Tau is a microtubule associated protein found in inclusions in tauopathies including Alzheimer's disease. One known cause of neurodegeneration is an excess of exon 10 inclusion in tau mRNA which is caused by several mutations in the MAPT gene, encoding tau. Processing of the Amyloid Precursor Protein (APP) generates P- amyloid (Ap) peptides which are deposited as amyloid plaques in AD brain. APP transcripts containing alternatively spliced exon 7 are increased in AD brain and processing of APP exon 7 containing protein isoforms may result in increased AP production. Recently, the DNA and RNA- binding protein, Tar DNA binding protein of 43 kDa (TDP-43) inclusions have been found in 20-35% of AD cases as well as other tauopathies. TDP-43 has roles RNA processing regulation including pre-mRNA splicing. Cytoplasmic inclusions ofTDP-43 result in a loss of nuclear TDP-43 and suggest a loss of TDP-43 function that may impair its role in multiple RNA processing events. Our hypothesis is that TDP-43 dysfunction affects tau and APP RNA processing either directly or indirectly. We analysed post-mortem from human brain tissue and found an increase in severity of tau pathology associated with the presence ofTDP-43 inclusions suggesting that TDP-43 dysfunction may modify tau pathology in AD brain. We found increased expression of tau exon 10 that correlated with increased expression of APP exon 7 in a subset of AD cases however there was no association between presence of TDP-43 inclusions and altered tau or APP splicing.

Parkinson’s disease (PD) is characterised by a progressive loss of dopaminergic neurones from the SNpc, leading to numerous downstream changes in the basal ganglia circuitry. Overactivity of the glutamatergic subthalamonigral pathway may underlie this continual degeneration of the nigrostriatal system. With this in mind, this thesis examined whether selective activation of group III metabotropic glutamate receptor subtypes may offer a novel strategy to halt persistent degeneration in PD. Initial distribution studies revealed mGlu4 and 7 group III mGlu receptor subtypes, demonstrated particularly intense immunoreactivity in the SNpc, suggesting these receptors may be ideally positioned to provide neuroprotective effects. Therefore, the first objective was to confirm this neuroprotective possibility using a broad spectrum agonist, L-AP4. Sub-chronic supranigral L-AP4 treatment mediated functional neuroprotection against a unilateral 6-OHDA lesion of the SN, confirmed by behavioural assessment and post-mortem analyses. Secondly, the pharmacological identity of the group III mGlu receptor mediating this protective effect was examined. To investigate mGlu4 receptors, the novel mGlu4 selective PAM VU0155041, was also shown to provide functional neuroprotection in the 6-OHDA rat model to an almost comparable level reached with L-AP4. Whilst these neuroprotective effects are likely mediated by an inhibition of glutamate to protect from glutamate-mediated excitotoxicity, VU015504 also led to a significant reduction in levels of GFAP and IBA-1 suggesting an additional anti-inflammatory action. Further studies revealed little evidence for co-localisation of mGlu4 receptors with GFAP in the SN suggesting this anti-inflammatory component likely reflects an indirect effect via stimulation of neuronal mGlu4 receptors. Finally, to investigate mGlu7 receptors, the selective allosteric agonist AMN082, was also shown to protect the nigrostriatal tract and demonstrate a degree of preservation of motor function. In contrast, mGlu8 receptor activation using the selective agonist DCPG, failed to protect the nigrostriatal tract or preserve motor behaviour. Collectively, these findings demonstrate that, of the group III mGlu receptors investigated, mGlu4 offers the most potential as a promising target for establishing disease modification in PD.

The number of neuroimaging studies has grown exponentially in recent years and their results are not always consistent. Meta-analyses are helpful to summarize this vast literature and also offer insights that are not apparent from the individual studies. While a number of suitable voxel-based meta-analytic methods for neuroimaging data had been developed at the time this thesis was conceived, they also suffered from a series of important drawbacks such as the separate analyses of positive (e.g. grey matter volume increases) and negative (e.g. grey matter volume reductions) findings, not accounting for the effect size, not taking sample size, intra-study variance or between-study heterogeneity into account, and not allowing combination of reported peak coordinates and statistical parametric maps. The aim of this thesis was the development of a series of voxel-based meta-analytic methods and software tools for neuroimaging studies, which overcame some of the limitations of previous methods. Specifically, this thesis includes: a) the development of a new voxel-based meta-analytic method, named signed differential mapping (SDM), which adopted and combined the various positive features of previous methods and also introduced a series of improvements and novel features; b) the subsequent development and adaptation of the method to allow addressing additional research questions, such as meta-analyses comparing several disorders, meta-analyses of white matter volume or fractional anisotropy studies, and combination of various imaging modalities; and c) examples of applications of these methods. The methods and software derived from this thesis have been well received by the field. As of September 2012, more than thirty meta-analyses using SDM have been published, and the first study introducing the methods has been cited more than a hundred times. Suggestions for future research and further methodological development are discussed.

Defects in axonal transport are an early pathological event in Alzheimer’s disease, suggesting that damage to the transport process could contribute to the disease. A large number of cargoes are transported through axons and mitochondria represent a particularly important cargo. This is because mitochondria need to be distributed to axonal regions where their functions in ATP synthesis and the regulation of calcium homeostasis are required. This is particularly important in synaptic regions where neurons have high energy and calcium-buffering requirements. As such, mitochondria are transported bi-directionally through axons and this movement is responsive to physiological stimuli. has been shown to disrupt axonal transport of mitochondria but these studies involved application of synthetic Ap to neuronal culture media. In this thesis, the effect of the expression of a familial Alzheimer’s disease mutant amyloid precursor protein (APP) that increases Ap production (the APP Swedish mutant) on axonal transport of mitochondria was studied. This approach represents a more physiological route for studying the effect of APP and Ap on axonal transport. Mitochondrial axonal transport was monitored in cultured neurons via time-lapse microscopy. Expression of APPswe led to a selective disruption of anterograde but not retrograde axonal transport of mitochondria. Mitochondria are transported anterogradely on the microtubule based molecular motor kinesin-1. Mitochondria attach to kinesin-1 via the outer mitochondrial membrane protein Mirol and the adaptor protein TRAK1. Defective mitochondrial transport induced by APPswe did not affect the binding of kinesin-1 to mitochondria. Rather, APPswe caused mitochondria with associated Mirol, TRAKl and kinesin-1 to detach from the microtubule rails. In order to gain insight into the molecular mechanisms that underlie the APPswe effect on transport and to test potential therapeutic approaches, a number of different Alzheimer’s disease-associated features were experimentally manipulated. In particular, inhibition of APP processing and A|3 production with the y-secretase inhibitor DAPT, inhibition of glycogen synthase kinase-3 and increased tubulin acetylation all rescued the APPswe-induced transport defect. These results provide novel insight into the mechanisms underlying defective axonal transport in Alzheimer’s disease and provide new information on how some proposed Alzheimer’s disease therapeutics might act at a molecular level.

Insulin resistance, part of the metabolic syndrome, is associated with type 2 diabetes mellitus (T2DM) and increased risk for Alzheimer’s disease (AD). The e4 allele of APOE is the greatest genetic risk factor for sporadic, late onset AD, and is also associated with risk for T2DM. The thiazolidinediones (TZDs), PPARy agonists, are peripheral insulin sensitisers used to treat T2DM and have been found to slow cognitive decline in mild to moderate AD patients. Since it is not yet clear how PPARy affect insulin signalling processes in AD, I investigated the effects of the TZD, pioglitazone, on Ap metabolism and tau phosphorylation in high fat diet (HFD)-induced insulin resistant mice carrying the human APOEeS or APOEe4 genes. HFD reduced tau phosphorylation at specific epitopes, independent of APOE genotype. Pioglitazone treatment reduced tau phosphorylation, in an APOE-dependent manner, with the APOEeS mice being most responsive. Examination of HFD effects on the cortical transcriptome revealed increased expression of the ABCA1 gene in mice lacking APOE and the ADRA2A gene in APOEe4 mice. ABCA1 and apoE are associated with Ap clearance in the brain, while ADRA2A suppresses insulin secretion and polymorphisms in this gene is associated with T2DM risk. Erk1/2, p38, JNK, and calcineurin were differentially expressed between APOEeS and APOEe4 mice on HFD indicating these kinases and phosphatases may contribute to the increased risk for AD imparted by APOEe4. I have identified APOE allele-dependent effects of PPARy agonists on tau phosphorylation and have identified diet and APOE allele-dependent effects on gene expression that relate to AD. Further work is needed to validate current findings and to further elucidate the mechanism. A better understanding of the relative importance of brain and peripheral insulin resistance will greatly improve our understanding of the disease process and also aide the rational design of therapeutics to halt its progression.

Pelizaeus-Merzbacher disease (PMD) is an X-linked neurological disorder characterised by dysmyelination of the central nervous system. The major molecular defect involved in PMD is a variably sized duplication of Xq22 including the PLP1 gene. Determining the copy number of PLP1 is the first line of enquiry when trying to establish a molecular diagnosis of PMD. Multiplex amplifiable probe hybridisation is a quantitative technique that has been explored as a method for diagnosing PLP1 duplications in PMD patients and female carriers as part of this project. The rearrangements in a number of PMD families were investigated using FISH, PCR and sequencing of chromosome breakpoints. One tandem duplication and two more complex rearrangements were characterised during this study. A bioinformatic investigation of sequences present at the breakpoints in these families, and the genomic sequence throughout Xq22, has provided some insights into possible mechanisms causing duplications of this region. Increased dosage of Xq26-27 has been associated with pituitary hypoplasia and a mutation involving the SOX3 gene, located at Xq27.1, has been reported in a family with growth hormone deficiency. Characterisation of a duplication involving SOX3 in a family with X-linked hypopituitarism has been carried out using a similar strategy to the PLP1 duplications. This SOX3 duplication is the smallest yet reported and only contains two other genes. This provides strong evidence for increased dosage of SOX3 being involved in the aetiology of X-linked hypopituitarism. Gene dosage is increasingly being recognised as a cause for human genetic disorders, and it is important for diagnosis that changes in gene dosage can be reliably detected. As more cases of human genetic disease involving gene dosage become apparent, it is clear that duplications are frequent occurrences in the human genome, which may often be undetected as a cause of human genetic variation and disease.

The use of transgenic models in recent years has shown that there is a relationship between PrP expression level and disease progression. Lower-than-normal expression leads to extended incubation period and clinical phase (Beuler, 1993; Manson et al., 1994). In contrast, overexpression shortens incubation period or generates novel neurological syndromes associated with degeneration of skeletal muscle, peripheral nerves and the CNS (Westaway et al., 1994). Natural differences in expression of PrP<SUP>C</SUP> resulting from a variety of mutations within the promoter, enhancer, intron or non-coding sequences could therefore play a part in determining whether a host is either susceptible or resistant to disease. This project was designed to investigate the potential of the sheep PrP gene 3'-untranslated region (UTR) to control expression of a reporter gene in neuroblastoma (N2a) cells of mouse origin. Interest in this region of the PrP gene results from sheep PrP genetic studies (Hunter et al., 1991), PrP mRNA (Hunter et al., 1994) and protein studies (Horiuchi et al., 1995). Sequence analysis of the PrP gene 3' UTR region showed that it was polymorphic and contained instability motifs and potential alternative polyadenylation signals. The deletion of 2.7 kb from the PrP gene 3' UTR was shown to result in altered expression of chloramphenicol acetyl transferase (CAT) at the post-transcriptional level in N2a lines but not in cell free systems. Further constructs were made by unidirectional deletions in the 3' UTR and results suggested that differences in the length of the 3' UTR can affect expression at the transcription and/or post transcriptional level. Additional experiments showed by deletion and site directed mutagenesis that an alternative polyadenylation signal ATTAAA at position 1525 can be used in 3' end processing of RNA in N2a cells. These findings may have implications both for the expression of the PrP gene in the natural host of scrapie and for development and progression of disease.

Stroke is a leading cause of morbidity, disability and cognitive impairment. Approximately, 25% of post stroke survivors develop dementia in the subsequent five years after initial cerebrovascular injury. However, the majority of individuals maintain their cognitive function. The cognitive function after stroke (CogFAST) study was established to assess the long term consequences of ischemic injury in the elderly, determine the types of dementia inherent in stroke survivors and to elucidate the pathological substrates of cognitive impairment. The aim of this specific study was to determine whether regenerative mechanisms, specifically angiogenesis and neurogenesis, could explain the variance in cognitive status of post stroke survivors; those who succumbed to dementia (PSD) compared to those who did not (PSND). We also compared subjects with Alzheimer’s disease (AD) and vascular dementia (VaD) and aged match controls. Quantitative histopathological methods were used to assess microvascular density and neurogenic markers in post-mortem human tissue. Vascular density was assessed using a morphometric technique based on stereology and found significant increases in both PSD and AD compared to all other groups. A significant increase in vessel diameter was also observed in PSND compared to other groups. Changes in expression of pro-angiogenic molecules hypoxic inducing factor 1α, integrin and vascular endothelial growth factor were assessed, with an increase reported in PSD and AD. Differences in expression of neurogenic markers in the dentate gyrus were noted, generally an increase was observed in a number of markers in VaD subjects compared to other groups. Significant decreases in expression in PSND and AD were also found compared to control groups. A reduction in expression was reported, in all dementia groups, for Hu C/D. Further analysis was performed using the neuronal marker Hu C/D in the hippocampus, which has been suggested to be involved in neuronal maintenance and plasticity. An increased in expression in the Cornu Ammonis 1 (CA1) in PSND subjects compared to all dementia groups. Significant reductions were also reported in VaD cases in a number of regions of the hippocampus compared to controls and PSND subjects. Although, significant differences were found between PSND and PSD cases, the data did not produce conclusive evidence that either angiogenesis or neurogenesis could explain the maintenance of cognitive function in PSND subjects. A number of similarities were also found between PSD and AD subjects, which may suggest an underlying similar mechanism.

Parkinson’s Disease (PD) is a progressive neurodegenerative condition characterised by deterioration of the dopaminergic (DA) neurons of the substantia nigra pars compacta (SNpc). The exact mechanism by which SNpc cell death in PD occurs is poorly understood but several lines of evidence implicate both environmental and genetic factors. Trichloroethylene (TCE) is an industrial solvent used as a degreasing agent and in dry cleaning. TCE is a major environmental contaminant in the air, the water system and soil and is therefore exposed at low levels to various groups in the population. There have been links between chronic exposure to TCE and Parkinson-like symptoms reported and in vivo experiments support this link. It has also been discovered that TCE can be converted, via chloral, to a potentially DA neurotoxin TaClo in man. This project investigated this link between TCE and PD and to elucidate any causative mechanisms. Cellular exposure paradigms were used to show neurotoxicity of TCE, chloral and TaClo in DA neurons. The mechanism of this cell death in TaClo was found to be necroptotic - and not apoptotic - in nature and involve induction of autophagy, DNA damage and an increase in reactive oxygen species (ROS) in exposed cells. A possible source of this ROS was suggested with the findings that TaClo significantly inhibits Complex I of the mitochondrial oxidative phosphorylation chain - an effect known to produce superoxide - and an increase in mitochondrial ROS seen in cells following TaClo treatment. The cell death induced by chloral was found to follow a different path, with neither apoptotic or necroptotic characteristics observed in exposed cells, and Complex I only inhibited at high doses. However, chloral was found to block the reduction of cytochrome c at lower doses, a property that may be involved in the neurotoxicity seen with chloral. Animal models of TCE, chloral and TaClo exposure found no significant motor or cognitive abnormalities in behavioural testing of either wild-type mice or rats or human A30P mutated α-synuclein overexpressing mice. However, TCE and TaClo exposed wild-type and A30P mice did show a significant decrease in DA neuronal number and density in the SNpc, suggesting both compounds are neurotoxic to DA neurons in vivo. An LC-MS/MS assay was developed to assess neurotransmitter levels in the brains of toxin exposed animals, however the method was not found to be consistently accurate and showed extreme variability in results. In conclusion, the main results of this thesis suggest that TCE does lead to DA neurodegeneration in the SNpc of exposed individuals, probably through metabolism to the neurotoxic compound TaClo. The neurotoxic properties of TaClo are DA specific and relatively potent. The mechanism of neurotoxicity is hypothesised to be through inhibition of mitochondrial Complex I, inducing increased ROS production and damage of intracellular organelles, DNA and proteins, which in turn leads to the activation of autophagy and PARP activation. This intracellular stress instigates RIP1 mediated necroptosis and death of the cells.

This thesis examines the application of cerebral diffusion weighted imaging (DWI) and short echo time (TE) proton magnetic resonance spectroscopy (1H-MRS) for the evaluation of patients with different forms of human prion disease. Human prion diseases are progressive, uniformly fatal neurodegenerative diseases and as treatments are developed, early diagnosis is essential. Particularly important is the diagnosis of presymptomatic cases and prediction of disease onset in these individuals. In this thesis I demonstrate that MRI measures of Apparent Diffusion Coefficient (ADC) at low and high b-value and short TE 1H-MRS are potential neuroimaging biomarkers of prion disease activity. I show that ex-vivo MRI at high field provides important insights into the microstructural changes underlying the sensitivity of some of these quantitative MRI methods to prion disease pathology. The findings presented here exemplify the potential of quantitative MRI in both increasing our understanding of the pathophysiology of prion diseases and in providing neuroimaging biomarkers which will be of great importance for the future evaluation of treatment efficacy.