Limits...
The effects of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function after glutamate excitotoxicity.

Wang X, Li H, Ding S - Int J Mol Sci (2014)

Bottom Line: In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function.Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity.Our findings provide insights into potential neuroprotective strategies in ischemic stroke.

View Article: PubMed Central - PubMed

Affiliation: Dalton Cardiovascular Research Center, Department of Bioengineering, University of Missouri, Columbia, MO 65211, USA. xw825@mail.missouri.edu.

ABSTRACT
NAD+ is an essential co-enzyme for cellular energy metabolism and is also involved as a substrate for many cellular enzymatic reactions. It has been shown that NAD+ has a beneficial effect on neuronal survival and brain injury in in vitro and in vivo ischemic models. However, the effect of NAD+ on mitochondrial biogenesis and function in ischemia has not been well investigated. In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function. Our results show that supplementation of NAD+ could effectively reduce apoptotic neuronal death, and apoptotic inducing factor translocation after neurons were challenged with excitotoxic glutamate stimulation. Using different approaches including confocal imaging, mitochondrial DNA measurement and Western blot analysis of PGC-1 and NRF-1, we also found that NAD+ could significantly attenuate glutamate-induced mitochondrial fragmentation and the impairment of mitochondrial biogenesis. Furthermore, NAD+ treatment effectively inhibited mitochondrial membrane potential depolarization and NADH redistribution after excitotoxic glutamate stimulation. Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity. Our findings provide insights into potential neuroprotective strategies in ischemic stroke.

Show MeSH

Related in: MedlinePlus

The time course of mitochondrial fragmentation after glutamate stimulation. (A) Maximal projection confocal images of primary cortical neurons transfected with mito-AcGFP1. Neurons were treated without or with 30 μM glutamate together with 3 μM glycine for 3, 6 and 24 h, respectively. The lower panels are the high resolution images of the boxed region in upper panels. The images at different time points were acquired from different neurons; (B–C) Frequency distribution and cumulative curves of mitochondrial length and area under different conditions. The values of histogram intervals (bins) are 0.25 μm for mitochondrial length and 0.5 μm2 for mitochondrial area. Distribution data showed that glutamate treatment caused significant increase in the number of shorter and smaller mitochondria and this effect was dependent on treatment time; (D) The summary of average mitochondrial length, area and density for each condition. Data are shown as mean ± SE; **p < 0.01, ANOVA test. Data were collected from 10–11 neurons grown on two glass coverslip per experimental condition and a total of 1169 mitochondria were measured.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4264177&req=5

ijms-15-20449-f003: The time course of mitochondrial fragmentation after glutamate stimulation. (A) Maximal projection confocal images of primary cortical neurons transfected with mito-AcGFP1. Neurons were treated without or with 30 μM glutamate together with 3 μM glycine for 3, 6 and 24 h, respectively. The lower panels are the high resolution images of the boxed region in upper panels. The images at different time points were acquired from different neurons; (B–C) Frequency distribution and cumulative curves of mitochondrial length and area under different conditions. The values of histogram intervals (bins) are 0.25 μm for mitochondrial length and 0.5 μm2 for mitochondrial area. Distribution data showed that glutamate treatment caused significant increase in the number of shorter and smaller mitochondria and this effect was dependent on treatment time; (D) The summary of average mitochondrial length, area and density for each condition. Data are shown as mean ± SE; **p < 0.01, ANOVA test. Data were collected from 10–11 neurons grown on two glass coverslip per experimental condition and a total of 1169 mitochondria were measured.

Mentions: Mitochondria are highly dynamic organelles with the length, size and shape controlled by fission and fusion [20,21]. The balance of fission and fusion is disrupted in neuronal injury and degeneration, thus causing morphological change and structural damage associated with mitochondrial dysfunction [22,23,24]. Mitochondrial fragmentation has been widely detected after apoptosis [25]. Here, we initially studied the time course of mitochondrial fragmentation after glutamate stimulation. Neurons were transfected with mitochondria-targeted AcGFP (mito-AcGFP) to visualize the morphology of mitochondria. Individual mitochondrion in the dendrites can be revealed by confocal microscopy and the length and area were analyzed by Metamorph software. Figure 3A shows mitochondrial images of individual neurons in control and treated with 30 μM glutamate and 3 μM glycine glutamate for different times. Our data show that mitochondrial fragmentation occurred after neurons were exposed to glutamate. Frequency distribution analysis shows that glutamate stimulation reduced mitochondrial length and area as compared with neurons in control conditions (Figure 3B, C, Figure S1). The average length and area of mitochondria were progressively decreased with the treatment time (Figure 3D). However, the density of the mitochondria along the dendrites did not change with glutamate treatment (Figure 3D). These results demonstrate that glutamate induces neuronal mitochondrial fragmentation in a time-dependent manner.


The effects of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function after glutamate excitotoxicity.

Wang X, Li H, Ding S - Int J Mol Sci (2014)

The time course of mitochondrial fragmentation after glutamate stimulation. (A) Maximal projection confocal images of primary cortical neurons transfected with mito-AcGFP1. Neurons were treated without or with 30 μM glutamate together with 3 μM glycine for 3, 6 and 24 h, respectively. The lower panels are the high resolution images of the boxed region in upper panels. The images at different time points were acquired from different neurons; (B–C) Frequency distribution and cumulative curves of mitochondrial length and area under different conditions. The values of histogram intervals (bins) are 0.25 μm for mitochondrial length and 0.5 μm2 for mitochondrial area. Distribution data showed that glutamate treatment caused significant increase in the number of shorter and smaller mitochondria and this effect was dependent on treatment time; (D) The summary of average mitochondrial length, area and density for each condition. Data are shown as mean ± SE; **p < 0.01, ANOVA test. Data were collected from 10–11 neurons grown on two glass coverslip per experimental condition and a total of 1169 mitochondria were measured.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4264177&req=5

ijms-15-20449-f003: The time course of mitochondrial fragmentation after glutamate stimulation. (A) Maximal projection confocal images of primary cortical neurons transfected with mito-AcGFP1. Neurons were treated without or with 30 μM glutamate together with 3 μM glycine for 3, 6 and 24 h, respectively. The lower panels are the high resolution images of the boxed region in upper panels. The images at different time points were acquired from different neurons; (B–C) Frequency distribution and cumulative curves of mitochondrial length and area under different conditions. The values of histogram intervals (bins) are 0.25 μm for mitochondrial length and 0.5 μm2 for mitochondrial area. Distribution data showed that glutamate treatment caused significant increase in the number of shorter and smaller mitochondria and this effect was dependent on treatment time; (D) The summary of average mitochondrial length, area and density for each condition. Data are shown as mean ± SE; **p < 0.01, ANOVA test. Data were collected from 10–11 neurons grown on two glass coverslip per experimental condition and a total of 1169 mitochondria were measured.
Mentions: Mitochondria are highly dynamic organelles with the length, size and shape controlled by fission and fusion [20,21]. The balance of fission and fusion is disrupted in neuronal injury and degeneration, thus causing morphological change and structural damage associated with mitochondrial dysfunction [22,23,24]. Mitochondrial fragmentation has been widely detected after apoptosis [25]. Here, we initially studied the time course of mitochondrial fragmentation after glutamate stimulation. Neurons were transfected with mitochondria-targeted AcGFP (mito-AcGFP) to visualize the morphology of mitochondria. Individual mitochondrion in the dendrites can be revealed by confocal microscopy and the length and area were analyzed by Metamorph software. Figure 3A shows mitochondrial images of individual neurons in control and treated with 30 μM glutamate and 3 μM glycine glutamate for different times. Our data show that mitochondrial fragmentation occurred after neurons were exposed to glutamate. Frequency distribution analysis shows that glutamate stimulation reduced mitochondrial length and area as compared with neurons in control conditions (Figure 3B, C, Figure S1). The average length and area of mitochondria were progressively decreased with the treatment time (Figure 3D). However, the density of the mitochondria along the dendrites did not change with glutamate treatment (Figure 3D). These results demonstrate that glutamate induces neuronal mitochondrial fragmentation in a time-dependent manner.

Bottom Line: In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function.Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity.Our findings provide insights into potential neuroprotective strategies in ischemic stroke.

View Article: PubMed Central - PubMed

Affiliation: Dalton Cardiovascular Research Center, Department of Bioengineering, University of Missouri, Columbia, MO 65211, USA. xw825@mail.missouri.edu.

ABSTRACT
NAD+ is an essential co-enzyme for cellular energy metabolism and is also involved as a substrate for many cellular enzymatic reactions. It has been shown that NAD+ has a beneficial effect on neuronal survival and brain injury in in vitro and in vivo ischemic models. However, the effect of NAD+ on mitochondrial biogenesis and function in ischemia has not been well investigated. In the present study, we used an in vitro glutamate excitotoxicity model of primary cultured cortical neurons to study the effect of NAD+ on apoptotic neuronal death and mitochondrial biogenesis and function. Our results show that supplementation of NAD+ could effectively reduce apoptotic neuronal death, and apoptotic inducing factor translocation after neurons were challenged with excitotoxic glutamate stimulation. Using different approaches including confocal imaging, mitochondrial DNA measurement and Western blot analysis of PGC-1 and NRF-1, we also found that NAD+ could significantly attenuate glutamate-induced mitochondrial fragmentation and the impairment of mitochondrial biogenesis. Furthermore, NAD+ treatment effectively inhibited mitochondrial membrane potential depolarization and NADH redistribution after excitotoxic glutamate stimulation. Taken together, our results demonstrated that NAD+ is capable of inhibiting apoptotic neuronal death after glutamate excitotoxicity via preserving mitochondrial biogenesis and integrity. Our findings provide insights into potential neuroprotective strategies in ischemic stroke.

Show MeSH
Related in: MedlinePlus