Neurofibromin 1 in mushroom body neurons mediates circadian wake drive through activating cAMP-PKA signaling.
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ABSTRACT:
SUBMITTER: Machado Almeida P
PROVIDER: S-EPMC8486785 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:The mushroom bodies (MBs), one of the main structures in the adult insect brain, play a critical role in olfactory learning and memory. Though historical genes such as dunce and rutabaga, which regulate the level of cAMP, were identified more than 30 years ago, their in vivo effects on cellular and physiological mechanisms and particularly on the Ca(2+)-responses still remain largely unknown. In this work, performed in Drosophila, we took advantage of in vivo bioluminescence imaging, which allowed real-time monitoring of the entire MBs (both the calyx/cell-bodies and the lobes) simultaneously. We imaged neuronal Ca(2+)-activity continuously, over a long time period, and characterized the nicotine-evoked Ca(2+)-response. Using both genetics and pharmacological approaches to interfere with different components of the cAMP signaling pathway, we first show that the Ca(2+)-response is proportional to the levels of cAMP. Second, we reveal that an acute change in cAMP levels is sufficient to trigger a Ca(2+)-response. Third, genetic manipulation of protein kinase A (PKA), a direct effector of cAMP, suggests that cAMP also has PKA-independent effects through the cyclic nucleotide-gated Ca(2+)-channel (CNG). Finally, the disruption of calmodulin, one of the main regulators of the rutabaga adenylate cyclase (AC), yields different effects in the calyx/cell-bodies and in the lobes, suggesting a differential and regionalized regulation of AC. Our results provide insights into the complex Ca(2+)-response in the MBs, leading to the conclusion that cAMP modulates the Ca(2+)-responses through both PKA-dependent and -independent mechanisms, the latter through CNG-channels.
| S-EPMC4596083 | biostudies-other
Project description:Serotonin (5-HT), norepinephrine and orexins (ORX) are the three best established mediators of wake-related activation of hypoglossal (XII) motoneurons that innervate the muscles of the tongue. Since the tongue's use is temporarily closely aligned with the rest-activity cycle, we tested whether expression of mRNA for relevant 5-HT, norepinephrine and ORX receptors varies in the XII nucleus with the rest-activity cycle. Adult rats (n=7-9/group) were decapitated at 8-9 am (near rest period onset) or at 6-7 pm (near active period onset). Tissue micropunches were extracted from medullary slices containing the XII motor and sensory external cuneate (ECN) nuclei. 5-HT2A, ?1-adrenergic and ORX type 2 receptor mRNAs were quantified using RT-PCR. Only 5-HT2A receptor mRNA levels differed between the two time points and were higher at the active period onset; no differences were detected in the ECN. Consistent with the mRNA results, 5-HT2A protein levels were also higher in the XII nucleus at the active period onset than at rest onset. Thus, the endogenous serotonergic excitatory drive to XII motoneurons may be enhanced through circadian- or activity-dependent mechanisms that increase the availability of 5-HT2A receptors prior to the active period. Conversely, reduced levels of 5-HT2A receptors during the rest-sleep period may exacerbate the propensity for sleep-disordered breathing in subjects with anatomically compromised upper airway.
| S-EPMC3778066 | biostudies-literature
Project description:Meteorin-like (METRNL) protein is a newly identified myokine that functions to modulate energy expenditure and inflammation in adipose tissue. Herein, we aim to investigate the potential role and molecular basis of METRNL in doxorubicin (DOX)-induced cardiotoxicity. METRNL was found to be abundantly expressed in cardiac muscle under physiological conditions that was decreased upon DOX exposure. Cardiac-specific overexpression of METRNL by adeno-associated virus serotype 9 markedly improved oxidative stress, apoptosis, cardiac dysfunction and survival status in DOX-treated mice. Conversely, knocking down endogenous METRNL by an intramyocardial injection of adenovirus exacerbated DOX-induced cardiotoxicity and death. Meanwhile, METRNL overexpression attenuated, while METRNL silence promoted oxidative damage and apoptosis in DOX-treated H9C2 cells. Systemic METRNL depletion by a neutralizing antibody aggravated DOX-related cardiac injury and dysfunction in vivo, which were notably alleviated by METRNL overexpression within the cardiomyocytes. Besides, we detected robust METRNL secretion from isolated rodent hearts and cardiomyocytes, but to a less extent in those with DOX treatment. And the beneficial effects of METRNL in H9C2 cells disappeared after the incubation with a METRNL neutralizing antibody. Mechanistically, METRNL activated SIRT1 via the cAMP/PKA pathway, and its antioxidant and antiapoptotic capacities were blocked by SIRT1 deficiency. More importantly, METRNL did not affect the tumor-killing action of DOX in 4T1 breast cancer cells and tumor-bearing mice. Collectively, cardiac-derived METRNL activates SIRT1 via cAMP/PKA signaling axis in an autocrine manner, which ultimately improves DOX-elicited oxidative stress, apoptosis and cardiac dysfunction. Targeting METRNL may provide a novel therapeutic strategy for the prevention of DOX-associated cardiotoxicity.
| S-EPMC7558217 | biostudies-literature
Project description:During fasting, mammals maintain normal glucose homeostasis by stimulating hepatic gluconeogenesis. Elevations in circulating glucagon and epinephrine, two hormones that activate hepatic gluconeogenesis, trigger the cAMP-mediated phosphorylation of cAMP response element-binding protein (Creb) and dephosphorylation of the Creb-regulated transcription coactivator-2 (Crtc2)--two key transcriptional regulators of this process. Although the underlying mechanism is unclear, hepatic gluconeogenesis is also regulated by the circadian clock, which coordinates glucose metabolism with changes in the external environment. Circadian control of gene expression is achieved by two transcriptional activators, Clock and Bmal1, which stimulate cryptochrome (Cry1 and Cry2) and Period (Per1, Per2 and Per3) repressors that feed back on Clock-Bmal1 activity. Here we show that Creb activity during fasting is modulated by Cry1 and Cry2, which are rhythmically expressed in the liver. Cry1 expression was elevated during the night-day transition, when it reduced fasting gluconeogenic gene expression by blocking glucagon-mediated increases in intracellular cAMP concentrations and in the protein kinase A-mediated phosphorylation of Creb. In biochemical reconstitution studies, we found that Cry1 inhibited accumulation of cAMP in response to G protein-coupled receptor (GPCR) activation but not to forskolin, a direct activator of adenyl cyclase. Cry proteins seemed to modulate GPCR activity directly through interaction with G(s)?. As hepatic overexpression of Cry1 lowered blood glucose concentrations and improved insulin sensitivity in insulin-resistant db/db mice, our results suggest that compounds that enhance cryptochrome activity may provide therapeutic benefit to individuals with type 2 diabetes.
| S-EPMC2952072 | biostudies-literature
Project description:Although the mammalian rest-activity cycle is controlled by a "master clock" in the suprachiasmatic nucleus (SCN) of the hypothalamus, it is unclear how firing of individual SCN neurons gates individual features of daily activity. Here, we demonstrate that a specific transcriptomically identified population of mouse VIP+ SCN neurons is active at the "wrong" time of day-nighttime-when most SCN neurons are silent. Using chemogenetic and optogenetic strategies, we show that these neurons and their cellular clocks are necessary and sufficient to gate and time nighttime sleep but have no effect upon daytime sleep. We propose that mouse nighttime sleep, analogous to the human siesta, is a "hard-wired" property gated by specific neurons of the master clock to favor subsequent alertness prior to dawn (a circadian "wake maintenance zone"). Thus, the SCN is not simply a 24-h metronome: specific populations sculpt critical features of the sleep-wake cycle.
| S-EPMC7803671 | biostudies-literature
Project description:Increasing evidence suggests an important role for VPAC2-activated signal transduction pathways in maintaining a synchronized biological clock in the suprachiasmatic nucleus (SCN). Activation of the VPAC2 signaling pathway induces per1 gene expression in the SCN and phase-shifts the circadian clock. Mice without the VPAC2 receptor lack an overt, coherent circadian rhythm in clock gene expression, SCN neuron firing rate, and locomotor behavior. Using a systems approach, we have developed a kinetic model integrating VPAC2 signaling mediated by the cyclic AMP (cAMP)/protein kinase A (PKA) pathway and leading to induced circadian clock gene expression. We fit the model to experimental data from the literature for cAMP accumulation, PKA activation, cAMP-response element binding protein phosphorylation, and per1 induction. By linking the VPAC2 model to a published circadian clock model, we also simulated clock phase shifts induced by vasoactive intestinal polypeptide (VIP) and matched experimental data for the VIP response. The simulated phase response curve resembled the hamster response to a related neuropeptide, GRP1-27, and light. Simulations using pulses of VIP revealed that the system response is extraordinarily robust to input signal duration, a result with physiologically relevant consequences. Lastly, simulations using varied receptor levels matched literature experimental data from animals overexpressing VPAC2 receptors.
| S-EPMC1367307 | biostudies-literature
Project description:Although the mammalian rest-activity cycle is controlled by a "master clock" in the suprachiasmatic nuclei (SCN) of the hypothalamus, it is unclear how firing of individual SCN neurons gates individual features of daily activity. Here, we demonstrate that a specific transcriptomically identified population of mouse VIP+ SCN neurons is active at the "wrong" time of the day -nighttime- when most SCN neurons are silent. Using chemogenetic and optogenetic strategies, we show that these neurons and their cellular clocks are necessary and sufficient to gate and time nighttime sleep, but have no effect upon daytime sleep. We propose mouse nighttime sleep, analogous to the human siesta, is a "hard-wired" property gated by specific neurons of the master clock to favor subsequent alertness prior to dawn (a circadian "wake maintenance zone"). Thus, the SCN is not simply a 24h metronome: specific populations sculpt critical features of the sleep-wake cycle.
2021-01-21 | GSE154038 | GEO
Project description:The blood-brain barrier (BBB) of Drosophila comprises a thin epithelial layer of subperineural glia (SPG), which ensheath the nerve cord and insulate it against the potassium-rich hemolymph by forming intercellular septate junctions (SJs). Previously, we identified a novel Gi/Go protein-coupled receptor (GPCR), Moody, as a key factor in BBB formation at the embryonic stage. However, the molecular and cellular mechanisms of Moody signaling in BBB formation and maturation remain unclear. Here, we identify cAMP-dependent protein kinase A (PKA) as a crucial antagonistic Moody effector that is required for the formation, as well as for the continued SPG growth and BBB maintenance in the larva and adult stage. We show that PKA is enriched at the basal side of the SPG cell and that this polarized activity of the Moody/PKA pathway finely tunes the enormous cell growth and BBB integrity. Moody/PKA signaling precisely regulates the actomyosin contractility, vesicle trafficking, and the proper SJ organization in a highly coordinated spatiotemporal manner. These effects are mediated in part by PKA's molecular targets MLCK and Rho1. Moreover, 3D reconstruction of SJ ultrastructure demonstrates that the continuity of individual SJ segments, and not their total length, is crucial for generating a proper paracellular seal. Based on these findings, we propose that polarized Moody/PKA signaling plays a central role in controlling the cell growth and maintaining BBB integrity during the continuous morphogenesis of the SPG secondary epithelium, which is critical to maintain tissue size and brain homeostasis during organogenesis.
| S-EPMC8390003 | biostudies-literature
Project description:Neural regulation of sleep and metabolic homeostasis are critical in many aspects of human health. Despite extensive epidemiological evidence linking sleep dysregulation with obesity, diabetes, and metabolic syndrome, little is known about the neural and molecular basis for the integration of sleep and metabolic function. The RAS GTPase-activating gene Neurofibromin (Nf1) has been implicated in the regulation of sleep and metabolic rate, raising the possibility that it serves to integrate these processes, but the effects on sleep consolidation and physiology remain poorly understood. A key hallmark of sleep depth in mammals and flies is a reduction in metabolic rate during sleep. Here, we examine multiple measures of sleep quality to determine the effects of Nf1 on sleep-dependent changes in arousal threshold and metabolic rate. Flies lacking Nf1 fail to suppress metabolic rate during sleep, raising the possibility that loss of Nf1 prevents flies from integrating sleep and metabolic state. Sleep of Nf1 mutant flies is fragmented with a reduced arousal threshold in Nf1 mutants, suggesting Nf1 flies fail to enter deep sleep. The effects of Nf1 on sleep can be localized to a subset of neurons expressing the GABAA receptor Rdl. Sleep loss has been associated with changes in gut homeostasis in flies and mammals. Selective knockdown of Nf1 in Rdl-expressing neurons within the nervous system increases gut permeability and reactive oxygen species (ROS) in the gut, raising the possibility that loss of sleep quality contributes to gut dysregulation. Together, these findings suggest Nf1 acts in GABA-sensitive neurons to modulate sleep depth in Drosophila.
| S-EPMC10763969 | biostudies-literature
Project description:Serotonin (5-HT) and dopamine are critical neuromodulators known to regulate a range of behaviors in invertebrates and mammals, such as learning and memory. Effects of both serotonin and dopamine are mediated largely through their downstream G-protein coupled receptors through cAMP-PKA signaling. While the role of dopamine in olfactory learning in Drosophila is well described, the function of serotonin and its downstream receptors on Drosophila olfactory learning remain largely unexplored. In this study we show that the output of serotonergic neurons, possibly through points of synaptic contacts on the mushroom body (MB), is essential for training during olfactory associative learning in Drosophila larvae. Additionally, we demonstrate that the regulation of olfactory associative learning by serotonin is mediated by its downstream receptor (d5-HT7) in a cAMP-dependent manner. We show that d5-HT7 expression specifically in the MB, an anatomical structure essential for olfactory learning in Drosophila, is critical for olfactory associative learning. Importantly our work shows that spatio-temporal restriction of d5-HT7 expression to the MB is sufficient to rescue olfactory learning deficits in a d5-HT7 null larvae. In summary, our results establish a critical, and previously unknown, role of d5-HT7 in olfactory learning.
| S-EPMC7718245 | biostudies-literature