LPS-Induced proNGF Synthesis and Release in the N9 and BV2 Microglial Cells: A New Pathway Underling Microglial Toxicity in Neuroinflammation

Abstract

PurposeWhile aberrant activation of microglial cells was evidently involved in neuroinflammation and neurotoxicity in the neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease, objective of study was to address if activated microglias deliver their effect by releasing pro-neurotrophins.Materials and methodsBy in vitro culture of N9 and BV2 cell lines and lipopolysaccharide (LPS) stimulation model, generation and release of proNGF, proBDNF and MMP-9 was studied in the activated microglial cells by immunocytochemistry, western blotting and bioassay methods.ResultsActivation of microglial cells was observed with obvious increasing iba1-immunoreactivity following LPS stimulation in cell culture. Synthesis and up-regulation of proNGF protein significantly occurred in N9 and BV2 cells 12h-48h after LPS exposure, whereas no significant changes of proBDNF and MMP9 were observed in these microglial cell lines with LPS insult. More interestingly, extracellular release or secretion of proNGF molecule was also detected in culture medium of N9 cells after LPS stimulation. Finally, bioassay using MTT, Hoechst/PI and TUNEL staining in SH-SY5Y cells further confirmed that proNGF treatment could result in apoptotic cell death but it did not significantly influence cell viability of SH-SY5Y cells.ConclusionsThis in vitro study revealed LPS-stimulated proNGF synthesis and release in activated N9/BV2 microglial cell lines, also suggesting that proNGF may appeal a new pathway or possible mechanism underlying microglial toxicity in the neuroinflammation and a potential target for therapeutic manipulation of the neurodegenerative diseases.

VEGF inhibition alters neurotrophin signalling pathways and induces caspase‐3 activation and autophagy in rabbit retina

“…The different proBDNF and proNGF expression patterns suggest diverse and independent mechanisms of regulation induced by anti-VEGF administration. This condition supports other findings, demonstrating that NGF and BDNF transcription, as well as proNGF and proBDNF expression, can be distinctly different in terms of time-course and spatial pattern, also following the same stimuli (Duan et al, 2013;Meyer, Matsuoka, Wetmore, Olson, & Thoenen, 1992;Noga et al, 2005). Furthermore, it is possible to speculate that proBDNF increase could represent a specific feedback mechanism to hinder the alterations induced by anti-VEGF agents which, unfortunately, may be not functional because of the downregulation of neurotrophins receptor expression/activation.…”

Resveratrol Induces the Expression of Interleukin-10 and Brain-Derived Neurotrophic Factor in BV2 Microglia under Hypoxia

“…Iba-1 protein is expressed in microglia [ 57 ], and is a microglia-specific calcium-binding protein that also participates in phagocytosis [ 58 ]. Ito et al [ 59 ] demonstrated that Iba-1 expression is related to microglial activation in the ischemic brain, and the physiological roles of activated microglia can be evaluated by determining the expression of Iba-1 [ 59 , 60 , 61 ]. Based on our results, we concluded that hypoxia led to the activation of microglia and that resveratrol (25 μM) reduced the activation of hypoxia-injured microglia.…”

Inducible overexpression of endothelial proNGF as a mouse model to study microvascular dysfunction

“…We previously reported that due to oxidative stress, processing and maturation of proNGF is impaired in the diabetic milieu, resulting in increased proNGF and decreased NGF levels in serum and ocular fluids from patients with proliferative diabetic retinopathy [5,6]. Furthermore, accumulation of proNGF has been reported in neurodegenerative diseases including Alzheimer’s Disease [11], Down’s syndrome [32] and myocardial infarction [33] as well as models of neuronal ischemic injury [34,35]. Therefore, there is a pressing need of rodent models that stably express a non-cleavable form of proNGF to better understand the impact of its accumulation in various tissues.…”