Characterization of novel neuropeptide proteolytic processing involved in dementia pathophysiology
Ethier EC., Quinn JP., Kandigian SE., Trombetta BA., Arnold SE., Carlyle BC.
BACKGROUND: Neuropeptides are neurotransmitter-like molecules with multiple roles in neuronal activity that are processed into functional peptides (aka proteoforms) by prohormone convertases and other currently unidentified proteases. Both neuropeptides and their proteoforms are implicated in the pathogenesis of different forms of dementia. The neuropeptide VGF is decreased in the cerebrospinal fluid (CSF) and brain of patients with Alzheimer's disease (AD) and other forms of dementia. VGF356-375 and VGF1-40 decrease in the CSF of patients with AD and frontotemporal dementia (FTD), respectively, compared to healthy age-matched controls. Although many such neuropeptide proteoforms are thought to be involved in dementia, current research has focused on the neuropeptides themselves, leaving the proteoforms and their physiological processing pathways understudied and their contribution to dementia pathogenesis unclear. METHOD: Using recombinant proteolysis assays, we aimed to investigate the proteolytic processing of several neuropeptides involved in dementia. Neuropeptides of interest were identified by mass spectrometry analysis of pooled CSF samples obtained from five AD patients and five healthy controls. Proteasix, the MEROPS, and the human protein atlas databases were used to identify novel neuronally-expressed proteases that may cleave these identified neuropeptides; proteases of interest were selected based on the predicted probability of cleavage. Recombinant protein assays and immunoblots with neuropeptide-specific antibodies were used to investigate the ability of these proteases to cleave the neuropeptides, and banding patterns were confirmed in triplicate. RESULT: Twenty-two neuronally expressed proteases previously linked to dementia were identified that may cleave the neuropeptides chromogranin A (CHGA), secretogranin-1 (SCG1), secretogranin-2 (SCG2), secretogranin-3 (SCG3), and VGF. The proteases a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), calpain-1 (CAPN1), cathepsin S (CTSS), granzyme A (GZMA), and matrix metalloprotease-3 (MMP-3) displayed high probability for neuropeptide proteolysis. These proteases cleaved the neuropeptides into N-terminal, mid-region, or C-terminal neuropeptide proteoforms, and all neuropeptide-protease combinations except the SCG3-CAPN1 pair demonstrated cleavage. CONCLUSION: Overall, this work provides mechanistic insight into neuropeptide processing and expands our understanding of neuropeptide proteoform involvement in dementia. Future projects aim to confirm and expand upon these results using mass-spectrometry, cell culture systems, and CSF and brain protein analysis.