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

<p>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.</p

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

<p>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.</p

Effect of creatine on intracellular ATP/Phosphocreatine content in hippocampal cells under oxidative stress.

<p>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.</p

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

<p>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.</p

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

<p>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.</p

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

<p>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.</p

Mitochondrial impairment and oxidative stress induced by α-Syn-accumulation is reduced by overexpression of Tom40.

<p>B103 neuroblastoma rat cells were infected with lentivirus encoding human α-Syn wild type (LV- α-Syn) or its mutated isoforms A53T (LV-α-Syn-A53T) and A30P (LV-α-Syn-A30P); Tom40 (LV-Tom40) or a combination of these vectors. (A) Immunohistochemical detection of mitochondria by in vivo labeling with MitoTracker. (C) In vivo detection of mitochondria-derived reactive oxygen species (ROS) in cytoplasm by CellROX fluorogenic probes. (E) Fluorescent immunolabeling of oxidative DNA lesions (anti-8-OHdG). (B, D and F) Pixel intensity analysis of the corresponding immunosignals. Scale bar represents 10 µm. *p<0.05 per Student’s <i>t</i> test.</p

Lentiviral delivery of Tom40 ameliorates α-Syn-induced mitochondrial alterations in transgenic mice.

<p>(A) Tom40 immunostaining in sagittal brain sections at the level of the hippocampus of non-transgenic (non-tg) controls and α-Syn-tg mice injected with control lentivirus (LV-control) or with lentiviral delivery of Tom40. (B) Stereological analysis of Tom40 immunoreactivity (optical density) showing increased signal after LV-Tom40 treatment. (C) Fluorescent immunolabeling of oxidative DNA lesions (anti-8-OHdG) in control and α-Syn tg mice treated with LV-control or LV-Tom40. Scale bar represents 100 µm in the upper panel and 15 µm in the lower panel. (D) Stereological analysis of anti-8-OHdG staining intensity reveals normal oxidative lesion load in tg animals overexpressing Tom40. (<b>E</b>) Quantification of ATP levels on brain homogenates from mice treated with LV-control or LV-Tom40. *p<0.05 per Student’s <i>t</i> test.</p

Tom40 is reduced in B103 cells overexpressing wild type and A53T-α-Syn.

<p>B103 neuroblastoma rat cells were infected with lentivirus encoding human α-Syn wild type (LV- α-Syn) or its mutated isoforms A53T (LV-α-Syn-A53T) and A30P (LV-α-Syn-A30P); Tom40 (LV-Tom40) or a combination of these vectors. (A) Western blot detection of α-Syn, Tom40 and Tom20 on whole cell lysates from infected B103 cells. (B) Densitometric analysis of Tom40 and Tom20 levels. (C) Efficient overexpression of Tom40 is achieved in B103 cells by infection with LV-Tom40. (D) Immunohistochemical detection of α-Syn and Tom40 in B103 cells infected with the corresponding lentiviral constructs. (E) Pixel intensity analysis of α-Syn levels, showing reduction of α-Syn in Tom40-overexpressing cells. *p<0.05 per Student’s <i>t</i> test. Scale bar represents 10 µm.</p

Tom40 overexpression reduces accumulation of α-Syn in neuronal cells.

<p>(A and B) Immunostaining of neuronal (anti-Neu N, upper panel) and glial (anti-GFAP, lower panel) cells in non-transgenic control animals and α-Syn tg mice that received LV-control or LV-Tom40 gene therapy. Scale bar represents 250 µm. (C and D) Stereological analysis of anti-NeuN and anti-GFAP-labeling, respectively (number of positive cells). (E) Effect of Tom40 overexpression on α-Syn levels: immunostaining of α-Syn on sagittal brain sections from non-transgenic (non-tg) controls, α-Syn-tg mice that received control lentivirus and α-Syn-tg mice injected with Tom40-lentivirus. Scale bar represents 250 µm in the upper panel and 50 µm in the lower panel. (F) Stereological analysis of α-Syn immunosignal (number of positive cells) showing reduced α-Syn levels on Tom40-overexpressing animals. *p<0.05 per Student’s <i>t</i> test.</p