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Parkinson’s disease (PD) is a neurodegenerative disorder characterized by the progressive loss of nigral dopaminergic (DA) neurons and the formation of Lewy bodies. Despite most cases being idiopathic, mutations in several genes have been implicated in familial forms of PD. In particular, recessive mutations in Parkin gene (PARK2) are the most common cause of young-onset inherited parkinsonism. Parkin is an E3 ubiquitin ligase involved both in the control of mitochondrial turnover and in the proteasome-dependent degradation of proteins, two pathways that have been causally linked to PD development. Although initially described as a recessive disorder, experimental evidence suggests that heterozygous Parkin mutations can exert dominant toxic effects causing neurodegeneration. In 2012, Ruffmann and colleagues identified the first pure heterozygous R275W Parkin patient with clinical features of typical late-onset PD and a diffuse Lewy body pathology. To assess the impact of R275W Parkin, we generated the first mouse line carrying Parkin R274W mutation, which corresponds to the human R275W substitution. Unlike Parkin deficient mouse models, both homo- and heterozygous R274W mice show an age-related motor impairment, degeneration of dopaminergic neurons and neuroinflammation. We detected structural and functional mitochondrial abnormalities related to PARIS-PGC-1α axis impairment in R274W+/+ mice brain and skeletal muscle. Strikingly, we noticed signs of protein aggregation in both R274W+/- and +/+ mice, while we identified bona fide Lewy bodies only in the midbrain of heterozygous mice. Additionally, in the brains of R274W mice we discovered overt abnormalities of the glymphatic system, the main route for brain waste clearance. Our preliminary observations suggest that Parkin influences aquaporin-4 (AQP4) localization. Altogether, our data suggest that R274W Parkin substitution behaves both as a loss ofand a gain of toxic function, highlighting a link between Parkin dominant toxicity and age-dependent motor impairment, neuroinflammation, DA neurons loss, glymphatic system dysfunctions and α-synuclein aggregation in vivo. Hence, our study provides a new robust mouse model to explore PD pathogenesis and glymphatic dysfunctions, offering the possibility to test novel therapeutic strategies with great predictivity.

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Protein misfolding and the accumulation of insoluble aggregates is a hallmark of several neurodegenerative disorders, including Alzheimer’s (AD) and Parkinson’s disease (PD). In AD and PD patients, extracellular protein deposits consisting of amyloid beta (Aβ) and intraneuronal inclusions composed of alpha synuclein (αS) are observed, respectively. Notably, the spatiotemporal patterning of soluble protein oligomers of αS and Aβ closely follow disease progression, giving support to an emerging role of soluble oligomers in PD and AD pathogenesis. However, the structural features underlying the toxicity of Aβ and αS oligomers remain elusive. This doctoral dissertation aims at elucidating the structural determinants of oligomer toxicity by focusing on the development and application of multidisciplinary approaches based primarily on solution NMR in combination with electron microscopy, multi-angle light scattering, fluorescence microscopy, wide-angle x-ray diffraction and cellular biophysics. Using this interdisciplinary approach, in chapters 2 and 3, we identify at atomic resolution the key structural elements that facilitate the colocalization, interaction and subsequent insertion of soluble Aβ oligomers into membranes, which ultimately result in the loss of membrane integrity. Notably, we show that small molecules, such as green tea catechins, remodel these structural features and effectively perturb the interactions with membranes. In chapter 4, we extend these analyses to αS and identify how the chaperone, Human Serum Albumin (HSA), remodels toxic αS oligomers into non-toxic species and breaks the catalytic cycle that generates new toxic oligomers. Lastly, in chapter 5, we describe a novel solution NMR approach to map at atomic resolution the sites of early self-association, with minimal bias from monomer dynamics, an effect that frequently dominates residue-dependent variations in solution NMR measurements. Overall, given that Aβ and αS are archetypical amyloidogenic proteins, we anticipate that the structure – toxicity relationships established herein, and the related experimental approaches may be transferrable to other amyloidogenic systems. / Dissertation / Doctor of Philosophy (PhD)

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Synucleins are highly conserved presynaptic proteins with unknown function. α-synuclein plays a key role regulating dopamine homeostasis and is intimately involved in Parkinson’s disease (PD) pathogenesis. However, the normal/pathological role of α-synuclein remains unidentified. Studies exploring its function are limited as current transgenic mouse models do not fully recapitulate PD pathology. This thesis reports the functional characterisation of two novel synuclein-based mouse models. I report the molecular and functional characterisation of transgenic mouse lines with wild-type or A30P-mutant human α-synuclein genomic locus carried within a bacterial artificial chromosome. SNCA-A30P^+Snca-/- mice exhibited a highly physiologically relevant expression pattern of the transgene, including expression in the substantia nigra pars compacta (SNpc) and a specific, age-related loss of TH^+ cells in the SNpc, the key region of preferential cell loss in PD, compared with non-transgenic Snca -/- littermate controls. Analysis of dopamine signalling using fast-scan cyclic voltammetry (FCV) showed young adult SNCA-A30P^+Snca-/- mice had an approximately 20% lower evoked extracellular dopamine concentration ([DA]o) compared with non-transgenic Snca -/- littermate controls, a decrease specific to the dorsal striatum. This difference diminished with age and could not be attributed to changes in dopamine reuptake/content. I detail the behavioural and neurochemical phenotype in mice lacking all three synucleins (α/β/γ). Functional compensation between synucleins emphasises the importance of studying their effects by removing all three proteins simultaneously. Triple-null mice exhibited hyperactivity in a novel environment reminiscent of a hyperdopaminergic-like phenotype, but showed no phenotype in anxiety or motor related tests. FCV revealed synuclein triple-null mice had a two-fold increase in [DA]o, specific to the dorsal striatum and not attributable to changes in dopamine reuptake/content, changes in striatal nicotinic receptor activity nor calcium-dependent changes in dopamine exocytosis. Together, the analysis from these two novel mouse models reveal synucleins play an important role in altering synaptic function in the dorsal striatum (the region selectively affected in PD) and contributes to growing evidence suggesting synucleins are negative regulators of synaptic dopamine release.

Alzheimer’s disease (AD) and Parkinson’s disease (PD) are defined by neurodegeneration and accumulations of misfolded proteins that spread through the brain in a well characterized manner. In AD these accumulations consist mainly of β-amyloid (Aβ) and tau, while in PD, α-synuclein (α-syn) make up the characteristic lewy pathology.     The general aim of this thesis was to investigate mechanisms associated with neurotoxic peptide activity by Aβ, tau and α-syn in relation to cellular degradation and transfer with a cell-to-cell transfer model system.    We found that intercellular transfer of oligomeric Aβ occurs independently of isoform. However, the amount of transfer correlates with each isoforms ability to resist degradation or cellular clearance. The Aβ1-42 isoform showed particular resistance to clearance, which resulted in higher levels of cell-to-cell transfer of the isoform and lysosomal stress caused by accumulation.    As Aβ accumulations can inhibit the proteasomal degradation we investigated how reduced proteasomal degradation affected neuron-like cells. We found increased levels of phosphorylated tau protein, disturbed microtubule stability and impaired neuritic transport after reduced proteasomal activity. These changes was partly linked to c-Jun and ERK 1/2 kinase activity.    We could also show that α-syn transferred from cell-to-cell in our model system, with a higher degree of transfer for the larger oligomer and fibrillar species. Similar to Aβ, α-syn mainly colocalized with lysosomes, before and after transfer.     Lastly, we have developed our cell-to-cell transfer system into a model suitable for high throughput screening (HTS). The type of cells have been upgraded from SH-SY5Y cells to induced pluripotent stem cells (iPSCs), with a differentiation profile more similar to mature neurons. The next step will be screening a small molecular library for substances with inhibitory effect on cell-to-cell transfer of Aβ peptides.     The importance of the degradative systems in maintaining protein homeostasis and prevent toxic accumulations in general is well known. Our findings shows the importance of these systems for neurodegenerative diseases and also highlight the link between degradation and cell-to-cell transfer. To restore or enhance the degradative systems would be an interesting avenue to treat neurodegenerative diseases. Another way would be to inhibit the transfer of misfolded protein aggregates. By using the HTS model we developed, a candidate substance with good inhibitory effect on transfer can hopefully be found.