Neurodegeneration in cerebellar granule cells of p/q type voltage gated calcium channel mutant leaner mice

Abstract

Mutations of the α1A subunit of CaV 2.1 voltage gated calcium (VGCC) channels are responsible for several inherited disorders affecting humans, including familial hemiplegic migraine, episodic ataxia type and spinocerebellar ataxia type. The leaner mouse also carries an autosomal recessive mutation in the α1A subunit of CaV 2.1 VGCCs, which, in the homozygous condition, results in a severe cerebellar atrophy and ataxia. The leaner mutation results in reduced calcium influx through CaV 2.1 VGCCs. To better understand cerebellar neurodegeneration and cerebellar dysfunction we focused our research on elucidating the relationship between mitochondrial function/dysfunction and calcium channel mutations. The aims of this dissertation were: 1) to estimate the extent of neuronal cell death, basal intracellular calcium and mitochondrial (dys)function in cerebellar granule cells (CGC) of adult leaner mice; 2) to analyze the role of the leaner calcium channel mutation on postnatal development of CGCs; and 3) to test whether inducing increased calcium influx by exposing cultured granule cells to potassium chloride can eliminate or reduce the CGC death. By using mechanism independent Fluoro-Jade staining and apoptosis specific TUNEL staining, we demonstrated that leaner CGC death continues into adulthood and the spatial pattern of granule cell death observed during postnatal development also continues into adulthood. The present investigation showed a reduced resting intracellular calcium in CGC from leaner mice as compared to age matched wild type mice, and tottering mice. The tottering mouse is another mutant mouse that carries a mutation in the α1A subunit of CaV 2.1 VGCCs like leaner mouse. However, these mice do not show any neurodegeneration and therefore they were used as a second control. Our results also showed that even though CGC of leaner mice have dysfunctional CaV2.1 channels, there is no change in depolarization induced Ca2+ influx, which suggests a functional compensation for CaV2.1 calcium channels by other VGCCs. Our results showed reduced mitochondrial membrane potential at the time of peak CGC death in leaner mice as compared to wild type CGCs and tottering CGCs. The results of this investigation suggest mitochondrial mediated but reactive oxygen species independent cell death in CGCs of leaner mice.