Mitochondria Tune NAD+ Levels for Cells Past Their Prime

Aging comes with loss. Hair becomes thinner, hearing duller, and memory scanter. One of the key regulators of total cellular metabolism, nicotinamide adenine dinucleotide (NAD⁺), also dwindles.¹ “In aging, you may have at most a 30-50 percent decline in NAD⁺ in different tissues, and we don’t really know which NAD⁺-dependent processes are likely to be susceptible to that,” said Joseph Baur, a physiologist and expert in NAD⁺ biology at the University of Pennsylvania Perelman School of Medicine. An electron acceptor essential to many metabolic processes, NAD⁺ also acts as a co-substrate for enzymes unrelated to redox, implicating NAD⁺ in DNA repair, signaling, and transcription.^1,2

Just as we need to learn to cope with aging, so do our cells. Mathias Ziegler, a medical biochemist at the University of Bergen, and his team are interested in how aging cells deal with NAD⁺ depletion. According to their research, recently published in Nature Metabolism, it’s an inside job.³

NAD⁺ is compartmentalized into organelles including mitochondria, peroxisomes, and endoplasmic reticulum,^4,5 and these partitioned pools regulate various physiological processes.⁶ Biologically, mitochondrial function and NAD⁺ levels both decrease with age. “We have generated a model as simple as a cell line to recapitulate these observations,” Ziegler said.

Rather than using pharmacological agents to acutely decrease NAD⁺ levels, Ziegler and his team developed a chronic NAD⁺ depletion model by overexpressing the catalytic domain of NAD⁺-consuming PARP1 (PARPcd) in distinct subcellular compartments in cell lines. This strategy allowed for constitutive NAD⁺ depletion, similar to what occurs in aging cells, to determine if NAD⁺ pools in different compartments are connected. They found that PARPcd-expressing cells handled NAD⁺ depletion well unless the mitochondrial NAD⁺ pool was targeted. When PARPcd was expressed in mitochondria, the researchers found defects in mitochondrial metabolism.

Given the sensitivity of the mitochondrial NAD⁺ pool, the researchers examined this compartment more closely. With the expression of PARPcd, mitochondrial NAD⁺ levels were lower than their wild-type counterparts, even when decreases in NAD⁺ originated in different compartments. This suggested that mitochondrial NAD⁺ was siphoned out to other organelles to cope with NAD⁺ depletion elsewhere. “We have segregated pools, and we don’t really know how they come about…We do know that they interact and that they steal from each other in a way,” Ziegler explained.

Considering the role of mitochondria as an NAD⁺ repository, the team investigated the maintenance of the large mitochondrial NAD⁺ pool.⁴ Mitochondria house an isoform of nicotinamide mononucleotide (NMN) adenylyltransferase, NMNAT3, an NAD⁺ biosynthesis enzyme. However, the mitochondrial NAD⁺ pool is generated by a recently identified mitochondrial NAD⁺ transporter,⁷ not NMNAT3,⁸ so NMNAT3’s function has remained elusive. “Everyone was scratching their heads a little bit about why do we have this then,” Baur, a previous collaborator of Ziegler’s who was not involved in this study, said regarding NMNAT3.

Ziegler’s group found that NMNAT3 maintains a reservoir of NMN, an NAD⁺ equivalent, inside mitochondria. Combined with transport of NAD⁺ into mitochondria, this compartment is poised with a high concentration of NAD⁺ and NAD⁺ equivalents to defend against cellular depletion of the cofactor. Baur, who was not involved in this study, was surprised by this result. “That’s I think the first plausible explanation for why we would retain NMNAT3 if it’s not actually used to generate the NAD⁺ pool in the first place,” Baur said.

The tools generated by Ziegler and his team establish mitochondria as a rheostat, or tuner, for cellular NAD⁺ levels, to buffer and compensate for cellular NAD⁺ loss. However, this organelle isn’t a cure-all in the search for the fountain of youth. Alterations in NAD⁺ homeostasis can affect age-related pathologies such as neurodegeneration and cancer,^1,2 but the mechanisms and implications of this remain unclear.⁹ Mitochondria represent a potential vulnerability that could be contributing to age-related diseases, motivating further study of compartmentalized NAD⁺ pools and NAD⁺ transport. For now, while some might try to delay the inevitable wrinkles and gray hair, people can rest assured that the mitochondria are battling against cellular NAD⁺ depletion in aging cells.