Cerebrospinal Fluid and Brain Proteoforms of the Granin Neuropeptide Family in Alzheimer’s Disease

The granin neuropeptide family is composed of acidic secretory signaling molecules that act throughout the nervous system to help modulate synaptic signaling and neural activity. Granin neuropeptides have been shown to be dysregulated in different forms of dementia, including Alzheimer’s disease (AD). Recent studies have suggested that the granin neuropeptides and their protease-cleaved bioactive peptides (proteoforms) may act as both powerful drivers of gene expression and as a biomarker of synaptic health in AD. The complexity of granin proteoforms in human cerebrospinal fluid (CSF) and brain tissue has not been directly addressed. We developed a reliable nontryptic mass spectrometry assay to comprehensively map and quantify endogenous neuropeptide proteoforms in the brain and CSF of individuals diagnosed with mild cognitive impairment and dementia due to AD compared to healthy controls, individuals with preserved cognition despite AD pathology (“Resilient”), and those with impaired cognition but no AD or other discernible pathology (“Frail”). We drew associations between neuropeptide proteoforms, cognitive status, and AD pathology values. Decreased levels of VGF proteoforms were observed in CSF and brain tissue from individuals with AD compared to controls, while select proteoforms from chromogranin A showed the opposite effect. To address mechanisms of neuropeptide proteoform regulation, we showed that the proteases Calpain-1 and Cathepsin S can cleave chromogranin A, secretogranin-1, and VGF into proteoforms found in both the brain and CSF. We were unable to demonstrate differences in protease abundance in protein extracts from matched brains, suggesting that regulation may occur at the level of transcription.

颗粒蛋白神经肽家族（granin neuropeptide family）是一类酸性分泌型信号分子，可在全神经系统中发挥功能，协助调控突触信号传递与神经活动。已有研究证实，该家族神经肽在包括阿尔茨海默病（Alzheimer’s disease, AD）在内的多种痴呆亚型中存在表达失调现象。近期研究表明，颗粒蛋白神经肽及其经蛋白酶剪切得到的生物活性肽（proteoforms，蛋白异构体），既可作为基因表达的强效调控因子，也可作为阿尔茨海默病突触健康的生物标志物。目前尚未直接探究人类脑脊液（cerebrospinal fluid, CSF）与脑组织中颗粒蛋白肽异构体的复杂组成情况。本研究建立了一种可靠的非胰蛋白酶质谱分析方法，可全面绘制并定量分析不同人群脑组织与脑脊液中的内源性神经肽蛋白异构体，这些人群包括确诊为阿尔茨海默病相关轻度认知障碍与痴呆的患者、健康对照个体、虽携带阿尔茨海默病病理却认知功能保留的"Resilient（认知储备型）"人群，以及认知受损但无阿尔茨海默病或其他明确病理特征的"Frail（脆弱型）"人群。本研究分析了神经肽蛋白异构体、认知状态与阿尔茨海默病病理指标之间的关联。相较于健康对照，阿尔茨海默病患者的脑脊液与脑组织中VGF蛋白异构体水平显著降低，而嗜铬粒蛋白A（chromogranin A）的部分特定蛋白异构体则呈现相反的变化趋势。为探究神经肽蛋白异构体的调控机制，本研究证实钙蛋白酶-1（Calpain-1）与组织蛋白酶S（Cathepsin S）可将嗜铬粒蛋白A、分泌粒蛋白-1（secretogranin-1）与VGF剪切为可在脑组织与脑脊液中检测到的蛋白异构体。本研究未在匹配脑组织的蛋白质提取物中检测到蛋白酶丰度的显著差异，提示其调控可能发生在转录水平。