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

导致疾病的半胱氨酸串蛋白-α 突变破坏 SNARE 依赖性溶酶体胞吐作用

• 批准号: 9326766
• 负责人: Nima Nick Naseri
• 金额: $ 4.4万
• 依托单位: WEILL MEDICAL COLL OF CORNELL UNIV
• 依托单位国家: 美国
• 财政年份: 2017
• 资助国家: 美国
• 起止时间: 2017-04-01 至 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9326766
• 关键词: Address; Adult; Affect; Alleles; Binding; Biochemical; Biological Assay; Brain; Cell membrane; Cell surface; Client; Co-Immunoprecipitations; Complex; DNA Sequence Alteration; Data; Defect; Degradation Pathway; Disease; Dominant-Negative Mutation; Exocytosis; Functional disorder; Future; Genes; Genetic; Glutamine; Homologous Gene; Hsc70 protein; Immunoblotting; Immunoprecipitation; Impairment; Ionomycin; Knock-out; LAMP-1; Lead; Lipofuscin; Lysosomal Storage Diseases; Lysosomes; Measures; Mediating; Membrane; Molecular; Molecular Chaperones; Mus; Mutate; Mutation; Neuronal Ceroid-Lipofuscinosis; Neurons; Pathologic; Pathology; Phenotype; Proteins; Role; S-nitro-N-acetylpenicillamine; SNAP receptor; Societies; Synapses; Testing; Therapeutic Intervention; Ubiquitination; Wild Type Mouse; alpha-SNAP; base; cysteine string protein; disease-causing mutation; effective therapy; experimental study; in vitro Model; insight; interdisciplinary approach; member; multicatalytic endopeptidase complex; mutant; novel; palmitoylation; prevent; synaptic function; synaptosomal-associated protein 25; synaptotagmin VII; syntaxin 4; tandem mass spectrometry; therapeutic evaluation

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.

项目总结/摘要 成人型神经元蜡样质脂褐质沉积症（ANCL）是一种致命的溶酶体储存疾病， 编码半胱氨酸串蛋白-α（CSPα）的基因中已知的显性突变：CSPα L115 R和CSPαL116Δ。 CSPα与SGT（富含谷氨酰胺的小分子三肽重复序列蛋白）形成伴侣复合物 和Hsp 70/Hsc 70（热休克蛋白/同源物70 kDa）来陪伴突触SNARE蛋白SNAP-25。它 令人惊讶的是，CSPα突变导致溶酶体病理学，因为它的作用仅在研究中得到阐明。 突触功能的背景。我最近发现，SNAP-23，SNAP-25的同源物，也是一个客户端的 CSPα/SGT/Hsc 70分子伴侣复合物。这种相互作用是通过（i）免疫沉淀来自 野生型小鼠脑，随后串联质谱鉴定SNAP-23，（ii）还原蛋白 SNAP-23在CSPα敲除（CSPα-/-）小鼠脑中的水平，和（iii）SNAP-23与 CSPα/SGT/Hsc 70伴侣蛋白复合物的每个成员。重要的是，SNAP-23介导Ca 2+依赖性 通过与VAMP-7和突触融合蛋白-4形成SNARE-复合物使溶酶体与质膜融合。在 为了支持这一功能，我发现CSPα-/-原发性 通过测量细胞内Ca 2+浓度升高后LAMP-1管腔结构域的细胞表面暴露， 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， 通过受损的SNAP-23功能在ANCL中的功能障碍和溶酶体病理学。关键差距仍然存在， 我们对CSPα突变如何导致ANCL病理级联反应的理解：a）ANCL如何 CSPα的突变影响SNAP-23的陪伴，和B）SNAP-23功能障碍如何导致溶酶体 病理学表现为脂褐素积聚。我的假设是CSPα中的ANCL突变阻止了 CSPα/SGT/Hsc 70复合物，伴随溶酶体SNARE蛋白SNAP-23，破坏 溶酶体胞吐作用并导致脂褐素积累。这一假设将使用 多学科方法，包括CSPα-/-小鼠的原代皮层神经元，生化测定和 慢病毒拯救实验。实验将通过两个拟议的具体目标进行： 将阐明ANCL突变如何影响CSPα对SNAP-23的伴侣作用：Aim 2将阐明ANCL如何影响SNAP-23的伴侣作用。 CSPα突变影响溶酶体胞吐作用，导致脂褐质沉积。实现这些目标将导致 详细了解ANCL的病理级联反应，为测试治疗方法开辟未来的途径 战略布局