Reelin is a target of polyglutamine expanded ataxin-7 in human spinocerebellar ataxia type 7 (SCA7) astrocytes

Abstract

Spinocerebellar ataxia type 7 (SCA7) is an autosomal-dominant neurodegenerative disorder that results from polyglutamine expansion of the ataxin-7 (ATXN7) protein. Remarkably, although mutant ATXN7 is expressed throughout the body, pathology is restricted primarily to the cerebellum and retina. One major goal has been to identify factors that contribute to the tissue specificity of SCA7. Here we describe the development and use of a human astrocyte cell culture model to identify reelin, a factor intimately involved in the development and maintenance of Purkinje cells and the cerebellum as a whole, as an ATXN7 target gene. We found that polyglutamine expansion decreased ATXN7 occupancy, which correlated with increased levels of histone H2B monoubiquitination, at the reelin promoter. Treatment with trichostatin A, but not other histone deacetylase inhibitors, partially restored reelin transcription and promoted the accumulation of mutant ATXN7 into nuclear inclusions. Our findings suggest that reelin could be a previously unknown factor involved in the tissue specificity of SCA7 and that trichostatin A may ameliorate deleterious effects of the mutant ATXN7 protein by promoting its sequestration away from promoters into nuclear inclusions.

Fig. 1.

Establishment of a recombinant lentiviral delivery system for the stable expression of ATXN7 in cultured human astrocytes. (A) The percentage of transduced cells (EGFP-positive) was similar to the percentage of cells expressing exogenous F-ATXN7 (Flag-positive). (B) Expression of exogenous ATXN7 proteins in WCE from SCA7 astrocytes was confirmed by immunoblot with anti-Flag antibodies at 3 and 6 d postinfection. Exogenous Flag-ATXN7 proteins were observed at expected molecular weights. SDS-insoluble aggregates (asterisk) were present at time points by 6 d postinfection in mutant SCA7 astrocytes. (C) Formation of NIs in cells expressing the polyQ ATXN7 protein was confirmed by immunofluorescence staining of infected astrocytes with anti-Flag antibodies. Cell bodies and nuclei were visualized with phalloidin (green) and Hoechst dye (blue), respectively. (D) GCN5 colocalization with NIs in mutant SCA7 astrocytes was observed by immunofluorescence staining of SCA7 astrocytes with anti-Flag and anti-GCN5 antibodies. Cell nuclei were visualized by Hoechst dye (blue).

Fig. 2.

Expression of polyQ ATXN7 results in the alternative regulation of multiple transcripts. (A) Several neural-associated genes are alternatively regulated in mutant SCA7 astrocytes. P values for all target genes shown (except GAPDH) were <0.01. (B) Down-regulation of the RELN transcript was verified with three real-time PCR primer sets. Error bars represent ± SEM (*P = 4.34 × 10⁻⁴³, **P = 3.35 × 10⁻³⁴, ***P = 1.14 × 10⁻²⁹, two-tailed Student t test). (C) Immunoblot of WCE demonstrates a reduction in RELN protein levels in mutant SCA7 astrocytes. Equal protein loading was verified relative to a cross-reacting band.

Fig. 3.

Polyglutamine expansion of ATXN7 alters chromatin occupancy and histone H2B monoubiquitination at the RELN promoter and increases global H2B monoubiquitination. (A) Polyglutamine expansion of ATXN7 resulted in a reduction of polyQ ATXN7 occupancy at the RELN promoter. (B) Although the change in ATXN7 occupancy on the RELN promoter did not substantially alter levels of H3K9/14 acetylation, (C) H2B monoubiquitination levels at the RELN promoter increased in mutant SCA7 astrocytes. (D) Global H3K9/14 acetylation was unchanged whereas global H2B monoubiquitination was increased in mutant SCA7 astrocytes. ChIP antibodies are shown on the x-axes of graphs in A–C. Histone H3 (A and B) and H2B (C) values shown were normalized to H3 and H2B occupancy within the β-actin promoter, respectively. Error bars represent ± SEM (*P = 3.62 × 10⁻³⁴, **P = 2.70 × 10⁻⁴, two-tailed Student t test).

Fig. 4.

Treatment with TSA, but not other HDACi, partially restores RELN transcript levels in mutant SCA7 astrocytes. (A) Treatment of mutant SCA7 astrocytes to 200 ng/mL TSA led to a significant increase in the amount of RELN transcript compared with vehicle. In contrast, exposure to sodium butyrate (B) or nicotinamide (C) failed to rescue RELN transcription. Error bars represent ± SEM (*P = 1.56 × 10⁻¹⁰, **P = 1.04 × 10⁻¹⁰, two-tailed Student t test).

Fig. 5.

Treatment with 200 ng/mL TSA alters ATXN7 protein levels, NI dynamics, and H2B monoubiquitination levels at the RELN promoter. (A) Levels of Flag-polyQ ATXN7 protein were increased in SCA7 astrocytes following 18 h of exposure to 200 ng/mL TSA as shown by immunoblotting WCE with anti-Flag antibodies. (B) TSA treatment increased the number of Flag-positive cells with NIs from 20.6% (vehicle) to 40.6% (P = 6.3 × 10⁻¹¹). (C) Histone H2B monoubiquitination in mutant SCA7 astrocytes were reduced to levels similar to those in vehicle-treated WT SCA7 astrocytes following 16 h of exposure to 200 ng/mL TSA (*P = 1.20 × 10⁻², two-tailed Student t test). Error bars represent ± SEM.