1. Jamie A. Johansen 1, D. Ashley Monks 3, S. Marc Breedlove 1,2, Cynthia L. Jordan 1,2 1 Neuroscience Program, 2 Department of Psychology Michigan State University; 3 Department of Psychology, University of Toronto at Mississauga Introduction Methods Results Increased Androgen Receptor Expression in Skeletal Muscle Fibers Leads to Fiber Loss The spinal nucleus of the bulbocavernosus (SNB) is a sexually dimorphic group of motoneurons that innervate sexually dimorphic perineal muscles, the bulbocavernosus (BC) and levator ani (LA). These muscles are highly androgen responsive; the size of LA muscle fibers is reduced by castration and restored with testosterone treatment in adulthood. In contrast, the extensor digitorum longus (EDL) is not responsive to androgens, showing no change in size after castration. These differences in androgen responsiveness parallel a difference in androgen receptor (AR) expression. LA muscle fibers contain much more AR immunoreactivity than EDL muscle fibers. Question: Does AR expression within muscle fibers account for differences in androgen responsiveness? We generated transgenic mice that over-express the rat AR selectively in muscle fibers using the human skeletal actin (HSA) promoter to ask whether EDL would now become androgen responsive. Surprisingly, the transgenic mice show a progressive loss of motor function, accompanied by muscle pathology. Unexpectedly, our mice develop an androgen-dependent motor dysfunction and muscle pathology. Transgenic males exhibit profound muscle pathology demonstrated by atrophic and angular-shaped fibers, and frequent internal nuclei. Transgenic males show profound losses in motor function, as evident in the Hang Test and Paw Print Analysis. Transgenic males show a 40-50% decrease in the number of EDL fibers. Testosterone induces a loss of motor function, accompanied by significant muscle pathology in transgenic females. However, in transgenic females, testosterone did not lead to the loss of muscle fibers seen in transgenic males. Technical support was provided by Cindy Knaff, Chas Jensen, Heather Malinowski, Nicole Timm, and Diane Redenius. These studies were funded by operating grant NIH NS- 045195 (CLJ), a grant from the MSU foundation (CJ, SMB) and a postdoctoral fellowship (DAM) from the Canadian Institutes of Health Research (CIHR). These results suggest that the wildtype AR in muscle fibers has an unexpected role in muscle health and disease. At sufficiently high levels, AR appears toxic to muscle fibers. The question remains whether high levels of AR in muscle fibers are also toxic to their innervating motor neurons. Because transgenic females treated with androgens experience a rapid decline in motor function, without a loss in the number of muscle fibers, muscle dysfunction rather than loss of fibers may underlie the decline in motor function. Ultimately this loss in cell function may lead to muscle fiber loss. Construct: AR cDNA was subcloned from pCMVAR. cDNAs were ligated into the NotI site of pBSX-HSA. Histology: Muscles were dissected from transgenic and wildtype males, placed in OCT filled cryomolds and flash frozen in liquid nitrogen. Muscles were then cryostat sectioned at 10um, and stained for hematoxylin and eosin (H&E), or nicotinamide adenine dinucleotide (NADH). Androgen Dependence: To look at androgen dependence, transgenic females were ovariectomized and given either a testosterone filled or blank Silastic capsule. Females were treated for 10 days and then muscles were harvested as above. Behavioral Testing: Hang Test - Mice were placed on a wire grid, and then turned upside down, and the time to fall was measured in seconds. Paw Print Analysis – Front feet were painted with non-toxic acrylic red paint, and hind feet were painted with blue. Mice were then guided to walk along a strip of filter paper. Figure 2. H&E staining of EDL muscles of wildtype (Wt) and transgenic (Tg) male mice indicate that Tg muscles contain both abnormally small, angular fibers (black arrow) and hypertrophied fibers (white arrow). Both fiber types contain internal nuclei (stars). Muscle fibers of transgenic mice display pathology. * * Over-expression of wildtype AR leads to a decrease in EDL muscle fiber number. Figure 3. Transgenic males show a profound loss in muscle fiber number, as compared to wildtype brothers (p=.0004). Fibers numbers are decreased by 40-50%. 0 200 400 600 800 1000 1200 Fiber Number WildtypeTransgenic * 0 Figure 6. Testosterone treated transgenic female mice do not show a decrease in muscle fiber numbers, even though they exhibit a profound loss in motor function. Fiber Number Wildtype Transgenic 0 200 400 600 800 1000 1200 Testosterone Blank However, T treatment does not decrease the number of EDL muscle fibers. Over-expression of wildtype AR in muscle fibers of transgenic male mice leads to a loss in motor function. Figure 1. Paw Print analysis of transgenic (Tg) males show a shortened stride compared to wildtype (Wt) males. This suggests a loss in motor function, strength and coordination. Stride Tg Wt Stride Figure 4. T induces a dramatic loss of motor function in transgenic females, as shown with the Hang test. As early as Day 3 of T treatment, female transgenic mice exhibit muscle weakness and are unable to perform the Hang test. * = Significantly different from Day 0, p <.003. Testosterone (T) induces a loss of motor function in previously asymptomatic transgenic females. Day 0 3 5 7 9 * -10 10 30 50 70 90 110 130 Transgenic, Testosterone Transgenic, Blank Wildtype, Testosterone Wildtype, Blank Hang Time (s) *** Transgenic males show a loss in motor function, striking muscle pathology and a significant loss of fibers. Testosterone induces in transgenic females a loss of motor function, striking muscle pathology but NOT a loss of fibers. Figure 5. NADH staining reveals that T treatment has marked effects on EDL muscles from Tg females including alterations in the internal architecture of fibers, and size and shape of the fibers. Note that ring fibers (arrows) are prevalent in the T- treated Tg muscle, and absent in blank-treated Tg muscle. T induces male-like pathology in muscles from transgenic females as revealed by NADH stain. Summary Significance Acknowledgments