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. 2009;11(1):28-42.
doi: 10.1007/s12017-009-8058-1. Epub 2009 Mar 14.

Nicotinamide prevents NAD+ depletion and protects neurons against excitotoxicity and cerebral ischemia: NAD+ consumption by SIRT1 may endanger energetically compromised neurons

Dong Liu  1 , Robert Gharavi, Michael Pitta, Marc Gleichmann, Mark P Mattson
Affiliations

Nicotinamide prevents NAD+ depletion and protects neurons against excitotoxicity and cerebral ischemia: NAD+ consumption by SIRT1 may endanger energetically compromised neurons

Dong Liu et al. Neuromolecular Med. 2009.

Abstract

Neurons require large amounts of energy to support their survival and function, and are therefore susceptible to excitotoxicity, a form of cell death involving bioenergetic stress that may occur in several neurological disorders including stroke and Alzheimer's disease. Here we studied the roles of NAD(+) bioenergetic state, and the NAD(+)-dependent enzymes SIRT1 and PARP-1, in excitotoxic neuronal death in cultured neurons and in a mouse model of focal ischemic stroke. Excitotoxic activation of NMDA receptors induced a rapid decrease of cellular NAD(P)H levels and mitochondrial membrane potential. Decreased NAD(+) levels and poly (ADP-ribose) polymer (PAR) accumulation in nuclei were relatively early events (<4 h) that preceded the appearance of propidium iodide- and TUNEL-positive cells (markers of necrotic cell death and DNA strand breakage, respectively) which became evident by 6 h. Nicotinamide, an NAD(+) precursor and an inhibitor of SIRT1 and PARP1, inhibited SIRT1 deacetylase activity without affecting SIRT1 protein levels. NAD(+) levels were preserved and PAR accumulation and neuronal death induced by excitotoxic insults were attenuated in nicotinamide-treated cells. Treatment of neurons with the SIRT1 activator resveratrol did not protect them from glutamate/NMDA-induced NAD(+) depletion and death. In a mouse model of focal cerebral ischemic stroke, NAD(+) levels were decreased in both the contralateral and ipsilateral cortex 6 h after the onset of ischemia. Stroke resulted in dynamic changes of SIRT1 protein and activity levels which varied among brain regions. Administration of nicotinamide (200 mg/kg, i.p.) up to 1 h after the onset of ischemia elevated brain NAD(+) levels and reduced ischemic infarct size. Our findings demonstrate that the NAD(+) bioenergetic state is critical in determining whether neurons live or die in excitotoxic and ischemic conditions, and suggest a potential therapeutic benefit in stroke of agents that preserve cellular NAD(+) levels. Our data further suggest that, SIRT1 is linked to bioenergetic state and stress responses in neurons, and that under conditions of reduced cellular energy levels SIRT1 enzyme activity may consume sufficient NAD(+) to nullify any cell survival-promoting effects of its deacetylase action on protein substrates.

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Figures

Fig. 1
Fig. 1
Activation of glutamate receptors results in decreased mitochondrial membrane potential, and reduced levels of NAD(P)H, NAD+, and SIRT1 deacetylase activity in cortical neurons. a Representative confocal images (top) and time-lapse traces (below) of NAD(P)H autofluorescence (blue) and the mitochondrial membrane potential (ΔΨm) indicator TMRE (red) prior to and during exposure to 100 μM glutamate. Values are average intensity change from baseline intensity (ΔF/Fo) (mean and SD of 4-5 independent experiments with 15-20 neurons analyzed in each experiment). b Total cellular NAD+ levels were measured by an enzymatic cycling assay at several time points after exposure to glutamate (100 μM) and NMDA (80 μM). c SIRT1 deacetylase activity at the indicated time points following exposure of neurons to glutamate and NMDA. d Survival of cortical neurons at the indicated time points following exposure of neurons to glutamate and NMDA. *P < 0.05, **P < 0.01 compared to the time 0 value
Fig. 2
Fig. 2
Excitotoxicity induced by glutamate and NMDA involves DNA damage and the accumulation of PAR polymers in the nuclei of cortical neurons. a Representative confocal images of TUNEL-positive (green) and PI-positive (red) cortical neurons at 6 h after exposure to glutamate (100 μM) and NMDA (80 μM). Cells were counterstained with Hoechst to show all cell nuclei. b and c Percentages of PI-positive and TUNEL-positive cells detected at 6 h after exposure to glutamate/NMDA. d Representative confocal images of poly (ADP-ribose) (PAR) polymer immunoreactivity in neurons in control cultures, and cultures that had been exposed to glutamate and NMDA for 6 h or 24 h. The lower right panel shows lack of staining of neurons (in a culture exposed to glutamate/NMDA for 24 h) when the primary antibody was omitted from the staining procedure. e Percentages of PAR polymer-positive neurons in control cultures (Con) and cultures that had been exposed to glutamate and NMDA alone or in combination with nicotinamide (2 mM) for 6 h or 24 h. f Cellular NAD+ levels in control cortical cultures, and cultures treated for 4 or 20 h with glutamate and NMDA (Glut/N) alone or in combination with 2 mM nicotinamide (Nic). *P < < 0.05, **P < 0.01
Fig. 3
Fig. 3
Nicotinamide treatment preserves levels of cellular NAD+ and SIRT1 in neurons subjected to an excitotoxic insult. a SIRT1 immunoreactivity in cultured cortical (upper panel) and hippocampal (lower panel) neurons. SIRT1 immunoreactivity was concentrated in the nucleus and so colocalized with Hoechst (DNA-binding dye) fluorescence. b SIRT1 deacetylase activities detected in different cell-free reactions or in nuclear proteins extracted from cortical cells that had been subjected to the indicated treatments. G/N glutamate and NMDA, Nic nicotinamide (2 mM). c Immunoblot showing SIRT1 and PARP-1 (full length PARP-1 band is shown) levels in cortical neurons treated with glutamate and NMDA alone or in combination with nicotinamide for 4 or 6 h. Blots were probed with β-actin. d Densitometric analysis (lower) of full length of PARP-1 and SIRT1 levels (normalized to the β-actin level) in cortical cells that had been exposed to glutamate/NMDA for 6 h. Values are the mean and SD of determinations from 4-6 cultures. *P < 0.05, **P < 0.01
Fig. 4
Fig. 4
Evidence that SIRT1 activity contributes to excitotoxic neuronal death. a SIRT1 deacetylase activities in cell extracts of neurons that had been exposed to the indicated concentrations of resveratrol or nicotinamide for 2 h. b Immunoblot showing SIRT1 levels in cortical neurons that had been treated for 2 h with the indicated concentrations of nicotinamide and sirtinol. c-e Survival of cortical neurons in cultures that had been treated with the indicated concentrations of sirtinol, NAD, NADH, or resveratrol, and then exposed to glutamate/NMDA for 24 h. f NAD+ levels in cortical neurons that had been treated with indicated concentration of resveratrol for 24 h. Values are the mean and SD of determinations made in at least four separate cultures. *P< 0.05, **P < 0.01
Fig. 5
Fig. 5
Levels of SIRT1 protein and enzymatic activity decrease transiently in the ischemic cerebral cortex following middle cerebral artery occlusion (MCAO). a Representative coronal sections stained with hematoxylin and eosin (H&E) showing the infarct, penumbra, and contralateral brain regions. b Autoradiographs showing the expression of glucose transporter 3 (GLUT3) mRNA detected by in situ hybridization with 35S labeled riboprobes in coronal brain sections from a sham-operated control mouse, and mice at 3 or 6 h after MCAO. Arrows point to the infarct area (3 h time point) and the penumbra (6 h time point). c SIRT1 deacetylase activities in the contralateral and ipsilateral cortex regions of brains collected at different time points after MCAO. d and e Representative immunoblots showing SIRT1 protein levels in the contralateral and ipsilateral regions of cerebral cortex at 3 h and 6 h post-MCAO, and densitometric quantification normalized to β-actin. There was an increase of SIRT1 levels in the contralateral and ipsilateral cortex compared to sham group, but the levels were decreased at 6 h post-MCAO. Values are the mean and SD of determinations made in at least three different experiments *P < 0.05, **P < 0.01
Fig. 6
Fig. 6
Nicotinamide treatment reduces ischemic brain injury in a permanent MCAO model of stroke. a Representative rostro-caudal series of TTC-stained coronal sections 24 after MCAO in a control mouse and a nicotinamide-treated mouse. b Quantification of infarct areas in saline-treated control mice and nicotinamide treated mice; values are the mean and SD (n = 5 or 6 mice per group). c Infarct volumes in saline-treated control mice and nicotinamide treated mice. *P < 0.05, **P 0.01. d The upper image shows a brain section from a sham-operated control mouse stained with neuronal marker NeuN (red) and counterstained with Hoechst (blue). The lower images are representative high magnification views of NeuN and Hoechst staining in the same region of the cerebral cortex from a sham-operated, a saline-treated, and a nicotinamide-treated mouse killed at 6 h after MCAO. Note that ischemia induced neuronal death (as indicated by decreased density of NeuN-positive cells) and nicotinamide treatment reduced the amount of neuronal loss. Scale bar = 50 μm. e Representative coronal sections showing TUNEL-positive cells (intense green fluorescent puncta) in the penumbra and infarct of the ipsilateral side of mouse brain 24 h after MCAO. Scale bar = 100 μm
Fig. 7
Fig. 7
Model of the neurodegenerative cascade of events in excitotoxicity and ischemic brain injury with a focus on the neuroprotective role of NAD+. During cerebral ischemia, decreased energy and oxygen supply results in compromised brain energy metabolism and plasma membrane depolarization, which causes excessive release of the excitatory neurotransmitter glutamate. Activation of glutamate receptors, particularly NMDA receptors, causes Ca2+ and Na+ influx. Increased Ca2+ concentrations in the cytosol and mitochondria results in bioenergetic and oxidative stress, and mitochondrial membrane permeability transition (MPT) pore opening. As the result of increased levels of reactive oxygen species (ROS), DNA strand breaks, PARP-1 activation and PAR polymer formation occur. The activities of PARP-1 and SIRT1 further deplete cellular NAD+ levels. Bioenergetic interventions, including nicotinamide and PARP-1 and SIRT1 inhibitors, can preserve cellular NAD+ levels and protect neurons against ischemic/excitotoxic damage and death
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