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Creatine protects against excitoxicity in an in vitro model of neurodegeneration.

Genius J, Geiger J, Bender A, Möller HJ, Klopstock T, Rujescu D - PLoS ONE (2012)

Bottom Line: Moreover, creatine effectively antagonized the H(2)O(2)-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant.Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible.In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT
Creatine has been shown to be neuroprotective in aging, neurodegenerative conditions and brain injury. As a common molecular background, oxidative stress and disturbed cellular energy homeostasis are key aspects in these conditions. Moreover, in a recent report we could demonstrate a life-enhancing and health-promoting potential of creatine in rodents, mainly due to its neuroprotective action. In order to investigate the underlying pharmacology mediating these mainly neuroprotective properties of creatine, cultured primary embryonal hippocampal and cortical cells were challenged with glutamate or H(2)O(2). In good agreement with our in vivo data, creatine mediated a direct effect on the bioenergetic balance, leading to an enhanced cellular energy charge, thereby acting as a neuroprotectant. Moreover, creatine effectively antagonized the H(2)O(2)-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant. Additionally, creatine mediated a direct inhibitory action on the NMDA receptor-mediated calcium response, which initiates the excitotoxic cascade. Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible. In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity.

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Effect of creatine on intracellular ATP/Phosphocreatine content in hippocampal cells under oxidative stress.Hippocampal cells (DIV 15) were challenged with hydrogen peroxide at rising concentrations in absence or presence of 5 mM creatine. After 24 h the cells were harvested for determination of intracellular ATP/PCr concentration, which was determined by luciferin/luciferase chemiluminescence and for measurement of total protein content, which served as a reference. Data are expressed as intracellular ATP concentration equivalents corrected for total protein +/− standard deviation. Each data point represents the mean of triplicates. The experiment was independently performed in triplicate. Unpaired Student's T-test was used for statistics. *denotes statistical significance at a level of p<0.01.
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pone-0030554-g003: Effect of creatine on intracellular ATP/Phosphocreatine content in hippocampal cells under oxidative stress.Hippocampal cells (DIV 15) were challenged with hydrogen peroxide at rising concentrations in absence or presence of 5 mM creatine. After 24 h the cells were harvested for determination of intracellular ATP/PCr concentration, which was determined by luciferin/luciferase chemiluminescence and for measurement of total protein content, which served as a reference. Data are expressed as intracellular ATP concentration equivalents corrected for total protein +/− standard deviation. Each data point represents the mean of triplicates. The experiment was independently performed in triplicate. Unpaired Student's T-test was used for statistics. *denotes statistical significance at a level of p<0.01.

Mentions: Hydrogen peroxide (H2O2) was added to the cell culture supernatant to induce oxidative stress. This condition led to a depletion of intracellular energy levels after 18 h of incubation (Fig. 3), along with enhanced LDH release into the supernatant (Fig. 4). Creatine at a concentration of 5 mM applied 3 h before H2O2 was added could maintain enhanced intracellular ATP/phosphocreatine concentrations as long as H2O2 concentrations remained well below 60 µM. Beyond this concentration energy levels were not altered by creatine pretreatment (Fig. 3). Unexpectedly, creatine aggravated H2O2-induced toxicity at high H2O2 concentrations and failed to reduce LDH release going along with H2O2 exposure, even at low concentrations (Fig. 4). In contrast, extracellular glutamate concentrations reflecting an overflow (and secondary hyperexcitability) which occurs along with oxidative stress were effectively reduced following creatine incubation (Fig. 5). Thus, creatine seems to efficiently interfere with this vicious circle which maintains the excitotoxic cascade after its initiation. Even under non-stressful baseline conditions glutamate concentrations remained reduced in creatine-treated hippocampal cell cultures. These effects were far less pronounced in mixed cortical cell cultures (data not shown). As glial cells were almost absent in our model the popular explanation for the H2O2 induced glutamate excess as an inhibition of redox-sensitive glutamate transporters leading to secondary pathology [23] seems to reflect only one partial aspect of the molecular mechanisms. The discrepancy between stabilization against secondary glutamate spillover and enhanced H2O2 toxicity in presence of creatine remains to be investigated. We tend to speculate that H2O2 neurotoxicity is not always necessarily due to the secondary glutamate excess, which was efficiently antagonized here.


Creatine protects against excitoxicity in an in vitro model of neurodegeneration.

Genius J, Geiger J, Bender A, Möller HJ, Klopstock T, Rujescu D - PLoS ONE (2012)

Effect of creatine on intracellular ATP/Phosphocreatine content in hippocampal cells under oxidative stress.Hippocampal cells (DIV 15) were challenged with hydrogen peroxide at rising concentrations in absence or presence of 5 mM creatine. After 24 h the cells were harvested for determination of intracellular ATP/PCr concentration, which was determined by luciferin/luciferase chemiluminescence and for measurement of total protein content, which served as a reference. Data are expressed as intracellular ATP concentration equivalents corrected for total protein +/− standard deviation. Each data point represents the mean of triplicates. The experiment was independently performed in triplicate. Unpaired Student's T-test was used for statistics. *denotes statistical significance at a level of p<0.01.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275587&req=5

pone-0030554-g003: Effect of creatine on intracellular ATP/Phosphocreatine content in hippocampal cells under oxidative stress.Hippocampal cells (DIV 15) were challenged with hydrogen peroxide at rising concentrations in absence or presence of 5 mM creatine. After 24 h the cells were harvested for determination of intracellular ATP/PCr concentration, which was determined by luciferin/luciferase chemiluminescence and for measurement of total protein content, which served as a reference. Data are expressed as intracellular ATP concentration equivalents corrected for total protein +/− standard deviation. Each data point represents the mean of triplicates. The experiment was independently performed in triplicate. Unpaired Student's T-test was used for statistics. *denotes statistical significance at a level of p<0.01.
Mentions: Hydrogen peroxide (H2O2) was added to the cell culture supernatant to induce oxidative stress. This condition led to a depletion of intracellular energy levels after 18 h of incubation (Fig. 3), along with enhanced LDH release into the supernatant (Fig. 4). Creatine at a concentration of 5 mM applied 3 h before H2O2 was added could maintain enhanced intracellular ATP/phosphocreatine concentrations as long as H2O2 concentrations remained well below 60 µM. Beyond this concentration energy levels were not altered by creatine pretreatment (Fig. 3). Unexpectedly, creatine aggravated H2O2-induced toxicity at high H2O2 concentrations and failed to reduce LDH release going along with H2O2 exposure, even at low concentrations (Fig. 4). In contrast, extracellular glutamate concentrations reflecting an overflow (and secondary hyperexcitability) which occurs along with oxidative stress were effectively reduced following creatine incubation (Fig. 5). Thus, creatine seems to efficiently interfere with this vicious circle which maintains the excitotoxic cascade after its initiation. Even under non-stressful baseline conditions glutamate concentrations remained reduced in creatine-treated hippocampal cell cultures. These effects were far less pronounced in mixed cortical cell cultures (data not shown). As glial cells were almost absent in our model the popular explanation for the H2O2 induced glutamate excess as an inhibition of redox-sensitive glutamate transporters leading to secondary pathology [23] seems to reflect only one partial aspect of the molecular mechanisms. The discrepancy between stabilization against secondary glutamate spillover and enhanced H2O2 toxicity in presence of creatine remains to be investigated. We tend to speculate that H2O2 neurotoxicity is not always necessarily due to the secondary glutamate excess, which was efficiently antagonized here.

Bottom Line: Moreover, creatine effectively antagonized the H(2)O(2)-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant.Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible.In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity.

View Article: PubMed Central - PubMed

Affiliation: Department of Psychiatry, Ludwig-Maximilians-University, Munich, Germany.

ABSTRACT
Creatine has been shown to be neuroprotective in aging, neurodegenerative conditions and brain injury. As a common molecular background, oxidative stress and disturbed cellular energy homeostasis are key aspects in these conditions. Moreover, in a recent report we could demonstrate a life-enhancing and health-promoting potential of creatine in rodents, mainly due to its neuroprotective action. In order to investigate the underlying pharmacology mediating these mainly neuroprotective properties of creatine, cultured primary embryonal hippocampal and cortical cells were challenged with glutamate or H(2)O(2). In good agreement with our in vivo data, creatine mediated a direct effect on the bioenergetic balance, leading to an enhanced cellular energy charge, thereby acting as a neuroprotectant. Moreover, creatine effectively antagonized the H(2)O(2)-induced ATP depletion and the excitotoxic response towards glutamate, while not directly acting as an antioxidant. Additionally, creatine mediated a direct inhibitory action on the NMDA receptor-mediated calcium response, which initiates the excitotoxic cascade. Even excessive concentrations of creatine had no neurotoxic effects, so that high-dose creatine supplementation as a health-promoting agent in specific pathological situations or as a primary prophylactic compound in risk populations seems feasible. In conclusion, we were able to demonstrate that the protective potential of creatine was primarily mediated by its impact on cellular energy metabolism and NMDA receptor function, along with reduced glutamate spillover, oxidative stress and subsequent excitotoxicity.

Show MeSH
Related in: MedlinePlus