Therapeutic potential of NAD⁺ for neurological diseases

A rapidly growing body of evidence has suggested that nicotinamide adenine dinucleotide (NAD⁺) and reduced nicotinamide adenine dinucleotide (NADH) play important roles in various key biological processes, including energy metabolism, mitochondrial function, calcium homeostasis, aging and cell death [1–3]. Recent studies have also found that NAD⁺ treatment can decrease oxidative cell death [1,2,4], and NAD⁺ can be transported across the plasma membranes into certain types of cells [5–8]. Latest findings have further demonstrated that NAD⁺ administration can profoundly decrease ischemic brain damage [9]. It is increasingly possible that NAD⁺ may be a novel drug for treating multiple neurological diseases.

There are several families of enzymes that catalyze various reactions by consuming NAD⁺, thus producing significant biological effects. The major NAD⁺-consuming enzymes include [1–3]:

•	Poly(ADP-ribose) polymerases (PARPs), which consume NAD+ to produce nicotinamide and poly(ADP-ribose) on target proteins;
•	Mono(ADP-ribosyl)transferases, which consume NAD+ to produce nicotinamide and mono-ADP-ribosylation of proteins;
•	Bifunctional ADP-ribosyl cyclases/cyclic ADP-ribose hydrolases, which consume NAD+ to both generate cyclic ADP-ribose and hydrolyze cyclic ADP-ribose into free ADP-ribose;
•	NAD+-dependent histone deacytalyses, in other words the silent information regulator (Sir)2 family of proteins or sirtuins, which consume NAD+ to deacylate histones, leading to the generation of acetyl-O-ADP-ribose and nicotinamide.

Increasing evidence has suggested that CD38 may be a main mediator of intracellular NAD⁺ levels under physiological conditions, while PARP-1 appears to be the key mediator of intracellular NAD⁺ levels when extensive DNA damage occurs [1,2].

Cumulative evidence has suggested that NAD⁺ may mediate cell death via multiple mechanisms:

•	First, NAD+ depletion could be the key mechanism for PARP-1 toxicity [2,4];
•	Second, the NADH/NAD+ ratio is a major index of cellular reducing power, which can modulate mitochondrial permeability transition (MPT) – a mediator of both apoptosis and necrosis under many conditions [10];
•	Third, NAD+ plays a key role in energy metabolism that could determine the mode of cell death;
•	Fourth, owing to the critical roles of calcium homeostasis in cell death, NAD+ may mediate cell survival by its extensive effects on calcium homeostasis [1];
•	Fifth, NAD+ may further mediate cell survival by affecting sirtuins that could play significant roles in cell survival and aging [1,2]

.PARP-1 could be the NAD⁺-dependent enzyme that is particulary important for understanding cell death. Oxidative stress and nitrosative stress play critical roles in many diseases [11–13]. A number of in vitro studies have indicated excessive PARP-1 activation as a key factor in mediating cell death induced by  oxidative and nitrosative stress, N-methyl-D-aspartate-induced excitotoxicity, oxygen–glucose deprivation and zinc [1–3]. Our in vitro studies and those of other researchers have found that MPT and apoptosis-inducing factor translocation are important steps linking NAD⁺ depletion to cell death [2,6,14].

Several lines of evidence have indicated that PARP-1 activation plays a key role in ischemic brain injury: increased PARP activities have been observed in both animal models of brain ischemia and in human brains after cardiac arrest [15]. Furthermore, both pharmacological and genetic inhibition of PARP-1 have been shown to significantly decrease infarct size in brains subjected to transient or permanent ischemia [16,17]. These findings, combined with the findings indicating critical roles of PARP-1 in ischemic damage of other organs [4], demonstrate that PARP-1 mediates ischemic brain injury.

A number of studies have indicated that oxidative stress plays a significant role in the pathogenesis of Parkinson’s disease (PD) and Alzheimer’s disease (AD) [18,19]. Thus, it is possible that PARP-1 may also mediate neuronal injury in PD and AD, which has been supported by increasing evidence [1]: PARP-1 activation appears to play a key role in neuronal death induced by 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine hydrochloride (MPTP), a model toxin for PD, in both in vitro and in vivo studies. Increased nuclear PARP activity has also been found in both the brains and peripheral cells of AD patients. Recent studies have suggested that PARP-1 activation mediates neuronal death in an in vitro model for AD and cumulative evidence has further indicated that PARP-1 activation plays significant pathological roles in traumatic brain injury, diabetes, hypoglycemic brain injury and shock and inflammation [2,4]. PARP-1 has increasingly become a promising therapeutic target for multiple diseases [2,4].

Several recent in vitro studies have suggested that NAD⁺ treatment can profoundly decrease cell death induced by various PARP activators, including peroxynitrite and the DNA alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine [6,8]. NAD⁺ treatment can also block multiple PARP-1-induced cellular alterations, including glycolytic inhibition, MPT and mitochondrial depolarization [6]. For a long time it was generally thought that NAD⁺ was not permeable to the plasma membranes of cells. However, recent studies have demonstrated that NAD⁺ treatment can increase intracellular NAD⁺ levels in both untreated cells and the cells exposed to PARP-1 activators [5–8]. Collectively, these studies have indicated that NAD⁺ treatment prevents PARP-1-induced cell injury by repleting intracellular NAD⁺ levels. Studies using astrocyte cultures suggest that NAD⁺ depletion mediates mitochondrial alterations, at least partially, by inhibiting glycolysis [6–8].

NAD⁺ decreases have been observed in ischemic brains, in which PARP-1 plays a key pathological role [17]. These results, combined with in vitro studies suggesting that NAD⁺ depletion mediates PARP-1 cytotoxicity, suggest that the NAD⁺ decreases may also contribute to PARP-1 toxicity in vivo. Several animal studies have further suggested that NAD⁺ loss may be a significant factor determining the relationships between PARP and ischemic brain damage, for example, in a mild ischemia model in which there was no NAD⁺ decrease, PARP activation could be beneficial by promoting DNA repair [20].

The latest study by Ying and colleagues has demonstrated that intranasal NAD⁺ administration can increase NAD⁺ levels in rat brains [9]. NAD⁺ administration 2 h after ischemic onset can decrease infarct formation by nearly 90% in a rat model of transient focal ischemia. Significant protective effects of NAD⁺ against ischemia/reperfusion-produced neurological deficits were also observed. These results provide the first evidence that NAD⁺ may be a novel drug for treating neurological diseases and NAD⁺ metabolism may be a new target for reducing brain injuries. The observation that NAD⁺ administration can prevent ischemic brain injury could provide an essential basis for further establishing the roles of altered NAD⁺ metabolism in ischemic brain injury.

There is evidence implicating NAD⁺ administration in reducing brain injury, not only in cerebral ischemia, but also in multiple other neurological diseases. Excessive PARP-1 activation plays a key role in brain ischemia, as well as in MPTP-induced parkinsonism and hypoglycemic brain injury [1,4]. PARP-1 has also been implicated in multiple sclerosis, AD, amyotrophic lateral sclerosis and Huntington disease [1,4]. Since NAD⁺ treatment provides the most profound protection in PARP-1-mediated cell injury in cell culture studies, with a long window of opportunity [6,8], it is possible that NAD⁺ administration may reduce brain injury in these diseases, at least partially, by decreasing PARP-1 toxicity. NAD⁺ may also produce protective effects by activating sirtuins, which have been suggested to be cytoprotective [1,2].

While many studies have demonstrated that PARP inhibitors can decrease cell injury under various pathological conditions [2,4], some studies have suggested that PARP inhibitors may also produce toxic effects. One of the major problems for PARP inhibitors in treating PARP-1-related diseases is that since PARP-1 is a key enzyme for repairing single-strand DNA damage [4], PARP-1 inhibition could compromise the DNA repair capacity of cells. Therefore, it is of great clinical significance to find new approaches that cannot only decrease PARP-1 toxicity at more delayed time points by blocking PARP-1-mediated downstream events, but also avoid the toxic effects produced by direct PARP-1 inhibition. Our latest studies have suggested that NAD⁺ administration may be one of these novel approaches [9].

NAD⁺ may have additional merits as a neuroprotective agent. First, in vitro studies show that NAD⁺ is the agent that can produce the greatest protective effects against PARP-1 cytotoxicity [6,8]; second, NAD⁺ is protective even when applied 3–4 h after PARP-1 activation [6], suggesting that NAD⁺ administration may have a long window of opportunity in decreasing ischemic brain injury; and, finally, in addition to preventing PARP-1 toxicity, it is likely that NAD⁺ may decrease cell death by other pathways, such as enhancing energy metabolism.

In summary, cumulating evidence has suggested that NAD⁺ administration may be a novel therapeutic strategy for ischemic brain damage. There is also evidence suggesting that NAD⁺ might be a new drug for multiple other neurological diseases. Since the finding regarding the therapeutic potential of NAD⁺ has only been reported in the very latest studies, many more future studies are critically needed to further determine the therapeutic potential of NAD⁺ for neurological diseases.

The following research directions may be of particular significance. First, to date, NAD⁺ administration has only been used in animal models of brain ischemia. It is necessary to determine NAD⁺ administration may also be beneficial in other neurological diseases. Second, because there has been very limited information regarding the effects of NAD⁺ administration in vivo, it is warranted to search for the mechanisms underlying the protective effects of NAD⁺ against cell injury in vivo. Finally, while considerable information has been obtained regarding the mechanisms of NAD⁺-treatment-produced cytoprotection in vitro, future studies are required to further elucidate these mechanisms. It may be of particular interest to determine the roles of sirtuins and NAD⁺-dependent calcium homeostasis-mediating factors in the protective effects of NAD⁺.

Future perspective

It is warranted to determine whether NAD⁺ administration may also be beneficial in other neurological diseases and to search for the mechanisms underlying the protective effects of NAD⁺ against cell injury.