Atlas of Circadian Metabolism Reveals System-wide Coordination and Communication between Clocks.

Metabolic diseases are often characterized by circadian misalignment in different tissues, yet how altered coordination and communication among tissue clocks relate to specific pathogenic mechanisms remains largely unknown. Applying an integrated systems biology approach, we performed 24-hr metabolomics profiling of eight mouse tissues simultaneously. We present a temporal and spatial atlas of circadian metabolism in the context of systemic energy balance and under chronic nutrient stress (high-fat diet [HFD]). Comparative analysis reveals how the repertoires of tissue metabolism are linked and gated to specific temporal windows and how this highly specialized communication and coherence among tissue clocks is rewired by nutrient challenge. Overall, we illustrate how dynamic metabolic relationships can be reconstructed across time and space and how integration of circadian metabolomics data from multiple tissues can improve our understanding of health and disease.

Dopamine D2 receptor signaling in the brain modulates circadian liver metabolomic profiles.

SignificanceWe analyzed the liver metabolome of mice deficient in the expression of the dopamine D2 receptor (D2R) in striatal medium spiny neurons (iMSN-D2RKO) and found profound changes in the liver circadian metabolome compared to control mice. Additionally, we show activation of dopaminergic circuits by acute cocaine administration in iMSN-D2RKO mice reprograms the circadian liver metabolome in response to cocaine. D2R signaling in MSNs is key for striatal output and essential for regulating the first response to the cellular and rewarding effects of cocaine. Our results suggest changes in dopamine signaling in specific striatal neurons evoke major changes in liver physiology. Dysregulation of liver metabolism could contribute to an altered allostatic state and therefore be involved in continued use of drugs.

The Brain's Reward System in Health and Disease.

Rhythmic gene expression is found throughout the central nervous system. This harmonized regulation can be dependent on- and independent of- the master regulator of biological clocks, the suprachiasmatic nucleus (SCN). Substantial oscillatory activity in the brain's reward system is regulated by dopamine. While light serves as a primary time-giver (zeitgeber) of physiological clocks and synchronizes biological rhythms in 24-h cycles, nonphotic stimuli have a profound influence over circadian biology. Indeed, reward-related activities (e.g., feeding, exercise, sex, substance use, and social interactions), which lead to an elevated level of dopamine, alters rhythms in the SCN and the brain's reward system. In this chapter, we will discuss the influence of the dopaminergic reward pathways on circadian system and the implication of this interplay on human health.

Differential regulation of striatal motor behavior and related cellular responses by dopamine D2L and D2S isoforms.

The dopamine D2 receptor (D2R) is a major component of the dopamine system. D2R-mediated signaling in dopamine neurons is involved in the presynaptic regulation of dopamine levels. Postsynaptically, i.e., in striatal neurons, D2R signaling controls complex functions such as motor activity through regulation of cell firing and heterologous neurotransmitter release. The presence of two isoforms, D2L and D2S, which are generated by a mechanism of alternative splicing of the Drd2 gene, raises the question of whether both isoforms may equally control presynaptic and postsynaptic events. Here, we addressed this question by comparing behavioral and cellular responses of mice with the selective ablation of either D2L or D2S isoform. We establish that the presence of either D2L or D2S can support postsynaptic functions related to the control of motor activity in basal conditions. On the contrary, absence of D2S but not D2L prevents the inhibition of tyrosine hydroxylase phosphorylation and, thereby, of dopamine synthesis, supporting a major presynaptic role for D2S. Interestingly, boosting dopamine signaling in the striatum by acute cocaine administration reveals that absence of D2L, but not of D2S, strongly impairs the motor and cellular response to the drug, in a manner similar to the ablation of both isoforms. These results suggest that when the dopamine system is challenged, D2L signaling is required for the control of striatal circuits regulating motor activity. Thus, our findings show that D2L and D2S share similar functions in basal conditions but not in response to stimulation of the dopamine system.

Dopamine D2 receptors in striatal output neurons enable the psychomotor effects of cocaine.

The psychomotor effects of cocaine are mediated by dopamine (DA) through stimulation of striatal circuits. Gabaergic striatal medium spiny neurons (MSNs) are the only output of this pivotal structure in the control of movements. The majority of MSNs express either the DA D1 or D2 receptors (D1R, D2R). Studies have shown that the motor effect of cocaine depends on the DA-mediated stimulation of D1R-expressing MSNs (dMSNs), which is mirrored at the cellular level by stimulation of signaling pathways leading to phosphorylation of ERKs and induction of c-fos Nevertheless, activation of dMSNs by cocaine is necessary but not sufficient, and D2R signaling is required for the behavioral and cellular effects of cocaine. Indeed, cocaine motor effects and activation of signaling in dMSNs are blunted in mice with the constitutive knockout of D2R (D2RKO). Using mouse lines with a cell-specific knockout of D2R either in MSNs (MSN-D2RKO) or in dopaminergic neurons (DA-D2RKO), we show that D2R signaling in MSNs is required and permissive for the motor stimulant effects of cocaine and the activation of signaling in dMSNs. MSN-D2RKO mice show the same phenotype as constitutive D2RKO mice both at the behavioral and cellular levels. Importantly, activation of signaling in dMSNs by cocaine is rescued by intrastriatal injection of the GABA antagonist, bicuculline. These results are in support of intrastriatal connections of D2R+-MSNs (iMSNs) with dMSNs and indicate that D2R signaling in MSNs is critical for the function of intrastriatal circuits.

Distinct cortical and striatal actions of a ß-arrestin-biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties.

The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed ß-arrestin2 (ßarr2)-biased D2R partial agonists to simultaneously target hyper- and hypodopaminergia. Using neuron-specific ßarr2-KO mice, we show that the antipsychotic-like effects of a ßarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-ßarr2 signaling. Furthermore, ßarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of ßarr2 and G protein-coupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that ßarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.

Selective Kv1.3 channel blocker as therapeutic for obesity and insulin resistance.

Obesity is an epidemic, calling for innovative and reliable pharmacological strategies. Here, we show that ShK-186, a selective and potent blocker of the voltage-gated Kv1.3 channel, counteracts the negative effects of increased caloric intake in mice fed a diet rich in fat and fructose. ShK-186 reduced weight gain, adiposity, and fatty liver; decreased blood levels of cholesterol, sugar, HbA1c, insulin, and leptin; and enhanced peripheral insulin sensitivity. These changes mimic the effects of Kv1.3 gene deletion. ShK-186 did not alter weight gain in mice on a chow diet, suggesting that the obesity-inducing diet enhances sensitivity to Kv1.3 blockade. Several mechanisms may contribute to the therapeutic benefits of ShK-186. ShK-186 therapy activated brown adipose tissue as evidenced by a doubling of glucose uptake, and increased ß-oxidation of fatty acids, glycolysis, fatty acid synthesis, and uncoupling protein 1 expression. Activation of brown adipose tissue manifested as augmented oxygen consumption and energy expenditure, with no change in caloric intake, locomotor activity, or thyroid hormone levels. The obesity diet induced Kv1.3 expression in the liver, and ShK-186 caused profound alterations in energy and lipid metabolism in the liver. This action on the liver may underlie the differential effectiveness of ShK-186 in mice fed a chow vs. an obesity diet. Our results highlight the potential use of Kv1.3 blockers for the treatment of obesity and insulin resistance.

Dual control of dopamine synthesis and release by presynaptic and postsynaptic dopamine D2 receptors.

Dysfunctions of dopaminergic homeostasis leading to either low or high dopamine (DA) levels are causally linked to Parkinson's disease, schizophrenia, and addiction. Major sites of DA synthesis are the mesencephalic neurons originating in the substantia nigra and ventral tegmental area; these structures send major projections to the dorsal striatum (DSt) and nucleus accumbens (NAcc), respectively. DA finely tunes its own synthesis and release by activating DA D2 receptors (D2R). To date, this critical D2R-dependent function was thought to be solely due to activation of D2Rs on dopaminergic neurons (D2 autoreceptors); instead, using site-specific D2R knock-out mice, we uncover that D2 heteroreceptors located on non-DAergic medium spiny neurons participate in the control of DA levels. This D2 heteroreceptor-mediated mechanism is more efficient in the DSt than in NAcc, indicating that D2R signaling differentially regulates mesolimbic- versus nigrostriatal-mediated functions. This study reveals previously unappreciated control of DA signaling, shedding new light on region-specific regulation of DA-mediated effects.

Regulatory roles of heterogeneous nuclear ribonucleoprotein M and Nova-1 protein in alternative splicing of dopamine D2 receptor pre-mRNA.

The dopamine D2 receptor (D2R) plays a crucial role in the regulation of diverse key physiological functions, including motor control, reward, learning, and memory. This receptor is present in vivo in two isoforms, D2L and D2S, generated from the same gene by alternative pre-mRNA splicing. Each isoform has a specific role in vivo, underlining the importance of a strict control of its synthesis, yet the molecular mechanism modulating alternative D2R pre-mRNA splicing has not been completely elucidated. Here, we identify heterogeneous nuclear ribonucleoprotein M (hnRNP M) as a key molecule controlling D2R splicing. We show that binding of hnRNP M to exon 6 inhibited the inclusion of this exon in the mRNA. Importantly, the splicing factor Nova-1 counteracted hnRNP M effects on D2R pre-mRNA splicing. Indeed, mutations of the putative Nova-1-binding site on exon 6 disrupted Nova-1 RNA assembly and diminished the inhibitory effect of Nova-1 on hnRNP M-dependent exon 6 exclusion. These results identify Nova-1 and hnRNP M as D2R pre-mRNA-binding proteins and show their antagonistic role in the alternative splicing of D2R pre-mRNA.

Contribution of Kv1.2 voltage-gated potassium channel to D2 autoreceptor regulation of axonal dopamine overflow.

Impairments in axonal dopamine release are associated with neurological disorders such as schizophrenia and attention deficit hyperactivity disorder and pathophysiological conditions promoting drug abuse and obesity. The D2 dopamine autoreceptor (D2-AR) exerts tight regulatory control of axonal dopamine (DA) release through a mechanism suggested to involve K(+) channels. To evaluate the contribution of Kv1 voltage-gated potassium channels of the Shaker gene family to the regulation of axonal DA release by the D2-AR, the present study employed expression analyses, real time measurements of striatal DA overflow, K(+) current measurements and immunoprecipitation assays. Kv1.1, -1.2, -1.3, and -1.6 mRNA and protein were detected in midbrain DA neurons purified by fluorescence-activated cell sorting and in primary DA neuron cultures. In addition, Kv1.1, -1.2, and -1.6 were localized to DA axonal processes in the dorsal striatum. By means of fast scan cyclic voltammetry in striatal slice preparations, we found that the inhibition of stimulation-evoked DA overflow by a D2 agonist was attenuated by Kv1.1, -1.2, and -1.6 toxin blockers. A particular role for the Kv1.2 subunit in the process whereby axonal D2-AR inhibits DA overflow was established with the use of a selective Kv1.2 blocker and Kv1.2 knock-out mice. Moreover, we demonstrate the ability of D2-AR activation to increase Kv1.2 currents in co-transfected cells and its reliance on Gßγ subunit signaling along with the physical coupling of D2-AR and Kv1.2-containing channels in striatal tissue. These findings underline the contribution of Kv1.2 in the regulation of nigrostriatal DA release by the D2-AR and thereby offer a novel mechanism by which DA release is regulated.

D2R signaling in striatal spiny neurons modulates L-DOPA induced dyskinesia.

Degeneration of dopaminergic neurons leads to Parkinson's disease (PD), characterized by reduced levels of striatal dopamine (DA) and impaired voluntary movements. DA replacement is achieved by levodopa treatment which in long-term causes involuntary movements or dyskinesia. Dyskinesia is linked to the pulsatile activation of D1 receptors of the striatal medium spiny neurons (MSNs) forming the direct output pathway (dMSNs). The contribution of DA stimulation of D2R in MSNs of the indirect pathway (iMSNs) is less clear. Using the 6-hydroxydopamine model of PD, here we show that loss of DA-mediated inhibition of these neurons intensifies levodopa-induced dyskinesia (LID) leading to reprogramming of striatal gene expression. We propose that the motor impairments characteristic of PD and of its therapy are critically dependent on D2R-mediated iMSNs activity. D2R signaling not only filters inputs to the striatum but also indirectly regulates dMSNs mediated responses.

Rapid-acting antidepressants and the circadian clock.

A growing number of epidemiological and experimental studies has established that circadian disruption is strongly associated with psychiatric disorders, including major depressive disorder (MDD). This association is becoming increasingly relevant considering that modern lifestyles, social zeitgebers (time cues) and genetic variants contribute to disrupting circadian rhythms that may lead to psychiatric disorders. Circadian abnormalities associated with MDD include dysregulated rhythms of sleep, temperature, hormonal secretions, and mood which are modulated by the molecular clock. Rapid-acting antidepressants such as subanesthetic ketamine and sleep deprivation therapy can improve symptoms within 24 h in a subset of depressed patients, in striking contrast to conventional treatments, which generally require weeks for a full clinical response. Importantly, animal data show that sleep deprivation and ketamine have overlapping effects on clock gene expression. Furthermore, emerging data implicate the circadian system as a critical component involved in rapid antidepressant responses via several intracellular signaling pathways such as GSK3ß, mTOR, MAPK, and NOTCH to initiate synaptic plasticity. Future research on the relationship between depression and the circadian clock may contribute to the development of novel therapeutic strategies for depression-like symptoms. In this review we summarize recent evidence describing: (1) how the circadian clock is implicated in depression, (2) how clock genes may contribute to fast-acting antidepressants, and (3) the mechanistic links between the clock genes driving circadian rhythms and neuroplasticity.

Inhibition of dopamine uptake by D2 antagonists: an in vivo study.

D(2)-like antagonists potentiate dopamine release. They also inhibit dopamine uptake by a mechanism yet to be clarified. Here, we monitored dopamine uptake in the striatum of anesthetized mice. The dopamine overflow was evoked by brief electrical stimulation of the medial forebrain bundle (four pulses at 100 Hz) and was monitored with carbon fiber electrodes combined with continuous amperometry. The decay phase of evoked overflows reflects dopamine half-life, which entirely depends on uptake. The D(2)-like antagonists haloperidol and eticlopride enhanced the half-life by 45% and 48%, respectively, a moderate effect as compared to the uptake blocker nomifensine (528%). Both D(2)-like antagonists did not affect dopamine uptake in mice lacking D(2) receptors. Inhibition of tonic dopamine release by gamma-butyrolactone did not mimic the enhancing effect of D(2) antagonists on dopamine half-life. However, prolonged stimulation boosted dopamine uptake and this effect was not observed after haloperidol treatment or in mice lacking D(2) receptors. Therefore, dopamine uptake is accelerated in conditions of excessive D(2) stimulation but not finely tuned in resting conditions. Inhibition of dopamine uptake by D(2) antagonists synergizes with the potentiation of dopamine release to strongly alter the phasic dopamine signaling.

Neurological defects in trichothiodystrophy reveal a coactivator function of TFIIH.

Mutations in the XPD subunit of the DNA repair/transcription factor TFIIH yield the rare genetic disorder trichothiodystrophy (TTD). Although this syndrome was initially associated with a DNA repair defect, individuals with TTD develop neurological features, such as microcephaly and hypomyelination that could be connected to transcriptional defects. Here we show that an XPD mutation in TTD mice results in a spatial and selective deregulation of thyroid hormone target genes in the brain. Molecular analyses performed on the mice brain tissue demonstrate that TFIIH is required for the stabilization of thyroid hormone receptors (TR) to their DNA-responsive elements. The limiting amounts of TFIIH found in individuals with TTD thus contribute to the deregulation of TR-responsive genes. The discovery of an unexpected stabilizing function for TFIIH deepens our understanding of the pathogenesis and neurological manifestations observed in TTD individuals.

Genetically determined interaction between the dopamine transporter and the D2 receptor on prefronto-striatal activity and volume in humans.

Dopamine modulation of neuronal activity during memory tasks identifies a nonlinear inverted-U shaped function. Both the dopamine transporter (DAT) and dopamine D(2) receptors (encoded by DRD(2)) critically regulate dopamine signaling in the striatum and in prefrontal cortex during memory. Moreover, in vitro studies have demonstrated that DAT and D(2) proteins reciprocally regulate each other presynaptically. Therefore, we have evaluated the genetic interaction between a DRD(2) polymorphism (rs1076560) causing reduced presynaptic D(2) receptor expression and the DAT 3'-VNTR variant (affecting DAT expression) in a large sample of healthy subjects undergoing blood oxygenation level-dependent (BOLD)-functional magnetic resonance imaging (MRI) during memory tasks and structural MRI. Results indicated a significant DRD(2)/DAT interaction in prefrontal cortex and striatum BOLD activity during both working memory and encoding of recognition memory. The differential effect on BOLD activity of the DAT variant was mostly manifest in the context of the DRD(2) allele associated with lower presynaptic expression. Similar results were also evident for gray matter volume in caudate. These interactions describe a nonlinear relationship between compound genotypes and brain activity or gray matter volume. Complementary data from striatal protein extracts from wild-type and D(2) knock-out animals (D2R(-/-)) indicate that DAT and D(2) proteins interact in vivo. Together, our results demonstrate that the interaction between genetic variants in DRD(2) and DAT critically modulates the nonlinear relationship between dopamine and neuronal activity during memory processing.

Getting specialized: presynaptic and postsynaptic dopamine D2 receptors.

Dopamine (DA) signaling controls many physiological functions ranging from locomotion to hormone secretion, and plays a critical role in addiction. DA elevation, for instance in response to drugs of abuse, simultaneously activates neurons expressing different DA receptors; how responses from diverse neurons/receptors are orchestrated in the generation of behavioral and cellular outcomes, is still not completely defined. Signaling from D2 receptors (D2Rs) is a good example to illustrate this complexity. D2Rs have presynaptic and postsynaptic localization and functions, which are shared by two isoforms in vivo. Recent results from knockout mice are clarifying the role of site and D2 isoform-specific effects thereby increasing our understanding of how DA modulates neuronal physiology.

Impaired light masking in dopamine D2 receptor-null mice.

Disruption of overt circadian rhythms can occur without influencing the endogenous pacemaker, the so-called 'masking' effect classically elicited by light. As the physiological pathways involved in light masking remain elusive, we analyzed mice lacking the dopamine D2 receptor. Although circadian rhythmicity was normal, D2R-null mice showed a markedly deficient light masking response, indicating that D2R-mediated signaling is an essential component of the neuronal pathways leading to light masking of circadian rhythms.

Cocaine-mediated circadian reprogramming in the striatum through dopamine D2R and PPARγ activation.

Substance abuse disorders are linked to alteration of circadian rhythms, although the molecular and neuronal pathways implicated have not been fully elucidated. Addictive drugs, such as cocaine, induce a rapid increase of dopamine levels in the brain. Here, we show that acute administration of cocaine triggers reprogramming in circadian gene expression in the striatum, an area involved in psychomotor and rewarding effects of drugs. This process involves the activation of peroxisome protein activator receptor gamma (PPARγ), a nuclear receptor involved in inflammatory responses. PPARγ reprogramming is altered in mice with cell-specific ablation of the dopamine D2 receptor (D2R) in the striatal medium spiny neurons (MSNs) (iMSN-D2RKO). Administration of a specific PPARγ agonist in iMSN-D2RKO mice elicits substantial rescue of cocaine-dependent control of circadian genes. These findings have potential implications for development of strategies to treat substance abuse disorders.

Dopaminergic Control of Striatal Cholinergic Interneurons Underlies Cocaine-Induced Psychostimulation.

Cocaine drastically elevates dopamine (DA) levels in the striatum, a brain region that is critical to the psychomotor and rewarding properties of the drug. DA signaling regulates intrastriatal circuits connecting medium spiny neurons (MSNs) with afferent fibers and interneurons. While the cocaine-mediated increase in DA signaling on MSNs is well documented, that on cholinergic interneurons (ChIs) has been more difficult to assess. Using combined pharmacological, chemogenetic, and cell-specific ablation approaches, we reveal that the D2R-dependent inhibition of acetylcholine (ACh) signaling is fundamental to cocaine-induced changes in behavior and the striatal genomic response. We show that the D2R-dependent control of striatal ChIs enables the motor, sensitized, and reinforcing properties of cocaine. This study highlights the importance of the DA- and D2R-mediated inhibitory control of ChIs activity in the normal functioning of striatal networks.