Excessive Lysosomal Exocytosis Triggers Pathogenic Mechanisms in Sialidosis Mice

描述(申请人提供)：影响溶酶体代谢的遗传损伤会改变细胞和组织的动态平衡，并影响多种生理过程，如溶酶体储存疾病(LSD)的复杂多器官表型所证明的那样。这项研究的长期范围是深入了解唾液酸病的发病机制的分子基础，严重的神经退行性LSD与溶酶体唾液酸酶NEU1缺陷有关。NEU1通过去除唾液酸端的唾液酸来启动唾液酸糖共轭化合物的水解反应。损失 NEU1活性的改变会导致其底物过度分解，进而改变其生化特性和功能。本应用的重点是剖析NEU1作为新发现的溶酶体胞吐作用(Lex)生理过程的负性调节因子的作用，并检验由NEU1功能丧失引起的Lex过多是涎症特征的全身和神经异常的常见致病决定因素的假说。这项研究的关键是发现NEU1控制着Lex的范围 通过调节其底物之一LAMP1的唾液酸含量。在Neu1r/r细胞中，被过唾液酸化的LAMP1标记的溶酶体更容易停靠在质膜上，并在钙内流时参与Lex。我们假设，溶酶体内容物过度释放到细胞外空间会改变细胞质膜和细胞外基质的组成，从而对许多器官的完整性和功能造成有害后果。我们建议在突变小鼠，即Neu1r/r小鼠，一种准确的唾液酸病临床前模型中，测试这一范式。在目标1中，我们将确定过量Lex下游的生化和分子效应，这些效应导致Neu1r/r小鼠肌肉结缔组织的进行性扩张和随之而来的肌肉退化。在目标2中，我们将确定那些导致Neu1r/r脑的海马区淀粉样变小体进行性形成的因素，这类似于阿尔茨海默病样神经退行性变的表型。在目标3中，我们提出了一系列生化方法来确定LAMP1是否在将溶酶体运输到质膜上起主要作用，如果是的话，这一过程是如何发生的。我们相信，这里提出的实验将深入了解溶酶体NEU1在基本溶酶体降解之外以前未被发现的功能。我们还预计，这些研究的结果将突出涎症发病机制的新方面，这些方面有可能推动这种毁灭性的儿童疾病的替代疗法的开发。

DESCRIPTION (provided by applicant): Genetic lesions affecting lysosomal metabolism alter cell and tissue homeostasis and affect a multitude of physiological processes, as documented by the complex multiorgan phenotypes of lysosomal storage diseases (LSDs). The long-term scope of this study is to gain insight into the molecular bases of the pathogenesis of sialidosis, severe neurodegenerative LSD linked to the deficiency of the lysosomal sialidase NEU1. NEU1 initiates the hydrolysis of sialo-glycoconjugates by removing their terminal sialic acids. The loss of NEU1 activity results in oversialylation of its substrates, which in turn, can change their biochemical properties and function. The focus of this application is to dissect the role of NEU1 as a newly identified negative regulator of the physiological process of lysosomal exocytosis (LEX) and to test the hypothesis that excessive LEX, resulting from NEU1 loss of function, is the common pathogenic determinant of the systemic and neurological abnormalities that are characteristic of sialidosis. Key to this study is the finding that NEU1 controls the extent of LEX by modulating the sialic acid content of one of its substrates, LAMP1. In Neu1r/r cells, lysosomes that are tagged with oversialylated Lamp1 are more prone to dock at the plasma membrane and engage in LEX upon calcium influx. We hypothesize that the excessive release of lysosomal contents into the extracellular space changes the composition of cells' plasma membranes and the extracellular matrix with deleterious consequences on the integrity and function of many organs. We propose to test this paradigm in a series of studies in mutant mice, namely Neu1r/r mice, an accurate preclinical model of sialidosis. In Aim 1, we will identify the biochemical and molecular effectors downstream of excessive LEX that cause the progressive expansion of muscle connective tissue and consequent muscle degeneration in Neu1r/r mice. In Aim 2, we will identify those factors that cause the progressive formation of amyloidogenic bodies in the hippocampal region of the Neu1r/r brain, which resembles the Alzheimer diseaserlike neurodegenerative phenotype. In Aim 3, we propose a series of biochemical approaches to determine whether LAMP1 plays a primary role in trafficking lysosomes to the plasma membrane and, if so, how this process occurs. We believe that the experiments proposed herein will give insight into previously undiscovered functions of lysosomal NEU1 beyond basic lysosomal degradation. We also expect that the results from these studies will highlight new aspects of the pathogenesis of sialidosis that have the potential to advance the development of alternative therapies for this devastating childhood disease.