Excessive Lysosomal Exocytosis Triggers Pathogenic Mechanisms in Sialidosis Mice

描述（由申请方提供）：影响溶酶体代谢的遗传病变改变细胞和组织稳态，并影响多种生理过程，如溶酶体贮积病（LSD）的复杂多器官表型所记录。本研究的长期范围是深入了解唾液酸中毒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NEU 1的缺乏有关。NEU 1通过去除末端唾液酸来启动唾液酸-糖缀合物的水解。损失 NEU 1活性的降低导致其底物的过度唾液酸化，这反过来可以改变其生化特性和功能。本申请的重点是剖析NEU 1作为一种新发现的溶酶体胞吐（LEX）的生理过程的负调节剂的作用，并检验这一假设，即NEU 1功能丧失导致的过量LEX是唾液酸沉积症特征性的全身和神经系统异常的常见致病决定因素。这项研究的关键是发现NEU 1控制LEX的程度， 通过调节其底物之一LAMP 1的唾液酸含量。在Neu 1 r/r细胞中，用过度唾液酸化的Lamp 1标记的溶酶体更容易停靠在质膜上，并在钙离子流入时参与LEX。我们推测，溶酶体内容物过度释放到细胞外空间改变了细胞质膜和细胞外基质的组成，对许多器官的完整性和功能产生有害后果。我们建议在突变小鼠（即Neu 1 r/r小鼠，一种准确的唾液酸中毒临床前模型）的一系列研究中测试这种模式。在目标1中，我们将确定过量LEX下游的生化和分子效应物，这些效应物导致Neu 1 r/r小鼠肌肉结缔组织的进行性扩张和随后的肌肉变性。在目标2中，我们将确定导致Neu 1 r/r脑海马区淀粉样小体逐步形成的因素，这类似于阿尔茨海默病样神经退行性表型。在目标3中，我们提出了一系列的生化方法来确定是否LAMP 1在运输溶酶体到质膜中起主要作用，如果是这样，这个过程是如何发生的。我们相信，本文提出的实验将深入了解以前未发现的功能，溶酶体NEU 1超越基本的溶酶体降解。我们还预计这些研究的结果将突出唾液酸中毒发病机制的新方面，这些方面有可能推动这种毁灭性儿童疾病替代疗法的开发。

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.