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

人胎盘抗原X-P2S(hPAX-P2S)免疫组织化学检测到的豆状小体(STB)是一种独特的非膜结合型神经质内包涵体，它还含有亨廷顿蛋白相关蛋白1(HAP1A/B)，HAP1A-cDNA3基因的导入可诱导培养细胞中出现类STB包涵体。数量惊人的STB优先分布在边缘和下丘脑区域，这些区域很少发生神经变性，而不是纹状体、丘脑和新皮质等神经变性的靶点。目前的研究首次提供了明确的证据，证明HAP1与脊髓性肌萎缩症(SBMA)来源的雄激素受体(AR)通过其配体结合结构域以一种多Q长度依赖的方式相互作用，并形成显著的包涵体隔离多Q-AR，并且双氢睾酮的加入使HAP1与ARQ25的结合强度比与ARQ65的结合强度降低得更明显。此外，SBMA突变体ARQ65诱导的APOP…通过与HAP1共转染可抑制更多的TOSIS，支持HAP1/STB保护假说，即HAP1/STB对神经退行性变具有保护作用。此外，在Western blotting中，首次证明抗hPAX-P2S抗血清能够识别HAP1A/B的C末端附近的HAP1^&lt；474-577&gt；(XP2S结构域)，并通过与HAP1^&lt；474-577&gt；的预吸附消除脑STB和HAP1A诱导的包涵体的hPAX-P2S免疫反应。这些发现清楚地表明，hPAX-P2S与STB组成的HAP1是相同的，HAP1诱导/免疫反应的包涵体对应于hPAX-P2S免疫反应的STB。此外，抗hPAX-P2S抗血清对HAP1B诱导/免疫反应的弥漫结构的染色很弱，表明XP2S结构域被弥漫结构中的一些分子覆盖，并在STB中被揭示，这对阐明STB的形成机制具有重要的线索。在免疫电子显微镜下，HAP1不仅定位于STB，而且与STB相邻的内质网样小管结构有关，但迄今没有任何针对细胞器标志物的抗体包括KDEL、GMP130、LAMP1、EEA1或Bcl2检测到STB。细胞器的起源尚未确定。较少

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