Elsevier

Neurobiology of Disease

Volume 20, Issue 1, October 2005, Pages 170-178
Neurobiology of Disease

Ectoine alters subcellular localization of inclusions and reduces apoptotic cell death induced by the truncated Machado–Joseph disease gene product with an expanded polyglutamine stretch

https://doi.org/10.1016/j.nbd.2005.02.011Get rights and content

Abstract

Protein misfolding is considered a key event in the pathogenesis of polyglutamine disease such as Machado–Joseph disease (MJD). Overexpression of chaperone proteins and the application of chemical chaperones are reported to suppress polyglutamine induced cytotoxicity in vitro and in vivo. The effects of compatible solutes, which are osmoprotectants in bacteria and possess the action in stabilizing proteins under stress, have not, to our knowledge, been studied. We explored the protective effects of the compatible solutes ectoine, hydroxyectoine, and betaine on apoptotic cell death produced by the truncated MJD gene product with an expanded polyglutamine tract in cultured neuro2a cells. Ectoine, but not hydroxyectoine or betaine, decreased large cytoplasmic inclusions and increased the frequency of nuclear inclusions. Immunoblot analysis showed that ectoine reduced the total amount of aggregates. Despite the presence of nuclear inclusions, apoptotic features were less frequently observed after ectoine application. Our findings suggest that ectoine, a natural osmoprotectant in bacteria, may function as a novel molecule protecting cells from polyglutamine-induced toxicity.

Introduction

Machado–Joseph disease (MJD) is an inherited neurodegenerative disorder caused by the CAG expansion in the coding region of the MJD1 gene (Kawaguchi et al., 1994). The normal gene product ataxin-3 is reported to be localized in the cytoplasm and nucleus (Paulson et al., 1997a, Tait et al., 1998). The pathological variant of ataxin-3 possessing an expanded polyglutamine stretch causes nuclear inclusions and cell death in vivo and in vitro (Evert et al., 1999, Ikeda et al., 1996, Jackson et al., 1998, Paulson et al., 1997b, Warrick et al., 1998, Yoshizawa et al., 2000). Misfolding of the protein fragment containing an expanded polyglutamine stretch is considered significant in producing aggregates and cell death (Kakizuka, 1998, Paulson, 1999, Perutz, 1999). The inhibition of protein misfolding is thus a hypothetical target for effective therapy in polyglutamine diseases. The overexpression of chaperone proteins, which recognizes misfolded proteins and suppresses protein aggregation, has been shown to reduce the rate of cell death in cultured cells and transgenic animals (Chai et al., 1999, Chan et al., 2000, Cummings et al., 1998, Kazemi-Esfarjani and Benzer, 2000, Kobayashi et al., 2000, Stenoien et al., 1999, Warrick et al., 1999). We previously studied the effects of low molecular weight compounds, such as the organic solvent dimethyl sulfoxide (DMSO), cellular osmolytes glycerol, and trimethylamine N-oxide (TMAO), which stabilize proteins in their native conformation, on aggregate formation and cell death induced by the truncated ataxin-3 with an expanded polyglutamine stretch in vitro (Yoshida et al., 2002). These reagents effectively suppressed aggregate formation and cell death, suggesting a rationale for the inhibition of protein misfolding. Our findings and those of others prompted us to explore the effects of other molecules that potentially influence the protein folding in polyglutamine-induced toxicity.

One such candidate is 2-methyl,4-carboxy-3,4,5,6-tetrahydropyrimidine, or ectoine. Ectoine, originally identified in halophilic eubacteria as a compatible solute, is a nonionic organic molecule of low molecular mass that serves as an osmoprotectant (Galinski et al., 1985). Ectoine was shown to be widely distributed among different bacteria and to be useful in preserving enzymatic activity against freeze–thawing, freeze–drying, and heat treatment through protein stabilization (Lippert and Galinski, 1992).

In this study, we examine the effects of several compatible solutes, including ectoine, on mutant ataxin-3 fragment-induced aggregate formation and apoptotic cell death. In visually examining cells expressing truncated ataxin-3 with 77 glutamines (Q77), we noted that ectoine reduced large cytoplasmic inclusions and increased the frequency of nuclear inclusions. Immunoblot analysis demonstrated a reduction in the total amount of aggregates composed of the mutant ataxin-3 fragment. Despite the increased frequency of nuclear inclusions, the integrity of nuclei appeared to be maintained. Judging from nuclear morphology, ectoine decreased apoptotic cell death. Annexin V staining confirmed the result obtained from nuclear morphology. Neither hydroxyectoine nor betaine showed similar effects. These results suggest that ectoine, a widely distributed natural osmoprotectant in bacteria, functions as a novel molecule protecting cells from polyglutamine-induced toxicity.

Section snippets

Reagents and plasmids

Ectoine, hydroxyectoine, betaine, and staurosporin were purchased from Sigma (Sigma, St. Louis, MO, USA). Sucrose was obtained from Wako (Wako, Osaka, Japan). Plasmid encoding N-terminal-truncated ataxin-3 with Q77 together with C-terminal myc and His epitopes was as described elsewhere (Yoshizawa et al., 2000). In this plasmid, 286 amino acid residues of ataxin-3 were deleted from the N-terminal side. The resultant protein was designated ΔN286 (Q77). The GFP tagged-ΔN286 (Q77)-expressing

Ectoine did not demonstrate cytotoxicity up to a concentration of 150 mM

We first studied the toxic effects of ectoine on neuro2a cells. As shown in Fig. 1 (A–H), ectoine did not demonstrate cytotoxicity up to a concentration of 150 mM. Cells in the presence of 150 mM of ectoine were slightly rounder than those without ectoine, but morphological changes were very subtle, so we decided to examine the effect of ectoine up to this concentration.

Ectoin increases inclusion frequency but decreases total aggregates produced by the ataxin-3 fragment with an expanded polyglutamine stretch

We used a plasmid encoding ΔN286(Q77), an ataxin-3 fragment with 77 polyglutamines together with an N-terminal deletion of 286

Discussion

Using the in vitro cellular model for MJD pathology, we demonstrated that ectoine reduced apoptotic features without decreasing inclusion frequency. Total aggregates decreased in immunoblot analysis. We previously reported that chemical chaperones-low molecular weight compounds that stabilize proteins in their native conformation-reduced aggregate formation and cytotoxicity induced by truncated expanded ataxin-3 in vitro (Yoshida et al., 2002). These compounds included the organic solvent

Acknowledgments

We thank Ms. Sumiko Nissato for her technical assistance. This study was supported in part by a grant from the Ministry of Education, Culture, Sports, Science and Technology of Japan and a grant from the University of Tsukuba, Japan.

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