RESUMO
RNA interference via the endogenous miRNA pathway regulates gene expression by controlling protein synthesis through post-transcriptional gene silencing. In recent years, miRNA-mediated gene regulation has shown potential for treatment of neurological disorders caused by a toxic gain of function mechanism. However, efficient delivery to target tissues has limited its application. Here we used a transgenic mouse model for spinal and bulbar muscular atrophy (SBMA), a neuromuscular disease caused by polyglutamine expansion in the androgen receptor (AR), to test gene silencing by a newly identified AR-targeting miRNA, miR-298. We overexpressed miR-298 using a recombinant adeno-associated virus (rAAV) serotype 9 vector to facilitate transduction of non-dividing cells. A single tail-vein injection in SBMA mice induced sustained and widespread overexpression of miR-298 in skeletal muscle and motor neurons and resulted in amelioration of the neuromuscular phenotype in the mice.
Assuntos
Regulação da Expressão Gênica/genética , Terapia Genética/métodos , MicroRNAs/genética , Doenças Neuromusculares/genética , Doenças Neuromusculares/terapia , Animais , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Doenças Neuromusculares/patologia , Roedores , SorogrupoRESUMO
Spinal and bulbar muscular atrophy (SBMA, also known as Kennedy's disease) is one of nine neurodegenerative disorders that are caused by expansion of polyglutamine-encoding CAG repeats. Intracellular accumulation of abnormal proteins in these diseases, a pathological hallmark, is associated with defects in protein homeostasis. Enhancement of the cellular proteostasis capacity with small molecules has therefore emerged as a promising approach to treatment. Here, we characterize a novel curcumin analog, ASC-JM17, as an activator of central pathways controlling protein folding, degradation and oxidative stress resistance. ASC-JM17 acts on Nrf1, Nrf2 and Hsf1 to increase the expression of proteasome subunits, antioxidant enzymes and molecular chaperones. We show that ASC-JM17 ameliorates toxicity of the mutant androgen receptor (AR) responsible for SBMA in cell, fly and mouse models. Knockdown of the Drosophila Nrf1 and Nrf2 ortholog cap 'n' collar isoform-C, but not Hsf1, blocks the protective effect of ASC-JM17 on mutant AR-induced eye degeneration in flies. Our observations indicate that activation of the Nrf1/Nrf2 pathway is a viable option for pharmacological intervention in SBMA and potentially other polyglutamine diseases.
Assuntos
Atrofia Bulboespinal Ligada ao X/genética , Curcumina/análogos & derivados , Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Transtornos Musculares Atróficos/genética , Fator 1 Relacionado a NF-E2/genética , Fator 2 Relacionado a NF-E2/genética , Receptores Androgênicos/genética , Fatores de Transcrição/genética , Expansão das Repetições de Trinucleotídeos/genética , Animais , Atrofia Bulboespinal Ligada ao X/tratamento farmacológico , Atrofia Bulboespinal Ligada ao X/patologia , Curcumina/administração & dosagem , Curcumina/química , Modelos Animais de Doenças , Drosophila melanogaster/genética , Técnicas de Silenciamento de Genes , Fatores de Transcrição de Choque Térmico , Humanos , Camundongos , Transtornos Musculares Atróficos/tratamento farmacológico , Transtornos Musculares Atróficos/patologia , Estresse Oxidativo/efeitos dos fármacos , Peptídeos/genética , Complexo de Endopeptidases do Proteassoma/efeitos dos fármacos , Agregação Patológica de Proteínas/genética , Dobramento de Proteína/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/administração & dosagemRESUMO
Spinal and bulbar muscular atrophy (SBMA) is a currently untreatable adult-onset neuromuscular disease caused by expansion of a polyglutamine repeat in the androgen receptor (AR). In SBMA, as in other polyglutamine diseases, a toxic gain of function in the mutant protein is an important factor in the disease mechanism; therefore, reducing the mutant protein holds promise as an effective treatment strategy. In this work, we evaluated a microRNA (miRNA) to reduce AR expression. From a list of predicted miRNAs that target human AR, we selected microRNA-298 (miR-298) for its ability to downregulate AR mRNA and protein levels when transfected in cells overexpressing wild-type and mutant AR and in SBMA patient-derived fibroblasts. We showed that miR-298 directly binds to the 3'-untranslated region of the human AR transcript, and counteracts AR toxicity in vitro. Intravenous delivery of miR-298 with adeno-associated virus serotype 9 vector resulted in efficient transduction of muscle and spinal cord and amelioration of the disease phenotype in SBMA mice. Our findings support the development of miRNAs as a therapeutic strategy for SBMA and other neurodegenerative disorders caused by toxic proteins.
Assuntos
Regulação para Baixo , Terapia Genética/métodos , MicroRNAs/genética , Atrofia Muscular Espinal/terapia , Receptores Androgênicos/genética , Regiões 3' não Traduzidas , Administração Intravenosa , Animais , Linhagem Celular , Dependovirus/genética , Modelos Animais de Doenças , Vetores Genéticos/administração & dosagem , Humanos , Células MCF-7 , Camundongos , Atrofia Muscular Espinal/genéticaRESUMO
Spinal and bulbar muscular atrophy is an X-linked motor neuron disease caused by polyglutamine expansion in the androgen receptor. Patients develop slowly progressive proximal muscle weakness, muscle atrophy and fasciculations. Affected individuals often show gynecomastia, testicular atrophy and reduced fertility as a result of mild androgen insensitivity. No effective disease-modifying therapy is currently available for this disease. Our recent studies have demonstrated that insulinlike growth factor (IGF)-1 reduces the mutant androgen receptor toxicity through activation of Akt in vitro, and spinal and bulbar muscular atrophy transgenic mice that also overexpress a noncirculating muscle isoform of IGF-1 have a less severe phenotype. Here we sought to establish the efficacy of daily intraperitoneal injections of mecasermin rinfabate, recombinant human IGF-1 and IGF-1 binding protein 3, in a transgenic mouse model expressing the mutant androgen receptor with an expanded 97 glutamine tract. The study was done in a controlled, randomized, blinded fashion, and, to reflect the clinical settings, the injections were started after the onset of disease manifestations. The treatment resulted in increased Akt phosphorylation and reduced mutant androgen receptor aggregation in muscle. In comparison to vehicle-treated controls, IGF-1-treated transgenic mice showed improved motor performance, attenuated weight loss and increased survival. Our results suggest that peripheral tissue can be targeted to improve the spinal and bulbar muscular atrophy phenotype and indicate that IGF-1 warrants further investigation in clinical trials as a potential treatment for this disease.