Functional and Molecular Characterization of Mechanoinsensitive "Silent" Nociceptors.

Mechanical and thermal hyperalgesia (pain hypersensitivity) are cardinal signs of inflammation. Although the mechanism underlying thermal hyperalgesia is well understood, the cellular and molecular basis of mechanical hyperalgesia is poorly described. Here, we have identified a subset of peptidergic C-fiber nociceptors that are insensitive to noxious mechanical stimuli under normal conditions but become sensitized to such stimuli when exposed to the inflammatory mediator nerve growth factor (NGF). Strikingly, NGF did not affect mechanosensitivity of other nociceptors. We show that these mechanoinsensitive "silent" nociceptors are characterized by the expression of the nicotinic acetylcholine receptor subunit alpha-3 (CHRNA3) and that the mechanically gated ion channel PIEZO2 mediates NGF-induced mechanosensitivity in these neurons. Retrograde tracing revealed that CHRNA3+ nociceptors account for ∼50% of all peptidergic nociceptive afferents innervating visceral organs and deep somatic tissues. Hence, our data suggest that NGF-induced "un-silencing" of CHRNA3+ nociceptors significantly contributes to the development of mechanical hyperalgesia during inflammation

Neuropathic pain caused by miswiring and abnormal end organ targeting

Nerve injury leads to chronic pain and exaggerated sensitivity to gentle touch (allodynia) as well as a loss of sensation in the areas in which injured and non-injured nerves come together1-3. The mechanisms that disambiguate these mixed and paradoxical symptoms are unknown. Here we longitudinally and non-invasively imaged genetically labelled populations of fibres that sense noxious stimuli (nociceptors) and gentle touch (low-threshold afferents) peripherally in the skin for longer than 10 months after nerve injury, while simultaneously tracking pain-related behaviour in the same mice. Fully denervated areas of skin initially lost sensation, gradually recovered normal sensitivity and developed marked allodynia and aversion to gentle touch several months after injury. This reinnervation-induced neuropathic pain involved nociceptors that sprouted into denervated territories precisely reproducing the initial pattern of innervation, were guided by blood vessels and showed irregular terminal connectivity in the skin and lowered activation thresholds mimicking low-threshold afferents. By contrast, low-threshold afferents-which normally mediate touch sensation as well as allodynia in intact nerve territories after injury4-7-did not reinnervate, leading to an aberrant innervation of tactile end organs such as Meissner corpuscles with nociceptors alone. Genetic ablation of nociceptors fully abrogated reinnervation allodynia. Our results thus reveal the emergence of a form of chronic neuropathic pain that is driven by structural plasticity, abnormal terminal connectivity and malfunction of nociceptors during reinnervation, and provide a mechanistic framework for the paradoxical sensory manifestations that are observed clinically and can impose a heavy burden on patients.The research leading to these results has received funding from the following sources: an ERC Advanced Investigator grant to R.K. (Pain Plasticity 294293); grants from the Deutsche Forschungsgemeinschaft to R.K. (SFB1158, projects B01, B06), to T.K. (SFB1158, project B08), to S.G.L. (SFB1158, project A01) and to V.G. (SFB1158, project A03); a grant to B.O. (project number 371923335); and grant CIDEGENT/2020/052 from Generalitat Valenciana to F.J.T. R.K. is a member of the Molecular Medicine Partnership Unit of the European Molecular Biology Laboratory and Medical Faculty Heidelberg. V.G. and T.A.N. were partially supported by a post-doctoral fellowship and physician scientist fellowship, respectively, from the Medical Faculty Heidelberg. D.M. was partially supported by a post-doctoral fellowship from Excellence Cluster CellNetworks. We acknowledge support from the Interdisciplinary Neurobehavioral Core (INBC) for the behavioural experiments, the data storage service SDS@hd and bwMLS&WISO HPC supported by the state of Baden-Württemberg and the German Research Foundation (DFG) through grants INST 35/1314-1 FUGG and INST 35/1134-1 FUGG, respectively.Peer reviewe

Production and characterisation of recombinant forms of human pulmonary surfactant protein C (SP-C): Structure and surface activity

Surfactant protein C (SP-C) is an essential component for the surface tension-lowering activity of the pulmonary surfactant system. It contains a valine-rich α helix that spans the lipid bilayer, and is one of the most hydrophobic proteins known so far. SP-C is also an essential component of various surfactant preparations of animal origin currently used to treat neonatal respiratory distress syndrome (NRDS) in preterm infants. The limited supply of this material and the risk of transmission of infectious agents and immunological reactions have prompted the development of synthetic SP-C-derived peptides or recombinant humanized SP-C for inclusion in new preparations for therapeutic use. We describe herein the recombinant production in bacterial cultures of SP-C variants containing phenylalanines instead of the palmitoylated cysteines of the native protein, as fusions to the hydrophilic nuclease A (SN) from Staphylococcus aureus. The resulting chimerae were partially purified by affinity chromatography and subsequently subjected to protease digestion. The SP-C forms were recovered from the digestion mixtures by organic extraction and further purified by size exclusion chromatography. The two recombinant SP-C variants so obtained retained more than 50% α-helical content and showed surface activity comparable to the native protein, as measured by surface spreading of lipid/protein suspensions and from compression π-A isotherms of lipid/protein films. Compared to the protein purified from porcine lungs, the recombinant SP-C forms improved movement of phospholipid molecules into the interface (during adsorption), or out from the interfacial film (during compression), suggesting new possibilities to develop improved therapeutic preparations

USH2A is a Meissner’s corpuscle protein necessary for normal vibration sensing in mice and humans

Fingertip mechanoreceptors comprise sensory neuron endings together with specialized skin cells that form the end-organ. Exquisitely sensitive, vibration-sensing neurons are associated with Meissner’s corpuscles in the skin. In the present study, we found that USH2A, a transmembrane protein with a very large extracellular domain, was found in terminal Schwann cells within Meissner’s corpuscles. Pathogenic USH2A mutations cause Usher’s syndrome, associated with hearing loss and visual impairment. We show that patients with biallelic pathogenic USH2A mutations also have clear and specific impairments in vibrotactile touch perception, as do mutant mice lacking USH2A. Forepaw rapidly adapting mechanoreceptors innervating Meissner’s corpuscles, recorded from Ush2a−/− mice, showed large reductions in vibration sensitivity. However, the USH2A protein was not found in sensory neurons. Thus, loss of USH2A in corpuscular end-organs reduced mechanoreceptor sensitivity as well as vibration perception. Thus, a tether-like protein is required to facilitate detection of small-amplitude vibrations essential for the perception of fine-grained tactile surfaces.The present study was funded by grants from the Deutsche Forschungsgemeinshaft (grant nos. SFB665-B6 to G.R.L., SFB1315 to J.F.A.P. and SFB1158-A01 to S.G.L.) and grants from the European Research Council (grant nos. 789128 to G.R.L. and ERC-2015-CoG-682422 to J.F.A.P.). Additional funding was from the Institute of Health Carlos III (Spanish Ministry of Science and Innovation, grant no. FIS PI16/00539 to J.M.).Peer reviewe

A Compilation of Global Bio-Optical In Situ Data for Ocean-Colour Satellite Applications

A compiled set of in situ data is important to evaluate the quality of ocean-colour satellite-data records. Here we describe the data compiled for the validation of the ocean-colour products from the ESA Ocean Colour Climate Change Initiative (OC-CCI). The data were acquired from several sources (MOBY, BOUSSOLE, AERONET-OC, SeaBASS, NOMAD, MERMAID, AMT, ICES, HOT, GeP&CO), span between 1997 and 2012, and have a global distribution. Observations of the following variables were compiled: spectral remote-sensing reflectances, concentrations of chlorophyll a, spectral inherent optical properties and spectral diffuse attenuation coefficients. The data were from multi-project archives acquired via the open internet services or from individual projects, acquired directly from data providers. Methodologies were implemented for homogenisation, quality control and merging of all data. No changes were made to the original data, other than averaging of observations that were close in time and space, elimination of some points after quality control and conversion to a standard format. The final result is a merged table designed for validation of satellite-derived ocean-colour products and available in text format. Metadata of each in situ measurement (original source, cruise or experiment, principal investigator) were preserved throughout the work and made available in the final table. Using all the data in a validation exercise increases the number of matchups and enhances the representativeness of different marine regimes. By making available the metadata, it is also possible to analyse each set of data separately. The compiled data are available at doi:10.1594/PANGAEA.854832 (Valente et al., 2015)

A compilation of global bio-optical in situ data for ocean-colour satellite applications - version three

A global in situ data set for validation of ocean colour products from the ESA Ocean Colour Climate Change Initiative (OC-CCI) is presented. This version of the compilation, starting in 1997, now extends to 2021, which is important for the validation of the most recent satellite optical sensors such as Sentinel 3B OLCI and NOAA-20 VIIRS. The data set comprises in situ observations of the following variables: spectral remote-sensing reflectance, concentration of chlorophyll-a, spectral inherent optical properties, spectral diffuse attenuation coefficient, and total suspended matter. Data were obtained from multi-project archives acquired via open internet services or from individual projects acquired directly from data providers. Methodologies were implemented for homogenization, quality control, and merging of all data. Minimal changes were made on the original data, other than conversion to a standard format, elimination of some points, after quality control and averaging of observations that were close in time and space. The result is a merged table available in text format. Overall, the size of the data set grew with 148 432 rows, with each row representing a unique station in space and time (cf. 136 250 rows in previous version; Valente et al., 2019). Observations of remote-sensing reflectance increased to 68 641 (cf. 59 781 in previous version; Valente et al., 2019). There was also a near tenfold increase in chlorophyll data since 2016. Metadata of each in situ measurement (original source, cruise or experiment, principal investigator) are included in the final table. By making the metadata available, provenance is better documented and it is also possible to analyse each set of data separately. The compiled data are available at https://doi.org/10.1594/PANGAEA.941318 (Valente et al., 2022)

A compilation of global bio-optical in situ data for ocean colour satellite applications – version three

A global in situ data set for validation of ocean colour products from the ESA Ocean Colour Climate Change Initiative (OC-CCI) is presented. This version of the compilation, starting in 1997, now extends to 2021, which is important for the validation of the most recent satellite optical sensors such as Sentinel 3B OLCI and NOAA-20 VIIRS. The data set comprises in situ observations of the following variables: spectral remote-sensing reflectance, concentration of chlorophyll-a, spectral inherent optical properties, spectral diffuse attenuation coefficient, and total suspended matter. Data were obtained from multi-project archives acquired via open internet services or from individual projects acquired directly from data providers. Methodologies were implemented for homogenization, quality control, and merging of all data. Minimal changes were made on the original data, other than conversion to a standard format, elimination of some points, after quality control and averaging of observations that were close in time and space. The result is a merged table available in text format. Overall, the size of the data set grew with 148 432 rows, with each row representing a unique station in space and time (cf. 136 250 rows in previous version; Valente et al., 2019). Observations of remote-sensing reflectance increased to 68 641 (cf. 59 781 in previous version; Valente et al., 2019). There was also a near tenfold increase in chlorophyll data since 2016. Metadata of each in situ measurement (original source, cruise or experiment, principal investigator) are included in the final table. By making the metadata available, provenance is better documented and it is also possible to analyse each set of data separately. The compiled data are available at https://doi.org/10.1594/PANGAEA.941318 (Valente et al., 2022)

Piezo2 channels: from structure to nociception

Resumen del trabajo presentado al VIII Congreso Red Española de Canales Iónicos, celebrado en Alicante del 24 al 27 de mayo de 2022.Piezo channels constitute a unique family of ion channels that transduce mechanical forces, including membrane deformation and stretch. While Piezo1 is broadly expressed, Piezo2 is present in mechanoreceptors and proprioceptors. In these afferent neurons, Piezo2 senses, with remarkable sensitivity, hair movements or the stretch of muscle fibers. However, the molecular substrates for such refined sensitivity and responsiveness to different mechanical stimuli are missing. Recent work from our group has highlighted the intracellular regions in Piezo2 architecture that underlay those fundamental properties. Furthermore, we show that Piezo2 is also present in a particular subtype of pain fibers known as the Silent Nociceptors. Notably, Piezo2 is responsible for the mechanically activated currents induced by inflammation in these fibers.Peer reviewe

Novel celular and molecular players on mechanosensation and pain

Trabajo presentado al Seminario de Unidad Neurobiología Celular y de Sistemas del Instituto de Neurociencias, celebrado online el 13 de abril de 2021.Peer reviewe