Epilepsy Models and Calcium Channel Function P/Q-type voltage-dependent calcium channel CACNA1A mutations cause dominantly inherited migraine, episodic ataxia, and cerebellar atrophy in humans and cause recessively inherited ataxia, episodic dyskinesia, cerebellar atrophy, and absence epilepsy in mice. The basis of these species differences and the disease mechanism(s) are not understood. To address this question and to identify required P/Q function in vivo, we created a germline Cacna1a null mutation (designated Cacna1aFcrtm1) by gene targeting. Null mice develop dystonia and late-onset cerebellar degeneration in a specific pattern. This indicates a requirement for P/Q function for survival in a subset of cerebellar neurons. Homozygous null mice completely lack P/Q-type channel activity, and they also lack w-CTx-MVIIC receptors, indicating that a single gene encodes P/Q channel activity. An increase of L- and N-type current densities is detected in P/Q-null granule cells. Heterozygous Cacna1aFcrtm1/+ mice are phenotypically normal, despite having a 50% reduction in current density, indicating that reduced current density is not itself sufficient to cause the pathophysiology of spontaneous mouse mutants with ataxia and seizures. It has been hypothesized that R-type Ca currents result from the expression of the Cacna1e gene. To test this hypothesis, we examined the properties of voltage-dependent Ca channels in mice in which the Cacna1e channel subunit had been deleted. Application of omega-conotoxin GVIA, omega-agatoxin IVA, and nimodipine to cultured cerebellar granule neurons from wild-type mice inhibited components of the whole-cell Ba current, leaving a """"""""residual"""""""" R current with an amplitude of approximately 30% of the total Ba current. A minor portion of this R current was inhibited by the Cacna1e-selective toxin SNX-482, indicating that it resulted from the expression of Cacna1e. However, the majority of the R current was not inhibited by SNX-482. The SNX-482-sensitive portion of the granule cell R current was absent from Cacna1e knockout mice. We also identified a subpopulation of dorsal root ganglion (DRG) neurons from wild-type mice that expressed an SNX-482-sensitive component of the R current. However, as with granule cells, most of the DRG R current was not blocked by SNX-482. We conclude from these experiments that there exists a component of the R current that results from the expression of the Cacna1e Ca channel subunit but that the majority of R currents must result from the expression of other Ca channel alpha subunits. Deafness Models Mutations at the waltzer (v) locus result in deafness and vestibular dysfunction due to degeneration of the neuroepithelium within the inner ear. We showed that waltzer encodes a novel cadherin (Cdh23), which is most closely related to the Drosophila fat protein. A single nucleotide deletion in the vJ allele and a single nucleotide insertion in the v allele are predicted to truncate each protein near the N-terminus and produce a functional null allele. In situ hybridization analysis showed that Cdh23 is expressed in the sensory hair cells of the inner ear, where it has been suggested to be a molecule critical for crosslinking of the stereocilia. In addition, Cdh23 is expressed in the utriculo-saccular foramen, the ductus reuniens, and Reissner's membrane, suggesting that Cdh23 may also be involved in maintaining the ionic composition of the endolymph. Finally, mutations in human CDH23 have recently been described for two loci, DFNB12 and USH1D, which cause nonsyndromic deafness, identifying waltzer as a mouse model for human hearing loss. Neural Degeneration Models Mice homozygous for the ataxia (axJ) mutation develop severe tremors by 2-3 weeks of age, followed by hind limb paralysis and death by 6 weeks of age. Recently, we have shown that axJ encodes ubiquitin-specific protease 14 (Usp14). USPs comprise a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates in Parkinson's disease (PD), spinocerebellar ataxia type 1 (SCA1), and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a short ubiquitin side chain, the removal of which may regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in axJ mice due to the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In situ hybridization studies show that Usp14 is expressed in a variety of neurons within the adult central nervous system (CNS), including the cerebral cortex, hippocampus, Purkinje cells, and deep cerebellar nuclei. However, unlike neurodegenerative disorders such as PD and SCA1 in humans or GAD in mice, there are no detectable ubiquitinated-protein aggregates nor is there neuronal cell loss in the axJ CNS. Instead, we identify synaptic connection defects in the axJ CNS, suggesting that USPs may be important factors in regulating the activity of synapses, possibly through neurotransmitter release.
