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Chronic apocynin treatment attenuates beta amyloid plaque size and microglial number in hAPP(751)(SL) mice.

Lull ME, Levesque S, Surace MJ, Block ML - PLoS ONE (2011)

Bottom Line: Despite in vitro analyses demonstrating that apocynin and DM ameliorate Aβ-induced extracellular superoxide production and neurotoxicity, both DM and apocynin failed to significantly affect learning and memory tasks or synaptic density in hAPP(751)(SL) mice.To discern how apocynin was affecting plaque levels (plaque load) and microglial number in vivo, in vitro analysis of microglia was performed, revealing no apocynin effects on beta-amyloid (Aβ) phagocytosis, microglial proliferation, or microglial survival.Despite absence of clear neuroinflammation, apocynin was still able to reduce both plaque size and microglial number, suggesting that apocynin may have additional therapeutic effects independent of anti-inflammatory characteristics.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Virginia Commonwealth University Medical Campus, Richmond, Virginia, United States of America.

ABSTRACT

Background: NADPH oxidase is implicated in neurotoxic microglial activation and the progressive nature of Alzheimer's Disease (AD). Here, we test the ability of two NADPH oxidase inhibitors, apocynin and dextromethorphan (DM), to reduce learning deficits and neuropathology in transgenic mice overexpressing human amyloid precursor protein with the Swedish and London mutations (hAPP(751)(SL)).

Methods: Four month old hAPP(751)(SL) mice were treated daily with saline, 15 mg/kg DM, 7.5 mg/kg DM, or 10 mg/kg apocynin by gavage for four months.

Results: Only hAPP(751)(SL) mice treated with apocynin showed reduced plaque size and a reduction in the number of cortical microglia, when compared to the saline treated group. Analysis of whole brain homogenates from all treatments tested (saline, DM, and apocynin) demonstrated low levels of TNFα, protein nitration, lipid peroxidation, and NADPH oxidase activation, indicating a low level of neuroinflammation and oxidative stress in hAPP(751)(SL) mice at 8 months of age that was not significantly affected by any drug treatment. Despite in vitro analyses demonstrating that apocynin and DM ameliorate Aβ-induced extracellular superoxide production and neurotoxicity, both DM and apocynin failed to significantly affect learning and memory tasks or synaptic density in hAPP(751)(SL) mice. To discern how apocynin was affecting plaque levels (plaque load) and microglial number in vivo, in vitro analysis of microglia was performed, revealing no apocynin effects on beta-amyloid (Aβ) phagocytosis, microglial proliferation, or microglial survival.

Conclusions: Together, this study suggests that while hAPP(751)(SL) mice show increases in microglial number and plaque load, they fail to exhibit elevated markers of neuroinflammation consistent with AD at 8 months of age, which may be a limitation of this animal model. Despite absence of clear neuroinflammation, apocynin was still able to reduce both plaque size and microglial number, suggesting that apocynin may have additional therapeutic effects independent of anti-inflammatory characteristics.

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Related in: MedlinePlus

Apocynin ameliorates LPS-induced TNFα production, but has no effect on microglial cell death or cell number in vitro.(A) Microglia-enriched cultures were treated with control media, lip polysaccharide (LPS; 10 ng/mL), and/or Apocynin (100 µM) for 24 hours. Tumor necrosis factor alpha (TNFα) levels in the supernatant were measured via ELISA. LPS (10 ng/mL) significantly increased levels of TNFα and pre-treatment with 100 µM Apocynin significantly reduced the amount of TNFα released by microglia. *p<0.05 vs. control; #p<0.05 vs. LPS, 1-way ANOVA with Bonferroni post-hoc test. (B) Apocynin does not protect against inflammation-induced cell death. Microglia-enriched cultures were treated with control media, 1000 ng/mL LPS, 100 µM Apocynin, or LPS and apocynin for 24 hours. After incubation, cell survival was measured with the MTT assay. 100 ng/mL LPS significantly reduced microglial cell survival (through inflammation-induced cell death), which is not rescued by apocynin). (C) Microglia-enriched cultures were treated with control media, 2 µM staurosporine (SS), 100 µM Apocynin, or SS and apocynin for 24 hours. After incubation, cell survival was measured with the MTT assay. Data show that 2 µM SS significantly reduces microglial cell survival (through apoptosis), and is not reversed by the addition of apocynin. *p<0.05 vs control,1-way ANOVA with Bonferroni post-hoc test. (D) Apocynin does not alter Aβ or LPS-induced increases in microglia number in vitro. Mixed neuron-glia cultures were treated with 2 µM Aβ, 10 ng/mL LPS and/or 100 µM apocynin for 24 hours. Cultures were then fixed and stained with IBA-1 antibody for microglia. The number of microglia was then counted in 9 representative areas per well. The number of microglia was significantly increased in cultures treated with LPS (196% of control) and Aβ (186% of control). Apocynin treatment did not prevent the increase in cell count caused by LPS or Aβ. Apocynin alone caused no significant change in cell count. *p<0.05 vs control, 1-way ANOVA with Bonferroni post-hoc test.
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pone-0020153-g005: Apocynin ameliorates LPS-induced TNFα production, but has no effect on microglial cell death or cell number in vitro.(A) Microglia-enriched cultures were treated with control media, lip polysaccharide (LPS; 10 ng/mL), and/or Apocynin (100 µM) for 24 hours. Tumor necrosis factor alpha (TNFα) levels in the supernatant were measured via ELISA. LPS (10 ng/mL) significantly increased levels of TNFα and pre-treatment with 100 µM Apocynin significantly reduced the amount of TNFα released by microglia. *p<0.05 vs. control; #p<0.05 vs. LPS, 1-way ANOVA with Bonferroni post-hoc test. (B) Apocynin does not protect against inflammation-induced cell death. Microglia-enriched cultures were treated with control media, 1000 ng/mL LPS, 100 µM Apocynin, or LPS and apocynin for 24 hours. After incubation, cell survival was measured with the MTT assay. 100 ng/mL LPS significantly reduced microglial cell survival (through inflammation-induced cell death), which is not rescued by apocynin). (C) Microglia-enriched cultures were treated with control media, 2 µM staurosporine (SS), 100 µM Apocynin, or SS and apocynin for 24 hours. After incubation, cell survival was measured with the MTT assay. Data show that 2 µM SS significantly reduces microglial cell survival (through apoptosis), and is not reversed by the addition of apocynin. *p<0.05 vs control,1-way ANOVA with Bonferroni post-hoc test. (D) Apocynin does not alter Aβ or LPS-induced increases in microglia number in vitro. Mixed neuron-glia cultures were treated with 2 µM Aβ, 10 ng/mL LPS and/or 100 µM apocynin for 24 hours. Cultures were then fixed and stained with IBA-1 antibody for microglia. The number of microglia was then counted in 9 representative areas per well. The number of microglia was significantly increased in cultures treated with LPS (196% of control) and Aβ (186% of control). Apocynin treatment did not prevent the increase in cell count caused by LPS or Aβ. Apocynin alone caused no significant change in cell count. *p<0.05 vs control, 1-way ANOVA with Bonferroni post-hoc test.

Mentions: We then focused in vitro analyses specifically on apocynin, which reduced both plaque size and microglial number in vivo. Specifically, we next addressed whether apocynin was able to ameliorate a generalized pro-inflammatory response from microglia. The pro-inflammatory cytokine response of primary microglia-enriched cultures was tested by measuring levels of TNFα following treatment with 10 ng/mL LPS and/or 100 µM apocynin. LPS significantly increased levels of TNFα at 24 hours after treatment (to 2907 pg/mL), and apocynin was able to significantly reduce this response (reduced to 1952 pg/mL), although levels did not return to that of control (4 pg/mL) (Figure 5) (p<0.05). These data confirm that if TNFα levels are elevated, in the very least, apocynin is able to reduce them in vitro.


Chronic apocynin treatment attenuates beta amyloid plaque size and microglial number in hAPP(751)(SL) mice.

Lull ME, Levesque S, Surace MJ, Block ML - PLoS ONE (2011)

Apocynin ameliorates LPS-induced TNFα production, but has no effect on microglial cell death or cell number in vitro.(A) Microglia-enriched cultures were treated with control media, lip polysaccharide (LPS; 10 ng/mL), and/or Apocynin (100 µM) for 24 hours. Tumor necrosis factor alpha (TNFα) levels in the supernatant were measured via ELISA. LPS (10 ng/mL) significantly increased levels of TNFα and pre-treatment with 100 µM Apocynin significantly reduced the amount of TNFα released by microglia. *p<0.05 vs. control; #p<0.05 vs. LPS, 1-way ANOVA with Bonferroni post-hoc test. (B) Apocynin does not protect against inflammation-induced cell death. Microglia-enriched cultures were treated with control media, 1000 ng/mL LPS, 100 µM Apocynin, or LPS and apocynin for 24 hours. After incubation, cell survival was measured with the MTT assay. 100 ng/mL LPS significantly reduced microglial cell survival (through inflammation-induced cell death), which is not rescued by apocynin). (C) Microglia-enriched cultures were treated with control media, 2 µM staurosporine (SS), 100 µM Apocynin, or SS and apocynin for 24 hours. After incubation, cell survival was measured with the MTT assay. Data show that 2 µM SS significantly reduces microglial cell survival (through apoptosis), and is not reversed by the addition of apocynin. *p<0.05 vs control,1-way ANOVA with Bonferroni post-hoc test. (D) Apocynin does not alter Aβ or LPS-induced increases in microglia number in vitro. Mixed neuron-glia cultures were treated with 2 µM Aβ, 10 ng/mL LPS and/or 100 µM apocynin for 24 hours. Cultures were then fixed and stained with IBA-1 antibody for microglia. The number of microglia was then counted in 9 representative areas per well. The number of microglia was significantly increased in cultures treated with LPS (196% of control) and Aβ (186% of control). Apocynin treatment did not prevent the increase in cell count caused by LPS or Aβ. Apocynin alone caused no significant change in cell count. *p<0.05 vs control, 1-way ANOVA with Bonferroni post-hoc test.
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pone-0020153-g005: Apocynin ameliorates LPS-induced TNFα production, but has no effect on microglial cell death or cell number in vitro.(A) Microglia-enriched cultures were treated with control media, lip polysaccharide (LPS; 10 ng/mL), and/or Apocynin (100 µM) for 24 hours. Tumor necrosis factor alpha (TNFα) levels in the supernatant were measured via ELISA. LPS (10 ng/mL) significantly increased levels of TNFα and pre-treatment with 100 µM Apocynin significantly reduced the amount of TNFα released by microglia. *p<0.05 vs. control; #p<0.05 vs. LPS, 1-way ANOVA with Bonferroni post-hoc test. (B) Apocynin does not protect against inflammation-induced cell death. Microglia-enriched cultures were treated with control media, 1000 ng/mL LPS, 100 µM Apocynin, or LPS and apocynin for 24 hours. After incubation, cell survival was measured with the MTT assay. 100 ng/mL LPS significantly reduced microglial cell survival (through inflammation-induced cell death), which is not rescued by apocynin). (C) Microglia-enriched cultures were treated with control media, 2 µM staurosporine (SS), 100 µM Apocynin, or SS and apocynin for 24 hours. After incubation, cell survival was measured with the MTT assay. Data show that 2 µM SS significantly reduces microglial cell survival (through apoptosis), and is not reversed by the addition of apocynin. *p<0.05 vs control,1-way ANOVA with Bonferroni post-hoc test. (D) Apocynin does not alter Aβ or LPS-induced increases in microglia number in vitro. Mixed neuron-glia cultures were treated with 2 µM Aβ, 10 ng/mL LPS and/or 100 µM apocynin for 24 hours. Cultures were then fixed and stained with IBA-1 antibody for microglia. The number of microglia was then counted in 9 representative areas per well. The number of microglia was significantly increased in cultures treated with LPS (196% of control) and Aβ (186% of control). Apocynin treatment did not prevent the increase in cell count caused by LPS or Aβ. Apocynin alone caused no significant change in cell count. *p<0.05 vs control, 1-way ANOVA with Bonferroni post-hoc test.
Mentions: We then focused in vitro analyses specifically on apocynin, which reduced both plaque size and microglial number in vivo. Specifically, we next addressed whether apocynin was able to ameliorate a generalized pro-inflammatory response from microglia. The pro-inflammatory cytokine response of primary microglia-enriched cultures was tested by measuring levels of TNFα following treatment with 10 ng/mL LPS and/or 100 µM apocynin. LPS significantly increased levels of TNFα at 24 hours after treatment (to 2907 pg/mL), and apocynin was able to significantly reduce this response (reduced to 1952 pg/mL), although levels did not return to that of control (4 pg/mL) (Figure 5) (p<0.05). These data confirm that if TNFα levels are elevated, in the very least, apocynin is able to reduce them in vitro.

Bottom Line: Despite in vitro analyses demonstrating that apocynin and DM ameliorate Aβ-induced extracellular superoxide production and neurotoxicity, both DM and apocynin failed to significantly affect learning and memory tasks or synaptic density in hAPP(751)(SL) mice.To discern how apocynin was affecting plaque levels (plaque load) and microglial number in vivo, in vitro analysis of microglia was performed, revealing no apocynin effects on beta-amyloid (Aβ) phagocytosis, microglial proliferation, or microglial survival.Despite absence of clear neuroinflammation, apocynin was still able to reduce both plaque size and microglial number, suggesting that apocynin may have additional therapeutic effects independent of anti-inflammatory characteristics.

View Article: PubMed Central - PubMed

Affiliation: Department of Anatomy and Neurobiology, Virginia Commonwealth University Medical Campus, Richmond, Virginia, United States of America.

ABSTRACT

Background: NADPH oxidase is implicated in neurotoxic microglial activation and the progressive nature of Alzheimer's Disease (AD). Here, we test the ability of two NADPH oxidase inhibitors, apocynin and dextromethorphan (DM), to reduce learning deficits and neuropathology in transgenic mice overexpressing human amyloid precursor protein with the Swedish and London mutations (hAPP(751)(SL)).

Methods: Four month old hAPP(751)(SL) mice were treated daily with saline, 15 mg/kg DM, 7.5 mg/kg DM, or 10 mg/kg apocynin by gavage for four months.

Results: Only hAPP(751)(SL) mice treated with apocynin showed reduced plaque size and a reduction in the number of cortical microglia, when compared to the saline treated group. Analysis of whole brain homogenates from all treatments tested (saline, DM, and apocynin) demonstrated low levels of TNFα, protein nitration, lipid peroxidation, and NADPH oxidase activation, indicating a low level of neuroinflammation and oxidative stress in hAPP(751)(SL) mice at 8 months of age that was not significantly affected by any drug treatment. Despite in vitro analyses demonstrating that apocynin and DM ameliorate Aβ-induced extracellular superoxide production and neurotoxicity, both DM and apocynin failed to significantly affect learning and memory tasks or synaptic density in hAPP(751)(SL) mice. To discern how apocynin was affecting plaque levels (plaque load) and microglial number in vivo, in vitro analysis of microglia was performed, revealing no apocynin effects on beta-amyloid (Aβ) phagocytosis, microglial proliferation, or microglial survival.

Conclusions: Together, this study suggests that while hAPP(751)(SL) mice show increases in microglial number and plaque load, they fail to exhibit elevated markers of neuroinflammation consistent with AD at 8 months of age, which may be a limitation of this animal model. Despite absence of clear neuroinflammation, apocynin was still able to reduce both plaque size and microglial number, suggesting that apocynin may have additional therapeutic effects independent of anti-inflammatory characteristics.

Show MeSH
Related in: MedlinePlus