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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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MyD88-dependent induction of neurotoxicity and glutaminase expression in microglia. (A) Neurotoxicity of MCM from wild type (WT) and MyD88-deficient microglia cultures either non-stimulated (MCM) or treated with necrotic HT22 neurons (MCM (Nec.)). (B) RT-PCR analysis of glutaminase expression of wild type or MyD88-deficient microglia treated or not treated with necrotic HT22 neurons. (C) Analysis of L-Glutamic acid levels in MCM of microglial cell cultures. Results are shown as mean ± SD and are representative of at least two experiments. *** p < 0.001 and * p < 0.05.
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Figure 4: MyD88-dependent induction of neurotoxicity and glutaminase expression in microglia. (A) Neurotoxicity of MCM from wild type (WT) and MyD88-deficient microglia cultures either non-stimulated (MCM) or treated with necrotic HT22 neurons (MCM (Nec.)). (B) RT-PCR analysis of glutaminase expression of wild type or MyD88-deficient microglia treated or not treated with necrotic HT22 neurons. (C) Analysis of L-Glutamic acid levels in MCM of microglial cell cultures. Results are shown as mean ± SD and are representative of at least two experiments. *** p < 0.001 and * p < 0.05.

Mentions: As we have demonstrated above, necrotic neurons activate microglia through a MyD88-dependent pathway. Consequently, we investigated whether the enhanced neurotoxicity of stimulated microglia was affected by the absence of MyD88. In contrast to wild type microglia, the increase in neurotoxicity associated with activation of microglia by necrotic neurons was completely abrogated in the absence of MyD88 expression in microglia (Fig 4A). When primary cerebellar neurons were cultured with 20% MCM from microglia pre-stimulated with necrotic neurons there was a 33% increase in cell death compared to cerebellar neurons cultured with MCM from non-stimulated cultures. In contrast, MCM from MyD88-deficient microglia treated with necrotic neurons did not show any increase in neurotoxicity. Since we have shown (Figure 3) that neurotoxicity is dependent on glutaminase activity, we analysed the mRNA expression levels of glutaminase and glutamate in supernatants of microglia from both wild type and MyD88-deficient microglia. We observed that the observed increase in neurotoxicity correlated with up-regulation of glutaminase mRNA expression in wild type microglia activated by necrotic neurons (Figure 4B). This increase was not observed in MyD88-deficient microglia, and this explains the inability of these microglia to produce enhanced neurotoxicity upon stimulation with necrotic neurons. Concomitantly, levels of glutamate were increased in MCM of wild type, but not MyD88-deficient, microglia stimulated with necrotic neurons 4C.


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)

MyD88-dependent induction of neurotoxicity and glutaminase expression in microglia. (A) Neurotoxicity of MCM from wild type (WT) and MyD88-deficient microglia cultures either non-stimulated (MCM) or treated with necrotic HT22 neurons (MCM (Nec.)). (B) RT-PCR analysis of glutaminase expression of wild type or MyD88-deficient microglia treated or not treated with necrotic HT22 neurons. (C) Analysis of L-Glutamic acid levels in MCM of microglial cell cultures. Results are shown as mean ± SD and are representative of at least two experiments. *** p < 0.001 and * p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
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Figure 4: MyD88-dependent induction of neurotoxicity and glutaminase expression in microglia. (A) Neurotoxicity of MCM from wild type (WT) and MyD88-deficient microglia cultures either non-stimulated (MCM) or treated with necrotic HT22 neurons (MCM (Nec.)). (B) RT-PCR analysis of glutaminase expression of wild type or MyD88-deficient microglia treated or not treated with necrotic HT22 neurons. (C) Analysis of L-Glutamic acid levels in MCM of microglial cell cultures. Results are shown as mean ± SD and are representative of at least two experiments. *** p < 0.001 and * p < 0.05.
Mentions: As we have demonstrated above, necrotic neurons activate microglia through a MyD88-dependent pathway. Consequently, we investigated whether the enhanced neurotoxicity of stimulated microglia was affected by the absence of MyD88. In contrast to wild type microglia, the increase in neurotoxicity associated with activation of microglia by necrotic neurons was completely abrogated in the absence of MyD88 expression in microglia (Fig 4A). When primary cerebellar neurons were cultured with 20% MCM from microglia pre-stimulated with necrotic neurons there was a 33% increase in cell death compared to cerebellar neurons cultured with MCM from non-stimulated cultures. In contrast, MCM from MyD88-deficient microglia treated with necrotic neurons did not show any increase in neurotoxicity. Since we have shown (Figure 3) that neurotoxicity is dependent on glutaminase activity, we analysed the mRNA expression levels of glutaminase and glutamate in supernatants of microglia from both wild type and MyD88-deficient microglia. We observed that the observed increase in neurotoxicity correlated with up-regulation of glutaminase mRNA expression in wild type microglia activated by necrotic neurons (Figure 4B). This increase was not observed in MyD88-deficient microglia, and this explains the inability of these microglia to produce enhanced neurotoxicity upon stimulation with necrotic neurons. Concomitantly, levels of glutamate were increased in MCM of wild type, but not MyD88-deficient, microglia stimulated with necrotic neurons 4C.

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