Transcriptomic data of human adrenocortical NCI-H295R cells treated with cortisol biosynthesis inhibitors.

Adrenal corticosteroid biosynthesis dysregulation can give rise to various pathological conditions, such as Cushing's syndrome, a disorder characterized by the sustained and excessive production of cortisol. Despite the development of several classes of steroidogenesis inhibitors to treat human diseases associated with cortisol overproduction, their use is limited by insufficient efficacy, adverse effects, and/or tolerability. Recently, we identified a series of benzimidazolylurea derivatives, including the representative compound CJ28, as novel cortisol biosynthesis inhibitors [1]. They significantly inhibited both basal and stimulated production of cortisol in NCI-H295R cells, a human adrenocarcinoma cell line. The inhibitory effects were attributed to both attenuated steroidogenesis and de novo cholesterol biosynthesis. Here, we provide transcriptomic (RNA-seq) data from adrenal cell cultures in response to treatment with either CJ28 or metyrapone (MET), an inhibitor of 11ß-hydroxylase). Total RNA was extracted from the cells treated with vehicle (0.1% DMSO), CJ28 (30 µM), or MET (30 µM) for 24 h. Primary sequence data were acquired using paired-end sequencing on an Illumina NovaSeq 6000 platform. The raw RNA-seq data have been deposited in the Gene Expression Omnibus (GEO) database (GSE236435). This dataset is a useful resource for providing valuable information on the gene expression networks underlying adrenocortical steroidogenesis.

Functional Characterization of Circadian Nuclear Receptors REV-ERBα and REV-ERBß in Human Osteosarcoma Cell Cultures.

REV-ERBα and its paralog, REV-ERBß, encoded by NR1D1 and NR1D2 genes, are key nuclear receptors that link the circadian timing system and metabolic homeostasis. Since heme is an endogenous ligand, REV-ERBs have been considered key components of the circadian molecular clock and can be pharmacologically targeted to treat various circadian rhythm-related diseases, such as cardiometabolic, inflammatory, and neuropsychiatric diseases, as well as cancer. REV-ERBs are believed to be functionally redundant and compensatory, although they often affect the expression of gene subsets in an isoform-specific manner. Therefore, this study aimed to identify the redundant and distinct roles of each isoform in controlling its target genes by comparing the transcriptome profiles of a panel of mutant U2OS human osteosarcoma cells in which either NR1D1 or NR1D2 was ablated. Indeed, our transcriptomic analyses revealed that most REV-ERB-regulated genes are controlled by redundant or even additive actions. However, the RNA expression profiles of each single mutant cell line also provide strong evidence for isoform-dependent actions. For example, REV-ERBα is more responsible for regulating the NF-κΒ signaling pathway, whereas a group of extracellular matrix components requires REV-ERBß to maintain their expression. We found that REV-ERBs have isoform-selective functions in the regulation of certain circadian output pathways despite their overlapping roles in the circadian molecular clock. Thus, the development of isoform-selective REV-ERB modulators can help treat metabolic disturbances and certain types of cancer.

Eye-Mimicked Neural Network Composed of Photosensitive Neural Spheroids with Human Opsin Proteins.

An in vitro model, composed of the short-wavelength human opsins and rhodopsins, is created. Two types of photosensitive neural spheroids are transfected for selective reaction under bluish-purple and green lights. These are employed to two devices with intact neuron and neural-spheroid to study the interaction. By photostimulation, the photosensitive spheroid initiated photoactivation, and the signal generated from its body is transmitted to adjacent neural networks. Specifically, the signal traveled through the axon bundle in narrow gap from photosensitive spheroid to intact spheroid as an eye-to-brain model including optic nerve. The whole process with photosensitive spheroid is monitored by calcium ion detecting fluorescence images. The results of this study can be applied to examine vision restoration and novel photosensitive biological systems with spectral sensitivity.

Identification of a novel class of cortisol biosynthesis inhibitors and its implications in a therapeutic strategy for hypercortisolism.

AIMS: Dysregulation of adrenocortical steroid (corticosteroids) biosynthesis leads to pathological conditions such as Cushing's syndrome. Although several classes of steroid biosynthesis inhibitors have been developed to treat cortisol overproduction, limitations such as insufficient efficacy, adverse effects, and/or tolerability still remain. The present study aimed to develop a new class of small molecules that inhibit cortisol production, and investigated their putative modes of action. MAIN METHODS: We screened an in-house chemical library with drug-like chemical scaffolds using human adrenocortical NCI-H295R cells. We then evaluated and validated the effects of the selected compounds at multiple regulatory steps of the adrenal steroidogenic pathway. Finally, genome-wide RNA expression analysis coupled with gene enrichment analysis was conducted to infer possible action mechanisms. KEY FINDINGS: A subset of benzimidazolylurea derivatives, including a representative compound (designated as CJ28), inhibited both basal and stimulated production of cortisol and related intermediate steroids. CJ28 attenuated the mRNA expression of multiple genes involved in steroidogenesis and cholesterol biosynthesis. Furthermore, CJ28 significantly attenuated de novo cholesterol biosynthesis, which contributed to its suppression of cortisol production. SIGNIFICANCE: We identified a novel chemical scaffold that exerts inhibitory effects on cortisol and cholesterol biosynthesis via coordinated transcriptional silencing of gene expression networks. Our findings also reveal an additional adrenal-directed pharmacological strategy for hypercortisolism involving a combination of inhibitors targeting steroidogenesis and de novo cholesterol biosynthesis.

Autophagy Modulation in Aggresome Formation: Emerging Implications and Treatments of Alzheimer's Disease.

Alzheimer's disease (AD) is one of the most prevailing neurodegenerative diseases in the world, which is characterized by memory dysfunction and the formation of tau and amyloid ß (Aß) aggregates in multiple brain regions, including the hippocampus and cortex. The formation of senile plaques involving tau hyperphosphorylation, fibrillar Aß, and neurofibrillary tangles (NFTs) is used as a pathological marker of AD and eventually produces aggregation or misfolded protein. Importantly, it has been found that the failure to degrade these aggregate-prone proteins leads to pathological consequences, such as synaptic impairment, cytotoxicity, neuronal atrophy, and memory deficits associated with AD. Recently, increasing evidence has suggested that the autophagy pathway plays a role as a central cellular protection system to prevent the toxicity induced by aggregation or misfolded proteins. Moreover, it has also been revealed that AD-related protein aggresomes could be selectively degraded by autophagosome and lysosomal fusion through the autophagy pathway, which is known as aggrephagy. Therefore, the regulation of autophagy serve as a useful approach to modulate the formation of aggresomes associated with AD. This review focuses on the recent improvements in the application of natural compounds and small molecules as a potential therapeutic approach for AD prevention and treatment via aggrephagy.

Anoctamin 1/TMEM16A in pruritoceptors is essential for Mas-related G protein receptor-dependent itch.

ABSTRACT: Itch is an unpleasant sensation that evokes a desire to scratch. Pathologic conditions such as allergy or atopic dermatitis produce severe itching sensation. Mas-related G protein receptors (Mrgprs) are receptors for many endogenous pruritogens. However, signaling pathways downstream to these receptors in dorsal root ganglion (DRG) neurons are not yet understood. We found that anoctamin 1 (ANO1), a Ca 2+ -activated chloride channel, is a transduction channel mediating Mrgpr-dependent itch signals. Genetic ablation of Ano1 in DRG neurons displayed a significant reduction in scratching behaviors in response to acute and chronic Mrgpr-dependent itch models and the epidermal hyperplasia induced by dry skin. In vivo Ca 2+ imaging and electrophysiological recording revealed that chloroquine and other agonists of Mrgprs excited DRG neurons via ANO1. More importantly, the overexpression of Ano1 in DRG neurons of Ano1 -deficient mice rescued the impaired itching observed in Ano1 -deficient mice. These results demonstrate that ANO1 mediates the Mrgpr-dependent itch signaling in pruriceptors and provides clues to treating pathologic itch syndromes.

Neural correlates of multidimensional motor outputs in an excitatory parafascicular-zona incerta circuit.

The parafascicular nucleus (Pf) in medial thalamus is interconnected with prefrontal cortex and basal ganglia. Though much research has determined its importance in cognitive regulation of behaviour, its projections to regions in subthalamus remain less known. Such connections include those to zona incerta (ZI), located immediately dorsal to subthalamic nuclei (STN) regulating motor output, and whose role in a motor context is only beginning to be investigated. We thus examined circuits from parafascicular (Pf) thalamus to ZI, and its activity during locomotion and spontaneous behaviours in mice. We found that a distinct group of CaMKIIα-positive excitatory parafascicular neurons, separated from VGLUT2-positive excitatory neurons, project widely into ZI, more than adjacent STN. Our results from fibre photometry and decoding with general linear model (GLM) indicate that PF-ZI pathways do not specifically correlate with amount of locomotion or movement velocity, but instead show more specified activity during relative directional changes of movements observed in turning, sniffing behaviours. These results hint at the PF-ZI pathway having a distinct role in directing action specificity and have implications for subcortical bases in dimensional control of behaviours.

Subanesthetic ketamine rapidly alters medial prefrontal miRNAs involved in ubiquitin-mediated proteolysis.

Ketamine is a dissociative anesthetic and a non-competitive NMDAR antagonist. At subanesthetic dose, ketamine can relieve pain and work as a fast-acting antidepressant, but the underlying molecular mechanism remains elusive. This study aimed to investigate the mode of action underlying the effects of acute subanesthetic ketamine treatment by bioinformatics analyses of miRNAs in the medial prefrontal cortex of male C57BL/6J mice. Gene Ontology and KEGG pathway analyses of the genes putatively targeted by ketamine-responsive prefrontal miRNAs revealed that acute subanesthetic ketamine modifies ubiquitin-mediated proteolysis. Validation analysis suggested that miR-148a-3p and miR-128-3p are the main players responsible for the subanesthetic ketamine-mediated alteration of ubiquitin-mediated proteolysis through varied regulation of ubiquitin ligases E2 and E3. Collectively, our data imply that the prefrontal miRNA-dependent modulation of ubiquitin-mediated proteolysis is at least partially involved in the mode of action by acute subanesthetic ketamine treatment.

Development of Non-Ethoxypropanoic Acid Type Cryptochrome Inhibitors with Circadian Molecular Clock-Enhancing Activity by Bioisosteric Replacement.

Circadian dysfunction is closely associated with an increased risk of various diseases. Considering that molecular clock machinery serves as an intrinsic time-keeping system underlying the circadian rhythm of biological processes, the modulation of the molecular clock machinery is an attractive therapeutic target with novel mechanisms of action. Based on the previous structure-activity relationship study of small molecule cryptochrome (CRY) inhibitors possessing an ethoxypropanoic acid moiety, non-ethoxypropanoic acid-type inhibitors have been developed by bioisosteric replacement. They were evaluated as potent and effective enhancers of E-box-mediated transcription, and, in particular, ester 5d and its hydrolysis product 2d exhibited desirable metabolic and pharmacokinetic profiles as promising drug candidates. Compound 2d directly bound to both CRY1 and 2 in surface plasmon resonance analyses, suggesting that the molecular target is CRY. Effects of compound 5d and 2d on suppressive action of CRY1 on CLOCK:BMAL1-activated E-box-LUC reporter activity revealed that both compounds inhibited the negative feedback actions of CRY on CLOCK:BMAL1. Most importantly, compounds 5d and 2d exhibited significant effects on molecular circadian rhythmicity to be considered circadian clock-enhancers, distinct from the previously developed CRY inhibitors possessing an ethoxypropanoic acid moiety.

Dying neurons conduct repair processes in the injured brain through osteopontin expression in cooperation with infiltrated blood monocytes.

The brain has an intrinsic capacity to repair injury, but the specific mechanisms are largely unknown. In this study, we found that, despite their incipient death, damaged neurons play a key repair role with the help of monocytes infiltrated from blood. Monocytes phagocytosed damaged and/or dying neurons that expressed osteopontin (OPN), with possible subsequent activation of their inflammasome pathway, resulting in pyroptosis. During this process, monocytes released CD63-positive exosome-like vesicles containing OPN. Importantly, following the exosome-like vesicles, neuron and astrocyte processes elongated toward the injury core. In addition, exosomes prepared from the injured brain contained OPN, and enhanced neurite outgrowth of cultured neurons in an OPN-dependent manner. Thus, our results introduce the concept that neurons in the injured brain that are destined to die perceive the stressful condition and begin the regeneration processes through induction of OPN, ultimately executing the repair process with the help of monocytes recruited from the circulation.

How Microglia Manages Non-cell Autonomous Vicious Cycling of Aß Toxicity in the Pathogenesis of AD.

Alzheimer's disease (AD) is a progressive neurodegenerative disease and a common form of dementia that affects cognition and memory mostly in aged people. AD pathology is characterized by the accumulation of ß-amyloid (Aß) senile plaques and the neurofibrillary tangles of phosphorylated tau, resulting in cell damage and neurodegeneration. The extracellular deposition of Aß is regarded as an important pathological marker and a principal-agent of neurodegeneration. However, the exact mechanism of Aß-mediated pathogenesis is not fully understood yet. Recently, a growing body of evidence provides novel insights on the major role of microglia and its non-cell-autonomous cycling of Aß toxicity. Hence, this article provides a comprehensive overview of microglia as a significant player in uncovering the underlying disease mechanisms of AD.

Programming effects of maternal stress on the circadian system of adult offspring.

Maternal stress has long-lasting influences on the brain functions of offspring, and several brain regions have been proposed to mediate such programming. Although perinatal programming of crosstalk between the circadian and stress systems has been proposed, the functional consequences of prenatal stress on the circadian system and the underlying mechanisms remain largely unknown. Therefore, we investigated whether exposing pregnant mice to chronic restraint stress had prolonged effects on the suprachiasmatic nucleus (SCN), which bears the central pacemaker for mammalian circadian rhythms, of offspring. SCN explants from maternally stressed mice exhibited altered cyclic expression patterns of a luciferase reporter under control of the mouse Per1 promoter (mPer1::LUC), which manifested as a decreased amplitude and impaired stability of the rhythm. Bioluminescence imaging at the single-cell level subsequently revealed that impaired synchrony among individual cells was responsible for the impaired rhythmicity. These intrinsic defects appeared to persist during adulthood. Adult male offspring from stressed mothers showed advanced-phase behavioral rhythms with impaired stability as well as altered clock gene expression in the SCN. In addition to affecting the central rhythm, maternal stress also had prolonged influences on the circadian characteristics of the adrenal gland and liver, as determined by circulating corticosterone levels and hepatic glycogen content, and on canonical clock gene mRNA expression in those tissues. Taken together, our findings suggest that the SCN is a key target of the programming effects of maternal stress. The widespread effects of circadian disruptions caused by a misprogrammed clock may have further impacts on metabolic and mental health in later life.

Real-Time Temporal Dynamics of Bicistronic Expression Mediated by Internal Ribosome Entry Site and 2A Cleaving Sequence.

Multicistronic elements, such as the internal ribosome entry site (IRES) and 2A-like cleavage sequence, serve crucial roles in the eukaryotic ectopic expression of exogenous genes. For utilization of multicistronic elements, the cleavage efficiency and order of elements in multicistronic vectors have been investigated; however, the dynamics of multicistronic element-mediated expression remains unclear. Here, we investigated the dynamics of encephalomyocarditis virus (EMCV) IRES- and porcine teschovirus-1 2A (p2A)-mediated expression. By utilizing real-time fluorescent imaging at a minute-level resolution, we monitored the expression of fluorescent reporters bridged by either EMCV IRES or p2A in two independent cultured cell lines, HEK293 and Neuro2a. We observed significant correlations for the two fluorescent reporters in both multicistronic elements, with a higher correlation coefficient for p2A in HEK293 but similar coefficients for IRES-mediated expression and p2A-mediated expression in Neuro2a. We further analyzed the causal relationship of multicistronic elements by convergent cross mapping (CCM). CCM revealed that in all four conditions examined, the expression of the preceding gene causally affected the dynamics of the subsequent gene. As with the cross correlation, the predictive skill of p2A was higher than that of IRES in HEK293, while the predictive skills of the two multicistronic elements were indistinguishable in Neuro2a. To summarize, we report a significant temporal correlation in both EMCV IRES- and p2A-mediated expression based on the simple bicistronic vector and real-time fluorescent monitoring. The current system also provides a valuable platform to examine the dynamic aspects of expression mediated by diverse multicistronic elements under various physiological conditions.

EWSR1, a multifunctional protein, regulates cellular function and aging via genetic and epigenetic pathways.

Ewing's sarcoma (EWS) is a bone cancer arising predominantly in young children. EWSR1 (Ewing Sarcoma breakpoint region 1/EWS RNA binding protein 1) gene is ubiquitously expressed in most cell types, indicating it has diverse roles in various cellular processes and organ development. Recently, several studies have shown that missense mutations of EWSR1 genes are known to be associated with central nervous system disorders such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Otherwise, EWSR1 plays epigenetic roles in gene expression, RNA processing, and cellular signal transduction. Interestingly, EWSR1 controls micro RNA (miRNA) levels via Drosha, leading to autophagy dysfunction and impaired dermal development. Ewsr1 deficiency also leads to premature senescence of blood cells and gamete cells with a high rate of apoptosis due to the abnormal meiosis. Despite these roles of EWSR1 in various cellular functions, the exact mechanisms are not yet understood. In this context, the current review overviews a large body of evidence and discusses on what EWSR1 genetic mutations are associated with brain diseases and on how EWSR1 modulates cellular function via the epigenetic pathway. This will provide a better understanding of bona fide roles of EWSR1 in aging and its association with brain disorders.

The Conserved Cys-2232 in Clostridioides difficile Toxin B Modulates Receptor Binding.

Clostridioides difficile toxins TcdA and TcdB are large clostridial glucosyltransferases which are the main pathogenicity factors in C. difficile-associated diseases. Four highly conserved cysteines are present in all large clostridial glucosyltransferases. In this study we focused on the conserved cysteine 2232 within the combined repetitive oligopeptide domain of TcdB from reference strain VPI10463 (clade I). Cysteine 2232 is not present in TcdB from hypervirulent strain R20291 (clade II), where a tyrosine is found instead. Replacement of cysteine 2232 by tyrosine in TcdBV PI10463 reduced binding to the soluble fragments of the two known TcdB receptors, frizzled-2 (FZD2) and poliovirus receptor-like protein-3/nectin-3 (PVRL3). In line with this, TcdBR20291 showed weak binding to PVRL3 in pull-down assays which was increased when tyrosine 2232 was exchanged for cysteine. Surprisingly, we did not observe binding of TcdBR20291 to FZD2, indicating that this receptor is less important for this toxinotype. Competition assay with the receptor binding fragments (aa 1101-1836) of TcdBV PI10463 and TcdBR20291, as well as antibodies newly developed by antibody phage display, revealed different characteristics of the yet poorly described delivery domain of TcdB harboring the second receptor binding region. In summary, we found that conserved Cys-2232 in TcdB indirectly contributes to toxin-receptor interaction.

Abrogation of the Circadian Nuclear Receptor REV-ERBα Exacerbates 6-Hydroxydopamine-Induced Dopaminergic Neurodegeneration.

Parkinson's disease (PD) is a neurodegenerative disease characterized by progressive degeneration of dopaminergic (DAergic) neurons, particularly in the substantia nigra (SN). Although circadian dysfunction has been suggested as one of the pathophysiological risk factors for PD, the exact molecular link between the circadian clock and PD remains largely unclear. We have recently demonstrated that REV-ERBα, a circadian nuclear receptor, serves as a key molecular link between the circadian and DAergic systems. It competitively cooperates with NURR1, another nuclear receptor required for the optimal development and function of DA neurons, to control DAergic gene transcription. Considering our previous findings, we hypothesize that REV-ERBα may have a role in the onset and/or progression of PD. In the present study, we therefore aimed to elucidate whether genetic abrogation of REV-ERBα affects PD-related phenotypes in a mouse model of PD produced by a unilateral injection of 6-hydroxydopamine (6-OHDA) into the dorsal striatum. REV-ERBα deficiency significantly exacerbated 6-OHDA-induced motor deficits as well as DAergic neuronal loss in the vertebral midbrain including the SN and the ventral tegmental area. The exacerbated DAergic degeneration likely involves neuroinflammation-mediated neurotoxicity. The Rev-erbα knockout mice showed prolonged microglial activation in the SN along with the overproduction of interleukin 1ß, a pro-inflammatory cytokine, in response to 6-OHDA. In conclusion, the present study demonstrates for the first time that genetic abrogation of REV-ERBα can increase vulnerability of DAergic neurons to neurotoxic insults, such as 6-OHDA, thereby implying that its normal function may be beneficial for maintaining DAergic neuron populations during PD progression.

Genetic Ablation of EWS RNA Binding Protein 1 (EWSR1) Leads to Neuroanatomical Changes and Motor Dysfunction in Mice.

A recent study reveals that missense mutations of EWSR1 are associated with neurodegenerative disorders such as amyotrophic lateral sclerosis, but the function of wild-type (WT) EWSR1 in the central nervous system (CNS) is not known yet. Herein, we investigated the neuroanatomical and motor function changes in Ewsr1 knock out (KO) mice. First, we quantified neuronal nucleus size in the motor cortex, dorsal striatum and hippocampus of three different groups: WT, heterozygous Ewsr1 KO (+/-), and homozygous Ewsr1 KO (-/-) mice. The neuronal nucleus size was significantly smaller in the motor cortex and striatum of homozygous Ewsr1 KO (-/-) mice than that of WT. In addition, in the hippocampus, the neuronal nucleus size was significantly smaller in both heterozygous Ewsr1 KO (+/-) and homozygous Ewsr1 KO (-/-) mice. We then assessed motor function of Ewsr1 KO (-/-) and WT mice by a tail suspension test. Both forelimb and hindlimb movements were significantly increased in Ewsr1 KO (-/-) mice. Lastly, we performed immunohistochemistry to examine the expression of TH, DARPP-32, and phosphorylated (p)-DARPP-32 (Thr75) in the striatum and substantia nigra, which are associated with dopaminergic signaling. The immunoreactivity of TH and DARPP-32 was decreased in Ewsr1 KO (-/-) mice. Together, our results suggest that EWSR1 plays a significant role in neuronal morphology, dopaminergic signaling pathways, and motor function in the CNS of mice.

Multimodal Regulation of Circadian Glucocorticoid Rhythm by Central and Adrenal Clocks.

Adrenal glucocorticoids (GCs) control a wide range of physiological processes, including metabolism, cardiovascular and pulmonary activities, immune and inflammatory responses, and various brain functions. During stress responses, GCs are secreted through activation of the hypothalamic-pituitary-adrenal axis, whereas circulating GC levels in unstressed states follow a robust circadian oscillation with a peak around the onset of the active period of a day. A recent advance in chronobiological research has revealed that multiple regulatory mechanisms, along with classical neuroendocrine regulation, underlie this GC circadian rhythm. The hierarchically organized circadian system, with a central pacemaker in the suprachiasmatic nucleus of the hypothalamus and local oscillators in peripheral tissues, including the adrenal gland, mediates periodicities in physiological processes in mammals. In this review, we primarily focus on our understanding of the circadian regulation of adrenal GC rhythm, with particular attention to the cooperative actions of the suprachiasmatic nucleus central and adrenal local clocks, and the clinical implications of this rhythm in human diseases.

Brain stimulation patterns emulating endogenous thalamocortical input to parvalbumin-expressing interneurons reduce nociception in mice.

BACKGROUND: The bursting pattern of thalamocortical (TC) pathway dampens nociception. Whether brain stimulation mimicking endogenous patterns can engage similar sensory gating processes in the cortex and reduce nociceptive behaviors remains uninvestigated. OBJECTIVE: We investigated the role of cortical parvalbumin expressing (PV) interneurons within the TC circuit in gating nociception and their selective response to TC burst patterns. We then tested if transcranial magnetic stimulation (TMS) patterned on endogenous nociceptive TC bursting modulate nociceptive behaviors. METHODS: The switching of TC neurons between tonic (single spike) and burst (high frequency spikes) firing modes may be a critical component in modulating nociceptive signals. Deep brain electrical stimulation of TC neurons and immunohistochemistry were used to examine the differential influence of each firing mode on cortical PV interneuron activity. Optogenetic stimulation of cortical PV interneurons assessed a direct role in nociceptive modulation. A new TMS protocol mimicking thalamic burst firing patterns, contrasted with conventional continuous and intermittent theta burst protocols, tested if TMS patterned on endogenous TC activity reduces nociceptive behaviors in mice. RESULTS: Immunohistochemical evidence confirmed that burst, but not tonic, deep brain stimulation of TC neurons increased the activity of PV interneurons in the cortex. Both optogenetic activation of PV interneurons and TMS protocol mimicking thalamic burst reduced nociceptive behaviors. CONCLUSIONS: Our findings suggest that burst firing of TC neurons recruits PV interneurons in the cortex to reduce nociceptive behaviors and that neuromodulation mimicking thalamic burst firing may be useful for modulating nociception.

The cryptochrome inhibitor KS15 enhances E-box-mediated transcription by disrupting the feedback action of a circadian transcription-repressor complex.

AIMS: We have previously identified a chemical scaffold possessing 2-ethoxypropanoic acid (designated as KS15) that directly binds to the C-terminal region of cryptochromes (CRYs: CRY1 and CRY2) and enhances E-box-mediated transcription. However, it is still unclear how KS15 impairs the feedback actions of the CRYs and which chemical moieties are functionally important for its actions. MAIN METHODS: The E-box-mediated transcriptional activities were mainly used to examine the effects of KS15 and its derivatives. Co-immunoprecipitation assays accompanied by immunoblotting were employed to monitor protein-protein associations. We also examined the effects of KS15 and selected derivatives on circadian molecular rhythms in cultured cells. KEY FINDINGS: The present study shows that KS15 inhibits the interaction between CRYs and Brain-Muscle-Arnt-Like protein 1 (BMAL1), thereby impairing the feedback actions of CRYs on E-box-dependent transcription by CLOCK:BMAL1 heterodimer, an indispensable transcriptional regulator of the mammalian circadian clock. Subsequent structure-activity relationship analyses using a well-designed panel of derivatives identified the structural requirements for the effects of KS15 on CRY-evoked regulation of E-box-mediated transcription. We found that KS15 and several derivatives significantly reduce the amplitude and delayed the phase of molecular circadian rhythms in fibroblast cultures. SIGNIFICANCE: Taken together, our results provide valuable information on the molecular mode-of-action as well as the chemical components of the CRYs inhibitor that pharmacologically impact on the transcriptional activity of the CLOCK:BMAL1 heterodimer.