Intermittent fasting reprograms the brain proteome to prevent synaptic degeneration and cognitive impairment in vascular dementia

Abstract

Rationale: Vascular dementia (VaD), driven by chronic cerebral hypoperfusion (CCH), leads to synaptic degeneration and cognitive decline, yet mechanisms linking vascular dysfunction to synaptic loss remain unclear. Intermittent fasting (IF) has emerged as a potential intervention, but its effects on synaptic integrity in VaD are unknown. This study aims to investigate the effects of IF against synaptic degeneration and cognitive impairment induced by CCH. Methods: Bilateral common carotid artery stenosis (BCAS) was employed to induce chronic CCH by placing 0.18 mm micro-coils around each common carotid artery in mice. To assess temporal differences, the coils remained in place for 1, 7, 14, or 30 days. IF was implemented for 16 hours daily over three months prior to BCAS induction. Cognitive impairment was evaluated using the Barnes maze test. White matter lesions (WMLs) and neuronal loss were assessed using Luxol fast blue and cresyl violet staining, respectively. Immunoblotting and immunohistochemistry were performed to quantify synaptic protein levels. Synaptic integrity was examined using transmission electron microscopy. Proteomic analysis of the hippocampus was conducted to investigate molecular adaptations to IF following CCH. Results: We demonstrate that a 16-hour IF regimen preserves cognitive function and synaptic density despite persistent hypoperfusion. Behavioral assays revealed that IF prevented spatial memory deficits in BCAS mice, while electron microscopy confirmed synaptic preservation without altering baseline architecture. Surprisingly, key synaptic protein levels remained unchanged, suggesting IF protects synaptic function rather than abundance. Proteomic profiling revealed dynamic hippocampal adaptations under IF, including upregulation of synaptic stabilizers, enhanced GABAergic signaling, and suppression of neuroinflammatory mediators. CCH induced microglial engulfment of synapses, suggesting a role in complement-mediated synaptic pruning. Temporal pathway analysis revealed IF's multi-phase neuroprotection: early synaptic reinforcement, mid-phase metabolic optimization, and late-phase suppression of chronic neuroinflammation. Conclusion: These findings establish IF as a potent modulator of synaptic resilience in VaD, acting through coordinated preservation of synaptic structure, inhibition of inflammatory synapse loss, and metabolic reprogramming. Our results highlight IF's potential as a non-pharmacological strategy to combat vascular cognitive impairment by targeting the synaptic vulnerability underlying dementia progression.

Keywords: cognitive impairment; intermittent fasting; neuronal death; synaptic loss; vascular dementia.

Figure 1

Intermittent fasting (IF) promotes metabolic switching and attenuates neuropathological alterations induced by chronic cerebral hypoperfusion (CCH). (A) Schematic outline of experimental groups, timeline, and design. Three-month-old male C57BL/6 mice were assigned to ad libitum (AL) or intermittent fasting (IF) groups; the IF group underwent daily 16-hour fasting for three months. At six months, both groups underwent either bilateral common carotid artery stenosis (BCAS) or sham surgery. Assessments included Barnes maze testing, neuropathology, synaptic protein quantification, synaptic structure and density analysis, and quantitative proteomics analysis of hippocampus. Analyses were performed at days 1, 7, 14, and 30 post-BCAS. Body weight and blood glucose and ketone levels were measured at experimental endpoint (B) Violin plots illustrate the physiological effect of IF compared to AL feeding. At the experimental endpoint, IF mice exhibited significantly lower body weight, reduced blood glucose levels, and elevated blood ketone levels relative to AL mice. n = 30-36 mice in each experimental group. ****P < 0.0001. (C and D) and (E and F) show representative luxol fast blue-stained sections and corresponding quantitative analyses of white matter lesions in the median (C and D) and paramedian (E and F) regions of the corpus callosum at the 7 and 30-day timepoints of the AL- CCH, IF-CCH and the respective sham mice. Scale bar: 50 μm. Lesions were characterized by myelin rarefaction and structural disruption. White matter disruption was graded: Grade 0 = no disruption; Grade 1 = disarranged nerve fibers; Grade 2 = marked vacuole formation; Grade 3 = loss of myelinated fibers. A clear reduction in WML severity was observed in IF mice compared to AL mice following CCH. (G and H) and (I and J) Representative crystal violet-stained images and quantification illustrating the loss of Nissl-positive neurons in the CA1 and CA3 regions of the hippocampus at days 7 and 30 post-CCH in AL mice. Scale bar: 100 μm. No significant differences were observed between IF-CCH and IF-sham groups. Notably, at day 7 post-CCH, IF-CCH mice exhibited a significantly higher number of neurons in CA1 and CA3 regions compared to AL-CCH mice (H and J). Data are represented as mean ± S.E.M. n = 5-6 mice in each experimental group. *P < 0.05; **P < 0.01; ***P < 0.001.

Figure 2

Intermittent fasting (IF) mitigates chronic cerebral hypoperfusion (CCH) induced cognitive deficits. (A and B) Barnes maze testing was conducted on AL and IF mice subjected to CCH. Learning ability was evaluated by measuring the latency to enter the target hole during the acquisition phase; AL-CCH mice exhibited significantly longer latencies compared to AL-sham controls on days 3 and 4 (A). No significant differences were observed between IF-CCH and IF-sham groups (A). Spatial memory retrieval was assessed on Day 15 using a reprobe test. AL-CCH mice demonstrated increased latencies (B) and reduced time spent in the target quadrant relative to AL-sham mice (B), indicative of memory impairment. In contrast, IF-CCH mice showed significantly reduced latencies (B) and increased time spent in the target quadrant compared to AL-CCH mice (B), reflecting improved spatial memory retention. Data represented as mean ± S.E.M. n = 13-18 mice per experimental group. (C-D) IF preserves Early Growth Response Protein 1 (EGR-1) expression in chronic cerebral hypoperfusion. Immunohistochemical analysis shows reduced EGR-1 expression in AL BCAS mice compared to IF BCAS mice in hippocampal CA3 and cortical regions following reprobe test. Scale bar: 20 μm. (D) Quantitative analysis reveals significantly higher EGR-1-positive cell counts in IF BCAS mice versus AL BCAS controls in the CA3 region. Data represent mean ± S.E.M. n = 5-6 mice in each experimental group. *p < 0.05; **P < 0.01.

Figure 3

Assessment of the modulatory effects of intermittent fasting on chronic cerebral hypoperfusion (CCH)-induced synaptic loss and ultrastructural alteration. (A and B) Representative transmission electron microscopy (TEM) images (A) and corresponding quantification (B) demonstrated significant decrease in synaptic density in AL-CCH mice in prefrontal cortex region at both day 7 and day 30 post-CCH, with no significant differences observed between IF-CCH and sham groups. (C and D) Ultrastructural analysis of the g-ratio indicated thinner cortical myelin sheaths (reflected by a higher g-ratio) in both AL-CCH and IF-CCH mice at day 30 compared to control (D). (E and F) Representative TEM images and quantification showed a marked increase in postsynaptic density (PSD) length (did not reach significant level) in both AL-CCH and IF-CCH groups only at day 7 post CCH compared to sham controls. (G and H) A significant increase in the number of mushroom-shaped spine in AL-CCH mice at day 30 post-CCH, with no difference observed between IF-CCH and sham. TEM images were taken at 10000 magnification, scale bar: 600 nm. (I and J) Representative IMARIS images and quantification illustrate a significant upregulation in microglial engulfment of the postsynaptic protein PSD95 in AL-CCH mice at day 7 post-CCH. Scale bar: 5 µm. Data are represented as mean ± S.E.M. n = 3-4 mice in each experimental group. *P < 0.05, **P < 0.01.

Figure 4

Temporal proteomic profiling of hippocampal tissue reveals synaptic alterations and pathway changes following chromic cerebral hypoperfusion (CCH). Heatmaps showing differentially expressed proteins in response to CCH (BCAS) at day 1 (A), day 7 (C), day 14 (E), and day 30 (G), with colours representing z-score normalized protein abundances (red: higher abundance; green: lower abundance). Boxplots illustrating changes in key proteins involved in synaptic transmission at, day 1 (B), day 7 (D), day 14 (F), and day 30 (H) in response to CCH compared the control sham. Pathway enrichment and molecular network analyses were conducted using Ingenuity Pathway Analysis (IPA). (I-L) Bar plots are showing significantly enriched canonical pathways in response to CCH at different time points. The Y-axis represents -log(p-value), and each bar corresponds to a pathway, with colour indicating predicted activation state based on the z-score, orange indicates predicted activation (positive z-score). Longer bars indicate greater statistical significance, with pathways ranked by significance. n = 5 mice in each experimental group. *P < 0.05, **P < 0.01.

Figure 5

Intermittent fasting (IF) changes the proteomic landscape of hippocampus. We first examined the effect of IF on the hippocampus under normal physiological conditions. (A and B) Bar plots showing the top enriched Gene Ontology (GO) terms across three categories—Biological Process (BP), Cellular Component (CC), and Molecular Function (MF)—modulated by IF. Bars represent normalized enrichment scores. Two analyses were performed: (A) using the entire proteomics dataset from AL-sham and IF-sham groups, and (B) using significantly differentially expressed proteins (DEPs). (C) Heatmap displaying DEPs in response to IF compared to AL. (D) Graphical summary of key canonical pathways and biological functions altered by IF, identified using Ingenuity Pathway Analysis (IPA). Predicted pathway activation states are indicated by z-scores (orange: activation; blue: inhibition). n = 20 mice in each experimental group.

Figure 6

Proteomic analysis uncovers the molecular mechanisms underlying intermittent fasting (IF) mediated protection against Chronic cerebral hypoperfusion (CCH) induced hippocampal pathology. Heatmaps of DEPs and boxplots showing expression of key synaptic transmission-related proteins in response to IF at days 1 (A and B), 7 (C and D), 14 (E and F), and 30 (G and H) post-CCH. The impact of IF under CCH conditions by analysing proteomic profiles from IF-CCH and AL-CCH groups. n =5 mice in each experimental group. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 7

Proteomic pathways underlying intermittent fasting (IF) mediated protection against Chronic cerebral hypoperfusion (CCH). Pathway enrichment and molecular network analyses were conducted using Ingenuity Pathway Analysis (IPA). (A-D) Canonical pathway enrichment analysis at indicated time points post-BCAS. Bar plots show significantly enriched pathways with color coding reflecting activation states (orange, activated; blue, inhibited). The Y-axis represents -log(p-value). Data compare IF-CCH versus AL-CCH proteomic profiles. n = 5 mice in each experimental group.