RESUMO
Arginine-rich dipeptide repeat proteins (R-DPRs), abnormal translational products of a GGGGCC hexanucleotide repeat expansion in C9ORF72, play a critical role in C9ORF72-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), the most common genetic form of the disorders (c9ALS/FTD). R-DPRs form liquid condensates in vitro, induce stress granule formation in cultured cells, aggregate, and sometimes coaggregate with TDP-43 in postmortem tissue from patients with c9ALS/FTD. However, how these processes are regulated is unclear. Here, we show that loss of poly(ADP-ribose) (PAR) suppresses neurodegeneration in c9ALS/FTD fly models and neurons differentiated from patient-derived induced pluripotent stem cells. Mechanistically, PAR induces R-DPR condensation and promotes R-DPR-induced stress granule formation and TDP-43 aggregation. Moreover, PAR associates with insoluble R-DPR and TDP-43 in postmortem tissue from patients. These findings identified PAR as a promoter of R-DPR toxicity and thus a potential target for treating c9ALS/FTD.
Assuntos
Demência Frontotemporal , Arginina , Proteína C9orf72/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dipeptídeos/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/metabolismo , Humanos , Poli Adenosina Difosfato RiboseRESUMO
A non-coding hexanucleotide repeat expansion in intron 1 of the C9orf72 gene is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (C9ALS/FTD), however, the precise molecular mechanism by which the C9orf72 hexanucleotide repeat expansion directs C9ALS/FTD pathogenesis remains unclear. Here, we report a novel disease mechanism arising due to the interaction of C9ORF72 with the RAB7L1 GTPase to regulate vesicle trafficking. Endogenous interaction between C9ORF72 and RAB7L1 was confirmed in human SH-SY5Y neuroblastoma cells. The C9orf72 hexanucleotide repeat expansion led to haploinsufficiency resulting in severely defective intracellular and extracellular vesicle trafficking and a dysfunctional trans-Golgi network phenotype in patient-derived fibroblasts and induced pluripotent stem cell-derived motor neurons. Genetic ablation of RAB7L1or C9orf72 in SH-SY5Y cells recapitulated the findings in C9ALS/FTD fibroblasts and induced pluripotent stem cell neurons. When C9ORF72 was overexpressed or antisense oligonucleotides were targeted to the C9orf72 hexanucleotide repeat expansion to upregulate normal variant 1 transcript levels, the defective vesicle trafficking and dysfunctional trans-Golgi network phenotypes were reversed, suggesting that both loss- and gain-of-function mechanisms play a role in disease pathogenesis. In conclusion, we have identified a novel mechanism for C9ALS/FTD pathogenesis highlighting the molecular regulation of intracellular and extracellular vesicle trafficking as an important pathway in C9ALS/FTD pathogenesis.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Demência Frontotemporal/metabolismo , Proteínas/metabolismo , Proteínas rab1 de Ligação ao GTP/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Transporte Biológico , Proteína C9orf72 , Células COS , Linhagem Celular , Chlorocebus aethiops , Expansão das Repetições de DNA , Fibroblastos/efeitos dos fármacos , Fibroblastos/patologia , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Humanos , Íntrons , Neurônios Motores/efeitos dos fármacos , Neurônios Motores/patologia , Neurônios/efeitos dos fármacos , Neurônios/patologia , Oligonucleotídeos Antissenso/farmacologia , Linhagem , Células-Tronco Pluripotentes/efeitos dos fármacos , Células-Tronco Pluripotentes/patologia , Proteínas/genética , Proteínas rab de Ligação ao GTP , Proteínas rab1 de Ligação ao GTP/genéticaRESUMO
An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons, decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis, reduced levels of the antiapoptotic protein Bcl-2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063-2078.