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

Abstract

Introduction

The PERIOD (PER)3 gene, a core component of the mammalian circadian clock, has been increasingly implicated in various physiological and behavioral processes. Notably, a deficiency in PER3 expression or function has been consistently associated with the manifestation of depression-like behaviors in experimental models. However, despite this observed correlation, the precise underlying molecular and cellular mechanisms that link PER3 deficiency to the development of depressive phenotypes have remained largely obscure and underexplored. This knowledge gap highlights a critical area for further investigation to better understand the pathophysiology of depression.

Objectives

Building upon the established association between PER3 deficiency and depression-like behaviors, the overarching objective of the current study was to comprehensively elucidate the specific role of PER3 in regulating these behaviors in mice. Furthermore, a key aim was to unravel the intricate molecular mechanisms through which PER3 exerts its influence on neural function and mood regulation. This mechanistic exploration is crucial for identifying potential therapeutic targets for depression.

Methods

To rigorously assess depression-like behaviors in the experimental mouse models, a battery of established and validated behavioral tests was employed. These included the sucrose preference test, which measures anhedonia (a core symptom of depression characterized by a loss of pleasure); the tail suspension test, which assesses behavioral despair and learned helplessness; and the forced swimming test, another widely used paradigm for evaluating behavioral despair. To investigate potential metabolic alterations underlying PER3 deficiency, metabolomic analysis was meticulously conducted on hippocampal tissue samples obtained from Per3 knockout mice. This analysis utilized advanced chromatography-mass spectrometry techniques, allowing for a comprehensive profiling of metabolic intermediates. Furthermore, to probe the direct molecular interactions involving PER3, its regulatory role on the expression of nicotinamide phosphoribosyltransferase (Nampt), a key enzyme in NAD+ biosynthesis, was thoroughly investigated. This was achieved through the application of sophisticated biochemical assays, including co-immunoprecipitation, to detect protein-protein interactions, and chromatin immunoprecipitation assays, to identify direct binding of PER3 to specific genomic regions.

Results

Metabolomic analysis of hippocampal tissues from Per3 knockout mice yielded compelling and consistent evidence that Per3 deficiency profoundly disrupts mitochondrial function. This disruption was characterized by several key metabolic perturbations: significantly reduced activities of critical enzymes within the tricarboxylic acid (TCA) cycle, including succinate dehydrogenase, citrate synthase, and α-ketoglutarate dehydrogenase, indicating impaired energy production. Furthermore, there was a discernible diminution in the expression levels of mitochondrial respiratory chain complexes I through V, suggesting impaired oxidative phosphorylation capacity. Critically, these mitochondrial dysfunctions were accompanied by significantly decreased levels of nicotinamide adenine dinucleotide (NAD)+, a vital coenzyme central to energy metabolism and redox reactions.

To test the functional significance of reduced NAD+ levels, supplementation with nicotinamide, a direct precursor to NAD+, was administered to the Per3 knockout mice. This intervention successfully rescued mitochondrial function, restoring key metabolic parameters, and remarkably, alleviated the observed depression-like behaviors in these mice. Similar beneficial effects, including improvements in both mitochondrial function and depressive phenotypes, were observed following the intraperitoneal administration of P7C3-A20, a known activator of NAMPT. Conversely, the co-administration of FK866, a specific inhibitor of NAMPT, effectively abolished these beneficial effects, unequivocally demonstrating that the observed improvements were dependent on NAMPT activity and, by extension, NAD+ production.

Delving into the mechanistic underpinnings, our molecular investigations revealed a novel regulatory mechanism: PER3 was found to directly regulate Nampt expression. This regulation occurred through PER3′s ability to bind to specific E-box elements located within the intronic regions of the Nampt gene. This binding interaction was found to occur in conjunction with BMAL1, another core circadian clock component, suggesting a collaborative role in transcriptional regulation. This PER3-BMAL1 interaction at the Nampt gene enhanced NAD+ production, which, in turn, activated SIRT3, a mitochondrial sirtuin known to play a crucial role in regulating mitochondrial function. The activation of SIRT3 then mitigated the observed mitochondrial dysfunction in Per3 knockout mice, thereby completing the mechanistic loop.

Conclusions

These comprehensive findings collectively uncover a novel and critically important mechanism by which PER3 ameliorates depressive behaviors. This mechanism involves the direct regulation of NAMPT-controlled NAD+ levels, which subsequently impacts and improves mitochondrial function. This intricate signaling cascade, involving PER3, NAMPT, NAD+, and SIRT3, highlights a fundamental role for PER3 in the pathophysiology of depression and underscores the critical importance of maintaining optimal energy metabolism in neuronal health and mood regulation. These insights open new avenues for therapeutic intervention targeting the PER3-NAMPT-NAD+-SIRT3 axis for the treatment of depression.

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

Declaration of Competing Interest

The authors explicitly declare that they have no known competing financial interests or any personal relationships that could be perceived to have influenced the work reported in this paper. This statement underscores the objectivity and integrity of the research presented.