Douglas A. Monks

Overexpression of wild-type androgen receptor in muscle recapitulates polyglutamine disease

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. These findings demonstrate that toxicity in skeletal muscles is sufficient to cause motoneuron disease and indicate that overexpression of the WT AR can exert toxicity comparable with the polyglutamine expanded protein. This model has two clear implications for Kennedy disease: (i) mechanisms affecting AR gene expression may cause neuromuscular symptoms similar to those of Kennedy disease and (ii) therapeutic approaches targeting skeletal muscle may provide effective treatments for this disease.

Androgen receptor immunoreactivity in skeletal muscle: Enrichment at the neuromuscular junction

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 (∼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. J. Comp. Neurol. 473:59–72, 2004.

Androgen receptor and Kennedy disease/spinal bulbar muscular atrophy,

Kennedy Disease/Spinal Bulbar Muscular Atrophy (KD/SBMA) is a progressive neurodegenerative disease caused by genetic polyglutamine expansion of the androgen receptor. We have recently found that overexpression of wildtype androgen receptor in skeletal muscle of transgenic mice results in a KD/SBMA phenotype. This surprising result challenges the orthodox view that KD/SBMA requires expression of polyglutamine expanded androgen receptor within motoneurons. Theories relating to the etiology of this disease drawn from studies of human patients, cellular and mouse models are considered with a special emphasis on potential myogenic contributions to as well as the molecular etiology of KD/SBMA.

Androgen-sensitivity of somata and dendrites of spinal nucleus of the bulbocavernosus (SNB) motoneurons in male C57BL6J mice.

In rats, androgens in adulthood regulate the morphology of motoneurons in the spinal nucleus of the bulbocavernosus (SNB), including the size of their somata and the length of their dendrites. There are conflicting reports about whether androgens exert similar influences on SNB motoneurons in mice. We castrated or sham-operated C57BL6J mice at 90 days of age and, thirty days later, injected cholera toxin conjugated horseradish peroxidase into the bulbocavernosus muscle (to label SNB motoneurons) on one side, and into intrinsic foot muscles contralaterally (to label motoneurons of the retrodorsolateral nucleus (RDLN)). Castrated mice had significantly smaller SNB somas compared to sham-operated mice while there were no differences in soma size of RDLN motoneurons. Dendritic length in C57BL6J mice, estimated in 3-dimensions, also decreased significantly after adult castration. In rats, androgens act directly through androgen receptors (AR) in SNB motoneurons to control soma size and nearly all SNB motoneurons contain AR. Since SNB somata in C57BL6J mice shrank after adult castration, we used immunocytochemistry to characterize AR expression in SNB cells as well as motoneurons in the RDLN and dorsolateral nucleus (DLN). A pattern of labeling matched that seen previously in rats: the highest percentage of AR-immunoreactive motoneurons are in the SNB (98%), the lowest in the RDLN (25%) and an intermediate number in the DLN (78%). This pattern of AR labeling is consistent with the possibility that androgens also act directly on SNB motoneurons in mice to regulate soma size in mice.

Got milk? Oxytocin triggers hippocampal plasticity

Giving birth improves spatial memory in rodents. A new study shows that oxytocin, which triggers birth and milk release, activates a signaling cascade involved in learning, and that this hormone is necessary and sufficient for memory improvement in mice that have had litters.

Steroid Receptor Coactivator-1 Is Not Required for Androgen-Mediated Sexual Differentiation of Spinal Motoneurons

Steroid receptor coactivator-1 (SRC-1) amplifies genomic steroid hormone signal transduction and has been implicated in steroid-mediated sexual differentiation of the mammalian nervous system. We investigated the possible effect of an SRC-1 null mutation on 2 morphological endpoints of androgenic signaling: the number and size of motoneurons within the spinal nucleus of the bulbocavernosus (SNB). In wild-type C57/BL6 mice, SRC-1 immunoreactive nuclei were observed within the SNB and one of its target muscles, the levator ani. However, SRC-1 null mice were indistinguishable from sex-matched wild-type littermates in both SNB number and cross-sectional area of SNB motoneurons. Similarly, we found no difference between SRC-1 null and wildtype littermates in the number or size of motoneurons in the retrodorsolateral nucleus, a motor pool that is not typically sexually differentiated in either number or size. These results demonstrate that SRC-1 is not essential for the development and maintenance of a sexually dimorphic neuromuscular system.

Non-neural androgen receptor promotes androphilic odor preference in mice

In mice, male-typical preference for female olfactory cues results largely from sexually differentiated testosterone production. It is currently unclear on which cells and tissues testosterone acts to produce male-typical preference for female olfactory cues. To further address the site of androgen action on olfactory preference, we have developed a loxP-based transgenic mouse that overexpresses androgen receptors (AR) only when activated by Cre. We used this transgene to overexpress AR globally in all tissues using a CMV-Cre driver and a Nestin-Cre driver to overexpress AR selectively in neural tissue. We then examined olfactory preference in transgenic and wildtype (Wt) littermates by simultaneously exposing animals to female-soiled, male-soiled and clean bedding. Ubiquitous overexpression of AR in CMV-AR mice increased preference for male bedding, whereas neural-specific AR overexpression in Nestin-AR transgenic mice did not differ from wildtype siblings in olfactory preference. Neural activation of olfactory brain areas in response to female-soiled bedding was also evaluated in these mice by measuring FOS immunoreactivity. This revealed a decrease in neural activity along the accessory olfactory pathway that accompanied the decrease in preference for female odors in CMV-AR males, compared to both Nestin-AR and Wt male siblings. Together, results indicate that androgens act via non-neural AR to mediate olfactory preference and neural responses to olfactory stimuli, and further suggest that AR in non-neural tissues can promote androphilic odor preferences in male mice.In mice, male-typical preference for female olfactory cues results largely from sexually differentiated testosterone production. It is currently unclear on which cells and tissues testosterone acts to produce male-typical preference for female olfactory cues. To further address the site of androgen action on olfactory preference, we have developed a loxP-based transgenic mouse that overexpresses androgen receptors (AR) only when activated by Cre. We used this transgene to overexpress AR globally in all tissues using a CMV-Cre driver and a Nestin-Cre driver to overexpress AR selectively in neural tissue. We then examined olfactory preference in transgenic and wildtype (Wt) littermates by simultaneously exposing animals to female-soiled, male-soiled and clean bedding. Ubiquitous overexpression of AR in CMV-AR mice increased preference for male bedding, whereas neural-specific AR overexpression in Nestin-AR transgenic mice did not differ from wildtype siblings in olfactory preference. Neural activation of olfactory brain areas in response to female-soiled bedding was also evaluated in these mice by measuring FOS immunoreactivity. This revealed a decrease in neural activity along the accessory olfactory pathway that accompanied the decrease in preference for female odors in CMV-AR males, compared to both Nestin-AR and Wt male siblings. Together, results indicate that androgens act via non-neural AR to mediate olfactory preference and neural responses to olfactory stimuli, and further suggest that AR in non-neural tissues can promote androphilic odor preferences in male mice.

Managing anabolic steroids in pre-hibernating Arctic ground squirrels: obtaining their benefits and avoiding their costs

Androgens have benefits, such as promoting muscle growth, but also significant costs, including suppression of immune function. In many species, these trade-offs in androgen action are reflected in regulated androgen production, which is typically highest only in reproductive males. However, all non-reproductive Arctic ground squirrels, irrespective of age and sex, have high levels of androgens prior to hibernating at sub-zero temperatures. Androgens appear to be required to make muscle in summer, which, together with lipid, is then catabolized during overwinter. By contrast, most hibernating mammals catabolize only lipid. We tested the hypothesis that androgen action is selectively enhanced in Arctic ground squirrel muscle because of an upregulation of androgen receptors (ARs). Using Western blot analysis, we found that Arctic ground squirrels have AR in skeletal muscle more than four times that of Columbian ground squirrels, a related southern species that overwinters at approximately 0°C and has low pre-hibernation androgen levels. By contrast, AR in lymph nodes was equivalent in both species. Brain AR was also modestly but significantly increased in Arctic ground squirrel relative to Columbian ground squirrel. These results are consistent with the hypothesis that tissue-specific AR regulation prior to hibernation provides a mechanism whereby Arctic ground squirrels obtain the life-history benefits and mitigate the costs associated with high androgen production.

Transcriptional Profile of Muscle following Acute Induction of Symptoms in a Mouse Model of Kennedy's Disease/Spinobulbar Muscular Atrophy

Background Kennedy’s disease/Spinobulbar muscular atrophy (KD/SBMA) is a degenerative neuromuscular disease affecting males. This disease is caused by polyglutamine expansion mutations of the androgen receptor (AR) gene. Although KD/SBMA has been traditionally considered a motor neuron disease, emerging evidence points to a central etiological role of muscle. We previously reported a microarray study of genes differentially expressed in muscle of three genetically unique mouse models of KD/SBMA but were unable to detect those which are androgen-dependent or are associated with onset of symptoms. Methodology/Principal Findings In the current study we examined the time course and androgen-dependence of transcriptional changes in the HSA-AR transgenic (Tg) mouse model, in which females have a severe phenotype after acute testosterone treatment. Using microarray analysis we identified differentially expressed genes at the onset and peak of muscle weakness in testosterone-treated Tg females. We found both transient and persistent groups of differentially expressed genes and analysis of gene function indicated functional groups such as mitochondrion, ion and nucleotide binding, muscle development, and sarcomere maintenance. Conclusions/Significance By comparing the current results with those from the three previously reported models we were able to identify KD/SBMA candidate genes that are androgen dependent, and occur early in the disease process, properties which are promising for targeted therapeutics.