Alterations in the expression levels of α-synuclein (SNCA) provide an important link between familial and sporadic forms of Parkinson’s disease (PD). Multiplications of the human wild-type SNCA locus give rise to early-onset autosomal-dominant forms of PD and elevated α-synuclein expression has been linked to an increased risk for late-onset sporadic PD. The identification of α-synuclein’s physiological and pathophysiological functions has been hindered by a lack of animal models that accurately recapitulate the key disease features. Traditional cDNA-based transgenic models fail to correctly reflect the spatiotemporal expression pattern of α-synuclein and consequently do not accurately model the disease. Bacterial artificial chromosome (BAC) technology allows transgene expression from the entire genomic locus under the control of native regulatory elements and therefore allows improved modelling of disease mechanisms and phenotypes. This thesis describes a longitudinal characterisation of the molecular and behavioural effects of overexpressing human wild-type α-synuclein in a novel BAC transgenic PD mouse model, the SNCA-OVX model. Firstly, the work investigates transgene expression and localisation, dopamine neuron loss, synaptic function and PD-related motor and non-motor phenotypes. At 3 months of age, deficits in gastrointestinal function were observed while normal levels of dopaminergic markers and neurons were maintained. At 18 months, mice displayed a 30% decrease in nigrostriatal dopamine neurons, accompanied by reduced motor coordination and function. This novel PD model, which reflects accurate transgene expression and displays progressive dopamine neuron loss accompanied by characteristic behavioural PD phenotypes, will aid the investigation of molecular disease mechanisms and the development of novel therapies. Secondly, this thesis describes the generation of Translating Ribosome Affinity Purification (TRAP) transgenic mice, which express an EGFP-tagged ribosomal protein L10a transgene under the control of the tyrosine hydroxylase promoter. Finally, double transgenic TH bacTRAP SNCA-OVX mice were generated to investigate changes in genome-wide gene expression in dopaminergic cell populations of SNCA-OVX mice to identify novel drug targets.

No description available.

Gastrointestinal (GI) alpha-synuclein (ASN) detection may represent a clinically useful biomarker of Parkinson's disease (PD), but this has been challenged by conflicting results of recent studies employing different immunohistochemical (IHC) methods and reporting diverse morphological patterns with variable biological interpretation. To increase sensitivity and specificity, we applied three different techniques to detect different possible conformations of ASN in GI tissue derived from biopsies of the GI tract, which were obtained from a longitudinally followed, clinically well-characterized cohort of PD subjects and healthy controls (HC) (Oxford Discovery study). With IHC, we used antibodies reactive for total (T-ASN-Abs), phosphorylated (P-ASN-Abs) and oligomeric (O-ASN-Abs) ASN; with the ASN Proximity Ligation Assay (AS-PLA), we targeted oligomeric ASN species specifically; finally, with the Paraffin-Embedded Tissue Blot (PET-Blot) we aimed to detect fibrillary conformations of ASN specifically. Optimisation and validation of the PET-Blot and PLA techniques was carried out with studies on brain tissue from subjects with ASN pathology, and these experiments were used to gain insight into morphology and distribution of different conformational variants of ASN in the brain of subjects with Lewy pathology. We specified all the detected morphological staining patterns with each technique interpreting them as pathologic or non-specific. Correlation to clinical symptoms was assessed to investigate the potential predictive or diagnostic value of specific staining patterns as biomarkers. A total of 163 GI tissue blocks were collected from 51 PD patients (113 blocks) and 21 healthy controls (50 blocks). In 31 PD patients, GI biopsies had been taken before PD diagnosis (Prodromal PD group); while in 20 PD patients biopsies were obtained after PD diagnosis (Manifest PD group). The majority of these tissues blocks were from large intestine (62%), followed by small intestine (21%), stomach (10%) and oesophagus (7%). With IHC, four ASN staining patterns were detected in GI tissue (Neuritic, Ganglionic, Epithelial, and Cellular), while two distinct staining patterns were detected with AS-PLA (cellular and diffuse signal) and with AS-PET-Blot (ASN-localised and peri-crypt signal). The level of agreement between different techniques was generally low, and no single technique or staining pattern was able to reliably distinguish PD patients (Prodromal or Manifest) from HC. Overall, our study suggests that even specific detection of ASN conformational variants currently considered pathologic was not adequate for the prediction of PD. Future studies with these or other novel techniques focusing on the upper part of the GI tract could overcome current limitations in sensitivity and specificity.

In certain neurodegenerative diseases such Dementia with Lewy Bodies (DLB), it is hypothesised that misfolded α-synuclein (α-syn) and β-amyloid both contribute to pathology. α-Syn and β-amyloid have been suggested to synergistically promote one another’s accumulation and aggregation, but the mechanisms are unknown. β-Amyloid is generated from β-/γ-secretase-mediated processing of the amyloid precursor protein (APP). This study investigated how α-syn overexpression in cells affects β-amyloid production from APP, using multiplex assays, luciferase reporter assays, and western blotting. Wildtype α-syn expression induces β-amyloid generation from APP in SH-SY5Y human neuroblastoma cells, and similar changes to APP processing occur in another neuronal cell model. Dominant-negative overexpression of α-syn mutants revealed that disrupting the N-terminal domain can increase APP amyloidogenic processing. Secretase enzymes that perform APP processing were next investigated. γ-Secretase activity, measured by a luciferase reporter, was not increased by α-syn overexpression. A higher ratio of β- to α-secretase processing was hypothesised, which led to expression and activity studies of the major β- and α-secretases, BACE1 and ADAM10 respectively. It was shown that the BACE1 protein expression is post-transcriptionally upregulated in α-syn cells, with increased APP cleavage in cells. ADAM10 protein expression is transcriptionally suppressed in wild-type α-syn cells, reducing total levels of catalytically active enzyme. However the change in ADAM10-mediated APP processing may be negligible since, critically, plasma membrane expression of ADAM10 appears to be maintained. To aid understanding of the mechanism that connects α-syn to APP processing, BACE1 expression was used in pharmacological studies of cell stress signalling. This approach revealed that in α-syn cells BACE1 lysosomal and/or proteasomal degradation may be disturbed. Additionally, BACE1 expression is induced by translational de-repression mediated by eIF2α ser-51 phosphorylation, which was increased in α-syn cells. Although preliminary, the data suggests a role for oxidative stress mediating the increased BACE1 expression in wild-type α-syn cells.

No description available.

Parkinson's Disease (PD) is the second most common neurodegenerative disorder, clinically characterized by severe motor impairment and pathologically characterized by progressive loss of the dopaminergic neurons of the substantia nigra pars compacta (SNpc) as well as the formation of cellular aggregate deposits called Lewy Bodies. While some advances have been made in understanding the molecular underpinnings of the disorder, the molecular implications of common genetic factors increasing risk for PD have not been adequately studied. First identified by GWA studies in 2009, the GAK/DGKQ/IDUA region on chromosome 4p16.3 shows significant genetic association to risk for PD, and the GAK protein has been shown to be associated with the primary component of Lewy Bodies, a-synuclein. In order to determine which gene in the 4p16.3 region may account for the genetic association to PD and to understand the molecular consequences of that association, post-mortem cortical brain tissue from 29 PD and 49 control patients was RNA-sequenced and differential exon usage in the context of disease and risk variant carrier status was analyzed. Exons in the 3' region of GAK were found to be associated to case status, and notably exon 25 expression in GAK was associated with both case status and the risk variant. This exon was further observed to be associated to several genes previously shown to interact with GAK, including SNCA, which codes for a-synuclein. As a proxy for expression of the 3' region of GAK, exon 25 was assessed for genome-wide association, and genes showing association to the exon were involved in pathways related to synaptic transmission and neuronal function. In order to validate these findings, microarray analysis of primary rat cortical neurons in which GAK expression was reduced by shRNA transduction was performed. GAK expression in rat neurons was significantly inversely correlated to endogenous SNCA expression, and also exhibited association to pathways involved in synaptic transmission and mitochondrial function. Together, these findings suggest aberrant GAK expression related to genetic risk to be an important factor in the pathogenesis of PD through GAK's influence on SNCA expression and through dysregulation of important neuronal pathways.

The biological basis of the clinical heterogeneity in Parkinson's Disease (PD) is unclear. It is likely to involve complex interactions between genetic and environmental factors and between a range of pathological processes, including protein homeostasis and immune system function. Microglial activation in the brain and peripheral innate immune changes are known to occur in PD. Recently genetic, animal and cellular studies have linked several innate immune related genes and proteins (e.g. HLADR, TREM2, TLR2, TLR4, caspase-1) to PD and provided evidence that they may have a role in PD pathogenesis. Alpha-synuclein is central to PD, with evidence from neuropathology, genetics and animal/cell models indicating that it plays a significant pathogenic role. There is developing evidence directly linking innate immune activity and alpha-synuclein pathology. For example, inflammation, particularly in response to microbial infection, is associated with increased alpha-synuclein accumulation in the periphery and activation of the innate immune inflammasome related caspase-1 leads to increased cleavage and aggregation of alpha-synuclein. Overall Hypothesis- "Parkinson's disease (PD) and its clinical heterogeneity are associated with systemic changes in innate immune and associated microbial factors and in alpha-synuclein". This was investigated from the perspective of an epidemiological study, a study of peripheral blood monocyte, innate immune/microbial markers and a cerebrospinal fluid (CSF) study in PD patients. *The epidemiological study, involved the longitudinal PICNICS cohort of 290 Idiopathic PD patients, and showed that the use of medication known to influence alpha-synuclein and immune function is associated with motor heterogeneity in PD. *The peripheral immune study involved 41 early PD patients and 41 age, gender and MAPT genotype matched paired controls, with the PD patients categorised into 2 groups based on the presence of previously identified clinical and genetic risk factors for the development of an early dementia (impaired semantic fluency, pentagon copying and MAPT H1/H1 haplotype). This study demonstrated that the phenotypic profile of peripheral monocytes and the level of serum alpha-synuclein and relevant innate immune and microbial markers do differ in early PD compared to controls and that there are differential changes in those patients at higher versus lower risk for early dementia. The systemic alpha-synuclein related changes appear to be present overall in PD patients compared to controls, while the more microbial/innate immune related changes appear to be more prominent in the dementia higher risk group. *The CSF study involved samples from 35 PD patients and has demonstrated evidence of relationships between neurodegeneration-linked CSF tau species and inflammatory cytokines, and between CSF alpha-synuclein and cognitive function, suggesting that these factors may be involved in PD heterogeneity within the central nervous system as well. Overall, these studies provide evidence that variations in alpha synuclein/ tau homeostasis and innate immune and microbial factors are related to PD and its clinical heterogeneity.

La maladie de Parkinson est l’une des plus répandue dans le monde. Des analyses post mortem ont mis en évidences des inclusions dans les cerveaux des patients, composées d’α-synucléine. Plusieurs études ont visé à identifier des composés capables d’inhiber l’agrégation de cette protéine, étant donné que cette agrégation est liée à sa toxicité. Néanmoins, de nombreux composés restent encore à être identifiés afin de mettre en évidence des structures moléculaires actives partagées qui pourrait mener à la synthèse d’un principe actif. Les stilbènes sont des composés phénoliques démontrant régulièrement des activités biologiques intéressantes pour la santé. Dans cette étude, nous étudions le comportement anti-agrégatif des stilbènes monomériques (resvératrol, picéatannol) et oligomèriques (ampélopsine, viniférine, et isohopeaphénol). Les résultats présentés mettent en évidence la capacité des stilbènes à inhiber l’agrégation de l’α-synucléine et fournissent des éléments pour comprendre ce mécanisme. / Parkinson’s disease is one of the most spread neurodegenerative diseases in the world. Post mortem analyses have put in evidence small inclusion bodies in patient’s brain, composed of α-synuclein fibrils. Several studies attempted then to identify compounds that could inhibit α-synuclein aggregation, as its aggregation is linked to its toxicity. Still, numerous active compounds need to be identified in order to put on relief shared active structures that could lead to a potent drug design. Stilbenes are phenolic compounds that often display interesting health-related biological activities. In this study, the anti-aggregative behavior of stilbenes monomers (resveratrol, piceatannol) and oligomers such as ampelopsin and isohopeaphenol was assessed. The results put on evidence stilbene propensity to inhibit α-synuclein aggregation and provide an insight into their inhibition mechanisms.

Les Glycosaminoglycannes (GAGs) sont une famille de polysaccharides principalement localisés au niveau de la matrice extracellulaire et de la membrane plasmique. Ils peuvent interagir avec des facteurs de croissance et des cytokines pour réguler leurs activités, participer à des transports protéiques à travers la membrane cellulaire, moduler les activités de certaines enzymes telles que les cathepsines (enzymes lysosomales). Toutes ces activités démontrent que les GAGs jouent des rôles primordiaux dans la régulation de la croissance, la différenciation, l'adhésion, l'inflammation et la mort cellulaire.L'implication de ces polysaccharides dans la régulation de l'apoptose via la voie mitochondriale n'a toujours pas été déterminée. Ici, nous démontrons dans un modèle cellulaire de fibroblastes de peau en culture primaire, soumis à un stress oxydatif par de l'H2O2, qu'un mimétique des GAGs, l'OTR4120, est capable de protéger la membrane du lysosome, de réduire le taux de ROS intracellulaires et d'inhiber la chute du potentiel de membrane mitochondrial et ainsi d'empêcher la libération du cytochrome c et l'activation des activités caspases-9 et -3 sans affecter la voie extrinsèque de l'apoptose. Les héparanes sulfates et les chondroïtines sulfates au contraire de l'héparine, ont montré un effet protecteur de l'apoptose en inhibant les protéines clefs de ce processus de mort cellulaire. Ainsi, les GAGs naturels et l'OTR4120 sont capables s'opposer à l'apoptose, en inhibant l'activité de la cathepsine D libérée dans le cytosol, empêchant ainsi l'activation de la voie intrinsèque de l'apoptose via la mitochondrie. Ces résultats ouvrent de nouveaux horizons notamment dans certaines maladies où le stress oxydatif est impliqué comme c'est le cas de certaines maladies neurodégénératives comme la maladie de Parkinson.La cause de la maladie de Parkinson (MP) qui affecte les neurones dopaminergiques demeure encore mystérieuse, bien que différentes preuves soutiennent les hypothèses impliquant des dysfonctionnements mitochondriaux et une accumulation d'α-synucléine comme étant les événements majeurs dans cette physiopathologie. Récemment, la cathepsine D a été impliquée dans des processus de mort cellulaire et montrée comme étant surexprimée dans des modèles de la MP. De plus, apparait être l'une des principales enzymes responsables de la dégradation de l'α-synucléine. Puisque les glycosaminoglycannes (GAGs) sont capables de réguler l'activité de la cathepsine D dans des cellules en culture dans une condition de stress, nous avons cherché à démontrer si GAGs pouvaient être localisés à un niveau intracellulaire, où ils pourraient interagir avec la cathepsine D et s'ils étaient capables de réguler l'accumulation/dégradation de l'α-synucléine. Ainsi nous avons mis en place un modèle cellulaire de la MP induit par le MPP+. Nous avons observé que l'expression génétique des enzymes de la biosynthèse des GAGs (HS2ST, HS6ST et CHST8) a été modifiée dans les cellules stressées par la neurotoxine et que leurs taux mesurés par leurs sulfates étaient augmentés plus précisément au niveau des HS. Au contraire, l'absence de GAGs sulfatés induit par le chlorate de sodium, un inhibiteur de la PAPs (donneur de sulfates), a permis d'augmenter l'activité de l'activité cathepsine D et également d'inhiber l'accumulation ou d'induire la dégradation de l'α-synucléine. Pour la première fois, il a été montré que les GAGs sont capables d'agir sur l'activité cathepsine D à l'intérieur de la cellule et de réguler l'accumulation/dégradation de l'α-synucléine. / Pas de résumé anglais

La maladie de Parkinson (MP) appartient à la famille des a-synucléinopathies. Ces pathologies ont en commun d'être liées à l'a-synucléine (a-syn), protéine neuronale dont les rôles précis sont encore mal définis. Dans un contexte où des pesticides, comme le paraquat, ont été liés épidémiologiquement au déclenchement précoce de la MP chez des agriculteurs, certaines substances sont devenues des substances de référence dans les études portant sur les synucléinopathies. Le paraquat induit une pathologie parkinsonienne comprenant des agrégations de l'a-syn ou des pertes spécifiques de neurones dopaminergiques. Dans nos études nous avons premièrement mis en place un modèle d'exposition par voie orale au paraquat en utilisant l'administration. Par la suite nous avons étudié les effets de cette administration orale au paraquat sur le système nerveux entérique présent dans l'intestin de souris C57Bl/6 et transgénique TgM83. Cette lignée transgénique possède la particularité de développer spontanément une synucléinopathie, simultanément dans l'intestin et le cerveau, présentant des dépôts d'a-syn phosphorylée. L'ingestion du paraquat induit une accélération de l'apparition de la synucléinopathie entérique. Dans une seconde étude biochimique, nous montrons que l'exposition orale au paraquat induit une augmentation globale des niveaux d'a-syn totale dans le système nerveux central et entérique des souris.Nous montrons ici les effets délétères de l'exposition orale au paraquat sur l'intestin en lien avec l'a-syn, par l'accélération d'une synucléinopathie mais aussi par l'induction d'une réponse générale, cérébrale et entérique, traduite par une hausse des niveaux d'a-syn / Parkinson's disease (PD) belongs to the a-synucleinopathies family. These pathologies have in common that they are linked to a-synuclein (a-syn), a neuronal protein whose precise roles are poorly defined. Some pesticides, such as paraquat, have been epidemiologically linked to the early onset of PD in farmers; some substances have become essential in the studies of synucleinopathies. Paraquat induces parkinsonian pathologies including a-syn aggregations or specific dopaminergic neurons loss. In our studies, we first set up a model of oral exposure to paraquat by drinking water. Subsequently, we studied the effects of this oral administration to paraquat on the enteric nervous system of the intestine of C57Bl/6 and TgM83 transgenic mice. This transgenic line has the peculiarity of simultaneously and spontaneously developing a synucleinopathy in the intestine and in the brain, displaying deposits of phosphorylated a-syn. The oral ingestion of paraquat induces acceleration in the appearance of enteric synucleinopathy. In a second biochemical study, we show that oral exposure to paraquat induces an overall increase in total a-syn levels in the central and enteric nervous systems of mice.Our results show the deleterious effects of paraquat oral administration on the intestine in relation to a-syn, by the acceleration of synucleinopathy but also by the induction of a widespread cerebral and enteric response, shown by an increase in cellular levels of a-syn