Validation of Six Commercial Antibodies for the Detection of Heterologous and Endogenous TRPM8 Ion Channel Expression

TRPM8 is a non-selective cation channel expressed in primary sensory neurons and other tissues, including the prostate and urothelium. Its participation in different physiological and pathological processes such as thermoregulation, pain, itch, inflammation and cancer has been widely described, making it a promising target for therapeutic approaches. The detection and quantification of TRPM8 seems crucial for advancing the knowledge of the mechanisms underlying its role in these pathophysiological conditions. Antibody-based techniques are commonly used for protein detection and quantification, although their performance with many ion channels, including TRPM8, is suboptimal. Thus, the search for reliable antibodies is of utmost importance. In this study, we characterized the performance of six TRPM8 commercial antibodies in three immunodetection techniques: Western blot, immunocytochemistry and immunohistochemistry. Different outcomes were obtained for the tested antibodies; two of them proved to be successful in detecting TRPM8 in the three approaches while, in the conditions tested, the other four were acceptable only for specific techniques. Considering our results, we offer some insight into the usefulness of these antibodies for the detection of TRPM8 depending on the methodology of choice

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

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

Biologic Therapy in Refractory Non-Multiple Sclerosis Optic Neuritis Isolated or Associated to Immune Mediated Inflammatory Diseases. A Multicenter Study

We aimed to assess the e cacy of biologic therapy in refractory non-Multiple Sclerosis (MS) Optic Neuritis (ON), a condition more infrequent, chronic and severe than MS ON. This was an open-label multicenter study of patients with non-MS ON refractory to systemic corticosteroids and at least one conventional immunosuppressive drug. The main outcomes were Best Corrected Visual Acuity (BCVA) and both Macular Thickness (MT) and Retinal Nerve Fiber Layer (RNFL) using Optical Coherence Tomography (OCT). These outcome variables were assessed at baseline, 1 week, and 1, 3, 6 and 12 months after biologic therapy initiation. Remission was defined as the absence of ON symptoms and signs that lasted longer than 24 h, with or without an associated new lesion on magnetic resonance imaging with gadolinium contrast agents for at least 3 months. We studied 19 patients (11 women/8 men; mean age, 34.8 13.9 years). The underlying diseases were Bechet?s disease (n = 5), neuromyelitis optica (n = 3), systemic lupus erythematosus (n = 2), sarcoidosis (n = 1), relapsing polychondritis (n = 1) and anti-neutrophil cytoplasmic antibody -associated vasculitis (n = 1). It was idiopathic in 6 patients. The first biologic agent used in each patient was: adalimumab (n = 6), rituximab (n = 6), infliximab (n = 5) and tocilizumab (n = 2). A second immunosuppressive drug was simultaneously used in 11 patients: methotrexate (n = 11), azathioprine (n = 2), mycophenolate mofetil (n = 1) and hydroxychloroquine (n = 1). Improvement of the main outcomes was observed after 1 year of therapy when compared with baseline data: mean SD BCVA (0.8 0.3 LogMAR vs. 0.6 0.3 LogMAR; p = 0.03), mean SD RNFL (190.5 175.4 m vs. 183.4 139.5 m; p = 0.02), mean SD MT (270.7 23.2 m vs. 369.6 137.4 m; p = 0.03). Besides, the median (IQR) prednisone-dose was also reduced from 40 (10?61.5) mg/day at baseline to. 2.5 (0?5) mg/day after one year of follow-up; p = 0.001. After a mean SD follow-up of 35 months, 15 patients (78.9%) achieved ocular remission, and 2 (10.5%) experienced severe adverse events. Biologic therapy is e ective in patients with refractory non-MS ON

Influence of MUC5B gene on antisynthetase syndrome

ABSTRACT: MUC5B rs35705950 (G/T) is strongly associated with idiopathic pulmonary fibrosis (IPF) and also contributes to the risk of interstitial lung disease (ILD) in rheumatoid arthritis (RA-ILD) and chronic hypersensitivity pneumonitis (CHP). Due to this, we evaluated the implication of MUC5B rs35705950 in antisynthetase syndrome (ASSD), a pathology characterised by a high ILD incidence. 160 patients with ASSD (142 with ILD associated with ASSD [ASSD-ILD+]), 232 with ILD unrelated to ASSD (comprising 161 IPF, 27 RA-ILD and 44 CHP) and 534 healthy controls were genotyped. MUC5B rs35705950 frequency did not significantly differ between ASSD-ILD+ patients and healthy controls nor when ASSD patients were stratified according to the presence/absence of anti Jo-1 antibodies or ILD. No significant differences in MUC5B rs35705950 were also observed in ASSD-ILD+ patients with a usual interstitial pneumonia (UIP) pattern when compared to those with a non-UIP pattern. However, a statistically significant decrease of MUC5B rs35705950 GT, TT and T frequencies in ASSD-ILD+ patients compared to patients with ILD unrelated to ASSD was observed. In summary, our study does not support a role of MUC5B rs35705950 in ASSD. It also indicates that there are genetic differences between ILD associated with and that unrelated to ASSD.We are indebted to the patients and healthy controls for their essential collaboration to this study. We also thank the National DNA Bank Repository (Salamanca) for supplying part of the control samples. This study was partially supported by grants from the Foundation for Research in Rheumatology (FOREUM). RL-M is a recipient of a Miguel Servet type I programme fellowship from the ‘Instituto de Salud Carlos III’ (ISCIII), co-funded by the European Social Fund (ESF, ‘Investing in your future’) (grant CP16/00033). SR-M is supported by funds of the RETICS Program (RD16/0012/0009), co-funded by the European Regional Development Fund (ERDF). VP-C is supported by a pre-doctoral grant from IDIVAL (PREVAL 18/01). VM is supported by funds of a Miguel Servet type I programme (grant CP16/00033) (ISCIII, co-funded by ESF). LL-G is supported by funds of PI18/00042 (ISCIII, co-funded by ERDF). OG is Staff Personnel of Xunta de Galicia (Servizo Galego de Saude, SERGAS) through a research-staff stabilization contract (ISCIII/SERGAS). OG,is member of RETICS Programme, RD16/0012/0014 (RIER: Red de Investigación en Inflamación y Enfermedades Reumáticas) via Instituto de Salud Carlos III (ISCIII) and FEDER. The work of OG (PI17/00409), was funded by Instituto de Salud Carlos III and FEDER. OG is a beneficiary of a project funded by Research Executive Agency of the European Union in the framework of MSCA-RISE Action of the H2020 Programme (Project number 734899). OG is beneficiary of a grant funded by Xunta de Galicia, Consellería de Educación, Universidade e Formación Profesional and Consellería de Economía, Emprego e Industria (GAIN), GPC IN607B2019/10

Differential contribution of TRPM8 and TRPA1 channels to the cold sensitivity of somatic and visceral neurons

Resumen del póster presentado al International Workshop on Chronic Pain and Itch: Mechanisms and Circuits, celebrado en Alicante (España) del 20 al 22 de octubre de 2021.Thermal signals provide essential information for the operation of interoceptive and exteroceptive neural circuits. They are essential for triggering thermally-driven reflexes and conscious behaviors. A fraction of cutaneous and visceral sensory endings are activated by cold temperatures. In comparison to somatic (DRG and TG) neurons, very little is known about the cellular and molecular mechanisms underlying cold sensitivity of visceral vagal neurons. We used pharmacological, molecular and genetic tools for a side-by-side characterization of cold-sensitive (CS) neurons in adult mouse trigeminal (TG) and vagal ganglia (VG). We found that CS neurons are more abundant in VG than in TG. In both ganglia, sensitivity to cold varied widely, with activation thresholds spanning a range ~15 ºC. In both ganglia, cold sensitivity was enhanced by the potassium channel blocker 4-AP. About 2/3 of CS neurons in VG co-express TRPA1 markers and cold-evoked responses are severely blunted in Trpa1 KO mice, with little impact of TRPM8 deletion or pharmacological TRPM8 blockade. Consistent with this result, by qPCR, we detected lower levels of TRPM8 in VG compared to TG. The number of TRPM8-positive neurons was low in VG and restricted to the rostral jugular ganglion. Using fluorescent retrograde markers, be identified vagal neurons projecting to different viscera. Airway-innervating vagal neurons showed enhanced cold sensitivity and a higher functional expression of TRPA1 compared to neurons innervating the stomach wall. In contrast, the majority of CS TG neurons co-express TRPM8 markers and their cold sensitivity is reduced after TRPM8 deletion or pharmacological blockade. However, pharmacological or genetic reduction of TRPA1 showed that these channels also contribute to the activation of low-threshold TG cold sensitive neurons. In summary, we found that TRPA1 and TRPM8 channels show differential roles in the cold sensitivity of somatic and visceral vagal neurons.Supported by Ministerio de Ciencia e Innovación, PID2019-108194RB-100 co-financed by the European Regional Development Fund (ERDF), the “Severo Ochoa” Program for Centers of Excellence in R&D SEV-2017-0723 and the International PhD Fellowships Program “La Caixa”-Severo Ochoa, Call 2015” (KGB) and predoctoral fellowship BES-2017-080782 (PHO).Peer reviewe

The role of TRPM8 and TRPA1 channels in cold sensitivity of trigeminal and vagal sensory neurons

Resumen del trabajo presentado al VIII Congreso Red Española de Canales Iónicos, celebrado en Alicante del 24 al 27 de mayo de 2022.KGB was supported by the International PhD Fellowships Program “La Caixa”-Severo Ochoa, Call 2015” and PHO is supported by MINECO predoctoral fellowship BES-2017-080782. Study supported by project PID2019-108194RB-I00 and co-financed by the European Regional Development Fund (ERDF) and the “Severo Ochoa” Program for Centers of Excellence in R&D (ref. SEV-2017-0723).Peer reviewe