Fig 2: Timeless ablation compromises hippocampal synaptic plasticity(A) Schematic of electrophysiological field recordings of Schaffer-collateral synapses in acute hippocampal slices.(B) Time course of LTP induced by theta-burst stimulation; representative traces (top). The number 1 denotes baseline (light color) and 2 denotes last 10 min (dark color).(C) Quantification of the last 10 min of field excitatory post-synaptic potentials (fEPSPs) revealed a significant reduction in LTP magnitude in CKO animals compared with CTRL (CTRL n = 9 slices/4 animals; CKO n = 12 slices/5 animals; *p = 0.033, Mann-Whitney test).(D) Input-output analysis of Schaffer-collateral fEPSPs revealed enhanced synaptic strength in the CKO condition compared with the CTRL group (CTRL n = 11 slices/4 animals; CKO n = 11 slices/5 animals; *p = 0.027, Mann-Whitney test).(E) Paired-pulse ratio analysis across varying interstimulus intervals showed no differences in presynaptic release (CTRL n = 9 slices/4 animals; CKO n = 10 slices/5 animals; N.S., unpaired t test). Individual data points represent the number of slices from the number of animals indicated (n). Experiments were performed at ZT12. Data are represented as the mean ± SEM. N.S. denotes non-significant, *p < 0.05.

Fig 3: Both our RNA-seq data and the bioinformation datasets analyses showed that TIM is associated with immunogenicity, especially with CD8+ T cells. a GSEA analysis of differential gene profiles between TIM siRNA transfected breast cancer MCF-7 cells and controls. There are immune-related pathways, lipid metabolism-related pathways, and other pathways. b Top 10 immune-related pathways in GSEA analysis with our RNA-seq data. c The detailed figure of top GSEA enrichments related to GO immune response to the tumor cell in MCF-7 siTIM group. d Comparison of lymphocyte infiltration signature score between TIM high and low expression groups with TCGA BRCA dataset. e GSEA analysis of differential gene profiles between the TIM high and low expression groups in the TCGA BRCA dataset. The GSEA enrichment circle plot showed the category of pathways, detailed pathway names, regulation tendency, and enrichment score. Three major types of pathways were enriched: DNA repair-, metabolism-, and immune-related pathways. f Immunophenogram for the visualization of terms determining antigen processing (MHC), immune stimulation (immunostimulators), and immune suppression (immunoinhibitors). g Volcano plot for the immune subsets in BRCA of TCGA dataset based on the MCPcounter immune score. h Boxplots of different T cell- or CD8+ T cell-associated functions based on the ssGESA immune algorithm of the TCGA dataset. i UMAP visualization of the total cells profiled in GSE161529, with each cell color-coded for the associated cell type. jTIM expression in each cell type in the UMAP visualization of GSE161529. k Villon plot of TIM expression in each cluster of cells of GSE161529 within the UMAP algorithm. Significance was tested as described in “Methods” section: *p-value < 0.05; **p-value < 0.01; ***p-value < 0.001; ****p-value < 0.0001. TIM TIMELESS, GSEA Gene Set Enrichment Analysis, BRCA breast cancer, MHC major histocompatibility complex, UMAP Uniform Manifold Approximation and Projection

Fig 4: TIMELESS is a chromatin-bound protein that is enriched in enhancer regions of synaptic plasticity genes and pathways in the hippocampus(A and B) GREAT (Genomic Regions Enrichment of Annotations Tool) analysis of TIMELESS ChIP-seq peaks in WT mouse hippocampal tissue. (A) Top seven pathways enriched for TIMELESS bound genes in the hippocampal region of WT mice using ingenuity pathway analysis (IPA). The significance threshold for pathway enrichment is p < 0.05 −log10(p value), corresponding to >1.3 on the x axis of the plot, marked by a red line. (B) Principal candidate peak genes overlapping with active enhancers in adult mouse hippocampus.51(C) Bigwig intensities of TIMELESS ChIP-seq assays adjacent to the Pde4b gene performed in hippocampal tissues from n = 3 independent samples (different shades of blue) and non-immunoprecipitated fractions (input, in gray). H3K27ac-bound regions are active sites of transcription, shown in red.51

Fig 5: Timeless deletion alters PDE4B expression, cAMP levels, and synaptic transmission in hippocampus(A) TIMELESS upregulates Pde4b transcription and thereby PDE4B protein levels in hippocampus. At ZT12, Pde4b mRNA levels and PDE4B protein levels are reduced in Timeless CKO relative to CTRL (qRT-PCR for mRNA: CTRL n = 9, CKO n = 5; ***p < 0.001, Mann-Whitney test. Western blot for protein: CTRL n = 7, CKO n = 7; **p = 0.014, Mann-Whitney test).(B) TIMELESS negatively regulates cAMP concentration in hippocampus. ELISA showed that cAMP concentration was elevated in hippocampus of CKO compared with CTRL (CTRL n = 5, CKO n = 6; *p = 0.027, Mann-Whitney test).(C) Schematic of PDE4B/cAMP signaling cascade in neurons.(D and E) Pharmacological treatments that target cAMP activation to promote synaptic potentiation demonstrated that TIMELESS can affect synaptic transmission. (D) CKO mice showed a moderate potentiation induced by bath application of 50 μM FSK (an adenylate cyclase activator) compared with the CTRL group, which displayed a dramatic transient decrease in neurotransmission. PDE4B-mediated cAMP degradation to AMP and generation of adenosine via 5′-nucleotidase can lead to suppression of neurotransmitter release,55,56 as seen in CTRL mice. Reduced PDE4B activity in CKO may explain the absence of transient depression in neurotransmission. (E) Quantification of mean fEPSP slope at 30–35 min of recording (CTRL n = 5 slices/4 animals; CKO n = 7 slices/4 animals; **p = 0.005, Mann-Whitney test).(F) Timeless deletion led to a reduced chemical potentiation induced by co-application of 50 μM FSK and 100 nM DPCPX (an A1 receptor antagonist) in nominal magnesium (FSK, DPCPX, 0 Mg2+) in slices from CKO mice relative to CTRL slices.(G) Quantification of mean fEPSP slope at 60–65 min of recording (CTRL n = 6 slices/4 animals; CKO n = 7 slices/4 animals; *p = 0.035, Mann-Whitney test). Individual points represent replicates from the number of animals indicated (n). All samples were collected and experiments performed at ZT12. Data are represented as the mean ± SEM. *p < 0.05, **p < 0.01, and ***p < 0.001. In (C), AC denotes adenylate cyclase, ATP denotes adenosine triphosphate, AMP denotes adenosine monophosphate, cAMP denotes cyclic AMP, PDE4B denotes phosphodiesterase 4B, FSK denotes forskolin, 5′NTE denotes 5′-nucleotidase, and DPCPX stands for the selective A1 adenosine receptor antagonist. In (D) and (F), representative traces of baseline fEPSPs are in light color and after treatment in dark color. The number 1 denotes baseline and 2 denotes treatment.