Modulating copper metabolism as a strategy to treat. neurodegenerative tauopathies

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1 Modulating copper metabolism as a strategy to treat neurodegenerative tauopathies Simon McKenzie-Nickson Submitted in total fulfilment of the requirements of the degree of Doctor of Philosophy March 2017 Department of Pharmacology and Therapeutics Bio21 Molecular Science and Biotechnology Institute Florey Institute of Neuroscience and Mental Health University of Melbourne i

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3 Abstract Transition metals such as iron and copper are essential for life and health and yet can cause toxicity through oxidative damage. Therefore, regulation of the levels and location of transition metals is of critical importance to both cellular and organism health. Dyshomeostasis of transition metals has been associated with age-related neurodegenerative diseases such as Alzheimer s disease (AD), Parkinson s disease (PD), and forms of Frontotemporal dementia (FTD) and thus correcting this dyshomeostasis is an attractive therapeutic target. Previous research from our laboratory has shown that a class of compounds, the Cu II bis(thiosemicarbazones), are efficacious in correcting the pathology in animal models of both AD and PD. The work outlined in this thesis focuses on gaining insight into the mechanism of action of the Cu II bis(thiosemicarbazone), glyoxalbis [N4-methylthiosemicarbazonato]Cu(II) (Cu II (gtsm)), in treating an animal model of AD. Additionally, this work aimed to build on these findings by testing the efficacy of Cu II (gtsm) in treating an animal model of FTD. Treatment of AD transgenic mice with Cu II (gtsm) improved the behavioural deficit seen in the animals in both the Morris water maze and in the Y-maze measures of spatial memory. Quantification of levels of amyloid-β in the brains of these mice revealed no changes in any detectable species. Treatment did however, decrease the levels of phosphorylated forms of Tau, one of the hallmarks of the disease. Analysis of Tau phosphatases and kinases revealed no changes in glycogen synthase kinase 3β, but did reveal an increase in the structural subunit of the Tau phosphatase, protein phosphatase 2A (PP2A). Based on these findings, efficacy of Cu II (gtsm) in treating the AD mice in this study is thought to be through an amyloid-β independent reduction in phosphorylated i

4 Tau through an increase in PP2A. Additionally, this study supports the concept of AD being an amyloid-β mediated tauopathy. Treatment of an FTD mouse model with Cu II (gtsm) improved the spatial memory deficit seen in the Morris water maze performance of these mice. Additionally, treatment reduced the strong hyperactivity phenotype and produced an anxiolytic effect in transgenic mice. Biochemically, treatment reduced Tau tangle load in the hippocampus and reduced a 100 kda dimer of Tau that was strongly correlated with behavioural deficits. As with the AD model, treatment increased the levels of the same subunit of PP2A. It was hypothesised that the efficacy of Cu II (gtsm) in treating FTD was again a reduction in pathological Tau via an increase in PP2A levels. Due to the ability of Cu II (gtsm) to increase cellular bioavailable copper, the compounds ability to treat the childhood disease, Menkes disease (MD), was also tested. Utilising the Mottled-Brindled (Mo/Br) mouse model (a naturally occurring mouse model with limited copper transporting ability due to mutant ATP7a) of MD, my work demonstrated that Cu II (gtsm) was a strong candidate for treating the disease. Treatment with Cu II (gtsm) both orally and via injection increased the levels of brain copper significantly more than copper salt treatment. The findings from this thesis suggest that increasing bioavailable copper has a similar mechanism of action in treating related tauopathies such as AD and FTD. Furthermore, the improvement in behavioural deficits over these two tauopathies suggests this compound could be effective in treating these diseases and validates increasing cellular copper as a clinical therapeutic strategy. Furthermore, the compound has shown promise in the treatment of MD which currently has no effective treatment. ii

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6 Declaration This is to certify that; I. the thesis comprises only my original work towards the PhD except where indicated in the Preface, II. III. due acknowledgment has been made in the text to all other material used, the thesis is less than 100,000 words in length, exclusive of tables, maps, bibliographies and appendices. Simon McKenzie-Nickson Department of Pharmacology and Therapeutics Bio21 Molecular Science and Biotechnology Institute The Florey Institute of Neuroscience and Mental Health The University of Melbourne; Parkville, Victoria 3052 X Simon McKenzie-Nickson iv

7 Preface I acknowledge that specific experiments carried out in this thesis were performed by others: The Cu II (gtsm) used was synthesised in the Department of Chemistry, University of Melbourne, by Dr Brett Patterson, Lachlan McInnes and Associate Professor Paul Donnelly. Gene expression analysis in chapter 2 was carried out by Dr Lesley Cheng. SELDI-TOF experiments in chapter 2 was carried out by Keyla Perez Behavioural experiments in chapter 2 were carried out with the help of Jacky Chan. Section 3.2 was carried out by Dr Lin hung with the help of Associate Professor Paul Adlard. Morris water maze search strategy in chapter 2 analysis was computed by Jake Rogers. Brain slicing for LA-ICP-MS was carried out by Mirjana Bogeski. LC-ICP-MS was carried out by Dr Adam Gunn and Dr Blaine Roberts. LA-ICP-MS was carried out by Dr Kai Kysenius. ICP-MS was carried out by Irene Volitakis. Enzyme assays in section 4.5 were carried out by James Hilton and Dr Steve Mercer in the lab of Dr Peter Crouch. Various steps in western blotting were carried out with help from Jacky Chan. v

8 Acknowledgements A body of work that extends over four years requires the support of an immense number of people, of which a several deserve special mention: My supervisors: Kevin Barnham, David Finkelstein, and Lin Hung First and foremost, thanks must go to my supervisors. Thank you for the coffees, beers, and guidance over the past 4 years. You have been instrumental to me completing this body of work of which I am very proud. Special thanks to Kevin for providing me with a fulfilling project, the resources to complete it, and helping me through the twists and turns of developing it and getting me to where I am. My PhD committee Laura Jacobson, our coffees and planning sessions were invaluable to my progress. Jim Camakaris, your knowledge of copper and passion for Menkes research helped a lot through the project but especially for the Menkes arm of the project. Peter Crack, you were all I could ask for in a Chair, thank you so much for all your help and mentoring. The oxidation biology group Thanks must go to Ashley Bush and his excellent team. Your support, training, and general conversations (looking at you Charlotte, Lisa, Jess, Lydia, and Amelia) over the years have been incredibly important to me and for that I thank you. vi

9 Paul Donnelly, Lachlan McInnes, and Brett Patterson Your synthesis of the compounds required, usually with far too little forewarning, have been crucial to the success of the animal trials I conducted, thank you. The Barnham lab The members of the Barnham lab, both past and present, have been a great team to work with! Andrew, Russell, Keyla, Laura J, Laura V, Leah, Jacky, Xiang, Vijaya, and all the past members, you have been a pleasure to work alongside and I am proud to call you all friends. Russell Down and Andrew Watt Special mention to my office-mates; thank you both for the friendship that developed from sharing an office with you both. From board meetings to the inception of the SAS, my PhD experience was enriched immensely by you both. Also, thank you Andrew for reviewing this thesis, especially in 2-days. Kai Kysenius Everyone needs a Finnish friend. From being my travel guide around Europe to helping brainstorm scientific ideas, I have appreciated your friendship immensely. Friends/family Of course, thanks must go to all my wonderful friends for helping me get through this. You are far too numerous to name but you know who you are, thank you for keeping me (in)sane throughout the last 4 years. Also, to my family, you have all been so vii

10 supportive as well, always asking how things are going and providing kind words of encouragement, especially from you Joy. Thank you, those words have helped more than you know. Thanks also needs to go to Jake, my brother in arms, thanks for all your help with the water maze work and being a great friend. Jan McKenzie To my ever-supportive mother, I wouldn t have and couldn t have made it this far without you. You are an incredible inspiration to me and your support of everything I do, both academic and non-academic, has made such a difference to my life. Thank you. Angela Taylor My incredible partner, thank you for coming along on this crazy ride. You ve stuck with me and supported me through the highs and the lows and now we are out the other side. Your support through the good times and the bad has been incredible and kept me on track. You re an amazing person and I m lucky to have you by my side. Trevor Laurence and Robin McKenzie You will never read the words within this thesis but know you both helped me write every one of them. I miss you both. Who dares wins. viii

11 Table of Contents Abstract...i Declaration... iv Preface...v Acknowledgements... vi Table of Contents... ix List of Figures... xv Chapter 1 Introduction Transition metals in biology Alzheimer s disease Biology of AD The quest for a therapy Frontotemporal dementia History of FTD Biology of Tau associated FTD Metals and FTD Bis(thiosemicarbazone) complexes Rationale of current study Chapter 2 Cu II (gtsm) treatment of the APP/PS1 model of Alzheimer s disease Introduction ix

12 2.2 Preliminary study of Cu II (gtsm) treatment of the APP/PS1 model of AD Effects of Cu II (gtsm) on APP/PS1 mouse behaviour Effects of Cu II (gtsm) on APP/PS1 biochemistry Summary of preliminary data Follow up trial of Cu II (gtsm) treatment of the APP/PS1 mouse model of AD Introduction Effects of Cu II (gtsm) on APP/PS1 behaviour Effects of Cu II (gtsm) on APP/PS1 biochemistry Effects of Cu II (gtsm) treatment on LC-ICP-MS profile Summary of follow up data Major conclusions from chapter 2 and discussion Cu II (gtsm) improves the behavioural phenotype of APP/PS1 mice Cu II (gtsm) does not alter the levels of amyloid-β species Cu II (gtsm) reduces Tau pathology in APP/PS1 mice Cu II (gtsm) does not affect kinase biology, but improves phosphatase levels Cu II (gtsm) increases PSD-95 levels Discussion and future directions Chapter 3 Cu II (gtsm) in a Tau-mediated model of Frontotemporal Dementia Introduction The rtg4510 mouse model of FTD x

13 3.2 Preliminary study of Cu II (gtsm) in the rtg4510 (P301L) mouse model of FTD Preface Introduction Effects of Cu II (gtsm) on rtg4510 behaviour Effects of Cu II (gtsm) on rtg4510 biochemistry Summary of Preliminary Data Cu II (gtsm) treatment of the rtg4510 (P301L) mouse model of FTD Preface Introduction Effects of Cu II (gtsm) on rtg4510 behaviour Effects of Cu II (gtsm) on rtg4510 biochemistry LC-ICP-MS analysis of the soluble copper proteome in rtg4510 mice Summary of follow up data Major conclusions from chapter 3 and discussion Cu II (gtsm) treatment improves the behavioural phenotype of rtg4510 mice Cu II (gtsm) reduces Tau pathology in rtg4510 mice Cu II (gtsm) increases levels of PP2A(A) in rtg4510 mice Cu II (gtsm) increases copper content of soluble proteins Discussion and future directions Chapter 4 - Cu II (gtsm) in Menkes disease (MD) xi

14 4.1 Introduction History of Menkes Disease The role of the Menkes protein (ATP7a) Treatment of Menkes Disease Copper metabolism Copper import into the cell Intracellular distribution The Mo/Br mouse model of MD Preface and rationale for MD pilot study Initial assessment of copper supplementation with Cu II (gtsm) in Mo/Br mice Treatment mediated changes in brain cuproenzyme activity Preface Superoxide dismutase Ceruloplasmin Dopamine-β-hydroxylase Lysyl-oxidases Assessment of oral bioavailability of Cu II (gtsm) in Mo/Br mice Major conclusions from chapter 4 and discussion Discussion and future directions Chapter 5 - Conclusions and future directions Future directions xii

15 Chapter 6 - Methods Chemicals Biochemistry Tissue homogenisation Protein quantification (BCA Assay) Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) Western blotting Transfer Immunoblotting Densitometry Sarkosyl extraction of Tau Inductively coupled plasma mass spectrometry (ICP-MS) Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) Liquid chromatography inductively coupled plasma mass spectrometry (LC- ICP-MS) RNA analysis Enzyme assays Caeruloplasmin Lysyl-oxidase (LOX) Dopamine-β-hydroxylase (DβH) xiii

16 6.9 Animal experiments Animal husbandry Animal dosing Y-maze Morris water maze Locomotor cell (open field) Rotarod (motor co-ordination) Culling and tissue collection Genotyping Statistics Bibliography xiv

17 List of Figures Figure 1.1. The natural history of Aβ deposition in sporadic Alzheimer's disease Figure 1.2. Schematic of proteolytic cleavage of APP Figure 1.3. Tau structure schematic... 9 Figure 1.4. Structures of two BTSC group family members. A) Cu II (gtsm) and B) Cu II (atsm) Figure 2.1. Cu II (gtsm) improves cognition in the Morris water maze probe trial Figure 2.2. Cu II (gtsm) does not alter Aβ species in APP/PS1 brain homogenate Figure 2.3. Levels of total Tau are unchanged in APP/PS1 transgenic mice Figure 2.4. Cu II (gtsm) reduces phosphorylated forms of Tau and decreases oligomeric Tau Figure 2.5. Levels of p-gsk are unchanged in APP/PS1 transgenic mice Figure 2.6. Cu II (gtsm) alters PP2A(A) subunit levels but not (B) or (C) Figure 2.7. Hippocampal mrna analysis of APP/PS1 mice reveals altered levels of expression of the 'A' and 'C' subunit of PP2A Figure 2.8. Cu II (gtsm) treatment did not improve performance of transgenic APP/PS1 mice during the acquisition phase of the Morris water maze Figure 2.9. Cu II (gtsm) did not improve performance of APP/PS1 transgenic mice in the Morris water maze probe trial Figure Cu II (gtsm) treatment rescues cognitive deficit on the Y-maze Figure Cu II (gtsm) did not impact performance in the locomotor cell (open field).48 Figure Cu II (gtsm) does not alter Aβ species in APP/PS1 brain homogenate Figure Total Tau levels are unaffected by both disease status and treatment Figure Cu II (gtsm) improves levels of pathological phospho-tau xv

18 Figure Vehicle treated APP/PS1 mice exhibit an increase in ptau202 which is decreased by drug treatment Figure The ratio of sarkosyl-soluble to insoluble Tau does not increase in transgenic mice and is unaffected by drug treatment in APP/PS1 mice Figure Cu II (gtsm) alters PP2A(A) subunit levels but not (B) or (C) Figure Cu II (gtsm) rescues the PSD-95 impairment in transgenic vehicle treated APP/PS1 mice while not affecting levels of synaptophysin or glial fibrillary acidic protein Figure Cu II (gtsm) increases copper content of a multitude of proteins Figure Cu II (gtsm) produces an increase in the iron content of ferritin Figure Cu II (gtsm) restores the Zn content of multiple peaks Figure Model of the link between MTHFR, folate, PP2A methylation and Tau phosphorylation Figure 3.1. Cu II (gtsm) improves rtg4510 transgenic mouse performance in the Y-maze Figure 3.2. Transgenic rtg4510 mice exhibit an increase in the number of total Y-maze entries Figure 3.3. Cu II (gtsm) treatment rescues probe trial deficit in rtg4510 mice Figure 3.4. Cu II (gtsm) treatment of rtg4510 mice decreases hippocampal neurofibrillary tangle load Figure 3.5. Cu II (gtsm) treatment of rtg4510 mice does not alter cortical neurofibrillary tangle load Figure 3.6. Cu II (gtsm) treatment does not alter total or phosphorylated Tau in rtg4510 mice xvi

19 Figure 3.7. Treatment of rtg4510 mice with Cu II (gtsm) rescues the decrease in the PP2A(A) subunit Figure 3.8. Cu II (gtsm) treatment rescues the strong anxiety-like and hyperactivity phenotype of transgenic rtg4510 mice Figure 3.9. Transgenic rtg4510 mice exhibit no deficit in Rotarod performance Figure Cu II (gtsm) does not alter the level of phosphorylated Tau or total levels of Tau in transgenic rtg4510 mice Figure Cu II (gtsm) rescues the dramatic increase in high molecular weight Tau in rtg4510 transgenic mice Figure High molecular weight Tau is positively associated with the hyperactivity phenotype in rtg4510 mice Figure LC-ICP-MS analysis of rtg4510 brain tissue shows increases in copper of multiple proteins Figure Iron content of ferritin is altered in rtg4510 transgenic mice Figure Zinc content of multiple protein peaks is altered in transgenic rtg4510 mice Figure 4.1. Copper homeostasis in mammalian cells Figure 4.2. Cu II (gtsm) produces a greater increase in brain copper than vehicle of CuCl2 treatment Figure 4.3. Cu II (gtsm) treatment produces a colour change in the coats of treated mice Figure 4.4. Homozygous Mo/Br brains contain very little copper as imaged with laser ablation inductively coupled plasma mass spectrometry Figure 4.5. Brain cuproenzyme activity is largely unaffected in homozygous Mo/Br mice xvii

20 Figure 4.6. Orally gavaged Cu II (gtsm) increases brain copper to a similar level as IP injection Figure 6.1. Diagram of transfer stack organisation Figure 6.2. Morris water maze room setup Figure 6.3. Morris water maze search strategy analysis xviii

21 Chapter 1 Introduction 1.1 Transition metals in biology Transition metals such as iron, zinc and copper are essential for life and play a role in multiple biological processes. These include the structural stability of proteins, acting as second messengers, and involvement in the catalytic activity of enzymes. The physical properties of copper and iron make them suitable to participate in reduction-oxidation (redox) reactions and enable the capture, transport and utilisation of oxygen (which is fundamental to aerobic life). However, this ability also means they can create oxidative stress through the production of free radicals. This toxic potential means that the cell has evolved complex and tightly controlled homeostatic mechanisms for regulating both the levels and location of these metals. The focus of this thesis is on the role of copper in two models of neurodegeneration, Alzheimer s disease (AD) and Frontotemporal dementia (FTD), and how pharmacologically manipulating copper bioavailability may yield improvements in disease progression. 1.2 Alzheimer s disease AD 1 is the worldwide leading cause of dementia, accounting for between 50-80% of cases. Dementia is a term used to describe a syndrome of symptoms including memory loss and cognitive deficits. A recent report estimated that the global economic burden of dementia is around US$818 billion (World Alzheimer Report 2015, which equates to 1.09% of the worlds gross domestic profit. With AD being the largest single contributor to this, much research has gone into the understanding and treatment of the disease. While the disease was 1

22 discovered over 100 years ago 1 it was until some 80 years later that real progress would be made in understanding the biology of the disease Biology of AD In the original report of the disease, German physician Alois Alzheimer described two hallmark changes in the brain of a 51-year-old female patient with a history of cognitive symptoms: the appearance of extracellular insoluble plaques largely comprised of a 4 kda peptide amyloid-β (Aβ) and intracellular neurofibrillary tangles (NFTs) comprised of a hyperphosphorylated form of the protein Tau 1. Clinically, the disease presents with symptoms ranging from becoming withdrawn and less spontaneous, getting lost in familiar places, and a general decline in cognitive ability, eventually progressing to more severe symptoms such as losing the ability to feed and swallow, severe memory deterioration and, in all cases, eventual death (usually by complications such as pneumonia 2 ). Diagnosis is largely a process of exclusion however recent advances in brain imaging has resulted in much more definitive diagnoses 3. There is currently no effective treatment for AD and current therapies are only symptomatic, efficacy varies greatly and they do not address the underlying cause of the disease. Advances in imaging and biomarker identification have indicated that the pathology begins some 20 years before a patient presents in the clinic with cognitive symptoms 4 providing a large therapeutic window. This natural history of AD progression is illustrated in figure 1.1 where measures of cortical Aβ (given as SUVR via brain imaging) are stratified based on clinical diagnosis and is depicted overtime illustrating amyloid accumulation (page 3). 2

23 Figure 1.1. The natural history of Aβ deposition in sporadic Alzheimer's disease. (A) While there were no significant differences in SUVR between participants with MCI and AD with high Aβ burden (2 31 [SD 0 43] for MCI+ and 2 33 [0 36] for AD+), the mean values for healthy controls with high 11 C-PiB retention (HC+) were significantly lower (1 98 [SD 0 24], *p=0 0002). (B) Aβ deposition follows sigmoidal kinetics over time, where it takes 12 years to go from a mean SUVR of 1 17 (SD 0 09) noted in healthy controls with low 11 C-PiB retention (HC ) to reach the 1 5 PiB SUVR threshold. It then takes another 19 years to go from the 1 5 SUVR to the mean SUVR of 2 33 (0 36) observed in established AD. As disease progresses, the rates of Aβ deposition start to slow, trending towards a plateau. The shaded area represents 95% CIs. The horizontal dashed line represents the SUVR threshold (>1 5 or < 1 5) discriminating between high or low 11 C- PiB retention. *Aβ accumulation begins. Aβ positivity threshold is crossed. Mean SUVR of established AD. Figure and legend adapted with permission 59. AD=Alzheimer's disease. MCI=mild cognitive impairment. 11 C-PiB=Carbon-11-labelled Pittsburgh compound B. SUVR=standardised uptake value ratio. Aβ=amyloid β. 3

24 Amyloid-β The Aβ peptide is the major component of the AD amyloid plaques and is generated from the proteolytic processing of a larger precursor parent protein termed amyloid precursor protein (APP). The protein can be cleaved by one of two mutually exclusive proteolytic pathways (figure 1.2; page 5) 5. The non-amyloidogenic pathway is initiated through cleavage via an α-secretase enzyme such as ADAM10 6. The amyloidogenic pathway begins with cleavage mediated by the β-secretase (BACE1) and subsequent cleavage by the γ-secretase complex. A variety of mutations within the APP gene can cause much more aggressive forms of AD with an earlier onset. The APP gene is located on chromosome 21 and as such, patients with Downs s syndrome (trisomy 21) develop AD at a much earlier age 7. Mutations in the APP gene generally influence the interaction of the APP protein with the proteolytic enzymes responsible for its cleavage. For example, a mutation within the β-secretase cleavage site of APP (this cut-site in concert with a cleavage mediated by the γ-secretase is responsible for the generation of the Aβ peptide) confers a phenotype whereby β-secretase has a much higher affinity for the APP substrate due to K595M596 being substituted for N595L596. This results in β-secretase cleaving APP 10 times faster in individuals with this genetic form of the disease compared to those with sporadic AD 8. Furthermore, a genetic polymorphism has been identified that actually reduces the risk of developing AD, the A637T mutation within APP lies adjacent to the β-secretase site and reduces Aβ production by around 40% 9. Another protein necessary for the generation of the Aβ peptide is the previously mentioned γ-secretase. The γ- secretase is a multi-protein complex and represents the final cleavage step to release Aβ. Mutations in two of the proteins involved in this complex were identified at around the same time, these proteins were presenilin 1 (PSEN1), located on chromosome and presenilin 2 (PSEN2), located on chromosome

25 Figure 1.2. Schematic of proteolytic cleavage of APP. The two possible mutually exclusive proteolytic cleavage pathways of APP. The amyloidogenic pathway requires the action of the β-secretase (BACE1) followed by subsequent cleavage by the γ-secretase complex (presenilin 1/2, nicastrin, PEN2, APH- 1) resulting in the generation of amyloid-β (Aβ), secreted amyloid precursor protein β (sappβ), and the APP intracellular domain (AICD). The non-amyloidogenic pathway requires the action of an α-secretase (ADAM 9, 10, or 17) and subsequent cleavage by the γ-secretase complex releasing the secreted amyloid precursor protein α (sappα). Figure adapted with permission from 12. APH-1: anterior pharynx defective, PEN2: presenilin enhancer 2. 5

26 In 1991, another chromosome was implicated as an AD risk factor. In a group of families that displayed familial AD, no linkage was found on chromosome 21 and so the group under took a genomic search for the linkage. They isolated a link to the proximal long arm of chromosome 19. Two years later a polymorphism was found in the predicted region. This polymorphism, termed ε4, was found in the gene encoding apolipoprotein E (APOE) 12,13. It was seen later that this mutation increased AD risk (20% - 90%) and reduced mean age of onset from 84 to 68 years of age 14. It is now thought that the APO family contributes to APP metabolism and is also involved in the clearance of Aβ from the brain via receptor mediated clearance across the blood brain barrier (reviewed extensively in 15 ). While the genetic component of AD comprises a small percentage of total AD cases (~1%), this important sub-population has provided vital information into the proteins and pathways involved in AD and led to the discovery of many of the proteins involved with AD. To date around 160 AD mutations have been identified (AD mutation database: The Aβ peptide is thought to be the toxic root cause of AD. However, despite much research into the expression patterns of APP expression, the functional sub-domains within the molecule itself, and in to the transport of APP to various parts of the cell, the precise function of the full-length APP protein is still unclear. There have, however, been roles found for APP in synaptic plasticity, long-term potentiation, cell-cell adhesion, and cell migration (reviewed in 5,16 ). As seen in figure 1.2 (page 5), APP cleavage can follow one of two mutually exclusive pathways. The pathway leading to the generation of Aβ (amyloidogenic pathway) 6

27 involves the action of β-secretase within the ectodomain of membrane bound APP which releases a peptide termed secreted amyloid precursor protein β (sappβ). This is followed by an intra-membrane cleavage by γ-secretase resulting in the release of Aβ and the amyloid intracellular domain (AICD). γ-secretase can further process the Aβ peptide to create Aβ peptides of varying length. The alternative non-amyloidogenic pathway involves α-secretase mediated cleavage in place of the β-secretase event, this cleaves APP within the Aβ sequence and hence precludes its formation. This pathway results in the generation of a secreted fragment known as secreted amyloid precursor protein α (sappα), which has been shown to possess neuroprotective, neurogenic and neurotrophic properties 17 23, in place of sappβ, and a small non-toxic peptide, p3, in place of Aβ. Following amyloidogenic processing, Aβ is secreted into the extracellular space, at pm concentrations, and has been implicated as being necessary for long term potentiation (LTP) 24. In the support of this is a study that found that Aβ is released during LTP induction 25. The predominant form of the peptide in the normal healthy brain is Aβ However, Aβ1-42 is the main component of the amyloid plaques seen in AD brains, this is due to Aβ1-42 having an increased susceptibility to aggregation and to accumulate in the extracellular space 27. Accumulation of Aβ and its oligomers in the extracellular space has a multitude of effects including inducing apoptosis 28 and excitotoxicity 29,30, inhibiting long term potentiation 31 33, and the peptide can also interfere with memory and cognition 29,34. Aβ oligomers also have the ability to interfere with endogenous signalling pathways, in particular, the phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) signalling pathway 32. This path is of interest as one of the downstream targets is glycogen synthase kinase (GSK-3β). GSK-3β is thought to be the main kinase responsible for the phosphorylation of Tau, which is thought to cause microtubule instability. The inhibitory 7

28 effects of Aβ on this cascade result in the hyperphosphorylation of Tau 35. This lends evidence to the Amyloid Cascade Hypothesis 36 as it places Aβ upstream of Tau hyperphosphorylation and aggregation. The fact that GSK-3β has been found to be the enzyme responsible for this 37 could place GSK-3β at the intersection between Aβ and Tau pathologies. In AD, Tau is the major component of the neurofibrillary tangles (NFTs); however, the protein also has a physiological role in the healthy brain. Tau is associated with microtubules (a structural component of the cytoskeleton), upon association with microtubules it is thought to stabilise the tubulin that makes up the microtubules. The Tau protein is encoded by a single gene, MAPT, which can be alternatively spliced to produce one of six major isoforms in the human brain 38,39 (figure 1.3; page 9). The action of the Tau protein is modified by many post-translational modifications such as phosphorylation, glycosylation, ubiquitination, deamidation, oxidation and tyrosine nitration (reviewed extensively in 40 ). Due to the role of hyperphosphorylated Tau in AD, phosphorylation tends to be the most studied modification Tau (MAPT) The single gene encoding Tau is located on chromosome 17q21.31 and spans 16 exons. Exons 2, 3 and 10 are alternatively spliced to give rise to the six different isoforms in the human brain 38. This differs from the peripheral nervous system which has additional Tau variants. The main difference in the brain Tau isoforms is the presence of three-four repetitive domains in the C-terminal end (see figure 1.3 for a schematic of Tau isoform structure), these are encoded by exons Additionally, there is the presence or absence of a number of 29 amino acid insertions (designated 0N, 1N, or 2N) at the amino terminal 8

29 end (these are encoded by exons 2 and 3) Expression of the different Tau isoforms is developmentally regulated and tissue specific 44. Figure 1.3. Tau structure schematic. Tau refers to a group of closely related proteins found primarily in the central nervous system (CNS). These proteins were initially identified when they co-purified with the microtubule subunit forming protein tubulin 45 and were hence classified as microtubuleassociated proteins (MAPs) that regulate microtubule stability 46. Human Tau proteins are encoded by the MAPT gene (chromosome 17) containing 16 exons that, with the exception of exons 4a, 6, and 8, are all transcribed to generate the primary Tau mrna 9

30 transcript. Exons -1 and 14 are not part of the final mrna product, while exons 2, 3, and 10 are subject to alternative splicing 47. The primary structure of human CNS tau protein isoforms, which range from 352 to 441 amino acids in length, is thus determined by the presence or absence of mrna elements encoded by these exons. All Tau proteins contain at least three amino acid repeat regions toward the C-terminus that are considered to be the primary sites of interactions with microtubules. Together with a central proline-rich region and a C-terminal flanking region, these repeat sequences form the microtubulebinding domain of tau. In some isoforms, this domain is extended by the inclusion of exon 10, which dictates the presence of a fourth microtubule-binding repeat region. In contrast, a stretch of ~120 amino acids constitutes a projection domain at the N- terminal region of Tau. This domain is modifiable by the inclusion of an amino acid sequence encoded by exon 2 or by exons 2 and 3. Thus, tau protein isoforms are generally denoted by their combination of N-terminal inserts and C-terminal repeats, of which there are six possibilities: 0N3R, 0N4R, 1N3R, 1N4R, 2N3R and 2N4R (full-length) tau. Figure and legend modified with permission from 48. Tau is a highly soluble protein that contains minimal secondary structure and hence is natively unfolded 49. Binding to microtubules however, can produce conformational changes in Tau resulting in ordered structure 50,51. Tau can be broadly divided into four domains based on residue composition including: The N-terminal projection domain, the proline rich domain, the microtubule binding region and the C-terminal domain. Several studies have indicated that when Tau is bound to microtubules, the N-terminal domain is facing away from the microtubule and can act as a linker region The proline-rich domain has been shown to have an important involvement in mediating cell signalling cascades such as the Src-kinases including Fyn 55 57, phosphatidylinositol and 10

31 phosphatidylinositol bisphosphate 58,59, and peptidyl-prolyl cis/trans isomerase Pin1 60. The repeat regions within the microtubule binding region both act as a regulator of microtubule polymerisation 61 64, but also act as a docking point for a multitude of proteins such as actin 65, histone deacetylase 6 66,67, ApoE 68, presenilin 1 69, and tau phosphatases 70. The role Tau plays in these interactions has been postulated to be a scaffold for signalling proteins mediating their subcellular distribution 71. Additionally, Tau can interact with other proteins implicated in protein misfolding diseases such as TDP-43 and α-synuclein. In the fully developed brain, Tau is mainly localised to axons, but can also be found in the somatodendritic compartment 72 or associated with the nucleolus 73. Phosphorylation of Tau can trigger missorting of Tau into the dendritic compartment through a mechanism involving altered microtubule binding The longest isoform of Tau contains 45 serine, 35 threonine and 5 tyrosine phosphate acceptor sites and most of these are within the microtubule binding repeats. Phosphorylation of Tau is known to negatively regulate its microtubule binding ability 46,78 81 except for position Thr50 which, in fact, positively regulates microtubule binding activity 82. In addition to regulating microtubule binding dynamics, phosphorylation at various residues also regulates subcellular localisation and the nature of the axonally transported cargo 83,84 as well as cargo destined for dendritic spines 84,85. Furthermore, phosphorylation regulates Tau s interaction with other proteins such as Src kinases 56,57,86,87 and Pin1 60. Kinase and phosphatase activity is balanced to regulate the phosphorylation state of Tau. A vast number of kinases have been implicated in Tau phosphorylation (table 1) in vitro, and there is evidence for the involvement of GSK3β and cyclin dependent kinase 5 (CDK5) in the normal brain Various phosphatases have shown in vitro activity in 11

32 dephosphorylating Tau ; however, protein phosphatase 2A (PP2A) has been shown to be the strongest in vivo, accounting for around 70% of tau phosphatase activity PP2A has also been implicated in the pathogenesis of AD and is reported to have reduced expression and activity 102,103. Additionally, pharmacological inhibition of PP2A in cell culture and rodents produces Tau pathology 101, PP2A is itself under control of a vast regulatory network of post-translational modifications and proteins (reviewed in 108,109 ). For example, methylation of Leu309 of the C subunit of PP2A enhances holoenzyme assembly (specifically AC core enzyme) and is essential for the incorporation of the PR55 isoform of the B subunit (this is relevant for Tau as the predominantly brain expressed PR55 B subunit is required for Tau dephosphorylation 110,111 ). Furthermore, methylated PP2A associates with Tau while demethylated PP2A is improperly localised and not associated with Tau 112. Phosphorylation at Tyr307 (adjacent to the methylation site) inhibits methylation and consequently holoenzyme formation 109. There are also endogenous inhibitors of PP2A such as inhibitor 1 of PP2A (I1PP2A), inhibitor 2 of PP2A (SET), and cancerous inhibitor of PP2A (CIP2A) which play roles in tumorigenesis (reviewed in 109 ). Table 1. Kinases involved in Tau phosphorylation. Kinase Reference Glycogen synthase kinase 3β 88,113 Cyclin-dependent kinase 2 89 Cyclin-dependent kinase 5 89,90 ERK/MAPK 46, Cyclic-AMP dependent protein kinase 119,120 Ca 2+ /Calmodulin dependent kinase 121 Microtubule-affinity regulating kinase 122,123 Src family kinases including Fyn 56,57,86 12

33 As described above, Tau can bind microtubules and regulate their stability. Tau s role in microtubule dynamics and stabilisation is thought to be through a stiffening effect. Through this role, Tau is involved in a wide range of important neuronal processes such as morphogenesis, differentiation, neurite polarity, axonal outgrowth, axonal elongation, neuronal plasticity 52, and axonal transport specifically by regulating kinesin and dynein-dynactin motors 134. Additionally, through interactions with tubulin, Tau induces nucleation, elongation and bundling of axonal microtubules 135,136. Under physiological conditions, greater than 80% of Tau is bound to microtubules 137. Despite this, cargo transport is unimpeded due to kiss-and-hopp binding dynamics where Tau is only bound for ~40 ms at a time 138. In addition to the canonical structural stabilisation role, Tau has also been implicated in non-microtubule associated roles. At the synapse, Tau has been shown to modulate signalling through muscarinic acetylcholine 139 and N-methyl-D-aspartate (NMDA) receptors 140 and at the post-synapse, Tau may act as a scaffold for multiple proteins involved with synaptic plasticity such as Fyn 140,141, postsynaptic density 95 (PSD-95) 142, GSK-3β 76, and via alterations in glutamatergic signalling through the NMDA and AMPA receptors 48. Tau could modulate LTP through its interaction with the NMDA receptor but also can modulate long-term depression (LTD), and perturbations in Tau s physiological state could, therefore, alter one or both processes, thereby contributing to the behavioural consequences of Tau hyperphosphorylation. Tau plays a major role in the neurodegeneration seen in AD. This is evidenced by the discovery that Tau deletion prevents the AD phenotype in a preclinical mouse model of the disease 140 and effectively places Tau as the downstream mediator of amyloid pathology. There is a growing body of evidence showing that Tau pathology in both 13

34 humans and mouse models correlates well with synaptic loss and behavioural abnormalities However, the precise role Tau plays in the disease pathogenesis remains unknown and the field is now focused on addressing this and assessing the potential for Tau directed drugs to treat the disease Neuroinflammation Neuroinflammation is a complex process that is induced in response to cellular insults such as stress, infection or neuronal injury. Neuroinflammation is implicated in many neurodegenerative diseases including AD. It involves the recruitment and proliferation of inflammatory cells such as microglia and astrocytes. The activation of these cells induces the release of chemokines, cytokines, complement and reactive oxygen species (ROS) from the cells. Upon the release of certain cytokines and chemokines, monocytes and lymphocytes are recruited and pass through the blood-brain barrier 148. Microglia have been shown to play a role in Aβ pathology. It has been shown that Aβ can attract and activate microglia, and microglia cultured from both AD and healthy brains, show significant chemotaxis towards Aβ deposits 149. Microglia also express a form of scavenger receptors that are able to bind Aβ and result in increased ROS production which may exacerbate pathology 150. Aβ can also induce expression of nitric oxide synthase (producing the nitric oxide free radical) which in turn destroys selected neuronal populations. This suggests that oxidative stress from this pathway may be responsible for at least some AD pathology 151. Microglia exposed to Aβ produce a wide variety of proinflammatory molecules, including: interleukins 1β, 6 and 8, tumour necrosis factor-α, macrophage inflammatory protein 1-α, monocyte chemo-attractant protein-1 as well as increased expression of major histocompatibility complex II molecules 149. Aβ can also activate microglia through the binding and activation of the receptors for advanced 14

35 glycosylation end-products (RAGE) and macrophage colony stimulating factor (M-CSF). Upon activation these receptors can activate intracellular signalling pathways leading to microglia activation 152. The chemotaxis of microglia towards Aβ plaques may in fact be the cells attempting phagocytosis-mediated plaque removal. Indeed, Aβ has been shown to induce a phagocytic response in microglia in a dose- and time-dependent manner 153. This has also been seen in vivo where microglia have been observed attempting to clear Aβ from the CNS following phagocytosis, and some of these have subsequently been found migrating to vessels and the ventricles, resulting in Aβ deposition in these areas 154. This suggests that the deposition of Aβ plaques in the vessels may in fact be due to deposition from microglial cells rather than initial aggregation events occurring there. It has also been shown that the phagocytic internalisation of Aβ is enhanced by oestrogen. Pre-treatment of microglia with oestrogen caused an increase in phagocytosis of Aβ and conversely treatment with an oestrogen receptor antagonist had the opposite effect 155. Microglia themselves have been observed to be a source of Aβ secretion in the presence of Aβ and pro-inflammatory stimuli 156. Conversely, microglia have been shown to secrete a protease, insulin degrading enzyme (IDE), which degrades many small peptides including Aβ and that microglia can control local levels of Aβ via the expression of this protease 157. Astrocytes have been implicated in AD pathology in a fashion similar to microglia. Astrocytes release a battery of pro-inflammatory molecules, many of them over-lapping with what microglia release 158,159. They have also been shown to be able to degrade Aβ 160. It has been suggested that while astrocytes are able to engulf Aβ, Aβ laden astrocytes are prone to burst and seed new Aβ plaques 161. Interestingly though, a comparison was 15

36 drawn between microglia and astrocytes and it was found that microglia drove plaque formation whereas astrocytes were responsible for plaque degradation Metals There is a wealth of data showing that metal homeostasis is disrupted in AD This is evidenced by both changes in cellular levels and location of metals such as copper, iron, and zinc The proteins involved in the pathogenesis of AD (APP, Aβ, and Tau) all interact with these metals and it is thought that aberrations in the levels of the proteins consequently results in aberrations in the distribution and levels of the metal ions they bind 171. It has been shown that APP has the ability to regulate cellular copper levels 172,173 and APP expression is influenced by both copper 172 and iron 174,175. Furthermore, decreases in intracellular copper promotes the production of Aβ through promotion of the amyloidogenic processing of APP 176,177. Paradoxically, high levels of copper seem to be associated with AD 178,179. Experimentally, rabbits fed a diet high in both copper and cholesterol show increased Aβ plaque formation and oxidative stress 180, and mouse models of AD exposed to copper show increased Aβ production and neuroinflammation 181. This study found that copper supressed the levels of the low-density lipoproteinrelated protein 1 (LRP1) in brain vasculature. LRP1 is typically associated with Aβ clearance and as such this finding may link dietary copper levels with AD pathology. Interestingly, this effect was only found with copper and not aluminium, zinc or iron 181. Assessment of changes in metal levels have resulted in consensus that while the metal content of plaques is increased in both AD patients and animal models of AD , the cellular levels of copper are actually decreased 168,169,186. Iron levels are also increased in certain regions of the AD brain 170, where they may act as a possible source of oxidative stress. In fact, changes in iron within the brains of 16

37 AD mouse models precede amyloid deposition 189 making in vivo assessment of iron a possible clinical biomarker 190. The increases in iron are particularly interesting given that iron can increase the levels of phosphorylated Tau Genomic analysis has also revealed that the iron binding protein transferrin and transferrin receptor alleles are risk factors for AD , further supporting a role for iron in AD pathogenesis. Taken together, these data provide a strong link between metal levels and AD pathology and how alterations in one could influence the other. The plaques which define AD pathology have been shown to contain high levels of metals and have earnt the term metal sinks. Analysis has shown levels as high as 1055 µm zinc, 940 µm iron, and 390 µm copper 199. Furthermore, iron, copper and zinc are enriched within the dystrophic neurites surrounding plaques 200 and with Tau NFT bearing neurons 183,187,201. The combination of enriched metal deposition within AD pathological hallmarks and evidence of decreased cellular metal content shows that the redistribution of metals in the AD-affected brain may be a core aspect of AD pathology and progression. Metals play a key role in intracellular signalling cascades. For example, a pool of intracellular copper is loaded into vesicles, by the copper transporter, ATP7a, and released post-synaptically via a calcium mediated event in response to NMDA receptor stimulation 202. This release protects neurons from excitotoxicity via an antagonistic action on NMDA glutamate receptors 203. Indeed, neurons isolated from animals with non-functional ATP7a copper transporters are exquisitely sensitive to excitotoxicity 202. Additionally, copper plays a role in regulation of the γ-amino butyric acid-a (GABAA) receptors which in turn modulates neuronal activity The inhibitory effects of copper of neuronal activity through NMDA and GABA receptors means that a copper 17

38 deficiency, or mislocalisation, would result in lowered control of neuronal activity and possibly lead to excitotoxicity. Copper has also been found to positively modulate the tropomyosin-receptor kinase B (TrkB) and brain derived neurotrophic factor (BDNF) signalling cascade through a copper mediated increase in phosphorylation of the TrkB receptor 207. Copper further increases TrkB/BDNF signalling by increasing the release of pro-bdnf and its processing to mature, active BDNF 207. TrkB/BDNF signalling is involved in a variety of neuronal pathways and is important for myelination Interestingly, a recent review has provided a compelling argument linking TrkB/BDNF signalling to AD pathology 213 with observations that tangle bearing neurons contain significantly less full-length BDNF, the levels of the TrkA, B and C receptors are down-regulated in the brains of AD patients, combined with observations that BDNF signalling can stimulate dephosphorylation of Tau and inhibition of BDNF signalling promotes Tau phosphorylation. These data together show the large role metals play in the development of AD pathogenesis. Not reviewed here, but extensively elsewhere 171 is the capability for metals to interact with the Aβ peptide and increase oxidative stress. Furthermore, given the key roles these metals play in neuronal signalling pathways, alterations in metal homeostasis, such as those seen in AD, has the potential to disturb multiple pathways key for neuronal health and synaptic function. This makes targeting the dysregulation of metals an attractive therapeutic strategy with delivery agents or chelators (see section ) The quest for a therapy Despite over 120 years of research into AD, there is still no effective therapy to treat the disease. Therapeutic strategies either aim for symptomatic relief (temporary symptom 18

39 relief, but no alteration in disease progression) or disease-modification (slowing or halting of disease progression, but may not provide symptomatic relief), or ideally a combination of both symptom relief and halting of disease progression. Of the current clinically approved AD drugs, three are in the same class (acetylcholine esterase inhibitors). Additionally, patients can also be prescribed memantine (an NMDA receptor antagonist) which is supposedly more effective in the moderate phase of the disease. These treatments are symptomatic only, provide little relief, are only effective for a short duration and fraught with major side-effects. As such, millions are spent each year attempting to tackle these problems. This section will briefly mention some of the strategies in treating the disease, for a more in depth review see Aβ targeted immunotherapy The most widely researched strategy to treat the disease stems directly from the amyloid cascade hypothesis which posits the Aβ peptide as the causative agent in the pathogenesis. A variety of immunotherapeutic strategies have been employed all aiming to remove Aβ (or specific forms of it) from the brain. This strategy has, thus far, been met with spectacular failure with most drugs struggling with efficacy or side-effects or both. For a review of these clinical trials and a critique of the methodology see 215. There has been a recently trialled drug, Aducanumab (Biogen), which has shown the most promise by both lowering cortical amyloid (in a dose-dependent manner) and improving cognition in patients 216. Aducanumab is being assessed further in an ongoing phase III trial β-secretase inhibitors Another strategy stemming from the amyloid cascade hypothesis is targeting the production of the Aβ peptide through inhibition of the enzyme that liberates the Aβ fragment from its membrane anchored parent protein. However, this approach has been 19

40 met with difficulties arising from the fact that the β-secretase in question (BACE) has over 100 other endogenous targets 217. Hence, trials of these compounds have been fraught with side-effects. Although one phase III trial was recently halted due to no observed efficacy, other trials our ongoing, with close attention being paid to potential side-effects such as alterations in myelination (which has been seen in mouse models 218,219 ) γ-secretase inhibitors Like β-secretase inhibition, γ-secretase inhibition is an attempt to prevent liberation of the Aβ peptide from membrane bound parent peptide. Unfortunately, like BACE, this enzyme has more targets than APP and one of them, notch, is a canonical, highly conserved signalling pathway involved in neuronal differentiation and neurogenesis. Several high-profile compounds have been removed from development due to a variety of notch related side-effects. There is development however, in inhibitors specific for APP processing Anti-aggregation There is growing evidence that it is not the Aβ monomer that is toxic, in fact it probably has a physiological role 24, but rather it is higher order oligomers that are cytotoxic 31,34, As such, there has been an interest in developing compounds that do not affect the production of Aβ, but act to prevent its aggregation. While this strategy is extremely attractive, it has proven technically difficult and most have not satisfied preclinical criteria of efficacy for clinical advancement. However, one compound, scyllo-inositol, made it to clinical trials. In a phase II clinical trial of mild-to-moderate patients the drug failed to meet primary end-points; however, it did reduce cerebrospinal fluid (CSF) levels of Aβ Additionally, there is a strategy to design antibodies that target the soluble oligomers to have the same effect. 20

41 Metal homeostasis As discussed in the metals section above, metal dyshomeostasis is involved in neurodegeneration and as such, strategies to rescue this are attractive. Metal protein attenuating complexes are a class of drugs that have attempted to prevent the interaction of Aβ with metal ions that promote aggregation. Preclinical evidence with a compound called PBT2 suggested this strategy would be efficacious A phase II clinical trial found dose-dependent reductions in CSF Aβ levels and cognitive improvements in two behavioural measures 228. However, a follow-up phase II trial showed an improvement in behavioural tests over the course of the trial, but these results did not differ significantly from placebo 229. This study remains unpublished. The drug is still being investigated as a therapy for Huntington s disease 230. Increasing bioavailable copper through supplementation of copper containing complexes has also been explored as a strategy to rectify metal dyshomeostasis with promising preclinical results 231. There are multiple strategies to treat AD, some dependent and some independent of the amyloid cascade hypothesis. Additionally, there has been some focus on drugs specifically targeting Tau aggregation which are not mentioned here. They have recently finished a phase II clinical trial which failed to meet primary endpoints; however, the sponsors are continuing development after an incorrectly controlled subgroup analysis showed an effect 232. Furthermore, Tau phosphorylation itself has been targeted in the clinic 233 through lithium treatment which has shown to be a GSK3β inhibitor 92,93. These studies have had limited success with all trials investigating GSK3β inhibition failing. 1.3 Frontotemporal dementia Frontotemporal dementia (FTD) is the second most common form of dementia in people under the age of 65 and accounts for roughly 5-15% of all dementia worldwide 234,

42 FTD has a clinical phenotype that differs somewhat from AD-related dementia in that there is deterioration in both behaviour and personality while memory is less affected 236. FTD can be broken down into the behavioural variant and primary aphasia variants (semantic dementia and progressive non-fluent aphasia) and often presents with motor symptoms and/or motor neuron disease 237,238. Given this variable clinical phenotype, it is not surprising to learn that the molecular and genetic basis for pathogenesis is equally as variable History of FTD FTD presents with a strong genetic component 239. The first gene to be causally linked to FTD was in 1998 where mutations in the microtubule-associated protein tau (MAPT) gene encoding the protein Tau were linked to familial cases Mutations in MAPT now account for up to 20% of FTD cases and over 40 mutations have been identified 243. MAPT mutations exhibit complete penetrance and account for nearly all Tau positive FTD 244. Over 80% of inherited FTD can be accounted for by mutations in C9OF72, Progranulin (GRN) and MAPT; however, other loci associated with Valosin-containing protein (VCP) and chromatin-modifying protein 2B (CHMP2B) have been identified (reviewed in 244 ). The discussion on the biology of FTD presented in this chapter will focus on Tau associated FTD; for a wide-ranging review on the pathogenesis involved with other forms of FTD see 234,237,239,243, Biology of Tau associated FTD The normal structure and biological function of Tau is discussed in section , here the tau mutations associated with Tau positive FTD are discussed. 22

43 There are currently over 50 mutations identified in the MAPT gene 245. Differing mutations produce a remarkably varied phenotype, even between family members carrying the same mutation Tau mutations have also been found in other neurological diseases such as Pick s disease, corticobasal degeneration and progressive supranuclear palsy A large number of the disease associated mutations are found in exons 9-12, these are the regions coding for repeat sequences (and adjacent introns) 255. Many of the mutations decrease the affinity of Tau for microtubules, hence preventing Tau from promoting microtubule formation/stabilisation (these are G272V, P301L, P301S, V337M, G389R and R406W) 43,249,254, Some (V337M, G772V, P301L) facilitate Tau phosphorylation either through making Tau a better substrate for Tau kinases or by reducing phosphatase action 259,260. For an extensive review of the mutations associated with tauopathies see 245, Metals and FTD As we have seen in section , transition metals have been reported to play a large role in the pathogenesis of AD, this is also true for tauopathies (such as Tau-positive FTD). Indeed, it has been noted that metals such as iron, copper and zinc can bind Tau directly and induce aggregation 191, There are two cysteine residues present in the microtubule binding domain of Tau which have the potential to be involved in metal binding, indeed mutation of these residues to alanine drastically reduces metal induced fibrillization 263. Additionally, there is evidence that if the Cys residues are mutated in a fly model, Tau toxicity is markedly reduced 264. Zinc can also influence the phosphorylation state of Tau through multiple pathways and can activate kinases such as P70S6 kinase and Raf/mitogen-activated protein kinase increasing phosphorylation 265,266. Zinc can also inhibit the action of PP2A 267,268 further increasing phosphorylation. 23

44 Tau toxicity has been reported to be influenced by copper through modulation of the Tau kinase, CDK However, many of these studies have looked at either in vitro studies or bulk levels of metal. As discussed in section , this is probably a simplistic view and unlikely to accurately represent the role of these metals in a physiological setting due to possible changes in subcellular location and changes in metalloprotein binding (for a discussion of copper binding proteins see section 4.2). 1.4 Bis(thiosemicarbazone) complexes Various aspects such as the chemistry, structure and pharmacology of these compounds have been extensively reviewed in several recent reviews Here, I will give a brief overview of the history and development of these compounds for the use in neurodegenerative diseases and their relevance to the work outlined in this thesis. This class of compounds has been of pharmacological interest due to their varying structures giving rise to different responses and manipulation of structure altering efficacy 273, but also to their wide range of medical applications such as antibacterial, anticonvulsant, tumour imaging and increasing bioavailable copper 270,271. This combined with the fact that these drugs are small, stable, orally bioavailable and, importantly, can cross the blood brain barrier 274 makes them very attractive candidates for treating neurological diseases. Bis(thiosemicarbazone) complexes (BTSCs) have a common central structure around a coordinated metal ion and varying side groups. A 2007 study showed differences in biological action determined by the nature of the side groups 273, of particular interest were two variants known as Cu II (gtsm) and Cu II (atsm). These two variants share most of their structure (including the coordinated copper ion), but have differing side chains (two 24

45 hydrogen atoms and two methyl groups on the diimine backbone respectively, see figure 1.4). The presence of electron-donating methyl groups on Cu II (atsm) increases the Cu II /Cu I reduction potential to 0.60 V compared to the Cu II /Cu I reduction of Cu II (gtsm) at V (compared to Ag/AgCl) 272,275. The effect of this difference is that Cu II (atsm) has decreased propensity to release the coordinated copper ion and a resulting increase in stability. This means that when these two compounds enter a normoxic cell, the Figure 1.4. Structures of two BTSC group family members. A) Cu II (gtsm) and B) Cu II (atsm) intracellular reductants such as glutathione and ascorbate are sufficient to reduce the copper present in Cu II (gtsm), but not Cu II (atsm) and as such Cu II (gtsm) releases copper more readily than Cu II (atsm). However, in challenged cells where reductive capacity is increased (such as hypoxia) the reductive potential of the cell is sufficient to reduce the copper coordinated in Cu II (atsm) and mediate its release 275. It is the hypoxic specific release of copper from Cu II (atsm) that has resulted in its development as a tumoral imaging agent 271. The capability for these compounds to release bioavailable Cu intracellularly combined with the copper dyshomeostasis present in neurodegenerative diseases generated interest 25

46 in testing the efficacy of these compounds in cellular 273 and animal models 231 of AD. In 2009, a study was published that assessed the efficacy of an oral, daily dosing regimen of Cu II (gtsm) in the AD mouse model, APP/PS This study showed that, along with increasing the copper content of cells, the treatment improved the behavioural deficits associated with the disease model. Treatment also improved disease associated impairments in the levels of phosphorylated GSK-3β, phosphorylated forms of Tau and reductions what appeared to be oligomeric Aβ. The authors hypothesised that the intracellular release of copper was restoring copper sensitive signalling pathways. Interestingly, Cu II (atsm) was ineffective at altering the disease phenotype, hypothesised to be due to its reduced capacity to release copper. This study illustrated that copper delivery by Cu II (gtsm) could be efficacious in improving the disease associated deficits in AD. 1.5 Rationale of current study To confirm the reproducibility of the above study, this thesis sought to both confirm the findings seen in the Crouch, Hung et al. study which showed that treatment of the APP/PS1 model of AD with Cu II (gtsm) improved cognition and reduced pathological Tau through what appeared to be an increase in bioavailable cellular copper 231. Furthermore, we sought to biochemically expand on the mechanism of action of the compound (Cu II (gtsm)) in treating the APP/PS1 mouse model of AD and to expand the behavioural assessment. To assess whether the drug s efficacy in improving pathological levels of Tau was specific for AD, the trial was to be followed up with another trial of the compound in a Tau-centric mouse model of FTD known as the rtg4510 model and to use a proteomic approach to investigate the mechanism of action in both tauopathies. 26

47 Chapter 2 Cu II (gtsm) treatment of the APP/PS1 model of Alzheimer s disease 2.1 Introduction Previous work from our lab has suggested that treating the APP/PS1 mouse model of AD with a daily dose of Cu II (gtsm) (see section 1.4 for a discussion on this group of compounds) is efficacious in relieving the cognitive deficits seen in the model and improves biochemical markers of the disease 231. This study found that when a cell culture model (SHSY-5Y neuroblastoma cells) is treated with the compound there is a substantial rise in intracellular copper and that this was much greater than in cells treated with the same dose of a related compound, Cu II (atsm). The animal trial in the study used a daily dose of Cu II (gtsm) via oral gavage at a dose of 10 mg/kg and behaviour was assessed in the Y-maze. Follow-up biochemistry was undertaken to assess Aβ and tau pathology. 5-6-month old APP/PS1 mice treated with the compound were found to have significantly better performance in the Y-maze compared with vehicle treated littermates and were statistically indistinguishable from wildtype mice. Additionally, Cu II (atsm) failed to achieve this improvement, further suggesting that it is the copper released from the drug that is responsible for efficacy. Biochemical analyses of brain homogenate revealed a significant decrease in the levels of phosphorylated Tau (epitope Ser404, which has shown to be enriched in paired helical filaments (the main component of pathologically folded Tau) 276 and has shown to be one of the earliest pathological events in AD pathogenesis 277 ) in the context of unchanged total levels of Tau. Additionally, the study found a decrease in total Aβ with what appeared to be a decrease in an oligomer resembling a trimer and that this correlated with the behavioural deficit of the animals in the Y-maze. From a mechanistic point of view, the authors noted an increase in the 27

48 phosphorylation of the Tau kinase, GSK3β (see section ), at the Ser9 epitope (which is inhibitory). The authors hypothesised that the intracellular release of copper activated signalling pathways which resulted in an inhibitory effect on GSK3β via an increase in Ser9 phosphorylation. 2.2 Preliminary study of Cu II (gtsm) treatment of the APP/PS1 model of AD Effects of Cu II (gtsm) on APP/PS1 mouse behaviour Based on the Crouch-Hung et al. study described above 231, the current study sought to assess the reproducibility of the effects and expand on the mechanism of action of the drug. The current study used the same mouse model as the Crouch, Hung et al. study. The APP/PS1 mouse (sourced from JAX Laboratories, USA) expresses a mutant form of APP containing the K670N and M671L (Swedish) mutations in combination with expressing the ΔE9 (exon 9 deletion) presenilin1 mutation. These animals have an increasing Aβ load in the cortex and hippocampus with progressing age. Amyloid plaques are observed as early as 4-months in these animals and plaque count increases with age Effects of Cu II (gtsm) on cognition (Morris water maze) To assess the efficacy of Cu II (gtsm) in reversing pathology, mice (APP/PS1 transgenic and wildtype littermates) were treated from 9-months. Both transgenic and wildtype mice were treated with either Cu II (gtsm) at a dose of 5 mg/kg or an equivalent volume of SSV (denoted as vehicle). Dosing was daily via oral gavage for 2-months, after which time the animals were culled as per section Prior to culling, animal behaviour was assessed in the Morris water maze (see section 6.9.4). Wildtype mice treated with Cu II (gtsm) did 28

49 not have altered performance in any aspect of the experiments when compared with vehicle treated wildtype mice and as such were excluded from analysis, this is consistent with previous data 231. Transgenic mice treated with vehicle performed poorly in the Morris water maze as measured during the recall (probe) phase of the trial. Figure 2.1, panel A shows that, during the probe trial, wildtype mice spent approximately 25 of the 60 second probe trial in the target quadrant while the transgenic mice treated with vehicle only spent around 7 seconds in the target quadrant (P = compared with wildtype mice, 1-way ANOVA LSD post hoc test). Treatment with Cu II (gtsm) significantly improved performance in the probe trial with treated animals spending a similar amount of time to wildtype mice in the target quadrant (P = compared with transgenic vehicle treated mice, 1-way ANOVA LSD post hoc test). These data suggest that Cu II (gtsm) treatment improves the cognitive deficits seen in the APP/PS1 model and this effect is specific for diseased animals as wildtype mice treated with Cu II (gtsm) show unaltered performance when compared with vehicle treated wildtype mice. When assessing the acquisition time (learning curve) throughout the training phase of the experiment (latency to escape by day) there did not appear to be apparent differences in the escape time between any of the groups (Figure 2.1, panel B). Recent research 279 has indicated that in fact latency is a poor indicator of spatial memory acquisition as animals can improve performance by adopting egocentric (hippocampal independent) search strategies rather than allocentric (hippocampal dependent) search strategies while having similar performance times. As such, the mouse movement (search) patterns should be interrogated to ascertain the search strategy as per 279 to indicate spatial memory acquisition. 29

50 Figure 2.1. Cu II (gtsm) improves cognition in the Morris water maze probe trial. A) Time spent in the target quadrant during the probe trial of the Morris water maze showed a significantly poorer performance in the transgenic vehicle treated group when compared with wildtype littermates. This effect was rescued with Cu II (gtsm) treatment. Statistical analysis via 1-way ANOVA, LSD post hoc, ** = P < 0.01, N=6-9. B) Acquisition curve displaying average escape latency by day for each group showed that there was no difference between the groups. Statistical analysis via 2-Way ANOVA found no significant interaction, but a significant effect (P < 0.001) for time. The results of the Morris water maze indicated that transgenic mice have poor spatial memory as evidenced by the low time spent in the target quadrant on the probe day. Treatment with Cu II (gtsm) improved the time spent in the target quadrant to be statistically indistinguishable from the performance of wildtype mice. There was no difference between the groups in the acquisition curves. The experiment was performed 30

51 before search a strategy analysis protocol was published and as such could not be performed. This is addressed in a subsequent study (see section ) Effects of Cu II (gtsm) on APP/PS1 biochemistry As stated in section 2.2.1, the APP/PS1 mouse model of AD relies on the overproduction of Aβ to produce AD-like pathology. Tau related pathology (hyperphosphorylation) has also been reported in these animals 280. Biochemical analysis of brain tissue began with assessment of changes to the levels and states of Aβ and Tau as these are the pathological features associated with AD Effects of Cu II (gtsm) on Amyloid-β To assess the levels of Aβ in the brain tissue of APP/PS1 mice, brain tissue was homogenised in homogenisation buffer (see section 6.2.1) and a sample of the whole brain homogenate was analysed via surface enhanced laser desorption ionisation time of flight - mass spectrometry (SELDI-TOF-MS) by Keyla Perez utilising an antibody targeting the N-terminal region of Aβ (WO2). The SELDI-TOF data showed that there was no change in any species of Aβ (figure 2.2). The Crouch-Hung et al. study found no changes in plaque load and the species identified as a trimer was not reproducible (personal communication, Kevin Barnham) therefore our data were consistent with no global alteration in amyloid accumulation. This suggests that the drugs effect is through a pathway downstream of Aβ levels, in alignment with the idea that AD is an Aβ mediated tauopathy. This finding was in contradiction to previous findings 231. However, the finding in this section was reproduced 31

52 in a follow up trial (see section ) utilising a double antibody mixture (W02 and 4G8) suggesting that Cu II (gtsm) reproducibly did not alter Aβ levels. Figure 2.2. Cu II (gtsm) does not alter Aβ species in APP/PS1 brain homogenate. A) Analysis of Aβ species via SELDI-TOF showed that no species of Aβ was changed by the drug treatment. Two-tailed unpaired Students T-Test found no differences in the groups (P > 0.05). B) Representative spectra. 32

53 Effects of Cu II (gtsm) on Tau To assess the effects of Cu II (gtsm) treatment on Tau biology, the phosphorylation of Tau was analysed via immunoblotting at multiple epitopes in addition to a sarkosyl-tau preparation (see section for methodology) being performed to assess levels of insoluble aggregated Tau. These animals do not develop NFT pathology; however, TBS-insoluble oligomeric Tau has not previously been examined in the APP/PS1 mouse model of AD. Tau hyperphosphorylation at epitopes Ser396 and Ser404 have been associated with AD Tau pathology 281. To assess Figure 2.3. Levels of total Tau are unchanged in APP/PS1 transgenic mice. phosphorylation at these epitopes, brain Levels of total Tau determined by homogenate samples were subjected to SDS- PAGE and immunoblotting (as per sections and respectively) and blotted using phosphorylation specific antibodies (see section 6.3). Total levels of Tau were not altered by treatment (figure 2.3). Figure 2.4, densitometry of immunoblots showed that the levels of total Tau were unchanged between genotype and drug treatment with representative blots below. Statistical analysis by 1-way ANOVA LSD post hoc, ns = not significant. N=4 in all groups. panel A indicates that at both Ser396 and Ser404 epitopes there was a 50% decrease in the levels of phosphorylated Tau (P =

54 and P = respectively, two-tailed Students t-test). These data are in line with what Crouch-Hung et al. found, with the addition of data showing a reduction in the Ser396 phosphorylation site suggesting that, in the context of unchanged total Tau levels, Cu II (gtsm) drug treatment was altering a component of the system regulating Tau Figure 2.4. Cu II (gtsm) reduces phosphorylated forms of Tau and decreases oligomeric Tau. A) Levels of phosphorylated Tau at residues Ser396 and Ser404 determined by densitometry of immunoblots showed that for both Ser396 and Ser404 epitopes, drug treatment reduced the levels of phosphorylation. * = P < 0.05 (Unpaired two-way Students T-test, N=5-7) with representative blots below corresponding to the epitope listed above. B) Densitometry analysis of sarkosyl extracted Tau from brain tissue showed that transgenic vehicle treated mice displayed an increase in the levels of oligomeric Tau relative to total levels of Tau and that Cu II (gtsm) treatment significantly reduced this. Densitometry values for sarkosyl-soluble and sarkosyl-insoluble tau are combined to create a ratio, this ratio was then normalised against the wildtype average to give a percentage of wildtype value. ** = P < 0.01, * = P < 0.05, 1-Way ANOVA LSD post hoc, N=

55 phosphorylation more generally (for a review of this system and the proteins involved see section ). Although insoluble Tau pathology has not previously been noted in the APP/PS1 mouse model of AD (previous studies have generally looked for NFT pathology immunohistochemically), early oligomeric Tau was assessed using a widely-used protocol utilising the detergent N- lauroylsarcosine as per 282. Incubation of TBS-insoluble fractions with this detergent results in aggregated Tau remaining in the pellet material while TBS-insoluble early oligomeric Tau was extracted into the soluble fraction. To quantify changes in this fraction, immunoblotting, using a total- Tau antibody (see section 6.3.2) densitometry values were utilised to create a ratio of sarkosyl-soluble Tau to sarkosylinsoluble Tau. To investigate whether or not Figure 2.5. Levels of p-gsk are unchanged in APP/PS1 transgenic mice. Levels of phospho-gsk (Ser9) determined by densitometry of immunoblots showed that the levels were unchanged between genotype and drug treatment with representative blots below. Statistical analysis by 1-way ANOVA LSD post hoc, ns = not significant. N=6 in all groups there were changes in this early oligomeric pool of Tau, we used a protocol based on 282 and detailed in section Figure 2.4, panel B shows that the APP/PS1 mouse AD model did in fact have an aggregated Tau 35

56 phenotype as illustrated by the ~175% increase in sarkosyl-tau (an increase in the levels of insoluble aggregated Tau) compared to wildtype (P = , 1-wy ANOVA LSD post hoc). Furthermore, drug treatment significantly reduced this back to levels comparable to wildtype (~110%, P = 0.047, 1-way ANOVA LSD post hoc). This, to our knowledge, is the first description of an aggregated Tau phenotype to be observed in the APP/PS1 mouse model of AD. 36

57 Effects of Cu II (gtsm) of protein phosphatase 2A (PP2A) Western blot analysis of brain tissue showed no alteration of the Ser9 phosphorylation epitope of GSK3β in these animals (figure 2.5). Given that Cu II (gtsm) treatment did not alter the levels of major Tau kinase the major Tau phosphatase, PP2A, was then examined. PP2A is responsible for around 70-80% of phosphate removal from Tau and has been implicated in AD pathology 97. To assess the effects of Cu II (gtsm) on PP2A biology, using a suite of antibodies outlined in appendix 2. PP2A is a multi-subunit 37

58 Figure 2.6. Cu II (gtsm) alters PP2A(A) subunit levels but not (B) or (C). Densitometry analysis of brain homogenate samples for A) PP2A(A), B) PP2A(B), C) PP2A(C) showed that Cu II (gtsm) treatment produced a significant increase in the levels of the PP2A(A) subunit while PP2A(B) and PP2A(C) levels remain unchanged. Transgenic vehicle treated mice showed no significant change when compared with wildtype mice (P>0.05). D) Representative immunoblots. * = P < 0.05, ns = not significant, 1-way ANOVA, LSD post hoc, N = 5-6 in all groups. holoenzyme and its activity is affected by methylation and phosphorylation. Antibodies targeted to total levels of all three subunits were used in the current study. 38

59 Figure 2.6, panels A, B and C show that there was not a statistically significant reduction in any of the subunits when comparing wildtype mice with vehicle treated transgenic mice; however, a large degree of variation in the wildtype was noted making interpretation difficult (for antibodies used see section 6.3.2). In the follow up study (section 2.3) a significant decrease in the A subunit was observed in transgenic mice when compared with wildtype mice. Treatment of transgenic mice with Cu II (gtsm) did however, produce a significant increase in the level of the A subunit of the PP2A Figure 2.7. Hippocampal mrna analysis of APP/PS1 mice reveals altered levels of expression of the 'A' and 'C' subunit of PP2A. Hippocampal mrna samples were subjected to RT-PCR to quantify the expression of PP2A subunits A (PPP2R1A), B (PP2R1B), and C (PPP2CA). Expression of mrna encoding the A and C subunit were decreased in transgenic mice compared to wildtype mice. Cu II (gtsm) produced an increase in the expression of the C subunit compared with vehicle treated transgenic mice. Statistical analysis by 1-way ANOVA LSD post hoc, * = P < N=4 in all groups. Data generated by Dr Lesley Cheng. 39

60 complex (P = 0.034, 1-way ANOVA LSD post hoc) while the B and C subunits remain largely unchanged. This may be due to either increased expression or decreased turnover Cu II (gtsm) does not affect gene expression of PP2A enzyme subunits To investigate the cause behind the increased levels of the PP2A(A) subunit, RNA was analysed from the brains of the treated mice to assess changes in gene expression of transcripts coding the PP2A subunits. RNA analysis was carried out as per section 6.7 by Dr Lesley Cheng at the Department of Biochemistry, University of Melbourne. Figure 2.7 shows the fold change in gene expression of transcripts encoding the A, B, and C subunits of PP2A. The analysis shows that transgenic mice have a marginally, but significantly lower expression of the A and C subunits (P < 0.05, 1-way ANOVA LSD post hoc). Cu II (gtsm) produced a significant increase in the C subunit (P = 0.043, 1-way ANOVA LSD post hoc), but not the A subunit (P = 0.30, 1-way ANOVA LSD post hoc). While these results were significant, their small magnitude is unlikely to result in significant changes in protein levels Summary of preliminary data Copper dyshomeostasis is known to be a feature of AD and the mislocalisation of copper could result in disruption of intracellular copper mediated signalling pathways. In light of this, modulation of copper (and indeed zinc) levels has been attempted through chelation and redistribution 224,226, and through copper delivery using the BTSC compounds Cu II (gtsm) and Cu II (atsm) 231. Both strategies have produced promising results in preclinical models of AD. Treatment of APP/PS1 mice with Cu II (gtsm) produced an improvement in the behavioural deficit seen in these mice and biochemically reduced classical AD biomarkers such as hyperphosphorylated Tau. Interestingly, in the Crouch, 40

61 Hung et al. study, Cu II (atsm) was unable to produce these effects suggesting that indeed the efficacy was due to the intracellular release of copper from Cu II (gtsm). This section sought to expand on the mechanistic understanding as to how Cu II (gtsm) treatment produced behavioural improvements in the APP/PS1 mouse model of AD. An 8-week treatment initiated post-symptomatically at a dose of 5 mg/kg rescued the cognitive deficits seen in the Morris water maze. This rescue was independent of Aβ levels and appeared to be through reducing pathological forms of Tau typically associated with the disease. The reductions in pathological Tau appeared concomitantly to an increase in the protein levels of a subunit of the major Tau phosphatase (PP2A) which I hypothesise results in higher levels of the complete holoenzyme complex. Given that this has been shown to be required for Tau dephosphorylation 87 this may be a mechanism by which treatment is reducing pathological Tau. 41

62 2.3 Follow up trial of Cu II (gtsm) treatment of the APP/PS1 mouse model of AD Introduction The pilot trial outlined in section 2.2 provided evidence that Cu II (gtsm) treatment can rescue the cognitive deficit observed in the Morris water maze of APP/PS1 mice. In addition to the behavioural improvements, treatment also reduced the levels of pathological phosphorylated Tau with evidence to suggest this was via an increase in the structural (A) subunit of the phosphatase PP2A. Due to tissue volume severely limiting the analysis, it was decided to carry out a follow-up trial with a larger number of animals in an attempt to further elucidate the mechanism by which Cu II (gtsm) treatment produced an increase in the levels of this subunit and to add further evidence to the reproducibility of both the behavioural and biochemical effects. The same strain of APP/PS1 mice were obtained from JAX Laboratories (USA) and the recommended age-matched controls from the same background strain were used as controls. Since the mice arrived singly housed, they continued to be housed singly with basic enrichment. As per section 2.2, mice were dosed with Cu II (gtsm) daily via oral gavage at 3.5 mg/kg (there was evidence that the newly synthesized batch of Cu II (gtsm) exhibited a small amount of toxicity at 5 mg/kg (weight loss)) to minimise discomfort to the animals, a lower dose was used for the trial). For the final 2-weeks treatment, the mice were dosed with isotopically enriched 65 Cu II (gtsm) for further analysis using LC-ICP-MS (section 6.6). Mouse behaviour was again assessed using the Morris water maze and the Y-maze in addition to assessing locomotor ability and anxiety in the locomotor cell (open field) and Rotarod. Following behavioural analysis, tissue extraction was again carried out as per section

63 2.3.2 Effects of Cu II (gtsm) on APP/PS1 behaviour Previous research has indicated that Cu II (gtsm) treatment of APP/PS1 mice can result in behavioural improvements in the Y-maze 231 and in the Morris water maze (section ). To this end, behaviour was assessed in both paradigms in the larger follow up trial Effects of Cu II (gtsm) on cognition (Morris water maze) To assess spatial memory formation, behaviour was assessed in the Morris water maze after 8-weeks of treatment. The Morris water maze was carried out as per section additionally, tracking software data and subsequent data analysis could perform search strategy analysis as per section Figure 2.8 shows the learning curves from the acquisition phase of the experiment, panel A shows that all three groups of animals had a similar learning curve during the acquisition phase and only on day six did wildtype performance significantly exceed transgenic animals (P < , 2-way ANOVA Tukey post hoc). However, search strategy analysis revealed that despite all animals performing similarly in panel A the strategies used to escape the maze were vastly different. Panel B, indicates that from day three a significantly higher proportion of wildtype mice adopted an allocentric search strategy to escape the maze and the magnitude of statistical significance of this difference grew with each following day. 43

64 Figure 2.8. Cu II (gtsm) treatment did not improve performance of transgenic APP/PS1 mice during the acquisition phase of the Morris water maze. Transgenic APP/PS1 mice and wildtype littermates were treated with Cu II (gtsm) for 8- weeks and behaviour was assessed in the Morris water maze. A) escape to latency (s) by day shows that all animals progressed in learning to escape the maze quicker with wildtype performance significantly exceeding transgenic by day 6. Statistics by 2-way ANOVA with Tukey post hoc, **** = P < B) using the algorithm outlined in section animals search strategy was dichotomised into either allocentric (hippocampal dependent) or non-allocentric strategies (non-hippocampal dependent). From day 3 a significantly higher percentage of wildtype mice adopted a allocentric search strategy while transgenic mice never exceeded 15-20% of animals using the strategy. Statistical analysis by 2-way ANOVA Tukey post hoc, * = P < 0.05, ** = P < 0.01, *** = P < N=7-13. All data is displayed as group mean +/- SEM. On day 7 of the protocol, the submerged maze was removed from the pool as per the protocol outlined in section during this time, tracking software analysed the amount of time spent in the quadrant of the former location of the platform. Figure 2.9 shows that when comparing the groups with one another, there is no significant difference between 44

65 the groups performance (P > 0.05, 1-way ANOVA, LSD post hoc). If one compares the groups performance against chance (figure 2.9, right panel), the wildtype mice displayed a significant (P = 0.017, 1-sample T-test vs chance) preference for the target quadrant indicating successful formation of a spatial memory while no significant difference was observed for both groups of transgenic mice. Figure 2.9. Cu II (gtsm) did not improve performance of APP/PS1 transgenic mice in the Morris water maze probe trial. Transgenic APP/PS1 mice and wildtype littermates were treated with Cu II (gtsm) for 8- weeks and behaviour was assessed in the Morris water maze. The first 30 seconds of the probe was analysed as platform seeking behaviour is extinguished early on once the previous platform location has been investigated. The panel on the left displays the data as a box and whisker plot, while the panel of the right displays the group mean +/- the 45

66 95% confidence interval. When comparing groups directly (left panel, 1-way ANOVA LSD post hoc, ns = not significant) there were no differences between the groups, however when performance is analysed with a 1-sample t-test (right panel, * = P < 0.05) against a hypothetical value of 7.5 s (chance) the wildtype mice have a higher preference for the target quadrant over and above chance. N= Effects of Cu II (gtsm) on cognition (Y-maze) Previous data from our group has found that Cu II (gtsm) treatment of transgenic APP/PS1 mice improved their performance in the Y-maze as measured by percentage entries into the novel arm 231. In this larger trial, we sought to again assess cognition in both the Y- maze and Morris water maze paradigms. Figure 2.10 shows the percentage entries into the novel arm for each group. The left panel indicates that transgenic mice perform significantly poorer than both wildtype mice (P = , 1-way ANOVA LSD post hoc) and Cu II (gtsm) treated transgenic mice (P = , 1-way ANOVA LSD post hoc). When these groups performance was compared to chance (right panel) both wildtype mice and Cu II (gtsm) treated mice performed significantly better than chance (P = and respectively, one sample t-test against a theoretical value of 33%) indicating a clear preference for the novel arm compared with both chance and intergroup analysis. The 95% confidence interval for the transgenic vehicle treated group overlapped with chance indicating no preference for the novel arm above chance (P = 0.13, one sample T- test against a theoretical value of 33%). 46

67 Figure Cu II (gtsm) treatment rescues cognitive deficit on the Y-maze. Transgenic APP/PS1 mice and wildtype littermates were treated with Cu II (gtsm) for 8- weeks and behaviour was assessed in the Y-maze Animals were allowed 5 minutes to explore the completely open maze 1 hr after a 10-minute training trial with the novel arm closed. The left panel of the figure depicts the % entries into the novel arm for each group as a box and whisker plot. Vehicle treated transgenic mice performed significantly worse than wildtype mice and Cu II (gtsm) treated transgenic mice. ** = P < 0.001, *** = P < , 1-way ANOVA LSD post hoc. The right panel shows the groups means +/- the 95% confidence interval. There is a dotted line at 33% to indicate performance no difference from chance and there is a dotted line at 50% to designate the performance of a cognitively healthy mouse. Both wildtype mice and Cu II (gtsm) treated transgenic mice performed significantly better than chance while vehicle treated transgenic mice 47

68 performed no different from chance. ** = P < 0.001, *** = P < , One sample t- test against chance (33%). N= Effects of Cu II (gtsm) on motor function and anxiety like behaviour (locomotor cell) In addition to the Morris water maze and the Y-maze assessment of behaviour, the animals were also assessed in the locomotor cell open field paradigm (as per section 6.9.5). Mice were allowed to explore the cell freely for 60 min while having their movement analysed. Figure Cu II (gtsm) did not impact performance in the locomotor cell (open field). Transgenic APP/PS1 mice and wildtype littermates were treated with Cu II (gtsm) for 8- weeks and motor performance was assessed in by a 60-min period in the locomotor cell (open field). Panel A) displays the total distance moved (mm) for each group as a box and whisker plot. Analysis with a 1-way ANOVA LSD post hoc revealed a significant difference between the drug treated transgenic mice and the vehicle treated transgenic mice; however, this was being driven by two outliers (signified as points on the plot) 48

69 which, when removed, eliminated the statistical significance. Additionally, panel B) displays the total resting tine of each group and a 1-way ANOVA LSD post hoc found no significant difference between the groups. To ascertain a measure of anxiety-like behaviour, C) shows the percentage of time the mice spent exploring the centre of the cell with no difference being found between groups (1-way ANOVA LSD post hoc). * = P < 0.05, 1-way ANOVA LSD post hoc. N=7-13. A measure of activity (and indeed hyperactivity) is the total distance covered by the mice during the 60-min trial. Figure 2.11, panel A shows that on initial analysis there was a significant difference between the vehicle treated transgenic mice and the Cu II (gtsm) treated transgenic mice (P = 0.043, 1-way ANOVA LSD post hoc). However, from the data it is noted that this was being driven by two outliers. Exclusion of these outliers from the analysis removes the statistical significance of the difference and as such the difference is interpreted as not being significant. An additional measure of hyperactivity is the time the mice spent at rest during the 60-min trial. This is displayed in Figure 2.11, panel B which shows no significant difference between the groups analysed (P > 0.05, 1- way ANOVA, LSD post hoc). Figure 2.11, panel C displays the percentage of time the mice spent exploring the centre of the cell, which is a measure of anxiety-like behaviour. 1-way ANOVA analysis with an LSD post hoc revealed no significant differences between any of the groups on this measure either which, at this age, is consistent with pervious work finding differences on this test emerging at months of age Effects of Cu II (gtsm) on APP/PS1 biochemistry To assess the reproducibility of the effects seen in section 2.2.2, Aβ and Tau protein levels were investigated. Animals were culled at the completion of the experiment (10-weeks of administration at the age of 11-months) as per section The mouse model used in 49

70 this section was the same as the model used in section 2.2 (APP/PS1, JAX Laboratories, USA) which is an Aβ driven mouse model. So, the first peptide to assess was Aβ Effects Cu II (gtsm) on Amyloid-β As per section , the levels of Aβ were assessed using SELDI-TOF mass spectrometry. This time, to increase sensitivity, a combination of W02 and 4G2 (50:50 mixture) anti-aβ antibodies was used as the immunocapture antibodies. Figure 2.12 shows the relative peak intensity of two species of Aβ (1-40 and 1-42). From the figure, it can be seen there is no significant differences between the groups, and statistical analysis confirms this (P > 0.05, Students unpaired T-test). This is in line with the results seen in which also found no difference in Aβ levels. While only a few key species are shown here, this effect was seen across every species the SELDI-TOF mass spectrometer detected (data not shown) confirming that drug treatment produces no alteration in Aβ levels in the APP/PS1 mice Effects of Cu II (gtsm) on Tau Next, the effects of Cu II (gtsm) treatment on Tau physiology was assessed by immunoblotting (see section 6.3) using a variety of anti-tau antibodies (see appendix 2 for a list of the antibodies used). The purpose of this was to assess total levels of Tau and changes in the phosphorylation state of Tau in addition to utilising a sarkosyl-tau prep (section 6.3.4) to assess changes in the aggregation state of Tau. 50

71 Figure Cu II (gtsm) does not alter Aβ species in APP/PS1 brain homogenate. A) Analysis of Aβ species via SELDI-TOF showing that no species of Aβ was changed by the drug treatment. Two-way unpaired Students T-Test found no differences in the groups (P>0.05), N=12-6. B) Representative brain homogenate spectra. 51

72 Initially, total Tau levels were assessed so that changes in phosphorylation state could be interpreted in the context of total Tau. Figure 2.13 shows that there was no alteration in the total levels of Tau between the wildtype and transgenic mice. Additionally, treatment with Cu II (gtsm) did not change the levels of Tau either. The levels of phosphorylated Tau at several epitopes were also assessed via immunoblotting. Ser396 and Ser404 are classical Alzheimer epitopes and Ser202 was included because of the finding in section that PP2A levels were increased. Ser202 is one of the sites exclusively acted upon by PP2A 284. In contrast to section 2.2, the phosphorylation at Ser396 was unaltered between any of the groups (figure 2.14, Figure Total Tau levels are unaffected by both disease status and treatment. Densitometry analysis of immunoblots probing for total Tau. Transgenic mice show no significant change in the levels of total Tau compared with wildtype mice panel A). Figure 2.14, panel B shows that Ser404 phosphorylation was significantly increased in the transgenic vehicle treated mice compared with the wildtype mice (P = , 1-way ANOVA, LSD post hoc) and treatment with Cu II (gtsm) brought this back to wildtype levels (P = , 1-way ANOVA LSD post hoc). 52

73 Figure Cu II (gtsm) improves levels of pathological phospho-tau. Levels of phospho-tau were determined by immunoblot densitometry before being normalised to the total levels of Tau measured in the respective sample. A) Phosphorylation at epitope Ser396 was not changed between groups. B) Phosphorylation at Ser404 was significantly raised in the transgenic vehicle treated mice and Cu II (gtsm) significantly reduced this level. C) Phosphorylation at Ser202 was also significantly raised in the vehicle treated mice and Cu II (gtsm) reduced this value. ** = P < 0.01, *** = P < , 1-way ANOVA LSD post hoc. N=6-8 in all groups. 53

74 Finally, Ser202 phosphorylation was also significantly increased in the transgenic vehicle treated mice compared to wildtype (P = , 1-way ANOVA LSD post hoc) and like the Ser404 levels, Cu II (gtsm) treatment reduced this to near wildtype levels (P = , 1-way ANOVA LSD post hoc). The Ser202 antibody also reacted with a band at 79 kda band of Tau which is traditionally termed the paired helical fragment epitope. Figure 2.14, panel C shows densitometry analysis of the band at 50 kda, as such an additional analysis was carried out on the band at 79 kda. Figure 2.15 shows the 79 kda band that reacts with the ptau202 antibody. Transgenic vehicle treated mice exhibit around a 50% increase in the levels of Ser202 phosphorylation relative to total Tau levels compared with wildtype mice (P = 0.029, 1-way ANOVA LSD post hoc). Cu II (gtsm) treatment reduced this level to the level of wildtype mice (P = 0.011, 1-way Figure Vehicle treated APP/PS1 mice exhibit an increase in ptau202 which is decreased by drug treatment. Immunoblot densitometry normalised to the total levels of Tau measured in the respective sample. Transgenic vehicle treated mice showed a ~50% increase in ptau202 that was reduced back to ~100% with Cu II (gtsm) treatment. * = P<0.05, 1- way ANOVA LSD post hoc. N=6-8. ANOVA LSD post hoc). 54

75 Tau pathology was also assessed with sarkosyl-tau preparation (section 6.3.4) to investigate changes in oligomeric Tau, such as those seen in section The preparation was carried out as before (section 6.3.4). To quantitate the differences, the Figure The ratio of sarkosyl-soluble to insoluble Tau does not increase in transgenic mice and is unaffected by drug treatment in APP/PS1 mice. A. Densitometry analysis of sarkosyl extracted Tau from brain tissue showed that neither genotype nor drug treatment produced an alteration in the ratio between the sarkosylsoluble Tau and the insoluble Tau. Statistical analysis by 1-way ANOVA LSD post hoc, ns = not significant. N=6-7. B. Representative blot running both pellet (insoluble) and soluble material on an SDS-PAGE gel. Gel layout as follows: lanes 1, 2, 7, 8 = Wildtype, lanes 3, 4, 9, 10 = Transgenic (vehicle treated), lanes 5, 6, 11, 12 = Transgenic (Cu II (gtsm) treated. The same layout follows for the soluble fractions. densitometry data from each immunoblot was combined to create a ratio of the supernatant value to the pellet value to create a sarkosyl-soluble: insoluble ratio. Figure 2.16 displays the calculated ratios as a percentage of wildtype and shows that there were 55

76 no significant alterations in the ratio between wildtype and vehicle treated transgenic mice (P = 0.58, 1-way ANOVA LSD post hoc) or between vehicle treated transgenic and Cu II (gtsm) treated transgenic mice (P = 0.16, 1-way ANOVA LSD post hoc) which contrasted with the result obtained in section Effects of Cu II (gtsm) on protein phosphatase 2A (PP2A) To see if the decreases in phosphorylation of Tau seen in section were associated with concomitant increases in PP2A, like those seen in section , the levels of the PP2A subunits were quantified via immunoblot densitometry. Brain homogenate samples were probed utilising not only antibodies against the levels of the A, B, and C subunits, but also antibodies specific for the phosphorylated and methylated forms of the C subunit as the activity and specificity of PP2A is highly influenced by these mutually exclusive post-translational modifications (for review see section ). Figure 2.17, panel A shows that the levels of the PP2A(A) subunit, like in section , were decreased in vehicle treated transgenic mice when compared with wildtype (P = 0.043, 1-way ANOVA LSD post hoc), and treatment with Cu II (gtsm) significantly restored the level to wildtype levels (P = way ANOVA LSD post hoc). Panels B and C show that neither the B nor the C subunit were affected by diseases status or drug treatment, consistent with the data generated in section

77 Figure Cu II (gtsm) alters PP2A(A) subunit levels but not (B) or (C). Densitometry analysis of brain homogenate samples for A) PP2A(A), B) PP2A(B), C) PP2A(C) showed that Cu II (gtsm) treatment produced a significant increase in the levels of the PP2A(A) subunit while PP2A(B) and PP2A(C) levels remained unchanged. Transgenic vehicle treated mice showed no significant change when compared with 57

78 wildtype mice (P > 0.05). D) Representative immunoblots for panels A, B, and C. Statistical analysis by 1-way ANOVA LSD post hoc ns = not significant, * = P < N=6-8 in all groups Effects of Cu II (gtsm) on synaptic health markers To ascertain the effects of both disease and drug treatment on markers of synaptic health, three markers were quantified using immunoblot densitometry. Brain homogenate samples were probed with antibodies against post synaptic density protein 95 (PSD-95), synaptophysin, and glial fibrillary acidic protein (GFAP). 58

79 Figure Cu II (gtsm) rescues the PSD-95 impairment in transgenic vehicle treated APP/PS1 mice while not affecting levels of synaptophysin or glial fibrillary acidic protein. Densitometry analysis of hippocampal tissue of A) post synaptic density -95 (PSD-95), B) synaptophysin, and C) glial fibrillary acidic protein (GFAP) show that vehicle treated transgenic animals displayed a significant reduction in the levels PSD-95 when compared to wildtype mice and that Cu II (gtsm) treatment restored this to the levels of wildtype mice. 59

80 Additionally, there was no significant changes noted the levels of synaptophysin and GFAP. D) representative immunoblots for panels A, B, and C. Statistical analyses by 1- way ANOVA LSD post hoc, ns = P > 0.05, * = P < 0.05, *** = P < N=6-8. Figure 2.18 displays the three synaptic health markers assayed. Panel A indicates that transgenic mice treated with vehicle exhibited a highly significant decrease (P = , 1-way ANOVA LSD post hoc) in the levels of PSD-95 in the hippocampus. Treatment of transgenic mice with Cu II (gtsm) improved the levels of PSD-95 back to wildtype levels (P = 0.015, 1-way ANOVA LSD post hoc). The levels of synaptophysin (panel B decreased in the transgenic vehicle treated mice, but did not reach statistical significance (P = 0.076, 1-way ANOVA LSD post hoc) and treatment with Cu II (gtsm) did not affect these levels (P = 0.85, 1-way ANOVA LSD post hoc). Unexpectedly, transgenic vehicle treated mice showed a decrease in the levels of GFAP (panel C) (P=0.033, 1-way ANOVA LSD post hoc) compared with wildtype while Cu II (gtsm) treatment produced no significant change in this level (P = 0.17, 1-way ANOVA LSD post hoc). Overall, however, the ANOVA did not reach statistical significance (P = 0.089) and as such this finding may represent an artefact. 60

81 Figure Cu II (gtsm) increases copper content of a multitude of proteins. Brain tissue extracted from transgenic mice (treated with vehicle or 65 Cu II (gtsm)) and wildtype littermates were homogenised and soluble fraction analysed by LC-ICP-MS. Blue, green and red lines show the 63 Cu trace for wildtype, vehicle treated transgenic, and Cu II (gtsm) treated transgenic mice respectivel: 63 Cu trace displayed as a function of time off the column. N= Effects of Cu II (gtsm) treatment on LC-ICP-MS profile To ascertain the effects of Cu II (gtsm) treatment on the copper content of soluble proteins, and to potentially identify targets of copper delivery, soluble fractions of the hippocampus were analysed by LC-ICP-MS (see section 6.6 for methodology). Figure 2.19 shows the copper profile across the genotype and treatment groups. The trace shows that vehicle treated transgenic mice showed a decrease in copper content in a number of copper peaks from 200 seconds to 420 seconds at which point the copper content was the same. The signal-to-noise ratio of the 65 Cu: 63 Cu ratio prevented meaningful analysis and so was excluded. 61

82 Figure Cu II (gtsm) produces an increase in the iron content of ferritin. Brain tissue extracted from transgenic mice (treated with vehicle or 65 Cu II (gtsm)) and wildtype littermates were homogenised and soluble fraction analysed by LC-ICP-MS. Blue, green and red lines show the 56 Fe trace for wildtype, vehicle treated transgenic, and Cu II (gtsm) treated transgenic mice respectively: 56 Fe trace displayed as a function of time off the column. N=6-13. The green line indicates that Cu II (gtsm) treatment produced an increase in copper content of all peaks on the spectra indicating all proteins on the column had an increased copper content. Furthermore, using the same technique to analyse the iron concentration of soluble proteins revealed that vehicle treated transgenic ferritin contained less iron than wildtype and that Cu II (gtsm) restored this to wildtype levels (figure 2.20). When analysing the zinc content of soluble proteins, figure 2.21 illustrates that Cu II (gtsm) restored the zinc deficiency seen in vehicle treated transgenic mice over multiple protein peaks, together with figure 2.19 and figure 2.20 shows a correction of metal dyshomeostasis. 62

83 Figure Cu II (gtsm) restores the Zn content of multiple peaks. Brain tissue extracted from transgenic mice (treated with vehicle or 65 Cu II (gtsm)) and wildtype littermates were homogenised and soluble fraction analysed by LC-ICP-MS. Blue, green and red lines show the 66 Zn trace for wildtype, vehicle treated transgenic, and Cu II (gtsm) treated transgenic mice respectively: 66 Zn ratio displayed as a function of time off the column. N= Summary of follow up data The follow-up study described above has shown that Cu II (gtsm) was efficacious in improving cognitive deficits in the APP/PS1 model of AD. Furthermore, improvements in Tau pathology were observed upon Cu II (gtsm) treatment as measured by phosphorylated forms of Tau. The improvements in Tau pathology were thought to be mediated by a Cu II (gtsm) mediated rescue of the levels of the A subunit of PP2A to around wildtype levels. Finally, there was also a significant drug mediated restoration in synaptic health as measured by the levels of PSD-95 in treated animals when compared with vehicle treated transgenic mice. 63

84 2.4 Major conclusions from chapter 2 and discussion The studies described in chapter 2 aimed to test the hypothesis generated in the previous work examining Cu II (gtsm) in APP/PS1 mice 231. This hypothesis was that Cu II (gtsm) improves the behavioural phenotype of the APP/PS1 mice through reductions in Tau pathology mediated by changes in GSK3β biology Cu II (gtsm) improves the behavioural phenotype of APP/PS1 mice The APP/PS1 mouse model of AD displays deficits in the tests of spatial memory such as the Morris water maze and the Y-maze 285. Previous work with the APP/PS1 from our lab had shown that Cu II (gtsm) treatment was efficacious in improving the spatial memory of transgenic APP/PS1 mice in the Y-maze 231. The initial study described here showed that Cu II (gtsm) improved the behavioural deficits in the Morris water maze (section 2.2.1) while the larger follow up study showed that Cu II (gtsm) treatment rescued deficits in the Y-maze, but not the Morris water maze (section 2.3.2). There were several factors that impacted the data generated in these two trials: the inherent variation (stress, anxiety, hypothermic impact) between animal behavioural studies 286,287, the natural genetic drift over time of inbred lines, and the fact that the trials were carried out in different locations means that behavioural studies analysed in isolation may have provided misleading results. However, together, the experiments described in the two trials above combined with the Crouch-Hung et al. study 231 have shown that Cu II (gtsm) consistently improved spatial memory performance across three separate studies. In an industry plagued with irreproducibility, this level of consistency was encouraging. Future drug discovery programs should aim to minimise forms of variation that could affect the behavioural data generated as this would increase consistency and allow direct comparison between trials. However, despite the variation in Tau phosphorylation phenotype, the data generated in 64

85 sections and support the hypothesis that Cu II (gtsm) treatment improves spatial memory deficits in APP/PS1 mice Cu II (gtsm) does not alter the levels of amyloid-β species SELDI-TOF analysis from the Crouch-Hung et al. study suggested possible alterations in the levels of oligomeric Aβ. To test this, brain samples were analysed from both the preliminary study (section ) and the follow-up study (section ). Both analyses showed that there was no difference in any Aβ species detected. These findings were in contrast with those observed previously and as such may suggest the previous finding was the result of non-specific binding or an artefact Cu II (gtsm) reduces Tau pathology in APP/PS1 mice In the Crouch-Hung et al, study, reductions in the levels of phosphorylated Tau were observed (Ser404 epitope). It was then hypothesised that in the trials described in this chapter, reductions in Tau pathology would also be observed. In the preliminary study described here (section 2.2) reductions in phosphorylated forms of Tau were seen at both the Ser396 and Ser404 epitopes, consistent with previous findings 231. In the follow-up study, no changes in the Ser396 epitope were noted, but reductions in the Ser404 and Ser202 epitopes were observed. Furthermore, the 79 kda Tau NFT band revealed by the Ser202 antibody was also significantly reduced. Variation in individual epitopes analysed may be due to the variation between the trials as described above (genetic drift and different location). However, the two studies described here both show consistent reductions in phospho-tau epitopes and reductions in the Ser404 epitope was consistently observed throughout. 65

86 APP/PS1 mice do not form NFT pathology 278. However, to our knowledge, an assessment of early oligomeric forms of Tau had not been undertaken in these mice before. To that end, a sarkosyl-tau prep was carried out to assess changes in sarkosylsoluble and insoluble Tau. The data in the preliminary study showed that increased levels of oligomeric Tau were indeed observable in APP/PS1 mice and importantly, Cu II (gtsm) treatment reduced this. It should be noted that this data was not normally distributed and was not replicated in the follow-up study raising the possibility that these findings were an artefact of the investigation. Additionally, the methodology produces inconsistent solubilisation of the pellet material and this may also be a source of variability. Follow up studies should include a larger number of animals and incorporate further optimisation to ensure consistent solubilisation. In both the studies described above and in the Crouch-Hung et al. study, no significant changes in total Tau were observed. Together this data, despite variation in the nature of phospho-tau species, supports the hypothesis that Cu II (gtsm) reduces Tau pathology as seen by reductions in the levels of phospho-epitopes of Tau Cu II (gtsm) does not affect kinase biology, but improves phosphatase levels Analysis of GSK3β in the initial study described here (section ) revealed no changes in the levels of phospho-gsk3β (Ser9). This result contrasts with the data generated in the Crouch-hung et al. study so to further elucidate what might be responsible for the reductions in phosphorylated Tau, the major Tau phosphatase, PP2A, was examined. A reduction in PP2A has been found in AD cases 103 and specific PP2A inhibition leads to AD-like pathology 105. Indeed, across both the preliminary and followup studies a significant drug-mediated increase in the structural subunit of the PP2A enzyme was observed while the B and C subunits remained largely unchanged. This result 66

87 was interpreted to mean that an increase in the structural subunit of the enzyme would stabilise the PP2A holoenzyme. A complete holoenzyme has been shown to be required for Tau dephosphorylation 87 and as such increased levels of a stabilised holoenzyme would result in increased Tau dephosphorylation Cu II (gtsm) increases PSD-95 levels In the follow-up study, several markers of synaptic health and inflammation were assessed (section ). A Cu II (gtsm) mediated rescue of PSD-95 levels was observed while no significant changes in synaptophysin or GFAP were observed. This is consistent with, in addition to improving Tau pathology, treatment also improves synaptic health. This increase is likely to contribute to improved cognitive performance Discussion and future directions Given that the two trials conducted (sections 2.2 and 2.3) show a degree in variation of the timeline of symptom onset and Tau pathology, it would be worthwhile to extend the length of the trial to determine the spectrum of changes in Tau pathology. For example, if aged longer would the animals from the second trial have developed an insoluble Tau phenotype and furthermore, would this phenotype worsen with age. Given the demonstrated drift in phenotype of the model, it would worthwhile longitudinally characterising the changes to better understand the pathology in these AD mouse models. Furthermore, the APP/PS1 mouse model of AD has not traditionally been used as a model of AD-related Tau pathology due to the lack of NFT generation in the model 288. The finding here that the phenotype involves an oligomeric form of Tau is novel and warrants a more thorough characterization of Tau species in future work. 67

88 PP2A has known to play a role in AD pathogenesis for some time 70,115,289 and its therapeutic potential has been highlighted previously 109. However, the enzymes biology and regulation is very complex 108,290, which means pharmacologically targeting the enzyme is problematic. One possible avenue of modulation that has been explored to some extent, is the concept of alteration of its methylation state via influencing the methylation cycle 291. Figure 2.22 shows the link between the methylation cycle and the methylation state of PP2A reproduced from

89 Figure Model of the link between MTHFR, folate, PP2A methylation and Tau phosphorylation. 5-MTHF, produced via the action of MTHFR, is essential for remethylation of homocysteine (Hcy) to methionine, the precursor of S-adenosylmethionine (SAM), the universal methyl donor. Methylation of PP2A catalytic subunit on Leu-309 by the dedicated SAM-dependent LCMT1 methyltransferase promotes the biogenesis of PP2A/Bα heterotrimers, the primary Tau Ser/Thr phosphatases in the brain. Common C677 T polymorphisms in the mthfr gene induce mild MTHFR deficiency, leading to reduced 5-MTHF levels. Folate deficiency promotes accumulation of Hcy, which may be converted back to SAH, a potent inhibitor of LCMT1 activity. MAT, methionine adenosine transferase; MS, methionine synthase; MTHF, methyltetrahydrofolate; SAHH, SAH hydrolase; THF, tetrahydrofolate. Figure and caption reproduced with permission from 288. While the figure focuses on the role of methyltetrahydrofolate, one can see many other points of intervention in this cycle. Interestingly, copper is important for this cycle at two places, both methionine synthase (MS) and SAH hydrolase (SAHH) are copper binding/requiring enzymes and homocysteine, and hence methionine, metabolism is disturbed in copper-deficient rats due to decreased MS activity 292. Furthermore, altering 69

90 the homocysteine metabolism cycle has been shown to impair PP2A functionality and induce Tau hyperphosphorylation 293. Methylation of PP2A is important for stability of the holoenzyme (decreased methylation correlates with decreases in the structural A subunit) and also for Tau dephosphorylation 291,294,295. Therefore, I hypothesise that delivered copper from Cu II (gtsm) improves the activity of copper dependent enzymes in the methylation cycle, thus increasing flux through the cycle. This then increases the activity of LCMT-1 and subsequently increases methylation of PP2A. To test this hypothesis, one could assay the methylation status of PP2A to quantify effects on methylation. Additionally, an interrogation of copper content of both MS and SAHH from both central nervous system and peripheral tissue would also show if treatment improved the copper content of these enzymes. A less direct measure would also be an assessment of changes in homocysteine levels in the blood. Blood drawn from mice in section 2.3 has been sent for metabolomic analysis to answer this question. The studies described in sections 2.2 and 2.3 confirm the view that PP2A is an attractive drug target for reducing pathological Tau phosphorylation in AD. Moreover, they show that PP2A activity can indeed be targeted with an exogenous compound. However, there are over 300 targets of PP2A within the cell 290 and as such modulation of enzyme activity could possibly result in off-target effects. Thus drug development programs targeting PP2A would need to carefully monitor possible complications such as increased rate of cancer (owing to the role of PP2A in the cell cycle 296 ). A global analysis of methylation in Cu II (gtsm) treated mice (such as DNA methylation or other methylated proteins) would also indicate if the increases in methylation are specific for PP2A or if methylation of a variety of targets is occurring and the possible ramifications of that. 70

91 Chapter 3 Cu II (gtsm) in a Tau-mediated model of Frontotemporal Dementia 3.1 Introduction The rtg4510 mouse model of FTD The rtg4510 mouse model of FTD is widely used as a versatile model of tauopathy 297. The genetics allow researchers temporal control over Tau expression owing to the tetracycline operon-responsive element upstream of the gene. Expression is driven by a second transgene containing the tetracycline-controlled transactivator under the control of the calcium/calmodulin-dependent protein kinase II. This means that Tau is constitutively expressed until expression is halted by the administration of the tetracycline analogue, doxycycline. These bi-transgenic mice express Tau (the 4-repeat tau isoform (4R0N), for a discussion around the Tau isoforms, see section ) at around 13 times the level of endogenous murine Tau and develop age-related Tau pathology, neuronal loss and behavioural impairments Tau pathology has been reported to begin early in these mice with tangle pathology being detectable from four months of age in the cortex and five to six months in the hippocampus. There is a 60% decrease in hippocampal (CA1) neurons by 5.5 months of age 297. Cortical cell loss follows at around 8-9 months of age with gross forebrain atrophy by 10-months 299. Electrophysiologically, reduced neocortical network activity is present from 5-months of age 298. While cortical slice records have actually shown increased activity 300 and that 71

92 these changes occur prior to notable Tau inclusions. Cognitive and motor impairments are notable from 2.5-months Preliminary study of Cu II (gtsm) in the rtg4510 (P301L) mouse model of FTD Preface This study was a pilot study to examine the efficacy of Cu II (gtsm) in treating the rtg4510 mouse model of FTD. The experiments listed in this section were not done by myself, but by Dr Lin Hung as a post-doctoral fellow in our laboratory. This is unpublished data and included as a section here to provide the framework for which section 3.3 is based upon. Data generated by Dr Hung are also credited in the appropriate figure legend Introduction Metal dyshomeostasis is a feature of many neurodegenerative diseases 163,270,302. Alterations in key transition metals such as copper, zinc and iron are thought to be responsible for some of the synaptic dysfunction seen in the disease and as such therapies targeted towards rectifying these imbalances have been an attractive approach to treating the disease. The BTSC class of compounds have shown that they alter metal homeostasis and improve the pathology in a range of neurodegenerative disease where metal dyshomeostasis is implicated 270. Cu II (gtsm) has been shown to consistently improve the behavioural deficits and reduce Tau pathology seen in the APP/PS1 model of AD (see 231 and Chapter 2). The rtg4510 mouse model of FTD over expresses human Tau carrying the P301L mutation associated with human FTD. These mice exhibit strong Tau pathology and behavioural deficits in spatial memory and locomotor activities 282,300,301, The 72

93 current study utilised this mouse model of FTD to test whether Cu II (gtsm) would also be efficacious in improving the phenotype of the rtg4510 tauopathy and whether the mechanism of action of Cu II (gtsm) would be similar when treating models of AD and FTD Effects of Cu II (gtsm) on rtg4510 behaviour The Y-maze and the Morris water maze tests (sections and respectively) were used with the aim of testing the animals spatial memory formation. The rtg4510 mouse model exhibits strong neurodegeneration and electrophysiological abnormalities in the hippocampus and not surprisingly age-related deficits in these tests of spatial memory 297. To test whether Cu II (gtsm) improved the spatial memory deficit seen in the model, 12- month old rtg4510 transgenic mice and wildtype littermates were dosed with 5 mg/kg daily by oral gavage for a period of 5-weeks. Mice were assessed in the Y-maze and Morris water maze. 73

94 Effects of Cu II (gtsm) on rtg4510 behaviour (Y-maze) Figure 3.1. Cu II (gtsm) improves rtg4510 transgenic mouse performance in the Y- maze. Data generated by Dr Lin Hung. 12-month old rtg4510 transgenic mice and wildtype littermates were treated with either vehicle or 5 mg/kg Cu II (gtsm) for 5-weeks and behaviour was analysed in the Y-maze. Transgenic mice had significantly poorer performance when measuring percentage entries into novel arm when compared to wildtype littermates. Cu II (gtsm) treatment improved this performance to statistically better than chance. Left panel displays data as a box and whisker plot, statistical analysis by 1-way ANOVA LSD post hoc, ns = not significant, *** = P < Right panel displays data as mean +/- 95% confidence interval, statistical analysis by 1-sample t-test against a hypothetical value of chance (33%), * = P < 0.05, *** = P < N=

95 The Y-maze test of spatial memory was carried out per the methodology listed in section Briefly, one hour after a 10- minute acquisition phase with one arm closed, animals were allowed 5-min to explore the maze with all arms open and entries into the novel arm were quantified by analysis software (Topscan, Cleversys, USA). Figure 3.1 shows the percentage entries into the novel arm were significantly decreased in vehicle treated transgenic mice when compared with wildtype mice (P = , 1-way ANOVA LSD post hoc). Cu II (gtsm) treatment of transgenic mice improved performance; however, this did not reach statistical significance when compared with vehicle treated transgenic mouse performance (P = 0.14, 1-way ANOVA LSD post hoc). When comparing the groups performance against chance (33% novel arm entries), both wildtype mice and Cu II (gtsm) treated mice display a preference for the Figure 3.2. Transgenic rtg4510 mice exhibit an increase in the number of total Y-maze entries. Data generated by Dr Ling Hung. 12- month old rtg4510 transgenic mice and wildtype littermates were treated with either vehicle or 5 mg/kg Cu II (gtsm) for 5- weeks and behaviour was analysed in the Y-maze. Transgenic mice (both vehicle and Cu II (gtsm) treated) had significantly increased number of total entries. Statistical analysis by 1-way ANOVA LSD post hoc, ns = not significant, *** = P < N=

96 novel arm (P = and P = respectively, 1-sample t-test against a hypothetical value of chance (33%)). Additionally, when one calculates the total number of entries into all the arms, one can gather information about the level of activity of the animals. Figure 3.2 shows that transgenic mice, regardless of drug treatment, showed a 2-3-fold increase in the number of total entries measured during the test (P < 0.001, 1-way ANOVA LSD post hoc) possibly indicating increased activity. 76

97 Effects of Cu II (gtsm) on rtg4510 behaviour (Morris water maze) While the Y-maze results were promising, a significant proportion of the literature has assessed the rtg4510 spatial memory deficit in the Morris water maze. So, as another measure of spatial memory, transgenic mice and wildtype littermates were also tested in the Morris water maze as per section Figure 3.3. Cu II (gtsm) treatment rescues probe trial deficit in rtg4510 mice. Data generated by Dr Ling Hung. 12-month old rtg4510 transgenic mice and wildtype littermates were treated with either vehicle or 5 mg/kg Cu II (gtsm) for 5-weeks and behaviour was analysed in the Morris water maze. A) Learning curve (mean +/- SEM) displaying latency to escape by day showed that wildtype mice improved their escape time at a significantly quicker rate than both Cu II (gtsm) and vehicle treated transgenic mice. Statistical analysis by 2-way ANOVA LSD post hoc, * = P < 0.05, **** = P < B) Box and whisker plot displaying time spent in the target quadrant during the probe trial showed vehicle treated mice performed significantly worse than wildtype littermates and treatment with Cu II (gtsm) rescued this deficit. Statistical analysis by 1- way ANOVA LSD post hoc, * = P < N =

98 Figure 3.3, panel A shows the acquisition curves for the groups in the Morris water maze. The wildtype group performed significantly better than the transgenic mice in their improvement in escape latency throughout the acquisition phase. Treatment with Cu II (gtsm) did not alter the transgenic mouse performance compared with vehicle treated transgenic vehicle treated mice. During the probe trial (figure 3.3, panel B), transgenic vehicle treated mice spent significantly less time in the target quadrant when compared with both wildtype mice and Cu II (gtsm) treated mice (P = and P = respectively, 1-way ANOVA LSD post hoc) the latter of which had similar performance to wildtype mice. These results are similar to those observed in the MWM in chapter Effects of Cu II (gtsm) on rtg4510 biochemistry The rtg4510 mouse model of FTD is driven by overexpression of a pathological form of Tau leading to extensive Tau pathology. The behavioural improvements in both the Y- maze and Morris water maze were promising so the next step was to assess changes in Tau physiology in the animals. To assess changes in Tau pathology, the brain tissue collected from the animals was assessed by immunohistochemistry to quantitate the level of neurofibrillary tangle formation in both the hippocampus and cortex. Subsequently, this brain tissue was also analysed by immunoblot to assess the levels of phosphorylated Tau Effects of Cu II (gtsm) on rtg4510 neurofibrillary tangle (NFT) counts Transgenic rtg4510 mice are known to develop NFT pathology throughout the hippocampus and cortex with increasing age 301. To examine changes in pathological Tau deposition in the current study, brain tissue was examined with immunohistochemistry staining for neurofibrillary tangles. Figure 3.4 shows that, as expected, in the hippocampus, transgenic mice exhibited a dramatic increase in the level of NFT staining. 78

99 Treatment with Cu II (gtsm) produced a significant decrease in the NFT load in the hippocampus compared to vehicle treated mice (P = , 1-way ANOVA LSD post hoc). Figure 3.5 shows that, like the hippocampus, there was a significant increase in the NFT staining in the cortex of vehicle treated transgenic mice and interestingly, unlike the hippocampus, treatment with Cu II (gtsm) did not significantly reduce these levels. 79

100 Figure 3.4. Cu II (gtsm) treatment of rtg4510 mice decreases hippocampal neurofibrillary tangle load. Data generated by Dr Lin Hung and Associate Professor Paul Adlard. Immunohistochemical staining for paired helical filaments (PHF) of Tau in A) wildtype, B) vehicle treated transgenic and C) Cu II (gtsm) treated transgenic mice in the hippocampus. D) Box and whisker plot showing quantitation of immunohistochemical counts. Transgenic mice exhibited a strong increase in PHF staining in the hippocampus compared with wildtype. Cu II (gtsm) treatment significantly reduced the NFT staining in the hippocampus. Statistical analysis by 1-way ANOVA LSD post hoc, ** = P < 0.01, *** = P < N=5-8. The decrease in tangle load in the hippocampus combined with increased hippocampal dependent behaviour (Y-maze and Morris water maze) suggested that aggregated Tau is 80

101 reduced and this could explain the behavioural improvements. Next, total levels of Tau and the phosphorylation state was assessed with immunoblotting. Figure 3.5. Cu II (gtsm) treatment of rtg4510 mice does not alter cortical neurofibrillary tangle load. Data generated by Dr Ling Hung and Associate Professor Paul Adlard. Immunohistochemical staining for paired helical filaments (PHF) of Tau in A) wildtype, B) vehicle treated transgenic and C) Cu II (gtsm) treated transgenic mice in the cortex. D) Box and whisker plot showing quantitation of immunohistochemical counts. Transgenic mice exhibit a strong increase in PHF staining in the hippocampus compared with wildtype. Cu II (gtsm) does not alter the NFT stain in the cortex. Statistical analysis by 1- way ANOVA LSD post hoc, ns = not significant, *** = P < N=

102 Effects of Cu II (gtsm) on total Tau and phospho-tau in rtg4510 mice. Immunoblotting and densitometry was used to assess changes in total Tau levels and the phosphorylation state of Tau in the animals. Figure 3.6, panel A shows a box and whisker plot of the total Tau levels. Figure 3.6. Cu II (gtsm) treatment does not alter total or phosphorylated Tau in rtg4510 mice. Data generated by Dr Lin Hung. Transgenic rtg4510 mice and wildtype littermates were treated with Cu II (gtsm) or vehicle for 5-weeks prior to sacrifice and tissue extraction. Brain tissue was analysed via immunoblot for the levels of total Tau and ptau396. A) Box and whisker plot showing a non-significant increase in the total levels of Tau in both groups of transgenic mice, Cu II (gtsm) did not alter the level of total Tau compared to vehicle treated transgenic mice. B) Levels of ptau396 normalised to the total Tau levels shows no significant difference between the groups analysed. C) Representative blots. Statistical analysis by 1-way ANOVA LSD post hoc, ns = not significant. N=

103 There was a non-significant increase in Tau in the vehicle treated mice (P = 0.064, 1-way ANOVA LSD post hoc). This increase was expected in this model; however, there was a large amount of variation present in the transgenic animals and as such the post hoc analysis did not reach statistical significance. Additionally, Cu II (gtsm) produced no change in the levels of total Tau. The levels of the ptau396 epitope were normalised to the total levels of Tau owing to the changes in total Tau levels between groups. Interestingly, there was no increase seen in the transgenic mice and correspondingly, Cu II (gtsm) treatment produced no alterations in this level Effects of Cu II (gtsm) on protein phosphatase 2A (PP2A) As seen in the previous chapter (sections and ), Cu II (gtsm) treatment produced an increase in the levels of the PP2A(A) subunit in transgenic APP/PS1 mice in the context of an improvement in Tau pathology. To assess the effects of treatment on PP2A levels, the protein levels of the three subunits were quantified with immunoblot densitometry. The levels of the PP2A(A) subunit (figure 3.7, panel A) were dramatically reduced in the vehicle treated transgenic mice (P = , 1-way ANOVA LSD post hoc), this level was increased to close to wildtype levels when treated with Cu II (gtsm) (P = 0.027, 1-way ANOVA LSD post hoc). The levels of the B and C subunit (figure 3.7, panels B and C) were not changed in the transgenic mice when compared with wildtype mice and treatment with Cu II (gtsm) did not significantly alter these levels. These data were consistent with the data generated in section

104 Figure 3.7. Treatment of rtg4510 mice with Cu II (gtsm) rescues the decrease in the PP2A(A) subunit. Data generated by Dr Lin Hung. Transgenic rtg4510 mice and wildtype littermates were treated with Cu II (gtsm) or vehicle for 5-weeks prior to sacrifice and tissue extraction. Brain tissue was analysed via immunoblot for the levels of the A, B and C subunits of PP2A. A) Vehicle treated transgenic mice exhibited a significant decrease in the A 84

105 subunit of PP2A while treatment with Cu II (gtsm) rescued this to wildtype levels. B) The PP2A(B) subunit level was not significantly altered between wildtype and transgenic mice and treatment with Cu II (gtsm) did not alter this. C) The PP2A(C) was not significantly altered between wildtype and transgenic mice and treatment with Cu II (gtsm) did not alter this. D) Representative blots. Statistical analysis by 1-way ANOVA LSD post hoc, ns = not significant, * = P < 0.05, *** = P < N= Summary of Preliminary Data As a result of the data generated in chapter 2, it was hypothesised that Cu II (gtsm) would be efficacious in treating the rtg4510 model of FTD by reducing pathological Tau through a PP2A mediated mechanism. This hypothesis was supported by the behavioural improvements in the Y-maze and Morris water maze described here. Cu II (gtsm) treatment also reduced the NFT tangle load in the hippocampus of transgenic mice, but not the cortex, suggesting a hippocampal mediated effect. Furthermore, analysis of PP2A biology produced data consistent with the Cu II (gtsm) mediated increase in the PP2A(A) described in chapter 2. The data generated in this preliminary study supported the hypothesis and warranted a follow-up study to further examine the therapeutic effects of Cu II (gtsm) in the rtg4510 model and expand on the behavioural analysis. 3.3 Cu II (gtsm) treatment of the rtg4510 (P301L) mouse model of FTD Preface The remainder of this chapter describes the follow-up study conducted by myself to address unanswered questions from the preliminary study described in section 3.2 and to expand on the behavioural analysis conducted. 85

106 3.3.2 Introduction To examine the ability of Cu II (gtsm) to treat the rtg4510 mouse model postsymptomatically and to expand on the behavioural domains tested, transgenic mice and their wildtype littermates were dosed with vehicle or Cu II (gtsm) at a dose of 5 mg/kg bodyweight daily by oral gavage. Treatment was initiated at 11-months of age and after 8-weeks of treatment, behaviour was assessed in the locomotor cell and the Rotarod. For the final 7 days of treatment, the animals were dosed with 65 Cu II (gtsm) for analysis via LC-ICP-MS (see section 6.6). Brain tissue was subsequently extracted for analysis by immunoblot for Tau pathology and synaptic health markers Effects of Cu II (gtsm) on rtg4510 behaviour The rtg4510 mouse model exhibits deficits in multiple modalities of cognition including spatial memory and hyperactivity 301,306,307. The experiments described in section 3.2 illustrate the ability of Cu II (gtsm) to rescue the cognitive phenotype of rtg4510 mice, to build on this result we assessed changes in the hyperactivity phenotype in the locomotor cell and on the Rotarod. Furthermore, section illustrates a hyperactivity phenotype in the Y-maze as measured but total number of arm entries Effects of Cu II (gtsm) on motor function and anxiety-like behaviour (locomotor cell) Previous research has indicated that the rtg4510 mouse model of FTD displays abnormalities in the locomotion and anxiety-like behaviour as measured in the locomotor cell (open field) test and that these abnormalities correlate well with Tau pathology 307. Both transgenic animals and wildtype littermates had their movement analysed in the locomotor cell for a period of 60 min as per section

107 Figure 3.8. Cu II (gtsm) treatment rescues the strong anxiety-like and hyperactivity phenotype of transgenic rtg4510 mice. Transgenic rtg4510 mice and wildtype littermates were treated with Cu II (gtsm) for 8- weeks and motor and anxiety-like behaviour was assessed in the locomotor cell (open field). Movement was tracked for a period of 60 min of free exploration of the cell. Data is displayed as a box and whisker plot. A) Total distance moved (mm) during the 60 min shows that vehicle treated transgenic mice displayed a strong increase in movement and Cu II (gtsm) treatment rescued this to almost wildtype levels. B) Total resting time (s) analysis indicated that vehicle treated transgenic mice rested approximately 50% of the time wildtype mice rested and treatment with Cu II (gtsm) significantly increased resting time. C) The time mice spent exploring the centre of the cell was converted to a percentage of the total time in the cell to give a measure of anxiety-like behaviour. Transgenic mice spent a significantly lower percent of total time exploring the centre of the cell compared to wildtype littermates, Cu II (gtsm) treated mice spent a significantly higher percentage of total time exploring the centre of the cell. Statistical analysis by 1-way ANOVA LSD post hoc, ** = P < 0.01, *** = P < 0.001, **** = P < N=

108 The hyperactive phenotype previously reported in 307 was replicated here. figure 3.8, panel A shows that vehicle treated transgenic mice had a 10-fold increase in total distance moved in the 60-min analysis period (P < , 1-way ANOVA LSD post hoc). Treatment of transgenic mice with Cu II (gtsm) significantly reduced the total distance moved (P = , 1-way ANOVA LSD post hoc) to a level only three times that of wildtype mice. The converse of total distance moved is the total time spent still, in line with the data from panel A, panel B shows that vehicle treated transgenic mice spent a significantly lower time resting (P < , 1-way ANOVA LSD post hoc) and treatment of transgenic mice with Cu II (gtsm) significantly increased the time resting (P < , 1-way ANOVA LSD post hoc). As a measure of anxiety-like behaviour, the time the animal spent in the centre of the cell (a pre-defined area in the analysis software) was converted to a percentage of the total time spent in the cell. Figure 3.8, panel C shows that transgenic vehicle treated mice spent much less time in the centre of the cell compared with wildtype littermates and Cu II (gtsm) treated transgenic mice (P = and P = respectively, 1-way ANOVA LSD post hoc) indicating an increased level of anxiety-like behaviour (thigmotaxic). These data indicate that transgenic rtg4510 mice displayed a strong hyperactivity phenotype as measured by total distance covered and the time spent resting which agrees with previous research 307. Transgenic rtg4510 mice also displayed increased anxietylike behaviour as measured by the percentage of time the mice spent exploring the centre of the cell, also in agreement with previous work with this model 307. Additionally, the data showed that treatment with Cu II (gtsm) significantly improved all the deficits measured in the locomotor cell (total distance, total resting time and anxiety- like behaviour) returning them to wildtype levels or very close to wildtype levels. 88

109 Effects of Cu II (gtsm) on motor function (Rotarod) Next, we sought to examine whether the motor changes seen in section were due to alterations in the motor capabilities of the animals or due to alterations in the neurological origin of the hyperactivity, i.e. was Cu II (gtsm) treatment altering the animals ability to move or altering the pathology driving the hyperactivity phenotype. To do this, animals motor capabilities and coordination was assessed in the Rotarod (see section 6.9.6). After an acclimation phase, animals had three five minute trials and their average latency to fall is displayed in figure 3.9. The figure shows that transgenic vehicle treated mice had a similar performance to both wildtype and treated transgenic mice (P = 0.58 and P = respectively, 1-way ANOVA LSD post hoc). Neither genotype nor treatment altered performance in the Rotarod. Figure 3.9. Transgenic rtg4510 mice exhibit no deficit in Rotarod performance. Three five minute trials were averaged to measure the average latency to fall off the Rotarod. Data is displayed as a box and whisker plot. Neither genotype nor drug treatment impacted animals performance on the Rotarod. Statistical analysis by 1-way ANOVA LSD post hoc, ns = not significant. N=

110 3.3.4 Effects of Cu II (gtsm) on rtg4510 biochemistry Given the highly significant results from the locomotor cell experiment and the observation that these behavioural alterations correlate with Tau pathology, it was hypothesised there would be strong effects on Tau pathology. Initially, the total levels of Tau were assessed to make sure any changes seen were not due to changes in total Tau. 90

111 Figure Cu II (gtsm) does not alter the level of phosphorylated Tau or total levels of Tau in transgenic rtg4510 mice. Transgenic rtg4510 mice and wildtype littermates were treated with Cu II (gtsm) or vehicle for 8-weeks prior to sacrifice and tissue extraction. Brain tissue was analysed via immunoblot for the levels of total Tau (A), ptau396 (B), and ptau404 (C). All panels show a dramatic increase in the levels of total Tau, ptau396 and ptau404 in vehicle treated transgenic mice, while treatment of transgenic mice with Cu II (gtsm) did not significantly alter levels of total Tau or the phosphorylated forms of Tau tested. D) shows 91

112 representative blots. Statistical analysis by 1-way ANOVA LSD post hoc, ns = P > 0.05, **** = P < N= Effects of Cu II (gtsm) on Tau The transgenic mice displayed a 250% increase in the levels of total Tau compared to wildtype mice (P < , 1-way ANOVA LSD post hoc) while treatment of transgenic mice did not alter the level of total Tau (figure 3.10, panel A). Assessment of the phosphorylation epitopes Ser396 and Ser404 revealed a large (500% and 750% respectively) increase in the levels of both these epitopes in the transgenic mice compared to wildtype littermates (P < , 1- way ANOVA LSD post hoc). Treatment with Cu II (gtsm) again failed to produce a significant change in the levels in either of Figure Cu II (gtsm) rescues the dramatic increase in high molecular weight Tau in rtg4510 transgenic mice. Transgenic rtg4510 mice and wildtype littermates were treated with Cu II (gtsm) or vehicle for 8-weeks prior to sacrifice and tissue extraction. Brain tissue was analysed via immunoblot for the levels of total Tau. Transgenic vehicle treated mice exhibited a significant increase in the levels of 100 kda Tau and Cu II (gtsm) treatment rescued this. Statistical analysis by 1-way ANOVA LSD post hoc, ** = P < 0.01, **** = P < N=

113 these epitopes as seen in figure 3.10, panels B and C. Previous work with this animal model found that a species of Tau with an SDS-PAGE molecular weight of 100 kda was a strong predictor of behavioural deficit 306. To that end, exposure times of blots were extended to reveal the band at 100 kda and band densitometry was quantitated. Figure 3.11 shows that, as expected, there as a significant increase in the levels of the 100 kda species of Tau in the vehicle treated transgenic mice (P < , 1-way ANOVA LSD post hoc) and Cu II (gtsm) treatment of transgenic mice significantly reduced this (P = , 1-way ANOVA LSD post hoc). Given that this higher molecular weight band had been found to correlate with behavioural deficits, the levels of the 100 kda species of Tau were correlated with the animals performance in the locomotor cell to assess the relationship between this species of Tau and the behavioural effects seen in section Figure 3.12, panel A shows that there was a significant positive relationship between the levels of high molecular weight (100 kda) Tau and the total distance moved in the locomotor cell experiment (Rsquare = 0.36, P = , linear regression analysis) while panel B shows that there was no relationship between the levels of physiological (50 kda) Tau and the distance moved in the locomotor cell (R-square = 0.062, P = 0.24, linear regression analysis), confirming the relationship was with high MW Tau specifically. Furthermore, figure 3.12, panel C displays the relationship between the levels of high molecular weight Tau and the percentage time the animals spent exploring the centre of the cell and while a relationship was observed it did not reach statistical significance (R-square = 0.099, P = 0.13, linear regression analysis). Panel D showed that again there was no relationship with physiological Tau and anxiety-like behaviour (R-square = , P = 0.82). These data 93

114 supported the previous research linking Tau pathology in rtg4510 mice with the hyperactivity and anxiety-like behavioural phenotype seen in transgenic mice. Figure High molecular weight Tau is positively associated with the hyperactivity phenotype in rtg4510 mice. Linear regression analysis (with trendline +/- 95% confidence interval) of the 100 kda Tau species with two modalities measured in the locomotor cell of rtg4510 transgenic mice and wildtype littermates. A) showed a positive relationship of the levels of high molecular weight Tau and the total distance moved in the locomotor cell while B) shows that this relationship was not true of physiological (50 kda) Tau. C) shows that there was a non-significant negative relationship between the levels of high molecular weight Tau and the amount of time the animals spent exploring the centre of the maze and D) shows that there was no relationship between physiological Tau (50 kda) and centre exploration 94

115 time. Statistical analysis via linear regression analysis, P < 0.05 is considered significant. N= LC-ICP-MS analysis of the soluble copper proteome in rtg4510 mice To gain insight into the potential protein targets of the bioavailable copper released into the cells, mice were dosed with isotopically enriched 65 Cu II (gtsm) for the final seven days of treatment. LC-ICP-MS analysis was then carried out on hippocampal tissue as per section 6.6. Figure 3.13 shows the LC-ICP-MS trace with data showing both the total 63 Cu (magenta, black, and brown traces) and the 65 Cu: 63 Cu ratio (blue, red, and green traces) for the groups analysed. The traces showing total 63 Cu indicates that there were subtle differences in copper content between different protein peaks, but the variation in the samples means none of 95

116 96

117 Figure LC-ICP-MS analysis of rtg4510 brain tissue shows increases in copper of multiple proteins. Brain tissue extracted from transgenic mice (treated with vehicle or 65 Cu II (gtsm)) and wildtype littermates were homogenised and soluble fractions analysed by LC-ICP-MS. Blue, green and red lines show the 65 Cu: 63 Cu ratio displayed as a function of time off the column and indicates two strong peaks of altered ratio as indicated by square brackets. Magenta, black and brown traces (+/- SEM) indicating total 63 Cu levels as a function of time showing areas of differing copper content between the groups. Orange trace indicates the location of superoxide dismutase 1 (SOD1). N=4-8. the differences reached statistical difference. The traces displaying the 65 Cu: 63 Cu ratios show that the ratio in wildtype and vehicle treated transgenic mice was consistent throughout the range while the 65 Cu II (gtsm) treated mice showed significant deviations at two peaks indicated by square brackets in figure The orange (SOD1) trace indicates that one of the areas of ratio deviation was consistent with the peak for SOD1 while the other area of ratio deviation was spread over an area containing multiple small molecular weight copper binding proteins confirming entry of exogenous copper into the copper metabolism pathways of the cell. Furthermore, metal homeostasis is shown to be disturbed in transgenic mice both in the case of iron (figure 3.14) and zinc (figure 3.15) and treatment with Cu II (gtsm) alters these levels in both cases further illustrating the link between the levels of these metals Summary of follow up data In the current study, aiming to build upon the data generated in section 3.2 and provide additionally mechanistic insight, Cu II (gtsm) treatment produced a highly significant 97

118 Figure Iron content of ferritin is altered in rtg4510 transgenic mice. Brain tissue extracted from transgenic mice (treated with vehicle or 65 Cu II (gtsm)) and wildtype littermates were homogenised and soluble fraction analysed by LC-ICP-MS. Blue, green and red lines show the 56 Fe trace for wildtype, vehicle treated transgenic, and Cu II (gtsm) treated transgenic mice respectively: 56 Fe trace displayed as a function of time off the column. N=4-8. reduction in both hyperactivity and anxiety-like behaviour. Cu II (gtsm) improved Tau pathology by reducing a 100 kda species of Tau, which based on linear regression analysis correlated significantly with the hyperactivity phenotype (independently of total Tau levels). 3.4 Major conclusions from chapter 3 and discussion The results described in chapter 2 generated the hypothesis that, based on those results, Cu II (gtsm) would be efficacious in treating another model of tauopathy. To test this, the rtg4510 model of FTD was treated with Cu II (gtsm). Furthermore, it was hypothesised that Cu II (gtsm) treatment would improve the behavioural phenotype of the model through a PP2A mediated reduction in Tau pathology. 98

119 Figure Zinc content of multiple protein peaks is altered in transgenic rtg4510 mice. Brain tissue extracted from transgenic mice (treated with vehicle or 65 Cu II (gtsm)) and wildtype littermates were homogenised and soluble fraction analysed by LC-ICP-MS. Blue, green and red lines show the 56 Fe trace for wildtype, vehicle treated transgenic, and Cu II (gtsm) treated transgenic mice respectively: 56 Fe trace displayed as a function of time off the column. N= Cu II (gtsm) treatment improves the behavioural phenotype of rtg4510 mice Due to advanced hippocampal pathology, rtg4510 mice display decreased performance in tests of spatial memory and performance can be improved if Tau pathology is reduced 297. In this study, it was shown that Cu II (gtsm) treatment of rtg4510 mice improved performance in both the Y-maze (figure 3.1) and the Morris water maze (figure 3.3). Furthermore, Cu II (gtsm) treatment resulted in a highly significant reduction in hyperactivity and anxiety-like behaviour (figure 3.8). These data support the hypothesis that Cu II (gtsm) would improve the behavioural phenotype of the model. Treatment improved both cognition (Y-maze and Morris water maze) and locomotor (hyperactivity) 99

120 phenotypes, both of which have linked to the degree of Tau pathology in the animals 297,301,307. Taken together, these data show that Cu II (gtsm) is effective in rescuing the behavioural phenotype of the rtg4510 mice Cu II (gtsm) reduces Tau pathology in rtg4510 mice The behavioural deficits in the rtg4510 mice are driven by Tau pathology, as shown by a recovery in performance once Tau expression is attenuated 297. Based on this it was hypothesised that owing to the efficacy in improving behaviour, Cu II (gtsm) treatment would also improve Tau pathology. Immunohistochemistry analysis revealed a significant reduction in the Tau pathology (NFT load) in the hippocampus of treated mice (figure 3.4) supporting the notion that Cu II (gtsm) would reduce Tau pathology. Additionally, immunoblot analysis found a significant Cu II (gtsm) mediated reduction in a higher MW species of Tau running at 100 kda (figure 3.11). Previous work has found that this 100 kda species of Tau correlates with behavioural deficits, our data supported this finding as a significant correlation between the level of this species of Tau and hyperactivity was observed (figure 3.12). The higher molecular weight band of Tau was found to be a dimer running at 100 kda, the group that identified this was unable to discover the functional significance of the species and whether it, itself was toxic or whether it was an indirect readout of another pathological process 306. Future experiments investigating the effects of stereotaxic injections of a purified form of this dimer could help uncover the toxic potential of the oligomer. The effects of Cu II (gtsm) on early oligomeric Tau in APP/PS1 mice (figure 2.4) taken with the data here suggest that Cu II (gtsm) treatment reduces oligomeric forms (sarkosyl-soluble Tau, NFT load and dimeric Tau). A systematic assessment of Tau phosphorylation utilising mass spectrometry would enable a fuller understanding of the changes occurring to Tau and 100

121 how this may impact both physiological function and give insight as to potential upstream targets (PP2A, GSK3β, CDK5 etc.) Cu II (gtsm) increases levels of PP2A(A) in rtg4510 mice Chapter 2 found that Cu II (gtsm) treatment of APP/PS1 mice produced an increase in the PP2A(A) subunit in both the preliminary study (figure 2.6) and the follow up study (figure 2.17). Based on this, it was hypothesised that Cu II (gtsm) would also produce an increase in the PP2A(A) subunit in the rtg4510 tauopathy. Indeed, as in section , transgenic rtg4510 mice had significantly lower levels of PP2A(A) than wildtype mice and Cu II (gtsm) treatment significantly increased this (figure 3.7). The reason why an increase in the A subunit resulted in improvements in Tau pathology is currently unknown. It would be reasonable to hypothesise that an increase in the A subunit (the structural subunit) of the holoenzyme would stabilise an active form of the holoenzyme and hence increase the engagement with Tau. It has been shown that an intact holoenzyme is required for Tau dephosphorylation 87,308 ; however, a deeper interrogation of PP2A biology would be required to confirm this. One could utilise mass spectrometry to identify the isoform of the various subunits to ascertain relative affinities for Tau, and a characterisation of enzyme activity towards Tau would provide further insight into this Cu II (gtsm) increases copper content of soluble proteins LC-ICP-MS (see section 6.6) analysis of hippocampal tissue from rtg4510 mice treated with either vehicle or Cu II (gtsm) showed incorporation of Cu II (gtsm) delivered copper into multiple proteins as indicated by multiple alterations of the 65 Cu: 63 Cu ratio in figure The first peak indicates a protein with a molecular weight matching that of SOD1. This is not surprising as SOD1 has an exceptionally high affinity for copper (see section 4.2) and a portion of any copper being delivered to a cell is likely to end up on SOD1. 101

122 The second, broader peak, corresponds to a variety of proteins of low molecular weight. Likely to be contained within this peak are copper binding proteins such as metallothionein, Hah1 and CCS; however, accurate identification of these proteins was not possible. However, the data does confirm that the delivered copper was indeed entering the cells copper metabolism pathway and confirmed that Cu II (gtsm) delivered bioavailable copper to cells within the brain. The LC-ICP-MS traces for iron (figure 3.14) and zinc (figure 3.15) illustrate metal dyshomeostasis in affected mice and furthermore that Cu II (gtsm) treatment is alters not only copper levels but also levels of iron and zinc providing evidence for the link between these metals. Alterations in the levels of these metals seen here combined with those observed in chapter 2 provide further evidence for the presence of metal dyshomeostasis in these two distinct but similar neurodegenerative models Discussion and future directions The data generated in the two rtg4510 dosing trials support the hypothesis that Cu II (gtsm) would be efficacious in treating the behavioural phenotype of the mice through a PP2A mediated reduction in Tau pathology. This chapter has shown that treatment with Cu II (gtsm) improved both cognitive and motor behaviour impairments and displayed a reduction in multiple forms of oligomeric Tau. Consistent with the hypothesis, drug treatment also produced an increase in the A subunit of PP2A. However, a causal link between the increase in PP2A(A) and the reduction in Tau pathology requires further experimentation. To further understand the mechanism by which Cu II (gtsm) was producing these effects would require a deep interrogation of the drug mediated changes in PP2A biology. This would include a full characterisation of the endogenous activators and inhibitors of the enzyme (see section or 96,109,290 ), the phosphorylation and 102

123 methylation state of the C subunit and identification/quantification of the isoforms to assess if there was an enrichment in isoforms with high affinity for Tau 87. Currently there are several strategies under investigation for treating Tau pathology such as utilisation of kinase inhibitors to prevent aggregation 309, small molecule mediated aggregation inhibition 310, enhancing processes to clear aggregated proteins such as the heat shock proteins 311, stabilisation of the microtubules 312,313 and in a similar strategy to that described in section , immunotherapy against Tau (or forms of Tau) For an extensive review of approaches to targeting Tau in neurodegenerative disease see 315,318,319. There are many different models of tauopathy tested; however, in the rtg4510 mouse model, the most promising strategy has been Tau immunisation 317. Interestingly, methylene blue which has been found to be successful in some models 320 has been shown to be unsuccessful in treating the rtg4510 model 321, illustrating differences in efficacy between mouse models. As such any therapy should be tested in multiple models to ensure reproducibility. Overall, the two studies described in chapter 3 support a PP2A mediated decrease in Tau pathology; however, more in depth analysis of Tau and PP2A biology will provide key insights to confirm this. This work also identified another strategy for treating Taumediated FTD and should be validated in other models of tauopathies to ensure reproducibility. 103

124 Chapter 4 - Cu II (gtsm) in Menkes disease (MD) 4.1 Introduction Menkes disease (MD) is a rare genetic x-linked disease affecting children resulting in a widespread copper depletion as a consequence of ineffective transport of copper from the diet and into cellular copper metabolism manifesting largely as neurological and connective tissue defects 322. An ideal treatment for this lethal disease would be an orally bioavailable compound that can deliver copper intracellularly. In addition to this it should also be able to cross the blood-brain barrier as this disease has severe neurological symptoms owing to almost complete brain copper depletion. Cu II (gtsm) has been shown to satisfy all these criteria and as such, this chapter describes a pilot study undertaken to assess the therapeutic potential for Cu II (gtsm) to treat MD History of Menkes Disease MD is an X-linked neurodegenerative disease of children. Clinically, the disease manifests as a failure to thrive, seizures, abnormally coloured and textured hair, hypotonia and ultimately death before teenage years. Early diagnosis can be difficult due to subtle disease manifestation early in life. The skin of neonates often appears loose, especially around the neck, trunk and axillae and newborns can exhibit other non-specific symptoms such as hypothermia, hypoglycaemia and jaundice. The disease is caused by mutations in the ATP7a gene encoding a transmembrane P-type ATPase copper transporter heavily involved in copper regulation and metabolism (also called the Menkes protein) due its capacity to pump copper across membranes Currently, the only treatment for the disease is daily copper-histidine injections for life with a better prognosis the earlier treatment is initiated (ideally within 2 weeks of birth) 326. This results in mild life extension (sometimes into adolescence) however with many neurological symptoms 104

125 remaining 326. The disease is considered rare with an incidence of ~1 in 100,000 live births. There is a diverse range of mutations causing MD The mutations causing MD consist of small deletions or insertions (22% of cases), splice junction mutations (18% of cases), nonsense mutations (18% of cases), missense mutations (17% of cases) and large gene deletions (17% of cases). The differing mutations in the ATP7a protein lead to differing reductions in copper transport and hence variations in severity. Additionally, there are other mutations in ATP7a which cause two related diseases occipital horn syndrome and ATP7a-related distal motor neuropathy. Typically, mutations causing MD result in ATP7a copper transport being reduced to 0-15% of healthy levels whereas mutations causing occipital horn syndrome and ATP7a-related distal motor neuropathy cause a reduction in ATP7a activity to 10-30% and 60-70% of healthy levels respectively The role of the Menkes protein (ATP7a) ATP7a is a multifunctional protein essential for copper homeostasis in the body. It is an eight-transmembrane protein with a large C-terminal tail. The N-terminal tail possesses six copper binding domains and copper is shuttled across the membrane upon ATP hydrolysis. Copper enters the body through enterocytes by the actions of divalent metal transporter 1 (DMT1) and copper transporter 1 (CTR1) and then enters circulation by the pumping action of ATP7a at the basolateral membrane. Once in the blood, copper is usually bound to albumin and transported to the liver where it can follow one of several fates. It is either stored within the liver (usually bound to small copper binding proteins called metallothioneins), re-entered into circulation (bound to the copper transport protein ceruloplasmin), or is excreted in the bile via the copper-transporting ATPase 2 (ATP7B, 105

126 also known as Wilsons protein, a distinct but highly similar protein to ATP7a). ATP7a is also present in the trans-golgi network where it is responsible for the incorporation of copper into cuproenzymes such as lysyl oxidase 345, peptidylglycine α-amidating monooxygenase 346,347, tyrosinase 348,349, extracellular super oxide dismutase 350, and dopamine β-hydroxylase 351 (see section 4.2 for a discussion on cellular copper metabolism). Importantly, when copper is elevated, ATP7a moves to the plasma membrane to efflux or facilitate systemic copper absorption 352. In addition to these functions, ATP7a has an important role in the central nervous system (CNS). Studies utilising the naturally occurring mouse model of Menkes disease (Mo/Br) identified a role for ATP7a in efflux of copper into the synapse. In a landmark study, Schlief et al. found that ATP7a is required for the efflux of an N-methyl-D-aspartate (NMDA) receptor-dependent pool of releasable copper and that this conferred resistance to excitotoxic insult via an antagonistic action on NMDA receptors 202. In this study, hippocampal neurons from the Mo/Br mice were markedly sensitive to NMDA receptor mediated excitotoxicity and had increased levels of caspase 3 activation and neuronal injury in vivo. The removal of protein from the media did not alter the level of protection afforded by the copper release suggesting that the copper was bound to an exchangeable pool of low affinity ligands such as amino acids, but not bound to a specific cuproenzyme in the synapse. Indeed, it has been shown that copper can act as a non-competitive inhibitor of NMDA receptors in cultured mouse and rat neurons 202. Furthermore, copper has been shown to reduce the repetitive firing of action potentials via potassium channels and voltage-gated sodium channels, ultimately inhibiting neurotransmitter release 353. Through these multiple pathways of reducing neuronal excitability, the authors suggest that this is the mechanism for the profound levels of neurodegeneration in both human disease and mouse models of the disease. 106

127 4.1.3 Treatment of Menkes Disease Peripheral injections of copper are not sufficient to restore physiological copper levels in the brain of MD patients owing to the lack of transport across the blood brain barrier. Early work with the mouse models of MD showed that there was some extension of life with CuCl2 injections and restoration of some copper dependent enzyme activity but this varied depending on the age of treatment Donsante et al. (2011) used Mo/Br mice on a C57BL/6 background obtained from JAX Laboratories, USA (the same strain used in the current study) and found that a peripheral (intraperitoneal) injection of copper (as CuCl2) was not sufficient to extend the life of treated mice more than 5 days over untreated mice and produced only a mild increase in brain copper 357. Indeed, human patients treated with copper-histidine peripheral injections suffered from on-going neurological conditions despite the extension of life afforded by the treatment 326. As such, there has been a focus on treatments to increase the copper concentration in the brain by using treatments increased the copper directly in the brain (either through adenoviral vector replacement of ATP7a 357,358 or through intracranial administration of copper) or by investigating copper containing compounds that can cross the blood brain barrier. The Cu II (btsc) compounds are viable candidates for increasing the levels of copper in the brains of MD patients given their ability to cross the blood brain barrier and be reduced to liberate bioavailable copper. Only one study to date has investigated the potential of these compounds to treat this disease 359. The study by Munakata et al. used the macular mouse model of MD (a more mild animal model of MD) and treated affected pups with Cu II (ptsm) on days 4, 10, and 17. At four weeks of age the animals were assessed for copper levels in various organs in addition to testing of the cuproenzyme, cytochrome c oxidase. The study used Cu II (ptsm) and CuCl2 as treatments and the appropriate vehicle controls. 107

128 The study found that while there were modest increases in copper levels in the brains of the treated animals, they had significantly improved cerebral and cerebellar cytochrome c oxidase activity when compared to CuCl2 treatment which suggests a more effective targeted delivery of copper to cellular enzymes in the brain. Additionally, Cu II (ptsm) produced significantly lower copper accumulation in the kidney which may be beneficial for long-term supplementation. These findings indicate that treatment of MD with additional BTSC derivatives is worth investigating. Other attempts at treating the disease have had limited success. Another study 357 used gene-therapy (by introducing non-mutant ATP7a expression in the choroid plexus) to attempt to correct the low levels of copper in the brain using the aggressive Mo/Br mouse model. They showed that at P12, untreated mice had around 25% of the copper in the brain compared to wildtype and gene-therapy combined with intracerebral injections of copper increased this to 50% of wildtype. They observed functional improvements in the form of an improved DOAPC:DHPG ratio which is an indirect measure of dopamine- β- hydroxylase activity. The gene-therapy treatment combined with intracerebral injections of copper did result in increased life expectancy; however, with median life span increased to 43 days (22% of treated animals survived to 300 days) compared with median survival of 13.3 days for untreated mice. Furthermore, treated mice gained weight that paralleled that of wildtype animals, despite starting at a lower level. Motor deficits as measured by the wire-hang test and rotarod, persisted through life (up to 300 days). The group hypothesised that delivery of copper to target enzymes in the secretory pathway (such as dopamine-β-hydroxylase) was likely via residual ATP7a activity, increased delivery to intracellular chaperones or via ATP7b. 108

129 Current treatment for MD is incredibly invasive and requires a significant commitment from both family and physician. In addition to this, the effectiveness of the therapy varies widely and depends on both the nature of the mutation and the age of diagnosis. These points illustrate a desperate need for novel treatment options that are less invasive, more efficacious or ideally both. This chapter describes the initial pilot study undertaken with an animal model of MD. 4.2 Copper metabolism The dietary requirement of copper for humans is 1-3 mg per day 360. Due to copper being both essential for life and potentially toxic, the movement of copper into, out of and throughout the cell (and indeed the organism) is under tight control. This section will describe the metabolism of copper in the cell and the main proteins involved in its control which are summarised in figure 4.1 adapted from Copper import into the cell Copper import into the cell is required for the correct and complete production of cuproenzymes and as such is under tight regulation. The first plasma membrane - associated copper transporter was discovered in S. cerevisiae, named copper transport protein 1 (Ctr1) 362. Indeed, S.cerevisiae cells lacking Ctr1 have altered mitochondrial energy production (lack of functional cytochrome-c-oxidase) and they also have altered iron metabolism 363. The presence of Ctr family proteins has been confirmed in mammals and humans and have striking sequence homology 364. The Ctr family of proteins all consist of an extracellular hydrophilic N-terminus rich in methionine amino acids, three transmembrane domains and a cytoplasmic C-terminus containing several highly conserved cysteine/histidine residues 365. The functional 109

130 complex exists as a homotrimer 366. Ctr1 preferentially binds Cu(I) over Cu(II) for transport and as such requires cell-surface metalloreductase activity for successful copper import (this can also be achieved using extracellular reductants such as ascorbate) The Met residues at the N-Terminus are important for facilitating copper intake in limiting conditions, but are not required for channel activity 370. Copper translocation is mediated by a strictly conserved MxxxM-sequence motif located in the second transmembrane 110

131 helix. This sequence provides ligands to facilitate Cu transport through the membrane Figure 4.1. Copper homeostasis in mammalian cells. Copper is taken up into brain cells by the copper transporter 1 (Ctr1). Also, the divalent metal transporter 1 (DMT1) may contribute to copper uptake. Since DMT1 and Ctr1 have been reported to prefer Cu + as substrate, an ecto-cuprireductase will provide the reduced copper species for uptake. Accumulated copper is sequestered by glutathione (GSH) or stored in metallothioneins (MT). In addition, copper is shuttled to its specific cellular targets by the copper chaperone for super oxide dismutase (CCS) to superoxide dismutase 111

132 1 (SOD1), by copper chaperone for cytochrome C oxidase 17 (Cox17) to synthesis of cytochrome C oxidase 1/2 (Sco1/2) and copper chaperone for cytochrome C oxidase 11 (Cox11) for subsequent incorporation into cytochrome C oxidase and by antioxidant protein 1 (Atox1) to ATP7A and ATP7B. Both, ATP7A and ATP7B transport copper into the trans-golgi network (TGN) for subsequent incorporation into copper-dependent enzymes such as tyrosinase (T), ceruloplasmin (Cp), dopamine-β-monoxygenase (DβM), peptidylglycine α-amidating monoxygenase (PAM) or lysyl oxidase (LOX). When the cellular copper level rises above a certain threshold, ATP7A and ATP7B translocate reversibly via vesicles toward the plasma membrane. ATP7A imports copper into vesicles for release by fusion with the plasma membrane and/or exports copper directly. Figure and caption adapted from 357. channel 370,371. Mutations in the putative binding cites (N-terminal methionine MPM motif, transmembrane MxxxM motif, or the C-terminal Cys/His residues) result in a different copper ion coordination 366. Mutational and structural studies thus far have created a translocation model where Cu(I) binds N-terminal methionine rich motifs extracellularly. Copper is then exchanged between highly specific Cu(I) binding sites involving conformational changes. The copper is subsequently transferred to the C-terminal Cys/His residues to act as donor sites for intracellular Cu binding proteins. This process of transfer of copper from Ctr1 to intracellular trafficking proteins is unclear, and it is not known whether the chaperones dock directly with Ctr1 or if there are other, yet unidentified, intermediates involved. The protein divalent metal transporter 1 (DMT1), while traditionally associated with iron uptake has also been implicated in copper uptake. DMT1 knock-out cells have reduced 112

133 uptake 372. Once in the cell, the copper enters one of several metabolic pathways (see figure 4.1) Intracellular distribution Copper chaperones Once copper has entered the cell via either CTR1 or DMT1, it binds to one of several copper binding chaperones which will determine where the copper is translocated (see figure 4.1). Cytochrome c oxidase copper chaperone (COX17) delivers copper to cytochrome C oxidase for a functional electron transport chain and COX17 deletions are embryonic lethal due to lack of cytochrome C oxidase activity 373. Copper chaperone for superoxide dismutase (CCS) mutant mice have decreased superoxide dismutase 1 (SOD1) activity, they are viable, but show reduced stress resilience and fertility consistent with the observed phenotype of a SOD1 knock-out mouse 374 confirming that CCS is a copper delivery chaperone for SOD1. Copper delivery to the mitochondria is controlled by an interplay of over eight copper chaperones involving COX17 and cytochrome C oxidase assembly protein (SCO1; reviewed in 375 ). Antioxidant 1 copper chaperone (ATOX1) is responsible for delivering copper to the secretory pathway via the ATPases, ATP7a and ATP7b 376. The ATOX1 knock-out mouse demonstrates characteristics of a peripheral copper deficiency, which is further exacerbated by a maternal ATOX1 deficiency establishing a possible role for ATOX1 in perinatal copper delivery 377. In addition to providing copper to the secretory pathway, ATOX1 can also move copper to the nucleus and affect cyclin D1 gene transcription Secretory pathway There are multiple enzymes in the cell for which copper is required. Several of these are processed through the Golgi and are destined for the secretory pathway such as tyrosinase, 113

134 peptidyl-α-monooxygenase (PAM), dopamine-β-hydroxylase (DBH), and ceruloplasmin. Indeed, X-ray fluorescence microscopy has revealed a kinetically labile pool of copper present in the Golgi 379. This copper makes its way to the Golgi via the chaperone ATOX1 (Hah1) 380 which binds one Cu(I) ion and transfers the copper to the P-type copper transporting ATPases, ATP7a and ATP7b, for incorporation into cuproenzymes 323,381. ATP7a and ATP7b are key regulators of copper homeostasis in the cell and their location within the cell is responsive to intracellular copper concentrations 382. The proteins can then pump the copper ions across a membrane into either the Golgi, into a vesicle or across the plasma membrane. It is via this last method that ATP7a translocates dietary copper across the basolateral membrane of enterocytes 383 and hence controls the levels of copper available to the body. ATP7b uses this mechanism to transport excess body copper into the bile for excretion out of the body from hepatocytes Ceruloplasmin and Hephaestin Ceruloplasmin is highly expressed in the liver and plays a role in iron homeostasis and distribution, providing an interface between copper and iron regulation 385. In fact, over 90% of circulating copper is bound to ceruloplasmin 386. Six copper atoms are required per protein for enzymatic activity 385. Mutations in the protein lead to a condition known as aceruloplasminemia which is characterised by iron accumulation in the brain and presents with anaemia and neurological dysfunction 387,388. Hephaestin is a membrane bound homologue of ceruloplasmin and plays a role in iron absorption in enterocytes 389. These two copper requiring proteins along with another multi-copper oxidase, zyklopen are responsible for systemic iron regulation, both in adults and during development 385,389,

135 Lysyl oxidase Lysyl oxidase is a copper requiring amine oxidase. It is responsible for the crosslinking of collagen and elastin, it does this through conversion of lysine residues to aldehydes which can then react with other similar species and cross link 391,392. This serves to increase the integrity and stability of connective tissue. Impairments in this purpose can be clearly seen in the copper deficient Menkes disease where sufferers show brittle hair and loose skin Dopamine-β-hydroxylase (DBH) and peptidylglycine α-amidating monooxygenase (PAM) DBH and PAM both have important roles in the central nervous system. DBH is responsible for catecholamine synthesis by converting dopamine to noradrenaline 393. Interestingly, perinatal copper deficiency has been reported to produce an increase in DBH levels and is probably a response to decreased copper 394. PAM is comprised of two subunits, a copper requiring peptidylglycine α-hydroxylating monooxygenase and a peptidyl-α-hydroxyglycine α-amidating lyase subunit who s sequential action is responsible for a myriad of active hormones such as vasopressin, gastrin, and cholecystokinin in the periphery and oxytocin, neuropeptide Y, and substance P in the central nervous system 395, Tyrosinase Tyrosinase is another copper requiring monooxygenase involved in the production of the skin pigment, melanin 397. This pigment is critical in protecting the organism from UV damage and deficiencies in the enzyme are exemplified by the copper deficiency disease, 115

136 Menkes disease (see section 4.1) where sufferers show hypopigmentation of the skin and hair The P-type copper transporting ATPases, ATP7a and ATP7b ATP7a and ATP7b are the largest contributors to copper levels in the cell and indeed the body. These P-type ATPases utilise, as the name suggests, ATP to transport ions across membranes and involve an autophosphorylation step. There are other P-type ATPases that exhibit the same mode of action, for example the Ca 2+ -ATPases, Na + /K + -ATPases and H + /K + -ATPases 398. While these different proteins have many differences in architecture, they share the same core structure. This starts with a cytosolic hydrophilic domain which contains the Actuator domain (A-domain) and the ATP binding domain. The ATP binding domain can be split into the phosphorylation domain (P-domain) and the Nucleotide binding domain (N-domain). The intramembrane channel is formed by several transmembrane helices and form the catalytic core. Transfer of copper begins with binding of ATP to the N-domain and metal binding to the transmembrane region. Following this, the enzyme is phosphorylated at the conserved aspartate residue present in the conserved DKTG sequence in the P-domain and this catalytic cycle follows classical E1/E2 dynamics 399,400. Following E1/E2 dynamics it is postulated that in the E1 state, the transporter has high affinity for the metal ions and faces the cytoplasm whereas in the E2 state the transporter has low affinity for the metal ions and faces the extracellular compartment (or intravesical compartment) and this results in release of the metal Copper sequestration Metallothioneins are a superfamily of low molecular weight metal binding proteins that have the ability to buffer metal levels in the cell and can act as sequestration mechanisms, but also have effects on gene transcription 401,402. It has also been suggested, that turnover 116

137 of these proteins can provide metals to the cellular metabolism in times of metal deficiency 403,404. This section has provided a brief overview of the major pathways in the cell that copper is be involved in; however, copper also has roles in intracellular signalling and mediating neuronal activity (see 203,344,360 for further review) The Mo/Br mouse model of MD Several models of Menkes disease exist containing different mutations within the ATP7a protein. The mottled-brindled model (Mo/Br) arose spontaneously on the C57BL strain in The model contains a six-base pair (in frame) deletion in exon 11. This mutation results in the removal of a leucine (position 799) and an alanine (position 800) from the native protein. The phenotype has many parallels to the human condition. Affected males have hypopigmentation of the fur, neurochemical abnormalities, curly vibrissae and exhibit signs of neurodegeneration as they age 354,355,406. Without intervention, affected males die at around days of age. Copper concentration is low in all organs except the gut mucosa, kidney and testis 407. Parenteral injection(s) of copper salts before 10 days of age is able to extend life and correct some of the phenotype Copper salt treatment can also restore copper concentrations to some organs and improve some cuproenzyme enzymatic deficiencies

138 4.3 Preface and rationale for MD pilot study As described above, there is currently no effective treatment for MD. A successful treatment would deliver copper into the cellular metabolism pathway and would not require functional ATP7a to distribute the copper throughout the body. Cu II (gtsm) fits these criteria; previous experiments 231,273 and the LC-ICP-MS experiments described in sections and show that Cu II (gtsm) treatment increases bioavailable copper and it has been shown be orally bioavailable and cross the blood-brain barrier 274. This makes Cu II (gtsm) an ideal candidate for the treatment of MD and as such a pilot trial was conducted to investigate this. The mice were purchased from JAX laboratories (USA) and a colony was established in the Florey Institute of Neuroscience and Mental Health animal facility for the generation of experimental animals. The Mo/Br homozygote pups are born with much lower weight than wildtype and heterozygote littermates and fail to thrive from that point, this results in a high rate of cannibalism of affected pups and an extremely low yield of experimental animals. Attempts were made to improve the breeding efficiency, these included: trio breeding, pair breeding, plugging and removing of male, sunflower seed supplementation, environmental enrichment, removal of light sources, removal of noise and foot traffic, and finally using surrogate mothers. All these measures were unsuccessful at increasing the breeding efficiency and as result and average of 0.8 Mo/Br homozygote mice survived to P7 and a substantial amount perished subsequently. This made the volume of data generated from the pilot study far less than anticipated. 118

139 4.4 Initial assessment of copper supplementation with Cu II (gtsm) in Mo/Br mice For the initial investigation of Cu II (gtsm) in Menkes mice, a protocol was developed to match that of previous studies that used copper chloride salts to improve symptoms. This was done to allow comparison with copper chloride treatment. This consisted of the administration of treatment at post-natal days 7 and 9 with 3.5 mg/kg of Cu II (gtsm). This low dose was used to limit toxicity to pups. On post-natal day 10, the mice were sacrificed and brain tissue was analysed by ICP-MS (section 6.4) to quantify metal levels in the brains of the animals. Figure 4.2 shows the copper levels (µg/g wet tissue) of the brains of wildtype mice, heterozygous littermates and homozygous littermates treated with either vehicle, CuCl2 or Cu II (gtsm). Heterozygote mice had approximately 50% of the copper of the wildtype littermates due to gene dosing of the protein (one functional copy and one non-function copy). Homozygous males had an extreme reduction in copper compared to wildtype mice (P < , 1-way ANOVA Dunnet s post hoc) which was to be expected 407. Interestingly CuCl2 treatment did not alter the levels of copper in the brain while Cu II (gtsm) treatment significantly increased the levels of copper in the brains to that of almost half of wildtype mice (P = , 1-way ANOVA Dunnet s post hoc). Upon treating homozygote mice with Cu II (gtsm), there was a change in coat colour observed within 16-hours. To document this, a group of four littermates were treated with Cu II (gtsm) or vehicle per the above schedule and photos were taken of the mice pretreatment and each day following treatment. 119

140 Figure 4.2. Cu II (gtsm) produces a greater increase in brain copper than vehicle of CuCl2 treatment Mice were treated on post-natal days 7 and 9 with either vehicle, CuCl2, or Cu II (gtsm) via an intraperitoneal injection. On post-natal day 10, mice were sacrificed and brain copper content was analysed by ICP-MS. Data represented as box and whisker plots show that homozygote mice exhibited a strong decrease in brain copper and only Cu II (gtsm) treatment significantly improved this. Statistical analysis by 1-way ANOVA Dunnet s post hoc, ns = not significant, ** = P < 0.01, **** = P < N=

141 121

142 Figure 4.3. Cu II (gtsm) treatment produces a colour change in the coats of treated mice. Sequential photos of homozygous male Mo/Br mice treated with either vehicle or Cu II (gtsm) on post-natal days 7 and 9. Cu II (gtsm) produced a darkening of the coat colour while vehicle treatment produced no change in coat colour. Figure 4.3 shows the colour change observed in the animals over time. The colour change was present in Cu II (gtsm) treated mice, but not vehicle treated mice from as little as 24- hours post treatment. To visualise the decreases in copper in the Mo/Br brain, brains of 10-day old mice (either wildtype or homozygous) were cut in 30 µm slices and mounted onto a glass slide. These slides were then ablated using LA-ICP-MS (section 6.5). Figure 4.4 displays LA-ICP- MS images for wildtype (72.3, 22.1, 26.1, 72,1) and homozygous Mo/Br (26.2, 77.3, 77.2, 72.4, 72.2) that showed an extreme depletion of copper in the brains of homozygous mice, agreeing with the LC-ICP-MS data in figure

143 Figure 4.4. Homozygous Mo/Br brains contain very little copper as imaged with laser ablation inductively coupled plasma mass spectrometry. 10-day old pups were sacrificed and brains fresh frozen and subsequently cut to 30 µm on a cryostat. Slices from position mm from bregma are shown for wildtype (72.3, 22.1, 26.1, 72.1) and homozygous Mo/Br (26.2, 77.3, 77.2, 72.4, 72.2) showing extremely low levels of copper in homozygous Mo/Br brains. 4.5 Treatment mediated changes in brain cuproenzyme activity The data from section 4.4 showing that Cu II (gtsm) delivered significantly more copper than vehicle and CuCl2 was very promising, but to assess if the delivered copper was functional, the activity of multiple cuproenzymes was assessed. 123

144 4.5.1 Preface The laboratory of Dr Peter Crouch in the Department of Pathology, University of Melbourne has expertise in assessing the enzymatic activity of cuproenzymes and, as such, a collaboration was formed with this lab. The enzymatic work described in this chapter was carried out by James Hilton and Dr Steve Mercer and figures are credited as required Superoxide dismutase 1 The quintessential cuproenzyme is the antioxidant enzyme SOD1 which catalyses the conversion of the free radical superoxide to hydrogen peroxide. The activity of SOD1 is dependent on a catalytic copper ion being incorporated into the holoenzyme 408. Due to technical difficulties with the assay this result was still pending at the time of writing Ceruloplasmin Ceruloplasmin is a copper binding enzyme that is involved in iron homeostasis throughout the body. It contains six copper ions and possesses copper-dependent oxidase activity 409. Figure 4.5, panel B shows the normalised ceruloplasmin activity measured and shows that the activity did not significantly differ between the wildtype and homozygote mice. Additionally, treatment did not significantly alter the activity of ceruloplasmin in the brain Dopamine-β-hydroxylase Dopamine-β-hydroxylase (DBH) is a copper-dependent enzyme important for neurotransmitter function. DBH catalyses the conversion of dopamine to noradrenaline. Human MD patients exhibit altered catecholamine metabolite ratios in the blood suggesting impaired function 326. When analysing the activity of DBH from brains of both 124

145 wildtype and Mo/Br mice, significantly increased percentage of DBH that is copperdeficient was observed (figure 4.5, panel C, P = 0.036, 1-way ANOVA Dunnet s post hoc). However, copper supplementation using either CuCl2 or Cu II (gtsm) did not significantly alter the percentage of copper deficient enzyme Lysyl-oxidases Lysyl-oxidases (LOX) are a family of enzymes involved in connective tissue formation through modification of collagen and elastin. These enzymes are copper-dependent and deficiencies in either the enzyme itself of supply of copper to the enzyme result in connective tissue abnormalities 410. Figure 4.5, panel D shows that the LOX activity was also unaltered in the homozygote Mo/Br mice compared to wildtype mice and treatment also did not affect the activity. 125

146 Figure 4.5. Brain cuproenzyme activity is largely unaffected in homozygous Mo/Br mice. Box and whisker plots for activity levels for four key cuproenzymes from Mo/Br and wildtype littermates whole brains. A) Ceruloplasmin activity was unaffected between any of the groups measured. B) LOX activity was not significantly altered between any of the 126

147 groups measured C) The percentage of DBH that is copper deficient was higher in Mo/Br mice compared with wildtype; however, copper supplementation by CuCl2 or Cu II (gtsm) did not improve this. Statistical analysis by 1-way ANOVA Dunnet s post hoc, * = P < N= Assessment of oral bioavailability of Cu II (gtsm) in Mo/Br mice The next important question was to ask if Cu II (gtsm) was orally bioavailable. Previous evidence in other mouse models of disease have shown that indeed the drug is bioavailable 231 ; however, it is important to assess if altering the function of ATP7a in the body would impair this. To assess this, a small pilot study was conducted; homozygous mice were dosed via oral gavage with a dose equal to those receiving the drug intraperitoneally (3.5 mg/kg). 24-hours after the dosing, the animals were sacrificed and the copper content of the brain was analysed by ICP-MS. Figure 4.6 shows that 3.5 mg/kg Cu II (gtsm) either orally or by IP produced comparable levels of copper in the brain 24-hours post treatment indicating equal bioavailability. 4.7 Major conclusions from chapter 4 and discussion Menkes disease is a widespread copper deficiency disease with many of the symptoms being attributable to deficiencies in key cuproenzymes. Owing to the ability of Cu II (gtsm) to deliver bioavailable copper to cells it was hypothesised that treatment of MD with Cu II (gtsm) would rescue the severe phenotype. Also, owing to the difficulty CuCl2 has crossing the blood-brain barrier, it was hypothesised that Cu II (gtsm) would deliver greater amounts to the brain. 127

148 ICP-MS analysis of brain tissue (figure 4.2) indicated that, as expected, CuCl2 produced no overall increase in copper in the brain due to the inability for copper to cross the blood-brain barrier. However, Cu II (gtsm) treated mice had almost double the copper in their brain as untreated animals. This confirmed that Cu II (gtsm) was able to deliver copper to the brain and in a manner superior to copper salt treatment. This also explained the inability for copper salt treatment to effectively treat the neurological symptoms in the human disease. Treatment also produced evidence that, at least peripherally, cuproenzyme function was being restored. This was evidenced by a coat change colour within 16 hours of treatment (figure 4.3). This provides evidence that the cuproenzyme, Figure 4.6. Orally gavaged Cu II (gtsm) increases brain copper to a similar level as IP injection. tyrosinase 355, had its function restored upon Homozygous Mo/Br mice were copper supplementation confirming that the delivered copper was, at least peripherally bioavailable. However, given that Cu II (gtsm) produced a significantly higher level of brain copper, it was hypothesised that treatment would produce improvements in brain cuproenzymes in addition to peripheral cuproenzymes. dosed with 3.5 mg/kg Cu II (gtsm) either by oral gavage or IP injection, Cu was quantified 24- hours later by ICP-MS. Oral gavage brought copper to a level comparable to that of IP injection. N=

149 To test this, brain tissue was utilised to test four major cuproenzymes activities. To our surprise, none of the cuproenzymes we tested (with the exception of DBH) were deficient in Mo/Br homozygous mice. It is not surprising then that treatment also did not produce alterations in the activity of these enzymes (figure 4.5). This was initially surprising, however, given the very high affinity for copper of SOD1 it is reasonable to assume that what copper does make it into the cell will end up bound to SOD1 until it is fully metallated. Furthermore, ceruloplasmin was loaded with copper by ATP7b 411, which is fully functional in these animals 338, and as such the copper loading pathway is unaffected for this protein. The LOX activity assay gave values which were remarkably low when compared with LOX isolated from peripheral tissue (personal communication, Dr Steve Mercer). This may indicate that this enzyme is not usually active in the brain and hence is unaffected by copper deprivation. DBH is loaded with copper by ATP7a and as such it would be expected that there would be abnormalities in the Mo/Br mice, additionally, there is evidence in humans that this enzyme is defective 326. Our data supported this, as DBH was significantly copper deficient compared with wildtype mice. Treatment was unable to rescue this deficiency. Treatment was given in two bolus doses over three days and the animals were sacrificed the following day therefore this may not have been enough time, or indeed copper, to produce a significant increase in activity. A longerterm study with more frequent doses would address this question. A preferable attribute for a drug is to be orally available to provide a less invasive treatment than an injection. This is especially true for MD where the current gold standard for treatment is daily injections of copper salts into the foot. This is invasive for the infant/child, requires daily practitioner access, and repeated injections could cause local inflammation. To that end, we tested the oral bioavailability of Cu II (gtsm) and found that 129

150 at the same dose it produced the same increase in brain copper as IP injection. This provides evidence that oral dosing was viable and effective with Cu II (gtsm) Discussion and future directions The data from this preliminary study has supported aspects of the initial hypothesis and raised questions about aspects of it. The data suggests that Cu II (gtsm) is superior to CuCl2 in achieving delivery of copper to the brain in Mo/Br mice; however, this increase did not affect the enzymatic activity of the cuproenzymes tested here. As described above, this may be due to the short dosing window and if more regular copper, over a greater length of time, were to be used perhaps there would have been increased activity of DBH. Additionally, there may be other molecular targets that the copper is interacting with to either initiate intracellular signalling cascades or perhaps other cuproproteins not measured in the current study (such as cytochrome-c-oxidase or peptidylglycine α- amidating monooxygenase). Furthermore, additional analysis is currently underway to assess cuproenzyme activity in the periphery of Mo/Br mice to assess the relative efficacy of CuCl2 compared with Cu II (gtsm). Cu II (gtsm) treatment improved brain copper over CuCl2 treatment and has shown evidence of improving the peripheral cuproenzyme deficiency combined with demonstrated oral bioavailability. This demonstrates that Cu II (gtsm) is not only a viable treatment for MD, but also has the benefit of being orally bioavailable. This preliminary study has shown that further work should be carried out to assess suitability for the clinic. Such work would include a dose-response study in combination with toxicity testing to establish efficacy and toxicity windows. Additionally, a long-term study with the Mo/Br model should be undertaken to assess changes in cognition later in life and whether Cu II (gtsm) could influence these phenotypes. Finally, a systematic assessment of 130

151 cuproenzyme biology in treated animals should be undertaken to assess efficacy and to predict if treatment may relieve the neurological symptoms. 131

152 Chapter 5 - Conclusions and future directions The BTSC class of compounds are excellent candidates for development as therapeutics. They are small, orally bioavailable, cross the blood-brain barrier and have been shown to be efficacious in treating a variety of neurodegenerative models including Alzheimer s disease (AD), Parkinson s disease (PD), and amyotrophic lateral sclerosis (ALS) (reviewed in 270 ). The two compounds of focus have been Cu II (atsm) and Cu II (gtsm) which differ slightly in structure, but differ largely in biological action (see discussion in section 1.4). Cu II (atsm) appears to influence nitrosative stress by neutralising the peroxynitrite radical and this is thought to be the mechanism of action in treating animal models of PD 412. Cu II (atsm) has also been shown to be very effective in treating animal models of ALS Interestingly, Cu II (gtsm) displays toxicity in treating PD (Hung et al. unpublished data). Furthermore, Cu II (gtsm) has been efficacious in treated a mouse model of AD ( 231 and chapter 2) while Cu II (atsm) has shown no efficacy in treating AD 231. This suggests distinct mechanisms of action between the two compounds. The work carried out for this thesis has sought to examine the mechanism of action of Cu II (gtsm) in treating the APP/PS1 mouse model of AD and build on the groups previous findings 231. A preliminary study followed by a larger follow-up trial were conducted to assess reproducibility of both behavioural improvements and alterations in key pathological biomarkers (Aβ and Tau). The data generated in chapter 2 show that Cu II (gtsm) consistently improves spatial memory performance and supports previous data 231. Biochemical analysis revealed improvements in Tau hyperphosphorylation and consistent increases in the structural subunit of the Tau phosphatase PP2A. Furthermore, SELDI-TOF analysis showed no alterations in any of the Aβ species detected. This 132

153 effectively placed Aβ upstream of Tau pathology and was consistent with the notion of AD being an Aβ driven tauopathy. Seeing that Cu II (gtsm) reduced Tau pathology in AD mice, it was hypothesised that treatment would also yield improvements in an animal model of other tauopathies such as FTD. The preliminary trial (conducted by Dr Lin Hung) and the follow-up study (conducted as part of this thesis) supported this hypothesis showing improvements in cognition and hyperactivity/anxiety. Consistent with the findings in the APP/PS1 mice, treatment with Cu II (gtsm) also reduced Tau pathology in the rtg4510 mice and increased levels of the structural subunit of PP2A. These data suggest that Cu II (gtsm) was effective in treating tauopathies through increasing the levels of the PP2A(A) subunit. We hypothesised that this increase in the structural subunit results in (or is the result of) a stabilisation of the PP2A holoenzyme and results in increased Tau dephosphorylation. PP2A deficits are indeed common to both diseases, PP2A levels and activity have been shown to be decreased in AD 96,103, the P301L mutation causing FTD results in decreased binding of PP2A to Tau and hence Tau hyperphosphorylation 256,416. The data produced here validates PP2A as a drug target for treating tauopathies and acts as a proof-of-principle that PP2A can be effectively targeted. This is especially important as strategies to target this enzyme are in their infancy utilising small molecule screens against proteins involved in the methylation of PP2A 109,417 Given the ability of Cu II (gtsm) to cross the blood-brain barrier and deliver copper to the brain, it was hypothesised that Cu II (gtsm) treatment of the copper deficiency disease, Menkes disease, would result in improvements in the phenotype. A preliminary study supported this hypothesis showing increased copper delivery to the brain and 133

154 improvements in hypopigmentation. No improvements in brain cuproenzyme activity were detected; however, given the short dosing schedule it is possible that more robust improvements would be seen over time with frequent dosing allowing adequate protein turnover. Furthermore, the fact that Cu II (gtsm) was shown to be orally bioavailable and improved brain copper levels to the same level as injected compounds makes this an extremely attractive therapeutic for the reduced invasiveness oral administration offers. With successful treatment of APP/PS1 mice in two trials and rtg4510 mice in two trials, this thesis shows the Cu II (gtsm) is viable candidate for further development for the treatment of tauopathies. The results in the Mo/Br mice also suggest further development for the treatment of Menkes Disease. 5.1 Future directions The link between increasing bioavailable copper and PP2A levels remains elusive. Our working hypothesis is that copper administration improves the copper status of enzymes involved in the methylation cycle. Through improving flux through this cycle (see Figure 2.22, page 69), the methylation status of PP2A is increased and thus the holoenzyme is stabilised (see the discussion in section 3.4.5). This hypothesis could be testing by assaying the methylation status of the PP2A enzyme, but also investigating aspects of the methylation cycle (such as homocysteine levels). A global assessment of methylation (such as DNA methylation) would also confirm changes in the methylation cycle. Furthermore, immunoprecipitation of PP2A holoenzyme complexes followed by mass spectrometric identification of subunit subtype would reveal whether there was an enrichment of holoenzyme and additionally whether there was an enrichment of subtypes resulting in increased Tau binding, such as the PR55 B subunit. 134

155 Development of Cu II (gtsm) in Menkes disease is an attractive prospect. Future work would involve ageing the animals past weaning with regular Cu II (gtsm) dosing followed by a histological assessment of the brain (to assess neuropathology) combined with an assessment of brain cuproenzyme activity. Currently, treatment of MD involves daily injections in affected children which results in mild improvements. Cu II (gtsm) improves brain copper beyond that of copper salts (the current gold standard of treatment) and is orally bioavailable. These findings suggest Cu II (gtsm) would be an excellent candidate for treating MD. 135

156 Chapter 6 - Methods 6.1 Chemicals The Cu II (gtsm) used was synthesised in the Department of Chemistry, University of Melbourne, by Brett Patterson, Lachlan McInnes and Paul Donnelly as described previously 418,419. All other chemicals were obtained from Sigma-Aldrich (Castle Hill, Australia) unless specified. 6.2 Biochemistry Tissue homogenisation Tissue weight was determined and a volume equal to four times the tissue weight was added of homogenisation buffer (tris buffered saline (TBS) supplemented with EDTAfree protease inhibitors and phosphatase inhibitor cocktail (Roche)). The tissue was then homogenised with a stick homogeniser on ice or by probe sonicator. The sample was then clarified by centrifugation for 10 minutes at 4 C at a speed of 13,000 RPM. The resulting TBS soluble fraction was pipetted into a clean 1.5 ml microcentrifuge tube for further analysis. Samples were then stored at -80 C Protein quantification (BCA Assay) Protein quantification was carried out using the Pierce BCA Protein Assay Kit (ThermoFisher Scientific) per manufacturer s instructions. Protein standards were created ranging from 2 mg/ml to 25 µg/ml using the kit provided bovine serum albumin (BSA) stock solution, this was diluted into the appropriate tissue storage buffer. The BCA reagent was prepared by mixing reagent A with reagent B (at a ratio of 50:1) and mixing 200 µl of this with 5 µl of sample (usually diluted 1:10 to bring into the working range 136

157 of the assay) in a 96-well plate (get brand). Following a 37 C incubation for 30 min, then an absorbance reading at 562 nm was taken in duplicate or triplicate Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) To be run on SDS-PAGE gels, samples were first diluted to 2 mg/ml in homogenisation buffer. 2X sample buffer (0.25 M Tris, 8% v/v SDS, 20% v/v glycerol, 0.4% bromophenol blue) was added at a ratio of 2:1. Samples were then boiled at 96 C for 5 minutes before being cooled on ice. Immediately prior to gel loading, samples were vortexed to ensure a homogenous mixture. Samples were pipetted into wells of BioRad TGX 4-20% gels. Gels were run at 200 V for ~30 min or until dye front had reached the end of the gel. Gels were then removed and exposed to UV light for 2.5 min in the BioRad MP Gel Imager to activate the gel and confirm correct running of the gel. 6.3 Western blotting Transfer Transfer of proteins from SDS-PAGE gel to nitrocellulose membrane was carried out as per manufacturer s instructions. Briefly, stacks were created (see Figure 6.1). The stack was then placed in the gel cassette and into the BioRad TurboBlot and run using the BioRad Mixed MW protocol (7 min at 2.5 A). The membrane was then rinsed in TBS and imaged using the BioRad MP Imager (this image was used as an indication of total 137

158 Figure 6.1. Diagram of transfer stack organisation. protein loaded and resulting densitometry values utilised to normalise immunoblot images to) Immunoblotting Membranes were in blocked in blocking solution (TBS with 0.1% Tween-20 and 5% low fat milk powder). Following blocking, membranes were incubated with primary antibody diluted in blocking solution. The membrane was then washed three times in TBS-T (TBS supplemented with 0.1% Tween-20) followed by incubation with the appropriate secondary antibody. Incubation times were either overnight at 4 C or for 2 hours at room temperature. Following incubation with the secondary antibody the membranes were washed three times with TBS-T and imaged using chemiluminescence using the BioRad MP Imager. Table 2. List of antibodies and concentrations used. Antibody Manufacturer Concentration Total Tau Dako 1:50,

159 p-tau 396 CST 1:2,000 p-tau 404 CST 1:2,000 p-tau 202 CST 1:1,000 Protein Phosphatase 2A(A) CST 1:1,000 Protein Phosphatase 2A(B) CST 1:10,000 Protein Phosphatase 2A(C) CST 1:1,000 Glycogen Synthase Kinase 3β CST 1:1,000 p-glycogen Synthase Kinase 3β-9 CST 1:1,000 Synaptophysin CST 1:1,000 Post synaptic density-95 CST 1:1,000 Glial acidic fibrillary protein CST 1:1, Densitometry Images were analysed using ImageLab software (BioRad, USA). Densitometry values were normalised to total protein values (obtained from BioRad MP imager above) to give an abstract normalised value. This was then normalised to the average value of the control group Sarkosyl extraction of Tau Brain tissue was first homogenised and clarified by centrifugation (see tissue section). The insoluble (pellet) fraction was then resuspended in 50 µl of cold buffer H (10 mm Tris-HCl, 1 mm EGTA, 0.8 M NaCl, 10% w/v sucrose, ph 7.4) and centrifuged at 27,200 x G (Beckman ultracentrifuge TLA-55 Rotor) for 20 min at 4 C. N-lauroylsarcosine was 139

160 then added to the supernatant (1% v/v) and this was incubated at 37 C with spinning for 90 min. Following the incubation step, the mixture was subjected to ultracentrifugation at 150,000 x G (Beckman ultracentrifuge TLA-55 Rotor) for 35 min at 20 C. The resulting supernatant was pipetted into a clean 1.5 ml Eppendorf tube and the pellet was resuspended in 10 µl 50 mm Tris-HCl buffer (or 0.5 µl per mg or starting tissue weight). To analyse this, the resulting supernatant and pellet samples were run on SDS-PAGE (as per the section and analysed via Western blot. The membrane was probed with the Total Tau antibody (see appendix 2) and for each sample the ratio of the pellet to the supernatant was calculated. This value was used for quantification and analysis. 6.4 Inductively coupled plasma mass spectrometry (ICP-MS) Samples of brain homogenate were placed into Eppendorf tubes and stored at 20 C. Samples were diluted in a 1% HNO3 solution prior to processing. The ICP-MS was performed using an Ultramass 700 (Varian, Victoria, Australia) set in peak-hopping mode, with spacing at AMU, 1 point per peak, 50 scans per replicate, at least three replicates per sample, and dwell time of 10,000 msec. Plasma flow was 15 litres/min, with auxiliary flow 1.5 litres/min. RF power was 1.2 kw. Sample was introduced using a glass nebuliser at a flow of 0.88 litres/min. The apparatus was calibrated using a 1% HNO3 solution containing Al, Fe, Co, Mn, Cu, and Zn at 5, 10, 50, and 100 ppb, with Y39 the internal standard for all isotopes of Cu and Z. 140

161 6.5 Laser ablation inductively coupled plasma mass spectrometry (LA- ICP-MS) Mice brain sections cut from Bregma mm were mounted on microscope slides and imaged for their metal concentration using LA-ICP-MS as described recently 420. Briefly, 25 mm thick cryostat sections were cut on microscope slides, air-dried overnight and placed in a 10 x 10 cm ablation cell together with matrix-matched elemental standards 421. The sections were ablated using a 30-mm square laser spot size at a speed of 120 mm/s scanning speed on a NWR213 ablation system (Kennelec Scientific, Mitcham, Victoria, Australia). Ablated material was transferred into the Agilent 8800 QQQ-ICP- MS (Mulgrave, Victoria, Australia) using an argon gas flow at 1.2 L/min and analysed for the following isotopes: 13C, 31P and 66Zn. Data were analysed using the Iolite analysis software (School of Earth Sciences, University of Melbourne) operating under the Igor Pro suite. Carbon (13C) and phosphorus (31P) channels were used for signal normalisation. 6.6 Liquid chromatography inductively coupled plasma mass spectrometry (LC-ICP-MS) Fresh ammonium nitrate running buffer (500 ml) was prepared and adjusted to ph by dropwise addition of concentrated ammonium hydroxide. The certified internal standard ion (CsCl) was added to a final concentration of 10 ppb and the solution was filtered through a 0.2 µm membrane. The size-exclusion column was equilibrated in running buffer at 25 C. PEEK tubing was connected exiting the UV detector to the ICP- MS nebuliser inlet Standards of SOD1 and FTN were prepared by diluting in TBS. SOD1 to a final concentration containing 200 ppb Cu and Zn, while FTN to a concentration containing 2000 ppb Fe. A metalloprotein standard curve for Cu and Zn (SOD1) and Fe 141

162 (FTN) by injecting a series of volumes for both protein standards (e.g. 1, 5, 10, 25 and 50 µl) was created. TBS phase tissue extract ( µg total protein) was injected ensuring the injection volume was kept minimal ( 50 µl). The samples were normalised to the same protein concentration (typically 1 3 mg/ml) to facilitate use of identical injection volumes and simplify data analysis. The HPLC system and size exclusion column was run at ml/min flow rate, column temperature maintained at C, and back-pressure below manufacturer suggested limits. UV absorbance was recorded at 280 nm (A280) and counts for the elements of interest and internal standard ion were collected. Data was plotted as counts per second (CPS) data for each element against the column elution time. 6.7 RNA analysis Total RNA was isolated from hippocampal sections using the mirneasy RNA kit (Qiagen), according to the manufacturer s protocol. RNA integrity was assessed by using the RNA Nano 6000 kit and the 2100 Bioanalyser (Agilent). RNA samples that had a ribosomal integrity number of at least 7 were selected for qrt-pcr analysis. Reverse transcription was performed on 1 µg of total RNA using the High Capacity cdna conversion kit (Applied Biosystems). cdna samples underwent qrt-pcr (TaqMan Fast Advanced Master Mix, Applied Biosystems) using LCMT1, PPME1, PPP2R1A and PPP2CA TaqMan Gene Expression Assays (Applied Biosystems) and run on the ViiA 7 Real-Time PCR System (Applied Biosystems). No template and reverse transcriptase controls were also prepared to ensure there was no background amplification. For data normalisation across samples, HPRT was used as an endogenous control gene. Normalisation of Ct values of each gene and determination of fold differences in gene expression (normalised to lesioned mice) was calculated by the 2-142

163 Ct method. Sample size of each group is indicated in the graphs under the respective bar area. 6.8 Enzyme assays Caeruloplasmin The purpose of this assay is to monitor the progression of ceruloplasmin ferroxidase activity in tissue samples through the production of holo diferric transferrin from apotransferrin. Iron exists typically in the form of Fe2+ (ferrous sulphate and ferrous ammonium sulphate), and ceruloplasmin is used to oxidise iron to the Fe3+ state, which is the state required for binding to transferrin. To measure this, a plate reader is utilised and set to 460nm, where activity can also be measured spectrophotometrically. 200mL of HEPES buffered saline (HBS) was prepared by dissolving g 4-(2- hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) and g NaCl2 in 150 ml of MilliQ (MQ) water then ph corrected to 7.2 using 1M NaOH. MQ water was then added until the buffer is made up to 200mL. MQ water was purged with N2 or He2 to mitigate non-specific iron oxidation prior to use. 1 ml of apotransferrin solution (250 µm) was prepared fresh by dissolving 19.62mg of human apotransferrin into 1 ml of MQ water. 50 ml of ferrous sulphate (1 mm) was prepared fresh by dissolving 13.9 mg of ferrous sulphate into 50 ml of MQ water. Reagents were added to a 96-well plate in the following order: 1. 50µL of MQ water 2. 10µL of HBS 3. 20µL of sample preparation 4. 40µL apotransferrin solution 143

164 5. 20µL ferrous sulphate/ferrous ammonium sulphate (addition of this initiates reaction). Activity was measured by monitoring the rate of change at 460 nm over 5 minutes, with a measurement every seconds. This allowed for the calculation of a linear range Lysyl-oxidase (LOX) Activity for LOX was measured from Triton-X soluble fractions using a fluorometric assay that detects the production of fluorescent resorufin from the substrate Amplex UltraRed (Thermo Fisher Scientific). An assay buffer was prepared from 50 mm sodium borate, 1 M urea and 10 mm CaCl2 made up in distilled H2O (ph 8.0), and this buffer was then used to make a 4 mm benzylamine (Sigma) solution. Equal volumes of Triton- X soluble sample in assay buffer and benzylamine solution were then loaded into a 96- well plate and incubated at 37 C for 30 minutes. A substrate mixture was prepared from 2 U ml -1 horseradish peroxidase and 40 M Amplex UltraRed made up in assay buffer. After incubation, each well was loaded with an equivalent volume of substrate mixture and LOX activity was determined via plate reader by measuring excitation and emission wavelengths of 544 nm and 590 nm, respectively. The end-point readings of relative fluorescence units were then used to calculate LOX activity with respect to equivalent amount of recombinant LOX protein (OriGene). Wells containing equivalent volumes of TBS (1x) supplemented with 1% Triton-X 100 in the absence of sample protein were also used to control for non-specific activity. 144

165 6.8.3 Dopamine-β-hydroxylase (DβH) Activity for D H was measured from Triton-X soluble fractions using tandem mass spectrometry (LC-MS/MS) to monitor enzymatic production of the D H product norepinephrine. Triton-X soluble samples were added to individual microfuge tubes then combined with a reaction mixture containing 200 mm sodium acetate, 30 mm N- ethylmaleimide, 5 M CuSO4, 50 L/mL catalase (Sigma), 10 mm sodium fumarate and 10 mm ascorbate made up in distilled H2O (ph 5.0). Following pre-incubation at 37 C for 5 minutes, reactions were initiated by adding 10 mm dopamine and then incubated at 37 C for 45 minutes. A known concentration of epinephrine was added to each sample tube as an internal standard, followed by 1 ml of 100 mm ammonium dihydrogen phosphate (ph 10) supplemented with 2% (v/v) stabiliser (0.5 M EDTA, 317 mg/ml sodium metabisulfite). Each sample was then subject to solid phase extraction using Bond Elut phenylboronic acid (PBA) 100 mg, 3 ml cartridges (Agilent). Cartridges were equilibrated with 1 ml acetonitrile followed by 5% (v/v) formic acid made up in methanol, then samples were added to the cartridges. After sample addition, a further 1 ml of 100 mm ammonium dihydrogen phosphate supplemented with 2% (v/v) stabiliser was administered to the cartridges. Next, the matrix was washed sequentially with 2 ml of 1% (v/v) ammonium hydroxide in 95% (v/v) methanol, 2 ml of 1% (v/v) ammonium hydroxide in 95% (v/v) acetonitrile, then 1% (v/v) ammonium hydroxide in 30% (v/v) acetonitrile. Once the matrix was dried under vacuum, analytes were eluted using 3 x 500 L aliquots of 5% (v/v) formic acid in methanol and then evaporated in a vacuum concentrator before being reconstituted in 0.3% (v/v) formic acid made up in distilled H2O. 145

166 LC-MS/MS analyses were performed using the 1100 series HPLC system (Agilent), and the 4000 QTRAP LC-MS/MS system (Sciex) equipped with a TurboIonSpray ion source. The system was run in Micro mode using a mix rate of 400 L/min, with the column compartment set to 50 C and samples kept at 20 C. Catecholamine analytes were separated using a Hypercarb column (150 mm x 1 mm, 5 m particle size, Thermo Fisher Scientific) at a flow rate of 50 L/min. Initial run conditions used 99% buffer A (0.3% (v/v) formic acid in dh2o) and 1% buffer B (100% acetonitrile) for 1 minute followed by a gradient to 25% buffer B within 20 minutes, then 80% buffer B within 2 minutes. Conditions were then held at 80% buffer B for 2 minutes before a return to 1% buffer B within 2 minutes and holding at 1% buffer B for 6 minutes. The QTRAP was set to positive ion mode using the multiple reaction monitoring (MRM) scan type, and conditions were spray voltage set to 4200V, source temperature set to 425 C, collision gas set to high, with source gas 1 and source gas 2 set to 20. A time of 100 ms was applied to each transition resulting in a duty cycle of seconds, with Q1 and Q3 resolutions set to Unit. Data were collected using the Analyst Build 5218 (Sciex) operating in MRM mode. Catecholamine analytes were quantified using the MultiQuant 2.1 (build ) software package (Sciex) through integration of signal peaks for norepinephrine, dopamine and epinephrine. Activity levels were calculated with respect to norepinephrine levels following reactions, and were presented as amount of norepinephrine produced per minute of reaction incubation per mg of sample protein 6.9 Animal experiments All animal experiments conformed to the Australian National Health and Medical Research Council published code of practice for animal research. Experimentation was 146

167 approved by the Florey Institute of Neuroscience and Mental Health ethics committee (ethics approval numbers , and ). All mice were obtained from Jax Laboratories (USA) unless otherwise stated with the genetics being elaborated on in specific chapters Animal husbandry Mice were kept in Techniplast IVC cages with free access to mouse chow and water. All animals were maintained on a 12-hr light-dark cycle. The cages were kept with a bed of sawdust and mouse were given a solid enclosure as enrichment along with paper Furl nesting material (Able Scientific ). Food and water levels were checked daily during weekdays by staff members of the Core Animal Services, Florey Institute of Neuroscience and Mental Health. After treatment initiation, animals weighed daily by experimenters (including weekends) to determine drug dose and to monitor adverse reactions to treatment. For the MD experiments, a colony was derived from Jax Laboratories (strain ID: ) and maintained in the breeding area in the Florey Institute of Neuroscience and Mental Health animal facility. All breeding and husbandry of the breeding colony was undertaken by staff members of the animal facility while maintenance of the colony for experimental purposes was carried out by myself Animal dosing Oral gavage dosing Prior to oral gavage, the needle was flushed, the mouse weighed and dose calculated. The mouse was then placed on a surface and gently scruffed to immobilise its body and head. Once immobilised, the gavage needle was gently inserted into the animal s mouth to the 147

168 depth of its oesophagus taking care not to enter the trachea. The correct volume of liquid was then injected slowly and the needle withdrawn. For adults, a 23-gauge gavage needle was used and for pups, and 29-gauge was used, these needles are repeat use and are stored in ethanol between uses Intraperitoneal dosing For intraperitoneal injections, the animal was weighed and the appropriate dose was calculated. The animal was then placed on a surface and gently scruffed. The mouse was then held belly up on a 45-degree angle with its head facing downwards to allow proximal movement of the internal organs. A 1 ml syringe with a 26-gauge needle was then inserted into the peritoneal space and the correct volume injected slowly and the needle slowly withdrawn as per Drug solubilisation Drugs were dissolved by probe sonication in Standard Suspension Vehicle (SSV; NaCl 0.9 % w/v, carboxy methyl cellulose 0.05 % w/v, Benzyl alcohol 0.05 % v/v, Tween % v/v). Sonication was carried out in 2-3 rounds of 10 second sonication set at 35-50% amplitude until mixture was homogenous. Mice were dosed at a specified dose in mg/kg and this was calculated daily from body weight. Vehicle treated mice were gavaged an equivalent volume of SSV Y-maze All Y-mazes were carried out during the light phase of the animals circadian cycle. The Y-maze is a uniform colour and consisted of three arms each with an angle of 120. The Y-maze arm measured 80 mm (width) x 300 mm (length) x 150 mm (height). At the outermost end of each arm, a laminated cue was placed above it. The animals were given 148

169 a minimum of one hour to acclimate to the conditions of the room (light/smells etc). After which time the mouse was placed in the home arm with access to the novel arm blocked off (giving the mouse access to the home arm and the familiar arm only). This was the acquisition phase and the mouse is allowed one hour to explore the maze. The mouse was then removed from the maze and placed in its home cage for one hour (giving a 1 hour inter-trial interval). The mouse was then placed back into the home arm, this time with access to the novel arm allowed. This was the test trial and the mouse was allowed five minutes to explore the maze. These trials were conducted in the mouse behaviour rooms in the Florey Institute of Neuroscience and Mental Health on a light setting of 5 and in silence, the external cues were randomised for each mouse. The sawdust was replaced between each mouse trial to remove any olfactory stimuli. Due to the naturally inquisitive nature of the mice, upon being exposed to a novel environment (the novel arm in the test trial) the mouse will preferentially explore it (spend ~50% of its time in the novel arm); however, when working memory is impaired, all arms (home, novel, and familiar) will appear as novel and hence the mouse will spend equal amounts of time in all three arms (~33%). During both trials, the mouse s movements were tracked in real time using digital tracking software (CleverSys, USA). The software used the mouse s XY coordinates combined with the dimensions of the maze to give information about the time spent in the sections of the maze (home arm, familiar arm, novel arm and centre). The run order was randomised prior to experimentation. 149

170 6.9.4 Morris water maze The Morris water maze used in here has been adapted from the original study 423 to reflect modern equipment, tracking software and search strategy analysis see 279. The Morris water maze protocol used in chapters 2.3 and chapter 3 was identical to the methodology used in 279. The protocol used in the current study was carried out with training from the lead author of the above paper, Mr Jake Rogers, and the identical room and cue setup was used (see figure 6.2). Figure 6.2. Morris water maze room setup. Morris water maze experimentation room drawn to scale for each cue saliency paradigm. The hidden platform position is visible in the SW quadrant of the pool, from which the four distal start locations (NW, N, E, and SE) can be extrapolated. The computer and the experimenter (not illustrated) were hidden behind a large curtain in the NE corner of the room during training in both paradigms. The high saliency cue paradigm, characterised by a significant increase in both the number and the size of 2D and 3D cues. This included revealing the heating lamps, holding containers and corridor to the room door by 150