X-linked spinal and bulbar muscular atrophy (SBMA or Kennedy's disease) is a progressive neuromuscular disorder characterized pathologically by degeneration of lower motor neurons. In 1991, expansion of a CAG trinucleotide repeat was identified in the coding region of the androgen receptor (AR) gene of SBMA patients. In addition to SBMA, seven other neurodegenerative disorders are caused by CAG repeat expansions that encode elongated polyglutamine tracts. Molecular and genetic studies of the CAG/polyglutamine repeat diseases suggest that the polyglutamine tract expansion has a toxic gain-of- function effect, the basis of which remains unknown. The purpose of this grant proposal is to determine the molecular basis of neuronal cell death in SBMA. To achieve this goal, we have initiated studies aimed at recapitulating AR polyglutamine neurotoxicity in mice and in cell culture. For producing an in vivo model of SBMA, we have generated AR yeast artificial chromosomes (YACs) carrying 100 CAG repeats. These AR YACs have been introduced into the mouse germline by fusing yeast cells carrying the AR YACs with mouse embryonic stem (ES) cells. In addition to an in vivo model of SBMA, we are attempting to develop in vitro models of AR polyglutamine neurotoxicity. We have demonstrated cellular toxicity of mutant AR expression constructs in HEK-293T cells, a non-neuronal cell line. We are attempting to extend this finding to motor neuron-like cell lines, MN-1 and NSC-34, and are also establishing primary cultures of cortical, hippocampal, and spinal motor neurons. Once we have produced accurate in vivo and in vitro models of SBMA, we will examine the role of apoptotic pathways in SBMA pathogenesis by evaluating whether caspase activation and/or p53 participate in AR polyglutamine neurotoxicity. We will use our in vivo and in vitro models to determine what functions of the AR protein are required for SBMA disease pathogenesis. Animal and cell culture models of AR polyglutamine neurotoxicity will allow us to identify alterations in gene expression in diseased motor neurons and cell lines by performing microarray expression comparisons. As SBMA, amyotrophic lateral sclerosis and autosomal spinal muscular atrophy all show motor neuron cell death, any toxic factors or degenerative pathways that we identify in our studies may be relevant to these and other motor neuron diseases. In addition to allowing us to track the molecular events that lead to neuronal cell death, animal and cell culture models of SBMA will allow us to design novel treatments and test potential therapies for this motor neuron disease.