Regular exercise ameliorates high-fat diet-induced depressive-like behaviors by activating hippocampal neuronal autophagy and enhancing synaptic plasticity

The study investigates how regular exercise counteracts depressive-like behaviors induced by long-term high-fat diet (HFD) consumption. HFD is linked to metabolic disturbances, neuroinflammation, reduced hippocampal volume, and impairments in neuronal and glial function, increasing vulnerability to cognitive and emotional disorders. HFD also suppresses neuronal autophagy in regions including the hippocampus, contributing to synaptic dysfunction and depressive phenotypes. Exercise is known to prevent depression and can activate autophagy, often via mTOR signaling modulation, thereby supporting neuroplasticity. The authors hypothesized that treadmill exercise alleviates HFD-induced depressive-like behavior by enhancing hippocampal synaptic plasticity through activation of neuronal autophagy mediated by the Wnt5a/CaMKII/mTOR pathway, with Wnt5a as a key upstream mediator.

Prior work shows chronic HFD elevates risk for metabolic disease and neurobehavioral impairments, including anxiety, anhedonia, and depression, potentially via neuroinflammation and impaired autophagy (e.g., hippocampus, hypothalamus, prefrontal cortex). Autophagy defects contribute to protein aggregate accumulation and neuronal degeneration; conversely, enhancing autophagy exhibits antidepressant-like effects in mice, and several antidepressants augment autophagy. Autophagy is integral to neuronal circuit remodeling, including axon growth, dendritic spine dynamics, synapse assembly, and vesicle turnover. Exercise confers systemic and neurological benefits, prevents depressive symptoms, and can induce autophagy by inhibiting mTOR signaling; previous work from the authors indicates treadmill training modulates mTOR to boost cortical synaptic plasticity and stress resilience. Wnt5a, a noncanonical Wnt ligand, regulates axon/dendrite growth and synaptogenesis, supports hippocampal dendritic stability, and signals via Ca2+/CaMKII pathways. These findings motivated testing whether exercise restores Wnt5a/CaMKII signaling to activate neuronal autophagy, enhance synaptic plasticity, and ameliorate HFD-induced depressive-like behaviors.

Animals: Six-week-old male C57BL/6J mice (19±1 g) housed under 12-h light/dark, 22±1 °C, food and water ad libitum. Ethics: Jinan University IACUC (IACUC-20221206-02). Design: Two experiments. - Experiment 1: n=36, randomized to Control, HFD, RUN (n=12/group). Control received standard chow; HFD and RUN received HFD (60% kcal from fat) for 8 weeks. RUN group performed treadmill running 1 h/day at 10 m/min (4–5 pm) during HFD. - Experiment 2: n=48, randomized to Control, HFD, RUN, RUN+Box5 (n=12/group). RUN+Box5 received exercise as above plus Wnt5a antagonist Box5 (1 mg/kg; P1216, Selleck). Behavioral assessments: Tail Suspension Test (TST), Forced Swimming Test (FST), Sucrose Preference Test (SPT), Open Field Test (OFT; center entries/duration, total distance, velocity). Body/metabolic measures: body weight trajectory; body size, liver index, epididymal fat weight, serum lipids. Histology and ultrastructure: Nissl staining for neuronal arrangement and counts; Golgi staining for dendritic arborization (Sholl analysis) and dendritic spine density in hippocampal CA1; transmission electron microscopy of CA1 neurons to quantify autophagosomes/autolysosomes and assess subcellular morphology. Molecular analyses: RNA-seq of hippocampal tissue with PCA, differential expression (Control vs HFD, HFD vs RUN), and GO; RT-qPCR validation of DEGs (including Wnt5a, Septin2, Scn3b, Zfp740) and synaptic markers/BDNF; immunofluorescence in CA1 for Wnt5a colocalization with NeuN; Western blots for Wnt5a, p-CaMKIIα/CaMKIIα, p-mTOR/mTOR, Beclin1, LC3B (I/II), p62, glutamate receptor subunits (NR2A, NR2B, GluR1, GluR2), synaptic proteins (PSD-95, synaptophysin), and co-immunoprecipitation for CaMKIIα–PSD-95 interaction. Electrophysiology: Whole-cell recordings of excitatory postsynaptic currents (EPSCs) in acute hippocampal CA1 slices (amplitude and frequency). Field potential recordings for LTP in the Schaffer collateral (CA3→CA1) pathway with high-frequency stimulation; analyze fEPSP slope and amplitude time courses and final 10-min averages. Pharmacology: Box5 to inhibit Wnt5a signaling in vivo during exercise. Statistics: SPSS 26.0, data as mean±SD. One-way ANOVA with Tukey (or Tamhane where appropriate); repeated-measures ANOVA for within-group; two-way ANOVA with Bonferroni for two-factor designs. Significance at P<0.05. Graphing with GraphPad Prism 9.0.

- Behavior and physiology: After 8 weeks HFD, mice showed increased immobility (TST, FST) and reduced sucrose preference; exercise (RUN) significantly reduced immobility and restored sucrose preference. OFT measures (center entries, center time, total distance, velocity) were decreased by HFD and restored by exercise. HFD increased body size, liver index, epididymal fat, and serum lipids; exercise mitigated these. Exercise reduced hippocampal neuroinflammation (e.g., lowered IL-6, TNF-α; increased IL-4, IL-10) and protected neuronal arrangement/number in hippocampus (notably CA1). - Transcriptomics: RNA-seq revealed clear group separation (PCA). Differential expression: Control vs HFD, 310 DEGs (92 up, 218 down in HFD); HFD vs RUN, 138 DEGs (69 up, 69 down in HFD vs RUN). Among top DEGs, Wnt5a was downregulated by HFD and restored by exercise. Wnt5a expression correlated with behavior: OFT (r=-0.9740, P<0.0001), TST (r=-0.9518, P<0.0001), FST (r=-0.9683, P<0.0001), SPT (r=0.9367, P=0.0002). - Wnt5a/CaMKII signaling and synaptic transmission: Immunofluorescence showed reduced neuronal Wnt5a in HFD CA1, restored by exercise. Western blots: HFD reduced Wnt5a and p-CaMKIIα; exercise reversed these. Whole-cell CA1 recordings showed exercise increased EPSC amplitude and frequency vs HFD, indicating enhanced synaptic transmission. - Autophagy and structural plasticity: EM revealed HFD caused chromatin irregularities, organelle swelling, and reduced autophagosomes; exercise increased autophagosomes/autolysosomes and normalized ultrastructure. Western blots: exercise decreased p-mTOR/mTOR and p62, increased Beclin1 and LC3B-II/LC3B-I, consistent with enhanced autophagy. Golgi staining: HFD reduced dendritic complexity and spine density in CA1; exercise restored branching (Sholl) and increased spine density. Synaptic markers: exercise increased glutamate receptor subunits (NR2A, NR2B, GluR1, GluR2) and synaptic proteins (PSD-95, SYN) reduced by HFD. Electrophysiology and structure together indicate exercise-enhanced synaptic plasticity. - Necessity of Wnt5a: Wnt5a antagonist Box5 abolished exercise benefits. Box5 suppressed exercise-induced increases in Wnt5a and p-CaMKIIα, reduced EPSC amplitude/frequency, decreased autophagosome numbers, reversed autophagy marker changes (raised p-mTOR/mTOR and p62; lowered Beclin1 and LC3B-II), decreased dendritic complexity/spine density, reduced CaMKIIα–PSD-95 binding (co-IP), and prevented exercise-mediated restoration of LTP (reduced fEPSP slope/amplitude during final 10 min). Overall, Wnt5a activation is necessary for exercise-induced neuronal autophagy and synaptic remodeling that ameliorate HFD-induced depressive-like behaviors.

The findings demonstrate that chronic HFD impairs hippocampal neuronal structure and function, elevates neuroinflammation, suppresses Wnt5a/Ca2+/CaMKII signaling, reduces neuronal autophagy, and weakens synaptic transmission and plasticity, culminating in depressive-like behaviors. Regular treadmill exercise counteracts these effects by restoring Wnt5a expression and CaMKII activation in CA1 neurons, suppressing mTOR signaling, and enhancing autophagy. This autophagy activation supports synaptic remodeling—greater dendritic complexity, higher spine density, increased synaptic receptor/protein levels—leading to stronger EPSCs and LTP and improved behavioral outcomes. Blocking Wnt5a with Box5 negates exercise-induced autophagy and synaptic benefits and prevents behavioral rescue, establishing Wnt5a as a necessary mediator linking exercise to neuronal autophagy and synaptic plasticity under HFD stress. These results integrate diet, Wnt5a/CaMKII/mTOR signaling, autophagy, and exercise into a coherent mechanism with relevance for preventing and treating diet-associated mood disorders.

This study identifies Wnt5a as an essential mediator through which regular exercise activates hippocampal CA1 neuronal autophagy, enhances synaptic plasticity, and alleviates HFD-induced depressive-like behaviors. Exercise reverses HFD-driven transcriptional, molecular, structural, and electrophysiological deficits via the Wnt5a/CaMKII/mTOR pathway. The work provides mechanistic insights into nonpharmacological intervention for diet-related neuropsychiatric conditions. Future research should delineate upstream regulators of Wnt5a during exercise, test cell type-specific contributions (e.g., astrocytes and microglia), employ genetic/autophagy-specific manipulations to establish necessity and sufficiency, and assess translational generalizability across sexes, ages, and exercise paradigms.

- Preclinical mouse model with only male C57BL/6J mice limits generalizability across sexes, strains, species, and ages. - Wnt5a inhibition used a systemic pharmacological antagonist (Box5), which lacks cell type specificity; direct genetic manipulations of Wnt5a or autophagy in defined neuronal populations were not performed. - Although data support Wnt5a/CaMKII/mTOR involvement, upstream exercise-induced regulators of Wnt5a were not identified, and the precise causal chain to mTOR suppression was not dissected. - The study focused primarily on neurons in CA1; potential roles of astrocytes and microglia in mediating exercise effects were not mechanistically tested. - Autophagy’s necessity was inferred from pathway modulation; direct autophagy blockade (e.g., genetic or flux-specific assays) was not employed to establish causal requirement.