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Insulin degrading enzyme as a therapeutic target for Parkinson’s disease
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RESUMO: A doença de Parkinson é a segunda doença neurodegenerativa mais comum. Esta doença apresenta várias
manifestações motoras que incluem tremor, bradicinésia e rigidez, possivelmente resultantes da perda de
neurónios dopaminérgicos na substantia nigra pars compacta. Uma característica subjacente à degeneração
destes neurónios é a acumulação e a agregação de alfa-sinucleína (aSyn). Estudos epidemiológicos recentes
concluíram que a diabetes mellitus tipo 2 (DM2) pode aumentar o risco de desenvolvimento de doença de
Parkinson até 380% (em particular, caso a DM2 tenha sido desenvolvida entre os 25-45 anos de idade). Além
disso, os doentes de Parkinson com diabetes apresentam uma progressão mais rápida dos problemas
motores bem como um declínio cognitivo mais acentuado. No entanto, o mecanismo molecular subjacente
a esta associação não é claro. Temos por hipótese que a enzima degradativa da insulina (IDE) possa ser
responsável por esta associação. A IDE apresenta capacidade degradativa para diferentes substratos,
incluindo a insulina ou o péptido beta-amilóide, mas não a aSyn. Curiosamente, nas células pancreáticas, a
IDE interage com a aSyn, impedindo a sua agregação e toxicidade. Além disso, no pâncreas, os níveis de IDE
correlacionam negativamente com os de aSyn. Portanto, acreditamos que a diabetes possa induzir uma
redução dos níveis de IDE também no cérebro, comprometendo consequentemente os seus efeitos
protetores contra a patogenicidade da aSyn. Nesta tese, o objetivo principal foi o de investigar se existe
alguma relação entre a desregulação da IDE e a patogenicidade da aSyn no cérebro. Para tal, utilizámos duas
estratégias. Por um lado, analisámos tecido cerebral provenientes de um modelo animal de (pré) diabetes.
Por outro, investigámos a possível interação entre a IDE e a aSyn num modelo celular de Sinucleinopatias.
Para tal, analisámos extratos proteicos provenientes de ratinhos C57BL/6J alimentados durante 12 semanas
com uma dieta controlada ou com uma dieta hiperlipidica (modelo pré-diabético-HFD). Verificámos que com
excepção do hipocampo, a diabetes diminui os níveis de IDE em todas as áreas cerebrais analisadas. Pelo
contrário, os níveis cerebrais de aSyn aumentam quer no hipocampo quer no cerebelo destes ratinhos (pré)
diabéticos. Além disso, constatámos que os níveis de IDE e de aSyn no cerebelo correlacionam-se
inversamente. Estas evidências corroboram a nossa hipótese de que a diabetes pode diminuir os níveis
cerebrais de IDE, aumentando potencialmente os níveis da aSyn. Para entender melhor a interação entre a
IDE e a aSyn, usámos um modelo in vitro de sinucleinopatias estabelecido, que se baseia na sobreexpressão
em células H4 da aSyn ou de uma variante de aSyn propensa a agregação (SynT). Observámos que quer a
forma catalítica activa da IDE (IDE-WT) quer a forma inactiva da IDE (E111Q) têm capacidade de diminuir a
citotoxicidade da aSyn, mas não da variante SynT. As nossas descobertas sugerem ainda que a capacidade
protectora da IDE ocorre de uma forma dependente da sua actividade, já que a IDE-WT é mais eficaz do que
a variante inactiva IDE-E111Q. Curiosamente, a IDE-WT e a IDE-E111Q aumentam a quantidade intracelular
de aSyn. Dado que a toxicidade da aSyn é suprimida, pensamos que a IDE consiga converter espécies
oligoméricas / tóxicas da aSyn em espécies não-tóxicas. De facto, observámos que a IDE-E111Q é capaz de
aumentar a solubilidade da SynT e tanto a IDE-WT como a IDE-E111Q favorecem a formação de inclusões
maiores de SynT, descritas como sendo menos tóxicas. Concluímos com este estudo que a IDE pode
desempenhar um papel importante no cérebro, suprimindo a patogénese da aSyn. Propomos assim que a
potenciação da IDE possa representar um alvo terapêutico relevante para o tratamento da doença de
Parkinson. Além disso, um tratamento que vise aumentar a capacidade da IDE cerebral também pode ter um
grande impacto no tratamento de doentes com DM2, de forma a reduzir o risco de desenvolverem a doença
de Parkinson, principalmente em indivíduos que apresentem maior comprometimento da IDE.
ABSTRACT: Parkinson’s disease is the second most common neurodegenerative disorder, characterized by motor features that include tremor, bradykinesia and rigidity, possibly resultant from the loss of dopaminergic neurons in the substantia nigra pars compacta. A major pathognomonic feature is the neuronal accumulation of aggregated alpha-synuclein (aSyn), known to trigger neurodegeneration. Recent cohort association studies concluded that type-2 diabetes mellitus (T2DM) increases up to 380% the risk of PD development (young individuals with T2DM). Moreover, T2DM is also associated with faster motor progression and cognitive decline of PD patients. However, the molecular link between this association is not clear. We hypothesize that a possible molecular player underlying both conditions is the insulin degrading enzyme (IDE). IDE presents degradative capacity for different substrates including insulin or amyloid-beta peptide, but not aSyn. Interestingly, in pancreatic cells, IDE interacts with aSyn, preventing its aggregation and toxicity. Moreover, there is an inverse correlation between IDE and aSyn levels in the pancreas. Therefore, we hypothesize that diabetes may induce IDE impairment in the brain, therefore compromising its protective effects against aSyn pathogenicity. In this thesis we aimed to establish a relationship between IDE dysregulation and aSyn pathogenicity, using a two-pronged strategy: analysis of brain samples from animal models of (pre)diabetes; evaluation of IDE-aSyn interplay in a cellular model of Parkinson’s disease. To that purpose, we analyzed protein extracts from brain tissue of WT mice under a control or a high-fat diet (HFD) for 12 weeks (prediabetic model). Remarkably, we showed that diabetes generally decreases the levels of IDE in all analyzed brain areas, at the exception of the hippocampus. In contrast, brain aSyn levels are increased in both hippocampus and cerebellum of these mice. Moreover, an inverse correlation between IDE and aSyn levels was observed (cerebellum). These findings support our hypothesis that diabetes may decrease IDE levels in the brain, and that aSyn levels inversely correlate with IDE levels. To better understand the interplay between IDE-aSyn, we used an established in vitro model of synucleinopathies, based on the overexpression of WT aSyn, or of an aggregation-prone variant of aSyn (SynT) in H4 cells. Notably, both catalytical active IDE-WT or catalytical inactive IDE-E111Q forms of IDE decrease the cytotoxicity of aSyn, but not of SynT. This confirms that IDE is able to reduce aSyn toxicity, and our findings suggest that it occurs in a catalytical-dependent manner. Intriguingly, both IDE-WT and IDE-E111Q increase the intracellular amount of aSyn. Since aSyn toxicity is suppressed, we hypothesize that IDE is diverting aSyn oligomeric/toxic species to more stabilized non-toxic species. In fact, we observed that IDE-E111Q is able to increase the solubility of SynT, and that both IDE-WT and IDE-E111Q favor the formation of larger inclusions of SynT, described to be less toxic. Altogether, we conclude that IDE may play an important role in the brain by suppressing aSyn pathogenesis, and that its potentiation could represent an appealing therapeutic target for Parkinson’s disease. Moreover, a brain-IDEtargeted treatment could also have a great impact in T2DM individuals, to control their high risk to develop PD, particularly of individuals with higher impairment of IDE.
ABSTRACT: Parkinson’s disease is the second most common neurodegenerative disorder, characterized by motor features that include tremor, bradykinesia and rigidity, possibly resultant from the loss of dopaminergic neurons in the substantia nigra pars compacta. A major pathognomonic feature is the neuronal accumulation of aggregated alpha-synuclein (aSyn), known to trigger neurodegeneration. Recent cohort association studies concluded that type-2 diabetes mellitus (T2DM) increases up to 380% the risk of PD development (young individuals with T2DM). Moreover, T2DM is also associated with faster motor progression and cognitive decline of PD patients. However, the molecular link between this association is not clear. We hypothesize that a possible molecular player underlying both conditions is the insulin degrading enzyme (IDE). IDE presents degradative capacity for different substrates including insulin or amyloid-beta peptide, but not aSyn. Interestingly, in pancreatic cells, IDE interacts with aSyn, preventing its aggregation and toxicity. Moreover, there is an inverse correlation between IDE and aSyn levels in the pancreas. Therefore, we hypothesize that diabetes may induce IDE impairment in the brain, therefore compromising its protective effects against aSyn pathogenicity. In this thesis we aimed to establish a relationship between IDE dysregulation and aSyn pathogenicity, using a two-pronged strategy: analysis of brain samples from animal models of (pre)diabetes; evaluation of IDE-aSyn interplay in a cellular model of Parkinson’s disease. To that purpose, we analyzed protein extracts from brain tissue of WT mice under a control or a high-fat diet (HFD) for 12 weeks (prediabetic model). Remarkably, we showed that diabetes generally decreases the levels of IDE in all analyzed brain areas, at the exception of the hippocampus. In contrast, brain aSyn levels are increased in both hippocampus and cerebellum of these mice. Moreover, an inverse correlation between IDE and aSyn levels was observed (cerebellum). These findings support our hypothesis that diabetes may decrease IDE levels in the brain, and that aSyn levels inversely correlate with IDE levels. To better understand the interplay between IDE-aSyn, we used an established in vitro model of synucleinopathies, based on the overexpression of WT aSyn, or of an aggregation-prone variant of aSyn (SynT) in H4 cells. Notably, both catalytical active IDE-WT or catalytical inactive IDE-E111Q forms of IDE decrease the cytotoxicity of aSyn, but not of SynT. This confirms that IDE is able to reduce aSyn toxicity, and our findings suggest that it occurs in a catalytical-dependent manner. Intriguingly, both IDE-WT and IDE-E111Q increase the intracellular amount of aSyn. Since aSyn toxicity is suppressed, we hypothesize that IDE is diverting aSyn oligomeric/toxic species to more stabilized non-toxic species. In fact, we observed that IDE-E111Q is able to increase the solubility of SynT, and that both IDE-WT and IDE-E111Q favor the formation of larger inclusions of SynT, described to be less toxic. Altogether, we conclude that IDE may play an important role in the brain by suppressing aSyn pathogenesis, and that its potentiation could represent an appealing therapeutic target for Parkinson’s disease. Moreover, a brain-IDEtargeted treatment could also have a great impact in T2DM individuals, to control their high risk to develop PD, particularly of individuals with higher impairment of IDE.
Descrição
Palavras-chave
Doença de Parkinson Diabetes Parkinson's disease Alpha-synuclein Type 2 Diabetes