Heteroplasmic mtDNA SNVs Disrupt Mitochondrial Nucleoid Architecture, Silencing TFEB‑Dependent Autophagy via Altered Metabolite‑Epigenetic Crosstalk

Hypothesis

Accumulation of heteroplasmic mitochondrial DNA single‑nucleotide variants (mtDNA‑SNVs) remodels mitochondrial nucleoid structure, impairing the release of retrograde metabolites (acetyl‑CoA, S‑adenosylmethionine) that normally sustain TFEB nuclear activity. This metabolite‑driven epigenetic insufficiency silences autophagy‑lysosomal gene promoters, establishing a feed‑forward loop that accelerates aging.

Mechanistic Model

1. mtDNA‑SNV‑induced nucleoid loosening – Variant mtDNA alters binding affinity of mitochondrial transcription factor A (TFAM) and nucleoid‑associated proteins, increasing nucleoid permeability.
2. Leakage of acetyl‑CoA and SAM – A more permeable nucleoid permits premature efflux of these key metabolites into the intermembrane space, where they are degraded or sequestered, lowering matrix concentrations.
3. Nuclear epigenetic impact – Reduced mitochondrial acetyl‑CoA diminishes histone acetylation at TFEB target promoters; lowered SAM limits histone and DNA methylation dynamics required for permissive chromatin states.
4. TFEB retention – Despite mTORC1 activity, insufficient acetyl‑CoA/SAM signaling fails to activate the acetyl‑transferase p300/CBP and methyltransferases that facilitate TFEB dephosphorylation and nuclear import, keeping TFEB cytoplasmic.
5. Autophagy collapse – Cytoplasmic TFEB cannot drive lysosomal biogenesis or autophagy gene expression, allowing damaged mitochondria to accumulate, further raising mtDNA‑SNV burden.

Testable Predictions

• Prediction 1: In cells or tissues with high heteroplasmic mtDNA‑SNV load, mitochondrial matrix acetyl‑CoA and SAM levels will be significantly lower than in SNV‑matched controls with homoplasmic wild‑type mtDNA.
• Prediction 2: Restoring matrix acetyl‑CoA (via mitochondria‑targeted acetyl‑CoA synthetase) or SAM (via mitochondria‑targeted methionine adenosyltransferase) will increase TFEB nuclear localization and rescue autophagy flux without altering mTORC1 activity.
• Prediction 3: Overexpressing TFAM to tighten nucleoid packaging in mtDNA‑mutator mice will reduce metabolite leakage, improve TFEB nuclear entry, extend healthspan, and delay the onset of age‑related phenotypes.
• Prediction 4: Chromatin immunoprecipitation sequencing (ChIP‑seq) for H3K27ac and H3K4me3 at TFEB‑dependent promoters will show decreased enrichment in high‑SNV contexts, reversible by metabolite supplementation.

Experimental Approach

1. Generate models: Use CRISPR‑free DdCBE base editors to introduce specific heteroplasmic mtDNA‑SNVs (e.g., m.5024C>T) in cultured human fibroblasts and in PolG mutator mice.
2. Metabolite quantification: Isolate mitochondria and measure matrix acetyl‑CoA and SAM via LC‑MS/MS; correlate with SNV heteroplasmy levels.
3. Rescue assays: Express mitochondria‑targeted acetyl‑CoA synthetase (Acetyl‑CoA synthetase 2) or SAM synthase (MAT2A) fused to a mitochondrial targeting sequence; assess TFEB subcellular fractionation, LC3‑II turnover, and lysosomal activity (LysoTracker).
4. Epigenetic read‑outs: Perform ChIP‑qPCR for H3K27ac/H3K4me3 at promoters of CLEAR network genes (e.g., LAMP1, CTSD) and RNA‑seq to quantify transcriptional output.
5. In vivo longevity: Treat PolG mutator mice with AAV9‑delivered mitochondria‑targeted metabolic enzymes; monitor grip strength, frailty index, and survival.

Potential Outcomes and Falsifiability

If metabolite supplementation or nucleoid tightening restores TFEB‑driven autophagy and improves healthspan, the hypothesis gains support. Conversely, if restoring matrix acetyl‑CoA/SAM fails to alter TFEB localization or autophagy despite confirmed metabolite replenishment, the model would be falsified, indicating that mtDNA‑SNVs act through alternative retrograde signals (e.g., ROS, calcium, or mtDNA‑derived nucleic acids) rather than metabolite‑epigenetic crosstalk.