AUTOPHAGY IN GLIA AND NEURONS

神经胶质细胞和神经元中的自噬

• 批准号: 2268229
• 负责人: WILLIAM A DUNN
• 金额: $ 13.26万
• 依托单位: UNIVERSITY OF FLORIDA
• 依托单位国家: 美国
• 财政年份: 1991
• 资助国家: 美国
• 起止时间: 1991-09-30 至 1995-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2268229
• 关键词: astrocytes; autophagy; cell population study; cerebral cortex; dogs; endoplasmic reticulum; hydrogen transporting ATP synthase; inclusion body; laboratory rat; membrane proteins; neuronal ceroid lipofuscinosis; neurons; protein degradation; tissue /cell culture

In neuronal ceroid lipofuscinosis (NCL), cerebral and retinal atrophy has been reported coincident with abnormal inclusion bodies found in astrocytes and neurons of the brain and pigmented epithelium of the retina. In addition to undegraded proteins, these structures contain lysosomal acid hydrolases, dolichyl oligosaccharides normally found in rough endoplasmic reticulum (RER), and the c-subunit of the mitochondrial ATP synthase. From our recent studies, it is likely that autophagy is responsible for the delivery of these cellular components to the inclusion bodies. Autophagy is a normal process whereby cellular components (ie, mitochondria) are sequestered within RER-derived vacuoles and ultimately degraded following fusion of these vacuoles with lysosomes. Therefore, we propose the following Primary Hypothesis: The accumulation of inclusion bodies within NCL cells is coincident with alterations in autophage-mediated protein degradation. To test our hypothesis, we will examine the autophagic response in astroglial and neuronal cells isolated from normal and NCL English setters. The rates of protein degradation in astroastroglial and neuronal cells from normal and NCL English setters will be measured under conditions whereby autophage is enhanced or inhibited (AIM #1). Cultured cells whose proteins have been previously labeled with 14C-valine will be incubated in media with or without amino acids and the release of acid-soluble radioactivity quantified over time. The rates of degradation will be correlated to the occurrence of the NCL inclusion bodies identified morphologically. The abnormal deposition of the inclusion bodies may be due to either an increase in autophagy or a decrease in intralysosomal proteolysis or both. Therefore, we will measure the rate of formation of autophagic vacuoles and the rate of intralysosomal degradation (AIM #2). The increase in the fractional volume represented by autophagosomes will be quantified morphometrically in respect to time of incubation with amino acid depleted medium. Relative rates of intralysosomal degradation will be estimated by measuring the release of 14C-valine from autophagolysosomes which had been isolated from cells previously incubated in amino acid depleted medium. Finally, we will compare the characteristics of autophagic vacuoles to those of inclusion bodies (AIM #3). Specifically, we will determine whether or not the inclusion bodies are acidic and contain lysosomal proteins (eg, cathepsin D, lysosomal membrane proteins, and superoxide dismutase) normally found in autophagolysosomes. Alternatively, we will evaluate whether or not the c-subunit of the ATP synthase, normally found in mitochondria and NCL inclusion bodies, is present in autophagosomes and autophagolysosomes. This information will allow us to evaluate the mode of entry (eg, autophagy) of the c-subunit into these inclusion bodies. The studies we propose will provide new insights into the mechanisms and regulation of protein degradation in normal and diseased astroglial and neuronal cells.

在神经元蜡样质脂褐质沉积症（NCL）中， 据报道，与发现的异常包涵体一致， 大脑的星形胶质细胞和神经元以及大脑的色素上皮 视网膜。 除了未降解的蛋白质外，这些结构还含有 溶酶体酸水解酶，长囊寡糖通常存在于 粗面内质网（RER），和C-亚单位的 线粒体ATP合酶。 从我们最近的研究来看， 自噬负责这些细胞成分的传递 到包涵体。 自噬是一个正常的过程， 成分（即线粒体）被隔离在RER衍生的 空泡，并最终降解后，这些空泡融合， 溶酶体 因此，我们提出以下基本假设： NCL细胞内包涵体的积累与 自噬介导的蛋白质降解的改变。 来测试我们 假设，我们将研究星形胶质细胞和 神经元细胞分离自正常和NCL英国塞特犬。 率 蛋白质降解的星形胶质细胞和神经元细胞从正常 和NCL英语二传手将在以下条件下进行测量， 自噬被增强或抑制（AIM#1）。 培养的细胞， 将孵育先前用14 C-缬氨酸标记的蛋白质 在含有或不含氨基酸的培养基中， 随时间定量的放射性。 降解速率将是 与鉴定的NCL包涵体的发生相关 形态学上。 包涵体的异常沉积可能是 由于自噬的增加或溶酶体内 蛋白水解或两者。 因此，我们将测量 自噬泡的数量和溶酶体内降解率（AIM #2）。 体积分数的增加由 自噬体将在形态学上相对于时间进行定量， 用氨基酸耗尽培养基孵育。 的相对速率 将通过测量以下物质的释放来估计溶酶体内降解 从细胞中分离的自噬溶酶体中的14 C-缬氨酸 先前在氨基酸耗尽的培养基中孵育。 最后我们将 比较自噬泡和包涵体的特征 体（AIM #3）。 具体来说，我们将确定是否 包涵体是酸性的并含有溶酶体蛋白（例如， 组织蛋白酶D、溶酶体膜蛋白和超氧化物歧化酶） 通常存在于自噬溶酶体中。 或者，我们将评估 无论是否是ATP合酶的C-亚基，通常存在于 线粒体和NCL包涵体，存在于自噬体中， 自噬溶酶体 这些信息将使我们能够评估模式 c亚单位进入这些包涵体的过程（如自噬）。 我们提出的研究将为机制提供新的见解， 调节正常和患病星形胶质细胞中的蛋白质降解， 神经元细胞