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Synucleinopathies are a group of diseases characterised by the presence of insoluble aggregated forms of α-synuclein. The most common of these diseases is Parkinson’s disease (PD) which affects approximately 1% of the UK population over the age of 60. Alpha-synuclein has also been linked to the disease through familial mutations and genome wide association studies as well as by its presence in sporadic cases. Although solid evidence exists for a role of α-synuclein in PD, it remains unclear as to how this protein exerts its toxicity on neurons and exactly how this leads to the cell death characteristic of this neurodegenerative disease. Alpha-synuclein belongs to a family of three proteins which also includes β- and γ-synuclein. These three proteins are highly homologous and evolutionarily conserved, however none of them have a well defined function. Evidence suggests a role for these proteins in synaptic vesicle dynamics but a more specific function remains to be unveiled. However, due to the considerable degree of homology across these three proteins, knockout models have been considered to allow functional compensation of the missing synuclein protein through one of the remaining family members. This has hindered studies from elucidating not only the role of α-synuclein but also β- and γ-synuclein. To overcome this problem triple synuclein knockout mice have been produced and characterised, as described in this thesis. As expected studies of these animals revealed no alterations in the number of dopaminergic neurons in either the substantia nigra pars compacta or ventral tegmental area. Despite this, a significant deficit in striatal dopamine concentrations was detected, regardless of the fact that the levels and function of tyrosine hydroxylase being normal. As well as this triple synuclein null mice were demonstrated to be hyperdopaminergic through various behavioural tests. Work employing physcostimulants and, through a collaboration, using fast scan cyclic voltametry suggested a role for these proteins in normal dopamine release dynamics at the level of the synaptic vesicle. A previous body of work has indicated that the loss of α- and/or γ-synuclein is able to provide a degree of resistance against the toxic affects of the dopaminergic neurotoxin MPTP. It was therefore hypothesised that the triple synuclein null animals would also display resistance to this toxin. However, these animals were shown to be more sensitive than wild type controls. Importantly it was apparent that animals lacking β-synuclein alone or in combination with other synucleins were the most sensitive to this toxin. Further work revealed a significant deficit in the ability of triple synuclein null mice to store dopamine in their synaptic vesicles. This may explain the sensitivity to MPP+, the active metabolite of MPTP, due to the fact it cannot be efficiently stored in synaptic vesicles, which restricts the toxins access to the mitochondria where it normally inhibits complex I, thus leading to cell death. When recombinant β-synuclein was reintroduced the deficit in synaptic vesicle dopamine uptake could be restored. However, β-synuclein can not do this alone and requires incubation with cytosolic factors, suggesting it acts as a chaperone in this role. This may explain why lines of synuclein null mice that specifically have the absence of β-synuclein apparently fair least well when exposed to MPTP. Finally, in order to assess the extent to which a loss of function role of α-synuclein leads to pathological alteration at the synapse an entirely novel conditional α-synuclein knockout mouse model was produced. Currently no ideal model exists to answer this question as conventional knockout models are based on the knockout of the protein in development. This may allow functional compensatory mechanisms to be established which can be overcome with a conditional knockout approach. As well as this it is important to assess this loss in an aged nervous system, as PD is a disease of aging. It is likely that, as α-synuclein forms insoluble Lewy bodies and undergoes abnormal posttranslational modifications, the amount of normally functioning protein at the synapse is depleted, therefore allowing a loss of function effect to develop. It is hoped this model will allow new insight into the early disease process. Overall this work further contributes to a body of evidence that suggests the synucleins play an important role in synaptic dopamine handling, particularly at the synaptic vesicle level. It is hoped that the newly established conditional α-synuclein knockout model will produce a new perspective on the loss of function role of α-synuclein in early disease development, an avenue that has yet to be fully explored.

Alzheimer's disease (AD) is an insidious age-related neurodegenerative disease and the most common type of dementia. AD neuropathology typically involves the accumulation of extracellular plaques as well as intracellular neurofibrillary tangles that mainly consist of amyloid-beta (Abeta) and hyperphosphorylated tau aggregates, respectively. Previous studies in animal models lacking the expression of Mapt, which encodes for microtubule-associated protein tau that is implicated in AD neuropathology, demonstrated that Abeta-driven toxicity is tau-dependent. However, inconsistent reports of Abeta-driven toxicity in Mapt homozygous knockout (Mapt-/-) animal models have emerged while a human MAPT-/- model remains unavailable to corroborate whether tau is essential to mediate the toxic phenotypes caused by Abeta in human genetic backgrounds. In this thesis, I describe the generation of the first human MAPT-/- induced pluripotent stem cell (iPSC) lines in collaboration with the Cowley research group in Oxford. Two isogenic panels of MAPT-/-, MAPT+/- and MAPT+/+ iPSC lines, which were derived from two parental iPSC lines of healthy individuals, were identified. These iPSC lines were differentiated into cortical neurons in parallel and these neurons exhibited similar levels of cortical marker and synapse expression regardless of their MAPT genotypes. However, tau ablation in the iPSC-derived cortical neurons was sufficient to cause neurite outgrowth impairments, alterations in mitochondrial transport along axons, and reduced electrophysiological activities. Upon the exposure to extrinsic Abeta insults in the forms of synthetic oligomers or AD brain-derived Abeta, the MAPT+/+ neurons experienced cytotoxicity, reduced number of anterograde motile mitochondria along axons, synapse loss, and hyperactivation in vitro. These adverse phenotypes could be reversed in the MAPT-/- neurons whereas Abeta-induced neurite outgrowth impairments were not rescued in the MAPT-/- neurons. Notably, having a reduced dosage of tau expression in the MAPT+/- neurons was enough to be protective against cell death caused by Abeta. The study on Abeta-driven toxicity was extended to iPSC lines derived from the sporadic AD patients who participated in the Deep and Frequent Phenotyping pilot cohort. Synapse losses caused by extrinsic Abeta insults were quantified in thirteen lines of patient iPSC-derived cortical neurons and these neurons displayed varying levels of synaptic vulnerability to Abeta across patient lines. Their individual vulnerability to Abeta in vitro was demonstrated to reflect clinical vulnerability to Abeta burden measured by cognitive readouts from the same patients in vivo, suggesting that AD modelling with iPSCs is a tractable method to study individual levels of disease vulnerability. Here I present the experimental evidence on the effects of tau deletion in human iPSC-derived cortical neurons and show that tau is required in certain aspects of Abeta-driven toxicity. The clinical relevance of studying Abeta-driven toxicity using sporadic AD patient iPSC-derived cortical neurons was also investigated by demonstrating that individual clinical vulnerability to Abeta can be modelled in vitro using these neurons.

Nigrostriatal dopamine neurons have long, highly arborised axons, indicating a requirement for axonal translation. The axonal 'translatome' will reflect the neuronal response to local stressors that precedes cell death in Parkinson's disease (PD) as well as revealing key transcriptional differences between protected and vulnerable dopaminergic populations. The translating ribosome affinity purification (TRAP) technique enables the capture of translating ribosomes specifically in dopaminergic neurons by expressing an enhanced green fluorescence protein (eGFP)-tagged ribosomal subunit driven by a cell type-specific promoter. We have produced a novel transgenic mouse line by crossing a human alphasynuclein overexpression model, previously generated by our lab, with a dopamine transporter (DAT)-Cre driven TRAP line. This thesis describes the collection and analysis of translating mRNA from dopaminergic cell bodies and terminals of 3 and 18-month control and Parkinsonian mice for sequencing. The expression features that best distinguished dopaminergic neurons from neighbouring cell types were first identified. This information was used to prioritise genes located within linkage regions of PD GWAS loci. Novel candidate genes were identified, including the Calcium Sensing Receptor. Comparison of axonal and somal samples demonstrated compartmental specialisation, with autophagic function, MAPK signalling, and markers of axonal homeostasis preferentially expressed in axons. Comparison between dorsal and ventral striatal axons identified a series of genes with greater dorsal expression and prior evidence of a role in axonal degeneration. These genes were considered putative markers of selective vulnerability. With age, coordinated changes in expression were observed in genes related to autophagy, oxidative stress and endocytic recycling. These changes were specific to the dopaminergic neuron and can be used to better understand the role of each gene in dopaminergic function. Results from this thesis have been collated and summarised in the form of a public dopaminergic expression atlas.

Parkinson's disease (PD) is the second most common neurodegenerative disorder worldwide. PD is characterised by the preferential loss of dopaminergic neurons in the Substantia Nigra pars compacta in the midbrain accompanied by progressive motor dysfunction. The precise aetiology of PD is unknown, however a causative role of Leucine-rich repeat kinase 2 (LRRK2) has been proposed. Mutations in the LRRK2 gene are the most frequent cause of familial PD and are also an independent risk factor for sporadic PD. Although the function of LRRK2 is not well characterised, a role of LRRK2 in the autophagy pathway has been suggested. The disruption of the autophagy pathway by LRRK2 pathogenic mutations has been described. However, the literature is often contradictory and the exact underlying mechanisms remain unknown. In this study, primary cortical cultures were generated from three bacterial artificial chromosome (BAC) transgenic (TG) rat models of PD harbouring either the whole human wild-type LRRK2 gene, the G2019S mutant (the most common LRRK2 mutation) or the R1441C mutant (the LRRK2 mutation leading to a more aggressive pathology). After characterising the autophagy pathway it was observed that the presence of either hWT-LRRK2 or LRRK2-G2019S inhibits autophagosome biogenesis in primary cortical cultures. hWT-LRRK2 and LRRK2-G2019S each localise to the Golgi apparatus where autophagy signalling complexes are situated, which may underlie the inhibitory effect on autophagosome biogenesis. The presence of LRRK2-R1441C, however, induces a lysosomal deficit that causes an accumulation in autophagosomes and decreased autolysosome maturation and lysosomal protein degradation. LRRK2-R1441C also increases lysosomal pH levels and causes lysosomal Ca2+ release deficits. Furthermore, hWT-LRRK2 and LRRK2 G2019S were found to bind to the a1 subunit of the v-type ATPase pump (vATPase a1) which is responsible for modulating lysosomal pH. Whereas LRRK2-R1441C showed a loss of binding capacity to this subunit, which was associated with a decrease in a1 subunit protein expression and cellular mislocalisation. Lastly, the Zn2+ ionophore, clioquinol, was able to rescue the LRRK2-R1441C-mediated lysosomal phenotypes through modulating lysosomal zinc levels and increased vATPase a1 expression which re-acidified lysosomes, corrected localised calcium release and increased autolysosome maturation. These data describe a novel functional link between LRRK2 and vATPase a1, and define critical mechanisms underlying the inhibition of autophagy by different pathogenic LRRK2 mutations. These findings demonstrate a novel mode of action by which drugs may rescue lysosomal dysfunction and alleviate blockage in autophagic flux. These results demonstrate the importance of LRRK2 in lysosomal biology, as well as the critical role of the lysosome in PD and the potential of lysosome-targeting compounds as a novel therapeutic for the disease.