Characterizing chromogranin and secretogranin proteoforms in dementia pathophysiology

Background Chromogranins and secretogranins are neurotransmitter‐like molecules (neuropeptides) that are processed into functional peptides (proteoforms) by proteases. Full‐length chromogranins and secretogranins and their peptide proteoforms are involved in diverse neuronal and synaptic functions, but their specific processing pathways and contribution to dementia remain largely unknown. This project aims to investigate chromogranin and secretogranin proteolytic processing and validate if specific proteoforms may be useful cerebrospinal fluid (CSF) biomarkers of synaptic health in dementia. Method Mass spectrometry (MS) of intact processed peptides was used to produce a detailed map of identified proteoforms arising from chromogranins and secretogranins in the CSF of 5 control and 5 Alzheimer’s disease (AD) patients. This CSF profile was compared against known processed peptides and those used in CSF‐targeted MS assays. Proteasix was used to identify potential proteases involved in the production of identified proteoforms and the human cell atlas was used to identify whether they were expressed in the brain. The cleavage specificity was assayed in vitro using recombinant proteins, analysed by gel electrophoresis and western blotting with chromogranin and secretogranin antibodies. Result Using tryptic versus non‐tryptic MS techniques, we detected distinct chromogranin and secretogranin proteoform profiles, suggesting that tryptic digestion underestimates the complexity of proteoform profiles. We are now developing a targeted MS assay to characterize the profile of chromogranin and secretogranin proteoforms in CSF samples spanning controls, different stages of AD and other neurodegenerative diseases. Using Proteasix; members of the calpain, cathepsin, caspase and MMP families were identified to cleave chromogranins and secretogranins. These protease families are known to be expressed in the brain as shown by the human cell atlas and have been previously identified to be dysregulated in dementia. Recombinant assays identified that these proteases can cleave chromogranins and secretogranins to produce different proteoforms. Conclusion Our results show that (i) standard tryptic digestion methods underestimate the proteoform complexity of chromogranins and secretogranins, and (ii) that chromogranins and secretogranins are proteolytically processed by proteases relevant to dementia pathophysiology. This work improves our mechanistic understanding of chromogranin and secretogranin processing and further establishes them as a potential synaptic health biomarker in dementia pathophysiology.