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...

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.

Recent studies suggest that, in female monkeys and rats, estrogens elicit dendritic spine synapse formation in the prefrontal cortex, an area that, similar to the hippocampus, plays a critical role in cognition. However, whether gonadal hormones induce synaptic remodeling in the male prefrontal cortex remains unknown. Here we report that gonadectomy reduced, whereas administration of 5␣-dihydrotestosterone or estradiol-benzoate to castrated male rats increased, the number of medial prefrontal cortical (mPFC) spine synapses, with estradiol-benzoate being less effective than 5␣-dihydrotestosterone. To investigate whether the androgen receptor contributes to the mediation of these changes, we compared the response of testicular feminization mutant (Tfm) male rats to that of wild-type animals. The number of mPFC spine synapses in gonadally intact Tfm rats and 5␣-dihydrotestosterone-treated castrated Tfm males was considerably reduced compared to intact wild-type animals, whereas the synaptogenic effect of estradiol-benzoate was surprisingly enhanced in Tfm rats. These data are consistent with the hypothesis that remodeling of spine synapses in the prefrontal cortex may contribute to the cognitive effect of gonadal steroids. Our findings in Tfm animals indicate that androgen receptors may mediate a large part of the synaptogenic action of androgens in the mPFC of adult males. However, because this effect of 5␣-dihydrotestosterone is not completely lost in Tfm rats, additional mechanisms may also be involved. (Endocrinology 148: 1963(Endocrinology 148: -1967(Endocrinology 148: , 2007 I T IS WELL DOCUMENTED that gonadal steroid hormones significantly influence cognitive performance in both humans and laboratory animals (1). Recent studies also suggest that remodeling and subsequent stabilization of dendritic spine synapses may represent a mechanism by which new memories are made and stored (2, 3). Consequently, it is believed that remodeling of spine synapses significantly contributes to the cognitive effect of gonadal hormones. Indeed, pioneering studies from Woolley and McEwen (4) have shown that, during the estrous cycle of rats, fluctuating levels of estradiol mediate a change in the density of spine synapses in the hippocampus that is critically involved in memory and cognitive functions. Another brain area that plays a crucial role in cognition, especially in working memory, is the prefrontal cortex (PFC) (5). The function and structure of PFC neurons is also affected by gonadal steroids. Estrogens improve PFC-related cognitive performance of ovariectomized nonhuman primates (6) and rats (7). In line with this, changing levels of circulating estrogens influence the total number of spines in layer I of Walker's area 46 in young female rhesus monkeys (8). Similarly, Wallace et al. (7) have shown that ovariectomy decreases the dendritic spine density of layer II/III pyramidal cells in the medial PFC (mPFC) of female rats, suggesting that changes in spine synapses may mediate the effect of estrogens on PFC-relat...

The effects of estradiol benzoate (EB), dihydrotestosterone (DHT), or the antiandrogen hydroxyflutamide on CA1 pyramidal cell dendritic spine synapses were investigated in adult male rats. To elucidate the contribution of the androgen receptor to the hormone-induced increase in hippocampal CA1 synapses, wild-type males were compared with males expressing the Tfm mutation, which results in synthesis of defective androgen receptors. Orchidectomized rats were treated with EB (10 microg/rat.d), DHT (500 mug/rat.d), hydroxyflutamide (5 mg/rat.d), or the sesame oil vehicle sc daily for 2 d and examined using quantitative electron microscopic stereological techniques, 48 h after the second injection. In wild-type males, DHT and hydroxyflutamide both induced increases in the number of spine synapses in the CA1 stratum radiatum, whereas EB had no effect. DHT almost doubled the number of synaptic contacts observed, whereas hydroxyflutamide increased synapse density by approximately 50%, compared with the vehicle-injected controls. Surprisingly, in Tfm males, the effects of EB, DHT, and hydroxyflutamide were all indistinguishable from those observed in wild-type animals. These observations demonstrate that Tfm male rats resemble normal males in having no detectable hippocampal synaptic response to a dose of EB that is highly effective in females. Despite the reduction in androgen sensitivity as a result of the Tfm mutation, hippocampal synaptic responses to both DHT and a mixed androgen agonist/antagonist (hydroxyflutamide) remain intact in Tfm males. These data are consistent with previous results suggesting that androgen effects on hippocampal spine synapses may involve novel androgen response mechanisms.

Axonal guidance involves extrinsic molecular cues that bind growth cone receptors and signal to the cytoskeleton through divergent pathways. Some signaling intermediates are deployed downstream of molecularly distinct axon guidance receptor families, but the scope of this overlap is unclear, as is the impact of embryonic axon guidance fidelity on adult nervous system function. Here, we demonstrate that the Rho-GTPase-activating protein ␣2-chimaerin is specifically required for EphA and not EphB receptor signaling in mouse and chick spinal motor axons. Reflecting this specificity, the loss of ␣2-chimaerin function disrupts the limb trajectory of extensor-muscleinnervating motor axons the guidance of which depends on EphA signaling. These embryonic defects affect coordinated contraction of antagonistic flexor-extensor muscles in the adult, indicating that accurate embryonic motor axon guidance is critical for optimal neuromuscular function. Together, our observations provide the first functional evidence of an Eph receptor-class-specific intracellular signaling protein that is required for appropriate neuromuscular connectivity.

Summary Anabolic androgenic steroid (AAS) abuse is widespread. Moreover, AAS are reinforcing, as shown by self-administration in rodents. However, the receptors that transduce the reinforcing effects of AAS are unclear. AAS may bind to classical nuclear androgen receptors (AR) or membrane receptors. We used two approaches to examine the role of nuclear ARs in AAS self-administration. First, we tested androgen self-administration in rats with the testicular feminization mutation (Tfm), which interferes with androgen binding. If nuclear ARs are essential for AAS self-administration, Tfm males should not self-administer androgens. Tfm males and wild-type (WT) littermates self-administered the non-aromatizable androgen dihydrotestosterone (DHT) or vehicle intracerebroventricularly (ICV) at fixed ratio (FR) schedules up to FR5. Both Tfm and WT rats acquired a preference for the active nose-poke during DHT self-administration (66.4±9.6 responses/4h for Tfm and 79.2±11.5 for WT responses/4h), and nose-pokes increased as the FR requirement increased. Preference scores were significantly lower in rats self-administering vehicle (42.3±5.3 responses/4h for Tfm and 19.1±4.0 responses/4h for WT). We also tested self-administration of DHT conjugated to bovine serum albumin (BSA) at C3 and C17, which is limited to actions at the cell surface. Hamsters were allowed to self-administer DHT, BSA and DHT-BSA conjugates for 15 days at FR1. The hamsters showed a significant preference for DHT (18.0±4.1 responses/4h) or DHT-BSA conjugates (10.0±3.7 responses/4h and 21.0±7.2 responses/4h), but not for BSA (2.5±2.4 responses/4h). Taken together, these data demonstrate that nuclear ARs are not required for androgen self-administration. Furthermore, androgen self-administration may be mediated by plasma membrane receptors.

The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic group of motoneurones that innervates the bulbocavernosus (BC) and levator ani (LA), skeletal muscles that attach to the base of the penis. In many species, including mice, rats and hamsters, the LA and BC have been found to be highly responsive to androgen and, in rats, these muscles mediate several effects of androgen on the SNB system. However, characterising the SNB system in mice is important because of the availability of genetic models in this species. In the present study, we examined AR expression in skeletal muscles of C57/BlJ6 adult male mice using immunoblotting and immunocytochemistry, comparing the BC/LA to the androgen-unresponsive extensor digitorum longus (EDL). We found similar differences in AR expression for these muscles in the mouse as previously reported for rats. In mice, the BC/LA contains more AR protein than does the EDL. At the cellular level, the LA contains a higher percentage of AR positive myonuclei and fibroblasts than does the EDL. Finally, AR expression is enriched at the neuromuscular junction of mouse LA fibres. The increased expression of AR in the LA compared to the EDL in both muscle fibres and fibroblasts indicates that each cell type may critically mediate androgen action on the SNB system in mice.

Background: Spinal bulbar muscular atrophy (SBMA) is caused by a CAG repeat expansion mutation in the androgen receptor (AR) gene, and mutant AR is presumed to act in motoneurons to cause SBMA. However, we found that mice overexpressing wild-type (wt) AR solely in skeletal muscle fibers display the same androgen-dependent disease phenotype as when mutant AR is broadly expressed, challenging the assumptions that only an expanded AR can induce disease and that SBMA is strictly neurogenic. We have previously reported that AR toxicity was ligand dependent in our model, and that very few transgenic (tg) males survived beyond birth. Methods: We tested whether the AR antagonist flutamide could block perinatal toxicity. tg males were treated prenatally with flutamide and assessed for survival and motor behavior in adulthood. Results: Prenatal treatment with flutamide rescued tg male pups from perinatal death, and, as adults, such perinatally rescued tg males showed an SBMA phenotype that was comparable to that of previously described untreated tg males. Moreover, tg males carrying a mutant endogenous allele for AR – the testicular feminization mutation (tfm) – and thus having functional AR only in muscle fibers nevertheless displayed the same androgen-dependent disease phenotype as adults. Conclusions: These mice represent an excellent model to study the myogenic contribution to SBMA as they display many of the core features of disease as other mouse models. These data demonstrate that AR acting exclusively in muscle fibers is sufficient to induce SBMA symptoms and that flutamide is protective perinatally.