Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in protein kinase Cγ (PKCγ). Interestingly, 18 of 22 mutations are concentrated in the C1 domain, which binds diacylglycerol and is necessary for translocation and regulation of PKCγ kinase activity. To determine the effect of these mutations on PKCγ function and the pathology of SCA14, we investigated the enzymological properties of the mutant PKCγs. We found that wild-type PKCγ, but not C1 domain mutants, inhibits Ca²⁺ influx in response to muscarinic receptor stimulation. The sustained Ca²⁺ influx induced by muscarinic receptor ligation caused prolonged membrane localization of mutant PKCγ. Pharmacological experiments showed that canonical transient receptor potential (TRPC) channels are responsible for the Ca²⁺ influx regulated by PKCγ. Although in vitro kinase assays revealed that most C1 domain mutants are constitutively active, they could not phosphorylate TRPC3 channels in vivo. Single molecule observation by the total internal reflection fluorescence microscopy revealed that the membrane residence time of mutant PKCγs was significantly shorter than that of the wild-type. This fact indicated that, although membrane association of the C1 domain mutants was apparently prolonged, these mutants have a reduced ability to bind diacylglycerol and be retained on the plasma membrane. As a result, they fail to phosphorylate TRPC channels, resulting in sustained Ca²⁺ entry. Such an alteration in Ca²⁺ homeostasis and Ca²⁺-mediated signaling in Purkinje cells may contribute to the neurodegeneration characteristic of SCA14.

Cerebellar Purkinje cells (PCs) express a large amount of the γ isoform of protein kinase C (PKCγ) and a modest level of PKCα. The PKCγ is involved in the pruning of climbing fiber (CF) synapses from developing PCs, and PKCα plays a critical role in long-term depression (LTD) at parallel fiber (PF)-PC synapses. Moreover, the PKC signaling in PCs negatively modulates the nonselective transient receptor potential cation channel type 3 (TRPC3), the opening of which elicits slow EPSCs at PF-PC synapses. Autosomal dominant spinocerebellar ataxia type 14 (SCA14) is caused by mutations in PKCγ. To clarify the pathology of this disorder, mutant (S119P) PKCγ tagged with GFP was lentivirally expressed in developing and mature mouse PCsin vivo, and the effects were assessed 3 weeks after the injection. Mutant PKCγ-GFP aggregated in PCs without signs of degeneration. Electrophysiology results showed impaired pruning of CF synapses from developing PCs, failure of LTD expression, and increases in slow EPSC amplitude. We also found that mutant PKCγ colocalized with wild-type PKCγ, which suggests that mutant PKCγ acts in a dominant-negative manner on wild-type PKCγ. In contrast, PKCα did not colocalize with mutant PKCγ. The membrane residence time of PKCα after depolarization-induced translocation, however, was significantly decreased when it was present with the mutant PKCγ construct. These results suggest that mutant PKCγ in PCs of SCA14 patients could differentially impair the membrane translocation kinetics of wild-type γ and α PKCs, which would disrupt synapse pruning, synaptic plasticity, and synaptic transmission.

The functional diversity of deubiquitinating enzymes (DUBs) is not well understood. The MJD family of DUBs consists of four cysteine proteases that share a catalytic “Josephin” domain. The family is named after the DUB ATXN3, which causes the neurodegenerative disease Machado-Joseph disease. The two closely related Josephin domain-containing (JosD) proteins 1 and 2 consist of little more than the Josephin domain. To gain insight into the properties of Josephin domains, we investigated JosD1 and JosD2. JosD1 and JosD2 were found to differ fundamentally in many respects. In vitro, only JosD2 can cleave ubiquitin chains. In contrast, JosD1 cleaves ubiquitin chains only after it is monoubiquitinated, a form of posttranslational-dependent regulation shared with ATXN3. A significant fraction of JosD1 is monoubiquitinated in diverse mouse tissues. In cell-based studies, JosD2 localizes to the cytoplasm whereas JosD1 preferentially localizes to the plasma membrane, particularly when ubiquitinated. The membrane occupancy by JosD1 suggests that it could participate in membrane-dependent events such as cell motility and endocytosis. Indeed, time-lapse imaging revealed that JosD1 enhances membrane dynamics and cell motility. JosD1 also influences endocytosis in cultured cells by increasing the uptake of endocytic markers of macropinocytosis while decreasing those for clathrin- and caveolae-mediated endocytosis. Our results establish that two closely related DUBs differ markedly in activity and function and that JosD1, a membrane-associated DUB whose activity is regulated by ubiquitination, helps regulate membrane dynamics, cell motility, and endocytosis.

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease characterized by various symptoms including cerebellar ataxia. Recently, several missense mutations in the protein kinase Cγ (γPKC) gene have been found in different SCA14 families. To elucidate how the mutant γPKC causes SCA14, we examined the molecular properties of seven mutant (H101Y, G118D, S119P, S119F, Q127R, G128D, and F643L) γPKCs fused with green fluorescent protein (γPKC-GFP). Wild-type γPKC-GFP was expressed ubiquitously in the cytoplasm of CHO cells, whereas mutant γPKC-GFP tended to aggregate in the cytoplasm. The insolubility of mutant γPKC-GFP to Triton X-100 was increased and correlated with the extent of aggregation. γPKC-GFP in the Triton-insoluble fraction was rarely phosphorylated at Thr⁵¹⁴, whereas γPKC-GFP in the Triton-soluble fraction was phosphorylated. Furthermore, the stimulation of the P2Y receptor triggered the rapid aggregation of mutant γPKC-GFP within 10 min after transient translocation to the plasma membrane. Overexpression of the mutant γPKC-GFP caused cell death that was more prominent than wild type. The cytotoxicity was exacerbated in parallel with the expression level of the mutant. These results indicate that SCA14 mutations make γPKC form cytoplasmic aggregates, suggesting the involvement of this property in the etiology of SCA14.