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

柱状体 (STB) 是一种独特的非膜结合神经细胞质包涵体，最初通过免疫组织化学检测到人胎盘抗原 X-P2S (hPAX-P2S)，还含有亨廷顿蛋白相关蛋白 1 (HAP1A/B)，并且HAP1A-cDNA 转染在培养细胞中诱导 STB 样内含物，大量 STB 优先分布在细胞中。神经变性很少发生的边缘和下丘脑区域，而不是神经变性的目标，包括纹状体、丘脑和新皮质。当前的研究首先提供了明确的证据，证明 HAP1 与源自脊髓和延髓肌肉的雄激素受体 (AR) 相互作用。萎缩（SBMA）通过其配体结合结构域以聚 Q 长度依赖性方式形成，并形成显着的包涵体隔离聚 Q-AR，并且添加二氢睾酮与 ARQ65 相比，更显着地降低 HAP1 与 ARQ25 的关联性。此外，与 HAP1 共转染可抑制 SBMA 突变体 ARQ65 诱导的细胞凋亡，支持“HAP1/STB 保护假说”，即 HAP1/STB 发挥作用。对神经退行性变的保护作用。此外，抗 hPAX-P2S 抗血清首次被证明可以识别HAPI^<474-577>（XP2S 结构域）在蛋白质印迹中靠近 HAP1A/B 的 C 末端，大脑 STB 的 hPAX-P2S 免疫反应和 HAP1A 诱导的内含物被证明可通过 HAP1^ 的预吸附而消除<474-577> 这些发现清楚地表明 hPAX-P2S 与 STB 构成的 HAP1 相同，并且HAP1 诱导/免疫反应性内含物对应于 hPAX-P2S 免疫反应性 STB。此外，抗 hPAX-P2S 抗血清对 HAP1B 诱导/免疫反应性弥散结构的免疫染色非常弱，表明 XP2S 结构域被弥散中的某些分子覆盖。 HAP1 的结构并在 STB 中公开，并且为阐明 STB 形成机制提供了重要线索。不仅局限于 STB，而且还与 STB 附近的内质网样管状结构相关，但迄今为止，任何针对细胞器标记物（包括 KDEL、GMP130、LAMP1、EEA1 或 Bcl2）的抗体均未检测到 STB。起源尚未确定。