Period3 modulates the NAD+-SIRT3 axis to alleviate depression-like behaviour by enhancing NAMPT activity in mice

Abstract

Introduction: PERIOD (PER)3 deficiency is associated with depression-like behaviors, but the underlying mechanisms remain unclear.

Objectives: This study aims to elucidate the role and mechanism of PER3 in regulating depression-like behaviors in mice.

Methods: Depression-like behaviors were assessed using the sucrose preference test, tail suspension test, and forced swimming test. Metabolomic analysis was conducted on hippocampal tissues from Per3 knockout mice using chromatography-mass spectrometry. The regulatory role of PER3 on the expression of nicotinamide phosphoribosyltransferase (Nampt) was investigated through co-immunoprecipitation and chromatin immunoprecipitation assays.

Results: Metabolomic analysis revealed that Per3 deficiency disrupts mitochondrial function, as evidenced by reduced activities of key tricarboxylic acidcycle enzymes (succinate dehydrogenase, citrate synthase, and α-ketoglutarate dehydrogenase), diminished expression of mitochondrial respiratory chain complexes I-V, and decreased nicotinamide adenine dinucleotide (NAD)⁺ levels in Per3 knockout mice. Supplementation with the NAD⁺ precursor nicotinamide rescued mitochondrial function and alleviated depression-like behaviors in Per3 knockout mice. Similar effects were observed with intraperitoneal administration of the NAMPT activator P7C3-A20, while these effects were abolished by the NAMPT inhibitor FK866. Mechanistically, PER3 was found to regulate Nampt expression by binding to E-box elements within its intronic regions in conjunction with BMAL1. This interaction enhanced NAD⁺ production, activating SIRT3 to mitigate mitochondrial dysfunction in Per3 knockout mice.

Conclusions: These findings uncover a novel mechanism by which PER3 ameliorates depressive behaviors through the regulation of NAMPT-controlled NAD⁺ levels and mitochondrial function, underscoring the critical role of PER3 in depression-related pathophysiology.

Keywords: Depression-like behavior; Energy metabolism; Mitochondrial complex; NAD(+); Per3; SIRT3.

Graphical abstract

Fig. 1

Metabolomics analysis in the hippocampus of Per3^−/− mice. (A) The PLS-DA model of GC–MS data derived from the hippocampus of WT group (blue dots) and Per3^−/− group (red dots). (B) The volcano plot of metabolites in the hippocampus between WT and Per3^−/− group. The red and blue dots expressed the up and down metabolites, and the grey indicate unchanged metabolites. (C, D) The quantity of altered metabolites and heat map of changed metabolites between WT and Per3^−/− group. (E, F) The analysis of significantly changed metabolites, and KEGG pathways between WT and Per3^−/− group. n = 4 per group.

Fig. 2

Effects of Per3 knockout on behaviors and mitochondrial bioenergetics in the hippocampus of mice. (A) NAD⁺ levels, (B-E) Activity of NAMPT, SDH, citrate synthase, α-ketoglutarate dehydrogenase, (F) mRNA levels of Uqcrc1, Uqcrc2, COX4 and ATP5, (G) Western blot analysis of oxidative phosphorylation system complexes (n = 3 per group), (H) ATP levels. Values represent the mean ± SEM. n = 8 in all groups in unspecified ones. *p < 0.05, **p < 0.002, ***p < 0.0002 vs. control groups.

Fig. 3

Effects of NAM supplementation on depression-like behavior and mitochondrial function in the hippocampus of Per3^−/− mice. (A) Experimental procedure diagram on Per3^−/− mice after NAM administration. (B) Sucrose preference test, (C, D) Immobility time in the TST, and TST. (E) NAD⁺ levels, (F) Activity of NAMPT, (G) Protein expression of NAMPT, (H) Activity of SDH, (I) Activity of citrate synthase, (J) Activity of a-ketoglutarate, (K) mRNA levels of Uqcrc1, Uqcrc2, COX4 and ATP5, (L) Western blot analysis of oxidative phosphorylation system complexes in hippocampus of mice (n = 3 per group), (M) ATP levels in the hippocampus of mice. Values represent the mean ± SEM. n = 8 in all groups in unspecified ones. *p < 0.05, **p < 0.002, ***p < 0.0002 vs. control groups.

Fig. 4

Effects of NAMPT inhibitor FK866 on depression-like behavior and mitochondrial function in NAM-supplemented Per3^−/− mice. (A) Sucrose consumption in the sucrose preference test, (B, C) Immobility time in the TST, and in the FST. (D-F) Activities of SDH, citrate synthase, and a-ketoglutarate, (G) mRNA levels of Uqcrc1, Uqcrc2, COX4 and ATP5, (H) Western blot analysis of oxidative phosphorylation system complexes (n = 3 per group), (I) ATP levels in the hippocampus of mice. Values represent the mean ± SEM. n = 8 in all groups in unspecified ones. *p < 0.05, **p < 0.002, ***p < 0.0002 vs. control groups.

Fig. 5

Effects of P7C3-A20 on depression-like behavior and mitochondrial function in Per3^−/− mice. (A) Sucrose consumption in the sucrose preference test, (B, C) Immobility time in the TST and the FST, (D-F) Activities of SDH,citrate synthase, and α-ketoglutarate, (G) mRNA levels of Uqcrc1, Uqcrc2, COX4 and ATP5, (H) Western blot analysis of oxidative phosphorylation system complexes in hippocampus of mice (n = 3 per group), (I) ATP levels in hippocampus of mice. Values represent the mean ± SEM. n = 8 in all groups in unspecified ones. *p < 0.05, **p < 0.002, ***p < 0.0002 vs. control groups.

Fig. 6

The expression of Nampt is directly modulated by PER3 and BMAL1. (A) Luciferase assay in HEK293T cells transfected with reporter plasmids containing Nampt WT E-box1 or reporter plasmid with E-box1 mutated sequence co-transfected with Per3 overexpression plasmid. (B) Luciferase assay in HEK293T cells transfected with reporter plasmids containing Nampt WT E-box2 co-transfected with Per3 overexpression plasmid. (C, D) Effects of E-box1 mutation and Per3 overexpression on Nampt mRNA and protein expression. (E) Co-IP assay was performed using anti-BMAL1, and anti-PER3 antibodies. (F, G) ChIP-re-ChIP analysis using antibodies against PER3, BMAL1, or IgG control at E-box1 site of Nampt gene in hippocampus primary cells isolated from WT and Per3 knockout mice. n = 8 in all groups in unspecified ones. Values represent the mean ± SEM. *p < 0.05, **p < 0.002, ***p < 0.0002 vs. control groups.

Fig. 7

Effects of Sirt3 on mitochondrial function in hippocampus primary cells. (A-C) Activity level of SIRT3, SIRT4 and SIRT5, (D) Activity of ATP synthase, (E) ATP levels in hippocampus primary cells. Values represent the mean ± SEM. n = 8 in all groups. *p < 0.05, **p < 0.002, ***p < 0.0002 vs. control groups.