Per2 Knockout Research Articles

Impact and Interrelationships of Striatal Proteins, EPHB2, OPRM1, and PER2 on Mild Cognitive Impairment.

With the global increase in life expectancy, there has been a rise in the incidence of cognitive impairments attributed to diverse etiologies. Notably, approximately 50% of individuals diagnosed with mild cognitive impairment (MCI) progress to dementia within 3years. However, the precise mechanisms underlying MCI remain elusive. Therefore, this study aimed to elucidate potential mechanisms implicated in MCI utilizing Per2 knockout (KO) mice, which have previously been shown to have cognitive deficits. Behavioral (Y-maze, Barnes maze) and molecular (electrophysiology, RNA sequencing, western blot, and immunofluorescence) experiments were conducted in Per2 KO and wild-type (WT) mice. Per2 KO mice exhibited impaired spatial working memory in the Y-maze and Barnes maze. However, there were no significant group differences in hippocampal long-term potentiation (LTP) between Per2 KO and WT mice, whereas striatal LTP in Per2 KO mice was lower compared to WT mice. In RNA sequencing analysis, 58 genes were downregulated and 64 genes were upregulated in the striatum of Per2 KO mice compared to WT mice. Among the differentially expressed genes, four genes (Chrm2, EphB2, Htr1b, Oprm1) were identified. Optimal expression levels of EPHB2 and OPRM1 were found to significantly enhance cognitive performance in mice. Additionally, Per2 KO mice exhibited reduced EPHB2-NMDAR-LTP and OPRM-mTOR signaling, along with elevated amyloid beta (Aβ) levels, when compared to WT mice. However, these alterations were reversed upon administration of morphine treatment. Striatal OPRM1-mTOR signaling, EPHB2-NMDAR-LTP signaling, and Aβ expression levels may exert a combined effect on MCI under the control of Per2 expression.

Augmentation of intracranial self-stimulation induced by amphetamine-like drugs in Period circadian regulator 2 knockout mice is associated with intracellular Ca2+ levels

Over the past decade, new psychoactive substances (NPS) have emerged in the illegal drug market and have continued to attract attention from the international community. Among these, amphetamine-like NPS, classified as stimulants, constitute a significant proportion. However, the pharmacological characteristics and mechanisms underlying addiction to amphetamine-like NPS remain poorly understood. Given that circadian rhythms are linked to the brain stimulation effects of methamphetamine (METH) and amphetamine, we investigated the effects of METH, 1-(4-methoxyphenyl)-N-methylpropan-2-amine (PMMA), and 1-(benzofuran-5-yl)-N-ethylpropan-2-amine (5-EAPB) on intracranial self-stimulation (ICSS) in wild-type (WT) or Period circadian regulator 2 knockout mice. Amphetamine-like drugs increase intracellular Ca2+ levels to provoke dopamine release, so we examined the impact of Per2 knockdown on intracellular Ca2+ levels in PC12 cells to elucidate a potential mechanism underlying NPS-induced ICSS enhancement. Our ICSS results showed that METH and PMMA significantly increased brain stimulation in Per2 knockout mice compared to WT mice. Similarly, METH and PMMA induced higher Ca2+ fluorescence intensity in Per2 knockdown PC12 cells than in control cells. In contrast, 5-EAPB did not produce significant changes in either ICSS or Ca2+ signaling. These findings suggest that Per2 plays a crucial role in the brain stimulation effects of amphetamine-like drugs through the regulation of intracellular Ca2+.

Sex-specific role for the clock protein PER1 in renal ET-1 regulation in salt-sensitive hypertension

Global knockout (KO) of the clock protein PERIOD1 (PER1) in male, but not female, C57BL/6J mice displayed both blunted blood pressure and urinary endothelin-1 (ET-1) rhythms. More recently, we showed that Dahl salt-sensitive (SS) Per1 KO male rats had exacerbated SS hypertension, alongside increased renal injury, renal ET-1 gene ( Edn1) expression, and ET-1 peptide expression from kidneys collected during the rat’s inactive period (2 PM). Additionally, we see increased long non-coding RNA EDN1-AS, which has been suggested to modulate ET-1 production. However, it is unknown whether PER1 regulates rhythms of ET-1 in SS rats and whether PER1 regulates ET-1 in a sex-dependent way. The study objective was to test the hypothesis that PER1 negatively regulates renal EDN1-AS and Edn1 to modulate ET-1 in a sex- and time-dependent manner. To test this hypothesis, kidneys were collected during rat’s active period (2 AM) from male and female SS Per1 KO and SS control rats on a normal salt (0.4% NaCl) or 3 weeks high salt (4% NaCl) diet ( N=5-6). Renal ET-1 protein levels were measured by ELISA and semi- and quantitative PCR was performed to quantify the levels of EDN1-AS and Edn1. Renal ET-1 levels were significantly higher at 2 AM than our previously reported data at 2 PM in both SS Per1 KO and control male rats ( p<0.0001). Renal ET-1 levels were significantly increased in male SS Per1 KO rats following a high salt diet ( p=0.0362) but not in females ( p=0.9522). This was also reflected in the Edn1 mRNA levels (male, p=0.0005; female, p=0.0814). EDN1-AS levels were also increased in males ( p=0.0123) but not in females. Interestingly, significant sex differences were seen in EDN1-AS levels, where EDN1-AS levels were higher in males than females following both low and high salt diets, suggesting a potential novel sex-dependent mechanism for ET-1 regulation. Together, these data suggest sex-specific action of PER1 in the regulation of ET-1 in a model of salt-sensitive hypertension. Future work aims to investigate blood pressure and renal injury in female SS Per1 KO rats and whether this apparent negative regulation of ET-1 by PER1 contributes to the SS hypertensive and renal injury phenotype seen in male SS Per1 KO rats. This work was supported by the National Institutes of Health grants R35 HL135749 (to A.S.), R01 342 DK109570 (to M.G. and A.S.), and Department of Veteran Affairs grants I01 BX004024 (to A.S.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The differential vulnerabilities of Per2 knockout mice to the addictive properties of methamphetamine and cocaine

With the pervasive occurrence of substance abuse worldwide, unraveling the neuropharmacology of drugs of abuse, such as psychostimulants, is undeniably essential. Mice lacking Period 2 (Per2), a gene associated with the biological time-regulating system or circadian rhythm, have been proposed as a potential animal model for drug abuse vulnerability, demonstrating a greater preference for methamphetamine (METH) reward than wild-type (WT) mice. However, the responses of Per2 knockout (KO) mice to the reinforcing effects of METH or other psychostimulants are yet to be established. In this study, the responses of WT and Per2 KO mice to various psychostimulants via intravenous self-administration were determined, along with their behaviors in METH- or cocaine (COC)-induced conditioned place preference and spontaneous locomotion in the open-field test. Per2 KO mice exhibited greater addiction-like responses to METH and 5-EAPB (1-(1-benzofuran-5-yl)-N-ethylpropan-2-amine), but their responses to COC and dimethocaine were comparable to WT mice, indicating a divergent influence of Per2 deficiency on abuse susceptibility to specific psychostimulants. To potentially define the underlying mechanism for this phenotype, 19 differentially expressed genes were identified, through RNA sequencing, which might respond specifically to repeated METH, but not COC, administration in the mouse striatum and were narrowed down to those previously associated with immediate early genes or synaptic plasticity. The correlation between locomotor activity and mRNA expression levels revealed a moderate correlation between METH-induced behavior and Arc or Junb expression in Per2 KO mice only, suggesting their essential role that may lead to the higher vulnerability of Per2 KO mice to METH, but not COC. These findings indicate a potentially unique effect of Per2 expression level on the involvement of Arc and Junb in determining specific vulnerabilities to drugs, and possibly including abuse potential.

Endogenous circadian reporters reveal functional differences of PERIOD paralogs and the significance of PERIOD:CK1 stable interaction

Adverse consequences from having a faulty circadian clock include compromised sleep quality and poor performance in the short-term, and metabolic diseases and cancer in the long-term. However, our understanding of circadian disorders is limited by the incompleteness of our molecular models and our dearth of defined mutant models. Because it would be prohibitively expensive to develop live animal models to study the full range of complicated clock mechanisms, we developed PER1-luc and PER2-luc endogenous circadian reporters in a validated clock cell model, U-2 OS, where the genome can be easily manipulated, and functional consequences of mutations can be accurately studied. When major clock genes were knocked out in these cells, circadian rhythms were modulated similarly compared with corresponding mutant mice, validating the platform for genetics studies. Using these reporter cells, we uncovered critical differences between two paralogs of PER. Although PER1 and PER2 are considered redundant and either one can serve as a pacemaker alone, they were dramatically different in biochemical parameters such as stability and phosphorylation kinetics. Consistently, circadian phase was dramatically different between PER1 and PER2 knockout reporter cells. Wefurther showed that the stable binding of casein kinase1δ/εto PER is not required for PER phosphorylation itself, but is critical for delayed timing of phosphorylation. Our system can be used as an efficient platform to study circadian disorders associated with pathogenic mutations and their underlying molecular mechanisms.

Impacts of Circadian Gene Period2 Knockout on Intestinal Metabolism and Hepatic Antioxidant and Inflammation State in Mice.

The period circadian regulator 2 (Per2) gene is important for the modulations of rhythmic homeostasis in the gut and liver; disruption will cause metabolic diseases, such as obesity, diabetes, and fatty liver. Herein, we investigated the alterations in intestinal metabolic and hepatic functions in Per2 knockout (Per2−/−, KO) and wild-type (Per2+/+, WT) mice. Growth indices, intestinal metabolomics, hepatic circadian rhythms, lipid metabolism, inflammation-related genes, antioxidant capacity, and transcriptome sequencing were performed after euthanasia. Data indicated that KO decreased the intestinal concentrations of amino acids such as γ-aminobutyric acid, aspartic acid, glycine, L-allothreonine, methionine, proline, serine, and valine while it increased the concentrations of carbohydrates such as cellobiose, D-talose, fucose, lyxose, and xylose compared with WT. Moreover, the imbalance of intestinal metabolism further seemed to induce liver dysfunction. Data indicated that Per2 knockout altered the expression of hepatic circadian rhythm genes, such as Clock, Bmal1, Per1, Per3, Cry1, and Cry2. KO also induced hepatic lipid metabolism, because of the increase of liver index and serum concentrations of low-density lipoprotein, and the upregulated expression of Pparα, Cyp7a1, and Cpt1. In addition, KO improved hepatic antioxidant capacity due to the increase activities of SOD and GSH-Px and the decrease in concentrations of MDA. Lastly, KO increased the relative expression levels of hepatic inflammation-related genes, such as Il-1β, Il-6, Tnf-α, Myd88, and Nf-κB p65, which may potentially lead to hepatic inflammation. Overall, Per2 knockout induces gut metabolic dysregulation and may potentially trigger alterations in hepatic antioxidant and inflammation responses.

Transcriptome analysis of clock disrupted cancer cells reveals differential alternative splicing of cancer hallmarks genes

Emerging evidence points towards a regulatory role of the circadian clock in alternative splicing (AS). Whether alterations in core-clock components may contribute to differential AS events is largely unknown. To address this, we carried out a computational analysis on recently generated time-series RNA-seq datasets from three core-clock knockout (KO) genes (ARNTL, NR1D1, PER2) and WT of a colorectal cancer (CRC) cell line, and time-series RNA-seq datasets for additional CRC and Hodgkin’s lymphoma (HL) cells, murine WT, Arntl KO, and Nr1d1/2 KO, and murine SCN WT tissue. The deletion of individual core-clock genes resulted in the loss of circadian expression in crucial spliceosome components such as SF3A1 (in ARNTLKO), SNW1 (in NR1D1KO), and HNRNPC (in PER2KO), which led to a differential pattern of KO-specific AS events. All HCT116KO cells showed a rhythmicity loss of a crucial spliceosome gene U2AF1, which was also not rhythmic in higher progression stage CRC and HL cancer cells. AS analysis revealed an increase in alternative first exon events specific to PER2 and NR1D1 KO in HCT116 cells, and a KO-specific change in expression and rhythmicity pattern of AS transcripts related to cancer hallmarks genes including FGFR2 in HCT116_ARNTLKO, CD44 in HCT116_NR1D1KO, and MET in HCT116_PER2KO. KO-specific changes in rhythmic properties of known spliced variants of these genes (e.g. FGFR2 IIIb/FGFR2 IIIc) correlated with epithelial-mesenchymal-transition signalling. Altogether, our bioinformatic analysis highlights a role for the circadian clock in the regulation of AS, and reveals a potential impact of clock disruption in aberrant splicing in cancer hallmark genes.

Per2 Expression Regulates the Spatial Working Memory of Mice through DRD1-PKA-CREB Signaling.

Several individuals worldwide show cognitive impairment due to various reasons, including a prolonged lifespan and an altered lifestyle. Various causes, such as broken circadian rhythms and dopamine-related factors, have been proposed to be involved in the development of cognitive impairment. However, the underlying pathways remain elusive. Humans with circadian misalignment often face cognitive impairments, and animals with mutations in circadian rhythm-related genes display impaired cognitive functions. To analyze this in detail, this study aimed to investigate the pathways potentially involved in cognitive impairment using Period2 (Per2) transgenic animals. Spatial working memory performance in Per2 knockout (KO) and wild-type mice was assessed using the Barnes maze and Y-maze. The dopamine-related protein expression levels in the hippocampus were measured by Western blotting and enzyme-linked immunosorbent assay (ELISA). Per2 KO mice exhibited impaired spatial working memory, and the expression levels of dopamine receptor D1 (DRD1), protein kinase A (PKA), and cAMP response element-binding protein (CREB) were higher in Per2 KO mice than in control mice. Additionally, DRD1 expression levels were inversely proportional to those of PER2. Thus, memory tests were again conducted after administration of the DRD1 antagonist SCH-23390. Per2 KO mice recovered from memory impairment, and the levels of PKA and CREB decreased after treatment. The effects of Aβ on memory in Per2 mice were also investigated, and we found the increased Aβ levels did not influence the memory performance of Per2 mice after SCH-23390 treatment. These results indicate that Per2 expression levels might influence spatial working memory performance via DRD1-PKA-CREB-dependent signaling.

Regulation of clock and clock-controlled genes during morphine reward and reinforcement: Involvement of the period 2 circadian clock.

Morphine abuse is a devastating disorder that affects millions of people worldwide, and literature evidence indicates a relationship between opioid abuse and the circadian clock. We explored morphine reward and reinforcement using mouse models with Per2 gene modifications (knockout (KO); overexpression (OE)). Mice were exposed to various behavioral, electroencephalographic, pharmacological, and molecular tests to assess the effects of morphine and identify the underlying mechanisms with a focus on reward and reinforcement and the corresponding involvement of circadian and clock-controlled gene regulation. Per2 deletion enhances morphine-induced analgesia, locomotor sensitization, conditioned place preference (CPP), and self-administration (SA) in mice, whereas its overexpression attenuated these effects. In addition, reduced withdrawal was observed in Per2 KO mice, whereas an augmented withdrawal response was observed in Per2 OE mice. Moreover, naloxone and SCH 23390 blocked morphine CPP in Per2 KO and wild-type (WT) mice. The rewarding (CPP) and reinforcing effects (SA) observed in morphine-conditioned and morphine self-administered Per2 KO and WT mice were accompanied by activated μ-opioid and dopamine D1 receptors and TH in the mesolimbic (VTA/NAcc) system. Furthermore, genetic modifications of Per2 in mice innately altered some clock genes in response to morphine. These findings improve our understanding of the role of Per2 in morphine-induced psychoactive effects. Our data and those obtained in previous studies indicate that targeting Per2 may have applicability in the treatment of substance abuse.

Normal Light-Dark and Short-Light Cycles Regulate Intestinal Inflammation, Circulating Short-chain Fatty Acids and Gut Microbiota in Period2 Gene Knockout Mice.

Regular environmental light–dark (LD) cycle-regulated period circadian clock 2 (Per2) gene expression is essential for circadian oscillation, nutrient metabolism, and intestinal microbiota balance. Herein, we combined environmental LD cycles with Per2 gene knockout to investigate how LD cycles mediate Per2 expression to regulate colonic and cecal inflammatory and barrier functions, microbiome, and short-chain fatty acids (SCFAs) in the circulation. Mice were divided into knockout (KO) and wild type (CON) under normal light–dark cycle (NLD) and short-light (SL) cycle for 2 weeks after 4 weeks of adaptation. The concentrations of SCFAs in the serum and large intestine, the colonic and cecal epithelial circadian rhythm, SCFAs transporter, inflammatory and barrier-related genes, and Illumina 16S rRNA sequencing were measured after euthanasia during 10:00–12:00. KO decreased the feeding frequency at 0:00–2:00 but increased at 12:00–14:00 both under NLD and SL. KO upregulated the expression of Per1 and Rev-erbα in the colon and cecum, while it downregulated Clock and Bmal1. In terms of inflammatory and barrier functions, KO increased the expression of Tnf-α, Tlr2, and Nf-κb p65 in the colon and cecum, while it decreased Claudin and Occludin-1. KO decreased the concentrations of total SCFAs and acetate in the colon and cecum, but it increased butyrate, while it had no impact on SCFAs in the serum. KO increased the SCFAs transporter because of the upregulation of Nhe1, Nhe3, and Mct4. Sequencing data revealed that KO improved bacteria α-diversity and increased Lachnospiraceae and Ruminococcaceae abundance, while it downregulated Erysipelatoclostridium, Prevotellaceae UCG_001, Olsenella, and Christensenellaceae R-7 under NLD in KO mice. Most of the differential bacterial genus were enriched in amino acid and carbohydrate metabolism pathways. Overall, Per2 knockout altered circadian oscillation in the large intestine, KO improved intestinal microbiota diversity, the increase in Clostridiales abundance led to the reduction in SCFAs in the circulation, concentrations of total SCFAs and acetate decreased, while butyrate increased and SCFAs transport was enhanced. These alterations may potentially lead to inflammation of the large intestine. Short-light treatment had minor impact on intestinal microbiome and metabolism.

Kidney-specific KO of the circadian clock protein PER1 alters renal Na+ handling, aldosterone levels, and kidney/adrenal gene expression.

PERIOD 1 (PER1) is a circadian clock transcription factor that is regulated by aldosterone, a hormone that increases blood volume and Na+ retention to increase blood pressure. Male global Per1 knockout (KO) mice develop reduced night/day differences in Na+ excretion in response to a high-salt diet plus desoxycorticosterone pivalate treatment (HS + DOCP), a model of salt-sensitive hypertension. In addition, global Per1 KO mice exhibit higher aldosterone levels on a normal-salt diet. To determine the role of Per1 in the kidney, male kidney-specific Per1 KO (KS-Per1 KO) mice were generated using Ksp-cadherin Cre recombinase to remove exons 2-8 of Per1 in the distal nephron and collecting duct. Male KS-Per1 KO mice have increased Na+ retention but have normal diurnal differences in Na+ excretion in response to HS + DOCP. The increased Na+ retention is associated with altered expression of glucocorticoid and mineralocorticoid receptors, increased serum aldosterone, and increased medullary endothelin-1 compared with control mice. Adrenal gland gene expression analysis revealed that circadian clock and aldosterone synthesis genes have altered expression in KS-Per1 KO mice compared with control mice. These results emphasize the importance of the circadian clock not only in maintaining rhythms of physiological functions but also for adaptability in response to environmental cues, such as HS + DOCP, to maintain overall homeostasis. Given the prevalence of salt-sensitive hypertension in the general population, these findings have important implications for our understanding of how circadian clock proteins regulate homeostasis.NEW & NOTEWORTHY For the first time, we show that knockout of the circadian clock transcription factor PERIOD 1 using kidney-specific cadherin Cre results in increased renal Na+ reabsorption, increased aldosterone levels, and changes in gene expression in both the kidney and adrenal gland. Diurnal changes in renal Na+ excretion were not observed, demonstrating that the clock protein PER1 in the kidney is important for maintaining homeostasis and that this effect may be independent of time of day.

Deletion of the clock gene Period2 (Per2) in glial cells alters mood-related behavior in mice

The circadian clock regulates many biochemical and physiological pathways, and lack of clock genes, such as Period (Per) 2, affects not only circadian activity rhythms, but can also modulate feeding and mood-related behaviors. However, it is not known how cell-type specific expression of Per2 contributes to these behaviors. In this study, we find that Per2 in glial cells is important for balancing mood-related behaviors, without affecting circadian activity parameters. Genetic and adeno-associated virus-mediated deletion of Per2 in glial cells of mice leads to reduced despair and anxiety. This is paralleled by an increase of the GABA transporter 2 (Gat2/Slc6a13) and Dopamine receptor D3 (Drd3) mRNA, and a reduction of glutamate levels in the nucleus accumbens (NAc). Interestingly, neuronal Per2 knock-out also reduces despair, but does not influence anxiety. The change in mood-related behavior is not a result of a defective molecular clock, as glial Bmal1 deletion has no effect on neither despair nor anxiety. Exclusive deletion of Per2 in glia of the NAc reduced despair, but had no influence on anxiety. Our data provide strong evidence for an important role of glial Per2 in regulating mood-related behavior.

Circadian Clock Factor PER1 is involved in Crosstalk between the Kidney and Adrenal Glands

Introduction Circadian clock proteins are transcription factors that play critical roles in the regulation of blood pressure, renal function, and hormone levels. Previously, we published that male mice with global knockout (KO) of the clock protein PER1 develop non-dipping hypertension in response to a high salt diet plus the mineralocorticoid DOCP (HSDOCP). HSDOCP treatment is a clinically relevant model of salt-sensitive hypertension. Global PER1 KO mice also exhibit inappropriately increased levels of aldosterone (Aldo) and decreased night:day ratio of urinary sodium (Na) excretion in response to HSDOCP. The objective of the present study was to examine the role of the kidney in PER1-mediated regulation of renal Na handling and Aldo levels. Hypothesis Kidney-specific KO of PER1 (KsPER1 KO) results in altered Aldo levels and decreased night:day rhythms of Na excretion in response to HSDOCP. Methods Ksp-cadherin Cre was used to generate KsPer1 KO mice. KO occurs in the distal nephron and collecting duct. KsPER1 KO (Cre +) mice were compared to control (CNTL) (Cre-) mice (all male; 2.5 months old). Baseline urine collections and plasma were collected on a normal salt diet (NS) and compared to similar samples collected after 3 days HS diet, followed by 3 days of HSDOCP treatment. Total RNA was isolated from adrenal glands (noon) for qPCR analysis. Results In contrast to global PER1 KO mice, the KsPER1 KO mice exhibited normal night:day urine Na excretion. However, KsPER1 KO mice exhibited increased Na retention vs CNTL (3.7 vs 2.7mEq, P<0.01). In response to HSDOCP, plasma Na significantly increased in KsPER1 KO (158 vs 164mM, P<0.05), but not in CNTL mice. HSDOCP treatment suppressed plasma Aldo in all mice, but KsPER1 KO mice displayed increased plasma Aldo vs. CNTL mice on both NS and HSDOCP treatment (NS: 21 vs. 12; HSDOCP: 4 vs. 2 pg/mL/gBW, P<0.01). Adrenal gland expression of the Aldo synthase gene, CYP11B2, was higher in KsPER1 KO vs CNTL (38%, P<0.05). Conclusions These results suggest that PER1 exerts kidney-specific action on renal Na handling independent of night:day effects. PER1 appears to be required for proper crosstalk between the kidney and adrenal gland to manage Aldo levels in response to HSDOCP. Given the prevalence of salt-sensitive hypertension in the general population, these findings have important implications for our understanding of how circadian clock proteins regulate homeostasis.

\xa0Knockout of the circadian gene, Per2, disrupts corticosterone secretion and results in depressive\u2010like behaviors and deficits in startle responses

BackgroundThe Period Circadian Regulator 2 (Per2) gene is important for the modulation of circadian rhythms that influence biological processes. Circadian control of the hypothalamus-pituitary-adrenal (HPA) axis is critical for regulation of hormones involved in the stress response. Dysregulation of the HPA axis is associated with neuropsychiatric disorders. Therefore, it is important to understand how disruption of the circadian rhythm alters the HPA axis. One way to address this question is to delete a gene involved in regulating a central circadian gene such as Per2 in an animal model and to determine how this deletion may affect the HPA axis and behaviors that are altered when the HPA axis is dysregulated. To study this, corticosterone (CORT) levels were measured through the transition from light (inactive phase) to dark (active phase). Additionally, CORT levels as well as pituitary and adrenal mRNA expression were measured following a mild restraint stress. Mice were tested for depressive-like behaviors (forced swim test (FST)), acoustic startle response (ASR), and pre-pulse inhibition (PPI).ResultsThe present results showed that Per2 knockout impacted CORT levels, mRNA expression, depressive-like behaviors, ASR and PPI. Unlike wild-type (WT) mice, Per2 knockout (Per2) mice showed no diurnal rise in CORT levels at the onset of the dark cycle. Per2−/− mice had enhanced CORT levels and adrenal melanocortin receptor 2 (Mc2R) mRNA expression following restraint. There were no changes in expression of any other pituitary or adrenal gene. In the FST, Per2−/− mice spent more time floating (less time struggling) than WT mice, suggesting increased depressive-like behaviors. Per2−/− mice had deficits in ASR and PPI startle responses compared to WT mice.ConclusionsIn summary, these findings showed that disruption of the circadian system via Per2 gene deletion dysregulated the HPA stress axis and is subsequently correlated with increased depressive-like behaviors and deficits in startle response.

Gene Expression Profiling in the Striatum of Per2 KO Mice Exhibiting More Vulnerable Responses against Methamphetamine.

Drug addiction influences most communities directly or indirectly. Increasing studies have reported the relationship between circadian-related genes and drug addiction. Per2 disrupted mice exhibited more vulnerable behavioral responses against some drugs including methamphetamine (METH). However, its roles and mechanisms are still not clear. Transcriptional profiling analysis in Per2 knockout (KO) mice may provide a valuable tool to identify potential genetic involvement and pathways in enhanced behavioral responses against drugs. To explore the potential genetic involvement, we examined common differentially expressed genes (DEGs) in the striatum of drug naïve Per2 KO/wild-type (WT) mice, and before/after METH treatment in Per2 KO mice, but not in WT mice. We selected 9 common DEGs (Ncald, Cpa6, Pklr, Ttc29, Cbr2, Egr2, Prg4, Lcn2, and Camsap2) based on literature research. Among the common DEGs, Ncald, Cpa6, Pklr, and Ttc29 showed higher expression levels in drug naïve Per2 KO mice than in WT mice, while they were downregulated in Per2 KO mice after METH treatment. In contrast, Cbr2, Egr2, Prg4, Lcn2, and Camsap2 exhibited lower expression levels in drug naïve Per2 KO mice than in WT mice, while they were upregulated after METH treatment in Per2 KO mice. qRT-PCR analyses validated the expression patterns of 9 target genes before/after METH treatment in Per2 KO and WT mice. Although further research is required to deeply understand the relationship and roles of the 9 target genes in drug addiction, the findings from the present study indicate that the target genes might play important roles in drug addiction.

Knockout of the circadian clock protein PER1 results in sex-dependent alterations of ET-1 production in mice in response to a high-salt diet plus mineralocorticoid treatment.

Previously, we showed that global knockout (KO) of the circadian clock transcription factor PER1 in male, but not female, mice fed a high-salt diet plus mineralocorticoid treatment (HS/DOCP) resulted in nondipping hypertension and decreased night/day ratio of sodium (Na) excretion. Additionally, we have shown that the endothelin-1 (ET-1) gene is targeted by both PER1 and aldosterone. We hypothesized that ET-1 would exhibit a sex-specific response to HS/DOCP treatment in PER1 KO. Here we show that male, but not female, global PER1 KO mice exhibit a decreased night/day ratio of urinary ET-1. Gene expression analysis revealed significant genotype differences in ET-1 and endothelin A receptor (ETA) expression in male, but not female, mice in response to HS/DOCP. Additionally, both wild-type and global PER1 KO male mice significantly increase endothelin B receptor (ETB) expression in response to HS/DOCP, but female mice do not. Finally, siRNA-mediated knockdown of PER1 in mouse cortical collecting duct cells (mpkCCDc14) resulted in increased ET-1 mRNA expression and peptide secretion in response to aldosterone treatment. These data suggest that PER1 is a negative regulator of ET-1 expression in response to HS/DOCP, revealing a novel mechanism for the regulation of renal Na handling in response to HS/DOCP treatment.

The Role of Circadian Clock Period 2 (per 2) gene and the expression of per2 related gene in atopic dermatitis animal model

Abstract Atopic dermatitis is a chronic, inflammatory skin disorder associated with defective skin barrier and epidermal hyper reactivity. AD is a Th2 mediated allergic disease. GATA3 is a transcription factor that plays an important role in Th2 differentiation. In this study we investigated the per2 gene role and the expression of per2 related genes in atopic dermatitis. We established the atopic dermatitis animal model in per2 knock out and C57BL/6 by 2,4-dinitrochlorobenzene (DNCB). A subset of mice in both per2 knock out and C57BL/6 were treated with saline as control group. The skin lesions severity score was alleviated in per2 knock out mice DNCB group than wild type group. We examined the TNF-α mRNA expression in skin samples that showed that per2 knock out mice protected from DNCB induced inflammation than wild type mice. We next examined BMAL1 mRNA expression in skin samples that showed BMAL1 functioned as anti-inflammatory molecule in per2 knock out mice and levels are high than wild type mice and GATA3 expression levels were not initiated in per2 knock out mice than wild type mice. According to these results we hypothesized per2 knock out mice protected from allergic atopic dermatitis by increasing anti-inflammatory molecule BMAL1 through supressing GATA3.

Knockout of Per1 Exacerbates the Hypertensive Phenotype of the Dahl Salt Sensitive Rat

The dynamic between blood pressure, its circadian rhythm, and the circadian clock genes has been considered as a factor in the subsequent renal injury that accompanies hypertension. Various circadian clock proteins such as BMAL1 and PERIOD (PER) function in a marriage of positive and negative feedback loops that control transcriptional regulation. Of particular interest is PER1 which naturally acts in the negative loop of the cycle as a repressor, but recent findings show that it may have additional roles as a transcription activator. PER1 has a role in renal sodium handling as a target of aldosterone, and it has been linked to the expression of the α‐subunit of the epithelial sodium channel (α‐ENaC). Investigators have found that knockout of PER1 in 129/sv mice causes a decrease in mean arterial pressure (MAP); however, when the gene is knocked out in the C57BL/6J mouse on a 4% NaCl diet (high salt, HS) with deoxycorticosterone pivalate (DOCP) treatment, there is a significant increase in MAP with animals displaying a “non‐dipping” pattern of MAP. The wildtype mice that were on HS with DOCP treatment had a 11.4 ± 1.9% decrease in MAP from night to day whereas the knockout mice with the same conditions had only a 5.7 ± 1.4% decrease. To test the contribution of PER1 in the development of salt‐induced hypertension, we created the Per1 knockout rat on the Dahl Salt‐Sensitive rat background (SS; knock out SSPer1−/−). Eight to 9‐week old SS and SSPer1−/− male rats were implanted with DSI telemetry units to measure blood pressure on a 4% NaCl diet (high salt, HS) for 3 weeks. MAP in SSPer1−/− rats was significantly higher compared to wild type SS rats (191 ± 7 vs. 168 ± 4 mmHg, P = 0.031; respectively) after 3 weeks of HS. We also revealed a significant increase in albuminuria in SSPer1−/− rats on HS compared to SS rats on HS (0.10 ± 0.01 vs. 0.05 ± 0.01 Alb/Cre Clearance ratio, P = 0.027; respectively). Knockout of Per1 in SS rats on HS also resulted in a significant increase in cholesterol (200.0 ± 11.8 vs. 135.0 ± 9.7 mg/Dl, P = 0.003; respectively) and alkaline phosphatase (37.5 ± 3.6 vs. 77.6 ± 3.3 U/L, P = &lt;0.001; respectively). Furthermore, SSPer1−/− rats after 3 weeks of HS diet had significant decreases in creatinine clearance compared to SS rats (65 ± 6 vs. 127 ± 24 mL/hour, P = 0.036; respectively). Here, we show that lack of PER1 causes a dramatization of the salt‐sensitive phenotype of the SS rat with a decline in renal function. These data propose that the role PER1 plays in sodium homeostasis in the kidney is pivotal for proper blood pressure regulation.Support or Funding InformationNational Institutes of Health Grants R35 HL135749 (AS) and R01 DK109570 (MLG) and by the American Heart Association 18PRE34030127 (DS)

Circadian gene PER1 senses progesterone signal during human endometrial decidualization.

Progesterone is an important hormone for female reproduction, however, how the fluctuation of progesterone acts upon reproductive processes remains largely unknown. Mounting evidence indicates a pivotal role of the circadian clock in sensing hormone dynamics for homeostatic regulation of physiological functions. Therefore, we sought to determine whether clock genes respond to progesterone signaling in female reproductive system. In this study, we tested the hypothesis that the circadian system could respond to progesterone signaling during human endometrial decidual transformation. The expression of the circadian gene PER1 increased immediately and remained elevated during human endometrial decidualization. The progesterone receptor activated PER1 transcription by directly binding to its promoter from the onset of the stromal proliferation-differentiation transition. PER1 knockout significantly downregulated the expression of some PGR target genes, and attenuated human endometrial decidual transformation by expediting FOXO1 protein degradation. In conclusion, progesterone could control the female reproductive process through sustained feedback from the circadian gene PER1, which is probably involved to P4-PR signaling responsiveness in the initiation and maintenance of decidualization.

Expression of Circadian Clock Components PER2 and BMAL1 are Altered During Infection of Helicobacter pylori

Background/HypothesisSeveral gastrointestinal functions have been demonstrated to be under circadian clock control and colorectal cancer tissues have been found to have a disruption in core circadian clock components BMAL1 and PER2. Those suffering from chronic Helicobacter pylori (H. pylori) infection are at a heightened risk of developing gastric adenocarcinoma. Compared to young individuals, the aged population suffer from an increased susceptibility to H. pylori infection as well as diminished expression of circadian clock components which could further predispose them to the development of gastric cancer. We therefore hypothesize that chronic H. pylori infection induces a hyperproliferative response in the gastric epithelium via BMAL1 activation resulting in a susceptibility to gastric cancer.MethodsGastric organoids (hFGOs) derived from primary gastric tissue of either aged (&gt; 55 years of age) or young (14 – 20 years) individuals were generated and subsequently infected with H. pylori. Analysis for proliferation and expression of circadian clock components BMAL1 and PER2 was performed for a period of 24 hours post‐infection. Organoids infected with H. pylori were analyzed for gastric cancer markers by qRT‐PCR.ResultsGastric fundic tissue obtained from individuals infected with H. pylori were found to have a significantly upregulated expression of cytoplasmic BMAL1. hFGOs exhibited a robust circadian expression of Bmal1, Per2, and phosphorylated (inactive) GSK‐3β. In comparison to uninfected controls, hFGOs infected with H. pylori displayed a heightened expression of BMAL1, phosphorylated GSK‐3β, and epithelial proliferation. Gastric organoids derived from of elderly patients was found to have a diminished rhythmic expression of clock genes Per2 and Bmal1. This dampening in expression of circadian clock components correlates with an increased expression of markers for intestinal metaplasia.ConclusionsPersistent infection by H. pylori inactivates GSK‐3β resulting in the activation of the BMAL1:CLOCK complex leading to a hyperproliferation in gastric epithelial cells. This mechanism could heighten the risk of developing gastric cancer in the aged population and in individuals with chronic H. pylori infection. Per2 knockout mice have been generated and infected with H. pylori in order to further understand this mechanism in vivo. Results are ongoing and pending.Support or Funding InformationNIH 1U19AI116491‐01This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.