Neuroprotection of microglial conditioned medium on 6‐hydroxydopamine‐induced neuronal death: role of transforming growth factor beta‐2

Abstract

Microglia, the immune cells of the CNS, play essential roles in both physiological and pathological brain states. Here we have used an in vitro model to demonstrate neuroprotection of a 48 h‐microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6‐hydroxydopamine, which induces a Parkinson‐like neurodegeneration, and to identify the protective factor(s). MCM nearly completely protects CGNs from 6‐hydroxydopamine neurotoxicity and at least some of the protective factor(s) are peptidic in nature. While the fraction of the medium containing molecules < 30 kDa completely protects CGNs, fractions containing molecules < 10 kDa or > 10 kDa are not neuroprotective. We further demonstrate that microglia release high amounts of transforming growth factor‐β2 (TGF‐β2) and that its exogenous addition to the fraction of the medium not containing it (< 10 kDa) fully restores the neuroprotective action. Moreover, MCM neuroprotection is significantly counteracted by an inhibitor of TGF‐β2 transduction pathway. Our results identify TGF‐β2 as an essential neuroprotective factor released by microglia in its culture medium that requires to be fully effective the concomitant presence of other factor(s) of low molecular weight.

Copper-Zinc Superoxide Dismutase (SOD1) Is Released by Microglial Cells and Confers Neuroprotection against 6-OHDA Neurotoxicity

“…As previously demonstrated [22,30], exposure of differentiated rat CGNs to the dopaminergic toxin 6-OHDA (20 µ M ) resulted in approximately 50% cell death after -24 h (fig. 1a).…”

“…1a, b), thus suggesting that neuroprotective substances released by microglia progressively accumulated in the medium. We previously demonstrated [22] that heat inactivation of the neuroprotective MCM, as well as its pre-treatment with peptidases/proteases, results in a significant decrease in MCM-mediated protection against 6-OHDA neurotoxicity in CGNs, leading to the idea that peptidic factor(s) contribute to neuroprotection. In order to identify the neuroprotective microglia-released proteic factors, we performed a proteomic analysis of 48-hour MCM.…”

“…This raises the obvious question of the actual contribution of the low SOD1 concentration present in the conditioned medium to its neuroprotective action. Explanation of this apparent contradiction likely resides in the fact that the low SOD1 concentration present in the conditioned medium could cooperate with other neuroprotective factors, such as the ones mentioned in the introduction (TGF-β2, apolipoprotein E, and plasminogen), to protect neurons [21,22,27]. Consequently, it is not surprising that when SOD1 is added as the only neuroprotective molecule to a nonconditioned medium higher concentrations are required.…”

“…In cultured dopaminergic mesencephalic neurons, microglia-derived plasminogen is neuroprotective through dopamine uptake [21]. In CGNs exposed to the dopaminergic toxin 6-hydroxydopamine (6-OHDA), we recently identified transforming growth factor-β2 (TGF-β2) as a neuroprotective molecule released by microglia [22]. In addition to constitutively released neuroprotective factors, exposure of microglia to media conditioned by apoptotic neurons may increase their neuroprotective action [16] as demonstrated by the release of immunoregulatory and pro-survival factors, such as prostaglandin E2, NGF, and TGF-β [23].…”

Neuroprotective role of γ‐enolase in microglia in a mouse model of Alzheimer's disease is regulated by cathepsin X

“…Microglial activation is regarded as a protective event in the neuroregenerative process (Streit, 2002;Polazzi et al, 2009), because activated microglia are capable of phagocytosis of amyloid-b deposits (Koenigsknecht & Landreth, 2004;Fuhrmann et al, 2010) and of releasing neurotrophic factors such as NGF, NT-3 and BDNF known to be neuroprotective (Nakajima et al, 2001;Schindowski et al, 2008). However, in several neuropathologies, where chronic inflammation is present, the inflammatory products derived from activated microglia may also promote neurodegeneration and contribute to neuronal loss (Gonz alez-Scarano & Baltuch, 1999).…”