Nicotinamide mononucleotide inhibits post-ischemic NAD⁺ degradation and dramatically ameliorates brain damage following global cerebral ischemia

Highlights

• Effect of treatment with nicotinamide mononucleotide on forebrain ischemia is examined.

• Nicotinamide mononucleotide administration ameliorates both histologic and neurologic outcome.

• Treated animals show normalized post-ischemic NAD levels and inhibition of poly-ADP-ribosylation.

Abstract

Nicotinamide adenine dinucleotide (NAD⁺) is an essential cofactor for multiple cellular metabolic reactions and has a central role in energy production. Brain ischemia depletes NAD⁺ pools leading to bioenergetics failure and cell death. Nicotinamide mononucleotide (NMN) is utilized by the NAD⁺ salvage pathway enzyme, nicotinamide adenylyltransferase (Nmnat) to generate NAD⁺. Therefore, we examined whether NMN could protect against ischemic brain damage. Mice were subjected to transient forebrain ischemia and treated with NMN or vehicle at the start of reperfusion or 30 min after the ischemic insult. At 2, 4, and 24 h of recovery, the proteins poly-ADP-ribosylation (PAR), hippocampal NAD⁺ levels, and expression levels of NAD⁺ salvage pathway enzymes were determined. Furthermore, animal's neurologic outcome and hippocampal CA1 neuronal death was assessed after six days of reperfusion. NMN (62.5 mg/kg) dramatically ameliorated the hippocampal CA1 injury and significantly improved the neurological outcome. Additionally, the post-ischemic NMN treatment prevented the increase in PAR formation and NAD⁺ catabolism. Since the NMN administration did not affect animal's temperature, blood gases or regional cerebral blood flow during recovery, the protective effect was not a result of altered reperfusion conditions. These data suggest that administration of NMN at a proper dosage has a strong protective effect against ischemic brain injury.

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.