Biochemical, ultrastructural, and reversibility studies on huntingtin filaments isolated from mouse and human brain
Biochemical, ultrastructural, and reversibility studies on huntingtin filaments isolated from mouse and human brain
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DOI：
10.1523/jneurosci.2365-04.2004
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发表时间：
2004-10-20
影响因子：
5.3
通讯作者：
Lucas, JJ
中科院分区：
文献类型：
--
作者：
Díaz-Hernández, M;Moreno-Herrero, F;Lucas, JJ
亨廷顿病 (HD) 和另外八种遗传性神经系统疾病是由 CAG 三联体重复扩增导致各自蛋白质中多聚谷氨酰胺序列扩增引起的。这些三联体CAG重复疾病的共同点是形成含有扩展的多聚谷氨酰胺序列的异常神经元内蛋白质内含物。据推测，这些聚集体有助于由构象毒性、其他含聚谷氨酰胺蛋白质的隔离或通过干扰某些酶活性引起的发病机制。由于难以将这些聚集物与大脑分离，测试这些假设受到了阻碍。在这里，我们报告，通过遵循基于高盐缓冲液均质化、非离子洗涤剂提取和梯度分级的方法，可以从 HD 的 Tet/HD94 条件小鼠模型的大脑中分离出聚谷氨酰胺聚集体。然后我们验证了该方法可以成功应用于死后 HD 大脑。人类和小鼠样本的免疫电子显微镜显示，内含物的稳定成分是含有突变亨廷顿蛋白和含有泛素的原纤维。原子力显微镜显示这些原纤维具有“串珠”形态。因此，它们类似于由重组突变体亨廷顿蛋白以及 Abeta 和 α-突触核蛋白淀粉样原纤维制成的体外组装的细丝。最后，通过关闭 HD 的 Tet/HD94 条件小鼠模型中的转基因表达，我们能够证明这些丝虽然在体外稳定，但在体内容易恢复，从而证明先前报道的泛素免疫反应性包涵体的逆转并不简单地反映了包涵体分解为其组成原纤维，并表明任何相关的构象或蛋白质隔离毒性也可能恢复。
Huntington's disease (HD) and eight additional inherited neurological disorders are caused by CAG triplet-repeat expansions leading to expanded polyglutamine-sequences in their respective proteins. These triplet-CAG repeat disorders have in common the formation of aberrant intraneuronal proteinaceous inclusions containing the expanded polyglutamine sequences. These aggregates have been postulated to contribute to pathogenesis caused by conformational toxicity, sequestration of other polyglutamine-containing proteins, or by interfering with certain enzymatic activities. Testing these hypotheses has been hampered by the difficulty to isolate these aggregates from brain. Here we report that polyglutamine aggregates can be isolated from the brain of the Tet/HD94 conditional mouse model of HD, by following a method based on high salt buffer homogenization, nonionic detergent extraction, and gradient fractionation. We then verified that the method can be successfully applied to postmortem HD brains. Immunoelectron microscopy, both in human and mouse samples, revealed that the stable component of the inclusions are mutant huntingtin-containing and ubiquitin-containing fibrils. Atomic-force microscopy revealed that these fibrils have a "beads on a string" morphology. Thus, they resemble the in vitro assembled filaments made of recombinant mutant-huntingtin, as well as the Abeta and alpha-synuclein amyloid protofibrils. Finally, by shutting down transgene expression in the Tet/HD94 conditional mouse model of HD, we were able to demonstrate that these filaments, although stable in vitro, are susceptible to revert in vivo, thus demonstrating that the previously reported reversal of ubiquitin-immunoreactive inclusions does not simply reflect disassembling of the inclusions into their constituent fibrils and suggesting that any associated conformational or protein-sequestration toxicity is also likely to revert.