Serine Palmitoyl Transferase and Hereditary Neuropathy

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DESCRIPTION (provided by applicant): Serine palmitoyltransferase (SPT) catalyzes the committed and rate-limiting step in sphingolipid synthesis. Mutations in the SPTLC1 gene, encoding the Sptlclp subunit of SPT, result in hereditary sensory neuropathy, type 1 (HSN1), the most common inherited neuropathy. Thus, aberrant sphingolipid synthesis may contribute to the pathophysiology of this disease. SPT contains at least one additional subunit, Sptlc2p, which forms a heterodimer with Sptlclp. HSN1 mutant Sptlclp proteins behave as dominant negative inhibitors of SPT activity by virtue of their ability to form catalytically inactive stable heterodimers. Our long-range goal is to identify the genes involved in sphingolipid synthesis and their role in human disease. Using a combination of model systems amenable to genetic and biochemical manipulation, we will determine how the mutations in SPT contribute to the pathophysiology of HSN1. Specifically we propose to: 1) isolate and characterize the mammalian SPT complex and identify the downstream components of the sphingosine biosynthetic pathway; 2) map the domains important for catalysis, heterodimerization, regulated Sptlc2p stability, and determine the topology of Sptlclp and Sptlc2p, and establish whether relocalization of SPT regulates activity; 3) elucidate the mechanism by which the HSN1 dominant negative mutations decrease SPT activity; and 4) construct transgenic mice expressing the SPTLC1-HSN1 dominant negative allele and an SPTLC1 "knock-out" mouse. SPT complexes will be immunopurified and their components identified by MALDI-TOF mass spectrometry. Two hybrid screens will be used to identify Sptlclp and Sptlc2p interacting proteins. Mapping of domains, determination of topology, intracellular localization, and characterization of dominant negative mutants will be performed using a combination of yeast genetics and expression of normal and mutant proteins in mammalian cells in which siRNAs are used to ablate endogenous gene expression. Mice transgenic for wildtype or mutant SPTLC1 will be characterized for behavioral, morphological, biochemical, and electrophysiological changes. Parallel analysis will be completed on SPTLC1 conditional knock-out mice. Taken together these studies will illuminate fundamental issues underlying the biology of sphingolipids, their regulation, and the disease process of HSN1.