The stress granule protein G3BP1 alleviates spinocerebellar ataxia-associated deficits

Koppenol et al. show that overexpression of G3BP1 in cell models of SCA2 and SCA3 leads to a reduction in ataxin-2 and ataxin-3 aggregation. G3BP1 lentiviral delivery reduces motor deficits and neuropathology in preclinical models, suggesting that G3BP1 may be a potential therapeutic target for polyQ disorders. Polyglutamine diseases are a group of neurodegenerative disorders caused by an abnormal expansion of CAG repeat tracts in the codifying regions of nine, otherwise unrelated, genes. While the protein products of these genes are suggested to play diverse cellular roles, the pathogenic mutant proteins bearing an expanded polyglutamine sequence share a tendency to self-assemble, aggregate and engage in abnormal molecular interactions. Understanding the shared paths that link polyglutamine protein expansion to the nervous system dysfunction and the degeneration that takes place in these disorders is instrumental to the identification of targets for therapeutic intervention. Among polyglutamine diseases, spinocerebellar ataxias (SCAs) share many common aspects, including the fact that they involve dysfunction of the cerebellum, resulting in ataxia. Our work aimed at exploring a putative new therapeutic target for the two forms of SCA with higher worldwide prevalence, SCA type 2 (SCA2) and type 3 (SCA3), which are caused by expanded forms of ataxin-2 (ATXN2) and ataxin-3 (ATXN3), respectively. The pathophysiology of polyglutamine diseases has been described to involve an inability to properly respond to cell stress. We evaluated the ability of GTPase-activating protein-binding protein 1 (G3BP1), an RNA-binding protein involved in RNA metabolism regulation and stress responses, to counteract SCA2 and SCA3 pathology, using both in vitro and in vivo disease models. Our results indicate that G3BP1 overexpression in cell models leads to a reduction of ATXN2 and ATXN3 aggregation, associated with a decrease in protein expression. This protective effect of G3BP1 against polyglutamine protein aggregation was reinforced by the fact that silencing G3bp1 in the mouse brain increases human expanded ATXN2 and ATXN3 aggregation. Moreover, a decrease of G3BP1 levels was detected in cells derived from patients with SCA2 and SCA3, suggesting that G3BP1 function is compromised in the context of these diseases. In lentiviral mouse models of SCA2 and SCA3, G3BP1 overexpression not only decreased protein aggregation but also contributed to the preservation of neuronal cells. Finally, in an SCA3 transgenic mouse model with a severe ataxic phenotype, G3BP1 lentiviral delivery to the cerebellum led to amelioration of several motor behavioural deficits. Overall, our results indicate that a decrease in G3BP1 levels may be a contributing factor to SCA2 and SCA3 pathophysiology, and that administration of this protein through viral vector-mediated delivery may constitute a putative approach to therapy for these diseases, and possibly other polyglutamine disorders.PPBI-POCI-01-0145-FEDER-022122info:eu-repo/semantics/publishedVersio

Estudo da potencial toxicidade do processo de silenciamento da ataxina 3 mutante humana mediado por shRNA lentiviral

Dissertação de Mestrado em Bioquímica, apresentada ao Departamento de Ciências da Vida da Faculdade de Ciências e Tecnologia da Universidade de Coimbra.Previamente, o nosso laboratório demonstrou a eficácia terapêutica do silenciamento génico da ataxina 3 mutante humana mediado por shRNA, usando lentivírus, no estriado e cerebelo de modelos animais da doença de Machado-Joseph (MJD). Tendo em conta as evidências crescentes de que o processo de silenciamento génico mediado por shRNAs pode conduzir a efeitos citotóxicos, decidiu-se averiguar a segurança da estratégia terapêutica anteriormente testada. Procedeu-se assim a uma avaliação da potencial toxicidade do shRNA lentiviral usado para o silenciamento da ataxina 3 mutante humana (shmutatx3), no estriado de murganhos. Com esse propósito foram analisados três aspectos: a integridade neuronal (através da análise imunohistoquímica da expressão de dois marcadores neuronais, DARPP-32 e NeuN, e da análise histoquímica com violeta de cresil), a resposta microglial (através da análise imunohistoquímica da expressão de um marcador microglial, iba-1) e a resposta astrocítica (através da análise imunohistoquímica e da análise por western blot da expressão de um marcador astrocítico, GFAP) no estriado de murganhos wild-type adultos em quatro time points após a injecção dos lentivírus: 2 semanas, 4 semanas, 8 semanas e 5 meses. O shmutatx3 parece apresentar toxicidade numa fase inicial, uma vez que os resultados referentes à avaliação da integridade neuronal indicam que a neurodegeneração às 2 semanas na injecção do LV-nlsLacZ-shmutatx3 (lentivírus que codificam o shmutatx3) é significativamente superior à que se verifica na injecção do LV-eGFP (lentivírus sem efeito terapêutico). Apesar da aparente toxicidade inicial, o shmutatx3 não é tóxico a longo prazo, uma vez que há uma recuperação funcional da zona lesada no estriado entre as 4 semanas e os 5 meses na injecção do LV-nlsLacZshmutatx3, que é quase completa aos 5 meses. Esse aspecto revela que existe uma adaptação neuronal à transcrição constitutiva do shmutatx3. Alguma da diferença em termos de neurodegeneração observada entre as injecções do LV-nlsLacZ-shmutatx3 e do LV-eGFP pode estar relacionada com o facto destes terem genes-repórter diferentes. Os resultados da avaliação da resposta microglial e da resposta astrocítica sugerem que a aparente toxicidade do shmutatx3 numa fase inicial, mas não a longo prazo, poderá derivar da prevalência alongada de uma microglia M1 e de um fenótipo pró-inflamatório dos astrócitos activados (fenótipos de defesa que podem ter um efeito neurotóxico se forem persistentes), que dá lugar à prevalência de uma microglia M2 e de um fenótipo anti-inflamatório dos astrócitos activados (fenótipos de reparação que têm um efeito neuroprotector e promotor do sprouting neuronal e da neurogénese) entre as 2 e as 4 semanas.Data from our laboratory has previously demonstrated the therapeutic efficacy of shRNA-mediated gene silencing of human mutant ataxin 3, using lentiviral vectors, in cerebellum and striatum of Machado-Joseph disease (MJD) animal models. Given the growing evidence that shRNAs-mediated gene silencing process can lead to cytotoxic effects, we decided to investigate the safety of our previously tested therapeutic strategy. Thus, in this study, we have evaluated the potential shRNA toxicity used for silencing human mutant ataxin 3 (shmutatx3), in the mouse striatum. For this purpose we analyzed three aspects: neuronal integrity (by immunohistochemical analysis of two neuronal markers, NeuN and DARPP-32, and histochemical analysis using cresyl violet), microglial response (by immunohistochemical analysis of a microglial marker, iba-1) and astrocytic response (by immunohistochemical analysis and western blot analysis of an astrocytic marker, GFAP) in the striatum of adult wild-type mice at four time points after injection of lentiviral vectors: 2 weeks, 4 weeks, 8 weeks and 5 months. The shmutatx3 seem to be toxic at an early-stage, since the results of neuronal integrity evaluation indicate that the neurodegeneration at 2 weeks in LV-nlsLacZshmutatx3 (lentiviruses encoding shmutatx3) injection is significantly higher than that found in LV-eGFP (lentiviruses without therapeutic effect) injection. Despite the apparent initial toxicity, shmutatx3 is non-toxic at long term, since there is a functional recovery of the injured area in the striatum between 4 weeks and 5 months in LVnlsLacZ-shmutatx3 injection, which is almost complete after 5 months. This aspect reveals that there is a neuronal adaptation to the constitutive transcription of shmutatx3. Some of the difference in the neurodegeneration observed between LVnlsLacZ-shmutatx3 and LV-eGFP injections may be related to the fact that these lentivirus have different reporter genes. The results of microglial response and astrocytic response suggest that the apparent toxicity of shmutatx3 at an early-stage, but not at long term, may derive from the elongated prevalence of a microglia M1 and a pro-inflammatory phenotype of activated astrocytes (defense phenotypes that may have a neurotoxic effect if they are persistent), which gives rise to the prevalence of a microglial M2 and an antiinflammatory phenotype of activated astrocytes (repair phenotypes that have a neuroprotective effect and promoting neuronal sprouting and neurogenesis) between 2 and 4 weeks

The autophagy‐enhancing drug carbamazepine improves neuropathology and motor impairment in mouse models of Machado–Joseph disease

Aims Machado-Joseph disease (MJD), or spinocerebellar ataxia type 3 (SCA3), is the most common autosomal dominantly-inherited ataxia worldwide and is characterised by the accumulation of mutant ataxin-3 (mutATXN3) in different brain regions, leading to neurodegeneration. Currently, there are no available treatments able to block disease progression. In this study, we investigated whether carbamazepine (CBZ) would activate autophagy and mitigate MJD pathology. Methods The autophagy-enhancing activity of CBZ and its effects on clearance of mutATXN3 were evaluated using in vitro and in vivo models of MJD. To investigate the optimal treatment regimen, a daily or intermittent CBZ administration was applied to MJD transgenic mice expressing a truncated human ATXN3 with 69 glutamine repeats. Motor behaviour tests and immunohistology was performed to access the alleviation of MJD-associated motor deficits and neuropathology. A retrospective study was conducted to evaluate the CBZ effect in MJD patients. Results We found that CBZ promoted the activation of autophagy and the degradation of mutATXN3 in MJD models upon short or intermittent, but not daily prolonged, treatment regimens. CBZ up-regulated autophagy through activation of AMPK, which was dependent on the myo-inositol levels. In addition, intermittent CBZ treatment improved motor performance, as well as prevented neuropathology in MJD transgenic mice. However, in patients, no evident differences in SARA scale were found, which was not unexpected given the small number of patients included in the study. Conclusions Our data support the autophagy-enhancing activity of CBZ in the brain and suggest this pharmacological approach as a promising therapy for MJD and other polyglutamine disorders.Fundação para Ciência e Tecnologia; Richard Chin and Lily Lock Machado-Joseph Disease Research Fund; American Portuguese Biomedical Research Fund (APBRF); National Ataxia Foundation (USA); European Union H2020 program, GA No.643417; EU Joint Program - Neurodegenerative Disease Research (JPND); Competitiveness Factors Operational Program (COMPETE 2020); ERDF through the Regional Operational Program Center 2020info:eu-repo/semantics/publishedVersio