Creatine protects against excitoxicity in an in vitro model of neurodegeneration

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.

Figure 1. Protective effect of creatine in hippocampal cell cultures exposed to glutamate.

Hippocampal cells (DIV 17) were incubated with rising concentrations of glutamate in absence or in presence of 5 mM creatine. After 24 h the LDH release into the cell culture supernatant was determined. Total protein of the lysed cell monolayer was used as a reference. Data are expressed as arbitrary units per mg protein +/− standard deviation. Each data point represents the mean of triplicates. Each experiment was independently performed in triplicate. Statistical analysis was performed by unpaired Student's T-test. *denotes statistical significance at a level of p<0.01.

Figure 2. Effect of creatine on intracellular ATP/Phosphocreatine content in hippocampal cells exposed to glutamate.

Hippocampal cells (DIV 17) were challenged with glutamate at rising concentrations in absence or presence of 5 mM creatine. After 24 h of incubation the cells were harvested and intracellular ATP/PCr concentration was determined by luciferin/luciferase chemiluminescence. Total protein content of the cell lysate was employed 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.

Figure 3. 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.

Figure 4. Protective effect of creatine in hippocampal cell cultures challenged with oxidative stress.

Hippocampal cells (DIV 15) were incubated with hydrogen peroxide in rising concentrations in absence or in presence of 5 mM creatine. After 24 h the LDH release into the cell culture supernatant was assessed. Total protein of the cell monolayer was used as a reference. Data are expressed as arbitrary units per mg protein +/− standard deviation. Each data point represents the mean of triplicates. Each experiment was independently performed in triplicate. Statistical analysis was performed by unpaired Student's T-test. *denotes statistical significance at a level of p<0.01.

Figure 5. Impact of creatine on glutamate efflux into the supernatant in hippocampal cell cultures exposed to hydrogen peroxide.

Hippocampal cells (DIV 15) were incubated with rising concentrations of hydrogen peroxide in absence or in presence of 5 mM creatine. After 24 h the glutamate release into the cell culture supernatant was enzymatically determined. Total protein of the lysed cell monolayer was used as a reference. Data are expressed as glutamate concentration per mg protein +/− standard deviation. Each data point represents the mean of triplicates. Each experiment was independently performed in triplicate. Statistical analysis was performed by unpaired Student's T-test. *denotes statistical significance at a level of p<0.01.

Figure 6. Impact of creatine pre-incubation on NMDA-triggered intracellular calcium rise in hippocampal cells.

Hippocampal cell cultures (DIV 18) were incubated with 5 mM of creatine for 18 h. Cells were harvested, dissociated and loaded with FURA PE-3/AM. Ca²⁺ ratiometry was performed in 0.5×10⁶ cells/ml at 37°C. After stable baseline ratios were achieved NMDA was added and the response was recorded for 400 seconds. Thapsigargin was added for SERCA inhibition. The tracings are representative for 5 individual experiments by calculating curve means. Data for intracellular Ca²⁺ are expressed in arbitrary units. The second tracing shows responses in creatine-pretreated cells, the first one has been acquired from control cells.