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

项目摘要/摘要 成人起病的神经元型蜡样脂褐素沉着症(ANCL)是一种致命性的溶酶体蓄积性疾病。 治疗方法不明，发病机制不明。拟议研究的目标是勾勒出以下几个方面： 半胱氨酸串蛋白α(CsPα)突变致ANCL的发病机制及药理学研究进展 Cally挽救分子功能障碍。最近，几项研究发现了导致ANCL的突变 编码Cspα的基因。然而，CSPα中ANCL突变究竟是如何导致溶酶体功能障碍和神经元的 死亡原因尚不清楚。此前，CspSNAP-25被证明是突触圈套蛋白α的伴侣。我们 现已发现SNAP-25同源物SNAP-23也由Cspα陪伴。重要的是，SNAP-23是一种 介导溶酶体胞吐的SNARE蛋白，提供了从CSPα功能障碍到 溶酶体病理学。我们已经获得了令人兴奋的初步数据，表明：a)CSPαDisrupt中的ANCL突变 它的棕榈酰化和膜结合，从而与SNAP-23结合；b)CSPα基因敲除小鼠 在脑和神经元中，SNAP-23蛋白水平显著降低，在溶酶体SNARE复合体中为- C)CSPα基因敲除小鼠脑内表现为早熟。 模拟脂褐素继而神经变性；以及d)SNAP-23(而不是SNAP-25)Re- 导致溶酶体胞吐，并导致脂褐素在WT神经元中积聚。这些数据导致了 中心假说是CSPANCL基因突变破坏了其对SNAP-23的伴随，导致了Ly-α的中断。 胞体胞吐，并导致ANCL的脂褐素沉积症，这些缺陷可以通过化学方法修复 监护人。为了验证这一假设，提出了三个具体的目标：目标1.确定ANCL如何突变 在CSP中，α影响SNAP-23的稳定性和溶酶体的胞吐作用。目的2.确定CSPα中的ANCL突变 导致溶酶体病理和神经变性，在体内的小鼠脑和在ANCL患者中-起源于 诱导神经元(INS)。目的3：确定拯救陷阱和溶酶体缺陷的药物策略。 这一建议的创新之处在于引入了a)Cspα在溶酶体胞吐中的一种新的神经保护功能， 通过伴随新的客户蛋白SNAP-23；b)溶酶体胞吐缺陷可以 促进溶酶体储存病理学；c)使用多学科方法，包括新的ANCL模型 (例如，通过CRISPR/CAS9基因编辑产生的具有同基因野生型INS的患者来源的INS，以及CSPα-/- 在体内慢病毒表达突变体CspαL115R或CspαL116del的小鼠)。这项建议具有重大意义，因为- 这些研究的完成有望在分子水平上描绘ANCL的病理级联。 水平(目标1)和体内(目标2)，并评价有潜力纠正分子- 大缺陷(S)(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研究。

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.