Intermittent fasting enhances long-term memory consolidation, adult hippocampal neurogenesis, and expression of longevity gene Klotho

The study addresses how intermittent fasting (IF) differs from daily calorie restriction (CR) in their effects on cognition and adult hippocampal neurogenesis (AHN). While both CR and IF extend lifespan/healthspan and improve learning and memory, prior literature often conflated the two regimens, making mechanisms unclear. Notably, IF can induce beneficial metabolic and neuroprotective effects without reducing total calorie intake. Given better adherence to IF versus long-term CR in humans, the authors directly compared IF (every-other-day feeding) to a matched 10% daily CR to test the hypothesis that IF more effectively enhances long-term memory and AHN, and to identify molecular mediators, focusing on the longevity gene Klotho (Kl), which is expressed in hippocampus and associated with cognition.

Background work shows CR (10–40% daily reduction) and various IF paradigms improve lifespan/healthspan and cognition across species. IF and CR induce adaptive cellular stress responses that promote neuronal resilience. Prior reports sometimes attributed benefits to CR when IF protocols were employed. IF has been shown to maintain body weight and yet elicit metabolic benefits comparable to 40% CR, including reduced insulin/glucose, and enhanced neuronal survival after excitotoxic challenge in hippocampal regions. Klotho, a longevity-associated gene primarily produced in kidney but also expressed in hippocampal dentate gyrus neurons, has been linked to cognitive performance in humans and rodents; emerging evidence suggests a role in regulating AHN.

Animals: Seventy-five 8-week-old female C57BL6 mice assigned to three groups: ad libitum (AL, n=25), calorie restriction (CR, 10% less than AL daily; n=25), and intermittent fasting (IF, every-other-day feeding yielding ~10% weekly reduction; n=25). Duration: 3 months. Subsets: gene expression (n=3/group), behavior (Morris water maze; AL n=5, CR n=10, IF n=10), histology with BrdU labeling (n=12/group; half culled at 24 h, half at 4 weeks post-injection). All procedures IACUC-approved. Behavior: Morris water maze (MWM) for spatial learning and memory. Acquisition: two trials/day across 8 days from different quadrants. Probe trials at 24 h and 10 days post-training to assess retention. BrdU labeling: For diet groups, daily i.p. BrdU 50 µg/g for 6 days; perfusion at 24 h or 4 weeks post last injection. For Kl mutant analysis, separate 8-week-old male and female kl/kl and wild-type mice received 3 BrdU injections (100 mg/kg) ~16 h apart; perfusion 4 weeks later. Histology: Perfusion fixation, sectioning at 40 µm, dentate gyrus (DG) sections collected. Immunohistochemistry for BrdU and DCX (neuroblast marker); dendritic morphometrics of DCX+ cells and septo-temporal distribution analyses. Unbiased stereology (optical fractionator) to quantify BrdU+ and DCX+ cells. Dorsal versus ventral hippocampus delineated per atlas coordinates (DH: −1.06 to −2.06 mm; VH: −3.08 to −3.80 mm from bregma). Gene expression and KL analysis: Genome-wide microarray on hippocampus to identify genes upregulated by IF versus CR and AL; pathway analysis via DAVID and Ingenuity Pathway Analysis. RT-qPCR validation of targets. KL protein immunoreactivity quantified by threshold-based image analysis on KL-immunostained sections (constant acquisition parameters) using Image Pro Plus. In vitro human hippocampal progenitor model: HPCOA07/03 cell line used to test roles of Kl in proliferation/differentiation/survival. Overexpression: Generated conditional KL-secreted overexpressing line (Klover) using Lenti-X Tet-On inducible system; induction with doxycycline (1 µg/ml). Assays under proliferative conditions (3 days) or during differentiation (induction concurrent with differentiation). Knockdown: Transfected cells with stealth siRNA against Kl (5 nM) using N-TER nanoparticle system; efficiency >80% using fluorescent control oligo; maintained under differentiating conditions for 7 days. Immunocytochemistry for neurogenesis markers; imaging via high-content platform and fluorescence microscopy; analysis in ImageJ. Statistics: Behavioral data analyzed by Two-Way ANOVA with Tukey’s post hoc; histology and RT-qPCR by One-Way ANOVA with Tukey’s post hoc; in vitro Klover assays by unpaired t-test; siRNA assays by One-Way ANOVA with Tukey’s multiple comparisons; kl/kl versus wild-type by unpaired t-test. Significance threshold p≤0.05.

- Caloric intake: IF mice consumed ~10% less weekly than AL, matching the 10% CR protocol. - Spatial learning: No group differences during MWM acquisition across 8 days. - Memory retention: 24-h probe trial showed no differences (F(6,88)=0.57, p=0.7528). At 10 days post-training, IF mice exhibited superior retention, spending 30% (vs AL) and 25% (vs CR) more time in the target quadrant (zone B); overall effect F(6,88)=2.424, p=0.0325. - Cell proliferation (BrdU 24 h): Significant group effect with higher BrdU+ cell counts in IF vs AL and CR (F(2,14)=21.02, p=0.0001). Mean ± SEM: AL 11,105 ± 494.4; CR 16,987 ± 762.4; IF 21,504 ± 1,747. - Neuroblasts: IF increased DCX+ neuroblasts in DG relative to AL and CR (qualitative summary; detailed counts not shown here). Dendritic analyses and septo-temporal distributions assessed (details in Supplementary). - Gene expression: Microarray identified Klotho (Kl) upregulated in hippocampus by IF but not by 10% CR; confirmed by RT-qPCR. KL immunoreactivity increased in IF hippocampus by threshold analysis. - In vitro human progenitors: Kl knockdown decreased neurogenesis; Kl overexpression (secreted KL) increased proliferation/differentiation markers, indicating a pro-neurogenic role of Kl. - Kl knockout mice: Histology revealed Kl is required for adult hippocampal neurogenesis, with pronounced effects in the dorsal hippocampus. Overall, IF was superior to matched 10% CR for enhancing long-term memory retention and AHN, with Kl identified as a candidate mediator.

The data directly demonstrate that IF (every-other-day feeding) enhances long-term memory consolidation beyond a matched 10% CR regimen, despite similar weekly caloric reduction, indicating regimen-specific effects rather than calorie amount alone. The concomitant increases in BrdU+ cells and DCX+ neuroblasts link the behavioral benefits to enhanced AHN. Transcriptomic and validation analyses identified Kl upregulation uniquely under IF, and functional assays showed Kl promotes neurogenesis in human hippocampal progenitors while its absence impairs AHN in mice, particularly in the dorsal hippocampus—an area implicated in spatial memory. Together, these findings suggest Kl as a mechanistic node through which IF augments AHN and cognition. Given the comparatively better adherence to IF-like eating patterns in humans, these results support exploring increased meal intervals as a translational strategy for cognitive enhancement and neuroprotection independent of stringent daily caloric restriction.

This study distinguishes IF from matched daily CR by showing that IF more robustly improves long-term spatial memory retention and enhances adult hippocampal neurogenesis. It identifies Klotho as a key molecular mediator upregulated by IF; Kl promotes neurogenesis in human progenitors and is required for AHN in vivo. These findings highlight IF as a promising dietary paradigm to boost cognitive function via AHN and position Kl as a target for interventions. Future research should delineate the signaling pathways linking IF to Kl expression, assess dose–response and timing parameters of IF, examine sex and age generalizability, and evaluate translational relevance in human trials, including biomarker (KL) monitoring.

- Species and sex: Primary in vivo experiments were conducted in female C57BL6 mice; generalizability to males and other strains/species requires confirmation. Kl knockout analyses included both sexes but with modest sample sizes. - Calorie restriction level: CR was limited to 10%; effects may differ at other CR levels (e.g., 30–40%). - Sample sizes: Some cohorts were small (e.g., MWM AL group n=5; gene expression n=3/group), which may limit power for certain comparisons. - Duration: Intervention lasted 3 months; longer-term effects and durability of cognitive benefits were not assessed. - Mechanistic depth: While Kl is implicated, upstream regulators and downstream pathways mediating IF-induced Kl expression and AHN enhancement were not fully characterized.