ABSTRACTThe overall goal of this proposal is to characterize a novel mouse model for Charcot-Marie Tooth (CMT) diseaseand define the effects on sphingolipid metabolism. CMT occurs in 1 out of 2500 people making it the mostcommonly inherited peripheral neuropathy producing motor and sensory deficits with onset occurring typicallyat middle age progressing with time (1 2). One variant of CMT CMT2F is caused by mutations in the heatshock protein B1 (HspB1) gene which encodes heat shock protein 27 (Hsp27) in humans and Hsp25 in mice.Hsp27 is a member of the class of small heat shock proteins and serves many functions including thermaltolerance chaperone activity protein degradation anti-apoptotic signaling cytoskeletal regulation andautophagy (3). Our lab has generated a novel genetically modified mouse model (GEMM) containing a pointmutation in HSP25 corresponding to the S135F mutation found in humans the most commonly reported CMT2Fmutation in the literature to date. This GEMM HspB1S139F was generated in collaboration with JacksonLaboratories using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)Cas9 technology. In cell culture models Hsp27 harboring the S135F mutation has been shown to bind and interact withceramide synthase 1 (CerS1) and modify CerS1 enzymatic activity by altering its cellular localization suggestingthat CerS1 activity may be altered in CMT2F pathology. CerS1 generates ceramide the central lipid insphingolipid metabolism which has many bioactive functions including growth arrest senescence apoptosisand autophagy [Reviewed in (4)]. CerS1 is highly expressed in the nervous system and when mutated in miceresults in neurodegenerative and ataxic phenotypes (5). Determining whether changes in sphingolipidbiosynthesis have direct roles in the pathology of CMT2F will further our understanding of CMT and offer noveltherapeutic targets to a disease with few therapeutic options.The goals of this proposal are innovative and significant as this will be the first study to directly explorethe role of this HspB1 mutation in the endogenous mouse protein in vivo defining the effects of thismutation on motor function pathology and sphingolipid metabolism.To this end we propose the following specific aims:Specific Aim 1: Establish HspB1S139F mice as a novel model to study CMT2F in vivo.Specific Aim 2. Establish the effects of the HspB1S139F mutation on sphingolipid metabolism in vivo.