Mutant PKCγ in Spinocerebellar Ataxia Type 14 Disrupts Synapse Elimination and Long-Term Depression in Purkinje Cells In Vivo
Mutant PKCγ in Spinocerebellar Ataxia Type 14 Disrupts Synapse Elimination and Long-Term Depression in Purkinje Cells In Vivo
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DOI：
10.1523/jneurosci.5530-10.2011
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发表时间：
2011-10
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影响因子：
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通讯作者：
A. Shuvaev;Hajime Horiuchi;T. Seki;H. Goenawan;T. Irie;Akira Iizuka;N. Sakai;H. Hirai
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作者：
A. Shuvaev;Hajime Horiuchi;T. Seki;H. Goenawan;T. Irie;Akira Iizuka;N. Sakai;H. Hirai
小脑浦肯野细胞（PCs）大量表达蛋白激酶C的γ亚型（PKCγ），并适度表达PKCα。PKCγ参与发育中的浦肯野细胞上攀缘纤维（CF）突触的修剪，而PKCα在平行纤维（PF） - 浦肯野细胞突触的长时程抑制（LTD）中起关键作用。此外，浦肯野细胞中的PKC信号传导对非选择性瞬时受体电位阳离子通道3型（TRPC3）产生负向调节作用，该通道的开放会在PF - 浦肯野细胞突触处引发缓慢的兴奋性突触后电流（EPSC）。常染色体显性遗传性脊髓小脑共济失调14型（SCA14）是由PKCγ突变引起的。为了阐明这种疾病的病理机制，我们将带有绿色荧光蛋白（GFP）标记的突变型（S119P）PKCγ通过慢病毒在体内表达于发育中和成熟的小鼠浦肯野细胞中，并在注射3周后评估其效果。突变型PKCγ - GFP在浦肯野细胞中聚集，但没有退化的迹象。电生理结果显示，发育中的浦肯野细胞上CF突触修剪受损，LTD表达失败，缓慢EPSC幅度增加。我们还发现突变型PKCγ与野生型PKCγ共定位，这表明突变型PKCγ对野生型PKCγ起显性负性作用。相比之下，PKCα不与突变型PKCγ共定位。然而，当存在突变型PKCγ构建体时，去极化诱导转位后PKCα在细胞膜上的停留时间显著减少。这些结果表明，SCA14患者浦肯野细胞中的突变型PKCγ可能差异性地损害野生型γ和α PKC的膜转位动力学，这将破坏突触修剪、突触可塑性和突触传递。
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 PCs in 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.