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

Pais TF, Figueiredo C, Peixoto R, Braz MH, Chatterjee S - J Neuroinflammation (2008)

Bottom Line: This response may lead to a deleterious type of microglial activation, which is often associated with neuroinflammation and neurotoxicity in several neuropathological conditions.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.This finding contributes to better understanding the mechanisms causing increased neuroinflammation and microglial neurotoxicity in a neurodegenerative environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal. tfariapais@gmail.com

ABSTRACT

Background: Microglia 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.

Methods: Primary 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.

Results: Necrotic 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) beta2 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.

Conclusion: These 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.

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Necrotic neurons enhance microglial-induced neurotoxicity mediated by NMDAR. (A) Neurotoxicity of MCM was assayed in cell cultures of CGN after 24 hours. MCM obtained from non-stimulated cells or after stimulation with necrotic neurons for 20 hours, MCM (Nec.), were added to a final concentration of 20% to neuronal cultures. Neuronal viability was analysed by PI incorporation after 24 hours. (B) Neuronal cell death was quantified after exposure to MCM of non-stimulated cells, MCM of microglial cells cultured in glutamine-free medium, MCM (- Glutamine), or cultured with a glutaminase inhibitor, MCM (+DON). (C) The NMDAR inhibitor, MK-801, was added to neuronal cultures at the same time as MCM and neuronal viability was assayed as described before. Results are shown as mean ± SD and are representative of at least two experiments. NS, non-significant. *** p < 0.001 and * p < 0.05.
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Figure 3: Necrotic neurons enhance microglial-induced neurotoxicity mediated by NMDAR. (A) Neurotoxicity of MCM was assayed in cell cultures of CGN after 24 hours. MCM obtained from non-stimulated cells or after stimulation with necrotic neurons for 20 hours, MCM (Nec.), were added to a final concentration of 20% to neuronal cultures. Neuronal viability was analysed by PI incorporation after 24 hours. (B) Neuronal cell death was quantified after exposure to MCM of non-stimulated cells, MCM of microglial cells cultured in glutamine-free medium, MCM (- Glutamine), or cultured with a glutaminase inhibitor, MCM (+DON). (C) The NMDAR inhibitor, MK-801, was added to neuronal cultures at the same time as MCM and neuronal viability was assayed as described before. Results are shown as mean ± SD and are representative of at least two experiments. NS, non-significant. *** p < 0.001 and * p < 0.05.

Mentions: Microglial cells, depending on their activation phenotype, secrete a variety of factors that include both neurotrophic factors such as bFGF and NGF and neurotoxic metabolites like NO and glutamate [11]. We assessed the neurotoxicity of microglia-conditioned medium (MCM), added at final concentration of 20%, obtained from untreated microglia or from microglia activated with necrotic neurons. Although MCM from untreated microglia exhibited some neurotoxicity, probably due to basal activation levels of this cell type [34], neuronal cell death was significantly increased by 21.5% (p < 0.005) when neurons were incubated with MCM from activated microglia (MCM (Nec.), Figure 3A). The neurotoxicity of MCM was due to the microglial conditioning since exposure of neurons to fresh medium at the same ratio resulted in a lesser, and non-significant, increase in cell death (13.4 ± 10.5% of neurons compared to 8.3 ± 3.0% in the absence of medium). We next investigated the basis for MCM-induced neurotoxicity. This was neither abolished when MCM was heat inactivated before exposing it to neurons, nor by inhibition of the microglial enzymes iNOS and IDO, which are responsible for the formation of two neurotoxic agents, NO and QA, respectively (data not shown). On the other hand, MCM obtained from microglial cells cultured in glutamine-free medium (- Glutamine) or in the presence of the glutaminase inhibitor, DON (+ DON), showed significantly reduced neurotoxicity (Figure 3B). These results suggested that cell death could be mediated by glutamate through NMDAR. To test this hypothesis, MCM from non-stimulated or stimulated microglia were added together with MK-801, an antagonist of NMDAR. In both conditions, the antagonist completely abolished the MCM-induced neurotoxicity, clearly showing that the mechanism of neurotoxicity mediated by microglia is NMDAR-dependent. Altogether these results show that necrotic neurons cause an over-activation of microglia with consequent NMDAR-mediated neurotoxicity, most likely mediated through increased production of glutamate.


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

Pais TF, Figueiredo C, Peixoto R, Braz MH, Chatterjee S - J Neuroinflammation (2008)

Necrotic neurons enhance microglial-induced neurotoxicity mediated by NMDAR. (A) Neurotoxicity of MCM was assayed in cell cultures of CGN after 24 hours. MCM obtained from non-stimulated cells or after stimulation with necrotic neurons for 20 hours, MCM (Nec.), were added to a final concentration of 20% to neuronal cultures. Neuronal viability was analysed by PI incorporation after 24 hours. (B) Neuronal cell death was quantified after exposure to MCM of non-stimulated cells, MCM of microglial cells cultured in glutamine-free medium, MCM (- Glutamine), or cultured with a glutaminase inhibitor, MCM (+DON). (C) The NMDAR inhibitor, MK-801, was added to neuronal cultures at the same time as MCM and neuronal viability was assayed as described before. Results are shown as mean ± SD and are representative of at least two experiments. NS, non-significant. *** p < 0.001 and * p < 0.05.
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Figure 3: Necrotic neurons enhance microglial-induced neurotoxicity mediated by NMDAR. (A) Neurotoxicity of MCM was assayed in cell cultures of CGN after 24 hours. MCM obtained from non-stimulated cells or after stimulation with necrotic neurons for 20 hours, MCM (Nec.), were added to a final concentration of 20% to neuronal cultures. Neuronal viability was analysed by PI incorporation after 24 hours. (B) Neuronal cell death was quantified after exposure to MCM of non-stimulated cells, MCM of microglial cells cultured in glutamine-free medium, MCM (- Glutamine), or cultured with a glutaminase inhibitor, MCM (+DON). (C) The NMDAR inhibitor, MK-801, was added to neuronal cultures at the same time as MCM and neuronal viability was assayed as described before. Results are shown as mean ± SD and are representative of at least two experiments. NS, non-significant. *** p < 0.001 and * p < 0.05.
Mentions: Microglial cells, depending on their activation phenotype, secrete a variety of factors that include both neurotrophic factors such as bFGF and NGF and neurotoxic metabolites like NO and glutamate [11]. We assessed the neurotoxicity of microglia-conditioned medium (MCM), added at final concentration of 20%, obtained from untreated microglia or from microglia activated with necrotic neurons. Although MCM from untreated microglia exhibited some neurotoxicity, probably due to basal activation levels of this cell type [34], neuronal cell death was significantly increased by 21.5% (p < 0.005) when neurons were incubated with MCM from activated microglia (MCM (Nec.), Figure 3A). The neurotoxicity of MCM was due to the microglial conditioning since exposure of neurons to fresh medium at the same ratio resulted in a lesser, and non-significant, increase in cell death (13.4 ± 10.5% of neurons compared to 8.3 ± 3.0% in the absence of medium). We next investigated the basis for MCM-induced neurotoxicity. This was neither abolished when MCM was heat inactivated before exposing it to neurons, nor by inhibition of the microglial enzymes iNOS and IDO, which are responsible for the formation of two neurotoxic agents, NO and QA, respectively (data not shown). On the other hand, MCM obtained from microglial cells cultured in glutamine-free medium (- Glutamine) or in the presence of the glutaminase inhibitor, DON (+ DON), showed significantly reduced neurotoxicity (Figure 3B). These results suggested that cell death could be mediated by glutamate through NMDAR. To test this hypothesis, MCM from non-stimulated or stimulated microglia were added together with MK-801, an antagonist of NMDAR. In both conditions, the antagonist completely abolished the MCM-induced neurotoxicity, clearly showing that the mechanism of neurotoxicity mediated by microglia is NMDAR-dependent. Altogether these results show that necrotic neurons cause an over-activation of microglia with consequent NMDAR-mediated neurotoxicity, most likely mediated through increased production of glutamate.

Bottom Line: This response may lead to a deleterious type of microglial activation, which is often associated with neuroinflammation and neurotoxicity in several neuropathological conditions.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.This finding contributes to better understanding the mechanisms causing increased neuroinflammation and microglial neurotoxicity in a neurodegenerative environment.

View Article: PubMed Central - HTML - PubMed

Affiliation: Instituto Gulbenkian de Ciência, Rua da Quinta Grande, 6, 2780-156 Oeiras, Portugal. tfariapais@gmail.com

ABSTRACT

Background: Microglia 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.

Methods: Primary 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.

Results: Necrotic 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) beta2 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.

Conclusion: These 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.

Show MeSH
Related in: MedlinePlus