Abstract
The cerebellum, the site where protein kinase C (PKC) was first discovered, contains the highest amount of PKC in the central nervous system, with PKCγ being the major isoform. Systemic PKCγ-knockout (KO) mice showed impaired motor coordination and deficient pruning of surplus climbing fibers (CFs) from developing cerebellar Purkinje cells (PCs). However, the physiological significance of PKCγ in the mature cerebellum and the cause of motor incoordination remain unknown. Using adeno-associated virus vectors targeting PCs, we showed that impaired motor coordination was restored by re-expression of PKCγ in mature PKCγ-KO mouse PCs in a kinase activity-dependent manner, while normal motor coordination in mature Prkcgfl/fl mice was impaired by the Cre-dependent removal of PKCγ from PCs. Notably, the rescue or removal of PKCγ from mature PKCγ-KO or Prkcgfl/fl mice, respectively, did not affect the CF innervation profile of PCs, suggesting the presence of a mechanism distinct from multiple CF innervation of PCs for the motor defects in PKCγ-deficient mice. We found marked potentiation of Ca2+-activated large-conductance K+ (BK) channel currents in PKCγ-deficient mice, as compared to wild-type mice, which decreased the membrane resistance, resulting in attenuation of the electrical signal during the propagation and significant alterations of the complex spike waveform. These changes in PKCγ-deficient mice were restored by the rescue of PKCγ or pharmacological suppression of BK channels. Our results suggest that PKCγ is a critical regulator that negatively modulates BK currents in PCs, which significantly influences PC output from the cerebellar cortex and, eventually, motor coordination.
Highlights
Protein kinase C (PKC) was originally identified by the late Yasutomi Nishizuka [1, 2], known as the father of protein kinase C (PKC) [3]
Adeno-associated virus serotype 9 (AAV9) vectors expressing enhanced green fluorescent protein (GFP)-porcine teschovirus-1–derived 2A peptide (P2A)PKCγ under the control of a Purkinje cells (PCs)-specific L7-4 promoter fused with minimal cytomegalovirus promoter sequence (L7-4-minCMV promoter) [16] with two different viral titers (1.6 × 109 or 1.6 × 1010 viral genome/mouse) were injected into the cerebellar cortex of postnatal day (P) 21 to 25 PKCγ-KO mice (Fig. 1 A and B)
In this study, using systemic and conditional PKCγ-deficient mice, we sought to clarify the physiological significance of PKCγ, a major isotype of classical PKCs (cPKCs), which is abundantly expressed throughout the dendrites and soma of PCs [8, 10]
Summary
Protein kinase C (PKC) was originally identified by the late Yasutomi Nishizuka [1, 2], known as the father of PKC [3]. In the 1970s, Nishizuka’s group used the bovine cerebellum to study the properties of cyclic guanosine monophosphate (cGMP)–dependent protein kinase. During this experiment, they identified a novel cyclic nucleotide–independent kinase [4], which required an unphysiologically high concentration of Mg2+ (50 to 100 mM), and it was initially named protein kinase M (PKM) [4]. Some genemodified mice were later discovered which showed overall normal motor coordination with persistent multiple CF innervation of PCs [14, 15]. Our results suggest that synaptically activated PKCγ in PCs plays a critical role in motor coordination by negative modulation of BK currents. M.K. is a guest editor invited by the Editorial Board
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