Cellular mechanism of Adult Onset Neuronal Ceroid Lipofuscinosis (ANCL) caused by mutations in cysteine string protein-alpha (CSPalpha).

项目总结/摘要 成人型神经元蜡样质脂褐质沉积症（ANCL）是一种致死性溶酶体沉积病， 治疗和未知的发病机制。拟议研究的目的是描绘莫- 半胱氨酸串蛋白-α（CSPα）突变引起ANCL的理论机制， 可以挽救分子功能障碍。最近，一些研究发现ANCL引起的突变， CSPα基因。然而，CSPα中的ANCL突变究竟如何导致溶酶体功能障碍和神经元损伤， 死亡仍不清楚。以前，CSPα被证明是突触陷阱蛋白SNAP-25的伴侣。我们 现在发现SNAP-25同源物SNAP-23也由CSPα陪伴。SNAP-23是一种 SNARE蛋白介导溶酶体胞吐作用，提供CSPα功能障碍与 溶酶体病理学我们已经获得了令人兴奋的初步数据，显示：a）CSPα中的ANCL突变破坏了 其棕榈酰化和膜缔合，以及因此其与SNAP-23的结合; B）在CSPα敲除小鼠中 在大脑和神经元中，SNAP-23蛋白水平显著降低，在溶酶体陷阱复合物中， c）CSPα基因敲除小鼠大脑显示出过早的适应性， 脂褐质的模拟，随后神经变性;和d）SNAP-23（而不是SNAP-25）的敲低， 诱导溶酶体胞吐作用并导致WT神经元中脂褐素积累。这些数据导致了 中心假设，CSPα中的ANCL突变破坏了SNAP-23的伴侣作用，导致ly- sosomal胞吐，并导致ANCL中的脂褐质沉积，这些缺陷可以通过化学治疗来挽救。 监护人为了检验这一假设，提出了三个具体目标：目标1。确定ANCL突变如何 CSPα影响SNAP-23稳定性和溶酶体胞吐作用。目标二。确定CSPα中的ANCL突变 导致溶酶体病理学和神经变性，在小鼠脑中体内和ANCL患者源性in- 诱导神经元（iNs）。目的3：确定拯救SNARE和溶酶体缺陷的药理学策略。 该提议在引入a）CSPα在溶酶体胞吐中的新的神经保护功能方面是创新的， 通过陪伴一种新的客户蛋白SNAP-23; B）一种新的概念，即溶酶体胞吐作用中的缺陷可以 有助于溶酶体储存病理学; c）使用多学科方法，包括ANCL的新模型 (e.g.患者来源的iN与通过CRISPR/Cas9基因编辑产生的同基因野生型iN，以及CSPα-/- 体内慢病毒表达ANCL突变体CSPα L115 R或CSPα L116 del的小鼠）。这一建议意义重大，因为- 这些研究的完成有望在分子水平上描述ANCL的病理级联反应， 水平（目标1）和体内（目标2），并评估有可能纠正分子水平（目标1）的药理学药物。 较大缺陷（目标3）。同时，这项工作将揭示一些基本的机制和功能， 神经元中溶酶体胞吐作用的发生。

PROJECT SUMMARY/ABSTRACT Adult onset neuronal ceroid lipofuscinosis (ANCL) is an invariably fatal lysosomal storage disease with no treatment and no known mechanism of pathogenesis. The objective of proposed studies is to delineate the mo- lecular mechanism of ANCL caused by mutations in cysteine string protein-α (CSPα), and then pharmacologi- cally rescue the molecular dysfunction. Recently, several studies have found ANCL-causing mutations in the gene encoding CSPα. Yet, exactly how ANCL mutations in CSPα lead to lysosomal dysfunction and neuron death remains unclear. Previously, CSPα was shown to chaperone the synaptic SNARE protein SNAP-25. We have now found that the SNAP-25 homolog SNAP-23 is also chaperoned by CSPα. Importantly, SNAP-23 is a SNARE protein which mediates lysosomal exocytosis, offering a direct connection from CSPα dysfunction to lysosomal pathology. We have obtained exciting preliminary data showing: a) ANCL mutations in CSPα disrupt its palmitoylation and membrane association, and thus its binding to SNAP-23; b) In CSPα-knockout mouse brains and neurons, there is significant reduction in SNAP-23 protein levels, in lysosomal SNARE-complex as- sembly, and in Ca2+-dependent lysosomal exocytosis; c) CSPα-knockout mouse brains reveal premature accu- mulation of lipofuscin followed by neurodegeneration; and d) Knockdown of SNAP-23 (not of SNAP-25) re- duces lysosomal exocytosis and leads to lipofuscin accumulation in WT neurons. These data have led to the central hypothesis that ANCL mutations in CSPα disrupt its chaperoning of SNAP-23, causing disruption of ly- sosomal exocytosis, and leading to lipofuscinosis in ANCL, and these defects can be rescued by chemical chaperones. To test this hypothesis, three specific aims are proposed: Aim 1. Determine how ANCL mutations in CSPα affect SNAP-23 stability and lysosomal exocytosis. Aim 2. Determine how ANCL mutations in CSPα lead to lysosomal pathology and neurodegeneration, in mouse brains in vivo and in ANCL patient-derived in- duced-neurons (iNs). Aim 3: Identify pharmacological strategies to rescue the SNARE and lysosomal defects. This proposal is innovative in introducing a) a new neuroprotective function of CSPα in lysosomal exocytosis, via chaperoning a new client protein, SNAP-23; b) a new concept that defects in lysosomal exocytosis can contribute to lysosomal storage pathology; c) using a multidisciplinary approach including new models of ANCL (e.g. patient-derived iNs with syngeneic wild type iNs generated via CRISPR/Cas9 gene-editing, and CSPα-/- mice lentivirally expressing ANCL mutants CSPαL115R or CSPαL116del in vivo). This proposal is significant be- cause completion of these studies is expected to delineate the pathological cascade of ANCL at the molecular level (Aim 1) and in vivo (Aim 2), and to evaluate pharmacological agents with potential to correct the molecu- lar defect(s) (Aim 3). At the same time, this work will reveal some of the fundamental mechanisms and func- tions of lysosomal exocytosis in neurons.