Constitutive, Basal, and β-Alanine-Mediated Activation of the Human Mas-Related G Protein-Coupled Receptor D Induces Release of the Inflammatory Cytokine IL-6 and Is Dependent on NF-κB Signaling

G protein-coupled receptors (GPCRs) have emerged as key players in regulating (patho)physiological processes, including inflammation. Members of the Mas-related G protein coupled receptors (MRGPRs), a subfamily of GPCRs, are largely expressed by sensory neurons and known to modulate itch and pain. Several members of MRGPRs are also expressed in mast cells, macrophages, and in cardiovascular tissue, linking them to pseudo-allergic drug reactions and suggesting a pivotal role in the cardiovascular system. However, involvement of the human Mas-related G-protein coupled receptor D (MRGPRD) in the regulation of the inflammatory mediator interleukin 6 (IL-6) has not been demonstrated to date. By stimulating human MRGPRD-expressing HeLa cells with the agonist β-alanine, we observed a release of IL-6. β-alanine-induced signaling through MRGPRD was investigated further by probing downstream signaling effectors along the Gαq/Phospholipase C (PLC) pathway, which results in an IkB kinases (IKK)-mediated canonical activation of nuclear factor kappa-B (NF-κB) and stimulation of IL-6 release. This IL-6 release could be blocked by a Gαq inhibitor (YM-254890), an IKK complex inhibitor (IKK-16), and partly by a PLC inhibitor (U-73122). Additionally, we investigated the constitutive (ligand-independent) and basal activity of MRGPRD and concluded that the observed basal activity of MRGPRD is dependent on the presence of fetal bovine serum (FBS) in the culture medium. Consequently, the dynamic range for IL-6 detection as an assay for β-alanine-mediated activation of MRGPRD is substantially increased by culturing the cells in FBS free medium before treatment. Overall, the observation that MRGPRD mediates the release of IL-6 in an in vitro system, hints at a role as an inflammatory mediator and supports the notion that IL-6 can be used as a marker for MRGPRD activation in an in vitro drug screening assay.

The importance of individual variation for the interpretation of behavioural studies: ethanol effects vary with basal activity level in zebrafish larvae

Standardization and reduction of variation is key to behavioural screening of animal models in toxicological and pharmacological studies. However, individual variation in behavioural and physiological phenotypes remains in each laboratory population and can undermine the understanding of toxicological and pharmaceutical effects and their underlying mechanisms. Here, we used zebrafish (ABTL-strain) larvae to explore individual consistency in activity level and emergence time, across subsequent days of early development (6–8 dpf). We also explored the correlation between these two behavioural parameters. We found inter-individual consistency over time in activity level and emergence time, but we did not find a consistent correlation between these parameters. Subsequently, we investigated the impact of variation in activity level on the effect of a 1% ethanol treatment, suitable for our proof-of-concept case study about whether impact from pharmacological treatments might be affected by inter-individual variation in basal locomotion. The inter-individual consistency over time in activity level did not persist in this test. This was due to the velocity change from before to after exposure, which turned out to be a dynamic individual trait related to basal activity level: low-activity individuals raised their swimming velocity, while high-activity individuals slowed down, yielding diametrically opposite response patterns to ethanol exposure. We therefore argue that inter-individual consistency in basal activity level, already from 6 dpf, is an important factor to take into account and provides a practical measure to improve the power of statistical analyses and the scope for data interpretation from behavioural screening studies.

Dichotomous dopaminergic and noradrenergic neural states mediate distinct aspects of exploitative behavioral states

The balance between exploiting known actions and exploring alternatives is critical for survival and hypothesized to rely on shifts in neuromodulation. We developed a behavioral paradigm to capture exploitative and exploratory states and imaged calcium dynamics in genetically identified dopaminergic and noradrenergic neurons. During exploitative states, characterized by motivated repetition of the same action choice, dopamine neurons in SNc encoding movement vigor showed sustained elevation of basal activity that lasted many seconds. This sustained activity emerged from longer positive responses, which accumulated during exploitative action-reward bouts, and hysteretic dynamics. Conversely, noradrenergic neurons in LC showed sustained inhibition of basal activity due to the accumulation of longer negative responses in LC. Chemogenetic manipulation of these sustained dynamics revealed that dopaminergic activity mediates action drive, whereas noradrenergic activity modulates choice diversity. These data uncover the emergence of sustained neural states in dopaminergic and noradrenergic networks that mediate dissociable aspects of exploitative bouts.

Protein Kinase C gamma Mutations Drive Spinocerebellar Ataxia Type 14 by Impairing Autoinhibition

Spinocerebellar ataxia type 14 (SCA14) is a neurodegenerative disease caused by germline mutations in the diacylglycerol (DG)/Ca2+-regulated protein kinase C gamma (PKCγ), leading to Purkinje cell degeneration and progressive cerebellar dysfunction. The majority of the approximately 50 mutations identified in PKCγ cluster to the DG-sensing C1 domains. Here, we use a FRET-based activity reporter to show that ataxia-associated PKCγ mutations enhance basal activity by compromising autoinhibition. Although impaired autoinhibition generally leads to PKC degradation, the C1 domain mutations protect PKCγ from phorbol ester-induced downregulation. Furthermore, it is the degree of disrupted autoinhibition, not changes in the amplitude of agonist-stimulated activity, that correlate with disease severity. Specifically, a SCA14 mutation in which phenylalanine 48 in the C1A domain is deleted had high basal activity both in cells and in vitro, yet was unresponsive to agonist stimulation. Validating that the pathology arises from disrupted autoinhibition, we show that the degree of impaired autoinhibition correlates inversely with age of disease onset in patients: mutations that cause high basal activity are associated with early onset, whereas those that only modestly increase basal activity, including a previously undescribed mutation D115Y, are associated with later onset. Molecular modeling indicates that almost all SCA14 mutations that are not in the C1 domains are at interfaces with the C1B domain, and bioinformatics analysis reveals that mutations in the C1B domain are under-represented in cancer. Thus, clustering of SCA14 mutations to the C1B domain provides a unique mechanism to enhance PKCγ basal activity while protecting the enzyme from downregulation.

Rhodobacter sphaeroides CarD Negatively Regulates Its Own Promoter

Bioinformatic analysis showed previously that a majority of promoters in the photoheterotrophic α-proteobacterium Rhodobacter sphaeroides lack the thymine at the last position of the -10 element (-7T), a base that is very highly conserved in promoters in bacteria other than α-proteobacteria. The absence of -7T was correlated with low promoter activity using purified R sphaeroides RNA polymerase (RNAP), but the transcription factor CarD compensated by activating almost all promoters lacking -7T tested in vitro , including rRNA promoters. Here we show that a previously uncharacterized R. sphaeroides promoter, the promoter for carD itself, has high basal activity relative to other tested R. sphaeroides promoters despite lacking -7T, and its activity is inhibited rather than activated by CarD. This high basal activity is dependent on a consensus extended -10 element (TGn) and specific features in the spacer immediately upstream of the extended -10 element. CarD negatively autoregulates its own promoter by producing abortive transcripts, limiting promoter escape and reducing full length mRNA synthesis. This mechanism of negative regulation differs from that employed by classical repressors, in which the transcription factor competes with RNA polymerase for binding to the promoter, and with the mechanism of negative regulation used by transcription factors like DksA/ppGpp and TraR that allosterically inhibit the rate of open complex formation. IMPORTANCE R. sphaeroides CarD activates many promoters by binding directly to RNAP and to DNA just upstream of the -10 element. In contrast, we show here that CarD inhibits its own promoter using the same interactions with RNAP and DNA used for activation. Inhibition results from increasing abortive transcript formation, thereby decreasing promoter escape and full-length RNA synthesis. We propose that the combined interactions of RNAP with CarD, with the extended -10 element, and with features in the adjacent -10/-35 spacer DNA stabilize the promoter complex, reducing promoter clearance. These findings support previous predictions that the effects of CarD on transcription can be either positive or negative, depending on the kinetic properties of the specific promoter.

Sex dimorphic changes in Trh gene methylation and thyroid-axis response to energy demands in maternally-separated rats

The hypothalamus-pituitary-thyroid (HPT) axis regulates energy balance through the pleiotropic action of thyroid hormones. HPT basal activity and stimulation by cold or voluntary exercise are repressed by previous chronic stress in adults. Maternal separation (MS) modifies HPT basal activity, we thus studied the response of the axis to energy demands and analyzed possible epigenetic changes on Trh promoter. Non-handled (NH) or MS male Wistar rats were cold exposed 1h at adulthood, only in NH rats Trh expression in the hypothalamic paraventricular nucleus (PVN) and serum TSH concentration were increased. Two weeks of voluntary exercise decreased fat mass, increased Trh expression, and thyroid hormones concentration changed proportionally to running distance in NH male rats and MS male rats. Although NH-females ran more than MS and much more than males, exercise decreased body weight and fat mass only in NH-rats with no change on any parameter of HPT axis, but increased Pomc expression in arcuate-nucleus of NH- whereas Npy in MS-females. Overall methylation pattern of PVN Trh gene promoter was similar in NH-males or -females; MS modified methylation of specific CpG sites, a thyroid-hormone receptor (THR) binding site present after the initiation site was hypomethylated in MS-males; in MS-females, the THR binding-site of the proximal promoter (site 4) and two sites in the first intron were hypermethylated. Our studies showed that, in a sex dimorphic manner, MS blunted the responses of HPT axis to energy demands in adult animals and caused methylation changes on Trh promoter that could alter T3 feedback.

Gating the pore of the calcium-activated chloride channel TMEM16A

The binding of cytoplasmic Ca2+ to the anion-selective channel TMEM16A triggers a conformational change around its binding site that is coupled to the release of a gate at the constricted neck of an hourglass-shaped pore. By combining mutagenesis, electrophysiology, and cryo-electron microscopy, we identified three hydrophobic residues at the intracellular entrance of the neck as constituents of this gate. Mutation of each of these residues increases the potency of Ca2+ and results in pronounced basal activity. The structure of an activating mutant shows a conformational change of an α-helix that contributes to Ca2+ binding as a likely cause for the basal activity. Although not in physical contact, the three residues are functionally coupled to collectively contribute to the stabilization of the gate in the closed conformation of the pore, thus explaining the low open probability of the channel in the absence of Ca2+.

Power and Coherence in the EEG of the Rat: Impact of Behavioral States, Cortical Area, Lateralization and Light/Dark Phases

The sleep-wake cycle is constituted by three behavioral states: wakefulness (W), non-REM (NREM) and REM sleep. These states are associated with drastic changes in cognitive capacities, mostly determined by the function of the thalamo-cortical system, whose activity can be examined by means of intra-cranial electroencephalogram (iEEG). With the purpose to study in depth the basal activity of the iEEG in adult rats, we analyzed the spectral power and coherence of the iEEG during W and sleep in the paleocortex (olfactory bulb), and in neocortical areas. We also analyzed the laterality of the signals, as well as the influence of the light and dark phases. We found that the iEEG power and coherence of the whole spectrum were largely affected by behavioral states and highly dependent on the cortical areas recorded. We also determined that there are night/day differences in power and coherence during sleep, but not in W. Finally, we observed that, during REM sleep, intra-hemispheric coherence differs between right and left hemispheres. We conclude that the iEEG dynamics are highly dependent on the cortical area and behavioral states. Moreover, there are light/dark phases disparities in the iEEG during sleep, and intra-hemispheric connectivity differs between both hemispheres during REM sleep.