Huntingtin in health and disease.
Huntingtin in health and disease.
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亨廷顿蛋白在健康和疾病中的作用。
DOI：
10.1172/jci17742
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发表时间：
2003
期刊：
影响因子：
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通讯作者：
Young,AnneB
中科院分区：
文献类型：
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作者：
Young,AnneB
N-甲基-D-天冬氨酸（NMDA）；神经元核内包涵体（NII）。聚合 (11, 27)。 HD 大脑的尸检研究还显示，与含有 P 物质、强啡肽和多巴胺 D1 受体的细胞相比，含有脑啡肽、腺苷 A2a 和多巴胺 D2 受体的投射神经元的损失存在差异 (29-31)。在青少年 HD 中，两种类型的纹状体投射神经元均受到同样的影响 (32)。在皮层中，较深层（第 V 层和第 VI 层）的神经元使用神经递质谷氨酸，形成核和神经突聚集体 (27)。 HD 中的聚集体首次在 HD 大脑体内活组织检查的电子显微镜研究中观察到 (33)。当时并未继续研究这一观察结果，但在 1996 年报道了第一只 HD 转基因小鼠 (28)（表达由亨廷顿蛋白启动子驱动的人类亨廷顿蛋白的第一个外显子）的研究时，人们记住了这一结果。这些小鼠发育正常，直到 5 周龄左右，此时它们开始体重减轻，并且在 Rotorod 测试中表现较差。脑重量和体重随后都会减少。这些动物患上糖尿病和颤抖，变得不那么活跃。它们最终垂死并在 13 周左右死亡 (34)。对这些动物大脑的广泛早期研究表明，没有像死后 HD 大脑中那样明显的神经化学异常。然而，电显微镜研究显示核内夹杂物。在转基因小鼠中，免疫细胞化学研究表明这些 NII 的 HD 蛋白和泛素呈阳性。此外，实际上这些所谓的 R6/2 小鼠大脑中的所有神经元都含有 NII (28)。这些研究促使科学家重新审视人脑的检查，其中也发现了 NII (11, 26)。人类 HD 大脑中 NII 的频率低于转基因小鼠，并且聚集体的出现如上所述。随后聚集体的形成被认为是HD发病机制的必要条件。随后出现的两篇论文表明，聚集体是一种附带现象，因为细胞死亡不一定是由神经元亨廷顿蛋白聚集引起的，但细胞死亡确实是在突变亨廷顿蛋白表达后发生的 (35, 36)。看来，为了具有毒性，突变蛋白必须进入细胞核，因为具有核输出信号的构建体可以减弱外显子 1 过度表达导致的死亡。此外，抑制半胱天冬酶可以使细胞免于死亡。聚集体导致细胞死亡这一假设的拥护者一直在各种体外和细胞培养测定中研究这种现象 (37, 38)。聚谷氨酰胺肽不可溶，需要与其他蛋白质结合才能在溶液中进行研究。紧密的聚集体显然形成极性拉链，通过氢键结合在一起 (39)。有些人还假设转谷氨酰胺酶将谷氨酰胺与赖氨酸共价连接 (40)。尚未有人充分纯化表达多聚谷氨酰胺片段的 N 末端片段以进行结晶。纯化的全长蛋白尚未分离。然而，可以通过将聚谷氨酰胺肽与 GST 蛋白（其序列可被胰蛋白酶破坏）融合来研究聚集 (41)。一旦蛋白质进入溶液，GST 蛋白质就会被胰蛋白酶裂解，然后可以通过过滤分析来跟踪聚集过程。聚集取决于聚谷氨酰胺重复序列的长度。转变似乎发生在 39-40 次重复的范围内。谷氨酰胺少于 39-40 个的肽的聚集能力不如重复次数超过 40 个的肽。细胞培养模型还显示出长度依赖性……
N-methyl-D-aspartate (NMDA); neuronal intranuclear inclusion (NII). aggregates (11, 27). Postmortem studies of HD brains also show differential loss of projection neurons containing enkephalin, adenosine A2a, and dopamine D2 receptors compared with cells containing substance P, dynorphin, and dopamine D1 receptors (29–31). In juvenile HD, both types of striatal projection neurons are equally affected (32). In the cortex, neurons in the deeper layers (layers V and VI), which use the neurotransmitter glutamate, develop nuclear and neurite aggregates (27). Aggregates in HD were first observed in an electromicroscopy study of in vivo biopsies of HD brains (33). This observation was not pursued at the time, but it was remembered in 1996 when studies of the first HD transgenic mouse (28)(expressing the first exon of human huntingtin driven by the huntingtin promoter) were reported. These mice develop normally until around 5 weeks of age, when they begin to lose weight and to perform less well on the Rotorod test. Both brain weight and body weight diminish subsequently. The animals develop diabetes and tremors and become less active. They are finally moribund and die at around 13 weeks (34). Extensive early studies of the brains of these animals showed no clear neurochemical abnormalities like those seen in postmortem HD brains. Electromicroscopy studies, however, showed intranuclear inclusions. In the transgenic mice, it then became obvious from immunocytochemical studies that these NIIs were positive for the HD protein and for ubiquitin. Furthermore, virtually all neurons in the brains of these so-called R6/2 mice contained NIIs (28). These studies led scientists to revisit the examination of human brains in which NIIs were also found (11, 26). The frequency of NIIs in human HD brains was lower than in the transgenic mice, and the aggregates appeared as described above. The formation of aggregates was subsequently thought to be the sine qua non of HD pathogenesis. Two papers then appeared that suggested that the aggregates were an epiphenomenon, since cell death did not necessarily result from neuronal huntingtin aggregation, yet cell death did arise after the expression of mutant huntingtin (35, 36). It appeared that, to be toxic, the mutant protein had to get into the nucleus, since constructs with nuclear-export signals attenuated death resulting from exon 1 overexpression. Furthermore, inhibition of caspases rescued cells from death. Advocates of the hypothesis that aggregates cause cell death have been studying the phenomenon in various in vitro and cell culture assays (37, 38). Polyglutamine peptides are not soluble and need to be tied to other proteins to be studied in solution. Tight aggregates apparently form into polar zippers that are held together by hydrogen bonding (39). Some have also hypothesized that transglutaminases link glutamines to lysines covalently (40). No one has yet purified enough of the N-terminal fragment expressing the polyglutamine stretch to perform crystallization. The purified fulllength protein has not been isolated. Aggregation, however, can be studied by fusing a polyglutamine peptide with a GST protein with a sequence that can be broken with trypsin (41). Once the protein is in solution, the GST protein is cleaved off with trypsin, and then the aggregation process can be followed by filter assays. Aggregation is dependent on the length of the polyglutamine repeat. A transition seems to occur in the range of 39–40 repeats. Peptides with fewer than 39–40 glutamines aggregate less robustly than peptides with more than 40 repeats. Cell culture models also show length-dependent …