**4. The Role of NADH in Cell Metabolism and Antioxidant Defense**

Metabolism is intimately associated with oxidative stress, since ATP production by mitochondria requires the reduction of oxygen to water, which is a major source of ROS. Enzymatic cofactors of energetic metabolism such as oxidized and reduced NAD (NAD+ and NADH, respectively), as well as their phosphorylated versions (NADP+ and NADPH), constitute key bridges between energy supply and the antioxidant defense of cells [30]. The availability of these cofactors is highly inter-related, and depending on the cellular context, their separate or combined measurement can be used to reveal redox homeostasis both in the cytosol and mitochondria [99]. We provide a brief overview of the main cellular sources and consumers of NAD+/NADH and their interplay with NADP+/NADPH levels with a special focus on neuronal cells.

The provision of NAD+ molecules in the body comes from de novo synthesis from tryptophan or via salvage pathways using nicotinamide (NAM) and nicotinamide riboside (NR) as precursors. The detailed pathways of NAD+ direct synthesis have been reviewed elsewhere [100]. In addition, the direct consumption of NAD+ is achieved mainly by the enzymatic activity of silent information regulator proteins or sirtuins (SIRTs) and poly (adenosine diphosphate-ribose) polymerases (PARPs). Sirtuins catalyze the deacetylation of target proteins by converting NAD+ into NAM and a *O*-Acyl ATP ribose. The activity of SIRTs has been profusely studied in the nucleus, where they control the function of different transcription factors and histone proteins to regulate cell senescence and neurodegeneration [101,102]. In addition, PARPs are enzymes that normally control DNA repair, whose overactivation under intense DNA oxidative damage may lead to cellular depletion of NAD+ and ATP. Both processes may promote cell death, potentially contributing to the pathogenesis of neurodegenerative disorders such as AD [103].

Different metabolic reactions determine the level and subcellular distribution of NADH. Accordingly, the synthesis of NADH from NAD+ in the cytosol is achieved by the glycolytic pathway, which generates two ATPs, two NADH, and two pyruvates as net yield per glucose. In addition, NADH is synthesized by two mitochondrial enzymes: pyruvate dehydrogenase (PDH), which produces acetyl-CoA entering to the tricarboxylic acid cycle (TCA), and malate dehydrogenase (MDH), which oxidates malate to generate oxaloacetate (part of TCA). The latter reaction may also occur in the cytosol in the opposite direction, leading to NADH consumption to sustain the malate shuttle towards mitochondria. Inside the mitochondria, NADH is oxidized to NAD+ by complex I (NADH: ubiquinone oxidoreductase) of the electron transport chain, donating its electrons to achieve oxidative phosphorylation and ATP synthesis. Importantly, in eukaryotic cells such as neurons, oxidation of NADH by complex I is the main source of ROS inside the cell [104]. In the cytosol, oxidation of NADH is produced by lactate dehydrogenase (LDH), which regenerates the NAD+ required for glycolysis to proceed. Indeed, the measurement of the NADH/NAD+ ratio may serve as an indicator of the balance between glycolysis and oxidative phosphorylation, which has been used for monitoring real time cellular metabolism [105]. Despite all these metabolic pathways that are present in astrocytes and neurons, both cell types differ in their metabolic profiles. For instance, astrocytes are richer in the expression of lactate dehydrogenase 5 (LDH5), which is better suited to produce lactate from pyruvate. On the contrary, neurons express more LDH1, which is more efficient at consuming lactate to produce pyruvate. These complementary molecular signatures are compatible with lines of evidence showing that neurons "outsource" glycolysis to astrocytes. As such, astrocytes behave as net sources of lactate, while neurons are net sinkers of this metabolite [106–109]. Importantly, cellular metabolism seems to be highly plastic and under some conditions, neurons can directly use glucose to perform glycolysis and all the subsequent metabolic steps [110,111].

The major cytosolic source of NADPH is the pentose phosphate pathway (PPP), which leads to the oxidative decarboxylation of glucose-6-phosphate (G6P) to produce NADPH and the ribose-5-phosphate sugar required for the synthesis of DNA and RNA [112]. The provision of NADPH obtained by neurons through PPP is relevant under oxidative stress. Indeed, it has been claimed that neurons may increase survival under oxidative stress conditions by diverting the metabolic flux of glucose from glycolysis to PPP in order to produce more NADPH and antioxidant power [113]. In addition, the subcellular levels of NADPH are replenished from the NADH pool by the action of the mitochondrial nicotinamide nucleotide transhydrogenase (NNT) [114]. Indeed, it has been estimated that half of the mitochondrial NADPH in the brain depends on the activity of NNT and interrupting its function may cause oxidative stress [99,115]. The abundance of NADPH is also partially determined by cytosolic as well as mitochondrial kinases (NAD kinases), which convert NAD+ into NADP+. In addition, two enzymes from the TCA cycle reduce NADP+ to NADPH inside the mitochondria, namely mitochondrial isocitrate dehydrogenase 2 (IDH2) and malic enzyme (ME1). Nevertheless, in the cytosol, there is another isocitrate dehydrogenase (IDH1) usually catalyzing the reaction in the opposite direction.

In general, while NADH levels are directly implicated in ATP and ROS synthesis, those of NADPH are directly involved in cellular antioxidant response and also in free radical generation by the enzyme NADPH oxidase [116]. However, given the metabolic conditions of brain cells, the role of NADPH would be predominantly antioxidant [99]. Accordingly, NADPH is used by glutathione reductase to reduce oxidized glutathione, and by thioredoxin reductase to reduce oxidized thioredoxin, which are major components of cellular ROS defense [117]. As both cytosolic and mitochondrial NADPH levels tightly depend on those of NADH, it follows that the concentration of both nucleotides determine ROS defense. Accordingly, it has been shown that the provision of NADH is required to support proper detoxification of peroxide from liver cells by mitochondria [117]. The main roles of NADH and NADPH in cell metabolism and antioxidant pathways are summarized in Figure 4.

Measuring NAD metabolism is of interest due to NAD's biological importance, and ties to human disease and normal aging. Different methods have been used to determine NAD metabolism. Some of them are highly sensitive, such as liquid chromatography mass spectrometry (LC/MS/MS). However, this approach only gives static information of a population of cells and is also invasive and destructive. Table 2 indicates some advantages

and disadvantages of different methods for quantifying NAD metabolism, highlighting the relevant contribution of FLIM.

**Figure 4.** Roles of NADH and NADPH in metabolism and antioxidant pathways. (**a**–**c**) Synthesis of NADH from NAD+ in (**a**) glycolysis, and (**c**) TCA cycle; NADPH from NADP+ in (**b**) PPP and (**c**) TCA cycle. (**d**) Synthesis of NADP+ from NAD+ by NAD+ kinase both in cytosol and mitochondria. (**e**) Oxidation of NADH by complex I is the main source of ROS inside the cell in addition to (**f**) the activation NOX2 that generates ROS through a reduction of oxygen using NADPH as the source of donor electrons. In brain cells, the role of NADPH is predominantly antioxidant; for instance, (**g**) NADPH is used by glutathione reductase to reduce oxidized glutathione, and by (**h**) thioredoxin reductase to reduce oxidized thioredoxin. (**i**) Under oxidative stress and DNA damage, PARP-1 is activated, and this is manifested by an increase in the consumption of NAD+ by PARP. (**j**) On the other hand, the enzymatic activity of SIRTs consumes NAD+. SIRTs catalyze the deacetylation of target proteins by converting NAD+ into NAM. Created with BioRender.com.


**Table 2.** Methods for measuring NAD+ and derivatives.


**Table 2.** *Cont.*
