Marked increase in cell permeability ascribed to open connexin (Cx)43 hemichannels is induced by metabolic inhibition (MI) of cortical astrocytes in culture, but the molecular mechanisms are not established. Dephosphorylation and͞or oxidation of Cx43 hemichannels was proposed as a potential mechanism to increase their open probability. We now demonstrate that MI increases the number of hemichannels on the cell surface assayed by biotinylation and Western blot, and that this change is followed by increased dephosphorylation and S-nitrosylation. The increase in rate of dye uptake caused by MI is comparable to the increase in surface expression; thus, open probability and permeation per hemichannel may be unchanged. Reducing agents did not affect dephosphorylation of Cx43 hemichannels but reduced dye uptake and S-nitrosylation. Uptake was also reduced by elevated intracellular but not extracellular levels of reduced glutathione. Moreover, nitric oxide donors induced dye uptake and nitrosylation of surface Cx43 but did not affect its abundance or phosphorylation. Thus, permeability per channel is increased, presumably because of increase in open probability. We propose that increased dye uptake induced by MI is mediated by an increased number of Cx43 hemichannels in the surface and is associated with multiple molecular changes, among which nitrosylation of intracellular Cx43 cysteine residues may be a critical factor. astroglia ͉ ischemia ͉ nitric oxide ͉ permeabilization

Summary Spinal and bulbar muscular atrophy (SBMA) is caused by the polyglutamine androgen receptor (polyQ AR), a protein expressed by both lower motor neurons and skeletal muscle. Although viewed as a motor neuronopathy, data from patients and mouse models suggest that muscle contributes to disease pathogenesis. Here we tested this hypothesis using AR113Q knock-in and human BAC transgenic mice that express the full-length polyQ AR and display androgen-dependent weakness, muscle atrophy and early death. We developed antisense oligonucleotides that suppressed AR gene expression in the periphery, but not CNS, following subcutaneous administration. Suppression of polyQ AR in the periphery rescued deficits in muscle weight, fiber size and grip strength, reversed changes in muscle gene expression, and extended lifespan of mutant males. We conclude that polyQ AR expression in the periphery is an important contributor to pathology in SBMA mice and that peripheral administration of therapeutics should be explored for SBMA patients.

X-linked spinal & bulbar muscular atrophy (SBMA) is characterized by adult-onset muscle weakness and lower motor neuron degeneration. SBMA is caused by CAG-polyglutamine (polyQ) repeat expansions in the androgen receptor (AR) gene. Pathological findings include motor neuron loss, with polyQ-AR accumulation in intranuclear inclusions. SBMA patients exhibit myopathic features, suggesting a role for muscle in disease pathogenesis. To determine the contribution of muscle, we developed a BAC mouse model featuring a floxed first exon to permit cell-type-specific excision of human AR121Q. BAC fxAR121 mice develop systemic and neuromuscular phenotypes, including shortened survival. After validating termination of AR121 expression and full rescue with ubiquitous Cre, we crossed BAC fxAR121 mice with Human Skeletal Actin-Cre mice. Muscle-specific excision prevented weight loss, motor phenotypes, muscle pathology, and motor neuronopathy, and dramatically extended survival. Our results reveal a crucial role for muscle expression of polyQ-AR in SBMA, and suggest muscle-directed therapies as effective treatments.

Macroautophagy (hereafter autophagy) is a key pathway in neurodegeneration. Despite protective actions, autophagy may contribute to neuron demise, when dysregulated. Here we considered X-linked spinal and bulbar muscular atrophy (SBMA), a repeat disorder caused by polyglutamine-expanded androgen receptor (polyQ-AR). We found that polyQ-AR reduced long-term protein turnover and impaired autophagic flux in motor neuron-like cells. Ultrastructural analysis of SBMA mice revealed a block in autophagy pathway progression. We considered the transcriptional regulation of autophagy, and observed a functionally significant physical interaction between transcription factor EB (TFEB) and AR. Normal AR promoted, but polyQ-AR interfered with TFEB transactivation. To evaluate physiological relevance, we reprogrammed patient fibroblasts to induced pluripotent stem cells, and then to neuronal precursor cells (NPCs). We compared multiple SBMA NPC lines, and documented metabolic and autophagic flux defects that could be rescued by TFEB. Our results indicate that polyQ-AR diminishes TFEB function to impair autophagy and promote SBMA pathogenesis.