Investigation into the pathogenesis of Spinocerebellar Ataxia Type 5
Clarkson, Yvonne Louise
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Mutations in SPTBN2, the gene encoding b-III spectrin, give rise to spinocerebellar ataxia type 5 (SCA5), an autosomal dominant neurodegenerative disease characterized by motor incoordination and cerebellar degeneration. The work reported in this thesis addressed possible mechanisms of disease pathogenesis using genetically modified mice lacking b-III spectrin (b-III-/-) and also investigated the normal function of b-III spectrin through identification of proteins that interact with its amino-terminus. Targeted recombination was successful in eliminating expression of full-length b-III spectrin but b-III spectrin lacking exons 2-6 ( 2-6 b-III spectrin) was found to be present at a low level in b-III-/- spectrin mice. To ascertain whether the novel truncated protein had any obvious gain-of-function or adverse property that would complicate analysis of b-III-/- spectrin mice the aberrant transcript 2-6 b-III spectrin was cloned and a number of in vitro experiments carried out. Protein stability, solubility, cellular localization, and functional assays indicated 2-6 b-III spectrin was less functional than full-length b-III spectrin, confirming the b-III-/- spectrin mouse could be considered a functional knockout.Analysis of b-III-/- spectrin mice revealed that from 18-weeks of age hind limb gait became progressively wider than age-matched wild-type (WT) controls and three behavioural tests (stationary rod, rotarod, and elevated beam) demonstrated a progressive impairment in motor performance and coordination. 3-week old b-III-/- spectrin mice performed worse on the rotating rod than age-matched controls but their performance at 3- and 5-rpm improved with consecutive days of testing. Only at 10-rpm did young b-III-/- spectrin mice fail to improve, whereas 6-month old b-III-/- spectrin mice were unable to stay on the rod even at 3-rpm. The ability to balance on a stationary rod was also worse at 6-months of age and the number of hindlimb slips made by b-III-/- spectrin mice on the elevated beam increased from 12-weeks of age. This progressive motor phenotype mirrors symptoms seen in SCA5 patients. In contrast heterozygous mice (b-III+/-) were shown not to develop an ataxic phenotype or display cerebellar degeneration, even at 2-years of age. Cell culture studies using one mutation (L253P) associated with SCA5 revealed that it interfered with protein trafficking from the Golgi apparatus and had a dominant-negative effect on WT function. Incubation at a lower temperature resulted in L253P b-III spectrin reaching the plasma membrane suggesting an altered protein conformation was responsible for the protein trafficking defect. The intracellular accumulation of proteins at the Golgi did not initiate the unfolded protein response. From this work it was concluded that the b-III-/- spectrin mouse is a new model of cerebellar ataxia and loss of b-III spectrin function underlies SCA5 pathogenesis. The results argued against haploinsufficiency and instead suggested disease-causing mutations have dominantnegative effects on WT function and indicate a deficit of cell membrane proteins could participate in SCA5 pathogenesis. Finally, using a yeast two-hybrid screen the amino terminus of b-III spectrin was found to interact with the carboxy-terminus of prosaposin (a neurotrophic factor) and clathrin light chain. The interactions were confirmed in mammalian cells suggesting neurite outgrowth and movement of membrane vesicles may be normal functions of b-III spectrin.