Necrotic neurons enhance microglial neurotoxicity through induction of glutaminase by a MyD88-dependent pathway

Teresa F Pais,Rui Peixoto,Sukalyan Chatterjee,Maria H Braz,Catarina Figueiredo

doi:10.1186/1742-2094-5-43

Teresa F Pais, Rui Peixoto + Show 3 more

Open Access

https://doi.org/10.1186/1742-2094-5-43

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Abstract

BackgroundMicroglia are macrophage-like cells that constantly sense the microenvironment within the central nervous system (CNS). In the event of neuronal stress or injury, microglial cells rapidly react and change their phenotype. This response may lead to a deleterious type of microglial activation, which is often associated with neuroinflammation and neurotoxicity in several neuropathological conditions. We investigated the molecular mechanisms underlying triggering of microglial activation by necrotic neuronal damage.MethodsPrimary cultures of microglia were used to study the effect of necrotic neurons on microglial inflammatory responses and toxicity towards cerebellar granule neurons (CGN). The mouse hippocampal cell line, HT22, was used in this study as the main source of necrotic neurons to stimulate microglia. To identify the signal transduction pathways activated in microglia, primary microglial cultures were obtained from mice deficient in Toll-like receptor (TLR) -2, -4, or in the TLR adapter protein MyD88.ResultsNecrotic neurons, but not other necrotic cell types, induced microglial activation which was characterized by up-regulation of: i) MHC class II; ii) co-stimulatory molecules, i.e. CD40 and CD24; iii) β2 integrin CD11b; iii) pro-inflammatory cytokines, i.e. interleukin 6 (IL-6), IL-12p40 and tumor-necrosis factor (TNF); iv) pro-inflammatory enzymes such as nitric oxide synthase (iNOS, type II NOS), indoleamine 2,3-dioxygenase (IDO) and cyclooxygenase-2 (COX-2) and increased microglial motility. Moreover, microglia-conditioned medium (MCM) obtained from cultures of activated microglia showed increased neurotoxicity mediated through the N-methyl-D-aspartate receptor (NMDAR). The activation of microglia by necrotic neurons was shown to be dependent on the TLR-associated adapter molecule myeloid differentiation primary response gene (MyD88). Furthermore, MyD88 mediated enhanced neurotoxicity by activated microglia through up-regulation of the expression and activity of glutaminase, an enzyme that produces glutamate, which is an NMDAR agonist.ConclusionThese results show that necrotic neurons activate in microglia a MyD88-dependent pathway responsible for a pro-inflammatory response that also leads to increased neurotoxic activity through induction of glutaminase. This finding contributes to better understanding the mechanisms causing increased neuroinflammation and microglial neurotoxicity in a neurodegenerative environment.

Highlights

Microglia are macrophage-like cells that constantly sense the microenvironment within the central nervous system (CNS)
The alpha chain of the αMβ2 integrin CD11b and the heat-stable antigen CD24 were increased, albeit to lesser degrees (~2 fold, p < 0.05 and p < 0.001 respectively) (Figure 1B). Other molecules such as the co-stimulatory molecules B7.1 and B7.2 and CD11c were not upregulated whereas CD45 and CD14 were poorly induced by necrotic cells (Figure 1B)
Some degree of stimulation was detectable with apoptotic HT22 cells, which is probably explained by the high percentage of secondary necrosis which we observed during incubation with microglia (40% after 24 hours)

Summary

Introduction

Microglia are macrophage-like cells that constantly sense the microenvironment within the central nervous system (CNS). Necrotic-like neuronal death is associated with the development of several pathologic conditions, including brain ischemia subsequent to stroke or trauma [1], infectiousdriven neuroinflammation, e.g. meningitis [2], HIV-associated dementia [3] and cerebral malaria [4], as well as noninfectious-driven neuroinflammation, e.g. multiple sclerosis (MS) [5]. In such pathological conditions microglia, the resident CNS macrophage equivalents, may contribute to neurotoxicity [6]. This may lead to unfettered microglial activation with deleterious consequences [8] such as increased production of the proinflammatory cytokines IL-1β, TNF, IL-6 and IL-12 [9], neurotoxins including the pro-oxidant molecules nitrogen oxide (NO) and reactive oxygen species (ROS) [10], and metabolites such as quinolinic acid and glutamate [11]

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