Les signatures histopathologiques de principales maladies neurodégénératives - maladie d'Alzheimer et la maladie de Parkinson - sont les enchevêtrements neurofibrillaires formés par la protéine tau et les corps de Lewy, formés par l'α-synucleine agrégée. Les mécanismes précis du repliement et de l'agrégation de ces protéines, pour la protéine tau comme pour l'α-synucleine, ne sont pas totalement compris à ce jour. Ici, nous nous sommes intéressés à cette question en utilisant des modèles in vitro et in vivo. En étudiant l'agrégation tau in vitro, nous avons mis en évidence un nouvel auto- assemblage réversible de tau, qui dépend de la température et de la présence d’ions zinc, et qui est a priori différent de l'agrégation de tau en présence d'inducteurs d'agrégation tels que l'héparine. Ce processus pourrait néanmoins être impliqué dans les premières étapes de l'agrégation pathologique de tau. Dans une deuxième partie nous avons développé des modèles murin pour étudier les dysfonctionnement de l’α-synucleine. Nous avons montré que l’α-synucleine est directement impliquée dans le développement embryonnaire de régions spécifiques du système nerveux, et qu'elle a des propriétés modulatrices seulement sur les neurones dopaminergiques de la substantia nigra, qui sont touchés dans la maladie de Parkinson.Les résultats obtenus dans nos études de deux protéines qui subissent une agrégation pathogène et forment des inclusions intracellulaires ont contribué à la compréhension des processus moléculaires et cellulaires associés à la dégénérescence neuronale, ce qui fournira de nouvelles pistes pour développer de nouvelles stratégies de thérapies de maladies neurodégénératives. / The histopathological hallmarks of the most common neurodegenerative diseases – Alzheimer’s disease and Parkinson’s disease are neurofibrillary tangles formed by tau protein and Lewy bodies inclusions formed by aggregated α-synuclein. The formation and accumulation of these proteins into inclusions cause functional disruptions of the cytoskeleton and leads to neuronal degeneration. The precise mechanisms of tau and synuclein misfolding and aggregation leading to those cellulare incluses, even though very studied, are not fully understood neither for tau protein nor for α-synuclein.Here we have addressed this question using both in vitro and in vivo models. Investigating tau aggregation in vitro, we have found a reversible self-assembly of tau, which depends on temperature and is induced by zinc ions, which is different from the tau aggregation in the presence of aggregation-inducers such as heparin. This process could be implicated in the first steps of tau pathological aggregation. In a second part, we have developed a mouse model for studying the α-synuclein dysfunction. We have shown that α- synuclein is directly involved in the embryonic development of the specific regions of the nervous system, and that it has modulating effect only on the populations of dopaminergic neurons of substantia nigra, which are affected in Parkinson’s disease.Results obtained in our studies of two proteins that undergo pathogenic aggregation and form intracellular inclusions contributed to understanding of molecular and cellular processes associated with neuronal degeneration, which is important for the development of new disease-modifying therapies of neurodegenerative disorders.

Parkinson's disease (PD) is a prevalent neurodegenerative disorder for which the greatest risk factor is age. Four to five percent of 85-year-olds suffer from this debilitating disease, which is characterized by the selective loss of dopaminergic neurons within the substantia nigra and the presence of protein aggregates known as Lewy bodies. While the etiology of this disease is still unknown, recent research implicates oxidative stress, activated microglia, and reactive dopamine (DA) metabolites to play a role in the initiation or progression of the disease. Activated microglia cause injury to dopaminergic neurons via a host of mechanisms, including reactive oxygen species production, release of cytokines, and phagocytic activity. Microglial activation has been detected in the brains of PD patients, but the source of this activation has not been elucidated. Previous research has shown electrophiles and endogenous neurotoxins to play a role in this microglial activation. The interaction between the neurotoxic metabolite of DA, 3,4-dihydroxyphenylacetaldehyde (DOPAL), and microglia has not been explored. DOPAL is a highly reactive, bifunctional electrophile produced by oxidative deamination of DA by monoamine oxidase (MAO). DOPAL is oxidized in the major metabolism pathway to 3,4-dihydroxyphenylacetic acid (DOPAC) by aldehyde dehydrogenase (ALDH). DOPAL has previously been shown to be 100-fold more toxic than DA in vitro and in vivo. Potent inhibition of the rate-limiting enzyme in DA biosynthesis, tyrosine hydroxylase, by DOPAL has been well-established. DOPAL-mediated aggregation of Α-synuclein, the primary component of PD-hallmark Lewy bodies, has been suggested but was further explored in this work. Results presented in this body of work include further determination of the aggregation of Α-synuclein by DOPAL, including evidence of covalent modification. The interaction of DOPAL with BV-2 microglia, an immortalized cell line, was addressed in depth through exploration of DOPAL catabolism, toxicity, and generation of an activational response. Metabolism of DOPAL to DOPAC was altered in activated microglia, with the production of DOPAC reduced by ~40%. Metabolism of DOPAL to DOPAC was also inhibited by both 4-hydroxynonenal and malondialdehyde, gold standards of lipid peroxidation. Both of these compounds were found to be significantly toxic to BV-2 cells at concentrations well below those considered toxic to dopaminergic cells. Alternatively, DOPAL and DA were found to be non-toxic to this cell line, while DOPAL was shown to be significantly toxic to dopaminergic cells at concentrations as low as 10 ΜM. Significant activation of BV-2 microglia by DOPAL was observed at 10 ΜM and above by release of TNF-Α. Morphological changes, release of IL-6, and changes in expression of COX-2 also indicated activation by DOPAL but not DA or DOPAC. BV-2-conditioned media, generated by incubation with DA, DOPAL, or DOPAC, was added to MN9D cells, and toxicity was measured by the MTT assay. BV-2 conditioned media generated by DOPAL incubation produced the greatest toxicity for MN9D cells. These results implicate DOPAL in dopaminergic cell death through microglial activation.

By using multidisciplinary analysis of CA inclusions in prostate glands of patients diagnosed with prostate cancer, we have revealed that their major components are the amyloid forms of S100A8 and S100A9 proteins associated with numerous inflammatory conditions and types of cancer. We have demonstrated that material closely resembling CA can be produced from S100A8/A9 in vitro and shows the characters of amyloids. This process is facilitated by calcium or zinc, both of which are abundant in ex vivo inclusions. These observations were supported by computational analysis of the S100A8/A9 calcium-dependent aggregation propensity profiles. We have found DNA and proteins from Escherichia coli in CA bodies, suggesting that their formation is likely to be associated with bacterial infection. CA inclusions were also accompanied by the activation of macrophages and by an increase in the concentration of S100A8/A9 in the surrounding tissues, indicating inflammatory reactions. These findings, taken together, suggest a link between bacterial infection, inflammation and amyloid deposition of pro-inflammatory proteins S100A8/A9 in the prostate gland, such that a self-perpetuating cycle can be triggered and may increase the risk of malignancy in the ageing prostate. We evaluated the autoimmune reactions to endocrine (insulin) and astrocytical (S100B) biomarkers in the blood sera of PD patients compared with healthy controls. Peripheral immune responses can be sensitive indicators of disease pathology. We found a statistically significant increase of the autoimmune responses to both antigens in patients compared with controls. Heterogeneity of the immune responses observed in patients may reflect the modulating effect of multiple variables associated with neurodegeneration and also changes in the basic mechanisms of individual autoimmune reactivity. We did not detect any pronounced immune reactions towards insulin amyloid fibrils and oligomers in patients, indicating that an amyloid-specific conformational epitope is not involved in immune recognition of this amyloid type. Immune reactions towards S100B and insulin may reflect the neurodegenerative brain damaging processes and impaired insulin homeostasis occurring in PD. Generated auto-antibodies towards the major amyloidogenic protein involved in PD Lewy bodies - a-synuclein and its amyloid oligomers and fibrils were measured in the blood sera of early and late PD patients and controls by using ELISA, Western blot and Biacore surface plasmon resonance analyses. We found significantly higher antibody levels towards monomeric a-synuclein in the blood sera of PD patients compared to controls, though the responses decreased with PD progression. There were no noticeable immune responses towards amyloid oligomers, but substantially increased levels of IgGs towards a-synuclein amyloid fibrils both in PD patients and controls, which subsided with the disease progression. Pooled IgGs from PD patients and controls interacted also with amyloid fibrils of Ab (1-40) and hen lysozyme, however the latter were recognized with lower affinity. This suggests that IgGs bind to amyloid conformational epitope, though displaying higher specificity towards human amyloid species associated with neurodegeneration. The findings suggest the protective role of autoimmunity in PD and therefore immune reactions towards PD major amyloid protein - a-synuclein can be used in treatment strategies and in diagnostics, especially in identifying early disease.

No description available.

Alpha-synuclein (AS) is a natively unfolded protein whose structure is extremely sensitive to its environment. The hallmark of Parkinson’s disease (PD) is aggregation and deposition of AS in inclusion bodies. Formation of misfolded AS monomers which are partially folded is the first and critical stage in fibrillation of AS and is a good target for designing therapeutic strategies. Characterization the biochemical properties of partially folded intermediates induced by fibrillization and anti- fibrillization agents will help to design drugs as new inhibitors of AS misfolding and aggregation. Nanopore analysis is an emerging technique for studying the molecular mechanism of protein misfolding. This technique was used to characterize the conformational change of AS in the presence of two groups of chemicals; anti-parkinsonian small molecules (dopamine and nicotine) and Parkinson’s developing toxin (Cu(II) and methamphetamine). Other biophysical techniques such as NMR spectroscopy and isothermal titration calorimentry (ITC) were able to confirm the nanopore analysis results and also to study other biophysical properties of the partially folded intermediates such as the binding constant of the interaction and the secondary structure content. The results from nanopore analysis showed that both groups of ligands shifted the blockade current peak of AS (centered at -86 pA) to lower blockade currents but in a different manner. Anti-parkinsonian drugs shifted the blockade current of AS to intermediate peaks between -40 to -80 pA but Parkinson developing toxins shifted the peak to a lower blockade current centered at -25 pA which suggests a more compact conformation. Thus nanopore analysis distinguished the different conformation induced by different ligands. Furthermore nanopore analysis with AS fragments showed that these ligands bind to different regions of AS. NMR spectroscopy of AS in the presence of dopamine and nicotine isomers was in agreement with the nanopore analysis and showed conformational changes of AS in a concentration dependent manner. CD spectroscopy results showed that the secondary structure of AS alone and in the presence of ligands was mostly random coil and suggests a loop formation model for the interaction of ligands with AS. The results of this thesis showed the application of nanopore analysis as a real-time and label-free technique to screen a library of ligands for designing misfolding inhibitors for PD treatment. The result of a synergic experiment with nicotine and caffeine showed that combination of these anti-parkinsonian small molecules would be a promising new drug for treatment of PD.

No description available.

The molecular mechanisms underlying the association between mutations in GBA1 and risk of developing the ‘synucleinopathy’ disorders Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) remain elusive. To better understand the precise molecular cascade that connects GBA1 mutations with α-synuclein dysregulation, a modified lipid extraction and HPTLC protocol was optimized to detect changes in levels of neutral sphingolipids (SLs) from neural cells and tissue expressing wild-type (WT) GBA1, mutant GBA1, or both. We demonstrate that mutant GBA1 does not confer a dominant-negative effect on WT GBA1-mediated activity; however, bona fide loss-of-enzymatic function mutation events led to the accumulation of lipid substrates in neural cells and tissue, and enhance α- synuclein/ubiquitin reactivity in brain tissue of mutant gba1 mice. Our HPLC-MS/MS data are consistent with other studies demonstrating that heterozygous GBA1 mutations do not lead to lipid accumulation, but may alter α-synuclein degradation through a yet-to-be defined novel gain-of-toxic function event.

Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy are disorders featuring accumulation of Lewy bodies in brain. The main component of these large insoluble intracellular inclusions is the presynaptic protein alpha-synuclein (α-synuclein). It is generally believed that α-synuclein monomers adopt an abnormal conformation that favors the formation of soluble oligomers or protofibrils and, eventually, insoluble fibrils depositing as Lewy bodies. Notably, the intermediately sized oligomers/protofibrils seem to have particular neurotoxic effects. Several factors may influence the formation of α-synuclein oligomers/protofibrils, e.g. the reactive aldehydes 4-hydroxy-2-nonenal (HNE) and 4-oxo-2-nonenal (ONE) formed during oxidative stress. The overall aims of this thesis were to investigate biophysical and biochemical properties of in vitro generated α-synuclein oligomers, characterize their functional effects on cell and animal disease models as well as to explore whether their formation could be prevented in a cell culture model for oligomerization.  Here, it was found that α-synuclein rapidly formed oligomers after incubation with both ONE and HNE. The resulting oligomers were stable and did not continue to form insoluble fibrils. By comparing HNE- and ONE induced α-synuclein oligomers biochemically they were both found to exhibit extensive β-beta sheet structure and had a molecular size of ~2000 kDa. However, they differed in morphology; the ONE induced α-synuclein oligomers described round amorphous species whereas the HNE induced α-synuclein oligomers appeared as elongated protofibril-like structures. Both these oligomers were cell internalized to varying degrees and induced toxicity in neuroblastoma cells. In addition, the ONE induced α-synuclein oligomers seemed to initiate aggregation of monomeric α-synuclein in vitro, but failed to do so in vivo. Finally, treatment of α-synuclein overexpressing cells with monoclonal antibodies specific for α-synuclein significantly reduced aggregation and lowered levels of the protein, suggesting increased turnover in these cells.  To conclude, this thesis has characterized different oligomeric α-synuclein species, which may have properties similar to soluble species central to the pathogenesis of Parkinson’s disease and other disorders with α-synuclein pathology. For therapeutic strategies it is important to selectively target such harmful protein species and avoid interaction with other forms of α-synuclein, which may have vital physiological cellular functions.

No description available.

No description available.