Fibroblast Growth Factor 2‐Mediated Regulation of Neuronal Exosome Release Depends on VAMP3/Cellubrevin in Hippocampal Neurons

Abstract

Extracellular vesicles (EVs) are endogenous membrane‐derived vesicles that shuttle bioactive molecules between glia and neurons, thereby promoting neuronal survival and plasticity in the central nervous system (CNS) and contributing to neurodegenerative conditions. Although EVs hold great potential as CNS theranostic nanocarriers, the specific molecular factors that regulate neuronal EV uptake and release are currently unknown. A combination of patch‐clamp electrophysiology and pH‐sensitive dye imaging is used to examine stimulus‐evoked EV release in individual neurons in real time. Whereas spontaneous electrical activity and the application of a high‐frequency stimulus induce a slow and prolonged fusion of multivesicular bodies (MVBs) with the plasma membrane (PM) in a subset of cells, the neurotrophic factor basic fibroblast growth factor (bFGF) greatly increases the rate of stimulus‐evoked MVB‐PM fusion events and, consequently, the abundance of EVs in the culture medium. Proteomic analysis of neuronal EVs demonstrates bFGF increases the abundance of the v‐SNARE vesicle‐associated membrane protein 3 (VAMP3, cellubrevin) on EVs. Conversely, knocking‐down VAMP3 in cultured neurons attenuates the effect of bFGF on EV release. The results determine the temporal characteristics of MVB‐PM fusion in hippocampal neurons and reveal a new function for bFGF signaling in controlling neuronal EV release.

FGF2 Affects Parkinson’s Disease-Associated Molecular Networks Through Exosomal Rab8b/Rab31

“…All animal experiments were conducted in accordance with the regulations of the local animal welfare committee. Primary hippocampal neurons were cultured in a round Nunc culture dish (Thermo Fisher Scientific) from embryonic day 18 Charles River (CD) rats as described previously in Kumar et al (2020). The EV pellets were harvested from the culture media of neurons treated with FGF2 (50 ng/ml) as per the guidelines provided by the International Society for Extracellular Vesicles (ISEV).…”

“…The information on EV isolation method is submitted to EV TRACK ( 1 EV-TRACK ID: EV200085). The isolated EV pellets were taken forward to perform MS analysis as described in Kumar et al (2020). The MS results of differential EV proteomic signature were then subjected to further bioinformatics analysis.…”

“…The abundant expression of Rab8b and Rab31 in EV pellets was confirmed via Western blot technique as described in Kumar et al (2020). In brief, blots were prepared using Mini-Protean Gels from Bio-Rad and Tris-glycine-based buffer.…”

“…Using at least three biological replicates, we recently assessed the global proteomic changes in EVs in response to treatment with FGF2 by performing a thorough MS analysis (Kumar et al, 2020). Out of 2258 differentially expressed proteins (DEPs), which were detected at least once in all samples, 705 were significantly expressed (FDR ≤ 0.05) and 441 proteins had a fold change of 1.5 or above (Kumar et al, 2020). Among the upregulated candidates, we found Rab8b and Rab31 as prominent members of the Rab protein family with an increased abundance in EV pellets (Supplementary Figure 1).…”

“…We recently demonstrated that fibroblast growth factor-2 (FGF2), a modulator of neuronal cholinergic activity, increases the release of EVs (Araujo et al, 1993;Kumar et al, 2020). In this recently published study, we performed mass spectrometry (MS) and found that FGF2 has significantly altered the EV proteome, leading to 441 proteins with a fold change of 1.5 and above.…”