(PREPARED BY APPLICANT): We have discovered that p-lll spectrin (SPTBN2) mutations cause spinocerebellar ataxia type 5 (SCA5) in an 11-generation American kindred and two additional independently reported SCA5 families. The American and a French family have separate in-frame deletions of 39 and 15 bp, respectively, in the third of 17 spectrin repeat motifs. A third mutation, found in a German family, is located in the calponin homology domain, a region known to bind actin, protein 4.1 and Arp1, which could cause generalized dysfunction of the membrane-cytoskeletal complex or deficits in dynein-dynactin mediated transport. Consistent with Purkinje cell degeneration in SCA5, P-lll spectrin is highly expressed in cerebellar Purkinje cells. Dramatic differences in the glutamate transporter EAAT4, a protein known to interact with p-lll spectrin, and the glutamate receptor delta 2 protein (GluR62) were found by Western and cell fractionation analysis of SCA5 autopsy tissue, suggesting the possibility that mutant p-lll spectrin disrupts the distribution or stability of these proteins. TIRF microscopy performed on cell lines transiently transfected with mutant or wildtype spectrin shows that mutant p-lll spectrin fails to stabilize EAAT4 at the plasma membrane. Our preliminary data show that p-lll spectrin mutations are a novel cause of neurodegenerative disease that may affect the stabilization or trafficking of membrane proteins. The goals of this application are to better understand the role of spectrin in ataxia, the normal function of P-lll spectrin, and the molecular consequences of the SCA5 mutations, including effects on EAAT4 and glutamate transport.
