A recent model of Parkinson’s disease (PD) suggests that the neuropathological, behavioural and cognitive symptoms progress in stages. There is substantial evidence for a prodromal stage of PD, during which time pre-motor symptoms develop. Rapid eye movement (REM) sleep behaviour disorder (RBD) is a risk factor for developing PD and may be part of the pre-motor stage. In both disorders, neuropathological α-synuclein aggregates are thought to be a direct cause of the resulting symptoms. One model has shown that in rats, proteasome inhibition produced by systemic exposure to environmental toxins results in α-synuclein pathology and motor behaviour dysfunction that mimics the progression of PD in humans. The present study examined the hypothesis that the systemic proteasome inhibition model would produce pre-Parkinsonian RBD-like pathology in rats. It was expected that sleep disturbances would be seen prior to behavioural disturbances in rats treated systemically with PSI (a proteasome inhibitor). Following baseline sleep recording and training on the inclined beam-traverse task, rats were injected with PSI (a proteasome inhibitor) or ethanol (control), 6 times over 2 wk. Sleep recording over 8 wk and behavioural testing over 16 wk provided no evidence of sleep disturbances or motor dysfunction. Post-mortem immunohistochemical analyses of brain tissue provided no evidence of PSI-associated α-synuclein aggregates in the locus coeruleus, subcoeruleus (dorsal part), or substantia nigra (areas involved in RBD and/or PD). These results did not provide support for RBD as a prodromal phase of PD within the systemic proteasome inhibitor-based model and add to a growing body of research reporting inconsistent findings using this model. We suggest that systemic PSI exposure in rats does not produce a viable model of RBD or PD. Whether RBD is an early symptom in the progression of PD remains to be established. / Thesis (Master, Neuroscience Studies) -- Queen's University, 2010-04-28 12:04:50.613

Abstract: Neurodegenerative diseases are associated with progressive loss of structure or function of neurons which results in cell death. Recent evidence indicate that all neurodegenerative disorders, sporadic or transmissible, may have a common pathological mechanism at the molecular level. This common feature consists of protein aggregation and accumulation of harmful aggregates in neuronal cells resulting in cellular apoptosis and neurotoxicity. Neurodegenerative diseases can affect abstract thinking, skilled movements, emotional feelings, cognition, memory and other abilities. This diverse group of diseases includes Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), prion diseases or transmissible spongiform encephalopathies (TSEs) and amyotrophic lateral sclerosis. In my project I worked on the molecular mechanism of protein aggregation, propagation and neurotoxicity in Parkinson's disease and prion disease. Prion disease and PD are associated with misfolding and aggregation of PrPc and a-Synuclein (a-Syn), respectively. Despite being two important neurodegenerative disorders, molecular mechanisms of a-Syn or PrPC aggregation and amyloidogenesis are still unclear in PD and prion disease. Furthermore, the toxic protein species in PD have not been characterized yet. In this study we characterize the mechanism of a-Syn and PrPc misfolding in a physiological-like cell free condition in the absence of a-Syn aggregates, PrPc ggregated isoform (Pre's), denaturants or acidic environment. A number of studies indicate that dimerization of PrPc or a-Syn may be a key step in the aggregation process. To test this hypothesis we verified if enforced dimerization of PrPc or a-Syn may induce a conformational change reminiscent of the conversion of PrPc or a-Syn to PrPR' or a-Syn aggregates, respectively. We used a well-described inducible dimerization strategy where a dimerizing domain called FK506-binding protein (Fv) was fused to PrPc or a-Syn in order to produce chimeric proteins Fv-PrP and a-SynF'''. A divalent ligand AP20187 was used to induce protein dimerization. Addition of AP20187 to recombinant Fv-PrP in physiological-like conditions resulted in a rapid conformational change characterized by an increase in beta-sheet (13-Sheet) structure and simultaneous aggregation of the proteins. However, non-dimerized PrP formed 13-Sheet conformation in very slower rates. In the presence of AP20187, we also report a rapid random coil into 13-sheet conformational transformation of a-SynF" within 24 h, whereas wild type a-Syn showed 24 h delay to achieve P-sheet structure after 48 h. Electron microscopy experiments demonstrated that dimerization induced amyloid fibril formation after 48 h for both Fv-PrP and a-Syr?", whereas in the absence of dimerizing ligand AP20187, PrP or a-Syn converted into amyloid fibrils after 3 days or even later. Dimerization-induced Fv-PrP aggregates were partially resistant to PK digestion which is a characteristics of the naturally occurring PrPR'. The rates of amyloidogenesis in the presence of dimerization was also characterized by Thioflavin T (ThT) fluorescence probing. Whereas the stable structure of Fv-PrP showed no ThT binding for over 60 h of incubation at 37°C, the addition of AP20187 to Fv-PrP resulted in a time-dependent increase in ThT binding. As for a-SynR, dimerization accelerated the rate of ThT binding and amyloid formation comparing to the slower amyloidogenesis rate of wild type a-Syn in the absence of dimerizer AP20187. The impact of dimerization on a-Syn aggregation was further determined by Fluorescence ANS probing, indicating a higher affinity of dimerization-induced a-SynF" aggregates for binding to ANS comparing to wild type a-Syn aggregates. These results indicate that dimerization increases the aggregation and amyloidogenesis processes for Fv-PrP and a-SynF". Both Fv-PrP and a-SynF" amyloids were successfully propagated in vitro by protein misfolding amplification (PMCA) cycle. These results ar in agreement with the theory that all protein aggregates in neurodegenerative diseases propagate with the same molecular mechanism. Neurotoxicity of recombinant Fv-PrP and a-SynF" aggregates was determined in cellulo and in vivo, respectively. Aggregates of Fv-PrP were toxic to cultured cells whilst soluble Fv-PrP and amyloid fibres were harmless to the cells. When injected to the mice brain, both a-Syni" and a-Syn pre-fibrillar aggregates internalized cells and induced neurotoxicity in the hippocampus of wild-type mice. These recombinant toxic aggregates further converted into non-toxic amyloids which were successfully amplified by PMCA method, providing the first evidence for the in vitro propagation of synthetic a-Syn aggregates. These results suggest an important role for protein dimerization in aggregation and amyloidogenesis, and therefore, in the pathology of PD and prion disease. The similarities between aggregation, amyloidogenesis and toxicity of PrPC and ct-Syn provide further evidence on the existance of a prion-like mechanism in all neurodegenerative disorders. // Résumé: Les maladies neurodégénératives sont associées à la perte progressive des propriétés structurales ou fonctionnelles des neurones, ce qui engendre la mort des cellules. De récentes études indiquent que tous les désordres neurodégénératifs, sporadiques ou transmissibles, peuvent avoir un mécanisme pathologique commun au niveau moléculaire. Ce dispositif commun se compose de l'agrégation de protéines, de la propagation des agrégats, et de l'accumulation d’agrégats toxiques dans les cellules neuronales, menant à l'apoptose et à la neurotoxicité cellulaire. Les maladies neurodégénératives peuvent affecter la pensée abstraite, les mouvements habiles, les sentiments émotifs, la connaissance, la Mémoire et d'autres capacités cognitives. Ce groupe divers de maladies inclut la maladie d'Alzheimer (AD), de Parkinson (PD), de Huntington (HD), les maladies à prions ou encéphalopathies spongiformes transmissibles (TSEs) et la sclérose latérale amyotrophique (ALS). [symboles non conformes]

La maladie de Parkinson (PD) et la démence à corps de Lewy (DLB) sont les deuxième et troisième maladies neurodégénératives les plus communes et font partie d’une classe de maladies appelées synucléinopathies. Les synucléinopathies sont associées à une pathologie liée à l’α-synucléine (α-syn) laquelle se caractérise par une accumulation de cette protéine dans les neurones, formant ainsi les corps de Lewy. L’α-syn pathologique se retrouve aussi sous forme d’oligomères et de fibrilles, qui sont toxiques pour les neurones et leurs synapses. L’une des premières anomalies observables chez les patients atteints de synucléinopathies est la dysfonction synaptique, souvent combinée à une perte de synapses. Il a été rapporté que les oligomères d’α-syn retrouvés au niveau des synapses précèdent la formation de corps de Lewy dans les neurones et leur transmission semble être associée à la progression des symptômes. Pourtant, les mécanismes moléculaires sous-jacents la dysfonction synaptique causée par l’α-syn restent inconnus. D’autre part, le fonctionnement normal des synapses est fortement régulé par une famille de protéines appelées organisateurs synaptiques. Les organisateurs synaptiques, incluant la protéine neurexine, sont des molécules d’adhésion cellulaire qui régulent la synaptogenèse, la plasticité, la libération des neurotransmetteurs et les fonctions cognitives. De plus, nous avons préliminairement montré que l’α-syn interagit avec l’isoforme β des neurexines (NRXs) (β-NRXs). Mon projet avait donc pour but de caractériser l’interaction α-syn/β-NRX et d’évaluer comment celle-ci contribue à la pathologie liée à l’α-syn. Nous avons émis l’hypothèse que cette interaction affecte la fonction synaptogénique liée aux NRXs et son trafic. Dans un premier temps, pour tester notre hypothèse, l’interaction α-syn/β-NRX a été évaluée grâce à des analyses de liaison à la surface cellulaire. Il a été constaté que les oligomères d’α-syn se lient fortement à NRX1,2β de manière dépendante du domaine riche en histidine (HRD), caractéristique de l’isoforme β, et cela sans perturber sa liaison à ses ligands endogènes postsynaptiques, neuroligine 1 (NLG1) et « leucine rich repeat transmembrane neuronal 2 » (LRRTM2). De plus, à travers des essais d’internalisation, nous avons observé que les oligomères d’α-syn altèrent le trafic de NRX1β en augmentant son internalisation de façon dépendante au HRD et altèrent également la différenciation NRX-dépendante de la synapse en synapse inhibitrice. Par conséquent, nous suggérons que cette internalisation accrue pourrait affecter la fonction synaptogénique associée aux NRXs. Ce travail contribue à une meilleure compréhension sur la façon dont l’α-syn provoque un dysfonctionnement synaptique, fournissant de nouvelles perspectives moléculaires et pharmacologiques sur les synucléinopathies. / Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are the second and the third most common neurodegenerative disorders and are part of a class of diseases called synucleinopathies. Synucleinopathies are associated with an α-synuclein (α-syn) pathology which shows an accumulation of α-syn in neurons, forming Lewy bodies. This pathological α-syn can form oligomers and fibrils, which are toxic for neurons and their synapses. One of the first changes to occur in patients’ brain with synucleinopathies is synaptic dysfunction often combined with synapse loss. Synaptic α-syn oligomers were revealed to precede the formation of Lewy bodies, and their transmission to other neurons to correlate with the progression of the symptoms. Yet, the molecular mechanisms underlying how α-syn leads to synaptic dysfunction are unknown. Synaptic function is highly regulated by a protein family called synaptic organizers. Synaptic organizers are cell adhesion molecules that regulate synaptogenesis, plasticity, neurotransmitter release, synaptic plasticity and cognitive functions. Of this family, we have found that α-syn interacts with the β-isoforms of the neurexins (NRXs) family members (β-NRXs). My project aimed to characterize α-syn/β-NRX interaction and to evaluate how this interaction contributes to α-syn pathology. We hypothesized that this interaction affects NRX trafficking and its synaptic function. Firstly, to test our hypothesis, the α-syn/β-NRX interaction was characterized by performing cell surface binding assays. I found that α-syn oligomers strongly bind to NRX1,2β in a histidine rich domain (HRD)-dependent manner, without disrupting NRX binding to its postsynaptic binding partners, neuroligin 1 (NLG1) and leucine rich repeat transmembrane neuronal 2 (LRRTM2). Moreover, using internalization assays, we discovered that α-syn oligomers impair NRX trafficking by increasing NRX1β internalization in an HRD-dependent manner and impair NRX-dependent inhibitory presynaptic differentiation. Thereby, we suggest that this increased internalization affects the inhibitory synaptogenic function of NRX-based synaptic organizing complexes. This work contributes to a better understanding of how α-syn causes synaptic dysfunction, providing promising new molecular mechanisms and pharmacological insights into synucleinopathies.

<p dir="ltr">My research projects focuses on the dual targeting of small molecules to abrogate aberrant α-syn, tau (2N4R), and p-tau (1N4R) aggregation and to reduce the spread of AD and related dementias. Not very many drug discovery programs focus on the specific isoforms of the tau protein. We established two series of compounds: aminoindole compounds connected by a carboxamide and triazine compounds connected by a triazine linker. Using biophysical methods we evaluated the effectiveness of both series of compounds in decreasing the amount of misfolded α-syn and tau protein in order to explore their anti-aggregation potential.</p><p><br></p>

Die Parkinson-Krankheit (PA) ist durch Fehlfaltung und Aggregation von Alpha-Synuklein (a-Syn) charakterisiert. Dieses zeigt Ähnlichkeiten mit dem Verhalten von Prionen bei transmissiblen spongiformen Enzephalopathien. Pathologische a-Syn Aggregate propagieren, wie Prione, über selbsttempliertes Seeding (Keimung). Aufgrund von Studien, die eine Übertragbarkeit der a-Syn Seeding-Aktivität nachgewiesen haben, war das Ziel dieser Dissertation die Risikobewertung potenzieller Übertragungen von a-Syn Seeds zu verbessern. Die Menge an Seeding-aktiven a-Syn in Hirn- und Magengewebe von PA-Patienten wurde durch Real-Time Quaking-Induced Conversion (RT-QuIC) Assays quantifiziert. Die Ergebnisse zeigten in beiden Geweben hohe Seeding-Titer, was die Notwendigkeit für effektive Inaktivierungsprotokolle unterstreicht. Die Quantifizierung des Effekts der Dampfsterilisation bei 134 °C ergab eine unerwartet hohe, Prion-übersteigende, Resistenz von PA-assoziiertem a-Syn gegen Dampfsterilisation. Die Verlängerung der Sterilisationsdauer führte zu einem identischen Reduktionsfaktor des Seeding-Titers. Fourier-Transform-Infrarot (FT-IR) Spektroskopie wurde genutzt, um eine Stammdifferenzierung zwischen a-Syn Aggregaten aus PA-assoziierten transgenen Mausmodellen mit den a-Syn-Mutationen A30P und A53T vorzunehmen, die für familiäre PA beim Menschen prädisponieren. Etablierte Protokolle zur Untersuchung von Prionstämmen (PS) wurden verfeinert um 5 hamsteradaptierte PS zu vergleichen: 263K, 22A-H, BSE-H, CWD-H, und ME7-H. Hier führte die Zugabe des Farbstoffs Kongo Rot zu spektralen Veränderungen in einigen PS und resultierte in der erfolgreichen Unterscheidung aller untersuchten PS. Mittels PMCA wurden die winzigen Mengen an a-Syn Aggregaten aus Maus-Hirngeweben für die anschließende FT-IR Spektroskopie vervielfältigt. Die Spektren der A30P und A53T a-Syn Aggregate wiesen allerdings keine Unterschiede auf, und der Zusatz von Kongo Rot hatte keinen Effekt auf die Spektren. / Parkinson’s Disease (PD) is characterized by misfolding and aggregation of Alpha-Synuclein (a-Syn), which shows similarities to the behavior of prions in transmissible spongiform encephalopathies (TSEs). Pathological a-Syn aggregates, like prions, propagate by self-templated seeding. Due to reports of transmissible seeding activity of a-Syn, the goal of this thesis was to improve risk assessment of potential a-Syn seed transmissions. Real-Time Quaking-Induced Conversion (RT-QuIC) assays were used to quantify seeding active a-Syn in brain and stomach tissue of PD patients. The results show high seeding titers in both tissues, highlighting the need for effective inactivation protocols. Quantification of the effects of steam sterilization at 134 °C on the seeding activity of cerebral PD-associated a-Syn revealed a surprisingly high, prion-exceeding, resistance to steam sterilization. Extended duration of steam sterilization has no further inactivation effect and led to an identical reduction factor of the seeding activity titer. Fourier-Transform Infrared (FT-IR) spectroscopy was used to attempt strain differentiation between a-Syn aggregates from PD-associated transgenic mouse models with the a-Syn A30P and A53T mutations which predispose for familial forms of PD in humans. Established protocols for prion strain examination were refined to compare five hamster-adapted prion strains: 263K, 22A-H, BSE-H, CWD-H, and ME7-H. Here, the addition of a dye (Congo Red) led to spectral changes of some prion strains and resulted in the successful distinction of all investigated prion strains. To increase the tiny amounts of a-Syn aggregates in the brain tissues of mice, PMCA was used to multiply these aggregates for subsequent FT-IR spectroscopy. However, the spectra of the A30P and A53T a-Syn aggregates were undistinguishable, and the addition of CR had no significant effect on the spectra.

Alterações morfológicas e funcionais ocorrem durante o envelhecimento, período da vida com maior incidência de doenças neurodegenerativas. No presente trabalho acompanhou-se a evolução dos grânulos de lipofucsina durante o envelhecimento para investigar alterações sinápticas, assim como proteínas associadas com doenças neurodegenerativas (alfa-sinucleína) e com o sistema ubiquitina-proteossoma em indivíduos de diferentes idades. No córtex temporal humano e de ratos determinou-se, nos diferentes grupos etários, seguindo a área, o número e as características dos grânulos de lipofucsina, o número de sinapses excitatórias, inibitórias e elétricas, os locais de contatos pós-sinápticos, o número de vesículas sinápticas por terminal e a expressão das proteínas alfa-sinucleína e ubiquitina. Amostras de córtex temporal humano de indivíduos com diferentes idades (20 - 28, 37 - 41 e 50 - 55 anos) foram coletadas de pacientes com epilepsia submetidos à lobectomia do lobo temporal. Amostras de ratos de 2, 6, 10 e 12 meses também foram coletadas. Foram utilizadas técnicas de microscopia de luz, eletrônica, confocal e western blots. Os dados obtidos de grânulos de lipofucsina são consistentes com outros estudos que observaram aumento dessa estrutura em mamíferos de maior idade. No entanto, os grânulos parecem crescer em volume, mas não em número, com aumento considerável da fração elétron lúcida (lipídica). Não houve diferença na expressão das proteínas alfa-sinucleína e ubiquitina entre os grupos das idades estudadas. A densidade sináptica foi similar entre os grupos experimentais, assim como o local de contato pós-sináptico. O aumento de vesículas elétron densas em sinapses inibitórias deve estar associado à demanda de neurotransmissores catecolaminérgicos. Estes resultados não expressam totalmente o processo de envelhecimento, pois as faixas etárias de humanos e ratos correspondem a uma idade ainda jovem. A coleta de material humano mais idoso foi impossibilitada pela faixa etária dos doentes submetidos à lobectomia. Os ratos do biotério da FMRP, não sobrevivem mais do que 12 meses em no nosso ambiente, incluindo manutenção dos animais isolados em racks. / Morphological and functional changes occur during the aging, period of life with increased incidence of neurodegenerative diseases. Following the evolution of the lipofuscin granules along three periods of life in humans and rats, the present work investigated synaptic changes, as well as proteins associated with neurodegenerative diseases (alpha-synuclein) and the ubiquitin-proteossoma system in individuals of different ages. The objectives of the study were to analyze the temporal cortex of humans and rats: the number of excitatory, inhibitory, and electric synapses, the site of postsynaptic contacts, the number of synaptic vesicles per terminal, and the expression of the proteins alpha-synuclein and ubiquitin following the size and features of the lipofuscin granules. Samples of temporal cortex of human subjects with different ages (20-28, 37-41 and 50-55 years) were collected from patients with epilepsy who underwent temporal lobectomy. Samples from rats of 2, 6, 10 and 12 months were also collected. Light, confocal, and electron microscopy, and western blots techniques were used as procedures. The data obtained on lipofuscin granules were coincident with other studies that observed a higher area occupied by this structure in older mammals. However, the granules seem to grow in volume, but not in number, with considerable increase of the electron lucid fraction (lipidic). There was no difference in the alpha-synuclein and ubiquitin expressions between the experimental groups. The synaptic densities were similar between the groups, as well as the postsynaptic contacts. Increase of the electron dense vesicles in inhibitory synapses, appeared to be associated with the demand of catecholamines. These results do not express totally the aging process, because the range of age used in humans, and rats belong to a young age. The human samples from older ages was difficult because, in general, of the age of the patients submitted to lobotomy. The rats of the FMRP bioterium do not survived more that 12 months in our environment, even in controlled conditions.

No description available.

Alterações morfológicas e funcionais ocorrem durante o envelhecimento, período da vida com maior incidência de doenças neurodegenerativas. No presente trabalho acompanhou-se a evolução dos grânulos de lipofucsina durante o envelhecimento para investigar alterações sinápticas, assim como proteínas associadas com doenças neurodegenerativas (alfa-sinucleína) e com o sistema ubiquitina-proteossoma em indivíduos de diferentes idades. No córtex temporal humano e de ratos determinou-se, nos diferentes grupos etários, seguindo a área, o número e as características dos grânulos de lipofucsina, o número de sinapses excitatórias, inibitórias e elétricas, os locais de contatos pós-sinápticos, o número de vesículas sinápticas por terminal e a expressão das proteínas alfa-sinucleína e ubiquitina. Amostras de córtex temporal humano de indivíduos com diferentes idades (20 - 28, 37 - 41 e 50 - 55 anos) foram coletadas de pacientes com epilepsia submetidos à lobectomia do lobo temporal. Amostras de ratos de 2, 6, 10 e 12 meses também foram coletadas. Foram utilizadas técnicas de microscopia de luz, eletrônica, confocal e western blots. Os dados obtidos de grânulos de lipofucsina são consistentes com outros estudos que observaram aumento dessa estrutura em mamíferos de maior idade. No entanto, os grânulos parecem crescer em volume, mas não em número, com aumento considerável da fração elétron lúcida (lipídica). Não houve diferença na expressão das proteínas alfa-sinucleína e ubiquitina entre os grupos das idades estudadas. A densidade sináptica foi similar entre os grupos experimentais, assim como o local de contato pós-sináptico. O aumento de vesículas elétron densas em sinapses inibitórias deve estar associado à demanda de neurotransmissores catecolaminérgicos. Estes resultados não expressam totalmente o processo de envelhecimento, pois as faixas etárias de humanos e ratos correspondem a uma idade ainda jovem. A coleta de material humano mais idoso foi impossibilitada pela faixa etária dos doentes submetidos à lobectomia. Os ratos do biotério da FMRP, não sobrevivem mais do que 12 meses em no nosso ambiente, incluindo manutenção dos animais isolados em racks. / Morphological and functional changes occur during the aging, period of life with increased incidence of neurodegenerative diseases. Following the evolution of the lipofuscin granules along three periods of life in humans and rats, the present work investigated synaptic changes, as well as proteins associated with neurodegenerative diseases (alpha-synuclein) and the ubiquitin-proteossoma system in individuals of different ages. The objectives of the study were to analyze the temporal cortex of humans and rats: the number of excitatory, inhibitory, and electric synapses, the site of postsynaptic contacts, the number of synaptic vesicles per terminal, and the expression of the proteins alpha-synuclein and ubiquitin following the size and features of the lipofuscin granules. Samples of temporal cortex of human subjects with different ages (20-28, 37-41 and 50-55 years) were collected from patients with epilepsy who underwent temporal lobectomy. Samples from rats of 2, 6, 10 and 12 months were also collected. Light, confocal, and electron microscopy, and western blots techniques were used as procedures. The data obtained on lipofuscin granules were coincident with other studies that observed a higher area occupied by this structure in older mammals. However, the granules seem to grow in volume, but not in number, with considerable increase of the electron lucid fraction (lipidic). There was no difference in the alpha-synuclein and ubiquitin expressions between the experimental groups. The synaptic densities were similar between the groups, as well as the postsynaptic contacts. Increase of the electron dense vesicles in inhibitory synapses, appeared to be associated with the demand of catecholamines. These results do not express totally the aging process, because the range of age used in humans, and rats belong to a young age. The human samples from older ages was difficult because, in general, of the age of the patients submitted to lobotomy. The rats of the FMRP bioterium do not survived more that 12 months in our environment, even in controlled conditions.