Mutant PKCgamma in spinocerebellar ataxia type 14 disrupts synapse elimination and long-term depression in Purkinje cells in vivo.

摘要

Cerebellar Purkinje cells (PCs) express a large amount of the gamma isoform of protein kinase C (PKCgamma) and a modest level of PKCalpha. The PKCgamma is involved in the pruning of climbing fiber (CF) synapses from developing PCs, and PKCalpha 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 PKCgamma. To clarify the pathology of this disorder, mutant (S119P) PKCgamma tagged with GFP was lentivirally expressed in developing and mature mouse PCs in vivo, and the effects were assessed 3 weeks after the injection. Mutant PKCgamma-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 PKCgamma colocalized with wild-type PKCgamma, which suggests that mutant PKCgamma acts in a dominant-negative manner on wild-type PKCgamma. In contrast, PKCalpha did not colocalize with mutant PKCgamma. The membrane residence time of PKCalpha after depolarization-induced translocation, however, was significantly decreased when it was present with the mutant PKCgamma construct. These results suggest that mutant PKCgamma in PCs of SCA14 patients could differentially impair the membrane translocation kinetics of wild-type gamma and alpha PKCs, which would disrupt synapse pruning, synaptic plasticity, and synaptic transmission.