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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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Microglial response to necrotic neurons is MyD88-dependent. (A) Microglial cells derived from MyD88-deficient mice were stimulated with necrotic HT22 cells for 24 hours and the expression of CD40 and MHC class II was assessed and compared to that of wild type cells. Pam3CSK4 is a TLR-2 ligand; ND, non-detectable. (B) The levels of IL-6 and IL-12 were quantified in the supernatants of microglial cell cultures by ELISA. (C) Induction of CD40 expression in TLR-2 or TLR-4 deficient microglia stimulated with necrotic neurons. LPS is a specific ligand for TLR-4. Results are shown as mean ± SD of three independent experiments or are representative of at least two experiments. *** p < 0.001; **p < 0.01 and * p < 0.05.
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Figure 2: Microglial response to necrotic neurons is MyD88-dependent. (A) Microglial cells derived from MyD88-deficient mice were stimulated with necrotic HT22 cells for 24 hours and the expression of CD40 and MHC class II was assessed and compared to that of wild type cells. Pam3CSK4 is a TLR-2 ligand; ND, non-detectable. (B) The levels of IL-6 and IL-12 were quantified in the supernatants of microglial cell cultures by ELISA. (C) Induction of CD40 expression in TLR-2 or TLR-4 deficient microglia stimulated with necrotic neurons. LPS is a specific ligand for TLR-4. Results are shown as mean ± SD of three independent experiments or are representative of at least two experiments. *** p < 0.001; **p < 0.01 and * p < 0.05.

Mentions: There is mounting evidence suggesting that "danger signals" released from necrotic cells can be recognized by PRR expressed on monocytes/macrophages and dendritic cells. These PRR include, but are not restricted to, members of the TLR family. MyD88 is an adapter protein required for signal transduction by TLRs, with the exception of TLR-3 [32]. We isolated microglial cells from MyD88-deficient mice to assess whether this pathway is involved in signal transduction pathways via which necrotic neurons activate microglial cells. Ablation of MyD88 in microglia cells suppressed microglial activation by necrotic neurons, as assessed by the expression of CD40 or MHC class II (Figure 2A). MyD88-deficient microglia could be activated by IFNγ but not by a specific ligand for TLR-2, Pam3CSK4. Increased IL-6 or IL-12 secretion was also abolished in MyD88-deficient microglia stimulated with necrotic neurons (Figure 2B). These results demonstrate that necrotic neurons are most likely recognized by PRRs expressed on microglia that signal via MyD88 to induce expression of pro-inflammatory genes associated with microglial activation.


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)

Microglial response to necrotic neurons is MyD88-dependent. (A) Microglial cells derived from MyD88-deficient mice were stimulated with necrotic HT22 cells for 24 hours and the expression of CD40 and MHC class II was assessed and compared to that of wild type cells. Pam3CSK4 is a TLR-2 ligand; ND, non-detectable. (B) The levels of IL-6 and IL-12 were quantified in the supernatants of microglial cell cultures by ELISA. (C) Induction of CD40 expression in TLR-2 or TLR-4 deficient microglia stimulated with necrotic neurons. LPS is a specific ligand for TLR-4. Results are shown as mean ± SD of three independent experiments or are representative of at least two experiments. *** p < 0.001; **p < 0.01 and * p < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Microglial response to necrotic neurons is MyD88-dependent. (A) Microglial cells derived from MyD88-deficient mice were stimulated with necrotic HT22 cells for 24 hours and the expression of CD40 and MHC class II was assessed and compared to that of wild type cells. Pam3CSK4 is a TLR-2 ligand; ND, non-detectable. (B) The levels of IL-6 and IL-12 were quantified in the supernatants of microglial cell cultures by ELISA. (C) Induction of CD40 expression in TLR-2 or TLR-4 deficient microglia stimulated with necrotic neurons. LPS is a specific ligand for TLR-4. Results are shown as mean ± SD of three independent experiments or are representative of at least two experiments. *** p < 0.001; **p < 0.01 and * p < 0.05.
Mentions: There is mounting evidence suggesting that "danger signals" released from necrotic cells can be recognized by PRR expressed on monocytes/macrophages and dendritic cells. These PRR include, but are not restricted to, members of the TLR family. MyD88 is an adapter protein required for signal transduction by TLRs, with the exception of TLR-3 [32]. We isolated microglial cells from MyD88-deficient mice to assess whether this pathway is involved in signal transduction pathways via which necrotic neurons activate microglial cells. Ablation of MyD88 in microglia cells suppressed microglial activation by necrotic neurons, as assessed by the expression of CD40 or MHC class II (Figure 2A). MyD88-deficient microglia could be activated by IFNγ but not by a specific ligand for TLR-2, Pam3CSK4. Increased IL-6 or IL-12 secretion was also abolished in MyD88-deficient microglia stimulated with necrotic neurons (Figure 2B). These results demonstrate that necrotic neurons are most likely recognized by PRRs expressed on microglia that signal via MyD88 to induce expression of pro-inflammatory genes associated with microglial activation.

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