Disease causing mutations in cysteine string protein-alpha disrupt SNARE-dependent lysosomal exocytosis

项目摘要/摘要 成人起病的神经元型蜡样脂褐素沉积症(ANCL)是一种致命性的溶酶体蓄积性疾病，由两种 已知的编码半胱氨酸串蛋白α(Cspα)基因的显性突变：CspαL115R和CspαL116Δ。 Cspα与SGT(富含谷氨酰胺的小分子四肽重复序列蛋白)形成伴侣复合体 Hsp70/Hsc70(热休克蛋白/同源70 kDa)伴侣突触圈套蛋白SNAP-25。它 令人惊讶的是Cspα的突变会导致溶酶体病理，因为它的作用只是在 突触功能的上下文。我最近发现SNAP-23是SNAP-25的同源物，也是 Cspα/SGT/Hsc70分子伴侣复合体。这种相互作用是通过(I)免疫沉淀CSPα从 野生型小鼠脑内SNAP-23的串联质谱学鉴定，(Ii)还原蛋白 CSPα基因敲除(CSPα-/-)小鼠脑内SNAP-23水平的研究 α/SGT/Hsc70分子伴侣复合体的每个成员。重要的是，SNAP-23介导钙依赖 通过与VAMP-7和Synaxin-4形成SNARE复合体，溶酶体与质膜融合。在……里面 支持这一功能，我已经发现在CSPα-/-原发病例中，钙依赖的溶酶体胞吐功能减弱 通过测量细胞内钙离子后LAMP-1腔结构域的细胞表面暴露来检测神经元 用离子霉素诱导。总之，这些初步数据得出了CSPα之间新的直接联系 通过SNAP-23功能受损导致ANCL的功能障碍和溶酶体病理。关键差距仍在 我们对CsPα突变如何导致ANCL病理级联的理解：A)ANCL是如何 Cspα突变影响SNAP-23的伴随，以及b)SNAP-23功能障碍如何导致溶酶体 伴有脂褐素蓄积的病理学。我的假设是CSPα中的ANCL突变阻止了 Cspα/SGT/Hsc70复合体伴随溶酶体套蛋白SNAP-23，破坏 溶酶体胞吐并导致脂褐素积聚。这一假设将使用 多学科方法，包括CSPα-/-小鼠的原代皮质神经元，生化分析和 慢病毒抢救实验。实验将通过两个拟议的具体目标进行：目标1 将阐明ANCL突变如何影响CSPα‘S陪同SNAP-23：AIM 2将阐明ANCL如何 Cspα突变影响溶酶体的胞吐作用，导致脂褐素沉积症。完成这些目标将导致 详细了解ANCL的病理级联，为未来的治疗试验开辟道路 战略。

PROJECT SUMMARY/ABSTRACT Adult onset neuronal ceroid lipofuscinosis (ANCL) is a fatal lysosomal storage disease caused by two known dominant mutations in the gene encoding cysteine string protein-α (CSPα): CSPαL115R and CSPαL116Δ. CSPα forms a chaperone complex with SGT (small glutamine-rich tetratricopeptide repeat-containing protein) and Hsp70/Hsc70 (heat shock protein/cognate 70 kDa) to chaperone the synaptic SNARE protein SNAP-25. It is surprising that mutations in CSPα lead to lysosomal pathology because its role has only been clarified in the context of synaptic function. I have recently found that SNAP-23, a homolog of SNAP-25, is also a client of the CSPα/SGT/Hsc70 chaperone complex. This interaction was found via (i) immunoprecipitation of CSPα from wild type mouse brain followed by tandem mass spectrometry identification of SNAP-23, (ii) reduced protein levels of SNAP-23 in CSPα knockout (CSPα-/-) mouse brains, and (iii) co-immunoprecipitation of SNAP-23 with each member of the CSPα/SGT/Hsc70 chaperone complex. Importantly, SNAP-23 mediates Ca2+-dependent fusion of lysosomes with the plasma membrane by forming a SNARE-complex with VAMP-7 and syntaxin-4. In support of this function, I have identified diminished Ca2+-dependent lysosomal exocytosis in CSPα-/- primary neurons by measuring cell surface exposure of the LAMP-1 luminal domain following intracellular Ca2+ induction with ionomycin. Altogether, these preliminary data draw a new and direct connection between CSPα dysfunction and lysosomal pathology in ANCL by means of impaired SNAP-23 function. Key gaps remain in our understanding of how mutations in CSPα cause the pathological cascade of ANCL: a) how ANCL mutations in CSPα affect chaperoning of SNAP-23, and b) how SNAP-23 dysfunction leads to lysosomal pathology with lipofuscin accumulation. My hypothesis is that ANCL mutations in CSPα prevent the CSPα/SGT/Hsc70 complex from chaperoning the lysosomal SNARE protein SNAP-23, disrupting lysosomal exocytosis and leading to lipofuscin accumulation. This hypothesis will be addressed using a multi-disciplinary approach including primary cortical neurons from CSPα-/- mice, biochemical assays and lentiviral rescue experiments. Experiments will be carried out by means of two proposed specific aims: Aim 1 will clarify how ANCL mutations affect CSPα’s chaperoning of SNAP-23: Aim 2 will elucidate how ANCL mutations in CSPα affect lysosomal exocytosis, leading to lipofuscinosis. Completion of these aims will lead to a detailed understanding of the pathological cascade of ANCL, opening future avenues for testing therapeutics strategies.