Physical exercise mediates a cortical FMRP-mTOR pathway to improve resilience against chronic stress in adolescent mice

The study investigates how aerobic exercise confers resilience against anxiety induced by chronic restraint stress (CRS) in adolescent mice. Prior work shows exercise alleviates anxiety-like behaviors and cognitive deficits, affecting neurogenesis, inflammatory cytokines, and cerebrovascular function. The precise molecular signaling underlying exercise-mediated anxiolysis remains unclear. The authors hypothesize that exercise activates the mTOR pathway in the medial prefrontal cortex (mPFC), thereby maintaining neuronal activity and promoting axonal myelination, which together prevent anxiety-like behaviors. They further propose that upstream regulation involves Fragile X mental retardation protein (FMRP) and RNA methylation, linking systemic metabolic effects of exercise to cortical signaling.

- Exercise reduces anxiety symptoms in humans and anxiety-like behaviors in animal models; proposed mechanisms include enhanced hippocampal neurogenesis, anti-inflammatory effects, and vascular changes. - Exercise modulates neurons, astrocytes, microglia, and oligodendrocytes; BDNF signaling is often implicated in exercise-induced neural plasticity. - The authors previously showed treadmill exercise activates mTOR in mouse motor cortex (naïve and cocaine-exposed), enhancing spinogenesis and neuronal activity. - Chronic stress can downregulate mTOR signaling; mTOR levels correlate with stress susceptibility in chronic mild stress models. - mPFC is critical for anxiety regulation, and axonal myelination correlates with cortical neuronal activity. - Literature supports that neuronal activity promotes oligodendrogenesis and adaptive myelination, and stress can impair myelination, particularly during early-life critical periods. These data motivate examining whether exercise engages mTOR in mPFC to modulate neuronal activity and myelination to relieve anxiety.

Animals: Male C57BL/6J mice, 4–6 weeks old, group-housed with ad libitum food/water, standard light-dark cycle. All procedures approved by Jinan University IACUC. Stress and exercise paradigms: - Chronic restraint stress (CRS): 3 h/day in restraint tube (7 p.m.–10 p.m.) for 14 consecutive days. - Treadmill exercise: 1 h/day (9 a.m.–10 a.m.) for 14 days at 10 m/min. Pharmacology: - BrdU: 50 mg/kg i.p. daily during the last 5 days for cell proliferation labeling. - Rapamycin: 150 mg/kg administered every 3 days during the 2-week exercise training to inhibit mTOR. - K255a: 10 µg/kg injected daily (compound included in methods; not central to reported results). Behavioral assays (conducted in light phase with 4-h intervals, ordered from least to most stressful): - Open field test (40×40×30 cm, 25 W lighting): 5-min exploration, EthoVision v7.0 analysis; metrics include total distance and central time. - Elevated plus maze (open/closed arms 30×5 cm; closed walls 15 cm): 5-min exploration, EthoVision analysis; metrics include total distance and open-arm duration. - Marble burying: 15 marbles (1.4 cm) on 3-cm bedding in standard cage; 10-min trial, marbles considered buried when ≥2/3 covered; three trials per mouse, averaged. Molecular assays: - RNA extraction (Triton Reagent), quantification (NanoDrop), cDNA synthesis (TaKaRa), qPCR with TB Green; primers for Fmr1, Tsc2, Raptor, Gapdh; 2^-ΔΔCt analysis. - Protein extraction with phosphatase/protease inhibitors, BCA quantification, SDS-PAGE, PVDF transfer, blocking (5% BSA), primary overnight at 4°C, secondary 2 h, imaging (Bio-Rad), densitometry (ImageJ). Targets: MBP, Akt/mTOR/S6 (total and phosphorylated), FMRP, Tubulin. Histology and imaging: - Perfusion fixation (PFA), 30% sucrose dehydration, 40-µm coronal sections, immunofluorescence with primary antibodies (per Table S1), secondary fluorophores, confocal imaging (Zeiss), quantification with ImageJ; 3 consecutive slices averaged. - Transmission electron microscopy (TEM): standard fixation (including osmium tetroxide), graded dehydration, embedding (EMbed 812), 80-nm sections, uranyl acetate and lead citrate staining, imaging (Hitachi) at 13,500× for axons; g-ratio calculated (axon diameter/myelinated fiber diameter) with ImageJ. Stereotaxic viral injections (mPFC prelimbic region AP +2.33 mm; ML ±0.1 mm; DV −1.2 mm): - AAV vectors (titer ~10^12 GC/ml), 100 nl per site, slow infusion, pipette held 8 min before retraction. - Genetic manipulations: shRNA knockdown of Raptor (mTOR complex component), Tsc2 (upstream inhibitor), Fmr1 (FMRP), and Alkbh5 (RNA demethylase). Neuron-targeting promoters (e.g., CaMKIIα) used where appropriate. - Chemogenetics: hM4Di (inhibitory) and hM3Dq (excitatory) DREADDs expressed bilaterally in mPFC; CNO administered to modulate neuronal activity during/after exercise paradigms. In vivo calcium imaging: - Surgical headplate installation and skull window (2 mm) over mPFC; epidural preserved; glass coverslip applied. - Two-photon imaging (Zeiss LSM780) at 920 nm, <25 mW, 2 Hz acquisition. ROIs over pyramidal neuron somata; ΔF/F0 computed using first 10% as baseline; events defined as >3× SD; motion correction with TurboReg. Statistics: - Normality tested; parametric tests (Student’s t-test, one-way ANOVA with Tukey post hoc) for normal data; nonparametric Kruskal–Wallis with Dunn’s post hoc for non-normal (e.g., calcium imaging). Significance P<0.05. Sample sizes guided by literature; N values in figure legends (e.g., behavior commonly N=7/group; imaging n=80 neurons from 4 mice/group). Blinded analysis for behavior and calcium imaging; experiments duplicated/triplicated.

- Exercise prevents CRS-induced anxiety-like behaviors without affecting locomotion: - Open field central time decreased by CRS and restored by exercise (ANOVA F(2,18)=21.95, P<0.0001); total distance unaffected (F(2,18)=0.079, P=0.9243). - Elevated plus maze open-arm time decreased by CRS and rescued by exercise (F(2,18)=44.95, P<0.0001); total distance unaffected (F(2,18)=0.1719, P=0.8434). - Marble burying increased by CRS and reduced by exercise (F(2,18)=19.63, P<0.0001). - Exercise preserves oligodendrogenesis and myelination in mPFC under CRS: - PDGFRα+ OPCs and CC1+ oligodendrocytes reduced by CRS and maintained with exercise; BrdU labeling indicates protected oligodendrogenesis. - MBP levels decreased by CRS and maintained by exercise (ANOVA F(2,12)=9.436, P=0.0034); TEM shows g-ratio changes consistent with preserved myelination (F(2,12)=9.691, P=0.0031). - Exercise activates the mTOR pathway in mPFC: - CRS downregulates p-Akt, p-mTOR, p-S6; exercise upregulates them alongside MBP increases (WB; N=4/group). - Rapamycin during exercise inhibits mTOR signaling, reduces MBP, decreases g-ratio, and abolishes anxiolysis (e.g., open field central time F(3,24)=9.695, P=0.0002; EPM open-arm time F(3,24)=11.34, P<0.0001; marble burying F(3,24)=5.181, P=0.0067); locomotion unaffected. - mPFC-specific Raptor knockdown (AAV-shRNA) suppresses p-Akt/p-mTOR/p-S6 and MBP, and abolishes exercise-induced anxiolysis (e.g., open field central time F(3,24)=18.47, P<0.0001; EPM open-arm time F(3,24)=16.48, P<0.0001). - Disinhibition of mTOR via Tsc2 knockdown restores mTOR activity and MBP in CRS mice and mimics exercise-induced anxiolysis (no effect in naïve mice). - mTOR controls neuronal activity and activity-dependent myelination: - Two-photon calcium imaging: CRS suppresses mPFC neuronal activity; exercise restores it; Raptor knockdown blocks this restoration (Kruskal–Wallis statistics: integrated calcium 234.4, P<0.0001; peak values 198.1, P<0.0001; frequency reduced by Raptor KD; n=80 neurons from 4 mice/group). - Chemogenetic inhibition (hM4Di+CNO) of mPFC neurons during exercise reduces cFos, lowers MBP, decreases Olig2+/PDGFRα+ cell densities, and abolishes anxiolysis (multiple ANOVAs P<0.0001); locomotion unaffected. Excitatory DREADD (hM3Dq) mimics exercise effects (supplementary data). - FMRP is an upstream inhibitor of mTOR modulated by exercise: - Proteomics identified Fmr1 among 200 DEGs regulated by CRS and exercise; FMRP protein and Fmr1 mRNA increased by CRS and decreased by exercise (ANOVAs: FMRP F(2,9)=5.337, P=0.0271; Fmr1 mRNA F(2,9)=9.904, P=0.0069). FMRP predominantly in NeuN+ neurons. - AAV-shRNA knockdown of Fmr1 in mPFC reactivates mTOR (↑p-mTOR, ↑p-S6), increases MBP, improves g-ratio, and prevents anxiety-like behaviors in CRS mice (e.g., open field central time F(3,24)=13.44, P<0.0001; EPM open-arm time F(3,24)=12.08, P<0.0001; marble burying F(3,24)=43.59, P<0.0001); no locomotor changes. - Exercise-driven RNA methylation suppresses FMRP to activate mTOR: - Prior m6A-seq data indicate Fmr1 transcript m6A is affected by exercise. Overexpressing Alkbh5 (RNA demethylase) in mPFC of exercised mice elevates FMRP and suppresses mTOR signaling, blocking anxiolysis (e.g., open field central time F(3,24)=13.09, P<0.0001; EPM open-arm time F(3,24)=20.14, P<0.0001; marble burying F(3,24)=160.7, P<0.0001). - Conversely, Alkbh5 knockdown in CRS mice suppresses FMRP, activates mTOR, and confers anxiolysis (supplementary). - SAM supplementation (oral or i.p.) in CRS mice suppresses FMRP, activates mTOR, and improves anxiety-like behaviors (open field central time F(2,18)=17.07, P<0.0001; EPM open-arm time F(2,18)=14.14, P=0.0002); locomotion unaffected.

Findings demonstrate that two weeks of aerobic treadmill exercise in adolescent mice protects against CRS-induced anxiety-like behaviors by preserving neuronal activity and activity-dependent myelination in the mPFC. Exercise activates the cortical mTOR pathway, which is necessary and sufficient for restoring myelination and anxiolysis under stress. Neuron-autonomous mTOR activation elevates neuronal calcium activity, which in turn promotes oligodendrogenesis and myelin integrity, linking circuit function to white matter plasticity. Upstream, exercise reduces neuronal FMRP levels via enhanced RNA m6A methylation, relieving FMRP’s negative regulation of mTOR. Genetic or pharmacological manipulations along this axis (Raptor/Tsc2, Fmr1, Alkbh5, SAM) recapitulate or block exercise effects, establishing a causal FMRP–mTOR pathway in mPFC that governs stress resilience. These results integrate metabolic/epigenetic consequences of exercise with cortical signaling and structural plasticity, expanding mechanistic understanding of exercise-mediated anxiolysis during adolescence.

This work identifies an exercise-driven FMRP–mTOR signaling pathway in mPFC neurons that maintains neuronal activity and axonal myelination, thereby conferring resilience to chronic stress-induced anxiety in adolescent mice. Exercise enhances brain RNA methylation to suppress FMRP, disinhibiting mTOR signaling, which supports activity-dependent myelination and anxiolytic behavior. The study establishes necessity and sufficiency of mTOR pathway modulation and neuronal activity for these effects and shows that targeting RNA methylation (e.g., via SAM) or genetic regulators (Fmr1, Tsc2) can mimic exercise benefits. Future research should delineate cell-type specific contributions (e.g., oligodendrocytes, interneurons), map broader upstream exercise-regulated networks affecting mTOR (e.g., BDNF/TrkB, AMPK), resolve developmental stage dependencies, and evaluate translational potential in humans.

- Rapamycin lacked tissue and cell specificity, potentially affecting peripheral tissues; genetic approaches localized effects but predominantly targeted neurons. - AAV vectors mainly influenced neurons rather than oligodendrocytes, limiting direct conclusions about oligodendroglial mTOR contributions. - Experiments were conducted in male adolescent mice; sex differences and other ages were not explored, affecting generalizability. - Behavioral and imaging endpoints were limited to mPFC; network-wide effects and other brain regions were not assessed. - While RNA methylation and FMRP were implicated, comprehensive mapping of all exercise-regulated upstream modulators of mTOR was beyond the scope. - Translation to human populations remains to be established.