incubated in the absence (control) or in the presence of 50 µm UCB plus 100 µm HSA, for 24 hr, at 37ºC. When appropriate, cells were pre-incubated with 50 µm GUDCA (from a 5 mm stock solution) or 10 ng/ml recombinant rat IL-10 (from a 50 µg/ml stock solution), 1 h prior to UCB addition. Parallel sets of experiments were performed where cells were incubated with UCB alone or in combination with 100 μm NAME or with 1 μm MK-801. At the end of the incubation period (considered as 4 DIV throughout the text) cellfree medium was removed and attached cells were fixed for 30 min with freshly prepared 4% paraformaldehyde in PBS, for immunocytochemical studies. In another set of experiments, incubation medium was removed and cells were additionally cultured until 18 DIV, to study the long-term effects of UCB exposure Neurite extension and ramification Neurite extension and ramification were assessed by the immunofluorescence detection of the cytoskeletal protein MAP-2, as described previously (Falcão et al., 2007). Briefly, cells were fixed as described above and a standard indirect immunocytochemical technique was carried out using a mouse anti-map-2 antibody (1:100) as the primary antibody and a horse FITC-labeled anti-mouse antibody (1:227) as the secondary antibody. Fluorescence was visualized using a Leica DFC490 camera adapted to an AxioSkope microscope. Green-fluorescence images of ten random microscopic fields (original magnification: 400X) were acquired per sample. Evaluation of neurite extension and number of nodes from individual neurons was achieved by manual tracing using ImageJ software (National Institutes of Health) Measurement of glutamate Glutamate content in incubation medium or in hippocampal slice homogenates was determined by an adaptation of the L-glutamic acid kit (Roche), using a 10-fold 113

142 Chapter IV volume reduction. The reaction was performed in a 96-well microplate and the absorbance measured at 490 nm. A calibration curve was used for each assay. All samples and standards were analyzed in duplicate and the mean value was used Evaluation of cell death Necrotic-like cell death was assessed by monitoring the cellular uptake of the fluorescent dye PI. Unpermeabilized adherent cells cultured on coverslips were incubated with a 75 μm PI solution for 15 min in the absence of light. Subsequently, cells were fixed with freshly prepared 4% (w/v) paraformaldehyde in PBS and the nuclei stained with Hoechst dye. Red-fluorescence and U.V. images of ten random microscopic fields (original magnification: 400X) were acquired per sample and the percentage of PI positive cells was counted Western blot assay Crude synaptosomes were obtained by differential centrifugation as described in Huttner et al. (1983). Total cell lysates were extracted from neurons using the Cell lysis buffer and centrifuged at 800 X g to separate P1 (nuclear and debris) and supernatant (S1) fraction. S1 fraction was centrifuged at 9200 X g to separate P2 (membranous fraction) and S2 (cytosolic fraction). Western blot assay was carried out as usual in our lab (Fernandes et al., 2006). Briefly, crude synaptosomal fractions were separated on a 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). Following electrophoretic transfer onto a nitrocellulose membrane and blocking with 5% milk solution, the blots were incubated with primary antibody overnight at 4ºC [anti- SNAP-25 (1:1000), anti-synaptophysin (1:2000) in TBS, or anti-β-actin (1:10000) in 5% (w/v) bovine serum albumin] and with horseradish peroxidase-labelled secondary antibody [anti-mouse (1:5000) or anti-rabbit (1:5000)] for 1 h at room temperature. Protein bands were detected by LumiGLO and visualized by autoradiography with Hyperfilm ECL Statistical Analysis Results of at least three different experiments, carried out in duplicate, were expressed as mean ± SEM. Differences between groups were determined by one-way ANOVA with Dunnett s or Bonferroni s multiple comparisons post tests, using Instat 3.05 (GraphPad Software, San Diego, CA). P <0.05 was accepted as statistically significant. 114

143 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia 3. Results 3.1. Microglia modulate UCB-induced glutamate release and NO production in organotypic-cultured hippocampal slices Activated microglia can produce a plethora of harmful products such as glutamate and NO in several disease models. Thus, we decided to evaluate glutamate and NO production in organotypic-cultured hippocampal slices upon UCB exposure and to determine if this production could be ascribed to microglia by the selective depletion of these cells.in homogenates prepared from hippocampal slices incubated with 50 μm UCB for 24 h, there was a significant increase in the amount of glutamate (3.8 fold, p<0.01, Figure IV.1A) when compared to slices not incubated with UCB. Interestingly, in microglia-depleted slices, the increase in glutamate induced by UCB was totally prevented (p<0.01), suggesting that glutamate accumulation was mainly due to resident microglia. The glutamate levels in the incubation media of UCB-treated slices (Fig. IV.1B) were also enhanced (1.4 fold, p<0.05, Figure IV.1B), as compared with nontreated slices, and the increase was even more pronounced in microglia-depleted slices (p<0.01). These findings suggest that microglia affects glutamate homeostasis leading to the accumulation of the neurotransmitter upon UCB exposure. Fig. IV.1. Microglia modulate UCB-induced glutamate release in organotypiccultured hippocampal slices. Hippocampal slices nondepleted or depleted in microglia, cultured for 3 days in vitro (DIV), were exposed for 24 h, to either no addition (control), or 50 µm UCB. Tissue concentrations of glutamate (A) or glutamate levels in incubation media (B) were evaluated using a colorimetric assay and expressed as mean ± SEM fold change compared with nondepleted in microglia, in the absence of UCB, from three independent experiments performed in duplicate. *p<0.05 and **p<0.01 vs. non-depleted and depleted in microglia, in the absence of UCB; p<0.05 and p<0.01 vs. UCB in nondepleted in microglia. 115

144 Chapter IV Furthermore, incubation of hippocampal slices with UCB also led to increased NO release into the culture medium (1.4 fold, p<0.05), but not in microglia-depleted slice cultures (p<0.05 vs. non-depleted slices) as depicted in Figure IV.2. Fig. IV.2. Microglia modulate UCB-induced NO production in organotypic-cultured hippocampal slices. Hippocampal slices non-depleted or depleted in microglia, cultured for 3 days in vitro (DIV), were exposed for 24 h, to either no addition or 50 µm UCB. NO production was estimated by the quantification of nitrite levels and expressed as mean ± SEM from three independent experiments performed in duplicate. *p<0.05 vs. nondepleted and depleted in microglia, in the absence of UCB; p<0.05 vs. UCB in nondepleted in microglia. Since glutamate and NO have already been implicated as mediators in UCBneurotoxicity (Brito et al., 2010; Grojean et al., 2000; McDonald et al., 1998; Vaz et al., 2010), as well as in other diseases, these findings suggest that microglial reactivity may be an additional intervenient in brain injury by UCB. Next we decided to further clarify how this increased production of glutamate and NO could be involved in UCB-induced nerve cell injury. To answer this question we used primary cultures of cortical neurons in order to better elucidate the molecular mechanisms involved NMDA receptors and NO are implicated in UCB-induced neurite impairment and in cell death Cortical neurons cultured for 3 DIV were exposed, during 24 h, to either no addition (control), or 50 µm UCB, in the absence or presence of 1 µm MK-801 (a NMDA glutamate-subtype receptor antagonist) or 100 µm L-NAME (a non-specific NO synthase inhibitor). Following UCB exposure, we started by evaluating neuronal demise by using PI followed by the determination of neurite extension and ramification as we wanted to 116

145 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia assess if glutamate and NO were responsible partners in neuritic arborisation impairment by UCB. As shown in Figure IV.3, either MK-801 or L-NAME added at the same time as UCB were able to totally prevent (p<0.01) the necrotic-like cell death that showed to duplicate by UCB over control values in immature neurons. Fig. IV.3. UCB-induced neuronal death is fully abrogated by MK-801 and L-NAME. Cortical neurons cultured for 3 days in vitro (DIV) were exposed for 24 h, to either no addition (control) or 50 µm UCB, in the absence or presence of 1 µm MK-801 or 100 µm L-NAME, and further incubated with 75 μm PI as described in Methods. (A) The percentage of PI-positive cells was calculated and expressed as mean ± SEM from three independent experiments performed in triplicate. (B) Representative results of one experiment are shown. Scale bar represents 40 µm. **p<0.01 vs. respective control; p<0.01 vs. UCB alone. 117

146 Chapter IV Moreover, the same was observed by either MK-801 or L-NAME in protecting the UCB-induced reduction in neurite extension and ramification provoked by UCB in both 4 DIV (MK-801, p<0.01 and p<0.05, respectively; L-NAME, p<0.01 for both) and 18 DIV neurons (MK-801, p<0.05 for both; L-NAME, p<0.05 and p<0.01, respectively), as shown in Figure IV.4. These results provide proof of concept that glutamate and NO participate in UCB-induced neurodevelopment abnormalities implicated in UCB-enduring harmful consequences and that microglia, as major sources of these neurotoxic molecules upon UCB stimulation, participate in its harmful effects. Based on the antioxidant properties of GUDCA in neurons (Brito et al., 2008a; Vaz et al., 2010) and on the immunosuppressive properties of both GUDCA and IL-10 (Fernandes et al., 2007) we decided to investigate their potentialities in saving neuritic arborisation from UCB harmful effects. Fig. IV.4. UCB-elicited impairment in neuritic outgrowth at 4 and 18 days in vitro (DIV) is mediated by NO and overstimulated NMDA receptors. Cortical neurons cultured for 3 DIV were exposed for 24 h, to either no addition (control) or 50 µm UCB, in the absence or presence of 1 µm MK-801 or 100 µm L-NAME as described in Methods. Incubation medium was removed and neurons were allowed to culture until 18 DIV. Neurite extension (A) and number of nodes (B) were identified by immunolabeling for MAP-2, quantified by ImageJ and expressed as arbitrary units (mean ± SEM). Representative results of one experiment regarding 4 DIV neurons is shown in C. Scale bar represents 40 µm. *p<0.05 and **p<0.01 vs. respective control; p<0.05 and p<0.01 vs. UCB alone. 118

147 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia 3.3. UCB-elicited accumulation of extracellular glutamate is reduced by both GUDCA and IL-10, but not abolished Taking into account the important role of glutamate NMDA receptors in UCB neurotoxicity portrayed by the above data, we then evaluated the effects of GUDCA and IL-10 upon UCB-induced glutamate release by cultured neurons. As shown in Figure IV.5, GUDCA significantly reduced (~30%, p<0.05) the efflux of glutamate by UCBtreated immature neurons, an effect even more marked (~45%, p<0.01) when we used IL-10. Nevertheless, although these compounds significantly drop the extracellular accumulation of glutamate they were unable to prevent it. Fig. IV.5. UCB-induced extracellular accumulation of glutamate is prevented by both GUDCA and IL-10. Cortical neurons cultured for 3 days in vitro (DIV) were exposed for 24 h, to either no addition (control) or 50 µm UCB, in the absence or presence of 50 µm GUDCA or 10 ng/ml IL-10. Results are mean ± SEM from three independent experiments performed in triplicate. **p<0.01 vs. respective control; p<0.05 and p<0.01 vs. UCB alone GUDCA or IL-10 pre-treatment counteracts impairment of neurite outgrowth and ramification, as well as cell death in UCB-treated neurons Considering the effects of UCB in neurons, above described, and the protective effects displayed by GUDCA and IL-10 in these cells we next investigated whether these compounds reveal ability to abrogate UCB-induced necrotic-like cell death in immature neurons, as well as neuritic changes. Both molecules besides abolishing cell-death (p<0.01, Figure IV.6) showed efficacy in preventing short-term deficits in neurite extension (GUDCA, p<0.05; IL-10, p<0.01) as depicted in Figure IV

148 Chapter IV Fig. IV.6. GUDCA or IL-10 pre-treatment counteracts cell death in UCB-treated immature neurons. Cortical neurons cultured for 3 days in vitro (DIV) were exposed for 24 h, to either no addition (control) or 50 µm UCB, in the absence or presence of 50 µm GUDCA or 10 ng/ml IL-10, and further incubated with 75 μm PI as described in Methods. (A) The percentage of PI-positive cells was calculated and expressed as mean ± SEM from three independent experiments performed in triplicate. (B) Representative results of one experiment are shown. Scale bar represents 40 µm. **p<0.01 vs. respective control; p<0.01 vs. UCB alone. The same was verified in the maintenance of neurite ramification, where significant protection was manifested by both GUDCA (p<0.05) and IL-10 (p<0.01) in 4 DIV neurons. Interestingly, even after removing the UCB stimulus and additionally culturing neurons until 18 DIV, the protective effects of GUDCA and IL-10 on UCBinduced long-term neuritic outgrowth impairment were still noticeable (p<0.05). 120

149 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia Fig. IV.7. GUDCA or IL-10 pre-treatment counteracts impairment of neurite outgrowth at 4 and 18 days in vitro (DIV) in UCB-treated immature neurons. Cortical neurons cultured for 3 DIV were exposed for 24 h, to either no addition (control) or 50 µm UCB, in the absence or presence of 50 µm GUDCA or 10 ng/ml IL-10 as described in Methods. Incubation medium was removed and neurons were allowed to culture until 18 DIV. Neurite extension (A) and number of nodes (B) were identified by immunolabeling for MAP-2, quantified by ImageJ and expressed as arbitrary units ± SEM. Representative results of one experiment regarding 4 DIV neurons is shown in C. Scale bar represents 40 µm. *p<0.05 and **p<0.01 vs. control; p<0.05 and p<0.01 vs. UCB alone UCB decreases the expression of pre-synaptic proteins and this event is abrogated by GUDCA, but not by IL-10 Previous results have proven that UCB can adversely affect spine formation and synapse establishment (Fernandes et al., 2009). Moreover, UCB-induced impairment of synaptic plasticity has already been acknowledged (Chang et al., 2009). Therefore, we decided to examine if UCB can modify the expression of proteins such as synaptophysin and SNAP-25, which are involved in synaptogenesis and synaptic vesicle assembly. Our results showed that UCB led to a significant reduction in both protein levels (0.9 fold, p<0.05 and 0.8 fold, p<0.01, respectively). In line with the aforementioned benefits, 121

150 Chapter IV GUDCA prevented the UCB synaptotoxicity from occurring (p<0.05), while a slight and non-significant effect was produced by IL-10 pretreatment (Figure IV.8). Fig. IV.8. UCB down-regulation of synaptophysin and SNAP-25 protein expression in immature neurons is prevented by GUDCA. Cortical neurons cultured for 3 days in vitro (DIV) were exposed for 24 h, to either no addition (control) or 50 µm UCB, in the absence or presence of 50 µm GUDCA or 10 ng/ml IL-10. Crude synaptosomal fractions were analyzed by western blotting with antibodies specific for synaptophysin and SNAP- 25. The intensity of the bands was quantified by scanning densitometry, standardized with respect to β-actin protein expression. Graph bars represent the fold change values (mean ± SEM) from three independent experiments performed in duplicate for synaptophysin (A) and SNAP-25 (B). Representative results of one experiment regarding synaptophysin and another concerning SNAP-25 are shown in (C). **p<0.01 vs. respective control; p<0.05 vs. UCB alone. Interestingly, GUDCA even revealed to up-regulate synaptophysin and SNAP-25 expression. Thus, GUDCA evidence to be a more promising therapeutical approach in hyperbilirubinemia than IL-10 due to its broader benefits and induced increase in presynaptic proteins that regulate synaptic vesicle docking, as well as membrane fusion and fission (Gray et al., 2010). 122

151 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia 3.6. Hampering of UCB-induced NO and glutamate production, as well as cell death by GUDCA was reproduced in hippocampal slices Given the promising protective effects of GUDCA regarding UCB neurotoxicity, verified for purified cultures, we became interest in returning to the singular physiological conditions provided by the hippocampal slice model to verify if the observed effects were reproduced in this more complex model. We were particularly interested in studying the pathways previously shown as activated in microglial cells upon UCB exposure, namely glutamate and NO production. Therefore, we assessed extracellular accumulation of glutamate and NO release, as described in Figure IV.9, and found that GUDCA was able to counteract their production (p<0.01). Fig. IV.9. GUDCA hampers UCBinduced NO and glutamate production in organotypiccultured hippocampal slices. Hippocampal slices cultured for 3 days in vitro were exposed for 24 h, to either no addition (control) or 50 µm UCB, in the absence or presence of 50 µm GUDCA. (A) Tissue concentrations of glutamate were evaluated using a colorimetric assay and expressed as mean ± SEM fold change compared with control conditions. (B) NO production was estimated by the quantification of nitrite levels and expressed as mean ± SEM from three independent experiments performed in duplicate. *p<0.05 and **p<0.01 vs. control; p<0.01 vs. UCB alone. Moreover, similar neuroprotective effects were demonstrated by the abrogation of PI uptake (p<0.01) in GUDCA-treated slices prior to UCB incubation (Figure IV.10), further highlighting the therapeutic potential of this particular bile acid. 123

152 Chapter IV Fig. IV.10. GUDCA hampers UCB-induced cell death in organotypic-cultured hippocampal slices. Hippocampal slices cultured for 3 days in vitro were exposed for 24 h, to either no addition (control), or 50 µm UCB, in the absence or presence of 50 µm GUDCA, and further incubated with 2 μg/ml PI as described in Methods. (A) Representative results of one experiment are shown. Scale bar represents 40 µm. (B) Graph bars represent the fold change values (mean ± SEM) from three independent experiments performed in duplicate. **p<0.01 vs. respective control; p<0.01 vs. UCB alone. 4. Discussion In this study we demonstrate, for the first time, that microglia modulate UCBinduced neurotoxicity in organotypic-cultured hippocampal slices. This modulation involves glutamate and NO as key mediators in the injurious effects prompted by UCB. Furthermore, our study depicts the neuroprotective properties of GUDCA and IL-10, revealing that their potential to prevent neuronal damage observed in pure neuronal cultures is reproduced in the more complex system of organotypic cultures. In addition, their ability to abrogate the neurodevelopmental abnormalities caused by neonatal 124

153 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia hyperbilirubinemia reinforces their possible use as new therapeutic approaches in this, still, life-threatening condition. Previous reports have shown that UCB interacts with whole nerve cell and mitochondrial membranes disrupting its redox status (Rodrigues et al., 2002b; Rodrigues et al., 2002c) and that several oxidative biomarkers are elicited in neuronal (Vaz et al., 2010) and astroglial cells (Brito et al., 2008b), as well as in synaptosomal membrane systems (Brito et al., 2004). Moreover, unpublished results from our group support the increase in NO production upon UCB exposure in neurons (personal communication Ana Rita Vaz, 2010) and microglia, where the production is further amplified by UCB-treated neuronal conditioned medium (unpublished results). Indeed, NO appears to be involved in numerous physiological and pathological processes (Bishop and Anderson, 2005). Several authors have evidenced that activated microglia releases NO following induction of nitric oxide synthase (Kawase et al., 1996) and that this radical mediates neural injury (Golde et al., 2002). On the other hand, the generation of an oxidative burst by microglia in response to injury may also constitute a measure to promote the clearance of damaged cells from the CNS (Kawase et al., 1996). The results displayed in this study support the role of glutamate and NO as important neurotoxic molecules. Moreover, as was shown in the ex vivo organotypic-cultured hippocampal slice model, their production upon UCB exposure may be attributed to microglial cells. Accordingly, blocking the action of both molecules abolished the toxic effects of UCB in our primary neuron culture model. However, the role of microglia in glutamate homeostasis upon UCB exposure seems to be more complex, as opposing effects were observed when evaluating glutamate content in tissue homogenates or incubation media from organotypic-cultured hippocampal slices. In fact, results from tissue samples point to microglia as the most important glutamate source, nevertheless, extracellular accumulation of glutamate in incubation media from microglia-depleted slices is enhanced, suggesting that microglia are actually promoting glutamate uptake. Indeed, activated microglia have been shown to up-regulate glutamate transporter-1 (GLT-1) in response to motoneuron injury in axotomized rat facial nucleus (Lopez-Redondo et al., 2000) and also as a defensive mechanism against herpes simplex virus infection (Persson et al., 2007). Additionally, inflammatory products such as tumor necrosis factor-α may similarly induce GLT-1 expression in microglia (Persson et al., 2005) while glutamine synthetase, the enzyme responsible for converting glutamate into the less toxic glutamine, is also expressed in activated microglia (Chretien et al., 2002; Rimaniol et al., 2000). This inducible profile of microglia contrasts with the constitutive expression of glutamate transporters in astrocytes and can be regarded as a compensatory mechanism to limit the deleterious 125

154 Chapter IV consequences of microglial activation in the brain (Gras et al., 2006). Our hypothesis is that microglia initially exerts a neuroprotective action towards UCB by promoting glutamate clearance, either by themselves or by engaging astrocytes in this function, as previously observed (Tilleux et al., 2009). However, despite microglial efforts to limit glutamate concentration, the ultimate outcome is an increase in its extracellular levels, possibly due to the failure of the clearance mechanisms or to the increased production by either glial or neuronal cells upon prolonged exposure times. A growing body of evidence underscores glutamate s key role in microglial neurotoxicity (Barger and Basile, 2001; Liang et al., 2008). Furthermore, several reports link oxidative stress and glutamate-mediated excitotoxicity (Bal-Price and Brown, 2001; Barger et al., 2007; Brito et al., 2010; Golde et al., 2002), which is consistent with our results demonstrating that glutamate and NO participate in UCB-induced neuronal demise. In fact, earlier studies have already reported that UCB-induced neurotoxicity is mediated by glutamate receptors (Grojean et al., 2000; Grojean et al., 2001; Hanko et al., 2006; Hoffman et al., 1996). Glutamate can cause alterations in dendritic outgrowth (Esquenazi et al., 2002; Mattson, 2008), even in immature neurons (Monnerie et al., 2003), as well as NO, that was shown to induce synapse loss (Sunico et al., 2010). Interestingly, our previous studies evidenced impairment of neurite extension and ramification in immature cortical neurons exposed to UCB that is sustained through cell maturation (Falcão et al., 2007). Changes in dendritic and axonal arborisation were also observed in UCB-treated immature hipppocampal neurons (Fernandes et al., 2009). Our novel results bridge those two findings, demonstrating a causal relation between UCB-induced impairment of neurite outgrowth, NMDA receptor overstimulation, and NO overproduction. This aspect gains additional relevance if we consider that glutamate efflux is enhanced in neurons and glial cells (Falcão et al., 2005; Fernandes et al., 2004) exposed to UCB, and its uptake inhibited in neurons (Silva et al., 1999; Silva et al., 2002). This finding is particularly important given the fact that neonatal hyperbilirubinemia has been associated to the development of mental disorders (Dalman and Cullberg, 1999) like schizophrenia (Hayashida et al., 2009; Miyaoka et al., 2000), and can impact on learning and memory (Zhang et al., 2003). UCB can adversely affect synapse establishment since it interferes with spine formation and reduces growth cone area (Fernandes et al., 2009). Accordingly, UCB exposure leads to impairment of neurotransmitter release in synaptic vesicle membranes (Roseth et al., 1998), modification of long-term synaptic plasticity (Chang et al., 2009), and decreased synaptic activity in hippocampal slices (Hansen, 1994). These studies are consistent with our results regarding decreased presynaptic protein expression upon UCB exposure. Actually, glutamate s implication in 126

155 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia neuritogenesis and synaptogenesis has already been acknowledged (Mattson et al., 1988). In this paper we report that UCB can lead to a diminished expression of the presynaptic proteins synaptophysin and SNAP-25. Synaptophysin is a calcium binding protein expressed on pre-synaptic vesicles and SNAP-25 is anchored in the synaptic terminal plasma membrane. These proteins are essential for synaptic vesicle fusion to the pre-synaptic plasma membrane, thus participating in synapse establishment and neurotransmitter release (Wang and Tang, 2006). A decrease in synaptic protein expression is regarded as a biomarker of altered neuronal development. In fact, synaptophysin expression is altered in epilepsy (Xu et al., 2009) and changes in proteins relevant to synaptic transmission and axonal transport have been coupled to neuroinflammation in Parkinson's disease (PD) (Chung et al., 2009). Cytoskeleton abnormal assembly, loss of dendrites and axons and impairment of neurotransmission can cause disruption of synaptic connectivity and instigate neurodegenerative diseases like Alzheimer s disease (Evans et al., 2008) or PD (Benitez-King et al., 2004), and psychiatric illnesses such as schizophrenia (Brennaman and Maness, 2008). GUDCA is the major product of ursodeoxycholic acid (UDCA) catabolism (Rudolph et al., 2002). UDCA and its conjugates have demonstrated neuroprotective effects by the stabilization of mitochondrial membranes (Solá et al., 2002), inhibition of mitochondrial swelling (Rodrigues et al., 2000b), cytochrome c release (Rodrigues et al., 2000a) and, ultimately, by the decrease of apoptotic cell death (Silva et al., 2001). UDCA oral administration induces a rapid and sustained decrease in plasma UCB concentrations in Gunn rats, the animal model used to study bilirubin encephalopathy (Cuperus et al., 2009). Recent reports have indicated potential benefits for GUDCA at preventing UCB-induced protein oxidation, lipid peroxidation, impairment of glutathione homeostasis and neuron cell death (Brito et al., 2008a), or mitochondrial respiratory chain dysfunction by UCB and restoration of cellular antioxidant potential (Vaz et al., 2010). Our group has also evidenced that both GUDCA and IL-10 can modulate astrocytic reactivity to UCB decreasing the elicited inflammatory properties and reducing cell death (Fernandes et al., 2007). The anti-inflammatory cytokine IL-10 has already been demonstrated to reduce glial activation (Ledeboer et al., 2000) and to exert neuroprotective effects (Bachis et al., 2001; Park et al., 2007). IL-10 is also able to downregulate microglial NO production, thus contributing to the recovery of neurite outgrowth (Rozenfeld et al., 2003). Our results show that UCB-induced deleterious effects on neuronal survival and neurite impairment are also prevented by GUDCA and IL-10. Protective properties of these compounds on the cellular efflux of glutamate, elicited by UCB, emphasize the protective effects observed since we have here demonstrated the 127

156 Chapter IV essential role of glutamate and NO in UCB-evoked alterations in network dynamics. To the best of our knowledge, only one other molecule, taurine, has evidenced protective effects against UCB-mediated neuronal damage (Zhang et al., 2010). It is also very important to acknowledge that neuroprotection by GUDCA in the purified neuronal culture, was kept in organotypic-cultured hippocampal slices, suggesting its usefulness in vivo. Given the fact that both GUDCA and IL-10 showed to prevent UCB-induced extracellular accumulation of glutamate, we expected that both molecules would exert protective effects on pre-synaptic protein expression. Surprisingly, only GUDCA proved to be effective at this level, which led us to the hypothesis that GUDCA might be acting on different mechanisms beyond glutamate-mediated neuronal damage. One possible explanation might be that GUDCA has recognized anti-oxidant properties (Brito et al., 2008a; Vaz et al., 2010) and, in fact, oxidative stress is associated with synaptogenesis and neuritogenesis alterations (Sunico et al., 2010), while IL-10 is more notorious for its anti-inflammatory capacities (Ledeboer et al., 2000). Moreover, GUDCA and IL-10 have also shown to modulate astroglial reactivity to UCB following different mechanisms (Fernandes et al., 2007). In conclusion, the results obtained in this study portray an interesting and complex role for microglia in glutamate homeostasis upon UCB exposure. These cells induce glutamate clearance as a first attempt to limit the excitotoxic neuronal damage but, on the other hand, also contribute for the pool of released glutamate culminating in an extracellular accumulation of this molecule. Moreover, our study evidences microglial cells as a source of NO upon UCB exposure. Our work is the first to establish the involvement of glutamate and NO in UCB-induced impairment of neurite extension and ramification, thus substantiating target-driven approaches which may reveal important in the prevention of long-term neurodevelopment disabilities. Most important is the neuroprotective effects of GUDCA and IL-10 in hyperbilirubinemia as they are able to prevent the UCB-induced increase in glutamate secretion and, through this or other mechanisms, counteract the deleterious effects of UCB on neuronal network dynamics and cell death. GUDCA has an additional benefit in preventing down-regulation of presynaptic protein expression, an event that may underlie UCB-evoked alterations in synaptic plasticity. In conclusion, our data reveal microglia, glutamate and NO as potential targets for more directed and effective early-therapeutic interventions, and GUDCA as a promising medicine in the management of neonatal hyperbilirubinemia. 128

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160 Chapter IV Rodrigues, C. M., Solá, S., Silva, R. F. M., Brites, D., 2000a. Bilirubin and amyloid-beta peptide induce cytochrome c release through mitochondrial membrane permeabilization. Mol Med. 6, Rodrigues, C. M., Stieers, C. L., Keene, C. D., Ma, X., Kren, B. T., Low, W. C., Steer, C. J., 2000b. Tauroursodeoxycholic acid partially prevents apoptosis induced by 3- nitropropionic acid: evidence for a mitochondrial pathway independent of the permeability transition. J Neurochem. 75, Roseth, S., Hansen, T. W., Fonnum, F., Walaas, S. I., Bilirubin inhibits transport of neurotransmitters in synaptic vesicles. Pediatr Res. 44, Rozenfeld, C., Martinez, R., Figueiredo, R. T., Bozza, M. T., Lima, F. R., Pires, A. L., Silva, P. M., Bonomo, A., Lannes-Vieira, J., De Souza, W., Moura-Neto, V., Soluble factors released by Toxoplasma gondii-infected astrocytes down-modulate nitric oxide production by gamma interferon-activated microglia and prevent neuronal degeneration. Infect Immun. 71, Rudolph, G., Kloeters-Plachky, P., Sauer, P., Stiehl, A., Intestinal absorption and biliary secretion of ursodeoxycholic acid and its taurine conjugate. Eur J Clin Invest. 32, Silva, R., Mata, L. R., Gulbenkian, S., Brito, M. A., Tiribelli, C., Brites, D., Inhibition of glutamate uptake by unconjugated bilirubin in cultured cortical rat astrocytes: role of concentration and ph. Biochem Biophys Res Commun. 265, Silva, R. F. M., Rodrigues, C. M., Brites, D., Bilirubin-induced apoptosis in cultured rat neural cells is aggravated by chenodeoxycholic acid but prevented by ursodeoxycholic acid. J Hepatol. 34, Silva, R. F. M., Rodrigues, C. M., Brites, D., Rat cultured neuronal and glial cells respond differently to toxicity of unconjugated bilirubin. Pediatr Res. 51, Silva, S. L., Vaz, A. R., Barateiro, A., Falcão, A. S., Fernandes, A., Brito, M. A., Silva, R. F. M., Brites, D., Features of bilirubin-induced reactive microglia: From phagocytosis to inflammation. Neurobiol Dis. 40, Solá, S., Brito, M. A., Brites, D., Moura, J. J., Rodrigues, C. M., Membrane structural changes support the involvement of mitochondria in the bile salt-induced apoptosis of rat hepatocytes. Clin Sci (Lond). 103, Soorani-Lunsing, I., Woltil, H. A., Hadders-Algra, M., Are moderate degrees of hyperbilirubinemia in healthy term neonates really safe for the brain? Pediatr Res. 50, Stoppini, L., Buchs, P. A., Muller, D., A simple method for organotypic cultures of nervous tissue. J Neurosci Methods. 37, Sunico, C. R., Gonzalez-Forero, D., Dominguez, G., Garcia-Verdugo, J. M., Moreno-Lopez, B., Nitric oxide induces pathological synapse loss by a protein kinase G-, Rho kinasedependent mechanism preceded by myosin light chain phosphorylation. J Neurosci. 30, Synowitz, M., Glass, R., Farber, K., Markovic, D., Kronenberg, G., Herrmann, K., Schnermann, J., Nolte, C., van Rooijen, N., Kiwit, J., Kettenmann, H., A1 adenosine receptors in microglia control glioblastoma-host interaction. Cancer Res. 66, Takeuchi, H., Jin, S., Suzuki, H., Doi, Y., Liang, J., Kawanokuchi, J., Mizuno, T., Sawada, M., Suzumura, A., Blockade of microglial glutamate release protects against ischemic brain injury. Exp Neurol. 214, Tilleux, S., Goursaud, S., Hermans, E., Selective up-regulation of GLT-1 in cultured astrocytes exposed to soluble mediators released by activated microglia. Neurochem Int. 55, Van Rooijen, N., Sanders, A., Liposome mediated depletion of macrophages: mechanism of action, preparation of liposomes and applications. J Immunol Methods. 174, Vaz, A. R., Delgado-Esteban, M., Brito, M. A., Bolanos, J. P., Brites, D., Almeida, A., Bilirubin selectively inhibits cytochrome c oxidase activity and induces apoptosis in immature cortical neurons: assessment of the protective effects of glycoursodeoxycholic acid. J Neurochem. 112, Wang, Y., Tang, B. L., SNAREs in neurons--beyond synaptic vesicle exocytosis (Review). Mol Membr Biol. 23, Weir, C., Millar, W. S., The effects of neonatal jaundice and respiratory complications on learning and habituation in 5- to 11-month-old infants. J Child Psychol Psychiatry. 38,

161 Microglia, glutamate and nitric oxide as key players in hyperbilirubinemia Xu, Z. C., Chen, Y. M., Xu, P., Liu, H., Xie, Y. L., Zeng, K. B., Epileptiform discharge upregulates p-erk1/2, growth-associated protein 43 and synaptophysin in cultured rat hippocampal neurons. Seizure. 18, Zhang, B., Yang, X., Gao, X., Taurine protects against bilirubin-induced neurotoxicity in vitro. Brain Res. 1320, Zhang, L., Liu, W., Tanswell, A. K., Luo, X., The effects of bilirubin on evoked potentials and long-term potentiation in rat hippocampus in vivo. Pediatr Res. 53, Zhang, S. C., Fedoroff, S., Neuron-microglia interactions in vitro. Acta Neuropathol. 91,

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163 Chapter V FINAL CONSIDERATIONS 135

164 136

165 Final Considerations 1. Concluding remarks and perspectives The general goal of this thesis was to explore if and how migroglial cells participate in the neurodegeneration due to UCB encephalopathy. In fact, previous studies had already demonstrated that UCB promoted a marked inflammatory response in astrocytes with the release of pro-inflammatory cytokines and glutamate, and that this inflammatory reaction was prompted by the activation of several signalling pathways (Fernandes et al., 2007a; Fernandes et al., 2006; Fernandes et al., 2004). Moreover, this immunostimulant effect was proven to be more relevant in astrocytes than in neurons exposed to UCB, while for this last cell type cell death by both necrosis and apoptosis was the most likely expected outcome (Falcão et al., 2005; Falcão et al., 2006). Nevertheless, and despite the notorious contribution of microglia to several neonatal pathological conditions such as hypoxic- ischemic injury (McRae et al., 1995; Vexler and Yenari, 2009), little was known about the involvement of microglia in UCB-elicited toxicity. Early reports had demonstrated that UCB may alter the function of various cells of the immune system (both in vivo and in vitro) (Vetvicka et al., 1985; Vetvicka et al., 1991). In addition, an increase in phagocytosis of both peripheral blood granulocytes and monocytes after UCB treatment was reported by Miler et al. (1985). Our group was the first to demonstrate that UCB also causes microglial activation characterized by morphological alterations, increased secretion of pro-inflammatory cytokines and glutamate as well as microglial cell death, after a short exposure period to UCB (Gordo et al., 2006). Indeed, when compared to other brain cells, microglia revealed to be the most responsive ones to UCB insult (Brites et al., 2009). However, a more detailed characterization of microglial reactivity profile to UCB was needed to further elucidate the molecular and cellular mechanisms of UCB-induced toxicity. Using an experimental model of isolated primary cultures of microglia we described the temporal profile of microglial reactivity towards UCB. We observed, for the first time, that UCB induces an increase in the phagocytic properties of microglia, followed by a shift into a rather inflammatory response with prolonged exposure time. So, we next characterized the inflammatory reaction elicited by UCB in those cells and found that MAPKs phosphorylation is an essential step for NF-κB nuclear translocation and that these events occur upstream of the release of inflammatory mediators such as TNF-α, IL-1β and IL-6, thus probably signalling this release. Moreover, additional inflammatory indicators were displayed by UCB-reactive microglial cells such as COX-2 up-regulation and MMP-2 and -9 increased activation. Furthermore, we provided evidence that UCBinduced cytokine secretion, particularly IL-1β, may participate in MMPs activation suggesting that these events might be reciprocally regulated. 137

166 Chapter V Interestingly, the alternation of the different activation stages of microglia along time in the presence of UCB, phagocytic and inflammatory, seem to indicate that microglia reacts towards UCB insult firstly with a phagocytic response, in an attempt to constrain the lesion extent and comprising a neuroprotective measure. Upon prolonged UCB exposure periods, either a shift on global microglia reaction occurs or two distinct sub-populations of microglial cells may co-exist, one directed at eliminating the damaged cells by phagocytosis, and another that engaged a more delayed inflammatory response. The fact that cell death by both apoptosis and necrosis arouses after the observed phagocytic response in microglia further corroborates the stated hypothesis. Accordingly, clearance of tissue debris performed by microglia following injury has been demonstrated in several disease models and might constitute a regenerative measure (Napoli and Neumann, 2009; Neumann et al., 2009). Our findings add on to the exciting concept that microglia displays functional plasticity (Graeber and Streit, 2009; Schwartz et al., 2006) and emphasize the role of these cells as active sensors of the brain that adapt their reactive phenotypes to the environmental circumstances (Hanisch and Kettenmann, 2007). Nevertheless, the brain is not compartmented and cellular interactions are essential regulators of many critical functions in the healthy and the diseased brain (Biber et al., 2007). Taking this into consideration, we found valuable to investigate how the interplay between microglia and other nerve cells could modulate the effects induced by UCB. In order to achieve this goal we used two different experimental models: conditioned media, to evaluate the effect of soluble factors, and mixed neuron-glia cultures, to assess proximity-dependent interactions. Our results showed that soluble factors released by astrocytes exposed to UCB dampen microglial production of inflammatory cytokines such as TNF-α and IL-1β, diminish MMP-9 activation and prevent cell death. Therefore, microglia, when isolated, respond intensively to UCB stimulation, but previously activated astrocytes seem to drive microglia to refrain its response. Other authors have postulated that this is a feasible mechanism to prevent excessive brain inflammation (Min et al., 2006). Interestingly, UCB-treated neuron conditioned medium produced a similar down-regulation on the production of inflammatory mediators by microglia but enhanced NO generation, MMP-9 activation and led to cell demise. In addition, we demonstrated that microglia s phagocytic abilities were further enhanced when these cells were exposed to conditioned medium derived from UCB-exposed neurons, instead of direct UCB stimulation. Such finding suggests that UCB-injured neurons might be signalling microglia to engage a phagocytic phenotype in an attempt to constrain lesion extent but also enhanced its inflammatory potential ultimately leading to 138

167 Final Considerations microglia demise. This hypothesis was further strengthened by the results regarding UCB-deleterious effects on neurite network and neuronal cell death when neurons were cultivated in close proximity with microglial cells. In fact, these studies showed that the previously observed UCB-induced neurite outgrowth impairment and cell demise (Falcão et al., 2007) were abrogated in the presence of microglia. This apparent protection could be the result of the phagocytic clearance of UCB-damaged neurons, similarly to the events described in other disease models as revealed by the phagocytosis of cellular debris or amyloid deposits by microglia in MS (Takahashi et al., 2005) and AD (Simard et al., 2006), respectively. The results obtained pertaining neuron-glia dynamics upon UCB exposure prompted us to explore the role of microglia in UCB-induced neurotoxicity in a more complex model, the organotypic slice cultures. These studies were performed in the hippocampus, one of the brain areas affected in kernicterus (Shapiro, 2003; Shapiro, 2005) that showed increased vulnerability to UCB s toxic effects as proven by the impairment of long-term synaptic plasticity found by Chang et al. (2009) in this brain area. By the selective depletion of microglia from organotypic-cultured hippocampal slices we were able to determine that these cells are the major sources of NO upon UCB stimulation and that they significantly interfere with glutamate homeostasis. The inducible expression of glutamate transporters (Lopez-Redondo et al., 2000; Persson et al., 2005) as well as the presence of glutamine synthetase in microglia (Rimaniol et al., 2000) has been previously reported. Our results suggest an initial neuroprotective role for microglia as glutamate scavengers. However, despite microglial efforts to limit glutamate concentration, the ultimate outcome is an increase in its extracellular levels and tissue accumulation, possibly due to the failure of the clearance mechanisms or to increased production by either glial or neuronal cells upon prolonged exposure times to UCB. Interestingly, glutamate had already been stressed out as an important mediator of UCB-induced neurotoxicity (Johnston, 2005) since its astrocytic uptake is inhibited in astrocytes (Silva et al., 1999) prolonging its presence in the synaptic cleft and engendering NMDA overactivation and consequent excitotoxic death (Grojean et al., 2000; Grojean et al., 2001; McDonald et al., 1998). Moreover, oxidative stress has also been demonstrated to be involved in UCB-induced injury (Brito et al., 2004; Brito et al., 2008; Vaz et al., 2010). More recently the two events were shown to be associated during UCB cytotoxicity since overactivation of glutamate receptors seems to mediate oxidative damage in neurons (Brito et al., 2010). In addition, oxidative stress was also shown to mediate UCB-induced degeneration of excitatory synaptic terminals in the auditory brainstem (Haustein et al., 2010). Therefore, we decided to explore the role of 139

168 Chapter V glutamate and NO in the impairment of neurite extension and ramification and in neuronal death in order to elucidate the molecular mechanisms triggering neurodevelopmental changes. Our studies clearly demonstrate the key role of NO and overactivation of NMDA receptors in UCB-induced impairment of neuritic outgrowth and neuronal demise, providing exciting new targets for the management of neonatal hyperbilirubinemia. Our results also indicated that UCB decreased the expression of presynaptic proteins, which are essential for synapse establishment and neurotransmitter release, and are consistent with the findings showing that UCB can also adversely affect synapse establishment (Fernandes et al., 2009), and cause alterations of synaptic plasticity (Chang et al., 2009). Abnormal cytoskeleton assembly, loss of dendrites and axons and impairment of neurotransmission can cause disruption of synaptic connectivity and instigate neurodegenerative diseases and mental disorders like schizophrenia (Benitez-King et al., 2004; Brennaman and Maness, 2008). Thus, association between neonatal hyperbilirubinemia and proneness to mental disorders in later life seems to be a reasonable possibility. In this study we also evaluated the therapeutic potential of GUDCA and IL-10 and found that these molecules were able to counteract the above mentioned deleterious effects prompted by UCB, namely network dynamics impairment and neuronal cell death, probably by the reduction of glutamate extracellular accumulation. In addition, GUDCA proved to be more effective than IL-10 in preventing the down-regulation of presynaptic proteins expression. Besides, GUDCA proved to also abrogate the injurious effect elicited by UCB not only in isolated neurons but also in the hippocampal slice culture model, thus reinforcing its potential as a therapeutic approach in the management of neonatal hyperbilirubinemia. Collectively, the data presented in this thesis (Fig. V.1) provide interesting findings regarding the implication of microglia in UCB-induced phenomena. Indeed, different reactive phenotypes are engaged by these cells depending on the duration and intensity of the toxic stimulus and on the modulation exerted by neighbouring cells, reinforcing their remarkable functional plasticity. 140

169 Final Considerations Isolated microglia Cell death UCB 0 h 15 min 30 min 2 h 4 h 8 h 12 h 24 h MAPKs NF κβ Phagocytosis Inflammation TNF α IL 1β A IL 6 COX 2 MMP 2 MMP 9 Conditioned media Mixed cultures UCB UCB B IL 1β IL 6 MMP 9 LDH leakage IL 1β and TNF α IL 6 MMP 9 LDH leakage Phagocytosis C Neurite outgrowth impairment Cell death Organotypic cultured hippocampal slices Isolated neurons UCB UCB NO Glu Neurite outgrowth impairment Cell death SNAP 25 Synaptophysin Glu D GUDCA IL 10 Fig.V.1. Schematic representation of microglia reactivity to unconjugated bilirubin. Isolated microglial cells present a dual phenotype, phagocytic and inflammatory, that switch along unconjugated bilirubin (UCB) exposure, with the release of several inflammatory mediators and activation of diverse upstream signalling pathways (A). In addition, astrocytes exposed to UCB dampen microglia activation while neurons signal a phagocytic phenotype in microglia and enhance its inflammatory potential leading to cell demise (B). Moreover, in UCB-stimulated neuron-microglia mixed cultures the neurotoxic effects of UCB are prevented, suggesting that stressed neurons promote a neuroprotective clearance function in microglia (C). Finally, microglia revealed to participate in glutamate homeostasis and to induce the release of nitric oxide (NO) in UCB-treated hippocampal slices (D), important molecules in UCB-induced neurotoxicity. Moreover, our results identify some of the therapeutic actions of interleukin (IL)-10 and glycoursodeoxycholic acid (GUDCA). 141