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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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Apocynin regulates microglial H2O2 production, but not Aβ phagocytosis.(A) Apocynin attenuates LPS-induced hydrogen peroxide (H2O2), as predicted. Microglia-enriched cultures were treated with Hank's balanced salt solution (HBSS), or HBSS with LPS (10 ng/mL), apocynin (100 µM), or the combination of apocynin (100 µM) and LPS for 3 hours. The level of H2O2 was then measured in each group and compared to control levels. Apocynin does significantly reduce LPS-induced increases in H2O2, returning levels to control values. *p<0.05 vs. control; #p<0.05 vs. LPS, 1-way ANOVA with Bonferroni post-hoc test. (B) Pre-treatment with 2 µM Aβ significantly reduces phagocytosis of fluorescent Aβ, and apocynin does not act to reverse this decrease. Microglia-enriched cultures were treated with control media, or media with β-amyloid (Aβ; 2 µM), apocynin (100 µM), or the combination of apocynin (100 µM) and Aβ (2 µM) for 24 hours. Fluorescently labeled Aβ (final concentration 0.1 µM) was then added to each well, and incubated with the cells for 6 hours to allow for phagocytosis of the fluorescent protein. The amount of phagocytosis of fluorescent Aβ was measured for each group and compared to control levels. *p<0.05 vs control, 1-way ANOVA with Bonferroni post-hoc test.
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pone-0020153-g004: Apocynin regulates microglial H2O2 production, but not Aβ phagocytosis.(A) Apocynin attenuates LPS-induced hydrogen peroxide (H2O2), as predicted. Microglia-enriched cultures were treated with Hank's balanced salt solution (HBSS), or HBSS with LPS (10 ng/mL), apocynin (100 µM), or the combination of apocynin (100 µM) and LPS for 3 hours. The level of H2O2 was then measured in each group and compared to control levels. Apocynin does significantly reduce LPS-induced increases in H2O2, returning levels to control values. *p<0.05 vs. control; #p<0.05 vs. LPS, 1-way ANOVA with Bonferroni post-hoc test. (B) Pre-treatment with 2 µM Aβ significantly reduces phagocytosis of fluorescent Aβ, and apocynin does not act to reverse this decrease. Microglia-enriched cultures were treated with control media, or media with β-amyloid (Aβ; 2 µM), apocynin (100 µM), or the combination of apocynin (100 µM) and Aβ (2 µM) for 24 hours. Fluorescently labeled Aβ (final concentration 0.1 µM) was then added to each well, and incubated with the cells for 6 hours to allow for phagocytosis of the fluorescent protein. The amount of phagocytosis of fluorescent Aβ was measured for each group and compared to control levels. *p<0.05 vs control, 1-way ANOVA with Bonferroni post-hoc test.

Mentions: We confirmed that apocynin was working as predicted by demonstrating that 30 minute pretreatment with apocynin will attenuate LPS-induced H2O2 production (Figure 4). To test a possible mechanism for the in vivo observation of decreased plaque size with apocynin treatment, the capability of microglial to phagocytize fluorescently labeled Aβ was tested in microglial-enriched primary cell cultures. Pre-treatment of cells with 2 µM Aβ for 24 hours prior to the addition of the fluorescent Aβ significantly reduces the phagocytosis capacity of microglia by 41% (p<0.05) (Figure 4). Co-treatment with 100 µM apocynin did not reverse the Aβ-induced decreases in fluorescent Aβ phagocytosis (Figure 4), supporting that superoxide and reactive oxygen species (e.g. ROS) do not mediate the Aβ-induced loss of phagocytic function. Thus, while loss of microglial phagocytic function has been implicated as a key component to the development of plaques and AD progression [58], [59], apocynin failed to ameliorate this response in vitro, indicating it is an unlikely mechanism in the in vivo effects on plaque size.


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 regulates microglial H2O2 production, but not Aβ phagocytosis.(A) Apocynin attenuates LPS-induced hydrogen peroxide (H2O2), as predicted. Microglia-enriched cultures were treated with Hank's balanced salt solution (HBSS), or HBSS with LPS (10 ng/mL), apocynin (100 µM), or the combination of apocynin (100 µM) and LPS for 3 hours. The level of H2O2 was then measured in each group and compared to control levels. Apocynin does significantly reduce LPS-induced increases in H2O2, returning levels to control values. *p<0.05 vs. control; #p<0.05 vs. LPS, 1-way ANOVA with Bonferroni post-hoc test. (B) Pre-treatment with 2 µM Aβ significantly reduces phagocytosis of fluorescent Aβ, and apocynin does not act to reverse this decrease. Microglia-enriched cultures were treated with control media, or media with β-amyloid (Aβ; 2 µM), apocynin (100 µM), or the combination of apocynin (100 µM) and Aβ (2 µM) for 24 hours. Fluorescently labeled Aβ (final concentration 0.1 µM) was then added to each well, and incubated with the cells for 6 hours to allow for phagocytosis of the fluorescent protein. The amount of phagocytosis of fluorescent Aβ was measured for each group and compared to control levels. *p<0.05 vs control, 1-way ANOVA with Bonferroni post-hoc test.
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Related In: Results  -  Collection

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pone-0020153-g004: Apocynin regulates microglial H2O2 production, but not Aβ phagocytosis.(A) Apocynin attenuates LPS-induced hydrogen peroxide (H2O2), as predicted. Microglia-enriched cultures were treated with Hank's balanced salt solution (HBSS), or HBSS with LPS (10 ng/mL), apocynin (100 µM), or the combination of apocynin (100 µM) and LPS for 3 hours. The level of H2O2 was then measured in each group and compared to control levels. Apocynin does significantly reduce LPS-induced increases in H2O2, returning levels to control values. *p<0.05 vs. control; #p<0.05 vs. LPS, 1-way ANOVA with Bonferroni post-hoc test. (B) Pre-treatment with 2 µM Aβ significantly reduces phagocytosis of fluorescent Aβ, and apocynin does not act to reverse this decrease. Microglia-enriched cultures were treated with control media, or media with β-amyloid (Aβ; 2 µM), apocynin (100 µM), or the combination of apocynin (100 µM) and Aβ (2 µM) for 24 hours. Fluorescently labeled Aβ (final concentration 0.1 µM) was then added to each well, and incubated with the cells for 6 hours to allow for phagocytosis of the fluorescent protein. The amount of phagocytosis of fluorescent Aβ was measured for each group and compared to control levels. *p<0.05 vs control, 1-way ANOVA with Bonferroni post-hoc test.
Mentions: We confirmed that apocynin was working as predicted by demonstrating that 30 minute pretreatment with apocynin will attenuate LPS-induced H2O2 production (Figure 4). To test a possible mechanism for the in vivo observation of decreased plaque size with apocynin treatment, the capability of microglial to phagocytize fluorescently labeled Aβ was tested in microglial-enriched primary cell cultures. Pre-treatment of cells with 2 µM Aβ for 24 hours prior to the addition of the fluorescent Aβ significantly reduces the phagocytosis capacity of microglia by 41% (p<0.05) (Figure 4). Co-treatment with 100 µM apocynin did not reverse the Aβ-induced decreases in fluorescent Aβ phagocytosis (Figure 4), supporting that superoxide and reactive oxygen species (e.g. ROS) do not mediate the Aβ-induced loss of phagocytic function. Thus, while loss of microglial phagocytic function has been implicated as a key component to the development of plaques and AD progression [58], [59], apocynin failed to ameliorate this response in vitro, indicating it is an unlikely mechanism in the in vivo effects on plaque size.

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