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FGF-2 released from degenerating neurons exerts microglial-induced neuroprotection via FGFR3-ERK signaling pathway.

Noda M, Takii K, Parajuli B, Kawanokuchi J, Sonobe Y, Takeuchi H, Mizuno T, Suzumura A - J Neuroinflammation (2014)

Bottom Line: Fibroblast growth factor-2 is secreted by neurons when damaged by glutamate or oligomeric amyloid β 1-42.FGF-2 enhances microglial migration and phagocytosis of neuronal debris, and is neuroprotective against glutamate toxicity through FGFR3-extracellular signal-regulated kinase (ERK) signaling pathway, which is directly controlled by Wnt signaling in microglia.FGF-2 secreted from degenerating neurons may act as a 'help-me' signal toward microglia by inducing migration and phagocytosis of unwanted debris.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. tmizuno@riem.nagoya-u.ac.jp.

ABSTRACT

Background: The accumulation of activated microglia is a hallmark of various neurodegenerative diseases. Microglia may have both protective and toxic effects on neurons through the production of various soluble factors, such as chemokines. Indeed, various chemokines mediate the rapid and accurate migration of microglia to lesions. In the zebra fish, another well-known cellular migrating factor is fibroblast growth factor-2 (FGF-2). Although FGF-2 does exist in the mammalian central nervous system (CNS), it is unclear whether FGF-2 influences microglial function.

Methods: The extent of FGF-2 release was determined by ELISA, and the expression of its receptors was examined by immunocytochemistry. The effect of several drug treatments on a neuron and microglia co-culture system was estimated by immunocytochemistry, and the neuronal survival rate was quantified. Microglial phagocytosis was evaluated by immunocytochemistry and quantification, and microglial migration was estimated by fluorescence-activated cell sorting (FACS). Molecular biological analyses, such as Western blotting and promoter assay, were performed to clarify the FGF-2 downstream signaling pathway in microglia.

Results: Fibroblast growth factor-2 is secreted by neurons when damaged by glutamate or oligomeric amyloid β 1-42. FGF-2 enhances microglial migration and phagocytosis of neuronal debris, and is neuroprotective against glutamate toxicity through FGFR3-extracellular signal-regulated kinase (ERK) signaling pathway, which is directly controlled by Wnt signaling in microglia.

Conclusions: FGF-2 secreted from degenerating neurons may act as a 'help-me' signal toward microglia by inducing migration and phagocytosis of unwanted debris.

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The neuroprotective effects of FGF-2 in neuron-microglia co-cultures. (A) Neuronal cultures were also treated with Glu and FGF-2. Neurons were stained with anti-MAP-2 antibody (green), and microglia were stained with a Cy5-conjugated anti-CD11b antibody (red). Scale bars, 50 μm. (B) The neuronal survival rate was calculated as the percentage of intact neurons in the treated sample relative to the untreated sample. The columns indicate mean with SEM from three independent experiments. * indicates significant differences compared with untreated neuronal cultures (***: P < 0.001); + indicates significant differences compared with untreated neuron–microglia co-cultures (+++: P < 0.001) by one-way ANOVA with Tukey’s post-hoc test. (C) After treatment with 20 μM Glu and 100 ng/ml FGF-2, the inhibitory effects of FGFR were evaluated using FGFR blockers or each anti-FGFR neutralizing antibody (PD, pan-FGFR blocker, 1 μM PD173074; SU, selective FGFR1 blocker, 500 nM SU11652; aR2, anti-FGFR2 antibody; aR3, anti-FGFR3 antibody; aR4, anti-FGFR4 antibody; aR5, anti-FGFR5 antibody; or isotype-matched IgG control). (D) The neuronal survival rate was calculated. The columns indicate the means with SEM from three independent experiments, each of which included the analysis of ten randomly selected fields. Significant differences compared with FGF-2-treatment were noted. **: P < 0.01, ***: P < 0.001 (one-way ANOVA with Tukey’s post-hoc test).
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Figure 3: The neuroprotective effects of FGF-2 in neuron-microglia co-cultures. (A) Neuronal cultures were also treated with Glu and FGF-2. Neurons were stained with anti-MAP-2 antibody (green), and microglia were stained with a Cy5-conjugated anti-CD11b antibody (red). Scale bars, 50 μm. (B) The neuronal survival rate was calculated as the percentage of intact neurons in the treated sample relative to the untreated sample. The columns indicate mean with SEM from three independent experiments. * indicates significant differences compared with untreated neuronal cultures (***: P < 0.001); + indicates significant differences compared with untreated neuron–microglia co-cultures (+++: P < 0.001) by one-way ANOVA with Tukey’s post-hoc test. (C) After treatment with 20 μM Glu and 100 ng/ml FGF-2, the inhibitory effects of FGFR were evaluated using FGFR blockers or each anti-FGFR neutralizing antibody (PD, pan-FGFR blocker, 1 μM PD173074; SU, selective FGFR1 blocker, 500 nM SU11652; aR2, anti-FGFR2 antibody; aR3, anti-FGFR3 antibody; aR4, anti-FGFR4 antibody; aR5, anti-FGFR5 antibody; or isotype-matched IgG control). (D) The neuronal survival rate was calculated. The columns indicate the means with SEM from three independent experiments, each of which included the analysis of ten randomly selected fields. Significant differences compared with FGF-2-treatment were noted. **: P < 0.01, ***: P < 0.001 (one-way ANOVA with Tukey’s post-hoc test).

Mentions: Next, we determined whether FGF-2 might exert microglial neuroprotection. As shown in Figure 3A,B, treatment with 20 μM glutamate induced apparent neuronal cell death in neuron-microglia co-cultures. The addition of 100 ng/ml FGF-2 significantly ameliorated neurotoxicity, while an anti-FGF-2 antibody canceled the effect. The addition of rat IgG (isotype-matched control for anti-FGF-2 antibody) had no effect on cell survival rate. In neuronal cultures, neuronal cell death was not ameliorated by FGF-2 treatment. There seems to be little difference in neuronal survival against Glu-induced excitotoxicity with or without microglia. We considered that the secreted level of FGF-2 from Glu-treated neurons might not reach the effective dose to enhance the neuronal survival. In addition, FGF-2 treatment suppressed the proinflammatory response of activated microglia through the inhibition of neurotoxic molecules, such as glutamate and NO (Additional file 1: Figure S1A,B). FGF-2 had no effect on microglial proliferation (Additional file 1: Figure S1C). FGF-2 dose-dependently enhanced the neuronal survival in the presence of microglia (Additional file 1: Figure S2).


FGF-2 released from degenerating neurons exerts microglial-induced neuroprotection via FGFR3-ERK signaling pathway.

Noda M, Takii K, Parajuli B, Kawanokuchi J, Sonobe Y, Takeuchi H, Mizuno T, Suzumura A - J Neuroinflammation (2014)

The neuroprotective effects of FGF-2 in neuron-microglia co-cultures. (A) Neuronal cultures were also treated with Glu and FGF-2. Neurons were stained with anti-MAP-2 antibody (green), and microglia were stained with a Cy5-conjugated anti-CD11b antibody (red). Scale bars, 50 μm. (B) The neuronal survival rate was calculated as the percentage of intact neurons in the treated sample relative to the untreated sample. The columns indicate mean with SEM from three independent experiments. * indicates significant differences compared with untreated neuronal cultures (***: P < 0.001); + indicates significant differences compared with untreated neuron–microglia co-cultures (+++: P < 0.001) by one-way ANOVA with Tukey’s post-hoc test. (C) After treatment with 20 μM Glu and 100 ng/ml FGF-2, the inhibitory effects of FGFR were evaluated using FGFR blockers or each anti-FGFR neutralizing antibody (PD, pan-FGFR blocker, 1 μM PD173074; SU, selective FGFR1 blocker, 500 nM SU11652; aR2, anti-FGFR2 antibody; aR3, anti-FGFR3 antibody; aR4, anti-FGFR4 antibody; aR5, anti-FGFR5 antibody; or isotype-matched IgG control). (D) The neuronal survival rate was calculated. The columns indicate the means with SEM from three independent experiments, each of which included the analysis of ten randomly selected fields. Significant differences compared with FGF-2-treatment were noted. **: P < 0.01, ***: P < 0.001 (one-way ANOVA with Tukey’s post-hoc test).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: The neuroprotective effects of FGF-2 in neuron-microglia co-cultures. (A) Neuronal cultures were also treated with Glu and FGF-2. Neurons were stained with anti-MAP-2 antibody (green), and microglia were stained with a Cy5-conjugated anti-CD11b antibody (red). Scale bars, 50 μm. (B) The neuronal survival rate was calculated as the percentage of intact neurons in the treated sample relative to the untreated sample. The columns indicate mean with SEM from three independent experiments. * indicates significant differences compared with untreated neuronal cultures (***: P < 0.001); + indicates significant differences compared with untreated neuron–microglia co-cultures (+++: P < 0.001) by one-way ANOVA with Tukey’s post-hoc test. (C) After treatment with 20 μM Glu and 100 ng/ml FGF-2, the inhibitory effects of FGFR were evaluated using FGFR blockers or each anti-FGFR neutralizing antibody (PD, pan-FGFR blocker, 1 μM PD173074; SU, selective FGFR1 blocker, 500 nM SU11652; aR2, anti-FGFR2 antibody; aR3, anti-FGFR3 antibody; aR4, anti-FGFR4 antibody; aR5, anti-FGFR5 antibody; or isotype-matched IgG control). (D) The neuronal survival rate was calculated. The columns indicate the means with SEM from three independent experiments, each of which included the analysis of ten randomly selected fields. Significant differences compared with FGF-2-treatment were noted. **: P < 0.01, ***: P < 0.001 (one-way ANOVA with Tukey’s post-hoc test).
Mentions: Next, we determined whether FGF-2 might exert microglial neuroprotection. As shown in Figure 3A,B, treatment with 20 μM glutamate induced apparent neuronal cell death in neuron-microglia co-cultures. The addition of 100 ng/ml FGF-2 significantly ameliorated neurotoxicity, while an anti-FGF-2 antibody canceled the effect. The addition of rat IgG (isotype-matched control for anti-FGF-2 antibody) had no effect on cell survival rate. In neuronal cultures, neuronal cell death was not ameliorated by FGF-2 treatment. There seems to be little difference in neuronal survival against Glu-induced excitotoxicity with or without microglia. We considered that the secreted level of FGF-2 from Glu-treated neurons might not reach the effective dose to enhance the neuronal survival. In addition, FGF-2 treatment suppressed the proinflammatory response of activated microglia through the inhibition of neurotoxic molecules, such as glutamate and NO (Additional file 1: Figure S1A,B). FGF-2 had no effect on microglial proliferation (Additional file 1: Figure S1C). FGF-2 dose-dependently enhanced the neuronal survival in the presence of microglia (Additional file 1: Figure S2).

Bottom Line: Fibroblast growth factor-2 is secreted by neurons when damaged by glutamate or oligomeric amyloid β 1-42.FGF-2 enhances microglial migration and phagocytosis of neuronal debris, and is neuroprotective against glutamate toxicity through FGFR3-extracellular signal-regulated kinase (ERK) signaling pathway, which is directly controlled by Wnt signaling in microglia.FGF-2 secreted from degenerating neurons may act as a 'help-me' signal toward microglia by inducing migration and phagocytosis of unwanted debris.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Neuroimmunology, Research Institute of Environmental Medicine, Nagoya University, Furo-cho, Chikusa-ku, Nagoya 464-8601, Japan. tmizuno@riem.nagoya-u.ac.jp.

ABSTRACT

Background: The accumulation of activated microglia is a hallmark of various neurodegenerative diseases. Microglia may have both protective and toxic effects on neurons through the production of various soluble factors, such as chemokines. Indeed, various chemokines mediate the rapid and accurate migration of microglia to lesions. In the zebra fish, another well-known cellular migrating factor is fibroblast growth factor-2 (FGF-2). Although FGF-2 does exist in the mammalian central nervous system (CNS), it is unclear whether FGF-2 influences microglial function.

Methods: The extent of FGF-2 release was determined by ELISA, and the expression of its receptors was examined by immunocytochemistry. The effect of several drug treatments on a neuron and microglia co-culture system was estimated by immunocytochemistry, and the neuronal survival rate was quantified. Microglial phagocytosis was evaluated by immunocytochemistry and quantification, and microglial migration was estimated by fluorescence-activated cell sorting (FACS). Molecular biological analyses, such as Western blotting and promoter assay, were performed to clarify the FGF-2 downstream signaling pathway in microglia.

Results: Fibroblast growth factor-2 is secreted by neurons when damaged by glutamate or oligomeric amyloid β 1-42. FGF-2 enhances microglial migration and phagocytosis of neuronal debris, and is neuroprotective against glutamate toxicity through FGFR3-extracellular signal-regulated kinase (ERK) signaling pathway, which is directly controlled by Wnt signaling in microglia.

Conclusions: FGF-2 secreted from degenerating neurons may act as a 'help-me' signal toward microglia by inducing migration and phagocytosis of unwanted debris.

Show MeSH
Related in: MedlinePlus