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. 2011 May;117(3):388-402.
doi: 10.1111/j.1471-4159.2010.07145.x. Epub 2011 Mar 14.

Oxidative Insults to Neurons and Synapse Are Prevented by Aged Garlic Extract and S-allyl-L-cysteine Treatment in the Neuronal Culture and APP-Tg Mouse Model

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Oxidative Insults to Neurons and Synapse Are Prevented by Aged Garlic Extract and S-allyl-L-cysteine Treatment in the Neuronal Culture and APP-Tg Mouse Model

Balmiki Ray et al. J Neurochem. .
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Abstract

Alzheimer's disease (AD) is one of the most common forms of dementia in the elderly. In AD patients, β-amyloid peptide (Aβ) plaques and neurofibrillary tangles are common features observed in the CNS. Aβ deposition results in the production of reactive oxygen species (ROS) leading to the hyperphosphorylation of tau that are associated with neuronal damage. Cholinesterase inhibitors and a partial NMDA receptor antagonist (memantine) have been identified as potential treatment options for AD. However, clinical studies have found that these drugs fail to prevent the disease progression. From ancient times, garlic (Allium sativum) has been used to treat several diseases. By 'aging' of garlic, some adverse reactions of garlic can be eliminated. Recent findings suggest that 'aged garlic extract' (AGE) may be a therapeutic agent for AD because of its antioxidant and Aβ lowering properties. To date, the molecular properties of AGE have been sparsely studied in vitro or in vivo. The present study tested specific biochemical and molecular effects of AGE in neuronal and AD rodent models. Furthermore, we identified S-allyl-L-cysteine (SAC) as one of the most active chemicals responsible for the AGE-mediated effect(s). We observed significant neuroprotective and neurorescue properties of AGE and one of its ingredients, SAC, from ROS (H(2)O(2))-mediated insults to neuronal cells. Treatment of AGE and SAC were found to protect neuronal cells when they were independently co-treated with ROS. Furthermore, a novel neuropreservation effect of AGE was detected in that pre-treatment with AGE alone protected ∼ 80% neuronal cells from ROS-mediated damage. AGE was also found to preserve pre-synaptic protein synaptosomal associated protein of 25 kDa (SNAP25) from ROS-mediated insult. For example, treatment with 2% AGE containing diet and SAC (20 mg/kg of diet) independently increased (∼70%) levels of SNAP25 and synaptophysin in Alzheimer's amyloid precursor protein-transgenic mice, of which the latter was significantly decreased in AD. Taken together, the neuroprotective, including preservation of pre-synaptic proteins by AGE and SAC can be utilized in future drug development in AD.

Figures

Fig. 1A
Fig. 1A. Differentiated PC12 cell co-treated with ROS and AGE: Cell viability assay
Differentiated PC12 cells (150,000/well) were co-treated with AGE (0.3% and 1.0%) and 200μM H2O2 for 48 hours. After harvest, the viability of cells of different treatment groups was measured by CTG assay. The numbers in the ‘X axis’ represent ‘relative luminescence unit’ and plotted relative to ‘vehicle without ROS’. Results showed a significant loss of viable cells with ROS (H2O2) treatment alone but increase in the viability of cells, which were co-treated with AGE and H2O2 versus ‘vehicle with ROS’. Fig. 1B: Differentiated PC12 cell co-treated with ROS and AGE: LDH assay Cellular toxicity was measured in the previous experiment with equal volume (15μl) of conditioned media samples. Secreted LDH level was measured and plotted. Results indicate that LDH release was significantly increased (damage) with ROS (H2O2) treatment but lowered in the AGE and H2O2 co-treatment groups versus ‘vehicle with ROS’. (In both Fig. 2A and 2B, the error bars represent mean and standard deviation of the experiment performed in triplicate). Fig. 1C: Differentiated PC12 cell co-treated with ROS and AGE: Cellular morphology Visualization of the morphology of the cells in the previous treatment group by immunocytochemistry (ICC) technique using α-tubulin (green) as a cytoskeleton marker, and DAPI (blue) as nuclear marker. Data revealed a significant loss of cells and altered cellular morphology in ‘vehicle with ROS’ group. Notably, AGE co-treatment with H2O2 increased the cell number and maintained the intercellular connections between the cells. Fig. 1D: Differentiated PC12 cell co-treated with ROS and SAC: Cell viability assay Differentiated PC12 cells (150,000/well) were co-treated with SAC (1μM and 2μM) and 200μM H2O2 for 48 hours as mentioned in Fig 1. After treatment, the viability of the cells of the treatment groups was measured by CTG assay Results showed a significant loss of viable cells with ROS (H2O2) treatment alone but increase in the viability of cells, which were co-treated with SAC and H2O2 versus ‘vehicle with ROS’. Fig. 1E: Differentiated PC12 cell co-treated with ROS and SAC: LDH assay Cellular toxicity (LDH assay) was measured with equal volume (15μl) of conditioned media samples. Results indicate that LDH release was significantly increased with ROS (H2O2) treatment and lowered in the SAC and H2O2 co-treatment groups versus ‘vehicle with ROS’. Fig. 1F: Differentiated PC12 cell co-treated with ROS and SAC: Cellular morphology Visualization of the morphology of the cells after SAC and ROS co-treatment by immunocytochemistry (ICC) technique using α-tubulin (green) as a cellular marker. Data revealed a significant loss of cells and altered cellular morphology in ‘vehicle with ROS’ group. SAC co-treatment with H2O2 increased the cell number and also maintained the intercellular connections between the cells.
Fig. 1A
Fig. 1A. Differentiated PC12 cell co-treated with ROS and AGE: Cell viability assay
Differentiated PC12 cells (150,000/well) were co-treated with AGE (0.3% and 1.0%) and 200μM H2O2 for 48 hours. After harvest, the viability of cells of different treatment groups was measured by CTG assay. The numbers in the ‘X axis’ represent ‘relative luminescence unit’ and plotted relative to ‘vehicle without ROS’. Results showed a significant loss of viable cells with ROS (H2O2) treatment alone but increase in the viability of cells, which were co-treated with AGE and H2O2 versus ‘vehicle with ROS’. Fig. 1B: Differentiated PC12 cell co-treated with ROS and AGE: LDH assay Cellular toxicity was measured in the previous experiment with equal volume (15μl) of conditioned media samples. Secreted LDH level was measured and plotted. Results indicate that LDH release was significantly increased (damage) with ROS (H2O2) treatment but lowered in the AGE and H2O2 co-treatment groups versus ‘vehicle with ROS’. (In both Fig. 2A and 2B, the error bars represent mean and standard deviation of the experiment performed in triplicate). Fig. 1C: Differentiated PC12 cell co-treated with ROS and AGE: Cellular morphology Visualization of the morphology of the cells in the previous treatment group by immunocytochemistry (ICC) technique using α-tubulin (green) as a cytoskeleton marker, and DAPI (blue) as nuclear marker. Data revealed a significant loss of cells and altered cellular morphology in ‘vehicle with ROS’ group. Notably, AGE co-treatment with H2O2 increased the cell number and maintained the intercellular connections between the cells. Fig. 1D: Differentiated PC12 cell co-treated with ROS and SAC: Cell viability assay Differentiated PC12 cells (150,000/well) were co-treated with SAC (1μM and 2μM) and 200μM H2O2 for 48 hours as mentioned in Fig 1. After treatment, the viability of the cells of the treatment groups was measured by CTG assay Results showed a significant loss of viable cells with ROS (H2O2) treatment alone but increase in the viability of cells, which were co-treated with SAC and H2O2 versus ‘vehicle with ROS’. Fig. 1E: Differentiated PC12 cell co-treated with ROS and SAC: LDH assay Cellular toxicity (LDH assay) was measured with equal volume (15μl) of conditioned media samples. Results indicate that LDH release was significantly increased with ROS (H2O2) treatment and lowered in the SAC and H2O2 co-treatment groups versus ‘vehicle with ROS’. Fig. 1F: Differentiated PC12 cell co-treated with ROS and SAC: Cellular morphology Visualization of the morphology of the cells after SAC and ROS co-treatment by immunocytochemistry (ICC) technique using α-tubulin (green) as a cellular marker. Data revealed a significant loss of cells and altered cellular morphology in ‘vehicle with ROS’ group. SAC co-treatment with H2O2 increased the cell number and also maintained the intercellular connections between the cells.
Fig. 1A
Fig. 1A. Differentiated PC12 cell co-treated with ROS and AGE: Cell viability assay
Differentiated PC12 cells (150,000/well) were co-treated with AGE (0.3% and 1.0%) and 200μM H2O2 for 48 hours. After harvest, the viability of cells of different treatment groups was measured by CTG assay. The numbers in the ‘X axis’ represent ‘relative luminescence unit’ and plotted relative to ‘vehicle without ROS’. Results showed a significant loss of viable cells with ROS (H2O2) treatment alone but increase in the viability of cells, which were co-treated with AGE and H2O2 versus ‘vehicle with ROS’. Fig. 1B: Differentiated PC12 cell co-treated with ROS and AGE: LDH assay Cellular toxicity was measured in the previous experiment with equal volume (15μl) of conditioned media samples. Secreted LDH level was measured and plotted. Results indicate that LDH release was significantly increased (damage) with ROS (H2O2) treatment but lowered in the AGE and H2O2 co-treatment groups versus ‘vehicle with ROS’. (In both Fig. 2A and 2B, the error bars represent mean and standard deviation of the experiment performed in triplicate). Fig. 1C: Differentiated PC12 cell co-treated with ROS and AGE: Cellular morphology Visualization of the morphology of the cells in the previous treatment group by immunocytochemistry (ICC) technique using α-tubulin (green) as a cytoskeleton marker, and DAPI (blue) as nuclear marker. Data revealed a significant loss of cells and altered cellular morphology in ‘vehicle with ROS’ group. Notably, AGE co-treatment with H2O2 increased the cell number and maintained the intercellular connections between the cells. Fig. 1D: Differentiated PC12 cell co-treated with ROS and SAC: Cell viability assay Differentiated PC12 cells (150,000/well) were co-treated with SAC (1μM and 2μM) and 200μM H2O2 for 48 hours as mentioned in Fig 1. After treatment, the viability of the cells of the treatment groups was measured by CTG assay Results showed a significant loss of viable cells with ROS (H2O2) treatment alone but increase in the viability of cells, which were co-treated with SAC and H2O2 versus ‘vehicle with ROS’. Fig. 1E: Differentiated PC12 cell co-treated with ROS and SAC: LDH assay Cellular toxicity (LDH assay) was measured with equal volume (15μl) of conditioned media samples. Results indicate that LDH release was significantly increased with ROS (H2O2) treatment and lowered in the SAC and H2O2 co-treatment groups versus ‘vehicle with ROS’. Fig. 1F: Differentiated PC12 cell co-treated with ROS and SAC: Cellular morphology Visualization of the morphology of the cells after SAC and ROS co-treatment by immunocytochemistry (ICC) technique using α-tubulin (green) as a cellular marker. Data revealed a significant loss of cells and altered cellular morphology in ‘vehicle with ROS’ group. SAC co-treatment with H2O2 increased the cell number and also maintained the intercellular connections between the cells.
Fig. 1A
Fig. 1A. Differentiated PC12 cell co-treated with ROS and AGE: Cell viability assay
Differentiated PC12 cells (150,000/well) were co-treated with AGE (0.3% and 1.0%) and 200μM H2O2 for 48 hours. After harvest, the viability of cells of different treatment groups was measured by CTG assay. The numbers in the ‘X axis’ represent ‘relative luminescence unit’ and plotted relative to ‘vehicle without ROS’. Results showed a significant loss of viable cells with ROS (H2O2) treatment alone but increase in the viability of cells, which were co-treated with AGE and H2O2 versus ‘vehicle with ROS’. Fig. 1B: Differentiated PC12 cell co-treated with ROS and AGE: LDH assay Cellular toxicity was measured in the previous experiment with equal volume (15μl) of conditioned media samples. Secreted LDH level was measured and plotted. Results indicate that LDH release was significantly increased (damage) with ROS (H2O2) treatment but lowered in the AGE and H2O2 co-treatment groups versus ‘vehicle with ROS’. (In both Fig. 2A and 2B, the error bars represent mean and standard deviation of the experiment performed in triplicate). Fig. 1C: Differentiated PC12 cell co-treated with ROS and AGE: Cellular morphology Visualization of the morphology of the cells in the previous treatment group by immunocytochemistry (ICC) technique using α-tubulin (green) as a cytoskeleton marker, and DAPI (blue) as nuclear marker. Data revealed a significant loss of cells and altered cellular morphology in ‘vehicle with ROS’ group. Notably, AGE co-treatment with H2O2 increased the cell number and maintained the intercellular connections between the cells. Fig. 1D: Differentiated PC12 cell co-treated with ROS and SAC: Cell viability assay Differentiated PC12 cells (150,000/well) were co-treated with SAC (1μM and 2μM) and 200μM H2O2 for 48 hours as mentioned in Fig 1. After treatment, the viability of the cells of the treatment groups was measured by CTG assay Results showed a significant loss of viable cells with ROS (H2O2) treatment alone but increase in the viability of cells, which were co-treated with SAC and H2O2 versus ‘vehicle with ROS’. Fig. 1E: Differentiated PC12 cell co-treated with ROS and SAC: LDH assay Cellular toxicity (LDH assay) was measured with equal volume (15μl) of conditioned media samples. Results indicate that LDH release was significantly increased with ROS (H2O2) treatment and lowered in the SAC and H2O2 co-treatment groups versus ‘vehicle with ROS’. Fig. 1F: Differentiated PC12 cell co-treated with ROS and SAC: Cellular morphology Visualization of the morphology of the cells after SAC and ROS co-treatment by immunocytochemistry (ICC) technique using α-tubulin (green) as a cellular marker. Data revealed a significant loss of cells and altered cellular morphology in ‘vehicle with ROS’ group. SAC co-treatment with H2O2 increased the cell number and also maintained the intercellular connections between the cells.
Fig. 2
Fig. 2. Differentiated PC12 cell co-treated with ROS and AGE: analysis of pre-synaptic protein SNAP25
Western immunoblot analysis of the pre-synaptic protein SNAP25 from the cell lysate of the experiment mentioned in Fig.1 showed significance increase in levels of SNAP25 in AGE (0.3% and 1.0%) and 200μM H2O2 co-treated differentiated PC12 cells versus ‘vehicle with ROS’. SNAP25 band densities were adjusted with the β-actin band signals and plotted relative to ‘vehicle without ROS’ with error bars, which represent mean and standard deviation of the experiment performed in triplicate.
Fig. 3A
Fig. 3A. Differentiated PC12 cell pre-treated with AGE and post-treated with ROS: Cell viability assay
A novel neuro-rescue property of AGE was shown in this CTG data, where differentiated PC12 cells were pre-treated with 0.3% and 1.0% AGE without the presence of H2O2 for 48 hours and then treated with 200μM of H2O2 alone for 24 hours. Cell viability was almost completely lost by ROS (H2O2) without any prior treatment of AGE. Notably, there was a significant increase in cell viability in the AGE pre-treated and H2O2 post-treated cells versus cells which were only post-treated with H2O2. Fig. 3B: Differentiated PC12 cell pre-treated with AGE and post-treated with ROS: Cellular morphology Cellular morphology of the previous experiment was studied by immunocytochemistry using α-tubulin (green) as a cytoskeleton marker and DAPI (blue) as nuclear marker. A gross reduction in cell number with complete loss of neuritic outgrowth was observed in the cells which were not pre-treated with AGE but post-treated with 200μM of H2O2 for 24 hours. AGE pre-treatment for 48 hours not only rescues neuronal cells from dying, but also preserves neurite connections even in the presence of ROS (H2O2).
Fig. 3A
Fig. 3A. Differentiated PC12 cell pre-treated with AGE and post-treated with ROS: Cell viability assay
A novel neuro-rescue property of AGE was shown in this CTG data, where differentiated PC12 cells were pre-treated with 0.3% and 1.0% AGE without the presence of H2O2 for 48 hours and then treated with 200μM of H2O2 alone for 24 hours. Cell viability was almost completely lost by ROS (H2O2) without any prior treatment of AGE. Notably, there was a significant increase in cell viability in the AGE pre-treated and H2O2 post-treated cells versus cells which were only post-treated with H2O2. Fig. 3B: Differentiated PC12 cell pre-treated with AGE and post-treated with ROS: Cellular morphology Cellular morphology of the previous experiment was studied by immunocytochemistry using α-tubulin (green) as a cytoskeleton marker and DAPI (blue) as nuclear marker. A gross reduction in cell number with complete loss of neuritic outgrowth was observed in the cells which were not pre-treated with AGE but post-treated with 200μM of H2O2 for 24 hours. AGE pre-treatment for 48 hours not only rescues neuronal cells from dying, but also preserves neurite connections even in the presence of ROS (H2O2).
Fig.4A
Fig.4A. Levels of SNAP25 in wild type versus APP-Tg mice brain lysate
Western immunoblotting with wild type and APP Tg mice brain lysates revealed a significant decrease in brain levels of the pre-synaptic protein SNAP25 in the untreated APP Tg mice versus wild type controls of the same age. SNAP25 band densities were adjusted with the corresponding β-actin band densities and plotted relative to ‘wild type controls’ (n=4 for wild type mice and n=5 for Tg mice). Fig. 4B: Levels of synaptophysin in wild type versus APP-Tg mice brain lysate: A similar significant decrease in the brain levels of another pre-synaptic protein synaptophysin was observed in the untreated Tg mice versus wild type controls of the same age. Synaptophysin band densities were adjusted with the corresponding β-actin band densities and plotted relative to ‘wild type controls’.
Fig.4A
Fig.4A. Levels of SNAP25 in wild type versus APP-Tg mice brain lysate
Western immunoblotting with wild type and APP Tg mice brain lysates revealed a significant decrease in brain levels of the pre-synaptic protein SNAP25 in the untreated APP Tg mice versus wild type controls of the same age. SNAP25 band densities were adjusted with the corresponding β-actin band densities and plotted relative to ‘wild type controls’ (n=4 for wild type mice and n=5 for Tg mice). Fig. 4B: Levels of synaptophysin in wild type versus APP-Tg mice brain lysate: A similar significant decrease in the brain levels of another pre-synaptic protein synaptophysin was observed in the untreated Tg mice versus wild type controls of the same age. Synaptophysin band densities were adjusted with the corresponding β-actin band densities and plotted relative to ‘wild type controls’.
Fig. 5A
Fig. 5A. Levels of SNAP25 in the brain lysate of AGE treated APP-Tg mice versus untreated APP-Tg mice
Western immunoblotting of the mice brain lysate showed a gradual time dependent decrease in brain levels of SNAP25 in untreated APP Tg mice, which were sacrificed after the 4-week experiment versus Tg mice sacrificed at the beginning of the 4-week experiment. Treatment of AGE significantly increases brain levels of SNAP25 versus untreated mice. There is no significant difference in brain levels of SNAP25 in AGE treated Tg mice sacrificed at the end of 4-week experiment versus untreated Tg mice sacrificed at the beginning of the experiment. Beta actin adjusted SNAP25 band densities were plotted relative to ‘untreated APP Tg mice’ sacrificed at the end of the experiment. (n=5 for untreated Tg mice; n=7 for AGE treated Tg mice and n=4 for untreated Tg mice sacrificed at the beginning of the 4-week experiment). Fig. 5B: Levels of synaptophysin in the brain lysate of AGE treated APP-Tg mice versus untreated APP-Tg mice: Similar results of SNAP25 were obtained in brain levels of synaptophysin as detected by Western immunoblotting. There is a significant increase in the levels of synaptophysin in AGE treated APP Tg mice versus untreated. A time dependent decrease in the levels of synaptophysin was also observed in Tg mice brain and no significant change was detected in the brain levels of synaptophysin between untreated Tg mice sacrificed at the beginning of the 4-week experiment versus AGE treated Tg mice sacrificed at the end of the experiment. Beta actin adjusted synaptophysin band densities were plotted relative to ‘untreated APP Tg mice’ sacrificed at the end of the experiment. Thus AGE treatment could prevent time-dependent decline of SNAP25 and synaptophysin in APP Tg mice brain.
Fig. 5A
Fig. 5A. Levels of SNAP25 in the brain lysate of AGE treated APP-Tg mice versus untreated APP-Tg mice
Western immunoblotting of the mice brain lysate showed a gradual time dependent decrease in brain levels of SNAP25 in untreated APP Tg mice, which were sacrificed after the 4-week experiment versus Tg mice sacrificed at the beginning of the 4-week experiment. Treatment of AGE significantly increases brain levels of SNAP25 versus untreated mice. There is no significant difference in brain levels of SNAP25 in AGE treated Tg mice sacrificed at the end of 4-week experiment versus untreated Tg mice sacrificed at the beginning of the experiment. Beta actin adjusted SNAP25 band densities were plotted relative to ‘untreated APP Tg mice’ sacrificed at the end of the experiment. (n=5 for untreated Tg mice; n=7 for AGE treated Tg mice and n=4 for untreated Tg mice sacrificed at the beginning of the 4-week experiment). Fig. 5B: Levels of synaptophysin in the brain lysate of AGE treated APP-Tg mice versus untreated APP-Tg mice: Similar results of SNAP25 were obtained in brain levels of synaptophysin as detected by Western immunoblotting. There is a significant increase in the levels of synaptophysin in AGE treated APP Tg mice versus untreated. A time dependent decrease in the levels of synaptophysin was also observed in Tg mice brain and no significant change was detected in the brain levels of synaptophysin between untreated Tg mice sacrificed at the beginning of the 4-week experiment versus AGE treated Tg mice sacrificed at the end of the experiment. Beta actin adjusted synaptophysin band densities were plotted relative to ‘untreated APP Tg mice’ sacrificed at the end of the experiment. Thus AGE treatment could prevent time-dependent decline of SNAP25 and synaptophysin in APP Tg mice brain.
Fig. 6A and Fig. 6B
Fig. 6A and Fig. 6B. Levels of SNAP25 and synaptophysin in the brain lysate of SAC treated APP-Tg mice versus untreated APP-Tg mice
Western immunoblot analyses of four months SAC (20mg/kg/day) treated APP-Tg mice brain lysate resulted in significant increase in the levels of pre-synaptic proteins SNAP25 and synaptophysin.
Fig. 6A and Fig. 6B
Fig. 6A and Fig. 6B. Levels of SNAP25 and synaptophysin in the brain lysate of SAC treated APP-Tg mice versus untreated APP-Tg mice
Western immunoblot analyses of four months SAC (20mg/kg/day) treated APP-Tg mice brain lysate resulted in significant increase in the levels of pre-synaptic proteins SNAP25 and synaptophysin.
Fig. 7
Fig. 7. Effect of SAC treatment in Y maze spontaneous exploration
Hippocampal-based spontaneous alterations and exploration behavior in Y maze after SAC treatment in Tg2576 mice. Note that, impaired exploratory behavior in Tgs fed with isocaloric control diet, and restored spontaneous exploration in Tgs fed with SAC diet.

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