Browsing by Subject "Circadian Rhythm"
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Item Analysis of Circadian Rhythm Using a Novel SCN-Specific Cre Transgenic Mouse Line(2010-07-12) Chang, Alexander S.; Yanagisawa, MasashiThe neurons that make up the suprachiasmatic nucleus (SCN) temporally organize behavior into circadian cycles of activity and rest. When dissociated, these neurons individually oscillate with various period, phase, and amplitude. These conflicting results can be reconciled if inter-neuronal networking in the SCN is required for a consolidated behavioral circadian rhythm. To test this hypothesis, a novel SCN-specific Cre transgenic mouse line, named NMS-Cre, was developed by inserting a bicistronic Cre expression cassette at the 3’-untranslated region of the Neuromedin S (NMS) gene. By crossing NMS-Cre line to a lox-STOP-lox diphtheria toxin receptor line, behavioral circadian rhythm was disrupted upon intraperitoneal injection of diphtheria toxin. A histological examination showed that diphtheria toxin injection eliminated ~85% of NMS-Cre containing neurons at the SCN. Next, I generated NMS-Cre mediated Bmal1 conditional knockout animals to study behavioral rhythm output when most of the SCN neurons are without a molecular oscillator. The NMS-Cre(+);Bmal1flox/flox animals have essentially normal circadian rhythm of locomotor activity. Then, I generated NMS-Cre mediated Vesicular GABA Transporter (VGAT) conditional knockout animals because GABA has long been suspected to play a role in behavioral circadian rhythm. The NMS-Cre;VGATflox/flox animals performed normally in behavioral circadian rhythm parameter such as free-running period, robustness, and phase response curve. These in vivo data demonstrated a model that intra-SCN neuronal network is required for behavioral circadian rhythm, and can be a conduit that mediates molecular clock outputs from a small number of SCN neurons. Despite the fact that virtually all SCN neurons are GABAergic, GABA is an unlikely transmitter for this intra-SCN networking. Finally, NMS knockout animals have a well-consolidated behavioral circadian rhythm. However, when subjected to photic phase advancement, NMS knockout animals shifted their activity onset time quicker than wild type control animals. In situ hybridization results ruled out that an altered response to light stimuli or dampened molecular clock oscillation in the SCN as the cause for the rapid phase shift. NMS knockout animals switching from constant illumination to constant dark environment are unable to return to the typical less than twenty-four hour free-running period. Therefore, NMS is involved in a circadian pacemaker function.Item Cellular Basis of Behavioral Circadian Rhythms in Mammals: The Role of Neuromedin S (Nms)-Producing Cells in the Suprachiasmatic Nucleus(2013-08-07) Lee, Ivan T.; Elmquist, Joel; Russell, David W.; Greene, Robert W.; Yanagisawa, Masashi; Takahashi, JosephBehavioral circadian rhythms in mammals are controlled by highly heterogeneous populations of neurons located in the suprachiasmatic nucleus (SCN). Lesion and transplantation studies have established that the SCN is both necessary and sufficient for the generation of daily rhythms in locomotion. It remains uncertain, however, whether this pacemaking property of the SCN is limited to certain subsets of cells or intrinsic to all neurons within the SCN. To dissect out the cellular properties of circadian rhythms, we utilized a BAC transgenic mouse line in which Cre recombinase (iCre) is driven by the promoter of neuromedin S (Nms), a neuropeptide that has restricted expression in ~40% of cells within the SCN. Using this cell-type specific driver, we genetically altered the molecular oscillation of Nms-positive cells by overexpressing the ClockΔ19 or the Period2 transgene. ClockΔ19 is a semi-dominant mutation that leads to lengthened behavioral circadian periods when expressed in the majority of SCN cells. Likewise, Period2, when overexpressed in all or almost all of the SCN neurons, lead to the loss of behavioral circadian rhythms. We found that, intriguingly, the transgenic expression of ClockΔ19 only in Nms-positive neurons leads to a lengthened period in circadian rhythms while the overexpression of Per2 in Nms-expressing neurons causes the loss of daily rhythms altogether, suggesting that behavioral rhythms can be controlled by the molecular oscillation of Nms-positive cells. Next, to ascertain whether Nms-expressing neurons are required for normal behavioral circadian rhythms, we utilized a tetanus toxin-based technology that permits the inducible and reversible inhibition of neurotransmission. Surprisingly, this genetic manipulation revealed that synaptic neurotransmission from Nms neurons is essential for the generation of behavioral circadian rhythms. Taken together, these results indicate that Nms marks a specialized subgroup of neurons that is both necessary and sufficient for the production of circadian rhythms in behavior.Item Code Within Codes: Codon Usage Regulate Protein Expression, Structure, and Function(2018-07-23) Fu, Jingjing; Jiang, Jin; Liu, Yi; Cobb, Melanie H.; Green, Carla B.Most amino acids are encoded by two to six synonymous genetic codons. Synonymous codons are not used with the same frequency in all organisms, and every organism has its own preferred codon usage bias. Codon usage bias has been shown to positively correlate with tRNA abundance, thus optimal codons are thought to be translated more efficiently and accurately. Consistent with this, strong codon usage biases have been shown to be important for the expression of highly expressed genes in different organisms, and codon optimization has been widely used to enhance heterologous protein expression. Therefore, codon usage can be an important determinant in gene expression. In addition, codon usage has been shown to influence translation elongation rate and protein structure by affecting the co-translational folding process in E. coli, fungi, and insects. In addition to its role in regulating protein translation, codon usage also has a major role in determining the level of gene expression through transcriptional and post-transcriptional processes. As such, gene codon usage has been proposed to be a code within the genetic code that can determine both gene expression levels and protein structures and therefore activity. However, the effects of codon usage in multi-tissue organisms, for example, animals and humans, are not clear. In the first part of the thesis, by codon-optimizing open reading frame of Drosophila period gene, I showed that dper codon usage is critical for its circadian clock function. Optimization of dper codon usage resulted in conformational changes of dPER protein, altered dPER phosphorylation profile and stability, and impaired dPER repressor function in the circadian negative feedback loop. In the second part of the thesis, I reported that changing rare codons to common in KRAS increased translation and mRNA levels. Regulation of mRNA levels is a major mechanism affecting KRAS levels, but the effect was not a product of mRNA stability, but instead transcriptional. Moreover, codon usage also had an impact on the structure of KRAS. Thus, the rare codon bias of KRAS effects more aspects of protein production and function than previously appreciated, which has important implications for other rare codon enriched mammalian genes.Item Modulation of Nocturnin Phosphatase Activity through the Disordered Amino Terminus(August 2021) Wickramaratne, Anushka Christobel; Hibbs, Ryan E.; Green, Carla B.; Takahashi, Joseph; Conrad, NicholasThe endogenous circadian clock controls the rhythmicity of behavioral and physiological processes and this is entrained by the daily fluctuations in light and dark. Nocturnin (Noct) is a rhythmically expressed gene regulated by the circadian clock that belongs to the CCR4 family of endonuclease-exonuclease-phosphatase (EEP) enzymes. Its expression is induced by acute stimuli and loss of Nocturnin (Noct-/-) in mice results in resistance to diet-induced obesity on a high fat diet and confers a protective effect to oxidative stress in HEK cells. Modeling of full-length Nocturnin reveals a partially structured amino terminus that is disparate from its CCR4 family members. I show that Nocturnin functions as a phosphatase, catalyzing the removal of the 2′-phosphate from NADP(H). High sequence conservation of the leucine zipper (LZ)-like motif, the only structural element in the amino terminus, highlights the potential importance of this domain in modulating phosphatase activity. I use in vitro biochemical and biophysical techniques to demonstrate that the amino terminus and the LZ-like domain are necessary for preserving the active site cleft in an optimal conformation to promote efficient turnover of the substrate. This modulation occurs in cis and is additionally pivotal in maintaining the stability and conformational integrity of the enzyme. These new findings suggest an additional layer of modulating the activity of Nocturnin in addition to its rhythmicity in order to provide fine-tuned control over cellular levels of NADPH. This lays the essential groundwork necessary to further understand the role of the partially structured amino terminus in metabolism and the oxidative stress response through regulation of NADP(H) and NAD(H) levels.Item A Multi-Organ Role for Nocturnin in Post-Transcriptional Regulation of RNA(2018-04-16) Onder, Yasemin; Huber, Kimberly M.; Green, Carla B.; Takahashi, Joseph; Yu, Gang; Mishra, PrashantNocturnin is an RNA-specific nuclease, a circadian deadenylase first discovered in the retina of Xenopus laevis, and is conserved among eukaryotes. Nocturnin is widely expressed in the brain and in the periphery and is also an immediate early gene that is acutely induced in response to various stimuli. This study investigates Nocturnin's potential role in post-transcriptional regulation of mRNAs in the mitochondria and in two different tissues: brain and brown adipose tissue (BAT). Nocturnin has a predicted mitochondrial-localization signal (MLS) surrounded by two potential translation initiation sites (AUG codons) raising the possibility of dual translation initiation sites. Here we demonstrate that Nocturnin is present in the mitochondria and that it exhibits mitochondrial or cytoplasmic localization via the use of alternative translation initiation sites. I also show that Nocturnin is acutely induced in response to cold and mitochondrial-encoded mRNAs in Noc-/- BAT exhibit impaired stability upon cold exposure. Global analysis of cold-induced changes in the transcriptome of Noc-/- and wild type (WT) mice reveal down-regulation of glycan biosynthesis genes in the Noc-/- BAT. Strikingly, metabolomics analysis demonstrates robust alterations in key tricarboxylic acid metabolites like pyruvate and succinate in the Noc-/- mice BAT in response to a prolonged cold exposure. In summary, we propose a model that Nocturnin acts as a metabolic switch in response to cold by diverting glucose and free fatty acids (FFA) to the mitochondria. In this study, Nocturnin's potential role in post-transcriptional regulation of synaptic plasticity is also investigated. Activity-dependent local protein translation in the dendrites is thought to be an important component of synaptic plasticity. The mechanism of how translation of different mRNAs is differentially regulated in the dendrites is yet not clear. Here I demonstrate that Nocturnin is present in the dendrites as well as in the post-synaptic density as observed in cultured neurons and brain slices from cortex and hippocampus. I investigated mGluR-LTD in hippocampal slices from Noc-/- and WT littermates and observed an attenuation of mGluR-LTD in Noc-/- mice in the first cohort, but this result was not replicated in a second cohort. Further studies are needed to elucidate Nocturnin's role in the synapse.Item Nocturnin Regulates Metabolic Flux Through Maintenance of Poly(A) Tail Length Dynamics(2016-07-26) Stubblefield, Jeremy Joseph; Elmquist, Joel; Green, Carla B.; Mangelsdorf, David J.; Takahashi, JosephCyclic processes in both behavior and physiology are aligned to the external environment through the circadian clock. Nocturnin (Noc) is a rhythmically expressed gene regulated in part by the circadian clock and intimately linked to the metabolic state of an organism. Loss of Nocturnin (Noc-/-) in mice results in resistance to diet-induced obesity. Encoding a deadenylase, NOC protein is thought to regulate mRNA turnover through its ability to remove Poly(A) tails from mRNA transcripts. Though NOC has been linked with lipid and glucose metabolism, its specific targets have not been identified. I explored NOC's role in metabolism by exposing Wildtype (WT) and Noc-/- mice to nutrient challenges consisting of High Fat Diet (HFD) and fasting/refeeding. I demonstrated that Noc can be acutely reduced with a fast and induced with refeeding in WT mice. Hepatic Noc expression oscillates in WT mice fed a High Fat Diet (HFD) but with increased amplitude. I performed mRNA-seq from livers of Wildtype (WT) and Noc-/- mice and identified significant upregulation of both cholesterol and bile acid synthesis genes in Noc-/- mice under basal and nutrient-challenged conditions. This dysregulation results in Noc-/- mice having significantly increased gallbladder volumes during times of fasting. I subjected WT and Noc-/- to hyperinsulinemic-euglycemic clamps and found that HFD-fed Noc-/- mice develop more severe insulin resistance than HFD-fed WT mice. Under insulin-stimulated (clamped) conditions, HFD-fed Noc-/- mice fail to suppress endogenous glucose production and have reduced whole-body glucose turnover. Additionally, regular chow (RC) fed Noc-/- mice exhibit insulin resistance only during the dark phase. This deficit in glucose/insulin sensitivity can be partially rescued in Noc-/- mice through transgenic overexpression of WT NOC, but not a catalytically dead mutant NOC that cannot function as a deadenylase. The deadenylase activity of NOC is thus important for these metabolic phenotypes and I found that genes associated with bile acid, cholesterol and glucose metabolism have altered Poly(A) tail length regulation in Noc-/- mice and thus represent possible targets of NOC. This new understanding of the relationship between Nocturnin, the circadian system and metabolism will help guide the treatment of conditions such as obesity and diabetes.Item Regulation of Circadian Genes by Cocaine in Striatal Regions and Their Role in Drug Reward(2012-07-10) Falcón-Morales, Edgardo; McClung, Colleen A.Disruptions in circadian rhythms are associated with neuropsychiatric disorders, including drug addiction. Indeed, mounting evidence reveals a role for the circadian clock in the regulation of drug reward and reward-related behaviors. Conversely, drugs of abuse are known to dysregulate circadian-associated processes and entrain locomotor behavior. The circadian clock is governed by a master pacemaker in the Suprachiasmatic Nucleus(SCN) of the anterior hypothalamus. However, the components of this circadian clock machinery are expressed throughout the brain and body, allowing for the occurrence of SCN-dependent or independent peripheral oscillators. One such brain circuit in which circadian genes are expressed is the mesolimbic dopaminergic pathway, containing brain regions such as the Nucleus Accumbens(NAc) and the Caudate Putamen (CP), among others. We set out to investigate how repeated cocaine exposure regulates core clock circadian genes in striatal regions and conversely, how core clock circadian genes regulate cocaine’s rewarding effects. Additionally, the potential regulation of rhythmic dopamine receptor expression directly by clock components and how their rhythmic expression is altered by repeated cocaine exposure was assessed. Chapter 3 determined circadian gene regulation in both the NAc and CP by cocaine. Not only did chronic cocaine upregulated a number of circadian genes at a specific timepoint, like Npas2 and the Per genes, but also altered or disrupted 24-hr rhythmic expression of these genes. Chapter 4 investigated the role of core circadian clock genes in cocaine reward, as measured by conditioned place preference. Npas2 mutant mice exhibited a decreased preference for cocaine, an effect that was recapitulated by viral-mediated Npas2 knockdown specifically in the NAc. Per mutant mice displayed an increase in cocaine preference. Knockdown of mPer1 and mPer2 in the NAc led to a trend towards increased preference. Chapter 5 identified a potential role for NPAS2 in the regulation of dopamine receptor rhythmic expression in the NAc, and that chronic cocaine disrupts this rhythmicity. These findings suggest an important role for Npas2 as mediator of cocaine responses in the NAc. Moreover, they further elucidate the bidirectional interactions between the circadian and reward systems, implicating the circadian control of the dopaminergic system in this interplay.Item The Role of CLOCK in Regulation of Dopamine Neurotransmission in the CLOCKdelta19 Mutant Mouse Model(2012-07-17) Spencer, Sade Monique; McClung, Colleen A.Mice with a mutation in the circadian gene Clock (Clockdelta19) display a behavioral profile which parallels a euphoric manic-like state including hyperactivity, disrupted activity rhythms, increased substance abuse vulnerability, and decreases in anxiety and depression-related behavior. The molecular clock has significant cross-talk with many of the brain’s neurotransmitter systems. The purpose of this dissertation is to characterize the role of CLOCK in regulating dopamine transmission in mood and reward-related circuits. We present a mechanism by which CLOCK regulates dopaminergic activity in the mesoaccumbens circuit and contributes to anxiety-related behavior. In vivo recording of ventral tegmental area (VTA) dopamine cells throughout the 24 hour cycle revealed that firing and bursting was elevated in Clockdelta19 mutants with the most significant deviations early in the light cycle. Mimicking this increase in dopaminergic activity using optogenetic targeting resulted in decreased anxiety-related behavior similar to the Clockdelta19 phenotype. Consistent with the electrophysiological findings, tyrosine hydroxylase (TH) mRNA and protein was elevated in the VTA in a daytime-specific manner leading to increased dopamine synthesis in the nucleus accumbens. CLOCK binding was detected at E-box elements within the TH promoter with greater enrichment observed during the light phase when TH expression is low. These results suggest a negative regulation of TH by CLOCK. To examine alterations in the nigrostriatal dopamine circuit, HPLC measurements of dopamine and metabolites were performed in the dorsal striatum revealing significant increases in DOPAC and HVA. Dopamine receptor agonists and antagonists were used to pharmacologically probe dopamine receptor function. An enhancement of the locomotor suppressing response to dopamine antagonists in Clockdelta19 mice suggested increased dopaminergic tone. Clockdelta19 mice were insensitive to the locomotor stimulating effects of a D1 agonist, but displayed increased levels of D1DR protein. Conversely, the Clockdelta19 mutants displayed enhanced locomotor suppression to a D2 agonist and a coincident increase in D2DR protein. Forskolin stimulation of cAMP resulted in blunted molecular responses in the Clockdelta19 mutants consistent with impairments in D1 signaling and/or enhancements in D2 signaling. In summary, normal CLOCK function appears to be involved in the regulation of dopamine transmission in the striatum.Item [Southwestern News](2003-07-03) Morrison, SusanItem [Southwestern News](2005-06-13) Hansard, Donna StephItem Spatiotemporal Regulation of the NADP(H) Phosphatase Nocturnin(August 2021) Laothamatas, Isara; Mishra, Prashant; Takahashi, Joseph; Conrad, Nicholas; Green, Carla B.Periodic changes in the environment are ubiquitous in the natural world. Among these, the most biologically relevant rhythm is the 24-hour geophysical day/night cycle. As an adaptive strategy, many organisms have evolved an endogenous biological clock to temporally organize their physiology and anticipate daily changes in the environment. At its core, the mammalian "circadian clock" is a molecular oscillator driven by a genetic transcription/translation feedback loop, which orchestrates the rhythmic expression of thousands of genes. An intimate link between circadian clocks and metabolism is established by the rhythmic transcription of output genes involved in almost every metabolic pathway. Among these oscillating genes, Nocturnin (also Noct; protein name: NOC) has one of the highest amplitude rhythms at the mRNA level. Mice with a loss-of-function in Noct possess metabolic phenotypes, where they are protected from high-fat diet-induced obesity and LPS-induced septic shock. However, the mechanism by which this occurs is not well-understood. Here, in collaboration with Green lab members and the Liou lab, I used both in vitro biochemical and in vivo cellular and mouse models to elucidate the molecular and physiological function of NOC. Even though NOC is highly-conserved with the endonuclease/exonuclease/phosphatase (EEP) domain-containing CCR4 family of deadenylases, we show that highly-purified recombinant NOC lacks ribonuclease activity. Instead, NOC catalyzes the dephosphorylation of NADP(H), and its activity level is associated with the cellular response to oxidative stress. Furthermore, we describe two isoforms of NOC and their spatiotemporal regulation in the mouse liver. Cytoplasmic NOC is constitutively-expressed throughout the day and associates externally with the endoplasmic reticulum and other membranes via N-terminal glycine myristoylation. In contrast, mitochondrial NOC levels are highly circadian with peak expression during the early dark phase. Overall, our work suggests that NOC links circadian clocks to metabolism by regulating local intracellular concentrations of NADP(H) in a manner that changes throughout the day.Item [UT News](1985-08-28) Harrell, AnnItem [UT Southwestern Medical Center News](2012-06-04) Wormser, DeborahItem [UT Southwestern Medical Center News](2007-03-19) Hansard, Donna StephItem [UT Southwestern Medical Center News](2009-06-08) McKenzie, AlineItem [UT Southwestern Medical Center News](2010-10-14) McKenzie, AlineItem [UT Southwestern Medical Center News](2010-07-13) McKenzie, Aline