PAK1 regulates ATXN1 levels providing an opportunity to modify its toxicity in spinocerebellar ataxia type 1

Abstract

Spinocerebellar ataxia type 1 (SCA1) is caused by the expansion of a trinucleotide repeat that encodes a polyglutamine tract in ataxin-1 (ATXN1). The expanded polyglutamine in ATXN1 increases the protein's stability and results in its accumulation and toxicity. Previous studies have demonstrated that decreasing ATXN1 levels ameliorates SCA1 phenotypes and pathology in mouse models. We rationalized that reducing ATXN1 levels through pharmacological inhibition of its modulators could provide a therapeutic avenue for SCA1. Here, through a forward genetic screen in Drosophila we identified, p21-activated kinase 3 (Pak3) as a modulator of ATXN1 levels. Loss-of-function of fly Pak3 or Pak1, whose mammalian homologs belong to Group I of PAK proteins, reduces ATXN1 levels, and accordingly, improves disease pathology in a Drosophila model of SCA1. Knockdown of PAK1 potently reduces ATXN1 levels in mammalian cells independent of the well-characterized S776 phosphorylation site (known to stabilize ATXN1) thus revealing a novel molecular pathway that regulates ATXN1 levels. Furthermore, pharmacological inhibition of PAKs decreases ATXN1 levels in a mouse model of SCA1. To explore the potential of using PAK inhibitors in combination therapy, we combined the pharmacological inhibition of PAK with MSK1, a previously identified modulator of ATXN1, and examined their effects on ATXN1 levels. We found that inhibition of both pathways results in an additive decrease in ATXN1 levels. Together, this study identifies PAK signaling as a distinct molecular pathway that regulates ATXN1 levels and presents a promising opportunity to pursue for developing potential therapeutics for SCA1.

Figure 1.

  Drosophila Pak3 modulates ATXN1 levels and suppresses ATXN1 [82Q]-induced neurotoxicity. (A) Representative immunoblot image and quantification of ATXN1 [82Q] levels in Pak3^RNAi-1 (Pak3^KK111386) or control Drosophila eyes showing reduction of ATXN1 levels upon Pak3 knockdown. ATXN1 [82Q] levels are normalized to LamC (n = 4, t-test, **P < 0.01. Error bars denote the SEM). (B) Scanning electron microscopy images of wild-type, SCA1 and SCA1/Pak3^RNAi-1 (Pak3^KK111386) Drosophila eyes. Pak3 knockdown (Pak3^KK111386) ameliorates ATXN1 [82Q]-induced toxicity (C) Cross-sections of the eye reveal increased thickness (i.e. less degeneration, arrow) of the retinal cell layer in SCA1 flies upon Pak3 knockdown (Pak3^KK111386). (D) Improved motor performance, as assessed by increased speed, of SCA1 flies upon Pak3 knockdown (Pak3^KK111386) in the nervous system (n = 30–60, non-linear ANOVA, ****P < 0.0001. Error bars denote the SEM).

Figure 2.

 PAK1 regulates ATXN1 levels in mammalian cells. (A)PAK1 knockdown using three different siRNAs in mRFP-ATXN1 [82Q]-IRES-YFP Daoy cell line reveals reduction in mRFP-ATXN1 [82Q]/YFP ratio by flow cytometry (n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001 ****P < 0.0001. Error bars denote the SEM). (B) Three different PAK1 siRNAs knockdown PAK1 and significantly reduce endogenous ATXN1 levels in Daoy cells (n = 3, one-way ANOVA, **P < 0.01, ***P < 0.001, ****P < 0.0001. Error bars denote the SEM) (C) Quantification of ATXN1 mRNA levels by qRT-PCR shows no significant change upon PAK1 knockdown (n = 3, t-test, **P < 0.01. Error bars denote the SEM). (D) Co-expression of GST-ATXN1 [82Q] with PAK1 or GFP control significantly increases ATXN1 levels upon PAK1 co-transfection (n = 3, t-test, **P < 0.01. Error bars denote the SEM). (E)PAK1 shRNA-mediated knockdown in mouse primary cerebellar neurons reduces Atxn1 levels (n = 3, t-test, *P < 0.05. Error bars denote the SEM).

Figure 3.

 PAK1 modulates ATXN1 independently of S776 phosphorylation. (A)PAK1 knockdown using siPAK1-1 significantly reduces mRFP-(S776A) ATXN1 [82Q] levels in a Daoy cell line stably expressing mRFP-(S776A) ATXN1 [82Q]-IRES-YFP by western blot (n = 3, one-way ANOVA, *P < 0.05, ****p < 0.0001. Error bars denote the SEM). (B) Co-expression of ATXN1 [82Q] or phospho-mutant S776A ATXN1 [82Q] with PAK1 or GFP control reveal that PAK1 modulates S776A ATXN1 [82Q] (n = 3, two-way ANOVA, ***P < 0.001; ns, not significant. Error bars denote the SEM). (C) MSK1, but not PAK1, phosphorylates ATXN1 [30Q] in in vitro³²P kinase assay. GTP is the co-factor for CDC42 activation that in turn activates PAK1 as depicted by the radioactive signal at a molecular weight below ATXN1 [30Q]. (D) Co-immunoprecipitation of endogenous CIC and 14–3-3ε with transfected flag-ATXN1 [30Q], but not myc-PAK1^KD (kinase dead, K299R) in HEK293T cells [*Light chain (LC) specific IgG, **heavy chain (HC) specific IgG]. (E)PAK1 knockdown (siPAK1-3) reduces endogenous ATXN1 levels in Daoy cells. However, upon addition of the proteasome inhibitor, MG132, the PAK1-dependent reduction of ATXN1 levels was abolished (n = 3, two-way ANOVA, *P < 0.05; ns, non-significant. Error bars denote the SEM).

Figure 4.

 Inhibition of PAK signaling reduces ATXN1 levels in cells and mice. (A) Treatment of mouse primary cerebellar neuron culture with FRAX486 (PAK1/2/3i), an inhibitor of Group I PAKs, results in a decrease of pPak1 (S144) and Atxn1 levels in a concentration-dependent manner (n = 3, one-way ANOVA, **P < 0.01, ****P < 0.0001. Error bars denote SEM). (B) PF03758309, panPAK inhibitor (panPAKi), modulates Atxn1 levels in a concentration-dependent manner in mouse cerebellar neuron culture (n = 3, one-way ANOVA, ***P < 0.001, ****P < 0.0001. Error bars denote the SEM). (C) Combinatorial delivery of MEK (PD0325901, MEKi) and panPAK (PF03758309, panPAKi) inhibitors in mRFP-ATXN1 [82Q]-IRES-YFP Daoy cell line additively reduces mRFP-ATXN1 [82Q] levels (n = 3, one-way ANOVA, *P < 0.05, ****P < 0.0001. Error bars denote the SEM). (D) Intraperitoneal administration of 15 mg/kg panPAK inhibitor every 8 h for 5 days in Atxn1^154Q/2Q mice reduce expanded Atxn1 [154Q] as wells as wild type, Atxn1 [2Q] levels in the cerebellum (n = 4, two-way ANOVA, **P < 0.01. Error bars denote the SEM).