Division of Cellular and Systems Medicine, School of Medicine, University of Dundee, Dundee, Scotland, UK.
The Euan Macdonald Centre, Edinburgh, UK.
Acta Neuropathol Commun. 2022 Oct 29;10(1):156. doi: 10.1186/s40478-022-01455-z.
Increasing evidence suggests synaptic dysfunction is a central and possibly triggering factor in Amyotrophic Lateral Sclerosis (ALS). Despite this, we still know very little about the molecular profile of an ALS synapse. To address this gap, we designed a synaptic proteomics experiment to perform an unbiased assessment of the synaptic proteome in the ALS brain. We isolated synaptoneurosomes from fresh-frozen post-mortem human cortex (11 controls and 18 ALS) and stratified the ALS group based on cognitive profile (Edinburgh Cognitive and Behavioural ALS Screen (ECAS score)) and presence of a C9ORF72 hexanucleotide repeat expansion (C9ORF72-RE). This allowed us to assess regional differences and the impact of phenotype and genotype on the synaptic proteome, using Tandem Mass Tagging-based proteomics. We identified over 6000 proteins in our synaptoneurosomes and using robust bioinformatics analysis we validated the strong enrichment of synapses. We found more than 30 ALS-associated proteins in synaptoneurosomes, including TDP-43, FUS, SOD1 and C9ORF72. We identified almost 500 proteins with altered expression levels in ALS, with region-specific changes highlighting proteins and pathways with intriguing links to neurophysiology and pathology. Stratifying the ALS cohort by cognitive status revealed almost 150 specific alterations in cognitively impaired ALS synaptic preparations. Stratifying by C9ORF72-RE status revealed 330 protein alterations in the C9ORF72-RE +ve group, with KEGG pathway analysis highlighting strong enrichment for postsynaptic dysfunction, related to glutamatergic receptor signalling. We have validated some of these changes by western blot and at a single synapse level using array tomography imaging. In summary, we have generated the first unbiased map of the human ALS synaptic proteome, revealing novel insight into this key compartment in ALS pathophysiology and highlighting the influence of cognitive decline and C9ORF72-RE on synaptic composition.
越来越多的证据表明，突触功能障碍是肌萎缩侧索硬化症（ALS）的核心和可能的触发因素。尽管如此，我们对 ALS 突触的分子特征仍然知之甚少。为了解决这一差距，我们设计了一个突触蛋白质组学实验，对 ALS 大脑中的突触蛋白质组进行了无偏评估。我们从新鲜冷冻的死后人类皮质（11 名对照和 18 名 ALS）中分离出突触小体，并根据认知特征（爱丁堡认知和行为 ALS 筛查（ECAS 评分））和是否存在 C9ORF72 六核苷酸重复扩展（C9ORF72-RE）对 ALS 组进行分层。这使我们能够使用基于串联质量标签的蛋白质组学评估区域差异以及表型和基因型对突触蛋白质组的影响。我们在突触小体中鉴定出超过 6000 种蛋白质，并通过稳健的生物信息学分析验证了突触的强烈富集。我们在突触小体中发现了 30 多种与 ALS 相关的蛋白质，包括 TDP-43、FUS、SOD1 和 C9ORF72。我们发现 ALS 中有超过 500 种表达水平改变的蛋白质，区域特异性变化突出了与神经生理学和病理学具有有趣联系的蛋白质和途径。根据认知状态对 ALS 队列进行分层，发现认知障碍 ALS 突触制剂中几乎有 150 种特定改变。根据 C9ORF72-RE 状态进行分层，在 C9ORF72-RE+ve 组中发现 330 种蛋白质改变，KEGG 途径分析突出了谷氨酸能受体信号传导相关的突触后功能障碍的强烈富集。我们通过 Western blot 和使用阵列断层扫描成像在单个突触水平验证了其中一些变化。总之，我们生成了人类 ALS 突触蛋白质组的第一个无偏图谱，揭示了 ALS 病理生理学这一关键隔室的新见解，并强调了认知能力下降和 C9ORF72-RE 对突触组成的影响。
