Targeted modification of the Per2 clock gene alters circadian function in mPer2luciferase (mPer2Luc) mice.
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ABSTRACT:
SUBMITTER: Ralph MR
PROVIDER: S-EPMC8191895 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In mammals, light information received by the eyes is transmitted to the pineal gland via the circadian pacemaker, i.e., the suprachiasmatic nucleus (SCN). Melatonin secreted by the pineal gland at night decodes night length and regulates seasonal physiology and behavior. Melatonin regulates the expression of the ?-subunit of thyroid-stimulating hormone (TSH; Tshb) in the pars tuberalis (PT) of the pituitary gland. Long day-induced PT TSH acts on ependymal cells in the mediobasal hypothalamus to induce the expression of type 2 deiodinase (Dio2) and reduce type 3 deiodinase (Dio3) that are thyroid hormone-activating and hormone-inactivating enzymes, respectively. The long day-activated thyroid hormone T3 regulates seasonal gonadotropin-releasing hormone secretion. It is well established that the circadian clock is involved in the regulation of photoperiodism. However, the involvement of the circadian clock gene in photoperiodism regulation remains unclear. Although mice are generally considered non-seasonal animals, it was recently demonstrated that mice are a good model for the study of photoperiodism. In the present study, therefore, we examined the effect of changing day length in Per2 deletion mutant mice that show shorter wheel-running rhythms under constant darkness followed by arhythmicity. Although the amplitude of clock gene (Per1, Cry1) expression was greatly attenuated in the SCN, the expression profile of arylalkylamine N-acetyltransferase, a rate-limiting melatonin synthesis enzyme, was unaffected in the pineal gland, and robust photoperiodic responses of the Tshb, Dio2, and Dio3 genes were observed. These results suggested that the Per2 clock gene is not necessary for the photoperiodic response in mice.
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Project description:Adaptive thermogenesis allows mammals to resist to cold. For instance, in brown adipose tissue (BAT) the facultative uncoupling of the proton gradient from ATP synthesis in mitochondria is used to generate systemic heat. However, this system necessitates an increase of the Uncoupling protein 1 (Ucp1) and its activation by free fatty acids. Here we show that mice without functional Period2 (Per2) were cold sensitive because their adaptive thermogenesis system was less efficient. Upon cold-exposure, Heat shock factor 1 (HSF1) induced Per2 in the BAT. Subsequently, PER2 as a co-activator of PPAR? increased expression of Ucp1. PER2 also increased Fatty acid binding protein 3 (Fabp3), a protein important to transport free fatty acids from the plasma to mitochondria to activate UCP1. Hence, in BAT PER2 is important for the coordination of the molecular response of mice exposed to cold by synchronizing UCP1 expression and its activation.
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Project description: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.
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Project description:Neurons in the mammalian suprachiasmatic nucleus (SCN) contain a cell-autonomous circadian clock that is based on a transcriptional-translational feedback loop. The basic helix-loop-helix-PAS proteins CLOCK and BMAL1 are positive regulators and drive the expression of the negative regulators CRY1 and CRY2, as well as PER1, PER2, and PER3. To assess the role of mouse PER3 (mPER3) in the circadian timing system, we generated mice with a targeted disruption of the mPer3 gene. Western blot analysis confirmed the absence of mPER3-immunoreactive proteins in mice homozygous for the targeted allele. mPer1, mPer2, mCry1, and Bmal1 RNA rhythms in the SCN did not differ between mPER3-deficient and wild-type mice. Rhythmic expression of mPer1 and mPer2 RNAs in skeletal muscle also did not differ between mPER3-deficient and wild-type mice. mPer3 transcripts were rhythmically expressed in the SCN and skeletal muscle of mice homozygous for the targeted allele, but the level of expression of the mutant transcript was lower than that in wild-type controls. Locomotor activity rhythms in mPER3-deficient mice were grossly normal, but the circadian cycle length was significantly (0.5 h) shorter than that in controls. The results demonstrate that mPer3 is not necessary for circadian rhythms in mice.
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Project description:BACKGROUND: Circadian oscillation of clock-controlled gene expression is mainly regulated at the transcriptional level. Heterodimers of CLOCK and BMAL1 act as activators of target gene transcription; however, interactions of PER and CRY proteins with the heterodimer abolish its transcriptional activation capacity. PER and CRY are therefore referred to as negative regulators of the circadian clock. To further elucidate the mechanism how positive and negative components of the clock interplay, we characterized the interactions of PER2, CRY1 and CRY2 with BMAL1 and CLOCK using a mammalian two-hybrid system and co-immunoprecipitation assays. RESULTS: Both PER2 and the CRY proteins were found to interact with BMAL1 whereas only PER2 interacts with CLOCK. CRY proteins seem to have a higher affinity to BMAL1 than PER2. Moreover, we provide evidence that PER2, CRY1 and CRY2 bind to different domains in the BMAL1 protein. CONCLUSION: The regulators of clock-controlled transcription PER2, CRY1 and CRY2 differ in their capacity to interact with each single component of the BMAL1-CLOCK heterodimer and, in the case of BMAL1, also in their interaction sites. Our data supports the hypothesis that CRY proteins, especially CRY1, are stronger repressors than PER proteins.
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Project description:Living in the earth's oxygenated environment forced organisms to develop strategies to cope with the damaging effects of molecular oxygen known as reactive oxygen species (ROS). Here, we show that Per2, a molecular component of the mammalian circadian clock, is involved in regulating a cell's response to oxidative stress. Mouse embryonic fibroblasts (MEFs) containing a mutation in the Per2 gene are more resistant to cytotoxic effects mediated by ROS than wild-type cells, which is paralleled by an altered regulation of bcl-2 expression in Per2 mutant MEFs. The elevated survival rate and alteration of NADH/NAD(+) ratio in the mutant cells is reversed by introduction of the wild-type Per2 gene. Interestingly, clock synchronized cells display a time dependent sensitivity to paraquat, a ROS inducing agent. Our observations indicate that the circadian clock is involved in regulating the fate of a cell to survive or to die in response to oxidative stress, which could have implications for cancer development and the aging process.
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Project description:The retina has an intrinsic circadian clock, but the importance of this clock for vision is unknown. Zebrafish offer many advantages for studying vertebrate vision and circadian rhythm. Here, we explored the role of zebrafish per2, a light-regulated gene, in visual behavior and the underlying mechanisms. We observed that per2 mutant zebrafish larvae showed decreased contrast sensitivity and visual acuity using optokinetic response (OKR) assays. Using a visual motor response (VMR) assay, we observed normal OFF responses but abnormal ON responses in mutant zebrafish larvae. Immunofluorescence showed that mutants had a normal morphology of cone photoreceptor cells and retinal organization. However, electron microscopy showed that per2 mutants displayed abnormal and decreased photoreceptor ribbon synapses with arciform density, which resulted in retinal ON pathway defect. We also examined the expression of three cone opsins by quantitative real-time PCR (qRT-PCR), and the expression of long-wave-sensitive opsin (opn1lw) and short-wave-sensitive opsin (opn1sw) was reduced in mutant zebrafish larvae. qRT-PCR analyses also showed a down-regulation of the clock genes cry1ba and bmal1b in the adult eye of per2 mutant zebrafish. This study identified a mechanism by which a clock gene affects visual function and defined important roles of per2 in retinal information processing.
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Project description:Inhibitor of DNA binding (Id) genes comprise a family of four helix-loop-helix (HLH) transcriptional inhibitors. Our earlier studies revealed a role for ID2 within the circadian system, contributing to input, output, and core clock function through its interaction with CLOCK and BMAL1. Here, we explore the contribution of ID4 to the circadian system using a targeted disruption of the Id4 gene. Attributes of the circadian clock were assessed by monitoring the locomotor activity of Id4-/- mice, and they revealed disturbances in its operation. Id4-mutant mice expressed a shorter circadian period length, attenuated phase shifts in responses to continuous and discrete photic cues, and an advanced phase angle of entrainment under a 12:12 light:dark cycle and under short and long photoperiods. To understand the basis for these properties, suprachiasmatic nucleus (SCN) and retinal structures were examined. Anatomical analysis reveals a smaller Id4-/- SCN in the width dimension, which is a finding consistent with its smaller brain. As a result of this feature, anterograde tracing in Id4-/- mice revealed retinal afferents innovate a disproportionally larger SCN area. The Id4-/- photic entrainment responses are unlikely to be due to an impaired function of the retinal pathways since Id4-/- retinal anatomy and function tested by pupillometry were similar to wild-type mice. Furthermore, these circadian characteristics are opposite to those exhibited by the Id2-/- mouse, suggesting an opposing influence of the ID4 protein within the circadian system; or, the absence of ID4 results in changes in the expression or activity of other members of the Id gene family. Expression analysis of the Id genes within the Id4-/- SCN revealed a time-of-day specific elevated Id1. It is plausible that the increased Id1 and/or absence of ID4 result in changes in interactions with bHLH canonical clock components or with targets upstream and/or downstream of the clock, thereby resulting in abnormal properties of the circadian clock and its entrainment.
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Project description:A single phase advance of the light:dark (LD) cycle can temporarily disrupt synchrony of neural circadian rhythms within the suprachiasmatic nucleus (SCN) and between the SCN and peripheral tissues. Compounding this, modern life can involve repeated disruptive light conditions. To model chronic disruption to the circadian system, we exposed male mice to more than a month of a 20-hr light cycle (LD10:10), which mice typically cannot entrain to. Control animals were housed under LD12:12. We measured locomotor activity and body temperature rhythms in vivo, and rhythms of PER2::LUC bioluminescence in SCN and peripheral tissues ex vivo. Unexpectedly, we discovered strong effects of the time of dissection on circadian phase of PER2::LUC bioluminescent rhythms, which varied across tissues. White adipose tissue was strongly reset by dissection, while thymus phase appeared independent of dissection timing. Prior light exposure impacted the SCN, resulting in strong resetting of SCN phase by dissection for mice housed under LD10:10, and weak phase shifts by time of dissection in SCN from control LD12:12 mice. These findings suggest that exposure to circadian disruption may desynchronize SCN neurons, increasing network sensitivity to perturbations. We propose that tissues with a weakened circadian network, such as the SCN under disruptive light conditions, or with little to no coupling, for example, some peripheral tissues, will show increased resetting effects. In particular, exposure to light at inconsistent circadian times on a recurring weekly basis disrupts circadian rhythms and alters sensitivity of the SCN neural pacemaker to dissection time.
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Project description:The hypothalamic suprachiasmatic nucleus (SCN), locus of the master circadian clock, bears many neuronal types. At the cellular-molecular level, the clock is comprised of feedback loops involving 'clock' genes including Period1 and Period2, and their protein products, PERIOD1 and PERIOD2 (PER1/2). In the canonical model of circadian oscillation, the PER1/2 proteins oscillate together. While their rhythmic expression in the SCN as a whole has been described, the possibility of regional differences remains unknown. To explore these clock proteins in distinct SCN regions, we assessed their expression through the rostro-caudal extent of the SCN in sagittal sections. We developed an automated method for tracking three fluorophores in digital images of sections triply labeled for PER1, PER2, and gastrin-releasing peptide (used to locate the core). In the SCN as a whole, neurons expressing high levels of PER2 were concentrated in the rostral, rostrodorsal, and caudal portions of the nucleus, and those expressing high levels of PER1 lay in a broad central area. Within these overall patterns, adjacent cells differed in expression levels of the two proteins. The results demonstrate spatially distinct localization of high PER1 vs. PER2 expression, raising the possibility that their distribution is functionally significant in encoding and communicating temporal information. The findings provoke the question of whether there are fundamental differences in PER1/2 levels among SCN neurons and/or whether topographical differences in protein expression are a product of SCN network organization rather than intrinsic differences among neurons.
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