Sex-specific disruptions in PKCγ signaling in a mouse model of spinocerebellar ataxia type 14
Sarah A. Wolfe, Yuliang Ma, Tomer M. Yaron-Barir, Carly Chang, Caila A. Pilo, Majid Ghassemian, Amanda J. Roberts, Sang Ryeul Lee, Benjamin A. Henson, Kristen Jepsen, Jared L. Johnson, Lewis C. Cantley, Susan S. Taylor, George Gorrie, Alexandra C. Newton
Sarah A. Wolfe, Yuliang Ma, Tomer M. Yaron-Barir, Carly Chang, Caila A. Pilo, Majid Ghassemian, Amanda J. Roberts, Sang Ryeul Lee, Benjamin A. Henson, Kristen Jepsen, Jared L. Johnson, Lewis C. Cantley, Susan S. Taylor, George Gorrie, Alexandra C. Newton
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Research Article Cell biology Neuroscience

Sex-specific disruptions in PKCγ signaling in a mouse model of spinocerebellar ataxia type 14

Abstract

Spinocerebellar ataxia type 14 (SCA14) is an autosomal dominant neurodegenerative disease caused by mutations in the gene encoding protein kinase C γ (PKCγ), a Ca2+- and diacylglycerol-dependent Ser/Thr kinase dominantly expressed in cerebellar Purkinje cells. These mutations impair autoinhibitory constraints to increase the basal activity of the kinase, resulting in deficits in the cerebellum that are not observed upon simple deletion of the gene, and severe ataxia. To better understand the impact of aberrant PKCγ signaling in disease pathology, we developed a knockin murine model of the SCA14 mutation ΔF48 in PKCγ. This fully penetrant mutation is severe in humans and is mechanistically informative, as it has high basal activity but is unresponsive to agonist stimulation. Genetic, behavioral, and molecular testing revealed that ΔF48 PKCγ mice have ataxia-related phenotypes and an altered cerebellar phosphoproteome driven primarily by enhanced Ca2+/calmodulin-dependent kinase 2 signaling, effects that were more severe in male mice. Analysis of existing human data revealed that SCA14 has a significantly earlier age of onset for males compared with females. Data from this clinically relevant mutation suggested that enhanced basal activity of PKCγ is sufficient to cause ataxia and that treatment strategies to modulate aberrant PKCγ may be particularly beneficial in males.

Authors

Sarah A. Wolfe, Yuliang Ma, Tomer M. Yaron-Barir, Carly Chang, Caila A. Pilo, Majid Ghassemian, Amanda J. Roberts, Sang Ryeul Lee, Benjamin A. Henson, Kristen Jepsen, Jared L. Johnson, Lewis C. Cantley, Susan S. Taylor, George Gorrie, Alexandra C. Newton

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Figure 6

ΔF48 PKCγ rewires the cerebellar phosphoproteome in SCA14 mice.

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ΔF48 PKCγ rewires the cerebellar phosphoproteome in SCA14 mice. Phosphop...
Phosphoproteomic analysis was carried out on protein extracted from whole cerebellar homogenate from all genotypes (WT, yellow; HET, red; HOM, purple) and sexes (n = 3 mice per group). (A) An upset plot summarizes the quantity of significantly different phosphopeptides and shared phosphopeptides identified between groups (P < 0.05). (B) Western blot analysis was performed on whole cerebellar homogenate (n = 8–10 mice per group) to assess MARCKS phosphorylation (Ser159/Ser163) [female (1-way ANOVA, F(2,23) = 2.4, P = 0.11; Tukey’s post hoc test, WT vs. HET P = 0.62, WT vs. HOM P = 0.48, HET vs. HOM P = 0.094), male (1-way ANOVA, F(2,26) = 3.1, P = 0.063; Tukey’s post hoc test, WT vs. HET P = 0.58, WT vs. HOM P = 0.051, HET vs. HOM P = 0.31)]. Bar graphs represent mean ± SEM. Significance determined by 1-way ANOVA with Tukey’s post hoc test. Volcano plots show log-transformed P values versus the log-transformed fold change quantified per change in phosphopeptide abundance in WT compared to (C) HET and (D) HOM in female mice. Color represents phosphopeptides with P value < 0.05. Dot plots indicate the top 10 most significantly enriched for gene ontologies from the differentially abundant phosphopeptides identified in (E) WT vs. HET, (F) WT vs. HOM, and (G) similarly altered phosphopeptides (defined as phosphopeptides significantly altered in HOM vs. WT with a corresponding trend, or significant change, in HET vs. WT) in female mice. Similarly altered phosphopeptides are shown in heatmaps by those that (H) increase or (I) decrease with increasing ΔF48 alleles in females. Dot plot color scale indicates P value, and dot size indicates number of genes per ontology (P < 0.05). Heatmap color scale indicates normalized phosphopeptides intensity, with values centered and scaled by row. Protein names accompanied by a number designate multiple phosphorylation sites for the same protein.