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A New Mouse Model Related to SCA14 Carrying a Pseudosubstrate Domain Mutation in PKC? Shows Perturbed Purkinje Cell Maturation and Ataxic Motor Behavior.

ABSTRACT: 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 ? (PKC?), a crucial signaling protein in Purkinje cells. It is still unclear whether increased or decreased PKC? 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, PKC?-A24E, known to induce a constitutive PKC? activation. In this protein conformation, the kinase domain of PKC? 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 PKC? protein expression in PKC?-A24E mice of either sex. Despite this reduction, there is clear evidence for an increased PKC activity in Purkinje cells from PKC?-A24E mice. Purkinje cells derived from PKC?-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 PKC?-A24E mice showed a dysregulation of related signaling pathways, such as mGluR1 or mTOR. Our results show that the induction of PKC? 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 PKC? cause one subtype of SCAs, SCA14. In this study, we have generated a knock-in mouse with a mutation in the pseudosubstrate domain of PKC?, which keeps PKC? in the constitutive active open conformation. We show that this mutation leading to a constant activation of PKC? 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.

SUBMITTER: Shimobayashi E

PROVIDER: S-EPMC7939089 | biostudies-literature | 2021 Mar

REPOSITORIES: biostudies-literature

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A New Mouse Model Related to SCA14 Carrying a Pseudosubstrate Domain Mutation in PKCγ Shows Perturbed Purkinje Cell Maturation and Ataxic Motor Behavior.

Shimobayashi Etsuko E Kapfhammer Josef P JP

The Journal of neuroscience : the official journal of the Society for Neuroscience 20210121 9

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 ...[more]

PMID: 33478986

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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:3-Phosphoinositide-dependent protein kinase-1 (PDK1) plays a critical role in the development of mammalian brain. Here, we investigated the role of PDK1 in Purkinje cells (PCs) by generating the PDK1-conditional knock-out mice (cKO) through crossing PV-cre or Pcp2-cre mice with Pdk1fl/fl mice. The male mice were used in the behavioral testing, and the other experiments were performed on mice of both sexes. These PDK1-cKO mice displayed decreased cerebellar size and impaired motor balance and coordination. By the electrophysiological recording, we observed the reduced spontaneous firing of PCs from the cerebellar slices of the PDK1-cKO mice. Moreover, the cell body size of PCs in the PDK1-cKO mice was time dependently reduced compared with that in the control mice. And the morphologic complexity of PCs was also decreased after PDK1 deletion. These effects may have contributed to the reduction of the rpS6 (reduced ribosomal protein S6) phosphorylation and the PKCγ expression in PDK1-cKO mice since the upregulation of pS6 by treatment of 3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-1, the agonist of mTOR1, partly rescued the reduction in the cell body size of the PCs, and the delivery of recombinant adeno-associated virus-PKCγ through cerebellar injection rescued the reduced complexity of the dendritic arbor in PDK1-cKO mice. Together, our data suggest that PDK1, by regulating rpS6 phosphorylation and PKCγ expression, controls the cell body maintenance and the dendritic development in PCs and is critical for cerebellar motor coordination.SIGNIFICANCE STATEMENT Here, we show the role of 3-phosphoinositide-dependent protein kinase-1 (PDK1) in Purkinje cells (PCs). The ablation of PDK1 in PCs resulted in a reduction of cell body size, and dendritic complexity and abnormal spontaneous firing, which attributes to the motor defects in PDK1-conditional knock-out (cKO) mice. Moreover, the ribosomal protein S6 (rpS6) phosphorylation and the expression of PKCγ are downregulated after the ablation of PDK1. Additionally, upregulation of rpS6 phosphorylation by3-benzyl-5-((2-nitrophenoxy) methyl)-dihydrofuran-2(3H)-1 partly rescued the reduction in cell body size of PCs, and the overexpression of PKCγ in PDK1-KO PCs rescued the reduction in the dendritic complexity. These findings indicate that PDK1 contributes to the maintenance of the cell body and the dendritic development of PCs by regulating rpS6 phosphorylation and PKCγ expression.

Dataset Information

A New Mouse Model Related to SCA14 Carrying a Pseudosubstrate Domain Mutation in PKC? Shows Perturbed Purkinje Cell Maturation and Ataxic Motor Behavior.

Publications

A New Mouse Model Related to SCA14 Carrying a Pseudosubstrate Domain Mutation in PKCγ Shows Perturbed Purkinje Cell Maturation and Ataxic Motor Behavior.

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