STAGE SPECIFIC EMBRYONIC ANTIGEN-1 GLYCOCONJUGATES IN DEVELOPING RAT CEREBELLUM

大鼠小脑发育阶段特异性胚胎抗原-1 糖复合物

• 批准号: 6301842
• 负责人: ROBERT H MC CLUER
• 金额: $ 11.91万
• 依托单位: EUNICE KENNEDY SHRIVER CTR MTL RETARDATN
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-12-01 至 2000-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6301842
• 关键词: adeno associated virus group; cerebellum; developmental neurobiology; embryo /fetus antigen; embryo /fetus cell /tissue; enzyme activity; fucose; genetic promoter element; glycolipids; glycoprotein biosynthesis; glycoproteins; glycosphingolipids; hexosyltransferase; immunocytochemistry; in situ hybridization; laboratory rat; lipid biosynthesis; newborn animals; northern blottings; polymerase chain reaction

Evidence that fucosylated glycoconjugates play a significant role in brain development is suggested by reports of severe mental retardation in patients that lack fucose residues. However, the mechanisms by which fucosylated glycoconjugates influence brain development are not known. The epitope 3-fucosyl-N-acetylactosamine (SSEA-1) is an excellent candidate to play a vital role in brain development, both because it is expressed in developing brain with temporal and spatial specificity, and because it has been shown to have a key role in cell-cell interactions in other tissues. Thus, we postulate that SSEA-1 plays a key role in the cell-cell interactions required for normal neural development. Our preliminary results indicate that SSEA-1 glycolipid expression in neonatal cerebellum correlates with granule cell migration, and the goal of this project is to elucidate the role of these molecules in this process. Specifically, we will characterize and measure SSEA-1 glycosphingolipids and proteins in developing rat cerebellum and relate these data to the stage and regional immunohistochemical appearance of this antigen. We also will characterize the fucosyltransferase activities involved in SSEA-1 glycoconjugate synthesis during cerebellar development, as our data suggests fucosyltransferase(s) play a critical role in controlling SSEA-1 glycolipid expression. Simultaneously, we will characterize the genes responsible for these fucosyltransferase activities and evaluate their regulation during development. Results obtained from these studies will then enable us to investigate the biological role of SSEA-1 glycoconjugates. We propose to determine whether overexpression, or blocking the accessibility, of SSEA-1 antigens in the neonatal cerebellum would result in anomalous behavior of developing granule cells. To cause over expression of SSEA-1, we propose to inject intracerebrally neonatal rats with AAV vectors carrying an epitope-tagged form of the relevant cloned rat fucosyltransferase under the control of a strong non-tissue specific promoter. To block the function of SSEA-1, we plan to inject irradiated hybridoma cells producing anti-SSEA-1 antibodies. These brains will then be examined histologically during subsequent development to establish the degree of fucosyltransferase and SSEA-1 expression and to determine whether perturbing SSEA-1 expression or availability changes the normal pattern of granule cell migration, parallel fiber extension and/or synaptogenesis.

岩藻糖基化糖复合物在 严重智力迟钝的报告表明大脑发育 缺乏岩藻糖残基的患者。 然而， 岩藻糖基化的糖缀合物影响脑发育是未知的。 表位3-岩藻糖基-N-乙酰乳糖胺（SSEA-1）是一种极好的抗肿瘤药物。 候选人在大脑发育中发挥至关重要的作用，因为它是 在发育中的大脑中表达，具有时间和空间特异性， 因为它在细胞间相互作用中起着关键作用 在其他组织中。 因此，我们假设SSEA-1在 正常神经发育所需的细胞间相互作用。 我们 初步结果表明，SSEA-1糖脂的表达， 新生儿小脑与颗粒细胞迁移相关， 这个项目的目的是阐明这些分子在这一过程中的作用。 过程 具体来说，我们将表征和测量SSEA-1 大鼠小脑发育过程中鞘糖脂和蛋白质变化及其相关性 这些数据的阶段和区域免疫组化表现， 这个抗原。 我们还将表征岩藻糖基转移酶 小脑期间参与SSEA-1糖缀合物合成的活动 发展，因为我们的数据表明岩藻糖基转移酶（s）发挥关键作用 控制SSEA-1糖脂表达的作用。 同时我们 将描述负责这些岩藻糖基转移酶的基因 活动，并评估其在发展过程中的监管。 结果 从这些研究中获得的信息将使我们能够调查 SSEA-1糖缀合物的生物学作用。 我们建议确定 无论是SSEA-1抗原的过表达还是阻断其可及性 新生儿小脑的异常会导致 发育中的颗粒细胞 为了引起SSEA-1的过度表达，我们建议 向新生大鼠脑内注射携带 表位标记形式的相关克隆大鼠岩藻糖基转移酶， 强非组织特异性启动子的控制。 阻止 SSEA-1的功能，我们计划将照射过的杂交瘤细胞 产生抗SSEA-1抗体。 这些大脑将被检查 在随后的发育过程中进行组织学检查，以确定 岩藻糖基转移酶和SSEA-1表达，并确定是否 干扰SSEA-1表达或可用性改变正常模式， 颗粒细胞迁移，平行纤维延伸和/或 突触发生