Sakamoto Masaki, Uchihara Toshiki, Nakamura Ayako, Mizutani Toshio, Mizusawa Hidehiro
Department of Neurology, Tokyo Metropolitan Neurological Hospital, Tokyo, Japan.
Acta Neuropathol. 2005 Oct;110(4):417-25. doi: 10.1007/s00401-005-1066-9. Epub 2005 Aug 25.
Alpha-synuclein (alphaS) and ubiquitin (Ub) are shared constituents of glial cytoplasmic inclusions (GCIs) and Lewy bodies (LBs), both composed of fibrillary structures. Staining profiles of GCIs were investigated with triple immunofluorescence involving immunostaining for alphaS and Ub, both amplified with catalyzed reporter deposition, and a fluorochrome, thiazin red (TR) that has an affinity to fibrillary structures. After observation for the triple-fluorescent images, the sections were subsequently stained with the Gallyas-Braak method. Sections of putamen, cerebellar white matter and motor cortex from patients suffering from multiple system atrophy (MSA) with varying duration of the disease (4-15 years) were quantified for these staining profiles of Gallyas-positive GCIs. Although most of GCIs were positive for Ub and variably positive for alphaS, they were consistently negative for TR. The result was opposite in LBs in Lewy body disease with variable affinity to TR, suggesting that the construction of GCIs is different from that of LBs. These four staining features (alphaS, Ub, TR and Gallyas) alone failed to exhibit apparent correlation with disease duration, lesion site or severity of degeneration as reported previously. The fraction of alphaS-negative and Ub-positive GCIs, however, linearly increased along the disease progression, while that of alphaS-positive and Ub-negative GCIs decreased in contrast. This reciprocal change suggests that alphaS immunoreactivity in GCIs is being replaced by Ub immunoreactivity during the disease progression, which resulted in the ultimate predominance of alphaS-negative and Ub-positive GCIs in the most advanced case. Interestingly, this predominance of alphaS-negative and Ub-positive GCIs was a feature of motor cortex, where degeneration usually remains mild in spite of robust appearance of Gallyas-positive GCIs. Another fraction, alphaS-positive and Ub-positive GCIs were frequent in cerebellar white matter, suggesting that GCI evolution is heterogeneous and dependent also on area examined. Progressive accumulation of Ub with concomitant disappearance of alphaS epitope and their colocalization, partly shared with LBs, may represent a process of GCI formation, possibly linked to an aspect of degeneration in MSA.
α-突触核蛋白（αS）和泛素（Ub）是神经胶质细胞质包涵体（GCI）和路易小体（LB）的共同组成成分，二者均由纤维状结构构成。采用三重免疫荧光法研究GCI的染色情况，该方法涉及对αS和Ub进行免疫染色（二者均通过催化报告沉积进行放大），以及使用对纤维状结构具有亲和力的荧光染料硫堇红（TR）。观察三重荧光图像后，随后用加利亚斯-布拉克法对切片进行染色。对患有多系统萎缩（MSA）且病程不同（4 - 15年）的患者的壳核、小脑白质和运动皮层切片进行加利亚斯阳性GCI的这些染色情况的定量分析。尽管大多数GCI对Ub呈阳性，对αS呈不同程度的阳性，但它们对TR始终呈阴性。在路易体病的LB中结果则相反，LB对TR具有不同的亲和力，这表明GCI的结构与LB不同。如先前报道，仅这四种染色特征（αS、Ub、TR和加利亚斯）未能显示出与病程、病变部位或变性严重程度有明显相关性。然而，αS阴性和Ub阳性的GCI比例沿疾病进展呈线性增加，而αS阳性和Ub阴性的GCI比例则相反下降。这种相互变化表明，在疾病进展过程中，GCI中的αS免疫反应性正被Ub免疫反应性所取代，这导致在最晚期病例中αS阴性和Ub阳性的GCI最终占主导地位。有趣的是，αS阴性和Ub阳性的GCI占主导是运动皮层的一个特征，尽管加利亚斯阳性的GCI外观明显，但该区域的变性通常仍较轻。另一部分，αS阳性和Ub阳性的GCI在小脑白质中很常见，这表明GCI的演变是异质性的，并且还取决于所检查的区域。Ub的逐渐积累伴随着αS表位的消失及其共定位，部分与LB共享，可能代表了GCI形成的一个过程，可能与MSA中的变性方面有关。
