RESUMO
Spinal and bulbar muscular atrophy (SBMA) is caused by expression of a polyglutamine (polyQ)-expanded androgen receptor (AR). The inefficient nuclear proteasomal degradation of the mutant AR results in the formation of nuclear inclusions containing amino-terminal fragments of the mutant AR. PA28γ (also referred to as REGγ) is a nuclear 11S-proteasomal activator with limited proteasome activation capabilities compared to its cytoplasmic 11S (PA28α, PA28ß) counterparts. To clarify the role of REGγ in polyQ-expanded AR metabolism, we carried out genetic and biochemical studies in cell models of SBMA. Overexpression of REGγ in a PC12 cell model of SBMA increased polyQ-expanded AR aggregation and contributed to polyQ-expanded AR toxicity in the presence of dihydrotestosterone (DHT). These effects of REGγ were independent of its association with the proteasome and may be due, in part, to the decreased binding of polyQ-expanded AR by the E3 ubiquitin-ligase MDM2. Unlike its effects in PC12 cells, REGγ overexpression rescued transgenic SBMA motor neurons from DHT-induced toxicity in a proteasome binding-dependent manner, suggesting that the degradation of a specific 11S proteasome substrate or substrates promotes motor neuron viability. One potential substrate that we found to play a role in mutant AR toxicity is the splicing factor SC35. These studies reveal that, depending on the cellular context, two biological roles for REGγ impact cell viability in the face of polyQ-expanded AR; a proteasome binding-independent mechanism directly promotes mutant AR aggregation while a proteasome binding-dependent mechanism promotes cell viability. The balance between these functions likely determines REGγ effects on polyQ-expanded AR-expressing cells.
RESUMO
Evidence from multiple animal models demonstrates that testosterone plays a crucial role in the progression of symptoms in spinal and bulbar muscular atrophy (SBMA), a condition that results in neurodegeneration and muscle atrophy in affected men. Mice bearing a transgene encoding a human androgen receptor (AR) that contains a stretch of 112 glutamines (expanded polyglutamine tract; AR112Q mice) reproduce several aspects of the human disease. We treated transgenic male AR112Q mice with testosterone for 6 months. Surprisingly, testosterone treatment of AR112Q males did not exacerbate the disease. Although transgenic AR112Q males exhibited functional deficits when compared with non-transgenics, long-term testosterone treatment had no effect on motor function. Testosterone treatment also failed to affect cellular markers of disease, including inclusion formation (the accumulation of large nuclear aggregates of mutant AR protein) and levels of unphosphorylated neurofilament heavy chain. These data suggest that the mechanism of disease in SBMA saturates at close to endogenous hormone levels and that individuals with SBMA who take, or have taken, testosterone for its putative therapeutic properties are unlikely to suffer adverse effects.
Assuntos
Atrofia Bulboespinal Ligada ao X/tratamento farmacológico , Atrofia Bulboespinal Ligada ao X/patologia , Progressão da Doença , Testosterona/uso terapêutico , Animais , Biomarcadores/metabolismo , Modelos Animais de Doenças , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Fenótipo , Falha de TratamentoRESUMO
An increasing number of neurodegenerative diseases are being linked to mutations in genes encoding proteins required for axonal transport and intracellular trafficking. A mutation in p150(Glued), a component of the cytoplasmic dynein/dynactin microtubule motor complex, results in the human neurodegenerative disease distal spinal and bulbar muscular atrophy (dSBMA). We have developed a transgenic mouse model of dSBMA; these mice exhibit late-onset, slowly progressive muscle weakness but do not have a shortened lifespan, consistent with the human phenotype. Examination of motor neurons from the transgenic model reveals the proliferation of enlarged tertiary lysosomes and lipofuscin granules, indicating significant alterations in the cellular degradative pathway. In addition, we observe deficits in axonal caliber and neuromuscular junction (NMJ) integrity, indicating distal degeneration of motor neurons. However, sciatic nerve ligation studies reveal that inhibition of axonal transport is not evident in this model. Together, these data suggest that mutant p150(Glued) causes neurodegeneration in the absence of significant changes in axonal transport, and therefore other functions of dynein/dynactin, such as trafficking in the degradative pathway and stabilization of the NMJ are likely to be critical in maintaining the health of motor neurons.
Assuntos
Lisossomos/fisiologia , Proteínas Associadas aos Microtúbulos/metabolismo , Neurônios Motores/fisiologia , Atrofia Muscular Espinal/fisiopatologia , Mutação de Sentido Incorreto , Degeneração Neural/fisiopatologia , Animais , Transporte Axonal , Modelos Animais de Doenças , Complexo Dinactina , Humanos , Camundongos , Camundongos Transgênicos , Proteínas Associadas aos Microtúbulos/genética , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/metabolismo , Degeneração Neural/genética , Degeneração Neural/metabolismo , Junção Neuromuscular , Subunidades Proteicas/genética , Subunidades Proteicas/metabolismoRESUMO
In polyglutamine diseases such as X-linked spinobulbar muscular atrophy (SBMA), it is unknown whether the toxic form of the protein is an insoluble or soluble aggregate or a monomer. We have addressed this question by studying a full-length androgen receptor (AR) mouse model of SBMA. We used biochemistry and atomic force microscopy to immunopurify oligomers soluble after ultracentrifugation that are comprised of a single approximately 50-kDa N-terminal polyglutamine-containing AR fragment. AR oligomers appeared several weeks prior to symptom onset, were distinct and temporally dissociated from intranuclear inclusions, and disappeared rapidly after castration, which halts disease. This is the first demonstration of soluble AR oligomers in vivo and suggests that they underlie neurodegeneration in SBMA.
Assuntos
Transtornos Musculares Atróficos/fisiopatologia , Receptores Androgênicos/química , Receptores Androgênicos/fisiologia , Envelhecimento , Animais , Modelos Animais de Doenças , Progressão da Doença , Feminino , Masculino , Camundongos , Transtornos Musculares Atróficos/patologia , Fragmentos de Peptídeos/análiseRESUMO
Neurons have extensive processes and communication between those processes and the cell body is crucial to neuronal function and survival. Thus, neurons are uniquely dependent on microtubule based transport. Growing evidence supports the idea that deficits in axonal transport contribute to pathogenesis in multiple neurodegenerative diseases. We describe the motor, cytoskeletal, and adaptor proteins involved in axonal transport and their interactions. Data linking disruption of axonal transport to diseases such as ALS are discussed. Finally, we explore the pathways that may cause neuronal dysfunction and death.
Assuntos
Esclerose Lateral Amiotrófica/metabolismo , Transporte Axonal/genética , Neurônios Motores/metabolismo , Degeneração Neural/genética , Doenças Neurodegenerativas/metabolismo , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Animais , Citoesqueleto/genética , Citoesqueleto/metabolismo , Complexo Dinactina , Dineínas/metabolismo , Humanos , Filamentos Intermediários/metabolismo , Cinesinas/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Mitocôndrias/metabolismo , Modelos Imunológicos , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/patologiaRESUMO
Transgenic models of neurodegenerative disease have proved uniquely powerful for delineating pathways of neuronal dysfunction and cell death. We have developed a transgenic model of the polyglutamine disease spinal and bulbar muscular atrophy (SBMA), an adult-onset, slowly progressive motor neuron disease caused by polyglutamine expansion in the androgen receptor (AR). Mice bearing a human AR with 112 glutamines reproduce many aspects of SBMA, including slowly progressive, gender-specific motor deficits, and neuronal intranuclear inclusions. Despite substantial motor deficits in male AR112Q mice, no motor neuron loss was observed, indicating that neuronal dysfunction, rather than neuronal death, is central to disease. Moreover, reduced levels of unphosphorylated neurofilament heavy chain (NF-H) were observed in motor neurons, suggesting a role for NF-H in SBMA neuronal dysfunction. The elimination of androgens by surgical castration of severely affected, aged 112Q male mice partially restored motor function as well as NF-H levels. These data suggest that hormone-based therapies designed to treat SBMA patients, even with advanced disease, are likely to be effective.
Assuntos
Castração , Transtornos Musculares Atróficos/fisiopatologia , Transtornos Musculares Atróficos/cirurgia , Proteínas de Neurofilamentos/metabolismo , Recuperação de Função Fisiológica , Animais , Encéfalo/patologia , Modelos Animais de Doenças , Progressão da Doença , Humanos , Coxeadura Animal/genética , Masculino , Camundongos , Camundongos Transgênicos , Atividade Motora/genética , Neurônios Motores/metabolismo , Neurônios Motores/patologia , Debilidade Muscular/genética , Transtornos Musculares Atróficos/genética , Peptídeos/genética , Fosforilação , Receptores Androgênicos/genética , Fatores Sexuais , Medula Espinal/patologia , Resultado do TratamentoRESUMO
Expansion of the CAG trinucleotide repeat encoding glutamine in the androgen receptor gene leads to spinobulbar muscular atrophy (SBMA), a neurodegenerative disorder in a family of polyglutamine diseases with enigmatic pathogenic mechanisms. One established property of glutamine residues is their ability to act as an amine accepter in a transglutaminase-catalyzed reaction, resulting in a proteolytically resistant glutamyl-lysine cross-link. To examine underlying disease mechanisms we investigated the relationship between polyglutamine-expanded androgen receptor and transglutaminase. We found androgen receptor N-terminal fragments are a substrate for transglutaminase. Western blots of the proteins following incubation with transglutaminase show that several different epitopes of the AR appear to be lost. We propose that this is due to the transglutaminase cross-linking of the AR, which interferes with antibody recognition. Furthermore, HEK GFP(u)-1 cells expressing polyglutamine-expanded androgen receptor and transglutaminase exhibit ligand-dependent proteasome dysfunction; this effect was not observed in the presence of cystamine, a transglutaminase inhibitor. In addition, transglutaminase-mediated isopeptide bonds were detected in brains of SBMA transgenic mice, but not in controls, suggesting involvement of transglutaminase-catalyzed reactions in polyglutamine disease pathogenesis. Our hypothesis is that cross-linked AR cannot to be degraded by the proteasome and obstructs the proteasome pore, preventing normal function. Because of the central role the ubiquitin-proteasome degradation system plays in fundamental cellular processes, any alteration in its function could cause cell death, ultimately contributing to SBMA pathogenesis.