Nicotinamide mononucleotide inhibits post-ischemic NAD+ degradation and dramatically ameliorates brain damage following global cerebral ischemia
Introduction
One of the pathologic outcomes after ischemic insult is the free radical induced DNA damage that activates the nuclear enzyme poly(ADP-ribose) polymerase 1 (PARP1) (Endres et al., 1997, Strosznajder et al., 2003), for review see (Chiarugi, 2005). This enzyme utilizes NAD+ as a substrate to form the poly-ADP-ribose (PAR) polymer. It has been proposed that uncontrolled PARP1 activation can deplete intracellular NAD+ and consequently ATP, leading to cell death (Lo et al., 1998, Szabo and Dawson, 1998). NAD+ is an important cofactor involved in multiple metabolic reactions (Brennan et al., 2006). NAD+ and NADH have central roles in cellular energy production as electron-accepting and electron-donating cofactors. Therefore, maintenance of normal cellular NAD+ levels is essential for tissue bioenergetic metabolism and several cell functions. A prominent role for NAD+ catabolism in cell death mechanisms is supported by the observation that following excitotoxic insult or in vivo models of brain ischemia, epilepsy and Alzheimer's disease, a significant decrease in total cellular NAD+ levels occurs prior to neuronal death (Endres et al., 1997, Liu et al., 2009).
NAD+ can be generated in cells either by de novo synthesis from tryptophan or it is re-synthesized from nicotinamide (Nam) via a salvage pathway (Belenky et al., 2007, Owens et al., 2013b). Majority of the NAD+ is replenished by the salvage pathway since Nam is the byproduct of the NAD+ catabolizing enzymes (Magni et al., 1999, Imai, 2009, Owens et al., 2013b). The salvage pathway represents two enzymatic reactions. In the first step, Nam is converted to nicotinamide mononucleotide (NMN) by nicotinamide phosphoribosyltransferase (Nampt) (Revollo et al., 2004), also known as pre-B-cell colony-enhancing factor (PBEF). NMN is then adenylylated to form NAD+ by nicotinamide nucleotide adenylyltransferase (Nmnat) (Belenky et al., 2007).
To maintain cellular NAD+ levels following an ischemic insult, one can either inhibit the NAD+ catabolizing enzymes (PARP1, CD38) or facilitate NAD+ generation by the salvage pathway via administration of a NAD+ precursor. It was shown that administration of Nam increases tissue NAD+ levels and improves bioenergetics following ischemia (Yang et al., 2002a). Alternatively, one can directly stimulate the NAD+ synthesis by administration of NMN that will not require the activity of Nampt, the rate-limiting enzyme in NAD+ synthesis. Additionally, NMN does not cause the same unfavorable flushing side effect associated with Nam activating the GPR109A receptor (Canto et al., 2012, Benyo et al., 2005).
Application of NMN leads to an increase in cellular NAD+ levels by a one-step enzymatic reaction where NMN is converted to NAD+ by Nmnat (Belenky et al., 2007). Incubation of brain sections with NMN prevented a rapid NAD+ catabolism in the tissue (Balan et al., 2010). Furthermore, decreased activity of Nampt enzyme can significantly aggravate post-ischemic brain damage (Zhang et al., 2010). Thus, heterozygous Nampt knockdown animals manifested aggravated brain damage following photothrombosis-induced focal ischemia (Zhang et al., 2010). Transgenic mice with neuron-specific overexepression of Nampt show reduced infarct size when compared to wild type animals (Jing et al., 2014). Similarly, the adverse effect of Nampt inhibitor FK866 was reversed by intraventricular NMN injection (Wang et al., 2011). Taken together, these data suggest that post-ischemic NMN administration could improve bioenergetic metabolism of the post-ischemic tissue and ameliorate the brain damage. Therefore, the objective of this study was to examine the effect of NMN on brain tissue NAD+ catabolism, PAR formation, and histologic and neurologic outcomes following transient global cerebral ischemia.
Section snippets
Animals and experimental groups
All the experimental procedures and treatment protocols were performed according to the laboratory practices described in (Lapchak et al., 2013), and (Landis et al., 2012). All mice procedures were conducted in AAALAC approved facilities following current ethical regulations from the Guide for the Care and use of Laboratory Animals of the National Institutes of Health international guidelines and were approved by the Animal Care and Use Committee of the University of Maryland Baltimore. Adult, 3
Assessment of physiological parameters
To assure that the blood gases are at physiological levels before and during induction of forebrain ischemia six mice were subjected to physiological study. The ventilator was adjusted to give pO2 and pCO2 values within normal levels. As Table 1 shows, the blood gas data, including pCO2, pO2, and pH showed normal physiological levels with a ventilator setting of 120 strokes per min and about 0.3 cm3 of stroke volume. There were no significant changes in blood gas levels during the ischemic
Discussion
We show here that administration of NMN following transient forebrain ischemia has a dramatic protective effect against the cell death of CA1 pyramidal neurons. This was reflected in complete recovery of post-ischemic neurological scores and in prevention of the ischemia-induced NAD+ catabolism. Furthermore, NMN treated animals did not show any increase in poly-ADP-ribosylation of hippocampal proteins.
Our model of forebrain ischemia utilizes increased levels of isoflurane as tool to reduce the
Conflict of interest
TK has a pending patent application for use of NMN to modulate NAD+ activity in neuropathological conditions. JHP, KO and AL declare no conflict of interest.
Ethical approval
All applicable international, national, and/or institutional guidelines for the care and use of animals were followed.
Acknowledgments
This work was supported by U.S. Veterans Affairs Merit grant BX000917 to TK.
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