Divalproex sodium modulates nuclear localization of ataxin-3 and prevents cellular toxicity caused by expanded ataxin-3

Abstract

Background & aims: Spinocerebellar ataxia type 3 (SCA3), also known as Machado-Joseph disease (MJD), is an autosomal dominantly inherited neurodegenerative disorder and the most common form of SCA worldwide. It is caused by the expansion of a polyglutamine (polyQ) tract in the ataxin-3 protein. Nuclear localization of the affected protein is a key event in the pathology of SCA3 via affecting nuclear organization, transcriptional dysfunction, and seeding aggregations, finally causing neurodegeneration and cell death. So far, there is no effective therapy to prevent or slow the progression of SCA3.

Methods: In this study, we explored the effect of divalproex sodium as an HDACi in SCA3 cell models and explored how divalproex sodium interferes with pathogenetic processes causing SCA3.

Results: We found that divalproex sodium rescues the hypoacetylation levels of histone H3 and attenuates cellular cytotoxicity induced by expanded ataxin-3 partly via preventing nuclear transport of ataxin-3 (particularly heat shock-dependent).

Conclusion: Our study provides novel insights into the mechanisms of action of divalproex sodium as a possible treatment for SCA3, beyond the known regulation of transcription.

Keywords: HDAC inhibitors; SCA3 treatment; divalproex sodium; nuclear localization; transcriptional dysfunction.

Figure 1

Divalproex sodium increases the level of acetylated histone H3 level in ataxin‐3‐transfected HEK293 cells. Normal (15CAG) or expanded (77CAG) ataxin‐3‐transfected cells were treated with divalproex sodium (Selleck chemical, 0.3 mmol/L in DMSO, (+)) or with DMSO alone (−) 48 h after transfection. The cells were treated with divalproex sodium or DMSO over 6, 12, and 24 h. The Western blot was probed with antiacetylated histone H3 (Abcam, Cambridge, UK), antihistone H3 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and antiataxin‐3 (1H9, Merck Millipore, Darmstadt, Germany). Antibodies include antihistone H3 and antiataxin‐3 are used as controls. The x‐axis shows the different treatment group. The y‐axis represents the acetylated histone H3 values normalized to histone H3. Error bars represent the standard error of the mean. Data represent three independent experiments (n = 3). The data with a normal distribution were analyzed with Student's t‐test. *P < 0.05

Figure 2

Divalproex sodium does not change the protein levels of ataxin‐3 in HEK293 cells. HEK293 cells were transfected with ataxin‐3^{15 CAG} or ataxin‐3^{77 CAG}. 48 h after transfection, cells were treated with divalproex sodium (at 0.3 mmol/L in DMSO for 6, 12, or 24 h; +) or DMSO alone as control (−). Western blots were probed with antiataxin‐3 (1H9, Merck Millipore) and antiactin (Sigma Aldrich, St. Louis, MO, USA) as a loading control. Error bars represent the standard error of the mean. Data represent three independent experiments (n = 3). The data in each time point with a normal distribution were analyzed with Student's t‐test

Figure 3

Divalproex sodium does not affect ataxin‐3^148Q aggregation as measured by filter trap assay. Three days after transfection, HEK293 cells expressing ataxin‐3^148Q or ataxin‐3^15Q were treated with DMSO alone as a control and divalproex sodium at 10 μmol/L in DMSO for 24 h. The untreated cells were used as blank control. Error bars represent standard error of the mean over four independent experiments (n = 4). Values with a normal distribution were assessed by Student's t‐test

Figure 4

Divalproex sodium alleviates heat‐shock‐induced nuclear uptake of ataxin‐3. A, Western blot of the subcellular fraction of CHO cells expressing ataxin‐3^77Q was probed with antiataxin‐3 (1H9, Merck Millipore), anti‐KDEL (cytoplasmic marker, Enzo Life Sciences, Farmingdale, NY, USA), and antilamin B1 (nuclear marker, Santa Cruz Biotechnology). Four groups of cells with different treatments are shown. Those without heat shock were treated with either divalproex sodium (0.3 mmol/L, 24 h; Dival) or DMSO as a control (Con). The heat‐shocked cells were pretreated with either divalproex sodium (0.3 mmol/L, 24 h; Dival + HS) or DMSO as control before heat shock at 43°C for 1.5 h (HS). B, Quantification of Western blot data showed that heat shock induces a strong shift of ataxin‐3 from the cytoplasm to the nucleus and significantly reduced levels of nuclear ataxin‐3 in cells pretreated with divalproex sodium before heat shock compared with the control. Error bars represent standard error of the mean in four independent experiments (n = 4). Values with a normal distribution were assessed by Student's t‐test. *P < 0.05, **P < 0.01, ***P < 0.001

Figure 5

Divalproex sodium alleviates the cytotoxicity induced by expanded ataxin‐3 as assessed by cell viability assay using the PrestoBlue cell viability reagent (Life Technologies). CHO cells expressing ataxin‐3^77Q were seeded in a 96 well plate and treated with various concentrations of divalproex sodium for 2 h (A,B) and 24 h (C,D). The x‐axis shows the log₁₀ of concentration of divalproex sodium. The y‐axes of the left diagrams (A,C) represent fluorescence values normalized to untreated cells. The diagrams on the right (B,D) show on the y‐axis values normalized to the DMSO‐treated group as sigmoidal curves. Error bars represent standard error of the mean in three independent experiments (n = 3). The data with a normal distribution were assessed by two‐way ANOVA. **P < 0.01, ***P < 0.001