Mutant PKCγ in Spinocerebellar Ataxia Type 14 Disrupts Synapse Elimination and Long-Term Depression in Purkinje CellsIn Vivo

Abstract

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

Transient Receptor Potential Channels as Drug Targets: From the Science of Basic Research to the Art of Medicine

“…Mutant PKCg in cerebellar Purkinje cells of patients with spinocerebellar ataxia type 14 could differentially impair TRPC3 function and disrupt synapse pruning, synaptic plasticity, and synaptic transmission (Shuvaev et al, 2011). Interestingly, a gain-of-function mutant in TRPC3 causes cerebellar ataxia in mice, the so-called Moonwalker (Mwk) mouse (Becker et al, 2009).…”

Increased protein kinase C gamma activity induces Purkinje cell pathology in a mouse model of spinocerebellar ataxia 14

“…It is interesting to note that other point mutations causing SCA often affect molecules in pathways related to the PKC pathway, in particular pathways which are involved in Ca 2+ -homeostasis such as IP3 receptor ITPR1 (Kasumu et al, 2012) or Ca 2 + -and K + -ion channels (reviewed by Schorge et al, 2010). It is also interesting to note that dendritic abnormalities and changes in the afferent innervation appear to be a common motif in many types of SCA (Duvick et al, 2010;Konno et al, 2013;Shuvaev et al, 2011;van de Leemput et al, 2007). In staggerer mutant mice which have a major loss of Purkinje cells which is related to the Purkinje cell loss in SCA1 (Serra et al, 2006) one major abnormality is a nearly complete loss of metabotropic glutamate receptor 1 (mGluR1) signaling (Mitsumura et al, 2011).…”

“…Nevertheless, there are also indications that despite an increased basal activity of mutated PKCγ, there might be a deficit to recruit downstream targets resulting in a certain loss of function (Verbeek et al, 2008). After viral transfection of a mutated form of PKCγ in vivo LTD was shown to be impaired and there were indications that the mutant PKCγ may interfere with normal endogenous PKC activity in a dominant negative manner (Shuvaev et al, 2011). Furthermore, another typical feature of mutated PKCγ proteins is an increased protein aggregation which may cause Purkinje cell death by ER-stress (Seki et al, 2005(Seki et al, , 2007.…”

Type-1 metabotropic glutamate receptor signaling in cerebellar Purkinje cells in health and disease

“…This mouse line has a threonine to alanine switch in TRPC3 that allows the cation channel to open under conditions of weaker mGluR1 activation 87 . On the other hand, a mouse model of SCA14 has larger mGluR1-mediated inward currents in Purkinje cells than do normal mice because of the failure to inactivate TRPC3 by mutant PKCγ 88 ( Table 2). These results suggest that increased Na + and Ca 2+ influx through TRPC3 channels disrupts normal functions of Purkinje cells and other cerebellar neurons, which causes ataxia.…”

“…Judging from the severe cerebellar dysfunctions of mGluR1-knockout mice, it is conceivable that mGluR1 loss-of-function underlies human ataxias 74– 78, 80, 82, 83 . However, it is important to note that mGluR1 gain-of-function has been reported in certain mouse models of human ataxias 84– 88 . Calcium overload to Purkinje cells due to excess mGluR1-mediated Ca 2+ release and/or TRPC3 channel activation is likely to cause cerebellar dysfunction.…”