Neurodegeneration in SCA14 is associated with increased PKC\u03b3 kinase activity, mislocalization and aggregation

Maggie M K Wong,Jane Vowles,Lauren M Watson,Andrea H Németh,Esther B E Becker,Geraint Fuller,Stephanie D Hoekstra,Sally A Cowley,Olaf Ansorge,Kevin Talbot

doi:10.1186/s40478-018-0600-7

Abstract

Spinocerebellar ataxia type 14 (SCA14) is a subtype of the autosomal dominant cerebellar ataxias that is characterized by slowly progressive cerebellar dysfunction and neurodegeneration. SCA14 is caused by mutations in the PRKCG gene, encoding protein kinase C gamma (PKCγ). Despite the identification of 40 distinct disease-causing mutations in PRKCG, the pathological mechanisms underlying SCA14 remain poorly understood. Here we report the molecular neuropathology of SCA14 in post-mortem cerebellum and in human patient-derived induced pluripotent stem cells (iPSCs) carrying two distinct SCA14 mutations in the C1 domain of PKCγ, H36R and H101Q. We show that endogenous expression of these mutations results in the cytoplasmic mislocalization and aggregation of PKCγ in both patient iPSCs and cerebellum. PKCγ aggregates were not efficiently targeted for degradation. Moreover, mutant PKCγ was found to be hyper-activated, resulting in increased substrate phosphorylation. Together, our findings demonstrate that a combination of both, loss-of-function and gain-of-function mechanisms are likely to underlie the pathogenesis of SCA14, caused by mutations in the C1 domain of PKCγ. Importantly, SCA14 patient iPSCs were found to accurately recapitulate pathological features observed in post-mortem SCA14 cerebellum, underscoring their potential as relevant disease models and their promise as future drug discovery tools.

Highlights

Spinocerebellar ataxia type 14 (SCA14) (OMIM 605361) most commonly represents with slowly progressive, relatively pure cerebellar ataxia characterized by gait disturbance, incoordination, mild dysarthria and nystagmus, with complex phenotypes such as myoclonus described in over a third of cases [9, 12]
We demonstrate that SCA14 patient induced pluripotent stem cells (iPSCs), in which Protein kinase C gamma (PKCγ) is expressed at levels more likely to be relevant to normal physiology compared with previous in vitro models, recapitulate pathological features observed in post-mortem SCA14 cerebellum
Reprogramming of donor fibroblasts to iPSCs was performed as described in the Additional file 1, and full characterization is provided in Additional file 1: Figure S2. iPSC lines from two age- and sex-matched healthy donors were used as controls (Additional file 1: Figure S2) and have been fully described elsewhere [16, 18]. iPSCs were maintained in feeder-free conditions on hESC-qualified Matrigel (Corning), in supplemented mTeSR (Stem Cell Technologies)

Summary

Introduction

Spinocerebellar ataxia type 14 (SCA14) (OMIM 605361) most commonly represents with slowly progressive, relatively pure cerebellar ataxia characterized by gait disturbance, incoordination, mild dysarthria and nystagmus, with complex phenotypes such as myoclonus described in over a third of cases [9, 12]. Brain MRI in SCA14 patients shows mild to severe cerebellar atrophy [9, 12], and loss of Purkinje cells has been described at post-mortem [7]. SCA14 is caused by mutations in the PRKCG gene encoding the conventional protein kinase C gamma (PKCγ), which is abundant in the Purkinje cells of the. Homozygous Prkcg knockout animals display only mild ataxia and show no loss of Purkinje cells [10, 22]. Overexpression studies in cell lines and animals have yielded conflicting cellular disease mechanisms including increased kinase function [1, 43], impaired kinase function [42], protein aggregation [36] and impaired ubiquitin proteasome degradation [38], as well as aggregation-independent pathologies [37]. There is a need for authentic SCA14 models to better understand the underlying disease mechanisms

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Conclusion