Cerebellar Purkinje cells (PCs) express two members of the classical protein kinase C (cPKC) subfamily, namely, PKCα and PKCγ. Previous studies on PKCγ knockout (KO) mice have revealed a critical role of PKCγ in the pruning of climbing fibers (CFs) from PCs during development. The question remains as to why only PKCγ and not PKCα is involved in CF synapse elimination from PCs. To address this question, we assessed the expression levels of PKCγ and PKCα in wild-type (WT) and PKCγ KO PCs using PC-specific quantitative real-time reverse transcription-polymerase chain reaction, western blotting, and immunohistochemical analysis. The results revealed that the vast majority of cPKCs in PCs were PKCγ, whereas PKCα accounted for the remaining minimal fraction. The amount of PKCα was not up-regulated in PKCγ KO PCs. Lentiviral expression of PKCα in PKCγ KO PCs resulted in a 10-times increase in the amount of PKCα mRNA in the PKCγ KO PCs, compared to that in WT PCs. Our quantification showed that the expression levels of cPKC mRNA in PKCγ KO PCs increased roughly from 1% to 22% of that in WT PCs solely through PKCα expression. The up-regulation of PKCα in PKCγ KO PCs significantly rescued the impaired CF synapse elimination. Although both PKCα and PKCγ are capable of pruning supernumerary CF synapses from developing PCs, these results suggest that the expression levels of cPKCs in PKCγ KO PCs are too low for CF pruning.
Keywords: cerebellum; climbing fiber; lentivirus; protein kinase C; purkinje cell; reverse transcription-polymerase chain reaction.
© 2017 International Society for Neurochemistry.