突变亨廷顿蛋白选择性损伤纹状体γ-氨基丁酸能投射神经元的分子机制研究

Huntington disease (HD) is an autosomal dominant neurodegenerative disorder caused by a mutation in a protein called huntingtin. No efficacious anti-neurodegenerative therapy for HD exists at present. The histopathological hallmark of HD is pronounced atrophy of the striatum. Within the striatum, there is differential involvement of subpopulations of striatal neurons.Striatal projection neurons are all GABAergic and morphologically characterized by a long axon, medium-sized cell bodies, and spiny dendrites. Intriguingly, striatal projection neurons represent the main and earliest striatal cell type affected in HD, whereas striatal interneurons are typically unaffected or only mildly affected at late stages of the disease. Althogh huntingtin is wildly expressed in and outside the nervous, but mutant huntingtin makes striatal projeciton neurons particularly vulnerable to cell death. The underlying molecular mechanisms remain unknown. Mutant huntingtin was reported to discupt fast axonal transport in drosophila, squid, and mice. Howerer, previous work did not resolve whether there is a enhanced sensitivity of striatal GABAergic projection neurons to axonal transport defects induced by mutant huntingtin. The enhanced sensitivity of striatal GABAergic projection neurons to axonal transport defects induced by mutant huntingtin may play important roles in the development of cell-type-specific defects in HD. In order to understand the the molecular mechanisms involved in the selective vulnerability of striatal GABAergic projection neurons in Huntington's disease, we try to use the HD knock-in mice and the GAD67-GFP mice which selectively express GFP in GABAergic neurons to study the vulnerability of the striatal GABAergic projection neurons to axonal transport defects induced by mutant huntingtin. Understanding the mechanism of the vulnerability of the striatal GABAergic projection neurons to axonal transport defects induced by mutant huntingtin could reveal novel pharmacological targets for HD.

亨廷顿舞蹈病（Huntington disease, HD）是一种常染色体显性遗传的神经变性疾病，目前无有效治疗方法。其特征性早期病理改变是纹状体γ-氨基丁酸（γ-aminobutyric acid, GABA）能投射神经元丢失。该病致病基因IT15基因编码的亨廷顿蛋白（Huntingtin，Htt）广泛表达于全身，而突变Htt仅选择性损伤纹状体GABA能投射神经元，其原因不清。我们的前期研究显示瞬时过表达突变Htt可部分损害体外培养的海马神经元的轴突转运。我们拟在前期研究基础上,利用HD基因敲入小鼠及能特异性显示GABA能神经元的转基因鼠，进一步研究纹状体GABA能投射神经元是否对突变Htt引起的轴突转运损害特别敏感从而表现为在HD中选择性丢失，并深入探讨其可能的分子机制。为HD发病原因及治疗提供新思路。

亨廷顿舞蹈病（Huntington disease, HD）是一种常染色体显性遗传的神经变性疾病，目前无有效治疗方法。其特征性早期病理改变是纹状体γ-氨基丁酸（γ-aminobutyric acid, GABA）能投射神经元丢失。该病致病基因IT15基因编码的亨廷顿蛋白（Huntingtin，Htt）广泛表达于全身，而突变Htt仅选择性损伤纹状体GABA能投射神经元，其原因不清。我们原代培养了不同脑区（皮层、海马、纹状体）的神经元，并瞬时转染野生型Htt质粒与突变Htt质粒。我们发现野生型Htt均匀表达在原代培养7天的海马神经元中，而突变Htt则在原代培养7天的海马神经元核内、核周、轴突内形成聚集体。进一步我们通过转染表达一个融合蛋白（红色荧光蛋白+线粒体基质定位信号）以显示活的线粒体，并利用激光共聚焦显微镜观察分析发红色荧光的线粒体在神经元轴突内的运动情况。我们发现野生型Htt的表达不改变线粒体的顺向与逆向轴突转运速度，然而聚集体突变Htt与可溶性突变Htt均显著减少线粒体的顺向与逆向轴突转运速度。而表达可溶性突变Htt与聚集体突变Htt这两组之间无显著性差异。这说明可溶性mHtt与聚集体mHtt均减低线粒体的轴突转运速度，而聚集体mHtt并没有比可溶性Htt影响得更明显。进一步分析可溶性与聚集体mHtt对线粒体运动性的影响。我们发现表达野生型Htt与对照组相比不改变静止线粒体的百分比，而可溶性mHtt与聚集体mHtt均显著增加了静止线粒体的百分比，且可溶性mHtt与聚集体mHtt两组之间无统计学差异。同时我们在原代培养的纹状体神经元中比较突变Htt与野生型Htt对原代培养的纹状体神经元线粒体轴突转运的影响，得到了与海马神经元中相类似的结果，表明可溶性mHtt与聚集体mHtt均减少线粒体轴突转运速度，降低线粒体的运动性，且可溶性mHtt与聚集体mHtt两者之间对线粒体轴突转运的破坏没有差异，进一步提示突变Htt对神经元轴突转运的破坏可能是疾病病理生理机制的早期事件，在疾病的发病机制中起着核心作用。