NEURONAL CELL DEATH IN A LATE-ONSET GENETIC DISORDER IN MICE

在识别特定的神经系统方面已经取得了重大进展 在阿尔茨海默病和相关疾病中退化;然而， 关于神经元的分子机制知之甚少 退化本身。我们最近的发现涉及蛋白质水解，我们 除了介导神经元的晚期阶段外， 变性，蛋白水解酶系统的活性改变可能是 代谢受损神经元的早期神经元反应，可能是 在神经元受到不可逆的损伤之前，我们 总体目标是研究形态学和分子方面的 神经元中的两个主要蛋白水解系统--溶酶体和 钙激活中性蛋白酶（CANP）系统-为了澄清 特异性蛋白酶改变与结构变化的关系 在正常衰老过程中伴随细胞萎缩和死亡， 小鼠迟发性神经退行性疾病。使用光学显微镜和 电子显微镜方法，我们将检查细胞和亚细胞 与小鼠退化进行性阶段相关的组织病理学 表达突变Mnd（运动神经元变性）。最近开发 抗主要脑蛋白酶CANP，组织蛋白酶B，D，G， 一个L--并针对CANP的两种特异性内源性抑制剂和一系列 非蛋白酶溶酶体酶将与 计算机图像分析，以评估内容和分布 这些抗原在正常外观和退化中的免疫细胞化学 脆弱细胞群中的神经元。选定的活动 溶酶体酶将通过酶细胞化学技术测定 应用于光和超微结构水平。改变的间接证据 将使用抗体探针寻找体内CANP活性，以怀疑 CANP的蛋白底物。3-30月龄小鼠的平行研究 将从正常老化过程中区分出退化特征， 阐明突变缺陷与年龄相关因素的相互作用。到 评估这些蛋白酶和调节蛋白，cDNA的合成 这些蛋白水解组分的cRNA探针将用于评估 在对照组、突变组和对照组的大脑中， 老年小鼠原位杂交。除了增加我们的 了解蛋白质水解的重要和复杂的过程，在正常 成熟和老化的神经元，这些研究将提供相关的信息 神经元功能障碍和细胞死亡的分子基础， 阿尔茨海默病和其他迟发性人类神经退行性疾病。

Major advances have been made toward identifying specific neuronal systems that degenerate in Alzheimer's disease and related disorders; however, little is known about the molecular mechanisms underlying the neuronal degeneration itself. Our recent findings implicate proteolysis, and we propose that, in addition to mediating late stages of neuronal degeneration, altered activity of proteolytic enzyme systems may be an early neuronal sponse in metabolically compromised neurons and may underlie functional deficits in neurons before they are irreversibly injured. Our overall objectives are to investigate morphologic and molecular aspects of the two major proteolytic systems in neurons--lysosomes and the calcium-activated neutral proteinase (CANP) system--in order to clarify relationship of specific proteinase alterations to the structural changes that accompany cell atrophy and death during normal aging and in a genetic, late-onset neurodegenerative disorder in mice. Using light microscopic and electron microscopic methods, we will examine the cellular and subcellular histopathology associated with progressive stages of degeneration in mice expressing the mutation Mnd (motor neuron degeneration). Recently developed antibodies against the major brain proteinases--CANP, cathepsins B, D, G, an L--and against two specific endogenous inhibitors of CANP and a series of non-proteinase lysosomal enzymes will be used in conjunction with computerized image analysis to evaluate the content and distribution of these antigens immunocytochemically in normal-appearing and degenerating neurons within vulnerable cell populations. The activities of selected lysosomal enzymes will be determined by enzyme cytochemical techniques applied the light and ultrastructural levels. Indirect evidence for altered CANP activity in vivo will be sought using antibody probes to suspected protein substrates of CANP. Parallel studies in mice at 3-30 months of age will distinguish degenerative features from processes of normal aging and clarify the interaction of the mutation defect with age related factors. To evaluate the synthesis of these proteinases and regulatory proteins, cDNA and cRNA probes to these proteolytic components will be used to assess levels of specific mRNAs within neurons in brains of control, mutant and aged mice by in situ hybridization. In addition to increasing our understanding of the important and complex process of proteolysis in normal mature and aging neurons, these studies will provide information pertinent to the molecular basis of neuronal dysfunction and cell death in Alzheimer's disease and other late-onset human neurodegenerative disorders.