DESCRIPTION (provided by applicant): Duchenne Muscular Dystrophy (DMD) is a devastating genetic muscular disorder of childhood manifested by progressive debilitating muscle weakness and wasting, and ultimately death in the second or third decade of life. We have made the surprising discovery that full-length dystrophin is expressed in activated satellite cells where it is required to establish Par complex mediated polarity. In the absence of dystrophin, the Par complex becomes dysregulated, the numbers of centrosomes becomes abnormally increased, and consequently satellite stem cells are unable to efficiently undergo asymmetric division. We hypothesize that this dysregulation of the Par complex impairs the satellite cell regenerative program and thus contributes to disease progression in DMD. We propose to investigate the molecular mechanisms through which dystrophin regulates the Par complex, characterize the biological consequences of the loss of polarity on the satellite cell regenerative program, and evaluate the ability of alternative regulatory pathways to restore asymmetric division. In Aim 1, the function of the Par complex in mdx satellite cells will be characterized. The axis of cell division in newly dividing satellite cells will be examined to determine whether the axis becomes randomized or otherwise altered. The composition of the Par complex will be investigated and the status of different modulators and effectors of polarity will be assessed. Finally, the findings will be validated in muscle biopsies obtained from DMD patients. These experiments will fully characterize the molecular nature of the polarity phenotype of dystrophin-deficient satellite cells and provide insight into the stem cell deficit in
DMD patients. In Aim 2, we will assess how this loss of polarity control perturbs satellite cell self-renewal, expansion and differentiation during regenerative myogenesis. The regenerative program in mdx mice will be characterized by morphometric and immunohistological analyses after acute injury. Satellite cell engraftment assays will be conducted to assess the performance of dystrophin-deficient satellite cells in a wild type versus a dystrophin- deficient environment. Satellite cell function in mice lacking Par1b and p38¿ will be characterized. These experiments will reveal the functional role for polarity control in the satellite cell regenerative program. InAim 3, the ability of alternative regulatory mechanisms to stimulate asymmetric cell division in mdx satellite cells will be investigated. We have found that EGF stimulates asymmetric division of mdx satellite cells. Therefore the mechanistic basis for this stimulation will be investigated, and
the ability of EGF to restore the regenerative program of dystrophin-deficient satellite cells will
be evaluated. These experiments will provide proof-of- concept that modulation of the mechanisms that regulate polarity may be amenable to apply as a therapeutic intervention for DMD. In conclusion, we have discovered a novel role for dystrophin in regulating muscle stem cell polarity. Our proposed experiments will elucidate the mechanisms regulating polarity in stem cells and provide important new insights into the molecular pathobiology contributing to the disease progression of DMD.