Fondazione Italiana Fegato ONLUS Annual scientific report of the Italian Liver Foundation November, 19 th 2015 Cristina Bellarosa, PhD Molecular mechanisms involved in bilirubin neurotoxicity
Authors Mohammed Qaisiya, PhD Post Doc Fellowship from AREA Science Park Cristina Brischetto Ungraduate Student from February to October 2015 Title of the thesis: «Molecular events associated to ER stress induction by unconjugated bilirubin in in vitro model» Cristina Bellarosa, PhD Senior Scientist at FIF Maternity leave from May to December 2015
Presentation s outline Introduction: Bilirubin Neurotoxicity Endoplasmic Reticulum UCB-induced ER stress Effect of UCB-induced ER-stress toward cell survival/cell death; Effect of UCB-induced ER-stress toward inflammation; UCB induced Autophagy and correlation with ER-stress.
Bilirubin metabolism Free Bilirubin (Bf) is the portion of Unconjugated Bilirubin (UCB) not bound to albumin is fat-soluble and toxic
Bilirubin Neurotoxicity Newbors could develop increased levels of unconjugated bilirubin (UCB) in the blood stream, clinically evident as physiological neonatal jaundice. This is a benign and transient phenomenon. Abnormal accumulation of bilirubin may cause bilirubin encephalopathy ranging from minimal and reversible neurological injury to severe and permanent condition leading to neurodevelopmental dysfunctions. Furthermore, infants affected by the genetic pathology of Crigler-Najjar I present high UCB plasma levels and are particularly exposed to bilirubin encephalopathy.
Bilirubin-induced cell injury UCB may act directly on the membranes; These membrane effect leads to glutamate excitotoxicity, mitochondrial energy failure and increased intracellular calcium concentration; Increased ica ++ concetration activates proteolytic enzymes, apoptotic pathways and necrosis. Watchko J.F., NeuroMolecular Medicine, 2006
Bilirubin-induced cell injury Neurons are more sensitive to UCB toxicity than glial cells and UCB toxicity is enhanced in young cells
Endoplasmic reticulum Protein synthesis, folding and maturation Storage of Ca2+ Lipid biosynthesis
Unfolded Proteins Response Collection of signaling pathways that: Monitors the ER conditions; Senses an insufficiency in the ER s proteing folding capacity; Communicates this information to gene expression programs. Walter P., Science, 2011
Bilirubin and Endoplasmic reticulum In the hepatocytes ER is exposed to concentrated UCB because this is the site for its glucuronidation. When internalized by hepatocytes, UCB is bound by the cytosolic protein ligandin and shuttled to the endoplasmic reticulum (ER) for conjugation. When bilirubin glucuronidation capacity is reduced, cytochrome P450 (CYP) driven bilirubin oxidation has been suggested to contribute significantly to the elimination of bilirubin. Cytochrome P450 (CYP) is a large family of well-conserved integral membrane proteins localized primarily in the membrane of the endoplasmic reticulum (ER), where these enzymes metabolize a variety of both endogenous and exogenous compounds ER is exposed to UCB Oakes G.H., J Biochem Molecular Toxicology, 2010 Abu-Bakar A., Toxicology and Applied Pharmacology, 2012 Gambaro S.E., Toxicokinetics and metabolism, 2014 Neve E.P., J Biochem Molecular Toxicology, 2010
ER as extremely sensitive compartment In order for proper protein folding to occur, the ER is extremely sensitive to changes in: cellular energy levels intracellular Ca 2+ levels intracellular redox state Bilirubin: impairs energy metabolism disrupts calcium homeostasis induces reactive oxigen species Bilirubin is a good candidate to generate ER-stress
Literature about Bilirubin-induced ER stress
Aim of our study To evaluate the effects of the toxic concentration of UCB toward ER stress and the related signaling associated with this event UCB?? ER-stress?? Cell survival/cell Death?? Autophagy Inflammation
In vitro comparative study SH-SY5Y cells: Species: HUMAN NEUROBLASTOMA (Undifferentiated) Derived from a metastatic bone marrow of a 4 years old female patient Received by: Stefano Gustincich (SISSA) GI-ME-N cells: Species: HUMAN NEUROBLASTOMA (Differentiated) Derived from a metastatic bone marrow of a 2 years old patient with stage IV neuroblastoma Received by: Maria Paola Nitti (UNIGE) U87 cells: Species: HUMAN ASTROCYTOMA Derived from brain of a male 44 years old patient with astrocytoma; classified as grade IV Received by: Elisabetta Ruaro (SISSA) SH-SY5Y GI-ME-N U87
Presentation s outline Introduction: Bilirubin Neurotoxicity Endoplasmic Reticulum UCB-induced ER stress Effect of UCB-induced ER-stress on cell survival/cell death; Effect of UCB-induced ER-stress on inflammation; UCB induced Autophagy and correlation with ER-stress.
Urra H., Biochimica et Biophysica Acta, 2013 CHOP s role in ER stress-induced apoptosis ER STRESS ADAPTATIVE RESPONSE/ CELL SURVIVAL CELL DEATH/ APOPTOSIS CHOP induction is one major event responsible for the switch between prosurvival and proapoptotic response.
Partial Conclusion 1 Neuronal cells are more susceptible to UCB toxicity than glial cells, although UCB accumulates in both cell types; UCB induces ER-stress in neuronal cells, not in glial cells; Undifferentiated neuronal cells are more sensitive to UCB toxicity than differentiated cells and it correlates with higher levels of CHOP mrna expression; ER-stress inibition and deletion of CHOP reduces UCB-induced cytotoxicity.
Presentation s outline Introduction: Bilirubin Neurotoxicity Endoplasmic Reticulum UCB-induced ER stress Effect of UCB-induced ER-stress on cell survival/cell death; Effect of UCB-induced ER-stress on inflammation; UCB induced Autophagy and correlation with ER-stress.
UCB Does UCB induce inflammation? Is it ER-stress related? 4-PBA sirna PERK PDTC and gene reporter TNFα and IL-8 Hotamisligil G.S, Cell, 2010
Partial Conclusion 2 UCB induces mrna expression and protein release of TNFα and IL-8; IL-8 mrna induction is regulated by NFкB and ERstress signaling via PERK.
Presentation s outline Introduction: Bilirubin Neurotoxicity Endoplasmic Reticulum UCB-induced ER stress Effect of UCB-induced ER-stress on cell survival/cell death; Effect of UCB-induced ER-stress on inflammation; UCB induced Autophagy and correlation with ER-stress.
Fondazione Italiana Fegato Italian Liver Foundation Does UCB induce Autophagy? Is it ER-stress related? UCB 4-PBA 2 1 Deegan S. et al.,biochem.biophys.res.commun,2014
Fondazione Italiana Fegato Italian Liver Foundation Partial Conclusion 3 UCB induces autophagy related genes (LC3, WIPI1, p62, FAM129A and DDIT) and increases the conversion of LC3-I to LC3-II; UCB induction of LC3 and p62 mrna occurs through ER stress;
UCB Final Conclusion ER-stress? Cell survival/cell Death Autophagy Inflammation ER responds to UCB load differentially modulating CHOP induction. Differentiated neuronal cells are able to adapt to UCB-induced ER-stress and to restore cell homeostasis, while UCB-induced ER-stress leads to cell death in the undifferentiated neuronal cells. UCB induced neurotoxicity in SH-SY5Y cells involves complex signaling that includes ER stress-dependent inflammation and autophagy ( to be confirmed)
Fondazione Italiana Fegato ONLUS Annual scientific report of the Italian Liver Foundation November, 19 th 2015 Cristina Bellarosa, PhD Molecular mechanisms involved in bilirubin neurotoxicity