Regulations of organellar Zn2+ homeostasis and dynamics by TRPML1 in neurons

不稳定的、无结合的锌离子在正常大脑功能中的重要性已被证明与 抑郁症、阿尔茨海默病等一系列神经系统疾病与细胞内锌离子分布的异常 阿尔茨海默病(AD)、衰老、粘膜病IV型(MLIV)和帕金森氏病。MLIV是一种溶酶体 具有神经退行性表型的储藏症在儿童时期发展起来。引起基因突变的 MLIV已在TRPML1基因中被鉴定，该基因编码一种对阳离子具有通透性的溶酶体通道 作为钙离子和锌离子。TRPML1功能丧失导致成纤维细胞溶酶体锌离子升高 MLIV患者。然而，这种锌离子的分子机制和生物影响都不是很明显。 不规范是可以理解的。我们的初步研究设计了一种新颖的传感器GZnP3，具有前所未有的 在纳米分子范围内的灵敏度，并提供了第一个深刻的证据，TRPML1可以动员锌从 神经元中的溶酶体和晚期内体进入胞浆。这种内溶酶体释放的锌离子在神经元中是独一无二的 神经突起中的锌离子信号比胞体中的信号强得多。此外，我们还揭示了纳米分子 锌离子可减少神经元的轴突运输。根据初步证据，我们推测 囊泡细胞器中锌离子释放受损可导致神经变性，并有助于 MLIV的发病机制。我们将利用MLIV细胞模型和我们独特的基因靶向探针来 测试我们的假设，并解决三个具体目标：(1)我们将量化和比较锌离子浓度 在正常细胞模型和人类MLIV细胞模型中建立的各种亚细胞隔间 成纤维细胞和大鼠海马神经元；(2)我们将利用我们的敏感探针来研究它们之间的相关性 MLIV病患者体内锌离子释放受损与转运机制的关系 (3)我们将检验我们的假设，即： TRPML1通过介导锌离子从溶酶体释放到胞浆来调节神经元的健康和功能： 释放的锌离子信号可以调节轴突运输，降低的溶酶体锌离子可以恢复自噬 溶酶体的功能。总的来说，这项拟议的研究将提供超灵敏的工具来监测细胞 锌离子动力学，更好地了解细胞器中锌离子分布和动力学的变化 在MLIV中，表征了溶酶体内锌释放与MLIV的相关性，并揭示了 TRPML1介导的锌离子动力学对神经元功能的影响。所有这些都将极大地扩展我们的 了解MLIV病的病理机制。

The importance of labile, unbound Zn2+ in normal brain function has been evidenced by the association of abnormal cellular Zn2+ distributions with a series of neurological diseases such as depression, Alzheimer's disease (AD), aging, Mucolipidosis type IV disease (MLIV), and Parkinson’s diseases. MLIV is a lysosomal storage disease with neurodegenerative phenotype developed in childhood. The genetic mutations causing MLIV have been identified in the gene TRPML1, which encodes a lysosomal channel permeable to cations such as Ca2+and Zn2+. Loss of TRPML1 function resulted in elevated lysosomal Zn2+ in the fibroblasts derived from MLIV patients. However, neither the molecular mechanisms nor the biological impacts of such Zn2+ dysregulations are understood. Our preliminary studies devised a novel sensor GZnP3 with unprecedented sensitivity in the nanomolar range and provided the first profound evidence that TRPML1 can mobilize Zn2+ from lysosomes and late endosomes to the cytosol in neurons. Such endolysosomal Zn2+ release is unique in neurons and yields much greater Zn2+ signals in neurites than in the soma. Furthermore, we revealed that nanomolar Zn2+ can reduce the axonal transport in neurons. Based on the preliminary evidence, we hypothesize that impaired Zn2+ release from the vesicular organelles can cause neurodegeneration and contribute to the pathogenesis of MLIV. We will utilize the MLIV cell models along with our unique genetically targeted probes to test our hypothesis and address three specific aims: (1) We will quantify and compare the Zn2+ concentrations among various subcellular compartments in normal and MLIV cell models that are established in human fibroblasts and rat hippocampal neurons; (2) We will utilize our sensitive probes to investigate the correlation between impaired endolysosomal Zn2+ release with MLIV disease and determine the transport mechanisms that concentrate high pools of Zn2+ into endolysosomal vesicles in neurons; (3) We will examine our hypothesis that TRPML1 regulates neuronal health and function by mediating Zn2+ release from lysosomes to the cytosol: the released Zn2+ signals can regulate axonal transport and reduced lysosomal Zn2+ can recover the autophagic function of lysosomes. Collectively, the proposed research will provide ultra sensitive tools for monitoring cellular Zn2+ dynamics, develop a better understanding about the changes in organellar Zn2+ distributions and dynamics in MLIV, characterize the correlation between endolysosomal Zn2+ release and MLIV, as well as reveal the impacts of TRPML1-mediated Zn2+ dynamics on neuronal function. All of the above will significantly expand our knowledge about the pathological mechanisms of MLIV disease.