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. 2013 Jun 20;498(7454):325-331.
doi: 10.1038/nature12204. Epub 2013 May 29.

RAS-MAPK-MSK1 pathway modulates ataxin 1 protein levels and toxicity in SCA1

Jeehye Park #  1   2   3 , Ismael Al-Ramahi #  1   2 , Qiumin Tan  1   2   3 , Nissa Mollema  4   5 , Javier R Diaz-Garcia  1   2 , Tatiana Gallego-Flores  1   2 , Hsiang-Chih Lu  2   6 , Sarita Lagalwar  4   5 , Lisa Duvick  4   5 , Hyojin Kang  1   2 , Yoontae Lee  1   2   3 , Paymaan Jafar-Nejad  1   2 , Layal S Sayegh  1   2 , Ronald Richman  1   2   3 , Xiuyun Liu  1   2   3 , Yan Gao  1   2 , Chad A Shaw  1 , J Simon C Arthur  7 , Harry T Orr  4   5 , Thomas F Westbrook  1   6   8 , Juan Botas  1   2 , Huda Y Zoghbi  1   2   3   6
Affiliations

RAS-MAPK-MSK1 pathway modulates ataxin 1 protein levels and toxicity in SCA1

Jeehye Park et al. Nature. .

Abstract

Many neurodegenerative disorders, such as Alzheimer's, Parkinson's and polyglutamine diseases, share a common pathogenic mechanism: the abnormal accumulation of disease-causing proteins, due to either the mutant protein's resistance to degradation or overexpression of the wild-type protein. We have developed a strategy to identify therapeutic entry points for such neurodegenerative disorders by screening for genetic networks that influence the levels of disease-driving proteins. We applied this approach, which integrates parallel cell-based and Drosophila genetic screens, to spinocerebellar ataxia type 1 (SCA1), a disease caused by expansion of a polyglutamine tract in ataxin 1 (ATXN1). Our approach revealed that downregulation of several components of the RAS-MAPK-MSK1 pathway decreases ATXN1 levels and suppresses neurodegeneration in Drosophila and mice. Importantly, pharmacological inhibitors of components of this pathway also decrease ATXN1 levels, suggesting that these components represent new therapeutic targets in mitigating SCA1. Collectively, these data reveal new therapeutic entry points for SCA1 and provide a proof-of-principle for tackling other classes of intractable neurodegenerative diseases.

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Figures

Figure 1
Figure 1. Integrative genetic screen identifies regulators of ATXN1(82Q) stability
a, Forward genetic screen strategy for modifiers of ATXN1(82Q) levels. mRFP–ATXN1(82Q)–IRES– YFP-expressing cells are used to assess the abundance of ATXN1(82Q) by monitoring the mRFP–ATXN1(82Q) to YFP fluorescence ratio. siRNAs that reduce ATXN1(82Q) protein lead to a decrease in mRFP–ATXN1(82Q) but not YFP. b, Primary screen result shows the effect on the average mRFP–ATXN1(82Q)/YFP ratio per kinase siRNAs tested. c, Schematic of the Drosophila screen for suppressors of ATXN1(82Q). Suppressor example is lic (homologue of human MEK3). d, Venn diagram of the identified gene candidates.
Figure 2
Figure 2. Modifiers shared between cell-based and in vivo screens
a, Histograms show the distribution of mRFP–ATXN1(82Q)/YFP ratio abundance in cells treated with indicated siRNA compared to control (n, number). b, Average mRFP–ATXN1(82Q)/YFP ratio change upon treatment with siRNAs hits (error bars: s.e.m. from triplicates, P < 0.05). c, Scanning electron microscopy (SEM) images of ATXN1(82Q) suppressors in Drosophila caused by reduced level of candidate genes in b. LOF, loss of function. DN, dominant negative. Names of human (h) homologues are noted in parentheses. d, Decreased levels of Drosophila MSK1, ERK and MEK homologues suppress motor impairment and improve survival. Error bars, s.e.m. e, Pathway analysis showing eight of the ten modifiers connected to MAPK pathway. Diamonds represent modifiers common to both screens.
Figure 3
Figure 3. Upstream MAPK pathway components regulate ATXN1 toxicity and levels
a, Decreased levels of Drosophila homologues of the MAPK upstream components suppress ATXN1(82Q)-induced eye degeneration. b, Decreased levels in the Drosophila homologues of FNTA, SOS, HRAS and RAF suppress ATXN1(82Q)-induced motor impairments. c, Change in mRFP–ATXN1(82Q)/YFP average fluorescence ratio upon treatment with indicated siRNAs. Error bars, s.e.m. from triplicates, P < 0.05. d, mRFP–ATXN1(82Q)/YFP ratio distribution in siRNA-treated whole-cell populations compared to control. e, Decreased levels of ATXN1(82Q) induced by RAS shRNA in Drosophila. f, MAPK pathway showing the ATXN1(82Q) modifiers and Fig. 5 inhibitors. Diamonds represent modifiers from the screen.
Figure 4
Figure 4. MSK1 phosphorylates ATXN1 at S776 and controls its stability
a, MSK1 phosphorylation consensus site, RXXS, found in ATXN1 across different species. b, In vitro kinase assay with purified GST–ATXN1(82Q) or GST–ATXN1(30Q) and GST–MSK1. c, Neuro2a western blot transfected with Myc-fused mouse Msk1 and GST–ATXN1(82Q) or ATXN1(82Q, S776A). Graphs (right) show signal quantification of the western blot (error bars denote s.e.m. from three independent experiments, *P < 0.05). WT, wild type. d, Immunodepletion of Msk1 from cerebellar extracts decreases the level of phospho-S776 ATXN1.*P < 0.05, **P < 0.01, Student’s t-test. e, Western blot shows that knockdown of MSK1 decreases ATXN1(82Q) level in fly heads of indicated genotypes (LaminC; loading control).
Figure 5
Figure 5. Pharmacological inhibition of the MAPK pathway decreases ATXN1 level
a, Western blots and quantifications show a decrease in ATXN1 levels from Daoy mRFP– ATXN1(82Q) cells upon treatment with the indicated inhibitors (PD184352, 10 μM; GW5704, 10 μM; Ro-31-8220, 1 μM; H89, 5 μM). Error bars indicate s.e.m. from three independent experiments. b, c, Dose–response graphs show a decrease in Atxn1 levels from mouse cerebellar slices upon treatment with the indicated inhibitors. Error bars indicate s.e.m. from three independent experiments. *P < 0.05, **P < 0.01, Student’s t-test. DMSO, dimethylsulphoxide.
Figure 6
Figure 6. Msk reduction rescues behavioural and pathological phenotypes in SCA1 mice
a, b, Western blot and quantification show decreased Atxn1 levels in Atxn1154Q/+ cerebella upon complete loss of Msk1 (4–5-week-old mice; error bars denote s.e.m. from n = 4 per genotype). c, Reduction of Msk1 and Msk2 improves motor performance of Atxn1154Q/+ (9–10 weeks old, see Supplementary Information for details, *P = 0.027). d, Partial loss-of-function of Msk1 alone or Msk1 and Msk2 rescues Purkinje cell (PC) loss of ATXN1(82Q) (B05) mice (12 weeks old; n = 3 per genotype, three sections per mice). Scale bars, 100 μm (left), 50 μm (right). *P < 0.05, **P < 0.01, ***P < 0.001, post-hoc Holm-Šídák test.
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