Requirement of an intact microtubule cytoskeleton for aggregation and inclusion body formation by a mutant huntingtin fragment

Huntington's disease is caused by the expansion of CAG repeats coding for a polyglutamine tract in the huntingtin protein. The major pathological feature found in Huntington's disease neurons is the presence of detergent-insoluble ubiquitinated inclusion bodies composed of the huntingtin protein. However, the mechanisms that underlie inclusion body formation, and the precise relationship between inclusion bodies and events that initiate toxicity, remain unclear. Here, we analyzed the effects of drugs or genetic mutations that disrupt the microtubule cytoskeleton in a Saccharomyces cerevisiae model of the aggregation of an amino-terminal polyglutamine-containing fragment of huntingtin exon 1 (HtEx1). Treatment of yeast with drugs that disrupt microtubules resulted in less than 2% of the detergent-insoluble HtEx1 observed in mock-treated cells and prevented the formation of large juxtanuclear inclusion bodies. Disruption of microtubules also unmasked a potent glutamine length-dependent toxicity of HtEx1 under conditions where HtEx1 exists in an entirely detergent-soluble nonaggregated form. Results from the yeast model paralleled those from neuronal pheochromocytoma cells, where disruption of microtubules eliminated the formation of juxtanuclear and intranuclear inclusion bodies by HtEx1. Our results suggest that active transport along microtubules may be required for inclusion body formation by HtEx1 and that inclusion body formation may have evolved as a cellular mechanism to promote the sequestration or clearance of soluble species of HtEx1 that are otherwise toxic to cells.

亨廷顿舞蹈症（Huntington's Disease）由亨廷顿蛋白（huntingtin protein）中编码多聚谷氨酰胺肽段的CAG重复序列扩增所引发。亨廷顿舞蹈症患者神经元的核心病理特征，是存在由亨廷顿蛋白构成的去垢剂不溶性泛素化包涵体。然而，包涵体形成的潜在分子机制，以及包涵体与引发毒性的事件之间的确切关联，目前仍未阐明。本研究以酿酒酵母（Saccharomyces cerevisiae）为模型，分析了破坏微管细胞骨架的药物或遗传突变，对亨廷顿外显子1氨基端含多聚谷氨酰胺片段（amino-terminal polyglutamine-containing fragment of huntingtin exon 1，以下简称HtEx1）聚集过程的影响。实验结果显示，使用破坏微管的药物处理酵母后，检测到的去垢剂不溶性HtEx1含量不足空白对照处理组细胞的2%，且可完全阻断大型核周包涵体的形成。此外，微管破坏还能在HtEx1完全以去垢剂可溶性非聚集形式存在的条件下，揭示出HtEx1强效的谷氨酰胺长度依赖性毒性。本研究在酵母模型中得到的结果，与神经元来源的嗜铬细胞瘤细胞中的实验结果高度吻合：在后者中，微管破坏可消除HtEx1形成核周及核内包涵体的能力。综上，本研究结果表明，沿微管的主动运输可能是HtEx1形成包涵体的必要条件；而包涵体的形成或许已进化为一种细胞保护机制，用于促进对原本对细胞具有毒性的可溶性HtEx1形式的隔离与清除。