D. Ashley Monks

Microarray Analysis of Gene Expression by Skeletal Muscle of Three Mouse Models of Kennedy Disease/Spinal Bulbar Muscular Atrophy

Background Emerging evidence implicates altered gene expression within skeletal muscle in the pathogenesis of Kennedy disease/spinal bulbar muscular atrophy (KD/SBMA). We therefore broadly characterized gene expression in skeletal muscle of three independently generated mouse models of this disease. The mouse models included a polyglutamine expanded (polyQ) AR knock-in model (AR113Q), a polyQ AR transgenic model (AR97Q), and a transgenic mouse that overexpresses wild type AR solely in skeletal muscle (HSA-AR). HSA-AR mice were included because they substantially reproduce the KD/SBMA phenotype despite the absence of polyQ AR. Methodology/Principal Findings We performed microarray analysis of lower hindlimb muscles taken from these three models relative to wild type controls using high density oligonucleotide arrays. All microarray comparisons were made with at least 3 animals in each condition, and only those genes having at least 2-fold difference and whose coefficient of variance was less than 100% were considered to be differentially expressed. When considered globally, there was a similar overlap in gene changes between the 3 models: 19% between HSA-AR and AR97Q, 21% between AR97Q and AR113Q, and 17% between HSA-AR and AR113Q, with 8% shared by all models. Several patterns of gene expression relevant to the disease process were observed. Notably, patterns of gene expression typical of loss of AR function were observed in all three models, as were alterations in genes involved in cell adhesion, energy balance, muscle atrophy and myogenesis. We additionally measured changes similar to those observed in skeletal muscle of a mouse model of Huntingtons Disease, and to those common to muscle atrophy from diverse causes. Conclusions/Significance By comparing patterns of gene expression in three independent models of KD/SBMA, we have been able to identify candidate genes that might mediate the core myogenic features of KD/SBMA.

Myocyte Androgen Receptors Increase Metabolic Rate and Improve Body Composition by Reducing Fat Mass

Testosterone and other androgens are thought to increase lean body mass and reduce fat body mass in men by activating the androgen receptor. However, the clinical potential of androgens for improving body composition is hampered by our limited understanding of the tissues and cells that promote such changes. Here we show that selective overexpression of androgen receptor in muscle cells (myocytes) of transgenic male rats both increases lean mass percentage and reduces fat mass. Similar changes in body composition are observed in human skeletal actin promoter driving expression of androgen receptor (HSA-AR) transgenic mice and result from acute testosterone treatment of transgenic female HSA-AR rats. These shifts in body composition in HSA-AR transgenic male rats are associated with hypertrophy of type IIb myofibers and decreased size of adipocytes. Metabolic analyses of transgenic males show higher activity of mitochondrial enzymes in skeletal muscle and increased O(2) consumption by the rats. These results indicate that androgen signaling in myocytes not only increases muscle mass but also reduces fat body mass, likely via increases in oxidative metabolism.

Recovery of function in a myogenic mouse model of spinal bulbar muscular atrophy.

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.

A tetracycline-inducible and skeletal muscle-specific Cre recombinase transgenic mouse

We have generated a transgenic mouse that expresses Cre recombinase only in skeletal muscle and only following tetracycline treatment. This spatiotemporal specificity is achieved using two transgenes. The first transgene uses the human skeletal actin (HSA) promoter to drive expression of the reverse tetracycline‐controlled transactivator (rtTA). The second transgene uses a tetracycline responsive promoter to drive the expression of Cre recombinase. We monitored transgene expression in these mice by crossing them with ROSA26 loxP‐LacZ reporter mice, which express β‐galactosidase when activated by Cre. We find that the expression of this transgene is only detectable within skeletal muscle and that Cre expression in the absence of tetracycline is negligible. Cre is readily induced in this model with tetracycline analogs at a range of embryonic and postnatal ages and in a pattern consistent with other HSA transgenic mice. This mouse improves upon existing transgenic mice in which skeletal muscle Cre is expressed throughout development by allowing Cre expression to begin at later developmental stages. This temporal control of transgene expression has several applications, including overcoming embryonic or perinatal lethality due to transgene expression. This mouse is especially suited for studies of steroid hormone action, as it uses tetracycline, rather than tamoxifen, to activate Cre expression. In summary, we find that this transgenic induction system is suitable for studies of gene function in the context of hormonal regulation of skeletal muscle or interactions between muscle and motoneurons in mice.

Sexual Differentiation of the Spinal Nucleus of the Bulbocavernosus Is Not Mediated Solely by Androgen Receptors in Muscle Fibers

The spinal nucleus of the bulbocavernosus (SNB) neuromuscular system is a highly conserved and well-studied model of sexual differentiation of the vertebrate nervous system. Sexual differentiation of the SNB is currently thought to be mediated by the direct action of perinatal testosterone on androgen receptors (ARs) in the bulbocavernosus/levator ani muscles, with concomitant motoneuron rescue. This model has been proposed based on surgical and pharmacological manipulations of developing rats as well as from evidence that male rats with the testicular feminization mutation (Tfm), which is a loss of function AR mutation, have a feminine SNB phenotype. We examined whether genetically replacing AR in muscle fibers is sufficient to rescue the SNB phenotype of Tfm rats. Transgenic rats in which wild-type (WT) human AR is driven by a human skeletal actin promoter (HSA-AR) were crossed with Tfm rats. Resulting male HSA-AR/Tfm rats express WT AR exclusively in muscle and nonfunctional Tfm AR in other tissues. We then examined motoneuron and muscle morphology of the SNB neuromuscular system of WT and Tfm rats with and without the HSA-AR transgene. We observed feminine levator ani muscle size and SNB motoneuron number and size in Tfm males with or without the HSA-AR transgene. These results indicate that AR expression in skeletal muscle fibers is not sufficient to rescue the male phenotype of the SNB neuromuscular system and further suggest that AR in other cell types plays a critical role in sexual differentiation of this system.

Overexpression of Androgen Receptors in Target Musculature Confers Androgen Sensitivity to Motoneuron Dendrites

The dendritic arbors of spinal motoneurons are dynamically regulated by a variety of factors, and several lines of evidence indicate that trophic interactions with the target musculature are of central importance. In highly androgen-sensitive motoneuron populations, androgens are thought to regulate motoneuron dendrites through their action at the receptor-enriched target musculature. Using rats transgenically modified to overexpress androgen receptor (AR) in skeletal muscle, we directly tested the hypothesis that the enhanced expression of AR in the target musculature can underlie the androgenic regulation of motoneuron dendritic morphology. The morphology of motoneurons innervating the quadriceps muscle was examined in wild-type (WT) rats as well as in rats that had been transgenically modified to overexpress ARs in their skeletal musculature. Motoneurons innervating the vastus lateralis muscle of the quadriceps in gonadally intact male rats, and castrated males with or without androgen replacement, were labeled with cholera toxin-conjugated horseradish peroxidase, and dendritic arbors were reconstructed in three dimensions. In WT rats, quadriceps motoneuron dendrites were insensitive to hormonal manipulation. In contrast, quadriceps motoneuron dendrites in gonadally intact transgenic males were larger than those of WT males. Furthermore, overexpression of ARs in the quadriceps muscle resulted in androgen sensitivity in dendrites, with substantial reductions in dendritic length occurring after castration; this reduction was prevented with testosterone replacement. Thus, it appears that the androgen sensitivity of motoneuron dendrites is conferred indirectly via the enrichment of ARs in the musculature.

Prenatal flutamide enhances survival in a myogenic mouse model of spinal bulbar muscular atrophy

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.

Effects of Bax gene deletion on social behaviors and neural response to olfactory cues in mice.

Bax is a pro‐death protein that plays a crucial role in developmental neuronal cell death. Bax−/− mice exhibit increased neuron number and lack several neural sex differences. Here we examined the effects of Bax gene deletion on social behaviors (olfactory preference, social recognition, social approach and aggression) and the neural processing of olfactory cues. Bax deletion eliminated the normal sex difference in olfactory preference behavior. In the social recognition test, both genotypes discriminated a novel conspecific, but wild‐type males and Bax−/− animals of both sexes spent much more time than wild‐type females investigating stimulus animals. Similarly, Bax−/− mice were more sociable than wild‐type mice in a social approach test. Bax deletion had no effect on aggression in a resident/intruder paradigm where males, regardless of genotype, exhibited a shorter latency to attack. Thus, the prevention of neuronal cell death by Bax gene deletion results in greater sociability as well as the elimination of sex differences in some social behaviors. To examine olfactory processing of socially relevant cues, we counted c‐Fos‐immunoreactive (Fos‐ir) cells in several nodes of the accessory olfactory pathway after exposure to male‐soiled or control bedding. In both genotypes, exposure to male‐soiled bedding increased Fos‐ir cells in the posterodorsal medial amygdala, principal nucleus of the bed nucleus of the stria terminalis and medial preoptic nucleus (MPN), and the response in the MPN was greater in females than in males. However, a reduction in Fos‐ir cells was seen in the anteroventral periventricular nucleus of Bax−/− mice.

Subcellular effects of myocyte-specific androgen receptor overexpression in mice

Although androgen receptor (AR) within myocytes is thought to mediate many of the effects of testosterone and other androgens on skeletal muscle, little is known about the functions of AR within these cells. We, therefore, studied the ultrastructure of skeletal muscle of HSA-AR transgenic (Tg) mice that overexpress AR selectively in myocytes and exhibit neuromuscular atrophy. We examined male HSA-AR mice from two different founding lines: L78 (lower copy number and less severe phenotype) and L141 (higher copy number and more severe phenotype) and compared these to wild-type (Wt) brothers. We also examined testosterone-treated female mice from these two lines and compared them both to their Wt sisters and to vehicle-treated controls. Ultrastructural examination of extensor digitorum longus sections using transmission electron microscopy revealed remarkably disorganized myofibrils in male Tg and testosterone-treated female Tg mice. Quantification of ultrastructural pathology indicated reduced myofibril width, hypertrophic and hyperplastic intermyofibrillar mitochondria, and pronounced glycogen accumulation in HSA-AR males of both lines. Reduced myofibrillar width and increases in mitochondrial number, size, and volume density were also observed in testosterone-treated HSA-AR females, although glycogen accumulation was not observed. Structural abnormalities in mitochondria were also associated with increases in electron transport chain activity and systemic resting metabolic rate, indicative of hypermetabolism. We find that overexpression of AR in myocytes of HSA-AR mice results in alterations in myofibrils, mitochondria, and glycogen. Alterations in myofibrils and mitochondria appear to result from acute actions of testosterone, whereas those on glycogen do not. Pathology of myofibrils and/or mitochondria may, therefore, mediate in part the neuromuscular atrophy observed in HSA-AR mice.

Tactile Stimulation During Artificial Rearing Influences Adult Function and Morphology in a Sexually Dimorphic Neuromuscular System

Maternal licking of rat pups affects the development of the spinal nucleus of the bulbocavernosus (SNB), a sexually dimorphic motor nucleus that controls penile reflexes involved with copulation. Maternal licking influences SNB motoneurons, with reductions in licking producing decreased SNB number, size, and dendritic length in adulthood. Reduced maternal licking also produces deficits in adult male copulatory behavior. In this experiment, we used an artificial rearing paradigm to assess the potential role of tactile stimulation in mediating the effects of maternal licking on the SNB neuromuscular system. During artificial rearing, pups were stroked with a paintbrush to mimic maternal licking, receiving low, medium, or high levels of daily stimulation. In adulthood, ex copula penile reflex behavior was tested and the morphology of SNB motoneurons assessed. SNB motoneurons were retrogradely labeled with cholera toxin‐conjugated HRP and dendritic arbor was reconstructed in three dimensions. Animals that received low levels of stimulation showed deficits in penile reflexes relative to maternally reared controls, including a longer latency to erection, fewer cup erections, and fewer erection clusters. SNB dendritic morphology was also shaped by stimulation condition, with animals that received low or medium levels of stimulation showing an average 27% reduction in dendritic length. In addition, several reflex behaviors were significantly correlated with dendritic length, including latency to first erection, percent of cup erections, and number of erection clusters. These results suggest that tactile stimulation provided by maternal licking mediates some of the effects of maternal care on the development of male copulatory behavior.