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

Calorie restriction (CR) and intermittent fasting (IF) are dietary interventions shown to extend lifespan and improve health across various species. While both methods improve learning and memory, the specific mechanisms and comparative benefits remain unclear. Previous research has often conflated CR and IF, leading to a lack of understanding of their distinct effects. A key study by Anson et al. (2003) demonstrated that IF, even without a reduction in overall calorie intake, yielded neuroprotective effects comparable to 40% CR, suggesting that mechanisms beyond simple calorie reduction are at play. This study aimed to directly compare the effects of IF and a matched 10% daily CR regimen on learning and memory in mice, focusing on adult hippocampal neurogenesis (AHN) and the role of the longevity gene *Klotho*. The choice of 10% CR was informed by the observation that IF mice consumed approximately 10% fewer calories weekly. *Klotho* was chosen due to its known expression in the hippocampus and association with cognitive function.

Numerous studies have reported the beneficial effects of CR and IF on various aspects of health, including inflammation, neurodegeneration, and brain plasticity. However, many studies using CR protocols to study these benefits actually employed intermittent fasting regimens. The study by Anson et al. (2003) was pivotal in distinguishing between the two interventions, demonstrating neuroprotection in IF mice without overall calorie reduction. While both CR and IF have shown cognitive benefits, the specific mechanisms and differences between these interventions remain under-investigated. This study aims to fill this gap by comparing IF and matched CR regimens, and exploring the potential role of Klotho in mediating these effects.

Seventy-five 8-week-old female C57BL6 mice were divided into three groups: ad libitum (AL), 10% CR, and IF (every-other-day feeding). The mice were maintained on these diets for three months. A subset of mice underwent gene expression analysis to study molecular mechanisms involved in AHN. The Morris water maze (MWM) was used to assess spatial learning and memory. Bromodeoxyuridine (BrdU) was administered to label proliferating cells, and subsequent histological analysis was performed to quantify adult hippocampal neurogenesis. Immunostaining was conducted to assess various markers related to neurogenesis. Microarray analysis was employed to identify differentially expressed genes between IF and CR groups, with particular focus on *Klotho*. In vitro experiments using human hippocampal neural progenitor cells (HPCOA07/03) were conducted to investigate the role of *Klotho* in neurogenesis, including experiments involving Klotho overexpression and knockdown. Stereological analysis was used to quantify the number of BrdU and doublecortin (DCX)-positive cells in the dorsal and ventral hippocampus. Statistical analysis was performed using appropriate methods, including Two-Way ANOVA and one-way ANOVA with post-hoc tests.

Intermittent fasting (IF) resulted in a 10% reduction in weekly caloric intake compared to ad libitum feeding. In the Morris water maze, while no differences were observed in the acquisition phase of spatial learning, IF mice showed significantly improved long-term memory retention (10 days post-training) compared to both ad libitum and 10% calorie-restricted mice. IF also led to a significant increase in the number of BrdU-labeled cells (proliferating cells) and neuroblasts in the hippocampus 24 hours post-BrdU injection, as well as a greater number of surviving neuroblasts 4 weeks post injection. Microarray analysis revealed that the longevity gene *Klotho* (*Kl*) was significantly upregulated in the hippocampus of IF mice only. In vitro studies using human hippocampal progenitor cells showed that *Kl* downregulation decreased neurogenesis, while *Kl* overexpression increased it. Histological analysis of *Kl* knockout mice confirmed that *Kl* is essential for adult hippocampal neurogenesis, particularly in the dorsal hippocampus.

This study demonstrates that IF, even without significant overall calorie reduction, is superior to a matched 10% daily CR regimen in enhancing long-term memory retention in mice. This superior enhancement is associated with increased adult hippocampal neurogenesis (AHN) and upregulation of the longevity gene Klotho. The in vitro and in vivo findings strongly support a critical role for Klotho in mediating the beneficial effects of IF on cognition. The results suggest that Klotho might be a novel therapeutic target for enhancing memory function and addressing age-related cognitive decline. The improved adherence to IF regimens compared to long-term CR makes IF a potentially more practical approach for promoting cognitive health in human populations.

This study provides strong evidence that intermittent fasting is a more effective dietary intervention than matched daily calorie restriction for improving long-term memory and promoting adult hippocampal neurogenesis in mice. The identification of Klotho as a key regulator of these effects opens new avenues for research into the mechanisms of cognitive enhancement and suggests potential therapeutic targets for age-related cognitive decline. Future research could explore the specific signaling pathways downstream of Klotho in the hippocampus and investigate the translational potential of IF and Klotho modulation for human cognitive health.

The study was conducted using only female mice, limiting the generalizability of findings to males. The duration of the dietary interventions (3 months) might not be sufficient to observe all long-term effects. The in vitro studies, though valuable, are not fully representative of the complex in vivo environment. Further research is needed to fully elucidate the precise mechanism by which Klotho mediates the effects of IF on neurogenesis and memory.