RESUMO
Dysfunctional mitochondria are linked to several neurodegenerative diseases. Metabolic defects, a symptom which can result from dysfunctional mitochondria, are also present in spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease, the most frequent, dominantly inherited neurodegenerative ataxia worldwide. Mitochondrial dysfunction has been reported for several neurodegenerative disorders and ataxin-3 is known to deubiquitinylate parkin, a key protein required for canonical mitophagy. In this study, we analyzed mitochondrial function and mitophagy in a patient-derived SCA3 cell model. Human fibroblast lines isolated from SCA3 patients were immortalized and characterized. SCA3 patient fibroblasts revealed circular, ring-shaped mitochondria and featured reduced OXPHOS complexes, ATP production and cell viability. We show that wildtype ataxin-3 deubiquitinates VDAC1 (voltage-dependent anion channel 1), a member of the mitochondrial permeability transition pore and a parkin substrate. In SCA3 patients, VDAC1 deubiquitination and parkin recruitment to the depolarized mitochondria is inhibited. Increased p62-linked mitophagy, autophagosome formation and autophagy is observed under disease conditions, which is in line with mitochondrial fission. SCA3 fibroblast lines demonstrated a mitochondrial phenotype and dysregulation of parkin-VDAC1-mediated mitophagy, thereby promoting mitochondrial quality control via alternative pathways.
Assuntos
Doença de Machado-Joseph , Ataxina-3/genética , Ataxina-3/metabolismo , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo , Canal de Ânion 1 Dependente de Voltagem/genética , Canal de Ânion 1 Dependente de Voltagem/metabolismoRESUMO
NOD-like receptors (NLR) are critical regulators of innate immune signaling. The NLR family consists of 22 human proteins with a conserved structure containing a central oligomerization NACHT domain, an N-terminal interaction domain, and a variable number of C-terminal leucine-rich repeats. Most NLR proteins function as cytosolic pattern recognition receptors with activation of downstream inflammasome signaling, NF-κB, or MAPK activation. Although NLRP10 is the only NLR protein lacking the leucine rich repeats, it has been implicated in multiple immune pathways, including the regulation of inflammatory responses toward Leishmania major and Shigella flexneri infection. In this study, we identify Abin-1, a negative regulator of NF-κB, as an interaction partner of NLRP10 that binds to the NACHT domain of NLRP10. Using S. flexneri as an infection model in human epithelial cells, our work reveals a novel function of NLRP10 in destabilizing Abin-1, resulting in enhanced proinflammatory signaling. Our data give insight into the molecular mechanism underlying the function of NLRP10 in innate immune responses.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas de Ligação a DNA/metabolismo , Disenteria Bacilar/imunologia , Células Epiteliais/fisiologia , Inflamação/imunologia , Shigella flexneri/fisiologia , Animais , Proteínas Reguladoras de Apoptose/genética , Células HEK293 , Humanos , Imunidade Inata , Inflamassomos/metabolismo , Camundongos , Camundongos Knockout , NF-kappa B/metabolismo , Ligação Proteica , Estabilidade Proteica , Transdução de SinaisRESUMO
Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph Disease, is a progressive neurodegenerative disorder characterized by loss of neuronal matter due to the expansion of the CAG repeat in the ATXN3/MJD1 gene and subsequent ataxin-3 protein. Although the underlying pathogenic protein expansion has been known for more than 20 years, the complexity of its effects is still under exploration. The ataxin-3 protein in its expanded form is known to aggregate and disrupt cellular processes in neuronal tissue but the role of the protein on populations of immune cells is unknown. Recently, mast cells have emerged as potential key players in neuroinflammation and neurodegeneration. Here, we examined the mast cell-related effects of ataxin-3 expansion in the brain tissues of 304Q ataxin-3 knock-in mice and SCA3 patients. We also established cultures of mast cells from the 304Q knock-in mice and examined the effects of 304Q ataxin-3 knock-in on the immune responses of these cells and on markers involved in mast cell growth, development and function. Specifically, our results point to a role for expanded ataxin-3 in suppression of mast cell marker CD117/c-Kit, pro-inflammatory cytokine TNF-α and NF-κB inhibitor IκBα along with an increased expression of the granulocyte-attracting chemokine CXCL1. These results are the beginning of a more holistic understanding of ataxin-3 and could point to the development of novel therapeutic targets which act on inflammation to mitigate symptoms of SCA3.
Assuntos
Doença de Machado-Joseph , Doenças Neurodegenerativas , Animais , Ataxina-3/genética , Ataxina-3/metabolismo , Humanos , Doença de Machado-Joseph/genética , Doença de Machado-Joseph/metabolismo , Doença de Machado-Joseph/patologia , Mastócitos/metabolismo , Camundongos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismoRESUMO
Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disorder resulting from an aberrant expansion of a polyglutamine stretch in the ataxin-3 protein and subsequent neuronal death. The underlying intracellular signaling pathways are currently unknown. We applied the Reverse-phase Protein MicroArray (RPMA) technology to assess the levels of 50 signaling proteins (in phosphorylated and total forms) using three in vitro and in vivo models expressing expanded ataxin-3: (i) human embryonic kidney (HEK293T) cells stably transfected with human ataxin-3 constructs, (ii) mouse embryonic fibroblasts (MEF) from SCA3 transgenic mice, and (iii) whole brains from SCA3 transgenic mice. All three models demonstrated a high degree of similarity sharing a subset of phosphorylated proteins involved in the PI3K/AKT/GSK3/mTOR pathway. Expanded ataxin-3 strongly interfered (by stimulation or suppression) with normal ataxin-3 signaling consistent with the pathogenic role of the polyglutamine expansion. In comparison with normal ataxin-3, expanded ataxin-3 caused a pro-survival stimulation of the ERK pathway along with reduced pro-apoptotic and transcriptional responses.
Assuntos
Ataxina-3/fisiologia , Doença de Machado-Joseph/fisiopatologia , Proteínas do Tecido Nervoso/fisiologia , Peptídeos/metabolismo , Fosfoproteínas/fisiologia , Transdução de Sinais/fisiologia , Animais , Apoptose , Ataxina-3/genética , Linhagem Celular , Fibroblastos , Quinase 3 da Glicogênio Sintase/fisiologia , Células HEK293 , Humanos , Camundongos , Camundongos Transgênicos , Fosfatidilinositol 3-Quinases/fisiologia , Análise Serial de Proteínas , Proteínas Proto-Oncogênicas c-akt/fisiologia , Serina-Treonina Quinases TOR/fisiologiaRESUMO
BACKGROUND & AIMS: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an autosomal dominantly inherited neurodegenerative disorder and the most common form of SCA worldwide. It is caused by the expansion of a polyglutamine (polyQ) tract in the ataxin-3 protein. Nuclear localization of the affected protein is a key event in the pathology of SCA3 via affecting nuclear organization, transcriptional dysfunction, and seeding aggregations, finally causing neurodegeneration and cell death. So far, there is no effective therapy to prevent or slow the progression of SCA3. METHODS: In this study, we explored the effect of divalproex sodium as an HDACi in SCA3 cell models and explored how divalproex sodium interferes with pathogenetic processes causing SCA3. RESULTS: We found that divalproex sodium rescues the hypoacetylation levels of histone H3 and attenuates cellular cytotoxicity induced by expanded ataxin-3 partly via preventing nuclear transport of ataxin-3 (particularly heat shock-dependent). CONCLUSION: Our study provides novel insights into the mechanisms of action of divalproex sodium as a possible treatment for SCA3, beyond the known regulation of transcription.