AUTOPHAGY IN GLIA AND NEURONS

神经胶质细胞和神经元中的自噬

• 批准号: 3417112
• 负责人: WILLIAM A DUNN
• 金额: $ 12.66万
• 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 1994-09-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3417112
• 关键词: astrocytes; autophagy; cell population study; cerebral cortex; dogs; 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.

神经性蜡样脂褐素沉着症(NCL)、脑和视网膜萎缩 据报道与在 大脑的星形胶质细胞和神经元以及有色上皮 视网膜。除了未降解的蛋白质外，这些结构还包括 溶酶体酸水解酶，通常在 粗面内质网(RER)和细胞的c-亚基 线粒体ATP合成酶。从我们最近的研究来看，很可能 自噬负责这些细胞成分的传递。 向包含体致敬。自噬是一个正常的过程，细胞 成分(即线粒体)被隔离在RER衍生的体内 液泡，并最终在这些液泡与 溶酶体。因此，我们提出以下基本假设： NCL细胞内包涵体的积聚与 自噬介导的蛋白质降解的变化。测试我们的 假设，我们将研究星形胶质细胞和 从正常和NCL英语二传种分离出神经细胞。房租 正常星形胶质细胞和神经细胞蛋白质降解的研究 和NCL英语二传手将在下列条件下进行测量 增强或抑制自动噬菌体(AIM#1)。培养细胞，其 以前用14C标记的蛋白质将被孵育 在含或不含氨基酸的介质中和酸溶的释放 放射性随着时间的推移而被量化。退化的速度将是 与已确定的NCL包涵体的出现相关 从形态上看。包涵体的异常沉积可能是 由于自噬增加或溶酶体内减少 蛋白质分解或两者兼而有之。因此，我们将测量形成率 自噬空泡的数量和溶酶体内降解率(AIM #2)。分数体积的增加由 自噬小体将根据时间以形态计量学的方式量化 用氨基酸耗尽的培养液孵育。的相对比率 溶酶体内降解将通过测量释放的 从细胞中分离的自噬溶酶体中的14C-Valine 先前在氨基酸耗尽的培养液中孵育。最后，我们会 比较自噬空泡和包裹体的特征 身体(目标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) 通常在自噬小体中发现。或者，我们将评估 无论三磷酸腺苷合成酶的c-亚基，通常存在于 线粒体和NCL包涵体，存在于自噬小体和 自噬溶酶体。这些信息将使我们能够评估该模式 C-亚基进入(例如，自噬)这些包涵体。 我们提出的研究将为我们提供新的机制和 正常和病变星形胶质细胞蛋白质降解的调节 神经细胞。