A role for transcriptional repressor methyl-CpG-binding protein 2 and plasticity-related gene serum- and glucocorticoid-inducible kinase 1 in the induction of inflammatory pain states

Activity-dependent changes in neurons of the rat superficial dorsal horn are crucial for the induction and maintenance of neuropathic and inflammatory pain states. To identify the molecular mechanisms underlying this sensitization of superficial dorsal horn neurons, we undertook a genome-wide microarray profiling of dorsal horn gene transcripts at various times after induction of peripheral inflammation of the rat ankle joint. At early time points, upregulation of gene expression dominated, but by 7 d, downregulation was predominant. Two to 24 h after inflammation, we identified a small number of highly upregulated transcripts previously shown to be repressed by the Methyl-CpG-binding protein 2 (MeCP2), including serum-and glucocorticoid-inducible kinase (SGK1) and FK 506 binding protein 5, genes known to be important in experience-dependent plasticity. A decrease in expression of SIN3A, a corepressor in the MeCP2 silencing complex, was also found after inflammation. Phosphorylation of MeCP2 regulates activity-dependent gene transcription, and crucially we found that MeCP2 was phosphorylated in lamina I projection neurons 1 h after induction of peripheral inflammation. Lamina I projection neurons have been shown to be essential for the development of most pain states. SGK1 protein was also localized, in part, to lamina I projection neurons, and its expression in the superficial dorsal horn increased after inflammation. Furthermore, antisense knock-down of SGK1 delayed the onset of inflammatory hyperalgesia by 24 h at least. Our results uncover an unexpected complexity in the regulation of gene expression, including the modulation of transcriptional repression, that accompanies development and maintenance of an inflammatory pain state

Zic2-Dependent Axon Midline Avoidance Controls the Formation of Major Ipsilateral Tracts in the CNS

SummaryIn bilaterally symmetric organisms, interhemispheric communication is essential for sensory processing and motor coordination. The mechanisms that govern axon midline crossing during development have been well studied, particularly at the spinal cord. However, the molecular program that determines axonal ipsilaterality remains poorly understood. Here, we demonstrate that ipsilateral neurons whose axons grow in close proximity to the midline, such as the ascending dorsospinal tracts and the rostromedial thalamocortical projection, avoid midline crossing because they transiently activate the transcription factor Zic2. In contrast, uncrossed neurons whose axons never approach the midline control axonal laterality by Zic2-independent mechanisms. Zic2 induces EphA4 expression in dorsospinal neurons to prevent midline crossing while Robo3 is downregulated to ensure that axons enter the dorsal tracts instead of growing ventrally. Together with previous reports, our data reveal a critical role for Zic2 as a determinant of axon midline avoidance in the CNS across species and pathways

Morphological and functional changes in TRPM8-expressing corneal cold thermoreceptor neurons during aging and their impact on tearing in mice

Morphological and functional alterations of peripheral somatosensory neurons during the aging process lead to a decline of somatosensory perception. Here, we analyze the changes occurring with aging in trigeminal ganglion (TG), TRPM8-expressing cold thermoreceptor neurons innervating the mouse cornea, which participate in the regulation of basal tearing and blinking and have been implicated in the pathogenesis of dry eye disease (DED). TG cell bodies and axonal branches were examined in a mouse line (TRPM8BAC-EYFP) expressing a fluorescent reporter. In 3 months old animals, about 50% of TG cold thermoreceptor neurons were intensely fluorescent, likely providing strongly fluorescent axons and complex corneal nerve terminals with ongoing activity at 348C and low-threshold, robust responses to cooling. The remaining TRPM81 corneal axons were weakly fluorescent with nonbeaded axons, sparsely ramified nerve terminals, and exhibited a low-firing rate at 348C, responding moderately to cooling pulses as do weakly fluorescent TG neurons. In aged (24 months) mice, the number of weakly fluorescent TG neurons was strikingly high while the morphology of TRPM81 corneal axons changed drastically; 89% were weakly fluorescent, unbranched, and often ending in the basal epithelium. Functionally, 72.5% of aged cold terminals responded as those of young animals, but 27.5% exhibited very low-background activity and abnormal responsiveness to cooling pulses. These morpho-functional changes develop in parallel with an enhancement of tear’s basal flow and osmolarity, suggesting that the aberrant sensory inflow to the brain from impaired peripheral cold thermoreceptors contributes to age-induced abnormal tearing and to the high incidence of DED in elderly people.This work was supported by grants FC-15-GRUPIN14–141 (Consejería de Economía y Empleo, Asturias, Spain),Fundación Ramón Areces, Caja Rural de Asturias, SAF2014–54518-C3-2-R, SAF2014– 54518-C3-1-R, SAF2017–83674-C2-2-R, SAF2017–83674-C2-1-R,SAF2016–77233-R (Ministerio de Economía, Industria y Competitividad, Spain and European Regional Development Funds, European Union)“Severo Ochoa” Program for Centers of Excellence in R&D (SEV-2013-0317)

Expression of the cold thermoreceptor TRPM8 in rodent brain thermoregulatory circuits

The cold- and menthol-activated ion channel transient receptor potential channel subfamily M member 8 (TRPM8) is the principal detector of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in non-neuronal tissues. Here, we show, by in situ hybridization (ISH) and by the analysis of transgenic reporter expression in two different reporter mouse strains, that TRPM8 is also expressed in the central nervous system. Although it is present at much lower levels than in peripheral sensory neurons, we found cells expressing TRPM8 in restricted areas of the brain, especially in the hypothalamus, septum, thalamic reticular nucleus, certain cortices and other limbic structures, as well as in some specific nuclei in the brainstem. Interestingly, positive fibers were also found traveling through the major limbic tracts, suggesting a role of TRPM8-expressing central neurons in multiple aspects of thermal regulation, including autonomic and behavioral thermoregulation. Additional ISH experiments in rat brain demonstrated a conserved pattern of expression of this ion channel between rodent species. We confirmed the functional activity of this channel in the mouse brain using electrophysiological patch-clamp recordings of septal neurons. These results open a new window in TRPM8 physiology, guiding further efforts to understand potential roles of this molecular sensor within the brain.Instituto de Salud Carlos III, Grant/Award Number: PI12/0058; National Institutes of Health, Grant/Award Number: ZIA DE000721-12; Ministerio de Ciencia e Innovación, Grant/Award Numbers: SAF2009-11175, SAF2010-14990-R, SAF2016-77233-R; Ministerio de Economía y Competitividad, Grant/Award Numbers: BES-2011-047063, BES-2017-080782; Severo Ochoa Programme for Centres of Excellence in R&D, Grant/Award Number: SEV-2017-0723 and cofinanced by the European Regional Development Fund; Generalitat Valenciana, Grant/Award Number: GRISOLIA/2008/02

The cold-sensing ion channel TRPM8 regulates central and peripheral clockwork and the circadian oscillations of body temperature

[Abstract] Aim: Physiological functions in mammals show circadian oscillations, synchronized by daily cycles of light and temperature. Central and peripheral clocks participate in this regulation. Since the ion channel TRPM8 is a critical cold sensor, we investigated its role in circadian function. Methods: We used TRPM8 reporter mouse lines and TRPM8-deficient mice. mRNA levels were determined by in situ hybridization or RT-qPCR and protein levels by immunofluorescence. A telemetry system was used to measure core body temperature (Tc). Results: TRPM8 is expressed in the retina, specifically in cholinergic amacrine interneurons and in a subset of melanopsin-positive ganglion cells which project to the central pacemaker, the suprachiasmatic nucleus (SCN) of the hypothalamus. TRPM8-positive fibres were also found innervating choroid and ciliary body vasculature, with a putative function in intraocular temperature, as shown in TRPM8-deficient mice. Interestingly, Trpm8-/- animals displayed increased expression of the clock gene Per2 and vasopressin (AVP) in the SCN, suggesting a regulatory role of TRPM8 on the central oscillator. Since SCN AVP neurons control body temperature, we studied Tc in driven and free-running conditions. TRPM8-deficiency increased the amplitude of Tc oscillations and, under dim constant light, induced a greater phase delay and instability of Tc rhythmicity. Finally, TRPM8-positive fibres innervate peripheral organs, like liver and white adipose tissue. Notably, Trpm8-/- mice displayed a dysregulated expression of Per2 mRNA in these metabolic tissues. Conclusion: Our findings support a function of TRPM8 as a temperature sensor involved in the regulation of central and peripheral clocks and the circadian control of Tc.Ministerio de Ciencia e Innovación (España); RT2018-099995-B100Ministerio de Ciencia e Innovación (España); AEI/10.13039/501100011033Generalitat Valenciana; PROMETEO/2021/031Ministerio de Asuntos Económicos y Transformación Digital (España); BES-2011-04706

Proinflammatory mediators modulate the heat-activated ion channel TRPV1 via the scaffolding protein AKAP79/150

Peer reviewedPostprin

Expression of the cold thermoreceptor TRPM8 in mouse brain circuits involved in thermal homeostasis

Resumen del trabajo presentado al VII Congreso Red Española Canales Iónicos, celebrado en Cáceres del 15 al 17 de mayo de 2019.The ion channel TRPM8 is the principal sensor of environmental cold in mammalian sensory nerve endings. Although it is mainly expressed in a subpopulation of peripheral sensory neurons, it has also been identified in certain non-neuronal tissues. Little is known about the expression of this thermosensitive ion channel in the central nervous system. The objective of this work was to study the expression and anatomical distribution of TRPM8 channels in mouse brain. We used RT-PCR and “in situ” hybridization (ISH). Furthermore, GFP immunohistochemistry was carried out in two transgenic TRPM8 reporter mouse models: TRPM8-green fluorescent protein (GFP) knock-in mice, Trpm8EGFPf and TRPM8-yellow fluorescent protein (YFP) transgenic mice, Trpm8BAC-EYFP+. Finally, we performed patch-clamp recordings in Trpm8BAC-EYFP+ septal neurons. We found that TRPM8 is expressed in mouse central nervous system, although with much lower levels of expression than in peripheral sensory ganglia. Positive cells were mainly identified in the preoptic hypothalamus, septal area, reticular thalamic nucleus and limbic regions with projections widely distributed within the brain and brainstem. Electrophysiological recordings in brain slices revealed the functionality of these ion channels. Our results showing expression of TRPM8 in the central nervous system open a new window in TRPM8 physiology. Further experiments are required to fully understand the potential roles of this molecular sensor within the brain.The study was supported by the projects SAF2009-11175 and PI12/0058 (RS), SAF2010-14990-R and SAF2016-77233-R (FV, AG), co-financed by the European Regional Development Fund (ERDF), the “Severo Ochoa” Programme for Centres of Excellence in R&D (SEV-2017-0723) and the IRP of the National Institute of Dental and Craniofacial Research, NIH (MAH). CF-P, PO and PH held predoctoral fellowships of the Generalitat Valenciana and Spanish MINECO (GRISOLIA/2008/025, BES-2011-047063 and BES-2017-080782).Peer reviewe

Increasing the expression of calcium-permeable TRPC3 and TRPC7 channels enhances constitutive secretion

The hTRPC [human TRPC (canonical transient receptor potential)] family of non-selective cation channels is proposed to mediate calcium influx across the plasma membrane via PLC (phospholipase C)-coupled receptors. Heterologously expressed hTRPC3 and hTRPC7 have been localized at the cell surface; however, a large intracellular component has also been noted but not characterized. In the present study, we have investigated the intracellular pool in COS-7 cells and have shown co-localization with markers for both the TGN (trans-Golgi network) and the cis-Golgi cisternae by immunofluorescence microscopy. Addition of BFA (Brefeldin A) to cells expressing hTRPC3 or hTRPC7 resulted in the redistribution of the Golgi component to the endoplasmic reticulum, indicating that this pool is present in both the Golgi stack and the TGN. Expression of either TRPC3 or TRPC7, but not TRPC1 or the cell surface marker CD8, resulted in a 2–4-fold increase in secreted alkaline phosphatase in the extracellular medium. Based on these results, we propose that an additional function of these members of the hTRPC family may be to enhance secretion either by affecting transport through the Golgi stack or by increasing fusion at the plasma membrane

Near-Membrane Dynamics and Capture of TRPM8 Channels within Transient Confinement Domains

The cold and menthol receptor, TRPM8, is a non-selective cation channel expressed in a subset of peripheral neurons that is responsible for neuronal detection of environmental cold stimuli. It was previously shown that members of the transient receptor potential (TRP) family of ion channels are translocated toward the plasma membrane (PM) in response to agonist stimulation. Because the spatial and temporal dynamics of cold receptor cell-surface residence may determine neuronal activity, we hypothesized that the movement of TRPM8 to and from the PM might be a regulated process. Single particle tracking (SPT) is a useful tool for probing the organization and dynamics of protein constituents in the plasma membrane.We used SPT to study the receptor dynamics and describe membrane/near-membrane behavior of particles containing TRPM8-EGFP in transfected HEK-293T and F-11 cells. Cells were imaged using total internal reflection fluorescence (TIRF) microscopy and the 2D and 3D trajectories of TRPM8 molecules were calculated by analyzing mean-square particle displacement against time. Four characteristic types of motion were observed: stationary mode, simple Brownian diffusion, directed motion, and confined diffusion. In the absence of cold or menthol to activate the channel, most TRPM8 particles move in network covering the PM, periodically lingering for 2–8 s in confined microdomains of about 800 nm radius. Removing cholesterol with methyl-beta-cyclodextrin (MβCD) stabilizes TRPM8 motion in the PM and is correlated with larger TRPM8 current amplitude that results from an increase in the number of available channels without a change in open probability.These results reveal a novel mechanism for regulating TRPM8 channel activity, and suggest that PM dynamics may play an important role in controlling electrical activity in cold-sensitive neurons