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.

神经肽类蛋白质家族由酸性分泌信号分子组成，这些分子在神经系统内发挥作用，有助于调节突触信号和神经活动。研究表明，神经肽类蛋白质在不同类型的痴呆症中，包括阿尔茨海默病（AD）中表现出失调。近期研究提出，神经肽类蛋白质及其由蛋白酶裂解的具有生物活性的肽段（蛋白亚型）可能既作为基因表达的强大驱动因素，又作为AD突触健康的生物标志物。然而，人类脑脊液（CSF）和脑组织中神经肽类蛋白亚型的复杂性尚未得到直接探讨。本研究开发了一种可靠的非酶解质谱法，用于全面映射和量化被诊断为轻度认知障碍和由AD引起的痴呆症患者，以及认知功能保留但存在AD病理学（“坚韧”）和认知功能受损但无AD或其他可辨识病理（“脆弱”）的个体脑和CSF中的内源性神经肽蛋白亚型，并与健康对照者进行比较。本研究揭示了神经肽类蛋白亚型、认知状态和AD病理学值之间的关联。与对照者相比，AD患者的CSF和脑组织中VGF蛋白亚型的水平降低，而选择性蛋白亚型来自嗜铬素A则表现出相反的效果。为了探讨神经肽类蛋白亚型调控机制，本研究表明，蛋白酶Calpain-1和Cathepsin S可以将嗜铬素A、分泌素-1和VGF裂解成在脑和CSF中发现的蛋白亚型。我们在匹配脑组织的蛋白质提取物中未能观察到蛋白酶丰度的差异，这表明调控可能发生在转录水平。