Loss of HAX-1 May Contribute to the Neurodegeneration Caused by a Kv3.3 Mutation

Abstract

Spinocerebellar Ataxia type 13 (SCA13) is caused by mutations in the KCNC3 gene, which encodes the voltage-dependent potassium channel Kv3.3. These channels are expressed at particularly high levels in cerebellum Purkinje cells. SCA13 is an autosomal dominant disease characterized primarily by degeneration of the cerebellum. We have found the Kv3.3 channel differs from other potassium channels in that it binds Hax-1, an anti-apoptotic protein that is absolutely required for the survival of cerebellar neurons. We have also found that depolarization of Kv3.3 channels directly activates Tank Binding Kinase 1 (TBK1), an enzyme that plays a key role in the formation of multivesicular bodies, autophagy and mitophagy. A disease-causing mutation, G592R Kv3.3, produces enhanced TBK1 activation both in cell lines and in the cerebellum of knock-in mice bearing this mutation. By electron microscopy, we find the enhanced activation of TBK1 in G592R Kv3.3 knock-in mice is associated with increased numbers of intracellular multivesicular bodies containing Hax-1, and increased levels of CD63, a molecular marker for these structures. We also found that the expression level of Hax-1 is reduced in G592R Kv3.3 knock-in mice by Western blotting. Mitochondrial function may also be impacted by the mutation because levels of mitofusin-1 and mitofusin-2, proteins that are required for fusion and maintenance of mitochondrial structure, are altered in the mutant mice. Using cell lines expressing the mutant channel, we have shown that the G592R Kv3.3-induced multivesicular bodies contain Hax-1. Inhibition of TBK1 in the cell lines prevents the increase in CD63 levels produced by the Kv3.3 mutation. Our findings suggest that Kv3.3 channels are directly coupled to pathways that regulate the trafficking of proteins into multivesicular bodies and that loss of the Hax-1 may contribute to the neurodegeneration caused by SCA13.