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

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

The emerging pharmacology of TRPM8 channels: Hidden therapeutic potential underneath a cold surface

14 p., 2 figures, 2 tables and references.Transient receptor potential melastatin 8 (TRPM8) is a non-selective cation channel activated by cold temperature and cooling agents. TRPM8 is expressed in a subpopulation of cold-sensitive sensory neurons, as well as in the male urogenital system. TRPM8 is markedly upregulated in prostate cancer and in other tumors such as breast adenocarcinoma and melanoma. Moreover, recent studies suggest the potential involvement of TRPM8 channels in the pathophysiology of cold nociception and cold allodynia. This has led to a strong interest in the pursuit of novel modulators of TRPM8 channels. This review highlights our current knowledge of TRPM8 pharmacology and modulation mechanisms, detailing structural features important for TRPM8 gating by different agonists, the mechanism of antagonism by different compounds and the potential relevance of TRPM8 for treatment of various pathological conditions.We also like to acknowledge funding from the Spanish MICINN projects BFU2007-61855 to F. Viana, CONSOLIDER-INGENIO 2010 CSD2007-0002 and from the Fundación Marcelino Botín to C. Belmonte.Peer reviewe

Guidance of retinal axons in mammals

In order to navigate through the surrounding environment many mammals, including humans, primarily rely on vision. The eye, composed of the choroid, sclera, retinal pigmented epithelium, cornea, lens, iris and retina, is the structure that receives the light and converts it into electrical impulses. The retina contains six major types of neurons involving in receiving and modifying visual information and passing it onto higher visual processing centres in the brain. Visual information is relayed to the brain via the axons of retinal ganglion cells (RGCs), a projection known as the optic pathway. The proper formation of this pathway during development is essential for normal vision in the adult individual. Along this pathway there are several points where visual axons face ‘choices’ in their direction of growth. Understanding how these choices are made has advanced significantly our knowledge of axon guidance mechanisms. Thus, the development of the visual pathway has served as an extremely useful model to reveal general principles of axon pathfinding throughout the nervous system. However, due to its particularities, some cellular and molecular mechanisms are specific for the visual circuit. Here we review both general and specific mechanisms involved in the guidance of mammalian RGC axons when they are traveling from the retina to the brain to establish precise and stereotyped connections that will sustain vision.The laboratory of EH is funded by the Spanish Ministry of Economy and Competitiveness under the following grants: Spanish Ministry of Economy and Competitiveness (BFU2016-77605), Generalitat Valenciana, PROMETEO Program (2012/026), ERA-Net Program (PCIN2015-192-C02-02); Fundació La Marató de TV3 (20142130), Tatiana Pérez de Guzmán el Bueno Foundation (201/C/2014) and European Research Council (no. 282329). We also acknowledge the financial support received from the Spanish Ministry of Economy and Competitiveness under the grant co-financed by the European Regional Development Fund (ERDF) and the “Severo Ochoa” Program for Centers of Excellence in R&D (SEV-2013-0317). The laboratory of LE is funded currently by the University of Aberdeen Development Trust, and an EastBio BBSRC PhD studentship.Peer reviewe

Pharmacological and functional properties of TRPM8 channels in prostate tumor cells

16 p., 7 figures, 1 table and references.Prostate cancer (PC) is a major health problem in adult males. TRPM8, a cationic TRP channel activated by cooling and menthol is upregulated in PC. However, the precise role of TRPM8 in PC is still unclear. Some studies hypothesized that TRPM8-mediated transmembrane Ca 2+ fluxes play a key role in cellular proliferation of PC cells. In contrast, other findings suggest that high TRPM8 levels may reduce the metastatic potential of PC cells. A detailed understanding of the response of TRPM8 channels to pharmacological modulators of their activity is relevant when considering potential therapies, targeting this ion channel to treat PC. We characterized the pharmacological and functional properties of native TRPM8 channels in four human prostate cell lines, PNT1A, LNCaP, DU145, and PC3, commonly used as experimental models of PC. PNT1A is a non-tumoral prostate cell line while the other three correspond to different stages of PC. Here, we show that cold- and agonist-evoked [Ca 2+] i responses in PC cells are much less sensitive to well-characterized agonists (menthol and icilin) and antagonists (BCTC, clotrimazole, and DD01050) of TRPM8 channels, compared to TRPM8 channels in other tissues, suggesting a different molecular composition and/or spatial organization. In addition, the forced overexpression of human TRPM8 facilitated the trafficking of TRPM8 channels residing in the endoplasmic reticulum to the plasma membrane, leading to a marked potentiation in the efficacy of the different blockers. These results predict that blockers of canonical TRPM8 channels may be less effective in halting proliferation of PC cells than expected.This work was supported by funds from the Spanish MICIIN: projects BFU2007-61855 to F.V and BFU2008-04425 and CONSOLIDER-INGENIO 2010 CSD2007-00023 to C.B. M.V. was the recipient of a predoctoral fellowship of the Spanish Government and C.M. holds a postdoctoral fellowship from FIS.Peer reviewe

Zic2 regulates Draxin to promote Neural Crest cells delamination

Resumen del póster presentado al 3rd AXON Meeting: “Circuits Development & Axon Regeneration”, celebrado en Alicante (España) del 11 al 13 de septiembre de 2019.También presentado al 5th Annual Retreat Developmental Neurobiology Unit, celebrado en el Instituto de Neurociencias de Alicante del 18 al 19 de febrero de 2019.The Neural Crest (NC) is a population of pluripotent cells that originate in the dorsal neuroephitelium of the neural tube and travel all over the embryo to contribute to the formation of many organs. The zinc finger transcription factor Zic2 participates in several steps of neural development including neural crest formation, being initially described as crucial for neural crest cells differentiation. However, Zic2 functional experiments in different species lead to disparate conclusions and today the precise role of this transcription factor in this process is still not well defined. Here, we first show that Zic2 is expressed in premigratory but not in migrating NC cells and demonstrate that, according to previous observations, downregulation of Zic2 in chick and mice produces a decreased number of migrating NC cells in the mesenchyme concomitant with an accumulation of NC cells at the dorsal tip of the neural tube. Conversely, Zic2 gain of function produces precocious exit of NC cells from the dorsal tube, indicating that Zic2 is necessary and sufficient to induce NC cells delamination. In addition, through an unbiased genome-wide screen we have identified the secreted molecule Draxin/Neucrin, an antagonist of the Wnt canonical signaling pathway, as a Zic2 target. Functional experiments in mouse and chick confirm that Zic2 regulates Draxin/Neucrin expression, which in turn contributes to regulate the onset of NC cells delamination. Together, these observations place Zic2 and its target Neucrin/Draxin as critical players on the delamination of NC cells.Peer reviewe

Transcriptional control of cholesterol biosynthesis in schwann cells by axonal neuregulin 1

A characteristic feature of many vertebrate axons is their wrapping by a lamellar stack of glially derived membranes known as the myelin sheath. Myelin is a cholesterol-rich membrane that allows for rapid saltatory nerve impulse conduction. Axonal neuregulins instruct glial cells on when and how much myelin they should produce. However, how neuregulin regulates myelin sheath development and thickness is unknown. Here we show that neuregulin receptors are activated by drops in plasma membrane cholesterol, suggesting that they can sense sterol levels. In Schwann cells neuregulin-1 increases the transcription of the 3-hydroxy-3-methylglutarylcoenzyme A reductase, the rate-limiting enzyme for cholesterol biosynthesis. Neuregulin activity is mediated by the phosphatidylinositol 3-kinase pathway and a cAMP-response element located on the reductase promoter. We propose that by activating neuregulin receptors, neurons exploit a cholesterol homeostatic mechanism forcing Schwann cells to produce new membranes for the myelin sheath. We also show that a strong phylogenetic correlation exists between myelination and cholesterol biosynthesis, and we propose that the absence of the sterol branch of the mevalonate pathway in invertebrates precluded the myelination of their nervous system.This work was supported in part by Spanish Ministry of Education and Science Grant SAF2004-01011 (to F. V.), “Instituto de Salud Carlos III” Grant PI05/0535, and “Conselleria de Salut de la Generalitat Valenciana” Grant AP-002/06 (to H. C.).Peer reviewe