Ultrastructural analysis and molecular screening of the neuronal cytoplasmic inclusion, "the stigmoid body"

神经元细胞质内含物“柱状体”的超微结构分析和分子筛选

基本信息

批准号：
17500231
负责人：
SHINODA Koh
金额：
$ 1.86万
依托单位：
Yamaguchi University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

The stigmoid body (STB), which was a distinct non-membrane-bound neurocytoplasmic inclusion originally detected by immunohistochemistry for human placental antigen X-P2S (hPAX-P2S), also contains huntingtin-associated protein 1 (HAP1A/B), and the HAP1A-cDNA transfection induces STB-like inclusions in cultured cells. A striking number of the STBs are preferentially distributed in the limbic and hypothalamic regions, where neurodegeneration rarely occurs, rather than the targets of neurodegeneration including the striatum, thalamus and neocortex. The current research first provided clear evidence that HAP1 interacts with androgen receptor (AR) derived from spinal-and-bulbar-muscular-atrophy (SBMA) via its ligand-binding domain in a polyQ-length-dependent manner and forms prominent inclusions sequestering polyQ-AR, and that addition of dihydrotestosterone reduces the association strength of HAP1 with ARQ25 more dramatically than that with ARQ65. Furthermore, SBMA-mutant-ARQ65-induced apop … More tosis was suppressed by cotransfection with HAP1, supporting "the HAP1/STB protection hypothesis" that the HAP1/STB plays a protective role against neurodegeneration.In addition, the anti-hPAX-P2S antiserum was first demonstrated to recognize HAPI^<474-577> (XP2S domain) near C-terminuses of HAP1A/B in Western blotting, and hPAX-P2S-immunoreactions of the brain STB and HAP1A-induced inclusions were shown to be eliminated by pre-adsorption with HAP1^<474-577>. These findings clearly indicated that hPAX-P2S is identical with STB-constituted HAP1 and that the HAP1-induced/immunoreactive inclusions correspond to the hPAX-P2S-immunoreactive STBs. In addition, the anti-hPAX-P2S antiserum far weakly immunostained HAP1B-induced/immunoreactive diffuse structures, suggesting that the XP2S domain is covered by some molecule in the diffuse structures and disclosed in the STB and have an important clue to elucidate mechanism of the STB formation. In immuno-electron microscopy, HAP1 is not only localized to the STB but also associated with endoplasmic-reticulum-like tubular structures adjacent to the STB, but the STB is not detected so far by any antibodies to organelle-markers including KDEL, GMP130, LAMP1, EEA1 or Bcl2. The organelle origin has yet to be determined. Less

柱头体（STB）是一种独特的非膜结合的神经细胞胞质包容，最初由人胎盘抗原X-P2S（HPAX-P2S）检测到，还包含Huntingtin相关的蛋白1（HAP1A/B）（HAP1A/B）（HAP1A/B），并含有hap1a-Cdna Transfection incultion coldution incultion incult incultion incult incult incult incult incult incult incultruist。显着数量的STB优选分布在边缘和下丘脑区域中，那里的神经退行性很少发生，而不是神经变性的靶标，包括纹状体，丘脑和新皮层。当前的研究首先提供了明确的证据，表明HAP1与脊髓和纤维 - 肌肉 - 肌肉 - 肌肉滋养（SBMA）通过其配体结合结构域以polyq-length依赖性的方式和杰出的包含形成序列的包含polyq-ar的雄激素受体（AR），并与二氢固醇的添加相互作用。 ARQ65。此外，SBMA-突变剂-ARQ65诱导的APOP…通过与HAP1进行共转染抑制了更多的Tosis，支持HAP1/STB的“ HAP1/STB保护假设”，HAP1/STB在抗神经变性中扮演保护作用。在蛋白质印迹中，HAP1A/B的C末端附近，以及通过HAP1^<474-577>的预吸收来消除脑STB和HAP1A诱导的夹杂物的HPAX-P2S-免疫反应。这些发现清楚地表明，HPAX-P2S与STB固定的HAP1相同，并且HAP1诱导/免疫反应性夹杂物对应于HPAX-P2S-免疫反应性STB。此外，抗HPAX-P2S抗血清差弱免疫染色的HAP1B诱导/免疫反应性弥漫性结构，这表明XP2S结构域在弥漫性结构中被某些分子覆盖，并在STB中披露，并在STB中披露，并具有重要的线索，以阐明STB组成的机制。在免疫电子显微镜中，HAP1不仅位于STB上，而且还与与STB相邻的内质 - 近后型管状管状结构有关，而且STB并未通过与包括KDEL，GMP130，LAMP1，LAMP1，EEA1，EEA1，EEA1，EEA1，EEA1或BCL2的细胞器标记的任何抗体检测到STB。细胞器起源尚未确定。较少的