Secretome analysis deciphers microglia-derived inflammatory signals and identifies an inducer of neuronal deficit

Microglia are dominant immune cells in central nervous system (CNS). Upon inflammatory stimulation, they secrete rich signaling proteins including classic cytokines to interfere with neighbouring neurons. Such deleterious signals have been linked to multiple neurodegenrative diseases. Deciphering the signals released from activated microglia thus holds promise in unveiling underlying mechanisms for neurodegenrative diseases.

To profile the microglia-derived signals, we first used secretome analysis to characterize the secretome of microglia cell line and primary murine microglia after Lipopolysaccharide (LPS) stimulation. The detected secretome repertoire consists of classic cytokines as well as proteins not previously associated with microglia activation, among which the top responder is a proteoglycan (PG4). Through biochemical manipulation with brefeldin A, we demonstrated that PG4 is secreted from microglia via the conventional secretory pathway Moreover, we confirmed that the secretion of PG4 also occurs in vivo by imunofluoresence, RT-qPCR analysis of mouse brain and ELISA analysis of cerebrospinal fluid (CSF) from mice treated with LPS by intraperitoneal (i.p.) and intracerebroventricular (i.c.v.) injection. Notably, clinical big data analysis of Alzheimer's patients also showed upregulation of PG4 in CSF and human autopsy brain tissue, suggesting its clinical relevance.

These findings propels us to explore the potential mechanistic association of PG4 with neurodegenerative diseases such as AD. We found that, upon administering neurons with exogenous recombinant protein PG4, neurite outgrowth was inhibited. We then used Target-Responsive Accessibility Profling (TRAP) approach, a target identification approach previously developed in our lab, to elucidate how PG4 regulates neuronal degeneration. We tentatively identified a neuronal membrane glycoprotein as a dose-responsive target of PG4. Knockdown of this target attenuated PG4-inducted axonal loss in cortical neurons.

Together, using secretome, chemoproteomics and molecular biology, we discover a signaling protein from activated microglia that exerts neurotoxicity. This finding may reveal novel pathological mechanisms of neuroinflammation-related CNS diseases and inspire therapeutic interventions.