Sequential versus Co-treatment of MitoQ and NMN to Enhance Autophagy, NAD⁺ Metabolism, and Mitochondrial Function in Cellular Senescence, with Exploratory Uncoupler–NMN Synergy in Vascular Aging

Abstract

Cellular senescence and mitochondrial dysfunction are interconnected hallmarks of aging that create a metabolically hostile environment characterized by NAD⁺ depletion and oxidative stress. The reduced NAD⁺/NADH ratio in senescent cells results from decreased respiratory capacity and CD38-mediated NAD⁺ consumption, while accumulated dysfunctional mitochondria produce excessive reactive oxygen species (ROS). Despite the therapeutic potential of NAD⁺ precursors and mitochondrial-targeted antioxidants individually, their combined efficacy remains unexplored. We will test the claim that MitoQ + NMN, given either simultaneously (co-treatment) or sequentially (MitoQ→washout→NMN), outperforms monotherapy in restoring cellular homeostasis; the null hypothesis is no improvement versus NMN alone. We will directly compare sequential dosing to co-treatment using a predeclared 0, 24, 48, and 72 hour washout grid with intracellular MitoQ quantified by targeted LC-MS/MS to confirm clearance. Using human dermal fibroblasts, we will induce senescence via H₂O₂ treatment, then randomize to vehicle, MitoQ 500 nM, NMN 1 mM, MitoQ+NMN co-treatment, and sequential MitoQ→NMN at each washout interval (0/24/48/72 h), with a TPP⁺ carrier control included. Primary outcomes include NAD⁺/NADH ratio quantification by LC-MS/MS, mitochondrial function via Seahorse XF analysis, and senescence markers (SA-β-gal, p16^INK4a). In an exploratory endothelial module, primary human endothelial cells are exposed to a low-dose mitochondrial uncoupler in combination with NMN (2×2 design) to quantify synergistic effects on mitochondrial quality control (PINK1/Parkin flux) and nitric oxide bioavailability. This addition mechanistically links mitochondrial quality control to vascular function, aligning the intervention with systemic vascular aging.

Introduction

Age-related diseases represent the leading causes of morbidity and mortality globally, with cellular senescence and mitochondrial dysfunction serving as common underlying mechanisms. Among the twelve hallmarks of aging, mitochondrial dysfunction significantly impacts aging because mitochondria regulate cellular energy, oxidative balance, and calcium levels. The accumulation of senescent cells contributes to tissue dysfunction through their senescence-associated secretory phenotype (SASP), which creates a pro-inflammatory microenvironment that accelerates aging processes. Recent epidemiological data indicate that interventions targeting these fundamental aging mechanisms could potentially prevent or delay multiple chronic diseases simultaneously, supporting the geroscience hypothesis that addressing biological aging directly impacts healthspan.

Biological Mechanisms

During aging, there is a specific loss of mitochondrial, but not nuclear, encoded OXPHOS subunits traced to an alternate PGC-1α/β-independent pathway of nuclear-mitochondrial communication induced by NAD⁺ decline and HIF-1α accumulation under normoxic conditions. This “pseudohypoxic” state disrupts mitochondrial biogenesis and function. In senescence, mTOR is persistently activated, which may be a consequence of elevated ROS from dysfunctional mitochondria, while AMPK activation promotes mitochondrial biogenesis partially by promoting SIRT1-dependent PGC-1α deacetylation through increasing intracellular NAD⁺/NADH ratio. The NAD⁺-sirtuin axis plays a crucial regulatory role, with SIRT3 serving as the main mitochondrial deacetylase, and its loss linked to insulin resistance, cancer, cardiovascular diseases, and neurodegeneration. Furthermore, telomere shortening interferes with NAD metabolism, causing CD38 NADase hyperactivation and NAD⁺ decline, limiting NAD bioavailability for PARP and sirtuin enzymes. Convergence of these pathways within the vasculature promotes endothelial senescence and SASP, impairs eNOS signaling, and accelerates arterial stiffness; thus, restoring mitochondrial quality control and NAD⁺ metabolism is predicted to ameliorate systemic vascular aging.

Current Treatment Limitations

Traditional antioxidant therapies have failed in clinical trials due to poor mitochondrial targeting and inability to address the NAD+ depletion that accompanies aging. While NAD+ precursor supplementation can increase NAD+ levels, senescent cells may initially use this NAD+ to support SASP production rather than cellular rejuvenation, as the low NAD+ state during aging may actually inhibit SASP development. Current single-agent approaches fail to address the bidirectional relationship between mitochondrial dysfunction and NAD+ depletion.

Rationale for Proposed Approach

Recent evidence shows that NAD+ supplementation with NMN prevents STING-induced senescence in CD8+ T cells by improving mitophagy and reducing mitochondrial dysfunction. Additionally, chronic MitoQ supplementation improves vascular function in healthy older adults by reducing mitochondrial reactive oxygen species. Because lowering mitochondrial ROS first should reduce NAD+ drain (PARP/CD38) and improve NMN utilization, we will explicitly include a co-treatment comparator and a predefined 0/24/48/72 h washout grid. Intracellular MitoQ will be quantified by targeted LC-MS/MS to verify clearance and justify the selected interval, making the timing claim falsifiable and aligned with the experimental design.

Hypotheses and Specific Aims

Specific Aim 1

To determine whether mitochondrial-targeted antioxidant pretreatment enhances NAD+ precursor efficacy

Hypothesis: MitoQ pretreatment will reduce mitochondrial ROS by >50%, creating a metabolic environment that enhances NMN-mediated NAD+ restoration
Approach: Sequential treatment protocol with a predeclared 0, 24, 48, and 72 hour washout grid verified by intracellular MitoQ LC-MS/MS
Expected outcome: 2-fold greater NAD+ increase versus NMN monotherapy

Specific Aim 2

To characterize the impact of sequential therapy on mitochondrial function and senescence markers

Hypothesis: Combined treatment will restore mitochondrial respiratory capacity and reduce senescence burden more effectively than monotherapies
Approach: Comprehensive bioenergetic profiling and senescence marker quantification
Expected outcome: >40% improvement in spare respiratory capacity and >35% reduction in senescent cell markers

Specific Aim 3

To evaluate optimal timing and dosing for the sequential intervention

Hypothesis: A 48-72 hour washout period between MitoQ and NMN will maximize therapeutic benefit
Approach: Time-course studies with multiple washout periods (24h, 48h, 72h, 96h)
Expected outcome: Identification of optimal treatment window for clinical translation

Specific Aim 4:

To test pharmacological uncoupler + NMN synergy on endothelial mitochondrial quality control and NO bioavailability.

Hypothesis: Low-dose uncoupling will reduce electron leak and ROS formation, while NMN restores sirtuin-dependent programs and mitophagy, yielding supra-additive improvements in mitophagy flux and NO bioavailability relative to monotherapies.
Approach: Minimal endothelial module (HUVECs; 2×2: vehicle, NMN, uncoupler, NMN+uncoupler) with predefined endpoints (PINK1/Parkin, eNOS^Ser1177/NO, ICAM-1/VCAM-1) and synergy analysis (Bliss independence; isobologram).

Methods

Cell Culture and Senescence Induction

Human dermal fibroblasts (CCD-1064Sk, ATCC) are cultured in DMEM (high glucose, 10% FBS, 1% penicillin–streptomycin, 2 mM L-glutamine) at 37 °C and 5% CO₂. Stress-induced premature senescence (SIPS) is generated using a multi-exposure H₂O₂ protocol: Days 1–5, 25 µM H₂O₂ for 2 h/day; Days 6–10, recovery; Days 11–15, 50 µM H₂O₂ for 2 h/day. Senescence is confirmed prior to randomization by ≥70% SA-β-gal positivity together with marker changes (↑p16^INK4a and/or ↑p21, ↓LMNB1, Ki-67 negativity).

Experimental Design

Groups: (i) young control (≤P5), (ii) SIPS + vehicle, (iii) MitoQ alone, (iv) NMN alone, (v) sequential MitoQ→NMN, (vi) simultaneous MitoQ+NMN, (vii) TPP+ carrier control (decyl-TPP, molar-matched to the TPP+ moiety in MitoQ), (viii) uncoupler alone (e.g., BAM15, 0.25–1.0 µM, low-toxicity range), (ix) NMN + uncoupler (doses as in monotherapies; synergy/endothelial module N ≥ 6 independent experiments; all other arms N ≥ 4).

Treatment groups:
- MitoQ: 500 nM for 48 h (≤0.1% DMSO).
- NMN: 1 mM for 14 days (refreshed at each medium change).
- Sequential regimen: MitoQ (48 h) → washout at 0, 24, 48, and 72 hours (96 hours exploratory) → NMN (14 days).
- Washout verification: ROS returning to pre-MitoQ baseline (MitoSOX and DCF at 0, 24, 48, 72, and 96 h post-removal) and intracellular MitoQ measured by targeted LC-MS/MS; LC-MS/MS verification is required to confirm clearance.

Procedures

NAD⁺ Quantification: Two parallel extracts: acid (NAD⁺) and alkaline (NADH); isotope dilution LC–MS/MS (¹³C₅-NAD⁺ and ²H-NADH) to correct interconversion, NMN, NR, and nicotinamide (NAM) are quantified (e.g., Waters ACQUITY UPLC-TQD) and normalized to DNA content (CyQUANT) or cell counts.
Mitochondrial Function: Seahorse XFe96 Cell Mito Stress Test measuring basal respiration, ATP-linked respiration, maximal respiration, and confirm uncoupling by an oligomycin-insensitive OCR increase at matched viability; include FCCP to define the ceiling for maximal respiration. Cells seeded at 2 × 10⁴/well; assay drugs are line-specifically titrated in preliminary runs. OCR is normalized to CyQUANT DNA content.
Senescence Markers: SA-β-galactosidase chromogenic assay (pH 6.0), automated counting >200 cells/replicate. RT-qPCR for p16^INK4a, p21, LMNB1 normalized to GAPDH, ACTB, 18S rRNA.
ROS Assessment: MitoSOX Red (5 µM) for mitochondrial superoxide; DCF-DA (10 µM) for intracellular ROS; flow cytometry with debris/doublet exclusion.
Mitochondrial Membrane Potential: TMRM (20–50 nM) with FCCP positive control and no-dye negative control to set gates; flow cytometric readout.
Mitophagy flux: PINK1/Parkin immunoblotting ± bafilomycin A1; optional mt-Keima reporter where available.
Endothelial NO: DAF-FM diacetate fluorescence with eNOS^Ser1177 immunoblot; nitrite (Griess) as confirmatory. Endothelial assays will be performed under laminar shear (≈12 dyn cm⁻², ibidi pump) for 4–6 h before NO/eNOS measurements to link outcomes to vascular function
Endothelial activation: ICAM-1/VCAM-1 quantification (ELISA or Western) ± TNF-α challenge (10 ng/mL, 4–6 h) to assess anti-inflammatory competence.

Outcome Measures

Primary Endpoints:

Autophagic flux: LC3-II and p62 turnover with and without bafilomycin A1
Mitochondrial function: spare respiratory capacity by Seahorse XF
NAD+/NADH ratio by LC-MS/MS

Secondary Endpoints:

Senescence burden: percent SA-β-gal positive and p16INK4a expression
SASP factors (IL-6, IL-8, MMP1) by ELISA
Mitochondrial membrane potential (TMRM)
Cell proliferation capacity (growth curves)
Sirtuin activity (SIRT1, SIRT3 deacetylase assays)

Exploratory Endpoints (Endothelial Module):

eNOS^Ser1177 phosphorylation, DAF-FM signal (NO bioavailability), and ICAM-1/VCAM-1 expression under basal and TNF-α conditions.

Quality-Control Endpoints (All Modules):

PINK1/Parkin levels and LC3-II/I (± bafilomycin A1) to index mitophagy flux.

Statistical Analysis

The unit of inference is the biological replicate (independent experiment using distinct passages or batches). Synergy arms have at least 6 independent experiments; all other arms have at least 4. Technical wells are not treated as independent in hypothesis tests. For single-time-point multi-group comparisons, one-way ANOVA with Tukey’s post hoc is performed on experiment-level means; factorial or time-course designs use two-way ANOVA or mixed-effects models with experiment as a random effect when appropriate. Multiple testing is controlled with Benjamini–Hochberg FDR (q = 0.05) across prespecified primary endpoints and contrasts. Effect sizes (Cohen’s d or partial η²) and 95% confidence intervals are reported; assumptions are checked via residual diagnostics with transformation or rank-based tests if violated.

Synergy analysis: For the endothelial 2×2 design, synergy is quantified by Bliss independence (ΔBliss with 95% bootstrap CIs) and Chou–Talalay isobolographic analysis on dose-titration subsets. Compute synergy only for dose conditions with ATP ≥ 85% of control and acceptable mitochondrial membrane potential by TMRM. Synergy is pre-specified as ΔBliss > 0 with FDR-adjusted q < 0.05. The primary translational success criterion is ΔBliss > 0 (FDR q < 0.05) and at least 20% increase in NO under shear versus NMN alone.

Ethics and Regulatory

All procedures conducted in accordance with institutional biosafety guidelines.

Budget Categories

Total Budget: $50,000

Reagents/Compounds (40%): $20,000
- MitoQ (Cayman Chemical): $3,000
- NMN (Sigma-Aldrich): $2,500
- Cell culture media, plastics, QC (incl. mycoplasma kits, STR): $4,000
- LC-MS standards & solvents: $2,500
- Antibodies (WB/IF, prioritized panel): $2,800
- Seahorse reagents (cartridges/buffers): $2,700
- Mild uncoupler (BAM15 or equivalent) for synergy assays: $1,200
- Primary HUVECs + endothelial media/consumables: $1,300
Core Facility Fees (30%): $15,000
- Seahorse XF analysis (≈10 plates): $6,700
- LC-MS/MS time (~20–40 h): $5,800
- Flow cytometry (≈50 h total): $2,500
Kits/Assays (20%): $10,000
- SASP ELISA panels (IL-6/IL-8/MMP1): $3,500
- ΔΨm/mitophagy reagents (TMRM, bafilomycin A1, FCCP, oligomycin, Rot/AA): $2,500
- Endothelial NO/activation assays (DAF-FM; ICAM-1/VCAM-1 ELISA): $2,500
- General assay consumables (qPCR enzymes/plates, SA-β-gal kits): $1,500
Personnel Support (10%): $5,000
- Research technician (0.1 FTE): $5,000

Timeline

Months 1–3 (Setup & validation): Reagent procurement and core bookings; optimize SIPS protocol; titrate Seahorse compounds; develop/lock LC-MS/MS method; verify MitoQ washout at 24/48/72 h; finalize SOPs and randomization; preregister analysis plan.
Months 4–6 (Cohort 1): Run first 1–2 independent experiments across all groups; interim QC (assay performance, variance checks); minor protocol adjustments if pre-specified. Endothelial module pilot: HUVEC 2×2 runs (N=1–2); feasibility for DAF-FM and ICAM-1/VCAM-1; preliminary ΔBliss estimates.
Months 7–9 (Cohort 2): Run remaining independent experiments; complete LC-MS/MS, ELISAs, flow cytometry; lock raw data and metadata. Endothelial module confirm: complete remaining 1–2 experiments; finalize synergy statistics.
Months 10–12 (Analysis & writing): Final statistics (ANOVA/mixed models, FDR), effect sizes/CIs; figures/tables; internal review; manuscript finalization and submission; archive data/code.

Quarterly Milestones:

Q1: Senescence model validated (≥70% SA-β-gal + markers); Seahorse/LC-MS methods locked; analysis plan preregistered; core time confirmed.
Q2: Cohort 1 completed; endothelial module feasibility achieved; predefined ΔBliss threshold and analysis code locked; budget check.
Q3: All experimental data collected; endothelial module data locked; synergy determination completed; data/metadata locked; draft figures complete.
- Go/no-go: If ΔBliss > 0 (FDR q < 0.05) and NO_under_shear ↑ ≥20% vs. NMN, prepare a short organoid/mouse feasibility
Q4: Final analysis complete; manuscript submitted.

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Sequential versus Co-treatment of MitoQ and NMN to Enhance Autophagy, NAD⁺ Metabolism, and Mitochondrial Function in Cellular Senescence, with Exploratory Uncoupler–NMN Synergy in Vascular Aging

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