? Charcot-Marie-Tooth disease (CMT) is the most common peripheral neuropathy with a prevalence of 1 in 2,500, but the molecular mechanisms underlying this disease remain unknown. Imaging studies of Schwann cells acquired from human patients and rodent disease models of CMT subtypes (CMT1A and CMT1B) revealed accumulations of peripheral myelin proteins, namely, peripheral myelin protein 22 (PMP22) and myelin protein zero (MPZ), to cytoplasmic inclusion bodies. These findings suggest that aberrant trafficking of PMP22 and MPZ are involved in CMT pathogenesis. However, little is known about the steps and the machinery involved in the intracellular trafficking of peripheral myelin proteins. Recently, mutations in SIMPLE, an ubiquitiously expressed protein of unknown function, are identified as the genetic defects responsible for a demyelinating form of CMT (CMT1C). SIMPLE contains a PSAP motif that binds Tsg101, a protein involved in the sorting of ubiquitinated cargo proteins to the lysosomal pathway for degradation. Preliminary studies demonstrated that SIMPLE colocalizes with Tsg101 at the early endosomes, implicating their physiological interaction in vivo. Together, this evidence suggest a role of SIMPLE in the regulation of endosome-to-lysosome trafficking. Furthermore, sequence analysis of SIMPLE reveals a RING finger motif, a predictive determinant of E3 ubiquitin-protein ligase activity. This finding raises the possibility that SIMPLE act as an E3 ubiquitin-protein ligase to ubiquitinate Tsg101 in order to regulate its function. Despite these possibilities, the ubiquitinating activity of SIMPLE has not been tested, and the role of SIMPLE in the regulation of endocytic trafficking remains unknown. This proposal will test the hypothesis that SIMPLE acts as an endosomal E3 ubiquitin-protein ligase to regulate endosome-to- lysosome trafficking, and that CMT1C mutations of SIMPLE disrupt this function.
The specific aims are to: 1) characterize the subcellular localization of SIMPLE and the impact of CMT1C mutations; 2) examine the E3 ubiquitin-protein ligase activity of SIMPLE and the effects of CMT1C mutations; 3) elucidate the functional role of SIMPLE in the regulation of endosome-to-lysosome trafficking. ?
Overall, these studies will assess the molecular pathways involved in a devastating disease that significantly decrease the quality of life. Current treatments such as physical therapy and pain medications are insufficient to battle worsening motor and sensory symptoms and do little to reverse or halt demyelination and axonal degeneration. Therefore, this project will examine the molecular basis of CMT in hopes of providing better treatments for these patients. ?
