Molecular and cellular mechanisms of spinocerebellar ataxia type 5 - β-III-spectrin is a key cytoskeletal protein that localizes to the soma and dendrites of cerebellar Purkinje neurons, and is required for dendritic arborization and signaling. Dominant mutations in the SPTBN2 gene encoding β-III-spectrin cause the neurodegenerative disorder spinocerebellar ataxia type 5 (SCA5). SCA5 causes degeneration of Purkinje cells, and an associated progressive gait and limb ataxia. There is no cure or therapy for SCA5. Many SCA5 missense mutations (including L253P) cluster within the β-III-spectrin actin- binding domain (ABD). Previously, we showed that increased actin binding is a shared molecular consequence of the ABD-localized mutations. Using novel Drosophila models, we demonstrated that a common cellular consequence of the ABD-localized mutations is reduced dendritic arborization. We further determined that truncation of the unique β-III-spectrin N-terminal domain (NTD), preceding the conserved ABD, rescues L253P-induced high-affinity actin binding and arbor defects. We also discovered that multiple phosphosites modulate the binding of wild-type β-III-spectrin to actin in vitro. However, it remains unclear how increased actin binding impairs the function of β-spectrin to support dendritic arborization. Moreover, the shared impact of SCA5 mutations to alter actin binding demands a better understanding of the mechanisms controlling β-spectrin-actin interactions. The objective of this application is to determine how SCA5-induced high-affinity actin binding leads to decreased dendritic arborization, and to elucidate mechanisms regulating spectrin-actin binding. In addition to the progress described above, using immunoprecipitation coupled to mass spectrometry, we identified numerous proteins in Drosophila neurons that physically associate with β- spectrin, including tropomyosin, myosins, kinases, phosphatases, and additional proteins. Intriguingly, tropomyosin, was recently shown to simultaneously bind actin and the unique β-spectrin NTD, suggesting tropomyosin is a key regulator of spectrin-actin binding. Further, tropomyosin loss-of-function alleles are known to increase dendritic arbor outgrowth. This suggests that modulation of tropomyosin, or other components of the interactome, may alleviate reduced arborization caused by SCA5 mutations. In this project we will define the Drosophila neuronal interactomes of the wild-type and L253P β-spectrin. We will use these interactomes and the well-developed Drosophila toolbox to guide a targeted genetic modifier screen for modulators of SCA5 arbor defects. Further, we will define in greater detail how the NTD, and its phosphorylation and binding to tropomyosin, regulates actin binding and SCA5 arbor defects. Numerous undergraduate students will participate in the execution of this project, providing important bench training for the next generation of problem solvers.
