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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 reduces NADPH oxidase activation and is neuroprotective in vitro.(A) Enriched microglia cultures were treated with media alone (Control), apocynin (10 µM), Dextromethorphan (DM, 10 µM), Aβ (2 µM), Apocynin + Aβ, and DM + Aβ. The production of extracellular superoxide was measured by the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1 at 30 minutes post-treatment. Results are mean ± SEM. Data are from four separate experiments. *p<0.05, compared with control cultures. (B) Apocynin and DM protect against Aβ-induced toxicity in cortical neuron-glia cultures.) Cortical neuron-glia cultures were treated with media alone (Control), Apocynin (10 µM), Dextromethorphan (DM, 10 µM), Aβ (2 µM), Apocynin + Aβ, and DM + Aβ. Toxicity was assessed by MTT 7 days later. Graphs show the results expressed as percentage of the control cultures and are the mean ± SEM from three independent experiments in triplicate. * p<0.05, control compared to treatment.
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pone-0020153-g003: Apocynin reduces NADPH oxidase activation and is neuroprotective in vitro.(A) Enriched microglia cultures were treated with media alone (Control), apocynin (10 µM), Dextromethorphan (DM, 10 µM), Aβ (2 µM), Apocynin + Aβ, and DM + Aβ. The production of extracellular superoxide was measured by the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1 at 30 minutes post-treatment. Results are mean ± SEM. Data are from four separate experiments. *p<0.05, compared with control cultures. (B) Apocynin and DM protect against Aβ-induced toxicity in cortical neuron-glia cultures.) Cortical neuron-glia cultures were treated with media alone (Control), Apocynin (10 µM), Dextromethorphan (DM, 10 µM), Aβ (2 µM), Apocynin + Aβ, and DM + Aβ. Toxicity was assessed by MTT 7 days later. Graphs show the results expressed as percentage of the control cultures and are the mean ± SEM from three independent experiments in triplicate. * p<0.05, control compared to treatment.

Mentions: To confirm that both apocynin and DM were capable of inhibiting NADPH oxidase at all, we next tested their ability to reduce the production of extracellular ROS and neurotoxicity in response to Aβ. Both apocynin and DM were able to reduce the production of Aβ-induced extracellular superoxide to nearly control levels in primary microglia cultures (Figure 3) and ameliorate Aβ-induced neurotoxicity in cortical mixed-neuron-glia cultures (Figure 3). Thus, in the presence of microglial NADPH oxidase activation in vitro, both compounds are able to reduce extracellular ROS and cellular damage. These findings further support that the inability of either DM or apocynin to reduce measures of oxidative stress and synaptic density may have been due to a lack of activation of NADPH oxidase and neuroinflammation in hAPP(751)SL mice at 8 months.


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 reduces NADPH oxidase activation and is neuroprotective in vitro.(A) Enriched microglia cultures were treated with media alone (Control), apocynin (10 µM), Dextromethorphan (DM, 10 µM), Aβ (2 µM), Apocynin + Aβ, and DM + Aβ. The production of extracellular superoxide was measured by the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1 at 30 minutes post-treatment. Results are mean ± SEM. Data are from four separate experiments. *p<0.05, compared with control cultures. (B) Apocynin and DM protect against Aβ-induced toxicity in cortical neuron-glia cultures.) Cortical neuron-glia cultures were treated with media alone (Control), Apocynin (10 µM), Dextromethorphan (DM, 10 µM), Aβ (2 µM), Apocynin + Aβ, and DM + Aβ. Toxicity was assessed by MTT 7 days later. Graphs show the results expressed as percentage of the control cultures and are the mean ± SEM from three independent experiments in triplicate. * p<0.05, control compared to treatment.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3105011&req=5

pone-0020153-g003: Apocynin reduces NADPH oxidase activation and is neuroprotective in vitro.(A) Enriched microglia cultures were treated with media alone (Control), apocynin (10 µM), Dextromethorphan (DM, 10 µM), Aβ (2 µM), Apocynin + Aβ, and DM + Aβ. The production of extracellular superoxide was measured by the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt, WST-1 at 30 minutes post-treatment. Results are mean ± SEM. Data are from four separate experiments. *p<0.05, compared with control cultures. (B) Apocynin and DM protect against Aβ-induced toxicity in cortical neuron-glia cultures.) Cortical neuron-glia cultures were treated with media alone (Control), Apocynin (10 µM), Dextromethorphan (DM, 10 µM), Aβ (2 µM), Apocynin + Aβ, and DM + Aβ. Toxicity was assessed by MTT 7 days later. Graphs show the results expressed as percentage of the control cultures and are the mean ± SEM from three independent experiments in triplicate. * p<0.05, control compared to treatment.
Mentions: To confirm that both apocynin and DM were capable of inhibiting NADPH oxidase at all, we next tested their ability to reduce the production of extracellular ROS and neurotoxicity in response to Aβ. Both apocynin and DM were able to reduce the production of Aβ-induced extracellular superoxide to nearly control levels in primary microglia cultures (Figure 3) and ameliorate Aβ-induced neurotoxicity in cortical mixed-neuron-glia cultures (Figure 3). Thus, in the presence of microglial NADPH oxidase activation in vitro, both compounds are able to reduce extracellular ROS and cellular damage. These findings further support that the inability of either DM or apocynin to reduce measures of oxidative stress and synaptic density may have been due to a lack of activation of NADPH oxidase and neuroinflammation in hAPP(751)SL mice at 8 months.

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