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Neurodegeneration in SCA14 is associated with increased PKC? kinase activity, mislocalization and aggregation.

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 (PKC?). 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 PKC?, H36R and H101Q. We show that endogenous expression of these mutations results in the cytoplasmic mislocalization and aggregation of PKC? in both patient iPSCs and cerebellum. PKC? aggregates were not efficiently targeted for degradation. Moreover, mutant PKC? 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 PKC?. 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.

SUBMITTER: Wong MMK

PROVIDER: S-EPMC6151931 | biostudies-literature | 2018 Sep

REPOSITORIES: biostudies-literature

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Neurodegeneration in SCA14 is associated with increased PKCγ kinase activity, mislocalization and aggregation.

Wong Maggie M K MMK Hoekstra Stephanie D SD Vowles Jane J Watson Lauren M LM Fuller Geraint G Németh Andrea H AH Cowley Sally A SA Ansorge Olaf O Talbot Kevin K Becker Esther B E EBE

Acta neuropathologica communications 20180924 1

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 cerebell ...[more]

PMID: 30249303

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Project description:Spinocerebellar ataxia type 14 (SCA14) is an autosomal neurodegenerative disease clinically characterized by progressive ataxia in the patient's gait, accompanied by slurred speech and abnormal eye movements. These symptoms are linked to the loss of Purkinje cells (PCs), which leads to cerebellar neurodegeneration. PC observations link the mutations in PRKCG gene encoding protein kinase C γ (PKCγ) to SCA14. Observations also show that the link between PKCγ and SCA14 relies on a gain-of-function mechanism, and, in fact, both positive and negative regulation of PKCγ expression and activity may result in changes in cellular number, size, and complexity of the dendritic arbors in PCs. Here, through a systems biology approach, we investigate a key question relating to this system: why is PKCγ membrane residence time reduced in SCA14 mutant PCs compared to wild-type (WT) PCs? In this study, we investigate this question through two contrasting PKCγ signaling models in PCs. The first model proposed in this study describes the mechanism through which PKCγ signaling activity may be regulated in WT PCs. In contrast, the second model explores how mutations in PKCγ signaling affect the state of SCA14 in PCs. Numerical simulations of both models show that, in response to extracellular stimuli-induced depolarization of the membrane compartment, PKCγ and diacylglycerol kinase γ (DGKγ) translocate to the membrane. Results from our computational approach indicate that, for the same set of parameters, PKCγ membrane residence time is shorter in the SCA14 mutant model compared to the WT model. These results show how PKCγ membrane residence time is regulated by diacylglycerol (DAG), causing translocated PKCγ to return to the cytosol as DAG levels drop. This study shows that, when the strength of the extracellular signal is held constant, the membrane lifetime of mutant PKCγ is reduced. This reduction is due to the presence of constitutively active mutant PKCγ in the cytosol. Cytosolic PKCγ, in turn, leads to phosphorylation and activation of DGKγ while it is still residing in the cytosol. This effect occurs even during the resting conditions. Thus, the SCA14 mutant model explains that, when both DAG effector molecules are active in the cytosol, their interactions in the membrane compartment are reduced, critically influencing PKCγ membrane residence time.

Project description:Spinocerebellar ataxias (SCAs) are diseases characterized by cerebellar atrophy and loss of Purkinje neurons caused by mutations in diverse genes. In SCA14, the disease is caused by point mutations or small deletions in protein kinase C γ (PKCγ), a crucial signaling protein in Purkinje cells. It is still unclear whether increased or decreased PKCγ activity may be involved in the SCA14 pathogenesis. In this study, we present a new knock-in mouse model related to SCA14 with a point mutation in the pseudosubstrate domain, PKCγ-A24E, known to induce a constitutive PKCγ activation. In this protein conformation, the kinase domain of PKCγ is activated, but at the same time the protein is subject to dephosphorylation and protein degradation. As a result, we find a dramatic reduction of PKCγ protein expression in PKCγ-A24E mice of either sex. Despite this reduction, there is clear evidence for an increased PKC activity in Purkinje cells from PKCγ-A24E mice. Purkinje cells derived from PKCγ-A24E have short thickened dendrites typical for PKC activation. These mice also develop a marked ataxia and signs of Purkinje cell dysfunction making them an interesting new mouse model related to SCA. Recently, a similar mutation in a human patient was discovered and found to be associated with overt SCA14. RNA profiling of PKCγ-A24E mice showed a dysregulation of related signaling pathways, such as mGluR1 or mTOR. Our results show that the induction of PKCγ activation in Purkinje cells results in the SCA-like phenotype indicating PKC activation as one pathogenetic avenue leading to a SCA.SIGNIFICANCE STATEMENT Spinocerebellar ataxias (SCAs) are hereditary diseases affecting cerebellar Purkinje cells and are a one of neurodegenerative diseases. While mutation in several genes have been identified as causing SCAs, it is unclear how these mutations cause the disease phenotype. Mutations in PKCγ cause one subtype of SCAs, SCA14. In this study, we have generated a knock-in mouse with a mutation in the pseudosubstrate domain of PKCγ, which keeps PKCγ in the constitutive active open conformation. We show that this mutation leading to a constant activation of PKCγ results in a SCA-like phenotype in these mice. Our findings establish the constant activation of PKC signaling as one pathogenetic avenue leading to an SCA phenotype and a mechanism causing a neurodegenerative disease.

Project description:Neurodegeneration with brain iron accumulation is a broad term that describes a heterogeneous group of progressive and invalidating neurologic disorders in which iron deposits in certain brain areas, mainly the basal ganglia. The predominant clinical symptoms include spasticity, progressive dystonia, Parkinson's disease-like symptoms, neuropsychiatric alterations, and retinal degeneration. Among the neurodegeneration with brain iron accumulation disorders, the most frequent subtype is pantothenate kinase-associated neurodegeneration (PKAN) caused by defects in the gene encoding the enzyme pantothenate kinase 2 (PANK2) which catalyzed the first reaction of the coenzyme A biosynthesis pathway. Currently there is no effective treatment to prevent the inexorable course of these disorders. The aim of this review is to open up a discussion on the utility of using cellular models derived from patients as a valuable tool for the development of precision medicine in PKAN. Recently, we have described that dermal fibroblasts obtained from PKAN patients can manifest the main pathological changes of the disease such as intracellular iron accumulation accompanied by large amounts of lipofuscin granules, mitochondrial dysfunction and a pronounced increase of markers of oxidative stress. In addition, PKAN fibroblasts showed a morphological senescence-like phenotype. Interestingly, pantothenate supplementation, the substrate of the PANK2 enzyme, corrected all pathophysiological alterations in responder PKAN fibroblasts with low/residual PANK2 enzyme expression. However, pantothenate treatment had no favourable effect on PKAN fibroblasts harbouring mutations associated with the expression of a truncated/incomplete protein. The correction of pathological alterations by pantothenate in individual mutations was also verified in induced neurons obtained by direct reprograming of PKAN fibroblasts. Our observations indicate that pantothenate supplementation can increase/stabilize the expression levels of PANK2 in specific mutations. Fibroblasts and induced neurons derived from patients can provide a useful tool for recognizing PKAN patients who can respond to pantothenate treatment. The presence of low but significant PANK2 expression which can be increased in particular mutations gives valuable information which can support the treatment with high dose of pantothenate. The evaluation of personalized treatments in vitro of fibroblasts and neuronal cells derived from PKAN patients with a wide range of pharmacological options currently available, and monitoring its effect on the pathophysiological changes, can help for a better therapeutic strategy. In addition, these cell models will be also useful for testing the efficacy of new therapeutic options developed in the future.

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Neurodegeneration in SCA14 is associated with increased PKC? kinase activity, mislocalization and aggregation.

Publications

Neurodegeneration in SCA14 is associated with increased PKCγ kinase activity, mislocalization and aggregation.

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