We created transgenic mice that overexpress WT androgen receptor (AR) exclusively in their skeletal muscle fibers. Unexpectedly, these mice display androgen-dependent muscle weakness and early death, show changes in muscle morphology and gene expression consistent with neurogenic atrophy, and exhibit a loss of motor axons. These features reproduce those seen in models of Kennedy disease, a polyglutamine expansion disorder caused by a CAG repeat expansion in the AR gene. Kennedy disease ͉ neuromuscular ͉ skeletal muscle ͉ spinal and bulbar muscular atrophy ͉ axonopathy A polymorphism in exon 1 of the androgen receptor (AR) gene, consisting of a variable number of glutamine (Q) repeats, affects AR function. Very long polyglutamine repeat (polyQ) tracts are associated with a progressive neuromuscular disease known as Kennedy disease (KD, or spinal bulbar muscular atrophy) (1). The etiological mechanism mediating polyQ toxicity is uncertain, but is generally thought to confer novel toxic functions to the protein, because expansion of polyQ tracts beyond 40 repeats in other proteins also cause neurodegenerative disease, including Huntington's disease (HD), and several autosomal dominant forms of spinocerebellar ataxia (SCA) (2). Histopathological studies of KD patients suggest ''neurogenic'' responses to denervation, and the etiology of this disease is therefore generally thought to begin with motoneuron pathology (3).Explicit mouse models of KD, in which polyQ AR alleles containing 60 CAG repeats or more are expressed, develop a disease phenotype that includes a marked reduction in body weight, kyphosis, and striking deficits in muscle strength and motor coordination (4-8). Androgen dependence, motoneuron and muscle pathology, and/or inclusions containing AR are also observed in these models (4,5,7,8). Our studies of AR in skeletal muscle (9, 10) led us to generate transgenic (Tg) mice in which AR is overexpressed solely in skeletal muscle fibers using an expression cassette containing the human skeletal ␣-actin (HSA) promoter. We discovered a striking phenotypic resemblance between these HSA-AR mice and mouse models of KD. This similarity is surprising given that the Q repeat in this AR transgene comprises only 22 glutamines and is expressed exclusively in skeletal muscle fibers and not in motoneurons. Results HSA Promoter Drives Transgene Expression Exclusively in SkeletalMuscle Fibers. We first validated our HSA expression cassette by generating HSA-LacZ (LacZ ϭ ␤-galactosidase gene) reporter mice [supporting information (SI) Fig. 5A]. Consistent with other expression cassettes containing the HSA promoter (11, 12) these reporter mice express ␤-gal specifically in skeletal muscle fibers, starting at embryonic day 9.5-10.5, with no detectable expression in other tissues, including the heart, viscera, fat or spinal cord ( SI Fig 5 B and C). We also created Tg mice in which a rat WT AR cDNA is driven by this same HSA expression cassette (SI Fig 6A), resulting in selective overexpression of AR in skeletal muscle fiber...

Steroids exert powerful effects on the brains and behavior of many species, but measures and manipulations of endocrine physiology in songbirds often reveal unexplained connections between steroids and the brain. The zebra finch song system, a sensorimotor neural circuit sensitive to steroids throughout life, organizes and functions largely in apparent independence from gonadally derived steroids. We tested the hypothesis that the zebra finch brain has the capacity for de novo steroidogenesis and that neurally synthesized steroids, neurosteroids, may impact the song system. Using multiple techniques, we demonstrate that the steroidogenic acute regulatory protein (StAR), cytochrome P450 side-chain cleavage (CYP11A1), and 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase, the first three factors in the steroidogenic pathway, are expressed in both developing and adult zebra finch brain. Detailed expression mapping at posthatch d 20 (P20) and adult reveals widespread area-specific expression and coexpression patterns for steroidogenic acute regulatory protein, CYP11A1, and 3beta-hydroxysteroid dehydrogenase/Delta5-Delta4 isomerase, which suggest neurosteroids may modulate multiple brain functions, including sensory and motor systems. Notably, whereas expression of other steroidogenic genes such as aromatase has been essentially absent from the song system, each of the major song nuclei express at least a subset of steroidogenic genes described here, establishing the song system as a potential steroidogenic circuit.

Potential cellular targets of androgen action within skeletal muscle of the rat were determined by comparing the cellular distribution of androgen receptor (AR)-positive nuclei in the highly androgen-responsive levator ani (LA) muscle with that of the relatively androgen-unresponsive extensor digitorum longus (EDL) muscle. We found that androgen responsiveness correlates with AR expression in muscle fibers and not in fibroblasts. Results indicate that a much higher percentage of myonuclei in the LA are AR(+) than in the EDL (74% vs. 7%), correlating with differences in androgen responsiveness. Both muscles contain an equivalent proportion of AR(+) fibroblasts (approximately 62%). AR(+) nuclei were not observed in terminal Schwann cells in either muscle. These results suggest that ARs within LA muscle fibers mediate the androgen-dependent survival and growth of the LA muscle and its motoneurons. We also observed an unexpected enrichment of AR(+) myonuclei and fibroblasts proximate to neuromuscular junctions, suggesting that ARs at muscle synapses may selectively regulate synapse-specific genes important for the survival and growth of motoneurons. Although castration reduced the proportion of AR(+) fibroblasts in both muscles, the proportion of AR(+) myonuclei was reduced only in the LA. As expected, testosterone treatment prevented these effects of castration but, unexpectedly, increased the proportion of AR(+) myonuclei in the EDL to above normal. These results suggest that how AR expression in skeletal muscle is influenced by androgens depends not only on the particular muscle but on the particular cell type within that muscle.

With this paper, we deliberately challenge the prevailing neurocentric theory of the etiology of spinal bulbar muscular atrophy (SBMA). We offer data supporting an alternative view that androgen receptor (AR) acts in skeletal muscles to cause the symptoms of SBMA. While SBMA has been linked to a CAG repeat expansion in the AR gene and mutant AR is presumed to act in motoneurons to cause SBMA, we find that over-expression of wild type AR solely in skeletal muscle fibers results in the same androgen-dependent disease phenotype as when mutant AR is broadly expressed. Like other recent SBMA mouse models, transgenic (tg) females in our model exhibit a motor phenotype only when exposed to androgens, and this motor dysfunction is independent of motoneuronal or muscle fiber cell death. Muscles from symptomatic females also show denervation-like changes in gene expression comparable to a knock-in model of SBMA. Furthermore, once androgen treatment ends, tg females rapidly recover motor function and muscle gene expression, demonstrating the strict androgen-dependence of the disease phenotype in our model. Our results argue that SBMA may be caused by AR acting in muscle.