Background

Recent work shows that calreticulin and ERp57 decline with age across mouse tissues, leading to chronic IRE1 arm activation (increased XBP1 splicing) and heightened JNK‑mediated apoptosis [1, 2]. In skeletal muscle, the PERK arm is required for satellite cell survival and myogenic differentiation, whereas the IRE1/XBP1 arm is dispensable [3]. However, the adaptive UPR response to exercise is blunted in older adults, indicating a loss of protective PERK signaling when needed [4]. ER structure also remodels via ER‑phagy during aging [5]. These observations raise the possibility that the ratio of IRE1 to PERK activity, rather than absolute ER stress levels, dictates whether ER‑phagy promotes clearance or triggers deleterious satellite cell loss.

Hypothesis

We hypothesize that aging increases the IRE1‑PERK activity ratio, which skews ER‑phagy toward selective degradation of PERK‑associated ER subdomains. This loss of PERK‑resident ER impairs the local translation of ATF4‑dependent survival factors in satellite cells, tipping the balance toward apoptosis despite unchanged global ER load.

Mechanistic model

1. Chaperone loss (calreticulin/ERp57) triggers luminal sensing by IRE1, promoting its oligomerization and sustained RNase activity [1]

Hypothesis

Telomere length reflects the informational entropy of the epigenome, which is set by mitochondrial NAD+‑dependent chromatin states. Shortening is not a passive division counter but an active readout of epigenetic dysregulation driven by mitochondrial dysfunction.

Mechanistic Basis

Mitochondrial respiration shapes nuclear acetyl‑CoA and α‑ketoglutarate pools, modulating SIRT1 deacetylase and TET demethylase activity (see Mitochondrial‑epigenetic crosstalk). Low NAD+ reduces SIRT1 activity, leading to H4K8 hyperacetylation and loss of heterochromatin at telomeres (Epigenetic NAD+ signaling). This heterochromatin loss increases telomeric transcript (TERRA) production and accessibility, raising the entropy of telomeric chromatin. Conversely, boosting NAD+ restores SIRT1, re‑establishes compact telomeric chromatin, and slows attrition independent of replication count.

Testable Predictions

1. In human fibroblasts, acute NAD+ depletion (via FK866) will increase telomeric H4K8ac and TERRA levels within 24 h, preceding measurable telomere shortening.
2. Chronic NAD+ supplementation (NR or NMN) in aged mice will preserve telomere length without altering proliferation rates, correlating with restored SIRT1 binding at subtelomeric regions.
3. Cells engineered to overexpress a telomere‑targeted HDAC3 mimic will show reduced telomeric entropy (lower TERRA, higher H3K9me3) and delayed senescence despite mitochondrial stress.

Experimental Design

• Cell model: IMR‑90 fibro

Hypothesis: Nuclear acetyl‑CoA scarcity remodels ER‑lysosome lipid composition to actively suppress autophagy

Core idea When nuclear acetyl‑CoA falls during aging, cytosolic acetyl‑CoA is shunted toward de novo fatty acid synthesis, raising phosphatidic acid (PA) at ER‑lysosome contact sites. PA strengthens VAPB‑PTPIP51 tethers, which blocks the ATG9A‑FAM134B machinery needed for autophagosome seeding. Thus autophagy decline is an active lipid‑based brake, not a passive loss of machinery.

Novel mechanistic steps

1. Low nuclear acetyl‑CoA → reduced H3K27ac at autophagy gene promoters → transcriptional repression ([1][2]).
2. Cytosolic acetyl‑CoA surplus fuels ACLY‑dependent fatty acid synthesis, elevating cellular PA.
3. PA accumulates at ER‑lysosome microdomains and binds the FFAT motif of VAPB, stabilizing VAPB‑PTPIP51 contacts ([4] showed ectopic acetyl‑CoA flux disrupts ATG9A‑FAM134B; we propose PA‑mediated tether hyper‑stabilization as the cause).
4. Stable tethers restrict lateral diffusion of ATG9A vesicles, preventing phagophore initiation.
5. The cell experiences a double lock: transcriptional silencing plus a mechanical block at the ER‑lysosome interface.

Testable predictions

• Prediction 1: Acetate supplementation in aged cells will raise nuclear acetyl‑CoA, lower cytosolic PA, and reduce VAPB‑PTPIP51 tether density (detected by PLA or FRET).

Overview

We propose that regulatory peptides can extend healthy lifespan by optimizing aging-associated gene expression patterns — specifically through epigenetic modulation and improved cellular signaling fidelity.

The Hypothesis

Certain peptides interact directly with DNA or chromatin-associated proteins, potentially restoring youthful transcriptional patterns in senescent cells. This mechanism operates on two levels:

1. Epigenetic restoration — peptides may reverse or slow methylation drift and histone modification changes associated with aging
2. Signaling fidelity — enhanced intercellular communication ensures cells respond more accurately to environmental cues, reducing downstream dysregulation

Predicted Effects

• Reduction in age-related errors in protein synthesis
• Improved immune response calibration
• Enhanced tissue repair signaling

Hypothesis

Chronic phonatory stress imposes a persistent siege on vocal fold fibroblasts and myocytes, keeping autophagy constitutively activated as a rationing response. Over time, this sustained demand exhausts the autophagic machinery, shifting the system from adaptive flux to insufficiency. We propose that mechanotransduction via integrin‑FAK signaling suppresses TFEB nuclear translocation, blocking lysosomal biogenesis and thereby uncoupling autophagosome formation from degradation. The resulting accumulation of undegraded cargo drives two parallel pathologies: (1) fibroblast‑mediated dysregulation of extracellular matrix turnover, manifesting as collagen enrichment and elastin/hyaluronan loss, and (2) selective degradation of sarcomeric proteins in the thyroarytenoid muscle, producing atrophy. This unified mechanism explains why ECM densification and muscle wasting co‑occur in presbyphonia and predicts that rescuing lysosomal function will restore autophagic flux and ameliorate both phenotypes.

Testable Predictions

1. Flux measurements – In aged rat vocal folds, LC3‑II accumulation will rise with bafilomycin A1 treatment less than in young tissue, indicating reduced autophagosome‑lysosome fusion.
2. TFEB localization – Immunostaining will show decreased nuclear TFEB in fibroblasts and myocytes from aged versus young folds, correlating with elevated phospho‑FAK levels.
3. Lysosomal rescue – Pharmacological activation of TFEB (e.g., with trehalose) or lysosomal gene overexpression will normalize collagen synthesis, increase elastin/hyaluronan, and p

Background

Age-related decline of HAS2 shifts hyaluronan synthesis from protective high‑molecular‑weight HA (HMW‑HA) to pro‑inflammatory low‑molecular‑weight fragments (LMW‑HA) [1,2,3]. LMW‑HA engages CD44, TLR2/4 and RHAMM, triggering sterile inflammation and, in chondrocytes and dermal fibroblasts, activates RhoA/ROCK signaling that drives cytoskeletal collapse, apoptosis or senescence [[1],[2],[3]]. Notably, CD44 can undergo ectodomain shedding, releasing an intracellular domain (CD44‑ICD) that translocates to the nucleus and influences gene expression [4]. Whether this nuclear pool directly represses HAS2 transcription remains unexplored.

Hypothesis

We propose that LMW‑HA–induced CD44 cleavage generates a nuclear CD44‑ICD that recruits histone deacetylases (HDAC1/2) to the HAS2 promoter, causing local chromatin condensation and transcriptional silencing. This creates a feed‑forward loop: reduced HAS2 diminishes HMW‑HA synthesis, elevating LMW‑HA levels, which further amplifies CD44‑ICD production and HAS2 repression.

Predictions

• Prediction 1: LMW‑HA treatment increases nuclear CD44‑ICD levels in chondrocytes and dermal fibroblasts.
• Prediction 2: Nuclear CD44‑ICD physically associates with HDAC1/2 at the HAS2 promoter, reducing histone H3 acetylation and HAS2 mRNA.
• Prediction 3: Pharmacological inhibition of HDACs or expres

Hypothesis\n\nWe hypothesize that combining galactooligosaccharides (GOS) supplementation with regular aerobic exercise produces a greater increase in hippocampal brain‑derived neurotrophic factor (BDNF) and a larger reduction in anxiety scores than either intervention alone, but only in participants whose baseline stool shows high relative abundance of Faecalibacterium prausnitzii.\n\n## Rationale\n\nPrebiotic fibers such as GOS raise colonic butyrate production [2], and butyrate crosses the blood‑brain barrier to inhibit histone deacetylases, thereby promoting BDNF transcription [4]. Aerobic exercise independently elevates circulating BDNF via muscle‑derived signals [6]. When both pathways are active, we expect additive or synergistic HDAC inhibition and neurotrophic signaling. F. prausnitzii is a major butyrate producer; individuals already rich in this taxon may generate more butyrate from a given GOS dose, amplifying the central effect [5]. In contrast, low F. prausnitzii may limit butyrate yield, blunting the synergy.\n\n## Testable Predictions\n\n1. In a randomized controlled trial, the GOS + exercise group will show a significantly larger increase in serum BDNF (proxy for central BDNF) after 8 weeks than the GOS‑only, exercise‑only, and placebo groups only among participants with baseline *F. praus

Hypothesis

Aged Paneth cells transition into a senescent state that directly drives the intestinal Wnt/β‑catenin gradient collapse by secreting Notum as a component of the senescence‑associated secretory phenotype (SASP). This hypothesis integrates three observations: (1) Paneth cell numbers remain stable or increase with age while Wnt3 output falls, (2) Notum inhibition rescues ISC function, and (3) DNA damage–p53 signaling is heightened in aged ISCs and niche cells. We propose that age‑related DNA damage in Paneth cells activates ATM‑p53, which in turn upregulates NF‑κB–dependent transcription of NOTUM, converting these cells into a maladaptive niche that paradoxically sustains its own numbers while suppressing stemness.

Key mechanistic steps

• Persistent oxidative stress or telomere attrition in Paneth cells triggers ATM activation → p53 stabilization.
• p53 cooperates with NF‑κB (activated by the same stress) to enhance NOTUM transcription and secretion.
• Secreted Notum deacetylates Wnt ligands (e.g., Wnt3) produced by Paneth and mesenchymal cells, preventing Frizzled/LRP6 engagement and flattening the Wnt gradient.
• The resulting low Wnt signaling in Lgr5+ ISCs diminishes β‑catenin–mediated transcription of stemness genes, reduces proliferation, and increases apoptosis.
• Compensatory Wnt production from subepithelial myofibroblasts is insufficient because Notum diffuses broadly, overriding local sources.

Testable predictions

1. Marker co‑localisation – In crypts from aged mice (≥20 mo), a subpopulation of Lysozyme+ Paneth cells will co‑express p21^

Hypothesis

Emodin’s primary action is EGFR inhibition, which suppresses the SASP without clearing senescent cells. If telomere length reflects an informational entropy metric rather than a simple division counter, then reducing the noise generated by SASP‑mediated DNA damage should lower the entropy load at telomeres, slowing their functional erosion. We propose that emodin, by dampening EGFR‑driven NF‑κB, MAPK, and NLRP3 signaling, decreases the production of oxidative and inflammatory molecules that contribute to stochastic DNA lesions and chromatin dysregulation at telomeres. This reduction in molecular noise translates to lower informational entropy at telomeric regions, preserving telomere‑associated shelterin complex stability and delaying the onset of a senescence‑associated secretory phenotype despite unchanged telomere repeat length.

Mechanistic Rationale

1. EGFR‑SASP axis: Emodin blocks EGFR phosphorylation, curbing downstream NF‑κB and MAPK pathways that drive IL‑6, IL‑8, and IL‑1β secretion (34).
2. SASP‑induced telomere stress: SASP factors stimulate NADPH oxidase activity and mitochondrial ROS, causing oxidative base damage and single‑strand breaks that are preferentially repaired at telomeres, increasing telomere‑associated γH2AX foci and altering the epigenetic state of subtelomeric chromatin (5 for macrophage polarization link).
3. Informational entropy: Telomeric DNA repeats function as a low‑co

Hypothesis

Escape genes on the X chromosome directly repress ileal Sgk1 expression, thereby limiting bile acid absorption and protecting against enterohepatic‑brain neurotoxicity. Females benefit from biallelic dosage of these repressors, while males, with a single X, exhibit higher Sgk1‑driven ASBT activity, increased bile acid accumulation in the hippocampus, and accelerated cognitive decline.

Mechanistic Rationale

1. X‑escape candidates – Genes such as Kdm6a (UTX) and Ddx3x escape X‑inhibition, encode chromatin modifiers, and are known to influence metabolic pathways (4).
2. Sgk1 regulation – Sgk1 transcription is modulated by histone acetylation at its promoter; KDM6A demethylates H3K27me3, fostering a repressive state, whereas loss of KDM6A activity leads to permissive chromatin and elevated Sgk1 (5).
3. Sex‑differential ileal expression – Single‑cell RNA‑seq of mouse ileum shows higher Kdm6a/Ddx3x transcript levels in XX enterocytes versus XY, correlating with lower Sgk1 mRNA (1).
4. Enterohepatic‑brain axis – Elevated ileal Sgk1 upregulates ASBT, increasing hepatic bile acid synthesis and circulating bile acids that cross the blood‑brain barrier, activate neuronal FXR, and trigger synaptic loss (5,[6](https://www.frontiersin.org/journals/neuroscience/articles/10.3389/fnins.20

Acarbose extends lifespan in male HET3 mice by ~22% while producing modest benefits in females, a disparity that parallels sex‑specific improvements in glucose tolerance and hepatic mTORC2/Akt/FOXO1a signaling【4】. Although acarbose lowers post‑prandial glucose and raises colonic short‑chain fatty acids (SCFAs) in both sexes【1】【2】, only males show enhanced insulin sensitivity, suggesting that the metabolic arm of longevity is gated by gonadal hormones. We hypothesize that the missing link is a testosterone‑dependent increase in intestinal expression of the SCFA receptor GPR41 (FFAR3), which amplifies propionate‑driven activation of hepatic mTORC2. In this model, acarbose‑induced propionate binds GPR41 on enteroendocrine L‑cells, triggering the release of serotonin and activation of vagal afferents that signal to the nucleus of the solitary tract. The vagal efferent output then increases hepatic insulin signaling via a cholinergic anti‑inflammatory cascade, leading to AKT phosphorylation at Ser473 specifically when androgen receptors are present in hepatocytes. Consequently, mTORC2 signaling rises, FOXO1a is phosphorylated and excluded from the nucleus, and gluconeogenic programs are repressed, yielding the observed male‑specific metabolic improvements. Females lack sufficient androgen‑receptor co‑activation, so despite comparable SCFA elevations, GPR41 signaling fails to potentiate mTORC2, explaining their muted response.

This hypothesis makes three falsifiable predictions: (1) Male mice lacking GPR41 in intestinal epithelium (Villin‑Cre;Gpr41^fl/fl) will lose acarbose‑indu

Hypothesis

JNK2 activity drives a temporal shift in AP-1 dimer composition from c-Fos/c-Jun to Fra1/JunB, which stabilizes H2A.J incorporation at SASP promoters and locks in chronic inflammation independent of upstream mitochondrial ROS.

Rationale

• Mitochondrial ROS initiates JNK activation, but sustained JNK signaling in senescence may become ROS‑independent through autocrine SASP loops 1.
• JNK1 and JNK2 have opposing roles in inflammatory disease 2, yet their isoform‑specific contributions to SASP are unknown.
• H2A.J accumulation is required for robust inflammatory gene expression 4 and could be recruited by specific AP-1 dimers.
• CD36 initiates SASP via Src–p38–NF-κB 3, providing an early signal that may feed into JNK2‑dependent chromatin remodeling.

We propose that after the acute phase, JNK2 preferentially phosphorylates JunB and Fra1, promoting their dimerization and binding to AP‑1 sites enriched in H2A.J‑containing nucleosomes. This complex recruits histone acetyltransferases that maintain an open chromatin state, thereby preserving SASP transcription even when mitochondrial ROS declines.

Predictions

1. In senescent cells, JNK2 knock‑down (but not JNK1) will reduce Fra1/JunB dimer levels and decrease H2A.J occupancy at IL6 and IL8 promoters.

Hypothesis: Chronic SASP signaling from senescent cells drives the formation of a HIF‑1α/NF‑κB heterocomplex that directly binds the NAMPT promoter, recruits DNMT3A, and imposes a repressive methylation lock that sustains NAD+ decline. Low NAD+ diminishes SIRT1 deacetylase activity, allowing HIF‑1α to accumulate even under normoxia; HIF‑1α then cooperates with NF‑κB p65 (activated by IL‑1β, TNF‑α, and IL‑6) to occupy cis‑regulatory elements upstream of NAMPT. This cooperative binding brings DNMT3A to the promoter, leading to CpG methylation and stable transcriptional silencing. The resulting NAD+ deficit further fuels mitochondrial ROS and SASP production, creating a self‑reinforcing loop that locks cells into a low‑energy, pro‑inflammatory state.

Key predictions:

1. In aged mouse liver and human adipose tissue, sequential ChIP‑reChIP will detect HIF‑1α and NF‑κB p65 co‑occupancy at the NAMPT promoter and an adjacent enhancer, accompanied by elevated DNMT3A binding and increased 5‑mC levels at specific CpGs.
2. Pharmacological inhibition of HIF‑1α (PX‑478) or selective blockade of IKKβ (thus NF‑κB) in 24‑month‑old mice will raise hepatic NAMPT mRNA by ≥2‑fold, elevate NAD+ concentrations to youthful levels, and reduce SASP cytokines (IL‑6, MCP‑1) without changing CD38 expression or activity.
3. CRISPR‑Cas9 deletion of a 12‑bp motif containing the predicted HIF‑1α/NF‑κB cooperative site in the NAMPT promoter of human iPSC‑derived fibroblasts will abolish age‑associated NAD+ decline after 60 days of culture, whereas a scrambled control edit will show the typical decline.

Hypothesis

Aged T cells release mitochondrial DNA (mtDNA) into the cytosol, activating the cGAS-STING pathway. This triggers a senescence-associated secretory phenotype (SASP) in the T cells themselves and in neighboring stromal cells, fueling inflammaging and tissue senescence. Inhibiting mtDNA release or cGAS-STING signaling in T cells will break this loop, reducing systemic senescence and improving tissue function independent of senolytics.

Mechanistic Rationale

• Aged T cells accumulate mitochondrial damage and exhibit defective mitophagy, leading to cytosolic mtDNA [2].
• Cytosolic mtDNA binds cGAS, producing cyclic GMP-AMP that activates STING, driving IRF3‑NF‑κB signaling and SASP cytokine production [4].
• SASP factors (IL-6, TNFα, CCL2) induce paracrine senescence in epithelial, endothelial, and fibroblast cells, expanding the senescent burden [3].
• STING activation also primes the NLRP3 inflammasome, amplifying IL-1β release and reinforcing inflammaging [1].
• Thymic involution limits naïve T cell renewal, increasing reliance on these dysfunctional memory T cells [5], thus sustaining the loop.

Experimental Design

Model: 20‑month‑old C57BL/6 mice. Interventions:

• Group A: T‑cell‑specific knockout of cGAS (using Cd4‑Cre cGAS^fl/fl).
• Group B: T‑cell‑specific overexpression of mitochondrial transcription factor A (TFAM) to enhance mtDNA packaging and limit release.
• Group C: Wild‑type littermates receiving vehicle.

Hypothesis

Transient OSKM expression creates a rejuvenation window (day 3‑13) where epigenetic age resets before somatic identity is lost (day 13‑28). We propose that the X‑chromosome enhancer landscape becomes a stochastic gate during the later sub‑phase (day 13‑20) that determines whether a cell can fully erase aging memory or retains it, thereby explaining the observed selection bias.

Mechanistic Basis

• p53 suppresses epigenetic noise by stabilizing chromatin; its activity declines as cells proliferate, peaking around day 13‑20 when p53‑dependent checkpoints are transiently relaxed.
• During this window, X‑linked enhancers—already prone to incomplete reinstallation—experience increased accessibility and stochastic methylation fluctuations.
• Cells that happen to lose inhibitory marks at age‑associated X‑enhancers proceed to a low‑noise, rejuvenated state; those that retain marks sustain epigenetic age memory and are eliminated via senescence/apoptosis.
• Lin28 overexpression, which accelerates division, amplifies this effect by further shortening the p53‑high phase, thereby increasing the probability of noise‑driven enhancer erasure.

Testable Predictions

1. Inhibiting p53 (e.g., with pifithrin‑α) specifically between day 13‑20 will increase X‑linked enhancer hypomethylation and raise the proportion of iPSCs that show complete erasure of fibroblast‑associated enhancer methylation.
2. Conversely, sustaining p53 activity (e.g., via MDM2‑siRNA knock‑down) during the same interval will diminish X‑linked enhancer noise, lower iPSC yield, and preserve donor a

Hypothesis

We propose that the transition from acute JNK‑AP‑1 protection to chronic inflammaging is governed by the kinetic accumulation of minority mitochondrial outer membrane permeabilization (miMOMP) events, which generate oscillatory bursts of mitochondrial ROS. These bursts modify redox‑sensitive cysteines on JNK‑activating scaffolds (e.g., MKK4/7) and on AP‑1 subunits themselves, shifting the dimer composition from c‑Jun homodimers that repress proliferation to c‑Jun/c‑Fos heterodimers that drive SASP transcription. In tissues where ASK1 preferentially signals through p38 MAPK, the phosphatase MKP‑1 is induced by p38 activity and accelerates JNK dephosphorylation, thereby raising the miMOMP threshold required for chronic JNK‑AP‑1 activation. Consequently, skin fibroblasts exhibit a lower miMOMP threshold and rapid SASP onset, whereas hepatocytes require sustained miMOMP stress before JNK‑AP‑1 dominates over ASK1‑p38 signaling.

Predictions

1. Single‑cell live imaging of mitochondrial ROS (using MitoSOX) combined with a FRET‑based JNK activity reporter will show that discrete ROS spikes precede sustained JNK phosphorylation only after a critical number of miMOMP events (≈3‑5 per cell) have occurred.
2. Mutating the redox‑sensitive Cys‑252 of c‑Jun to serine will lock AP‑1 in the homodimeric state, preventing SASP induction even when miMOMP is experimentally increased (e.g., by low‑dose BAX activator).
3. Pharmacological inhibition of MKP‑1 in hepatocytes will sensitize ASK1‑p38‑dominant cells to miMOMP‑driven JNK‑AP‑1 activation, converting their SASP profile to

Intermittent, low‑dose IGF-1 administration aligned with the nadir of endogenous GH secretion can improve mitochondrial genome stability in older adults without provoking chronic insulin resistance. We're proposing that brief IGF-1 spikes activate the IGF‑1R/PI3K/Akt pathway transiently, which in turn phosphorylates and activates AMPK through LKB1 when cellular ATP dips during the trough of GH secretion. Activated AMPK phosphorylates POLG and TFAM, boosting mitochondrial replication and repair of lesions 5. This repair shift reduces heteroplasmy load and lowers senescence markers p16 and p21, mirroring the acute protective effect observed in vitro 2. Because the IGF-1 signal is short‑lived, downstream mTORC1 activity doesn't sustain the proliferative shift that drives insulin resistance; instead, the concurrent rise in AMPK suppresses S6K1, preserving insulin signaling 3. Sex differences may emerge because estrogen amplifies AMPK activation, predicting a larger benefit in post‑menopausal women receiving estradiol replacement alongside the IGF-1 pulse regimen.

To test this hypothesis we propose a randomized, double‑blind, crossover trial in adults aged 65‑80 with baseline mtDNA heteroplasmy >5 % (measured by duplex sequencing). Participants receive either subcutaneous recombinant IGF-1 (20 µg) or placebo at 02:00 h, when endogenous GH is at its lowest, three times per week for 12 weeks, separated by a 12‑week washout. Primary outcomes are

Hypothesis

HER2‑low breast and gastric cancers display a metabolic shift that increases reliance on glutamine‑derived α‑ketoglutarate to sustain TCA cycle activity when HER2 signaling's dampened. This glutamine dependence creates a synthetic lethal interaction with glutaminase inhibition, which can be anticipated by measuring plasma levels of glutamate, glutamine and downstream TCA intermediates.

Mechanistic Rationale

Multi‑omics analyses show that ERBB2/HER2 expression inversely correlates with glycolysis and alanine‑aspartate‑glutamate pathways in tumor tissue and bloodERBB2/HER2 negatively correlates with immune infiltration and AAG/GG metabolic pathways, with metabolomic blood validation. When HER2 signaling's low, cells compensate by upregulating amino acid transporters (e.g., SLC1A5) and GLS to funnel glutamine into the TCA cycle via α‑ketoglutarate, thereby maintaining ATP production and redox balanceMulti-omics integration captures functional and phenotypic cancer traits beyond genetic alterations. This compensatory flux is not captured by genomics alone but emerges in proteogenomic and metabolomic layers, especially in the phosphorylation status of GLS and the acetylation of mitochondrial enzymes. AI‑driven fusion models that prioritize genomics for therapy prediction may miss this metabolic vulnerability because they underweight metabolomic features[Multi-omics integration captures functional and phenotypic cancer traits beyond gene

Hypothesis

Age‑related loss of excitatory cholinergic neurons in the myenteric plexus results from a preferential decline in mitochondrial complex I activity within this subpopulation, rendering them vulnerable to oxidative stress and calcium dysregulation. Preserved nitrergic neurons maintain complex I function but develop structural abnormalities due to chronic nitric‑oxide‑mediated protein modifications.

Mechanistic Rationale

Cholinergic myenteric neurons exhibit higher basal firing rates and rely heavily on oxidative phosphorylation to sustain acetylcholine synthesis and vesicular release【1】. This metabolic profile makes them uniquely sensitive to defects in complex I, the primary entry point for electrons into the electron transport chain. Age‑associated accumulation of mitochondrial DNA mutations or oxidative damage to complex I subunits would therefore impair ATP production disproportionately in cholinergic cells, triggering energetic failure, axonal degeneration, and ultimately cell death.

Nitrergic neurons, by contrast, utilize a greater proportion of glycolytic ATP and possess upregulated antioxidant enzymes (e.g., SOD2, glutathione peroxidase) that buffer reactive oxygen species generated during nitric oxide synthesis【2】. This metabolic bias spares them from outright loss but exposes them to persistent S‑nitrosylation of cytoskeletal proteins (e.g., tubulin, neurofilaments), leading to axonal swelling and structural compromise without triggering apoptosis.

The distal colon shows the steepest cholinergic decline because its myenteric plexus receives the l