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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 induce expression of activation markers by microglia. (A) Microglia were stimulated with necrotic HT22 neurons at a ratio of 1:1 for 24 hours and the expression of several activation markers analysed by FACS. The staining intensity is represented on histogram overlays for the mentioned proteins in non-stimulated microglia (grey filled), and in microglia stimulated either with IFN-γ (100 U/ml) (dashed line) or with necrotic neurons (solid line). (B) These results are shown as fold increases ("fold activation") of the Geo Mean when divided by the average expression of triplicates of untreated microglia. The fold increase in the expression of the different markers was compared to the effect on B7.1 expression. Necrotic neurons were significantly more efficient than apoptotic HT22 cells in inducing CD40 expression by microglia (graph on the right). (C) Histogram overlays for CD40 staining intensity of microglia cultured in medium only (grey filled), stimulated with necrotic HT22 neurons (solid line), or incubated with different necrotic cell types: histogram plot on the left, primary cerebellar granule neurons (CGN) (dashed line), astrocyte-enriched cell population (dotted line); histogram plot on the right, EL-4 (dotted line) and WHEI-164 (dashed line) cell lines. (D) Microglial cells were stimulated for 24 hours with necrotic neurons or with IFN-γ (10 U/ml), and the expression of COX-2, IDO and iNOS was analysed by RT-PCR. (E) Microglial cells were plated in an insert containing a membrane with a cut-off of 8 μm. The number of microglial cells migrated through the membrane was quantified 3 hours after adding necrotic HT22 neurons (50 μg/ml) to the lower chamber. ATP (100 μM) was used as a positive control for chemotaxis. Data are representative of at least two experiments done in triplicate. *** p < 0.001; **p < 0.01 and * p < 0.05.
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Figure 1: Necrotic neurons induce expression of activation markers by microglia. (A) Microglia were stimulated with necrotic HT22 neurons at a ratio of 1:1 for 24 hours and the expression of several activation markers analysed by FACS. The staining intensity is represented on histogram overlays for the mentioned proteins in non-stimulated microglia (grey filled), and in microglia stimulated either with IFN-γ (100 U/ml) (dashed line) or with necrotic neurons (solid line). (B) These results are shown as fold increases ("fold activation") of the Geo Mean when divided by the average expression of triplicates of untreated microglia. The fold increase in the expression of the different markers was compared to the effect on B7.1 expression. Necrotic neurons were significantly more efficient than apoptotic HT22 cells in inducing CD40 expression by microglia (graph on the right). (C) Histogram overlays for CD40 staining intensity of microglia cultured in medium only (grey filled), stimulated with necrotic HT22 neurons (solid line), or incubated with different necrotic cell types: histogram plot on the left, primary cerebellar granule neurons (CGN) (dashed line), astrocyte-enriched cell population (dotted line); histogram plot on the right, EL-4 (dotted line) and WHEI-164 (dashed line) cell lines. (D) Microglial cells were stimulated for 24 hours with necrotic neurons or with IFN-γ (10 U/ml), and the expression of COX-2, IDO and iNOS was analysed by RT-PCR. (E) Microglial cells were plated in an insert containing a membrane with a cut-off of 8 μm. The number of microglial cells migrated through the membrane was quantified 3 hours after adding necrotic HT22 neurons (50 μg/ml) to the lower chamber. ATP (100 μM) was used as a positive control for chemotaxis. Data are representative of at least two experiments done in triplicate. *** p < 0.001; **p < 0.01 and * p < 0.05.

Mentions: Neuronal cell injury has been previously shown to be associated with up-regulation of several molecules involved in immunological responses of microglia [7]. We investigated the modulation of several cell activation markers [7] by microglial cells responding to necrotic neuronal HT22 cells, a mouse hippocampal cell line [27], using FACS analysis (Figure 1A). Microglial cells responded to necrotic HT22 cells with a 2–6 fold increase in the expression of CD40 and of MHC class II (Figure 1A). The alpha chain of the αMβ2 integrin CD11b and the heat-stable antigen CD24 were also 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 up-regulated whereas CD45 and CD14 were poorly induced by necrotic cells (Figure 1B). Apoptotic HT22 neuronal cells induced a lower increase in CD40 expression in microglial cells when compared to necrotic HT22 cells (Figure 1B, right graph). However, 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) (data not shown). Primary cerebellar granule neurons (CGN), but not astrocytes, mimicked the effect of necrotic HT22 cells by inducing CD40 expression (Figure 1C, left panel) and TNF secretion by microglia (71.5 ± 17.6 pg/ml compared to non-detectable values in non-stimulated microglia or microglia incubated with astrocytes). In addition, necrotic cells obtained from mouse EL-4 (lymphoma) or WHEI 164 (fibrosarcoma) cell lines were unable to trigger CD40 expression (Figure 1C, right panel) or TNF (non-detectable values) in microglia. We further evaluated the pro-inflammatory phenotype of microglia upon stimulation with necrotic neurons. Necrotic neurons induced 11- and 9-fold increases in secretion of IL-12p40 and IL-6, respectively (Table 1). Production of TNF and of NO, estimated by the nitrite concentration in the supernatant of microglial cell cultures, were significantly increased by necrotic neurons although ~10-fold lower when compared with the levels induced by other stimuli such as IFN-γ or LPS (data not shown). IL-1β was not induced in microglia by necrotic neurons (Table 1). Furthermore, the mRNA expression levels of enzymes induced during neuroinflammatory conditions triggered by infection or brain injury, such as iNOS [28], IDO [29] and COX-2 [30], were increased in microglia by necrotic neurons (Figure 1D). Finally, necrotic neurons, when compared to a chemotactic stimulus such as ATP [31], induced significantly greater migration of microglial cells, (p < 0.01) seeded on a membrane insert, towards a lower chamber containing necrotic HT22 cells (Figure 1E). In summary, a clear pro-inflammatory phenotype is induced in microglia upon stimulation with necrotic neurons, as is increased microglial motility.


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 induce expression of activation markers by microglia. (A) Microglia were stimulated with necrotic HT22 neurons at a ratio of 1:1 for 24 hours and the expression of several activation markers analysed by FACS. The staining intensity is represented on histogram overlays for the mentioned proteins in non-stimulated microglia (grey filled), and in microglia stimulated either with IFN-γ (100 U/ml) (dashed line) or with necrotic neurons (solid line). (B) These results are shown as fold increases ("fold activation") of the Geo Mean when divided by the average expression of triplicates of untreated microglia. The fold increase in the expression of the different markers was compared to the effect on B7.1 expression. Necrotic neurons were significantly more efficient than apoptotic HT22 cells in inducing CD40 expression by microglia (graph on the right). (C) Histogram overlays for CD40 staining intensity of microglia cultured in medium only (grey filled), stimulated with necrotic HT22 neurons (solid line), or incubated with different necrotic cell types: histogram plot on the left, primary cerebellar granule neurons (CGN) (dashed line), astrocyte-enriched cell population (dotted line); histogram plot on the right, EL-4 (dotted line) and WHEI-164 (dashed line) cell lines. (D) Microglial cells were stimulated for 24 hours with necrotic neurons or with IFN-γ (10 U/ml), and the expression of COX-2, IDO and iNOS was analysed by RT-PCR. (E) Microglial cells were plated in an insert containing a membrane with a cut-off of 8 μm. The number of microglial cells migrated through the membrane was quantified 3 hours after adding necrotic HT22 neurons (50 μg/ml) to the lower chamber. ATP (100 μM) was used as a positive control for chemotaxis. Data are representative of at least two experiments done in triplicate. *** p < 0.001; **p < 0.01 and * p < 0.05.
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Figure 1: Necrotic neurons induce expression of activation markers by microglia. (A) Microglia were stimulated with necrotic HT22 neurons at a ratio of 1:1 for 24 hours and the expression of several activation markers analysed by FACS. The staining intensity is represented on histogram overlays for the mentioned proteins in non-stimulated microglia (grey filled), and in microglia stimulated either with IFN-γ (100 U/ml) (dashed line) or with necrotic neurons (solid line). (B) These results are shown as fold increases ("fold activation") of the Geo Mean when divided by the average expression of triplicates of untreated microglia. The fold increase in the expression of the different markers was compared to the effect on B7.1 expression. Necrotic neurons were significantly more efficient than apoptotic HT22 cells in inducing CD40 expression by microglia (graph on the right). (C) Histogram overlays for CD40 staining intensity of microglia cultured in medium only (grey filled), stimulated with necrotic HT22 neurons (solid line), or incubated with different necrotic cell types: histogram plot on the left, primary cerebellar granule neurons (CGN) (dashed line), astrocyte-enriched cell population (dotted line); histogram plot on the right, EL-4 (dotted line) and WHEI-164 (dashed line) cell lines. (D) Microglial cells were stimulated for 24 hours with necrotic neurons or with IFN-γ (10 U/ml), and the expression of COX-2, IDO and iNOS was analysed by RT-PCR. (E) Microglial cells were plated in an insert containing a membrane with a cut-off of 8 μm. The number of microglial cells migrated through the membrane was quantified 3 hours after adding necrotic HT22 neurons (50 μg/ml) to the lower chamber. ATP (100 μM) was used as a positive control for chemotaxis. Data are representative of at least two experiments done in triplicate. *** p < 0.001; **p < 0.01 and * p < 0.05.
Mentions: Neuronal cell injury has been previously shown to be associated with up-regulation of several molecules involved in immunological responses of microglia [7]. We investigated the modulation of several cell activation markers [7] by microglial cells responding to necrotic neuronal HT22 cells, a mouse hippocampal cell line [27], using FACS analysis (Figure 1A). Microglial cells responded to necrotic HT22 cells with a 2–6 fold increase in the expression of CD40 and of MHC class II (Figure 1A). The alpha chain of the αMβ2 integrin CD11b and the heat-stable antigen CD24 were also 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 up-regulated whereas CD45 and CD14 were poorly induced by necrotic cells (Figure 1B). Apoptotic HT22 neuronal cells induced a lower increase in CD40 expression in microglial cells when compared to necrotic HT22 cells (Figure 1B, right graph). However, 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) (data not shown). Primary cerebellar granule neurons (CGN), but not astrocytes, mimicked the effect of necrotic HT22 cells by inducing CD40 expression (Figure 1C, left panel) and TNF secretion by microglia (71.5 ± 17.6 pg/ml compared to non-detectable values in non-stimulated microglia or microglia incubated with astrocytes). In addition, necrotic cells obtained from mouse EL-4 (lymphoma) or WHEI 164 (fibrosarcoma) cell lines were unable to trigger CD40 expression (Figure 1C, right panel) or TNF (non-detectable values) in microglia. We further evaluated the pro-inflammatory phenotype of microglia upon stimulation with necrotic neurons. Necrotic neurons induced 11- and 9-fold increases in secretion of IL-12p40 and IL-6, respectively (Table 1). Production of TNF and of NO, estimated by the nitrite concentration in the supernatant of microglial cell cultures, were significantly increased by necrotic neurons although ~10-fold lower when compared with the levels induced by other stimuli such as IFN-γ or LPS (data not shown). IL-1β was not induced in microglia by necrotic neurons (Table 1). Furthermore, the mRNA expression levels of enzymes induced during neuroinflammatory conditions triggered by infection or brain injury, such as iNOS [28], IDO [29] and COX-2 [30], were increased in microglia by necrotic neurons (Figure 1D). Finally, necrotic neurons, when compared to a chemotactic stimulus such as ATP [31], induced significantly greater migration of microglial cells, (p < 0.01) seeded on a membrane insert, towards a lower chamber containing necrotic HT22 cells (Figure 1E). In summary, a clear pro-inflammatory phenotype is induced in microglia upon stimulation with necrotic neurons, as is increased microglial motility.

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