Step 10: Mitochondrial Dynamics and Biogenesis

1 Normal Function and ATP Accounting

Mitochondria undergo continuous fusion (mediated by MFN1/2 and OPA1) and fission (mediated by DRP1 and FIS1). Biogenesis is driven by PGC-1$\alpha$. Quality control integrates both processes: impaired segments are isolated by fission and cleared by mitophagy, while new organelles replace them. Direct ATP yield per cycle is zero (this is a capacity setter, not a producer); gated yield depends on the number and quality of functional mitochondria available over longer timescales. Failure reduces total cellular respiratory capacity across Steps 3–9 and is diagnostically difficult because the effect is longitudinal.

2 Documented ME/CFS Findings and Failure Modes

IgG immune complexes from ME/CFS patient serum cause mitochondrial fragmentation (shift toward fission) in endothelial cells (Section No Direct Measurement of Intramuscular T3 in ME/CFS in Chapter Energy Metabolism and Mitochondrial Function) (Liu et al. 2026). Direct measurement of DRP1/FIS1 versus MFN1/2/OPA1 balance in ME/CFS patient tissue has not been performed; the fragmentation is inferred from morphological observation.

Hypothesis: PGC-1α Suppression Blocks Mitochondrial Biogenesis Recovery

Certainty: 0.45. (0.40→0.45: two independent lines—SNS/PKA→AMPK suppression via Brown2018AMPK and NAD+→SIRT1→PGC-1α deacetylation via the sirtuin hub Replication Status and Limitations—converge on PGC-1α functional suppression through different mechanisms [autonomic signaling and metabolic co-substrate depletion] from different labs using different methods.) Chronic neuroinflammation and elevated TNF-$\alpha$ documented in ME/CFS are predicted to suppress PGC-1$\alpha$ — the master regulator of mitochondrial biogenesis — thereby preventing compensatory mitochondrial proliferation in response to energy deficits . PGC-1$\alpha$ also transcriptionally upregulates MnSOD, meaning its suppression would simultaneously impair ROS clearance (consistent with the Shankar 2025 MnSOD deficit ) and prevent biogenesis recovery after exertion-induced mitochondrial damage. In ME/CFS, where exercise triggers damage but PEM prevents the adaptive exercise response, PGC-1$\alpha$ may be caught in a state where it cannot be upregulated (because exercise is contraindicated) yet is chronically suppressed (by inflammation).

Four lines of indirect evidence now constrain this hypothesis:

PBMC null result: Castro-Marrero et al. measured PGC-1$\alpha$ protein by immunoblotting in CFS PBMCs ($n \approx 23$ CFS, $n \approx 15$ FM, $n \approx 15$ controls) and found no significant reduction in CFS versus controls — while PGC-1$\alpha$ and TFAM were both significantly reduced in fibromyalgia (Castro-Marrero et al. 2013). CoQ10 was reduced in both conditions. This is the only published measurement of PGC-1$\alpha$ protein in ME/CFS samples. The null result may reflect tissue specificity: PBMCs are not the relevant compartment for a biogenesis hypothesis centred on skeletal muscle and neurons.
Idiopathic chronic fatigue muscle: Wawrzyniak et al. performed vastus lateralis biopsies in sedentary older adults ($n = 20$ with idiopathic chronic fatigue vs $n = 28$ non-fatigued, mean age 72.4) and found PGC-1$\alpha$ protein reduced 37.4% ($p < 0.05$), NRF-1 reduced 19.4%, and SIRT3 reduced 18.1% (Wawrzyniak et al. 2016). Cytochrome c oxidase activity was reduced 18.4%. TFAM was not significantly changed — raising questions about PGC-1$\alpha$→TFAM pathway coupling. This is not ME/CFS (idiopathic fatigue in elderly, age and sedentary status confound), but it is the only study demonstrating the PGC-1$\alpha$→NRF-1→TFAM cascade in muscle of chronically fatigued individuals.
Compensatory mRNA upregulation: Wang et al. found that WASF3 overexpression in ME/CFS muscle ($n = 14$ biopsies vs $n = 10$ controls) paradoxically upregulated PGC-1$\alpha$ mRNA alongside respiratory complex genes, despite decreased respiration (Wang et al. 2023). This represents a failed compensatory response: the cell attempts biogenesis but cannot overcome WASF3-mediated disruption of respiratory supercomplex assembly. The distinction between PGC-1$\alpha$ mRNA (upregulated) and protein (unmeasured in ME/CFS muscle) is critical — post-translational regulation via the AMPK/SIRT1 axis determines whether PGC-1$\alpha$ is functionally active.
Upstream AMPK impairment: Brown et al. showed that AMPK activation and glucose uptake are impaired in primary skeletal muscle cells from ME/CFS patients in response to electrical pulse stimulation (exercise mimetic), but pharmacological AMPK activators (metformin, compound 991) rescued both deficits (Brown et al. 2018). The defect is proximal to AMPK, not in AMPK itself. AMPK is the major upstream activator of the AMPK→SIRT1→PGC-1$\alpha$ biogenesis axis. Combined with the Fluge 2016 finding that SIRT4 (which opposes SIRT1/PGC-1$\alpha$) is upregulated in ME/CFS PBMCs , this suggests a dual brake: impaired activation of the pro-biogenesis AMPK/SIRT1 arm plus active suppression via the SIRT4 arm.

Synthesis: The picture is more nuanced than simple PGC-1$\alpha$ suppression, and two competing interpretations must be distinguished. In PBMCs, PGC-1$\alpha$ protein is normal; in ME/CFS muscle, PGC-1$\alpha$ mRNA is compensatorily upregulated but the upstream activating cascade (AMPK→SIRT1→deacetylation) is impaired. Interpretation A (post-translational inactivation): PGC-1$\alpha$ protein in ME/CFS skeletal muscle may be normal or even elevated, but its acetylation state (marking it as inactive) will be increased, and its functional output (TFAM, NRF-1, cytochrome c levels) will be reduced. This predicts benefit from AMPK activators (metformin) or SIRT1 activators (resveratrol). Interpretation B (WASF3-downstream block): PGC-1$\alpha$ signaling is intact and its acetylation ratio is normal, but the new mitochondria it produces are immediately dysfunctional because WASF3 disrupts respiratory supercomplex assembly (Wang et al. 2023). Under this interpretation, the mRNA upregulation reflects a genuine compensatory response that is working correctly but cannot overcome the downstream block; AMPK/SIRT1 activators would be ineffective because the bottleneck is not at PGC-1$\alpha$. These two interpretations have opposite therapeutic implications and could be distinguished by measuring PGC-1$\alpha$ acetylation ratio alongside WASF3 levels and supercomplex assembly in the same muscle biopsies.

Testable prediction: PGC-1$\alpha$ protein and mRNA will be measurable in ME/CFS skeletal muscle biopsies. The critical measurement is not total PGC-1$\alpha$ but its acetylation ratio (inactive:active) and its functional output (TFAM, NRF-1, cytochrome c, MnSOD protein). Under Interpretation A, pharmacological AMPK activators (metformin; requires renal monitoring, contraindicated at eGFR $< 30$) or SIRT1 activators (resveratrol) at sub-exercise doses should reduce PGC-1$\alpha$ acetylation and partially restore biogenesis markers without requiring physical exertion. Under Interpretation B, these agents would be ineffective — the critical discriminating experiment.

Limitation: PGC-1$\alpha$ protein has not been directly measured in ME/CFS skeletal muscle. The PBMC null result (Castro-Marrero et al. 2013) and the muscle mRNA upregulation (Wang et al. 2023) suggest that the failure mode is post-translational (acetylation/phosphorylation) rather than transcriptional — but this remains a hypothesis.

Speculation: The Biogenesis Trap: ME/CFS Cells Waste Energy on Dysfunctional Mitochondrial Production

Certainty: 0.25. PGC-1$\alpha$ mRNA is upregulated in ME/CFS muscle (Wang et al. 2023), yet AMPK activation is impaired (Brown et al. 2018), SIRT4 opposes SIRT1 , and WASF3 disrupts respiratory supercomplex assembly (Wang et al. 2023). This combination creates a biogenesis trap — a metabolic Sisyphus:

The cell correctly senses mitochondrial insufficiency and transcribes more PGC-1$\alpha$.
The PGC-1$\alpha$ protein is hyperacetylated (inactive) because SIRT1 cannot deacetylate it (NAD⁺ depletion in skeletal muscle — not PBMCs, where Heng et al. found NAD⁺ elevated; see The Dual-Compartment NAD⁺ Paradox: PBMCs and Muscle Are Dysregulated in Opposite Directions — per NAD⁺–Sirtuin–Acetylation Hub: Unified Upstream Regulator of G22, G34, and G39).
Even the fraction that is active drives production of new mitochondria that are immediately dysfunctional because WASF3 prevents Complex IV supercomplex assembly.
The cell expends energy on biogenesis but receives no functional return.

If the fraction of functional new mitochondria is sufficiently low (i.e., the biosynthetic ATP cost exceeds the functional ATP return from partially assembled organelles), this could be worse than no biogenesis at all, because: (a) lipid and protein synthesis for new membranes consumes ATP; (b) importing nuclear-encoded mitochondrial proteins via TIM/TOM complexes consumes ATP; (c) new dysfunctional mitochondria generate additional ROS (electrons that cannot be efficiently passed to oxygen at Complex IV back up through the chain, increasing electron leak at Complexes I and III); (d) the cell cannot distinguish functional from dysfunctional new mitochondria for mitophagy (they may initially retain membrane potential).

Testable prediction: ME/CFS muscle will show elevated markers of active biogenesis (mitochondrial protein import machinery, cardiolipin synthesis intermediates) alongside decreased functional respiratory capacity per mitochondrion (citrate synthase-normalised Complex IV activity) — a signature of “spinning wheels.” The WASF3/ER-stress axis should be addressed before stimulating PGC-1$\alpha$/biogenesis; otherwise, more biogenesis amplifies ROS and energy waste.

Competing explanation: The three components (WASF3, AMPK impairment, SIRT4 upregulation) may be independent consequences of chronic inflammation or metabolic stress rather than forming a coordinated “trap.” Under this simpler model, PGC-1$\alpha$ mRNA upregulation is a genuine compensatory response that partially works (producing some functional mitochondria), WASF3 is an ER-stress epiphenomenon, and AMPK impairment is upstream of biogenesis entirely. The trap model predicts simultaneous elevation of biogenesis markers with decreased per-organelle function; the independent model predicts variable patterns depending on which component dominates.

Limitation: The biogenesis trap combines three findings from different tissue compartments and different studies into a single model. No study has simultaneously measured all components in the same ME/CFS tissue sample. The composite cannot be falsified by a negative result in any single component — only a study measuring WASF3 + PGC-1$\alpha$ acetylation + supercomplex assembly + functional respiratory capacity in the same biopsies could test the integrated model.

2.1 Sympathetic Nervous System as an Upstream AMPK Suppressor

The upstream AMPK impairment documented by Brown et al. (Brown et al. 2018) (point 4 above) may have a specific autonomic origin. Sympathetic nervous system activation elevates intracellular cAMP via $\beta$-adrenergic receptors, activating protein kinase A (PKA). PKA directly phosphorylates AMPK$\alpha$ at Ser485/491, inhibiting its activation by LKB1 (Park, Lee, and Kim 2023). In a state of chronic sympathetic dominance—characteristic of ME/CFS autonomic dysfunction (Chapter Cardiovascular Dysfunction)—sustained PKA activity would tonically suppress AMPK, blocking the AMPK → SIRT1 → PGC-1$\alpha$ biogenesis axis described above .

Park et al. (2023) further demonstrate that AMPK’s role in autophagy is more nuanced than previously understood: during acute energy depletion, AMPK actually suppresses autophagy (via ULK1 inhibition) to preserve cellular viability, only permitting autophagy during recovery (Park, Lee, and Kim 2023). In ME/CFS, where energy depletion may be chronic rather than transient, this dual role could create a paradox: AMPK is both tonically suppressed by sympathetic PKA and functionally programmed to suppress autophagy during the energy deficit it is experiencing. The result is a double brake on mitophagy and mitochondrial quality control.

This SNS → PKA → AMPK suppression mechanism connects to the broader autonomic aging framework (Chapter Cardiovascular Dysfunction Section Post-Exercise HRR as Autonomic Recovery Window — Extension to Non-Exercise Stressors): reduced vagal tone simultaneously removes the cholinergic anti-inflammatory brake on NF-$\kappa$B , while sympathetic overdrive suppresses the AMPK-dependent quality control machinery responsible for clearing damaged mitochondria. The two arms of autonomic imbalance converge on the same outcome: accumulation of dysfunctional mitochondria and sustained inflammaging.

The Brown et al. finding that pharmacological AMPK activators (metformin, compound 991) rescued the deficit (Brown et al. 2018) suggests that the AMPK machinery itself is intact—the problem is upstream signaling. If sympathetic PKA is the upstream suppressor, then interventions reducing sympathetic tone ($\beta$-blockers, vagal nerve stimulation) could theoretically restore AMPK activity without requiring direct pharmacological activation. This is speculative: no study has tested whether autonomic modulation improves AMPK phosphorylation in ME/CFS tissue.

2.2 mTOR/AMPK Balance and Autophagy Regulation

The AMPK/mTOR axis forms a bistable homeostatic switch: when cellular energy is abundant (low AMP/ATP ratio), mTORC1 is active, promoting anabolic processes (protein synthesis, ribosome biogenesis) while phosphorylating ULK1 at Ser757 to inhibit autophagy initiation (Rachakatla and Kalashikam 2022). When energy is scarce (high AMP/ATP), AMPK activates and simultaneously inhibits mTORC1 (via phosphorylation of RAPTOR at Ser792 and TSC2) and directly activates ULK1 at Ser317/Ser777 to trigger autophagosome formation (Rachakatla and Kalashikam 2022). The two pathways converge on ATG13: mTORC1 phosphorylates ATG13 at Ser258 to prevent ULK1/ATG13/FIP200 complex assembly — the rate-limiting step for autophagosome nucleation (Mannick and Lamming 2023).

In ME/CFS, this balance may be chronically tilted toward mTOR dominance through two converging mechanisms: (1) sustained sympathetic nervous system activation suppresses AMPK via PKA-mediated phosphorylation of AMPK$\alpha$ at Ser485/491 (described above), removing the primary brake on mTORC1; (2) elevated inflammatory cytokines (TNF-$\alpha$, IL-6) activate PI3K/AKT signaling upstream of mTORC1, providing a parallel activation route independent of energy status. The net effect is a cell locked in “growth mode” despite energy stress — unable to recycle damaged mitochondria and protein aggregates via autophagy.

The Drosen et al. (2025) murine model provides direct causal evidence for this cascade in a PEM-like context: genetic inactivation of ATG13 (mimicking the mTOR-driven phosphorylation block) triggers M1 macrophage infiltration into muscle tissue via STAT3 activation, producing IL-6 and RANTES (CCL5), followed by progressive demyelination of muscle-serving nerves (Drosen et al. 2025). The mice develop reproducible exercise intolerance with symptom worsening 24–48 hours after exertion — mirroring the characteristic PEM time course. An extension study by the same group (Toriola et al. 2026) confirmed that ATG13 depletion also impairs mitochondrial energy metabolism, increases oxidative stress, and polarizes macrophages to the M1 inflammatory mode (Toriola et al. 2026).

The mTOR/AMPK imbalance also directly impacts mitochondrial quality control through PGC-1$\alpha$ regulation: mTORC1 hyperactivation suppresses PGC-1$\alpha$ expression, while AMPK and SIRT1 activate it — creating a coordinated block on both mitophagy (removal of damaged mitochondria) and biogenesis (production of new mitochondria) (Huang et al. 2025). This dual blockade would explain the paradoxical observation in ME/CFS of both impaired AMPK activation and upregulated PGC-1$\alpha$ mRNA (Wang et al. 2023): the cell senses mitochondrial insufficiency and transcribes compensatory PGC-1$\alpha$, but mTOR-driven translational preference and SIRT1 deacetylase insufficiency render the PGC-1$\alpha$ protein functionally inactive.

ATG13 as Circulating Inflammatory Signal and Autophagy/Mitophagy Dysregulation in ME/CFS documents the downstream consequence of this block: ATG13 accumulates extracellularly and acts as a DAMP, while impaired mitophagy sustains oxidative stress. The convergence of the mTOR/AMPK/ATG13 pathway with the SNS→PKA→AMPK suppression described above and the NAD⁺-sirtuin-acetylation hub (Section Replication Status and Limitations) suggests a hierarchically organized failure: autonomic imbalance suppresses AMPK → mTORC1 is disinhibited → ATG13 is phosphorylated → autophagy and mitophagy are blocked → damaged mitochondria accumulate → ROS and mtDNA release activate innate immunity → inflammation further suppresses AMPK — closing a self-reinforcing loop.

Clinical translation. The mTOR pathway is pharmacologically tractable: rapamycin (sirolimus) binds FKBP12 to form a complex that allosterically inhibits mTORC1 (Mannick and Lamming 2023). Ruan et al. (2025) reported improved fatigue and PEM with low-dose weekly rapamycin in an uncontrolled pilot study of 86 ME/CFS patients, with exploratory biomarker changes (reduced pSer258-ATG13, increased BECLIN-1) consistent with restored autophagy (Ruan et al. 2025). However, this is an uncontrolled observation — the ~34% responder rate documented in the treatment map is provisional, and no randomized controlled trial has been completed. Bar-Tana (2025) has proposed mTORC1 syndrome (TorS) as a unifying paradigm for post-acute infection syndromes including ME/CFS (Bar-Tana 2025), though this remains a hypothesis paper without primary data.

Mechanistic extension — purine metabolism as an mTORC1 downstream effector. Gile et al. (2026) extended the rapamycin investigation beyond autophagy markers to examine purine metabolism in the same Phase II cohort (Gile et al. 2026). LCMS-based purine metabolomics revealed that rapamycin differentially regulates the enzymatic conversion of inosine monophosphate (IMP) to xanthosine-5-monophosphate (XMP) and hypoxanthine. Flow cytometry confirmed that rapamycin reduces IMP dehydrogenase (IMPDH) activity — the rate-limiting enzyme for de novo guanine nucleotide synthesis — thereby limiting IMP → XMP flux. This is mechanistically coherent: mTORC1 regulates IMPDH expression and purine biosynthesis via ATF4-mediated transcriptional control of the mitochondrial one-carbon pathway (established in cancer biology; Gile et al. situate their findings within this framework (Gile et al. 2026)). In ME/CFS, chronic mTORC1 hyperactivation would be predicted to drive IMPDH overactivity, producing purine intermediates that generate oxidative stress when metabolized. Purine supplementation experiments in PBMCs induced mitochondrial oxidative stress; rapamycin pre-treatment partially mitigated this effect. Seahorse OCR measurements confirmed improved mitochondrial respiratory capacity (basal, ATP-linked, maximal, and spare capacity) in responder PBMCs after 90 days of rapamycin therapy. This finding should be interpreted with caution: an uncontrolled design with 53% attrition and molecular results biased toward responders means the purine→mitochondrial axis represents one plausible mechanism, not an established causal pathway.

Clinical consequence — purine-driven microglial inflammation. Extracellular purines signal through purinergic receptors on microglia; purine accumulation from IMPDH overactivity could activate microglial P2X7 receptors, triggering NLRP3 inflammasome assembly and IL-1$\beta$ release. Gile et al. (2026) reported reduced microglial-signature inflammatory markers in PBMC flow cytometry from rapamycin-treated samples, providing preliminary evidence — from an uncontrolled cohort — that mTORC1 inhibition may attenuate inflammation via a purine-mediated mechanism. This connects the mTOR/autophagy framework to the neuroinflammatory and NLRP3/PEM hypotheses already discussed in the metabolic danger signal model (Chapter Symptom-Producing Mechanisms in ME/CFS) and the endothelial senescence-SASP loop (Senescent Endothelial Cell Burden as a Central Maintaining Mechanism in Chapter Immune System Dysfunction), placing mTORC1 as a proposed central hub that coordinates both mitochondrial quality control and neuroinflammation through distinct downstream pathways — autophagy (via ATG13) and purine metabolism (via IMPDH). Consequence: The purine metabolism axis reveals a second mTORC1-driven pathology pathway in ME/CFS — parallel to the autophagy block. This explains how mTORC1 can simultaneously impair mitochondrial respiration (via purine-mediated oxidative stress) and prevent clearance of damaged mitochondria (via ATG13-mediated autophagy inhibition). It also identifies IMPDH as a drug target downstream of mTORC1 that could in principle be modulated without the immunosuppressive risks of direct mTORC1 inhibition, though IMPDH inhibitors carry their own risk profile (mycophenolate: GI toxicity, myelosuppression, teratogenicity — pregnancy Category D) and any advantage over rapamycin remains entirely hypothetical. (Severity applicability: unknown — Gile 2026 cohort not stratified by severity.)

Warning: Rapamycin Is an Immunosuppressant — Research-Stage Only

Rapamycin is an FDA-approved immunosuppressant with known risks including opportunistic infections, hyperlipidaemia, impaired wound healing, and interstitial pneumonitis. It is a CYP3A4 and P-gp substrate with major drug-drug interactions. The Ruan et al. (2025) study (Ruan et al. 2025) lacked a control group, exposing findings to placebo effect, regression to the mean, and natural PEM fluctuation. No clinical recommendation can be made pending a randomized controlled trial. Off-label use without medical supervision is dangerous.

Warning: Falsifiability of the mTOR/AMPK/Autophagy Cascade

The causal chain described above (SNS → PKA → AMPK inhibition → mTORC1 disinhibition → ATG13-Ser258 phosphorylation → autophagy block → mitophagy failure → ROS/mtDNA release) is presented as a mechanistic framework synthesizing evidence from multiple systems (autonomic, metabolic, immune). The following predictions would individually falsify specific links; a coordinated study measuring all nodes in the same ME/CFS samples would provide a decisive test of the full cascade:

AMPK$\alpha$ Ser485/491 phosphorylation (the PKA site) will be elevated in ME/CFS PBMCs vs controls and will correlate with ULK1 Ser757 phosphorylation (the mTORC1 site) — linking SNS-driven AMPK suppression to mTORC1 activation at the autophagy gate.
mTORC1 activity (pS6K, p4E-BP1) will be elevated in ME/CFS tissue and will inversely correlate with autophagic flux (LC3-II turnover in bafilomycin-blocked cells).
The chain is falsified at its core if (a) mTORC1 activity is normal in ME/CFS despite elevated AMPK Ser485/491 phosphorylation, or (b) autophagy flux is normal in ME/CFS despite elevated mTORC1 activity, or (c) interventions that reduce mTORC1 (rapamycin, metformin) fail to improve autophagy flux despite reducing mTORC1 activity.
Extension — purine metabolism predictions. IMPDH activity should be elevated ≥1.5-fold in ME/CFS PBMCs vs controls and should correlate with mTORC1 activity (pS6K, r ≥ 0.5). Rapamycin should reduce IMPDH activity by ≥30% from baseline and normalize the IMP/XMP ratio (LS mean ratio < 1.5 vs ≥ 2.0 at baseline) in responder PBMCs. The purine → mitochondrial axis is falsified if rapamycin reduces IMPDH activity without improving any OCR parameter, or if IMP/XMP ratio normalization does not predict OCR improvement within patients (r < 0.3). The P2X7-mediated microglial link requires a specific antagonist test: a P2X7 antagonist (e.g., A438079) or NLRP3 inhibitor (MCC950) should block the microglial inflammatory response to purine supplementation in ME/CFS PBMCs at concentrations that do not affect IMPDH activity. This link is falsified if P2X7/NLRP3 blockade does not attenuate purine-induced microglial inflammation, or if the microglial anti-inflammatory effect of rapamycin persists unchanged after P2X7 receptor blockade.

3 mtDNA Alterations and Impaired Mitophagy

Mitochondrial DNA (mtDNA) is vulnerable to damage:

Mutations: Point mutations accumulate with oxidative stress
Deletions: Large deletions impair multiple ETC components
Copy number: Altered mtDNA copy number in some ME/CFS studies
Heteroplasmy: Mixture of normal and mutant mtDNA

3.1 Impaired Mitophagy

Mitophagy removes damaged mitochondria:

PINK1/Parkin pathway: Marks damaged mitochondria for degradation
Impaired clearance: May allow dysfunctional mitochondria to persist
Accumulation: Damaged mitochondria continue producing ROS
Quality control failure: Network of damaged organelles

4 Circulating Cell-Free Mitochondrial DNA and Mitophagy Signalling

Circulating cell-free mitochondrial DNA (ccf-mtDNA) is released from cells during both physiological processes (exercise, mitophagy) and pathological states (cell death, inflammation). Its levels therefore reflect the balance between mitochondrial turnover, damage, and clearance. In acute SARS-CoV-2 infection, markedly elevated ccf-mtDNA signals massive mitochondrial release during cell death and predicts adverse outcomes. By contrast, Matits et al. ($n = 228$; EPILOC cohort) reported nominally lower ccf-mtDNA in Long COVID patients compared to recovered controls, though the finding did not reach statistical significance after full covariate adjustment ($p = 0.089$; partial $\eta^2 = 0.01$–$ 0.02$; $p = 0.038$ only in a sensitivity analysis excluding high-CRP outliers). A preliminary case series (Szögi et al. ; $n = 5$ Long COVID, $n = 5$ ciliary dyskinesia controls) also reported reduced ccf-mtDNA alongside mitochondrial ultrastructural abnormalities, though the extremely small sample and non-standard controls preclude generalization.

Hypothesis: Impaired Mitophagy Traps Damaged Mitochondria in Long COVID

Certainty: 0.35. If ccf-mtDNA is genuinely reduced in Long COVID (a finding that did not reach conventional significance after full covariate adjustment in the sole adequately powered study), one interpretation is that impaired mitophagy prevents clearance and extracellular release of damaged mitochondria, causing their intracellular accumulation. This would produce chronic cellular stress and energy deficits without the external damage signal seen in acute infection . This connects to the mitophagy coordination pathway proposed in Section Endogenous Ouabain Depletion: A Missing Regulatory Signal for Na⁺/K⁺-ATPase?. If the ERR$\alpha$/ULK1/DRP1 fission-mitophagy-biogenesis cycle is disrupted, damaged organelles persist. However, the deconditioning alternative has not been excluded—physically active individuals have higher ccf-mtDNA, and Long COVID patients are typically less active than recovered controls.

Testable prediction: Muscle biopsy from Long COVID patients with low ccf-mtDNA should show increased mitochondrial PINK1/Parkin co-localization (stalled mitophagy) compared to recovered controls with normal ccf-mtDNA. Falsified if mitophagy markers are normal or reduced despite low ccf-mtDNA.

In ME/CFS, Tsilioni et al. found exosome-associated mtDNA elevated after exercise —a different compartment, different timing, and potentially a different mechanism (active exosomal secretion rather than passive release from cell death). Quantitative details (sample size, effect size) for the Tsilioni study are not available from the abstract, and the finding has not been independently replicated; it should be weighted accordingly. Whether resting ccf-mtDNA is also altered in ME/CFS is unknown (see ccf-mtDNA as a Potential Long COVID vs ME/CFS Distinguishing Biomarker in Section Speculative Cross-Disease Connections for the cross-disease comparison).

Post-hoc observation: IgG immune complexes and the mitophagy block. The following connection was constructed after both the Liu and Matits findings were available; it was not predicted by either study and should be read as a hypothesis-generating observation, not as a deduction. The IgG immune complex finding (Section No Direct Measurement of Intramuscular T3 in ME/CFS above) could in principle relate to the impaired mitophagy hypothesis. Liu et al. (Liu et al. 2026) showed that IgG from ME/CFS patients induces mitochondrial fragmentation without depolarizing the fragments—spare respiratory capacity was maintained or increased. Since the canonical PINK1/Parkin mitophagy pathway requires depolarization, IgG-induced fragments that retain membrane potential might evade this clearance route, potentially contributing to intracellular accumulation. However, this reasoning has several unverified steps: (a) PINK1/Parkin dynamics have not been measured in IgG-treated cells; (b) PINK1-independent mitophagy pathways (BNIP3, FUNDC1) could clear polarised fragments; (c) the Liu experiment used ME/CFS sera while the Matits finding is in Long COVID patients; (d) the HUVEC model may not represent whole-body ccf-mtDNA dynamics. The connection remains speculative and requires direct experimental testing before it can be considered mechanistically supported.

Deconditioning as a causal feedback loop, not merely a confound. Physical activity is a potent stimulus for mitophagy via AMPK activation. The deconditioning commonly seen in ME/CFS and Long COVID patients may therefore not simply confound the ccf-mtDNA measurement—it may mediate the mitophagy impairment. Reduced activity → reduced AMPK-stimulated mitophagy → accumulation of damaged mitochondria → further impaired energy production → further reduced activity capacity. This creates a self-reinforcing loop in which deconditioning is both a consequence and a cause of mitochondrial quality control failure. Strict pacing, while essential for preventing PEM-mediated kindling (Chapter Neurological and Neurocognitive Dysfunction), may paradoxically worsen this mitophagy deficit. Whether a “therapeutic window” exists—activity sufficient to maintain AMPK-mediated mitophagy signaling without crossing the PEM threshold—is an open question with direct clinical implications (see Chapter Lifestyle and Non-Pharmacological Interventions for pacing recommendations).

Warning: Deconditioning–Mitophagy Loop Does Not Justify Graded Exercise Therapy

The observation that reduced physical activity may impair AMPK-mediated mitophagy does not justify graded exercise therapy (GET), which operates at intensities far above any plausible mitophagy-only threshold and carries well-documented risk of harm in ME/CFS patients. The “therapeutic window” concept—if one exists—would involve minimal activity levels (e.g., gentle movement well below the PEM threshold), not structured exercise programmes. This distinction is critical: the deconditioning loop is a mechanistic observation, not a clinical recommendation.

Clinical translation: None of the pathways described in this subsection (ccf-mtDNA, mitophagy, PINK1/Parkin) are currently testable or targetable in clinical practice. ccf-mtDNA measurement is a research-only assay. No approved drug specifically targets mitophagy in ME/CFS or Long COVID. The one provisional clinical implication is that strict rest-only approaches may have a theoretical cost; discuss gentle movement within the patient’s individual PEM threshold with a pacing specialist (Chapter Lifestyle and Non-Pharmacological Interventions). For severe or very severe patients who are bedbound, this consideration does not apply—rest is essential.

Warning: Replication Status and Limitations

The mitochondrial fragmentation effect was predominantly observed in female patients; male-specific mechanisms remain unexplored. The HUVEC model relies heavily on glycolysis, which may mask oxidative phosphorylation effects that would manifest in more OXPHOS-dependent tissues (skeletal muscle, neurons). The IgG purification method did not effectively capture pentameric IgM, which may carry additional pathogenic activity. Not all ME/CFS patients’ IgG induced fragmentation, consistent with disease heterogeneity. Independent replication is pending.

5 Research Gaps at Step 10

Five gaps at Step 10, all concerning the longitudinal capacity-setting machinery: (G39) PGC-1$\alpha$ protein acetylation state and functional output in ME/CFS skeletal muscle — PGC-1$\alpha$ mRNA is paradoxically upregulated (Wang et al. 2023), protein is normal in PBMCs (Castro-Marrero et al. 2013), and upstream AMPK is impaired (Brown et al. 2018), but the critical muscle-biopsy measurement of PGC-1$\alpha$ post-translational state has never been performed; (G40) DRP1/FIS1 vs MFN1/2/OPA1 balance (fission/fusion tone); (G41) direct mitophagy flux in patient cells (PINK1/Parkin co-localization, LC3-II turnover); (G42) mtDNA copy number in skeletal muscle (inconsistent findings from blood; muscle is the relevant tissue); (G43) TFAM expression (mitochondrial transcription factor A, downstream of PGC-1$\alpha$).

Step 10 (Dynamics and biogenesis) research gap prioritization. Step 10 failure is longitudinal — it reduces total mitochondrial capacity over days to weeks rather than per-cycle output.
Gap	Essentiality	Worst-case impact	Tractability	Therapeutic leverage	Measurement	Priority
G39: PGC-1α acetylation state and functional output in muscle	High — master transcription factor gating every biogenesis target (TFAM, NRF-1, MnSOD, cytochrome c, etc.); mRNA upregulated (Wang et al. 2023) but protein normal in PBMCs (Castro-Marrero et al. 2013) — post-translational state is the critical unknown	Longitudinal; halving PGC-1α activity halves new mitochondrial synthesis rate	Hard — muscle biopsy Western + acetylation-specific IP + qPCR for targets	Near-term — metformin (AMPK; Brown 2018 showed rescue (Brown et al. 2018)), low-dose resveratrol (SIRT1), AICAR	Muscle biopsy (Western + acetylation IP)	1
G40: DRP1/FIS1 vs MFN1/2/OPA1 balance (fission/fusion tone)	High — determines whether damaged segments are isolated for mitophagy or kept in the network	Kinetic; affects quality-control efficiency	Hard — muscle biopsy IF + Western	Long-term — Mdivi-1 is research-only	Muscle biopsy (IF + Western)	2
G41: Direct mitophagy flux (PINK1/Parkin, LC3-II turnover)	High — the only mechanism for removing damaged mitochondria; Hochecker 2025: LC3-II elevated ~20% in ME/CFS PBMCs (trend $p = 0.054$) but without lysosomal inhibitors the direction of flux (increased initiation vs stalled completion) cannot be resolved (Hochecker et al. 2025); PINK1/Parkin/BNIP3 not yet measured	Determines whether damaged mitochondria accumulate (G39 + G40 interaction)	Hard — PBMC mitophagy flux assay exists but specialist; requires bafilomycin A1 or chloroquine block for flux measurement	Near-term — urolithin A (OTC, phase 1–2 in sarcopenia; not yet phase 3)	Blood draw (PBMC flux assay, specialist)	1
G42: Skeletal muscle mtDNA copy number	Medium — proxy for total mitochondrial content	~proportional to mtDNA depletion	Moderate — muscle biopsy qPCR is standard	Long-term	Muscle biopsy (qPCR)	2
G43: TFAM expression	Medium — downstream of PGC-1α; G39 covers most of the signal	Overlaps with G39	Moderate	Long-term	Muscle biopsy	3

G39 (PGC-1α) remains the highest-priority Step 10 gap, though the evidence landscape has shifted from “entirely unstudied” to “partially characterized with contradictory signals.” Castro-Marrero 2013 found PGC-1$\alpha$ protein normal in CFS PBMCs (Castro-Marrero et al. 2013); Wawrzyniak 2016 found it reduced 37% in idiopathic chronic fatigue muscle (not ME/CFS) (Wawrzyniak et al. 2016); the WASF3 study found PGC-1$\alpha$ mRNA upregulated in ME/CFS muscle (Wang et al. 2023); and Brown 2018 showed impaired upstream AMPK signaling that could prevent PGC-1$\alpha$ functional activation (Brown et al. 2018). The resolution likely lies in post-translational regulation: PGC-1$\alpha$ may be present but inactive (hyperacetylated) in ME/CFS muscle due to the dual brake of impaired AMPK/SIRT1 activation and active SIRT4 suppression . What is needed: a muscle biopsy study measuring not just total PGC-1$\alpha$ protein but its acetylation state and functional outputs (TFAM, NRF-1, cytochrome c). Step 10 is conditionally essential: cells can run on existing mitochondria for days without biogenesis, but sustained dysfunction depletes capacity non-linearly via a damaged-mitochondria accumulation cycle — a pattern consistent with the progressive capacity loss proposed for post-exertional malaise.

6 The NAD⁺–Sirtuin–Acetylation Hub

The preceding sections treat NAD⁺ depletion (G22, Step 6), NRF2 pathway dysfunction (G34, Step 9), and PGC-1$\alpha$ hyperacetylation (G39, Step 10) as separate research gaps. A post-hoc observation: these three gaps could be mechanistically linked through a single upstream regulator — NAD⁺-dependent sirtuin activity — if tissue NAD⁺ depletion proves real. This convergence rests on well-established enzymology (the sirtuin-substrate relationships are documented in non-ME/CFS biology), though the specific claim that these connections are operative in ME/CFS tissue remains a hypothesis.

SIRT1 deacetylates PGC-1$\alpha$ at multiple lysine residues, converting it from an inactive hyperacetylated form to the active transcription factor that drives mitochondrial biogenesis . SIRT1 has also been reported to deacetylate NRF2, promoting its nuclear retention and transcriptional activity on antioxidant response elements, though the directness and significance of this interaction remain debated (SIRT1 may also modulate NRF2 indirectly via KEAP1 or other regulators). SIRT3, a mitochondrial matrix sirtuin, deacetylates and activates isocitrate dehydrogenase 2 (IDH2), MnSOD, and components of Complex I — the last of which directly affects electron transport chain flux. All three sirtuins require NAD⁺ as a co-substrate (not merely a cofactor — NAD⁺ is consumed stoichiometrically, one molecule per deacetylation event, producing nicotinamide and O-acetyl-ADP-ribose).

In ME/CFS, three independent NAD⁺-consuming mechanisms are plausible, though their tissue-specific operation remains largely inferred rather than directly measured:

PARP hyperactivation (inferred): The Shankar 2025 MnSOD deficit in lymphocytes predicts increased mitochondrial superoxide → oxidative DNA damage → PARP1 activation → NAD⁺ consumption. A single DNA double-strand break can consume hundreds of NAD⁺ molecules through PARP poly-ADP-ribosylation. PARP activity has not been directly measured in ME/CFS tissue.
CD38 upregulation (inferred from immunology): CD38 is the dominant NAD⁺-consuming enzyme in most tissues and is strongly upregulated by inflammatory cytokines (TNF-$\alpha$, IFN-$\gamma$) — both chronically elevated in ME/CFS. CD38 also degrades the NAD⁺ precursor NMN, creating a double block on the salvage pathway. Note that CD38 upregulation in PBMCs would also consume NAD⁺ there — yet Heng et al. found PBMC NAD⁺ elevated (Heng et al. 2025), suggesting that if CD38 is active in PBMCs, its consumption is outpaced by ETC-driven NAD⁺ backup in that compartment.
Kynurenine pathway diversion (documented in plasma): Tryptophan is the sole substrate for de novo NAD⁺ synthesis via the kynurenine pathway. IDO1 upregulation by IFN-$\gamma$ diverts tryptophan toward quinolinic acid (neurotoxic) rather than completing the synthesis to NAD⁺ — documented in ME/CFS plasma metabolomics (Germain et al. 2022) (Groven et al. 2021), though the tissue-specific impact on muscle or neuronal NAD⁺ pools is unmeasured.

Hypothesis: NAD⁺–Sirtuin–Acetylation Hub: Unified Upstream Regulator of G22, G34, and G39

Certainty: 0.40. (0.35→0.40: the SNS→AMPK→mTORC1→autophagy axis provides independent convergence—from a different lab [Brown 2018] using different methods [electrical pulse stimulation AMPK phosphorylation]—on the same endpoint of PGC-1α functional suppression. The autonomic route converges with the metabolic co-substrate route, strengthening the hub model through parallel independent evidence.) NAD⁺ depletion in energy-intensive tissues (skeletal muscle, neurons — not PBMCs, where Heng et al. found NAD⁺ elevated (Heng et al. 2025), consistent with substrate backup rather than depletion) simultaneously inactivates SIRT1, SIRT3, and shifts the SIRT1/SIRT4 balance toward the inhibitory SIRT4 arm . The downstream consequences propagate through four documented pathways:

PGC-1$\alpha$ hyperacetylation (G39): SIRT1 cannot deacetylate PGC-1$\alpha$ → biogenesis is blocked despite compensatory mRNA upregulation (Wang et al. 2023) → mitochondrial mass declines over weeks (Step 10).
NRF2 functional suppression (G34): SIRT1 cannot deacetylate NRF2 → reduced nuclear retention and ARE binding → antioxidant target genes (MnSOD, GPX4, NQO1) are under-expressed even if NRF2 protein is present (Davis et al. 2025) (Step 9).
Complex I and TCA cycle impairment: SIRT3 cannot deacetylate Complex I subunits (NDUFA9) and IDH2 → reduced electron transport and TCA flux → lower ATP and more ROS (Steps 4–5).
PDC regulation via SIRT4: When SIRT1 activity falls but SIRT4 remains active (as documented in ME/CFS PBMCs ), SIRT4 opposes energy metabolism through at least two mechanisms: ADP-ribosylation of glutamate dehydrogenase (reducing glutamine-derived TCA anaplerosis) and lipoamidase activity that removes lipoyl groups from the E2 subunit of PDC → reduced pyruvate entry into the TCA cycle (Step 3).

This model predicts that G22, G34, and G39 are not independent gaps but three readouts of a single upstream failure. Resolving G22 (measuring tissue NAD⁺ and PARP/CD38 activity) would therefore provide mechanistic context for all three.

Testable predictions: + NAD⁺ in ME/CFS skeletal muscle will be reduced (opposite to the PBMC elevation found by Heng et al. (Heng et al. 2025)), predicting a tissue-compartment dissociation. + The muscle NAD⁺/NADH ratio will correlate inversely with PGC-1$\alpha$ acetylation ratio in the same biopsies — the tighter the correlation, the stronger the evidence that NAD⁺ depletion is the rate-limiting step for SIRT1-mediated PGC-1$\alpha$ activation. + NR or NMN supplementation (which raises tissue NAD⁺) should simultaneously reduce PGC-1$\alpha$ acetylation, increase NRF2 target gene expression, and improve Complex I-linked respiration in ME/CFS patient-derived cells — all three effects from a single intervention targeting the hub. + A factorial RCT (NAD⁺ precursor $\\times$ AMPK activator) should show synergistic rather than additive effects on biogenesis markers, because AMPK and SIRT1 mutually activate each other (AMPK increases SIRT1 activity by upregulating NAMPT, raising the NAD⁺ pool; SIRT1 deacetylates LKB1, which activates AMPK).

Falsifiability: This hub model would be falsified if (a) muscle NAD⁺ is normal in ME/CFS despite PGC-1$\alpha$ hyperacetylation, indicating a SIRT1-independent mechanism for the acetylation; or (b) NR/NMN supplementation raises tissue NAD⁺ but does not change PGC-1$\alpha$ acetylation or NRF2 target expression, indicating that NAD⁺ is not rate-limiting for sirtuin activity in this context; or (c) PGC-1$\alpha$ acetylation is normal in ME/CFS muscle, eliminating the need for a sirtuin-based explanation.

Competing explanation: A simpler model does not require NAD⁺ depletion as a unifying hub. Chronic inflammation — well-documented in ME/CFS — can independently suppress PGC-1$\alpha$ transcription (via NF-$\kappa$B), impair NRF2 signaling (via inflammatory pathway crosstalk), and damage Complex I (via ROS) through their own well-characterized regulatory mechanisms. Under this three-cause model, G22, G34, and G39 are genuinely independent and do not require a shared upstream regulator. The hub model would be preferred over this simpler alternative only if co-measurement shows that NAD⁺ depletion correlates more tightly with all three downstream readouts than inflammatory markers do — i.e., if a multivariate model with NAD⁺ as predictor outperforms one using TNF-$\alpha$/IL-6 as predictors for the combined PGC-1$\alpha$ acetylation + NRF2 target + Complex I activity outcome.

Limitation: The PBMC NAD⁺ elevation (Heng et al. 2025) and the predicted muscle NAD⁺ depletion have never been measured in the same cohort. The tissue-compartment dissociation is a prediction — specifically for skeletal muscle — not an observation; if muscle NAD⁺ proves normal, the model does not retreat to “the relevant tissue is neurons” but is falsified (criterion a above). SIRT1 has numerous substrates beyond PGC-1$\alpha$ and NRF2 (p53, FOXO3, NF-$\kappa$B), and the acetylation changes may reflect a broader regulatory shift rather than a specific biogenesis/antioxidant mechanism. The hub model risks unfalsifiable circularity if every downstream effect is attributed to NAD⁺ without specifying which effects would disprove the connection. This model was assembled post-hoc from pre-existing gap analyses; it should be evaluated as a hypothesis-generating framework, not as a confirmed mechanistic finding.

References

Bar-Tana, Jacob. 2025. “mTORC1 Syndrome (TorS): Unifying Paradigm for PASC, ME/CFS and PAIS.” Journal of Translational Medicine 23 (1): 325. https://doi.org/10.1186/s12967-025-06293-w.

Brown, Audrey E., Beth Dibnah, Elizabeth Fisher, Julia L. Newton, and Mark Walker. 2018. “Pharmacological Activation of AMPK and Glucose Uptake in Cultured Human Skeletal Muscle Cells from Patients with ME/CFS.” Bioscience Reports 38 (3): BSR20180242. https://doi.org/10.1042/BSR20180242.

Castro-Marrero, Jesús, Mario D. Cordero, Naia Sáez-Francàs, Cecilia Jiménez-Gutiérrez, Francisco Javier Aguilar-Montilla, Luisa Aliste, and José Alegre-Martín. 2013. “Could Mitochondrial Dysfunction Be a Differentiating Marker Between Chronic Fatigue Syndrome and Fibromyalgia?” Antioxidants & Redox Signaling 19 (15): 1855–60. https://doi.org/10.1089/ars.2013.5346.

Davis, Leah, Megan Higgs, Alice Snaith, Tiffany A. Lodge, Jessica Strong, José Antonio Espejo-Oltra, Slawomir Kujawski, et al. 2025. “Dysregulation of Lipid Metabolism, Energy Production, and Oxidative Stress in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome, Gulf War Syndrome and Fibromyalgia.” Frontiers in Neuroscience 19: 1498981. https://doi.org/10.3389/fnins.2025.1498981.

Drosen, ME, S Bulbule, G Gottschalk, D Peterson, LA Allen, L Arnold, and A Roy. 2025. “Inactivation of ATG13 Stimulates Chronic Demyelinating Pathologies in Muscle-Serving Nerves and Spinal Cord.” Immunologic Research 73 (1): 27. https://doi.org/10.1007/s12026-024-09557-7.

Germain, Arnaud, Ludovic Giloteaux, Gregory E Moore, Susan M Levine, Joy K Chia, Betsy A Keller, John Stevens, et al. 2022. “Plasma Metabolomics Reveals Disrupted Response and Recovery Following Maximal Exercise in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.” JCI Insight 7 (9): e157621. https://doi.org/10.1172/jci.insight.157621.

Gile, Brooke, Sarojini Bulbule, Mubaraq A. Toriola, Brian T. Ruan, Shabnam Marium, Anna Benko, Stephanie Grach, et al. 2026. “Association of Rapamycin Treatment with the Modulation of Purine Metabolism, Reduced Microglial Inflammatory Responses, Improved Mitochondrial Energy Metabolism, and Alleviation of Fatigue Symptoms in ME/CFS Subjects: Pilot Findings from Phase-II Observational Study.” Journal of Translational Medicine 24 (1): 921. https://doi.org/10.1186/s12967-026-08575-3.

Groven, Nina, Solveig Klæbo Reitan, Egil A Fors, and Ismail Cuneyt Guzey. 2021. “Kynurenine Metabolites and Ratios Differ Between Chronic Fatigue Syndrome, Fibromyalgia, and Healthy Controls.” Psychoneuroendocrinology 131: 105287. https://doi.org/10.1016/j.psyneuen.2021.105287.

Heng, Ruiwen Benjamin, Bavani Gunasegaran, Shivani Krishnamurthy, Sonia Bustamante, Ananda Staats, Sharron Chow, Seong Beom Ahn, et al. 2025. “Mapping the Complexity of ME/CFS: Evidence for Abnormal Energy Metabolism, Altered Immune Profile, and Vascular Dysfunction.” Cell Reports Medicine 6 (12): 102514. https://doi.org/10.1016/j.xcrm.2025.102514.

Hochecker, Bianca, Daniel Bizjak, Jürgen M Steinacker, et al. 2025. “Mitophagy Markers in Chronic Fatigue Syndrome and Post-COVID Syndrome.”

Huang, Y, C Wang, H Cui, G Sun, X Qi, and X Yao. 2025. “Mitochondrial Dysfunction in Age-Related Sarcopenia: Mechanistic Insights, Diagnostic Advances, and Therapeutic Prospects.” Frontiers in Cell and Developmental Biology 13: 1590524. https://doi.org/10.3389/fcell.2025.1590524.

Liu, Zhihang, Claudia Hollmann, Shreya Kalanidhi, Aeneas Grothey, Jan Lamerding, Ritika Bhargava, Hannah Graßhoff, et al. 2026. “Immunoglobulin G Complexes from Post-Infectious ME/CFS, Including Post-COVID ME/CFS Disrupt Cellular Energetics and Alter Inflammatory Marker Secretion.” Brain, Behavior, and Immunity – Health 52: 101187. https://doi.org/10.1016/j.bbih.2026.101187.

Mannick, JB, and DW Lamming. 2023. “Targeting the Biology of Aging with mTOR Inhibitors.” Nature Aging 3 (6): 642–60. https://doi.org/10.1038/s43587-023-00416-y.

Park, Ji-Man, Da-Hye Lee, and Do-Hyung Kim. 2023. “Redefining the Role of AMPK in Autophagy and the Energy Stress Response.” Nature Communications 14 (1): 2994. https://doi.org/10.1038/s41467-023-38401-z.

Rachakatla, A, and RR Kalashikam. 2022. “Calorie Restriction-Regulated Molecular Pathways and Its Impact on Various Age Groups: An Overview.” DNA and Cell Biology 41 (5): 459–68. https://doi.org/10.1089/dna.2021.0922.

Ruan, BT, S Bulbule, B Gile, A Reyes, B Chheda, L Bateman, J Bell, et al. 2025. “Low-Dose Rapamycin Alleviates Clinical Symptoms of Fatigue and PEM in ME/CFS Patients via Improvement of Autophagy: A Pilot Study.” Journal of Translational Medicine 23: 1148. https://doi.org/10.1186/s12967-025-07213-8.

Toriola, MA, E Timlin, S Bulbule, G Gottschalk, D Peterson, L Arnold, and A Roy. 2026. “Genetic Depletion of the Early Autophagy Protein ATG13 Impairs Mitochondrial Energy Metabolism, Augments Oxidative Stress, Induces the Polarization of Macrophages to the M1 Inflammatory Mode, and Compromises Myelin Integrity in Skeletal Muscle.” Inflammation Research. https://doi.org/10.1007/s00011-025-02158-6.

Wang, Ping-yuan, Jin Ma, Young-Chae Kim, et al. 2023. “WASF3 Disrupts Mitochondrial Respiration and May Mediate Exercise Intolerance in Myalgic Encephalomyelitis/Chronic Fatigue Syndrome.” Proceedings of the National Academy of Sciences 120 (34): e2302738120. https://doi.org/10.1073/pnas.2302738120.

Wawrzyniak, Nicholas R., Anna-Maria Joseph, David G. Levin, Daniel M. Gundermann, Christiaan Leeuwenburgh, Todd M. Manini, and Thomas W. Buford. 2016. “Idiopathic Chronic Fatigue in Older Adults Is Linked to Impaired Mitochondrial Content and Biogenesis Signaling in Skeletal Muscle.” Oncotarget 7 (33): 52695–709. https://doi.org/10.18632/oncotarget.10685.