Age-related loss of dopaminergic function tracks NAD+ decline but does not follow a simple linear dose‑response. We propose that NAD+ depletion initiates a reversible metabolic shift early in life, yet once mitochondrial DNA (mtDNA) damage accumulates beyond a critical threshold, NAD+ repletion alone cannot restore complex I activity or dopaminergic tone. This two‑hit hypothesis predicts a biphasic curve: (1) In young‑mid adulthood, falling NAD+ reduces SIRT1 deacetylase activity, stabilizes HIF‑1α, and induces a pseudohypoxic state that transiently suppresses oxidative phosphorylation while preserving mtDNA integrity; NAD+ precursors reverse these changes and rescue dopamine synthesis. (2) With advancing age, persistent NAD+ deficit impairs deacetylation of PGC‑1α and TFAM, compromising mitochondrial biogenesis and increasing mtDNA replication errors. The resulting mtDNA lesions and deletions create a self‑ sustaining defect in complex I that is independent of NAD+ levels. Consequently, NAD+ supplementation improves metabolic markers but fails to restore neuronal firing or dopamine release unless mtDNA damage is concurrently reduced.

Testable predictions:

• Measure mtDNA copy number, lesion frequency, and heteroplasmy in substantia nigra pars compacta (SNpc) of mice across the lifespan and correlate with regional NAD+ levels. Expect a nadir in NAD+ preceding a sharp rise in mtDNA damage around 18‑20 months.
• Administer NAD+ precursors (NR or NMN) to two age cohorts: early‑treated (6 months) and late‑treated (22 months). Early treatment should preserve dopamine turnover, motor performance, and complex I activity; late treatment should normalize NAD+ and SIRT1 activity yet show no improvement in complex I kinetics or dopamine release unless combined with a mtDNA‑targeted intervention (e.g., mitoTALEN-mediated deletion of damaged mtDNA).
• Use cybrid models where SNpc-derived mtDNA with high damage load is transferred into nucleo‑identical cells; these cybrids should resist NAD+‑mediated rescue of respiration, confirming that mtDNA genotype dictates the threshold.
• Pharmacologically inhibit HIF‑1α during the early NAD+ decline phase; this should blunt the pseudohypoxic shift and delay the onset of mtDNA damage, extending the window of NAD+ responsiveness.

If data show that NAD+ restoration only rescues dopaminergic function when mtDNA damage remains below a defined threshold, the hypothesis validates the idea that aging‑related dopaminergic decline is not a programmed ambition downgrade but a damage‑limited metabolic failure. Conversely, if NAD+ repletion fully restores function regardless of mtDNA lesion burden, the two‑hit model would be falsified, supporting a purely metabolic recalibration view.