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 4

PKCγ and PKCα are gene dose–dependently reduced in SCA14 mice.

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PKCγ and PKCα are gene dose–dependently reduced in SCA14 mice. Western b...
Western blot analysis of whole cerebellar homogenate from all genotypes (WT, yellow; HET, red; HOM, purple) and sexes (n = 8–10 mice per group) was performed to assess cerebellar levels of (A) PKCγ [female (1-way ANOVA, F(2,26) = 31.14, P = 1.26 × 10–7; Tukey’s post hoc test, WT vs. HET P = 0.0031, WT vs. HOM P < 0.00001, HET vs. HOM P = 0.00057), male (1-way ANOVA, F(2,24) = 101.20, P = 2.02 × 10–12; Tukey’s post hoc test, WT vs. HET P = 0.00057, WT vs. HOM P < 0.00001, HET vs. HOM P < 0.00001)], and (D) PKCα [female (1-way ANOVA, F(2,25) = 0.76, P = 0.48), male (1-way ANOVA, F(2,26) = 4.4, P = 0.022; Tukey’s post hoc test, WT vs. HET P = 0.82, WT vs. HOM P = 0.023, HET vs. HOM P = 0.081)]. Fluorescence immunohistochemistry was performed on fixed sagittal brain sections from all genotypes (WT, HET, HOM) and sexes (n = 3–6 mice). (B) Representative images show intensity and localization of PKCγ in whole cerebellum (scale bars: 1 mm, respective color scale indicates relative intensity). Since staining is multiplexed, and representative images are shown from the same sections as Figure 3 but in different channels (see Supplemental Figure 4 for examples of independent and merged images). (C) Quantification of the PCL and ML (n = 3–6 mice) measured the intensity of localized PKCγ [female (1-way ANOVA, F(2,10) = 11, P = 0.0033; Tukey’s post hoc test, WT vs. HET P = 0.18, WT vs. HOM P = 0.0025, HET vs. HOM P = 0.036), male (1-way ANOVA, F(2,12) = 20, P = 0.00015; Tukey’s post hoc test, WT vs. HET P = 0.044, WT vs. HOM P = 0.00010, HET vs. HOM P = 0.0092)]. (E) Representative images show intensity and localization of PKCα in whole cerebellum (scale bars: 1 mm, respective color scale indicates relative intensity). (F) Quantification of the PCL and ML (n = 3–6 mice) measured the intensity of localized PKCα. Bar graphs show mean ± SEM normalized to WT. Significance was determined by 1-way ANOVA with Tukey’s post hoc test. *P < 0.05; **P < 0.01; ***P < 0.001; ***P < 0.0001.