Snow A D, Sekiguchi R T, Nochlin D, Kalaria R N, Kimata K
Department of Pathology, University of Washington, Seattle 98195.
Am J Pathol. 1994 Feb;144(2):337-47.
Previous studies have shown the basement membrane form of heparan sulfate proteoglycan (HSPG) known as perlecan, co-localized to beta-amyloid protein (A beta)-containing amyloid deposits in brains of patients with Alzheimer's disease (AD) and Down's syndrome. Although HSPG was localized to diffuse A beta plaques in hippocampus, amygdala, and neocortex, it is not known whether they are present in diffuse A beta plaques in cerebellum. In the present study, Alcian blue staining and immunocytochemical techniques were used to determine whether highly sulfated glycosaminoglycans (GAGs) and/or HSPG (perlecan) were also present in diffuse A beta plaques of cerebellum. Tissues from cases of AD were examined for the co-localization of highly sulfated GAGs, HSPGs, and A beta in diffuse plaques in cerebellum in comparison with hippocampus. Consecutive serial sections of AD brain tissue were stained or immunostained with 1) the modified Bielschowsky stain; 2) a polyclonal antibody directed against synthetic A beta (1-40); 3) Congo red; 4) Alcian blue (pH 5.7) with varying concentrations of magnesium chloride for identification of sulfated and highly sulfated GAGs; and 5) polyclonal and monoclonal antibodies recognizing either the core protein or a specific GAG epitope on perlecan. All cases (7 of 7) of AD contained diffuse A beta plaques in the cerebellum as identified by positive Bielschowsky staining and A beta immunoreactivity. None of these cases demonstrated positive Alcian blue staining (at 0.3 and 0.7 mol/L MgCl2), HSPG, or HS GAG immunoreactivity in the same diffuse cerebellar plaques on adjacent serial sections. However, Alcian blue staining, HSPG, and/or HS GAG immunoreactivity were observed in blood vessel walls, choroid plexus, and within Purkinje cells, suggesting that the techniques used were reliable and specific. In cerebellum, all plaques containing amyloid cores that were Congo red-positive were also positive for highly sulfated GAGs (by Alcian blue staining at 0.7 mol/L MgCl2) and HSPG (both core protein and GAG chain) immunoreactivity. Even though HSPG immunoreactivity was not present in cerebellar diffuse plaques, all cases (4 of 4) examined demonstrated HSPG (both core protein and GAG chain) immunoreactivity in diffuse A beta plaques in hippocampus. Therefore, by Alcian blue staining and immunocytochemical methods, highly sulfated GAGs and HSPGs are not present in A beta diffuse plaques in cerebellum. Since previous studies indicate that the cerebellum contains relatively few amyloid-containing plaques in comparison with diffuse plaques, these studies suggest that HSPG may be an essential component needed for amyloid formation and/or persistence in brain as observed in cortical areas.(ABSTRACT TRUNCATED AT 400 WORDS)
先前的研究表明，硫酸乙酰肝素蛋白聚糖（HSPG）的基底膜形式即基底膜聚糖，与阿尔茨海默病（AD）和唐氏综合征患者大脑中含β淀粉样蛋白（Aβ）的淀粉样沉积物共定位。尽管HSPG定位于海马体、杏仁核和新皮质中的弥漫性Aβ斑块，但尚不清楚它们是否存在于小脑中的弥漫性Aβ斑块中。在本研究中，使用阿尔新蓝染色和免疫细胞化学技术来确定高度硫酸化的糖胺聚糖（GAG）和/或HSPG（基底膜聚糖）是否也存在于小脑的弥漫性Aβ斑块中。将AD病例的组织与海马体进行比较，检查小脑弥漫性斑块中高度硫酸化的GAG、HSPG和Aβ的共定位情况。AD脑组织的连续系列切片用以下方法进行染色或免疫染色：1）改良的 Bielschowsky 染色；2）针对合成 Aβ（1-40）的多克隆抗体；3）刚果红；4）不同浓度氯化镁的阿尔新蓝（pH 5.7），用于鉴定硫酸化和高度硫酸化的GAG；5）识别基底膜聚糖核心蛋白或特定GAG表位的多克隆和单克隆抗体。所有AD病例（7例中的7例）小脑均含有弥漫性Aβ斑块，通过 Bielschowsky 染色阳性和Aβ免疫反应性得以确认。在相邻系列切片的相同小脑弥漫性斑块中未发现这些病例有阿尔新蓝染色阳性（0.3和0.7 mol/L MgCl2）、HSPG或HS GAG免疫反应性。然而，在血管壁、脉络丛和浦肯野细胞内观察到阿尔新蓝染色、HSPG和/或HS GAG免疫反应性，这表明所使用的技术是可靠且特异的。在小脑中，所有含有刚果红阳性淀粉样核心的斑块对高度硫酸化的GAG（通过0.7 mol/L MgCl2的阿尔新蓝染色）和HSPG（核心蛋白和GAG链）免疫反应性也呈阳性。尽管HSPG免疫反应性不存在于小脑弥漫性斑块中，但所有检查的病例（4例中的4例）海马体弥漫性Aβ斑块中均显示出HSPG（核心蛋白和GAG链）免疫反应性。因此，通过阿尔新蓝染色和免疫细胞化学方法，小脑Aβ弥漫性斑块中不存在高度硫酸化的GAG和HSPG。由于先前的研究表明，与弥漫性斑块相比，小脑中含淀粉样蛋白的斑块相对较少，这些研究表明HSPG可能是大脑皮质区域中观察到的淀粉样蛋白形成和/或持续存在所需的重要成分。（摘要截短于400字）
