The overall goal of this proposal is to characterize a novel mouse model for Charcot-Marie Tooth (CMT) disease and define the effects on sphingolipid metabolism. CMT occurs in 1 out of 2,500 people, making it the most commonly inherited peripheral neuropathy, producing motor and sensory deficits with onset occurring typically at middle age progressing with time 1,2. One variant of CMT, CMT2F, is caused by mutations in the heat shock 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 thermal tolerance, chaperone activity, protein degradation, anti-apoptotic signaling, cytoskeletal regulation, and autophagy 3. Our lab has generated a novel genetically modified mouse model (GEMM) containing a point mutation in HSP25, corresponding to the S135F mutation found in humans, the most commonly reported CMT2F mutation in the literature to date. This GEMM, HspB1S139F was generated in collaboration with Jackson Laboratories using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)?Cas9 technology. In cell culture models, Hsp27 harboring the S135F mutation has been shown by our long time collaborators at Stony Brook to bind and interact with ceramide synthase 1 (CerS1) and modify CerS1 enzymatic activity by altering its cellular localization, suggesting that CerS1 activity may be altered in CMT2F pathology. CerS1 generates ceramide, the central lipid in sphingolipid metabolism, which has many bioactive functions including growth arrest, senescence, apoptosis, and autophagy [Reviewed in 4]. CerS1 is highly expressed in the nervous system and when mutated in mice results in neurodegenerative and ataxic phenotypes 5. Determining whether changes in sphingolipid biosynthesis have direct roles in the pathology of CMT2F will further our understanding of CMT and offer novel therapeutic 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 explore the role of this HspB1 mutation in the endogenous mouse protein in vivo, defining the effects of this mutation 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.
Charcot-Marie-Tooth (CMT) Disease occurs in 1 out of 2,500 people, making it the most common inherited peripheral neuropathy. CMT was initially defined clinically by its alternate name, Hereditary Motor and Sensory Neuropathy, as patients typically present with gait abnormalities, symmetric distal polyneuropathy, hammer toes, (pes cavus) and diminished sensation and deep tendon reflexes. More than 70 genes have been implicated in CMT, highlighting its genetic heterogeneity. Current treatments for CMT, including CMT2F, caused by mutation in heat shock protein 27 (Hsp27), are extremely limited. Our lab has generated a novel genetically modified mouse model (GEMM) containing a point mutation in HSP27, corresponding to the S135F mutation found in humans. The goals of this proposal are innovative and significant as this will be the first study to directly explore the role of this HspB1 mutation in vivo, characterizing effects of this mutation on motor function, pathology and sphingolipid metabolism.
