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. 2018 Aug:116:93-105.
doi: 10.1016/j.nbd.2018.05.002. Epub 2018 May 11.

Reduction of protein kinase A-mediated phosphorylation of ATXN1-S776 in Purkinje cells delays onset of Ataxia in a SCA1 mouse model

Judit M Pérez Ortiz  1 , Nissa Mollema  2 , Nicholas Toker  3 , Carolyn J Adamski  4 , Brennon O'Callaghan  2 , Lisa Duvick  2 , Jillian Friedrich  2 , Michael A Walters  5 , Jessica Strasser  5 , Jon E Hawkinson  5 , Huda Y Zoghbi  4 , Christine Henzler  6 , Harry T Orr  7 , Sarita Lagalwar  8
Affiliations

Reduction of protein kinase A-mediated phosphorylation of ATXN1-S776 in Purkinje cells delays onset of Ataxia in a SCA1 mouse model

Judit M Pérez Ortiz et al. Neurobiol Dis. 2018 Aug.

Abstract

Spinocerebellar ataxia type 1 (SCA1) is a polyglutamine (polyQ) repeat neurodegenerative disease in which a primary site of pathogenesis are cerebellar Purkinje cells. In addition to polyQ expansion of ataxin-1 protein (ATXN1), phosphorylation of ATXN1 at the serine 776 residue (ATXN1-pS776) plays a significant role in protein toxicity. Utilizing a biochemical approach, pharmacological agents and cell-based assays, including SCA1 patient iPSC-derived neurons, we examine the role of Protein Kinase A (PKA) as an effector of ATXN1-S776 phosphorylation. We further examine the implications of PKA-mediated phosphorylation at ATXN1-S776 on SCA1 through genetic manipulation of the PKA catalytic subunit Cα in Pcp2-ATXN1[82Q] mice. Here we show that pharmacologic inhibition of S776 phosphorylation in transfected cells and SCA1 patient iPSC-derived neuronal cells lead to a decrease in ATXN1. In vivo, reduction of PKA-mediated ATXN1-pS776 results in enhanced degradation of ATXN1 and improved cerebellar-dependent motor performance. These results provide evidence that PKA is a biologically important kinase for ATXN1-pS776 in cerebellar Purkinje cells.

Keywords: ATXN1-S776; Ataxia; Ataxin-1; Cerebellum; PKA; Phosphorylation; Polyglutamine; Protein stability; Purkinje cells; SCA1; cAMP-dependent protein kinase.

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Conflict of interest statement

Competing interests

None declared

Figures

Figure 1
Figure 1. Rapid in situ clearance of ATXN1 following inhibition of pS776 by a small molecule kinase inhibitor
(A) DAOY cells stably-expressing RFP-ATXN1[82Q] were treated with DMSO vehicle for 0 or 6 minutes and imaged for brightfield (upper row) and RFP (lower row). (B) DAOY cells stably-expressing RFP-ATXN1[82Q] were treated with 10 nM staurosporine (STR) for 0-6 min while imaged for brightfield and RFP signals on a heated stage. (C) Graphic representation of decrease in RFP-ATXN1[82Q] over time seen in (A) and (B).
Figure 2
Figure 2. The small molecule kinase inhibitor GSK690693 inhibits Atxn1-S776 phosphorylation and promotes clearance of Atxn1 in Atxn166Q/2Q mouse cerebellar slices and in SCA1 patient iPSC-derived neuronal cells
(A) GSK690693, H89 and SB757651 reduced S776 phosphorylation of exogenous GST-ATXN1[82Q] protein in mouse cerebellar extract containing the endogenous kinase. GSK690693 (IC50 = 0.3 μM) decreased phosphorylation with greater efficiency than H89 (IC50 = 2.8 μM) or SB757651 (IC50 = 3.0 μM). (B) 10 μM GSK690693 blocks phosphorylation of PKA substrates and decreases levels of Atxn1[66Q] and Atxn1[2Q] in cultured cerebellar slices from Atxn166Q/2Q mice. N = 5 mice/condition. (C) GSK690693 at 10 μM decreases endogenous human ATXN1 in cultured SCA1 patient iPSC-derived neuronal cells. N is indicated within bars. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p <0.0001.
Figure 3
Figure 3. Decreased ATXN1-S776 cerebellar phosphorylation induced by a M120A mutation in Prkaca
(A) Outline of the strategy used to create the CαM120 mouse. CαLoxM120A mice were obtained, crossed to Sox2Cre mice (Jackson laboratories), and bred to homozygosity. (B-E) CαM120A/M120A mice crossed to Pcp2-ATXN1[30Q];Atxn1−/− mice. (C) Western blot of relevant proteins in a cerebellar extract of 4-6 week old ATXN1[30Q];Atxn1−/− ;CαM120A/M120A mice. (D) Quantification of the results in (C). (E) Western blot of ATXN1[30Q];Atxn1−/− and ATXN1[30Q];Atxn1−/−;CαM120A/M120A -cerebellar extracts for PKA phospho-substrates as revealed using a pan-PKA-phospho-specific antibody. (F-I) CαM120A/M120A mice crossed to Pcp2-ATXN1[82Q];Atxn12Q/2Q mice. (G) Western blot of relevant proteins in a cerebellar extract of 4-5 week old ATXN1[82Q];CαM120A/M120A mice. (H) Quantification of the results in (G). (I) Western blot of ATXN1[82Q] and ATXN1[82Q];CαM120A/M120A cerebellar extracts for PKA phospho-substrates as revealed using a pan-PKA-phospho-specific antibody. Biological replicates (n) are indicated in bars (D and H). Data are represented as mean, ± SEM. Student’s t-test. * p < 0.05, ** p < 0.01, *** p < 0.001, p < 0.0001.
Figure 4
Figure 4. PKA CαM120A reduction delays onset of cerebellar motor deficits in ATXN[82Q];CαM120A/M120A mice
(A) Balance beam assessment of 6-7 week old wt/FVB, ATXN1[82]/+ and ATXN[82Q];CαM120A/M120A mice on a 10 mm round beam and 8 mm square beam. (B). Rotarod assessment of wt/FVB, ATXN1[82] and ATXN[82Q];CαM120A/M120A mice at 7-8 weeks. Data are represented as mean, ± SEM. Two-way ANOVA. n=10 per group. * p < 0.05, ** p < 0.01.
Figure 5
Figure 5. WGCNA Pink-ataxia gene network
(A) Pink Module eigengene changes in wt/FVB, ATXN1[82]/+ and ATXN1[82Q];CαM120A/M120A mice at 8-9 wks of age. (B) Cytoscape plot of Pink module Eigengene illustrates genes with highest connectivity (hubs) within the Pink-ataxia network. The color of the circles represents the log2 fold change; genes that are red are up in ATXN[82Q]/+ (with ataxia) relative to wt/FVB (without ataxia), and those that are green are down in ATXN[82Q]/+ relative to wt/FVB. The size of the circles represents the absolute value of the log2 fold change. The thickness of the line around each circle represents the log10 of the adjusted p-value (a thick border line means highly significant). The top 10 genes with the highest intramodular connectivity are listed in the table. Module connectivity measures the sum of the connections of a gene to the other genes in the module, whereby higher connectivity indicates higher correlation with every other gene. (C) IPA canonical pathway analysis of the WGCNA Pink-ataxia module is notable for G protein-coupled signaling pathways of the Gq type.
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