Calmodulin Supports TRPA1 Channel Association with Opioid Receptors and Glutamate NMDA Receptors in the Nervous Tissue

Supplementary Materials: The following are available online at https://www.mdpi.com/1422-006 7/22/1/229/s1, Figure S1: TRPA1 association with MORs in spinal cord in HINT1-/- and 1R-/- mice. Figure S2: The HINT1 protein or 1R does not support the MOR association with the Nt or Ct regions of TRPA1 channels. Figure S3: CaM mediates the TRPA1 Ct association with MOR in the absence of Ca2+. Figure S4: Pharmacological modulation of TRPA1 associations with MORs and glutamate NMDARs. Figure S5: Formalin-induced inflammatory pain alters TRPA1 associations with MORs and NMDARs. Figure S6: TRPA1 associations with opioid receptors and NMDARs in the CCI model of neuropathic pain.We would like to thank Gabriela de Alba and María José López for their excellent technical assistanceTransient receptor potential ankyrin member 1 (TRPA1) belongs to the family of thermo TRP cation channels that detect harmful temperatures, acids and numerous chemical pollutants. TRPA1 is expressed in nervous tissue, where it participates in the genesis of nociceptive signals in response to noxious stimuli and mediates mechanical hyperalgesia and allodynia associated with different neuropathies. The glutamate N-methyl-d-aspartate receptor (NMDAR), which plays a relevant role in allodynia to mechanical stimuli, is connected via histidine triad nucleotide-binding protein 1 (HINT1) and type 1 sigma receptor (σ1R) to mu-opioid receptors (MORs), which mediate the most potent pain relief. Notably, neuropathic pain causes a reduction in MOR antinociceptive efficacy, which can be reversed by blocking spinal NMDARs and TRPA1 channels. Thus, we studied whether TRPA1 channels form complexes with MORs and NMDARs that may be implicated in the aforementioned nociceptive signals. Our data suggest that TRPA1 channels functionally associate with MORs, delta opioid receptors and NMDARs in the dorsal root ganglia, the spinal cord and brain areas. These associations were altered in response to pharmacological interventions and the induction of inflammatory and also neuropathic pain. The MOR-TRPA1 and NMDAR-TRPA1 associations do not require HINT1 or σ1R but appear to be mediated by calcium-activated calmodulin. Thus, TRPA1 channels may associate with NMDARs to promote ascending acute and chronic pain signals and to control MOR antinociception.MICINN Plan Nacional I+D+i RT 2018-093677B-100University of Granada PPJIB2019.11MECD FPU 15/02356 FPU16/0321

Estudio limnológico de varios sistemas lóticos y lénticos aledaños a la reserva Yotoco Valle del Cauca, Colombia

Este trabajo es un estudio limnológico básico de cinco cuerpos de agua de la cuenca alta del río Cauca. Dos de ellos fueron ambientes lénticos (la laguna de Sonso y el embalse Calima) y tres fueron lóticos (el río Calima y las quebradas La Berreadera y El Boleo). Se ubican en los municipios de Buga, Yotoco y Calima en el Valle del Cauca, Colombia. Los muestreos se hicieron los días 15 y 16 de octubre de 2016. Como principal objetivo se buscó caracterizar cada uno de los lugares de muestreo en cuanto a sus variables morfológicas, fisicoquímicas y biológicas, por medio de la evaluación de distintas variables, tanto in situ como en laboratorio. Todo lo anterior permitió determinar las características generales y algunas específicas de las condiciones actuales de cada ecosistema acuático evaluado. Tales particularidades podrían ser relevantes en estudios de línea base, impacto ambiental, bioindicación y concientización ambiental

4to. Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad. Memoria académica

Este volumen acoge la memoria académica de la Cuarta edición del Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad, CITIS 2017, desarrollado entre el 29 de noviembre y el 1 de diciembre de 2017 y organizado por la Universidad Politécnica Salesiana (UPS) en su sede de Guayaquil. El Congreso ofreció un espacio para la presentación, difusión e intercambio de importantes investigaciones nacionales e internacionales ante la comunidad universitaria que se dio cita en el encuentro. El uso de herramientas tecnológicas para la gestión de los trabajos de investigación como la plataforma Open Conference Systems y la web de presentación del Congreso http://citis.blog.ups.edu.ec/, hicieron de CITIS 2017 un verdadero referente entre los congresos que se desarrollaron en el país. La preocupación de nuestra Universidad, de presentar espacios que ayuden a generar nuevos y mejores cambios en la dimensión humana y social de nuestro entorno, hace que se persiga en cada edición del evento la presentación de trabajos con calidad creciente en cuanto a su producción científica. Quienes estuvimos al frente de la organización, dejamos plasmado en estas memorias académicas el intenso y prolífico trabajo de los días de realización del Congreso Internacional de Ciencia, Tecnología e Innovación para la Sociedad al alcance de todos y todas

Morfogénesis de los túbulos dentinales: un modelo matemático Morphogenesis of dentinal tubules: a mathematical model

La diferenciación mesenquimal a odontoblasto es un proceso complejo que determina la formación de los túbulos dentinales. Este proceso involucra una cuidadosa y regulada secuencia de cambios en el comportamiento de las células mesenquimales, coordinados por la expresión de diferentes factores moleculares, entre ellos, principalmente, el Noggin y BMP2. En este artículo se simula la formación de los túbulos dentinales a partir de un modelo matemático de reacción difusión que es solucionado por el método de los elementos finitos.Mesenchymal differentiation into odontoblasts is a complex process determining the formation of dentinal tubules. The process involves a carefully regulated sequence of changes in the behavior of mesenchymal cells, coordinated by the expression of various molecular factors, particularly Noggin and BMP2. In this paper the formation of dentinal tubules is simulated using a reaction-diffusion mathematical model solved by the finite element method

The ¿1 Receptor and the HINT1 Protein Control ¿2¿1 Binding to Glutamate NMDA Receptors: Implications in Neuropathic Pain

Nerve injury produces neuropathic pain through the binding of ¿2¿1 proteins to glutamate N-methyl-D-aspartate receptors (NMDARs). Notably, mice with a targeted deletion of the sigma 1 receptor (¿1R) gene do not develop neuropathy, whereas mice lacking the histidine triad nucleotide-binding protein 1 (Hint1) gene exhibit exacerbated allodynia. ¿1R antagonists more effectively diminish neuropathic pain of spinal origin when administered by intracerebroventricular injection than systemically. Thus, in mice subjected to unilateral sciatic nerve chronic constriction injury (CCI), we studied the participation of ¿1Rs and HINT1 proteins in the formation of ¿2¿1-NMDAR complexes within the supraspinal periaqueductal gray (PAG). We found that ¿1 peptides required ¿1Rs in order to interact with the NMDAR NR1 variant that contains the cytosolic C1 segment. ¿1R antagonists or low calcium levels provoke the dissociation of ¿1R-NR1 C1 dimers, while they barely affect the integrity of ¿1-¿1R-NR1 C1 trimers. However, HINT1 does remove ¿1 peptides from the trimer, thereby facilitating the subsequent dissociation of ¿1Rs from NMDARs. In ¿1R-/- mice, CCI does not promote the formation of NMDAR-¿2¿1 complexes and allodynia does not develop. The levels of ¿2¿1-¿1R-NMDAR complexes increase in HINT1-/- mice and after inducing CCI, degradation of ¿2¿1 proteins is observed. Notably, ¿1R antagonists but not gabapentinoids alleviate neuropathic pain in these mice. During severe neuropathy, the metabolism of ¿2¿1 proteins may account for the failure of many patients to respond to gabapentinoids. Therefore, ¿1Rs promote and HINT1 proteins hinder the formation ¿2¿1-NMDAR complexes in the PAG, and hence, the appearance of mechanical allodynia depends on the interplay between these proteins.This work was supported by MICINN Plan Nacional I+D+i [grant number RT 2018-093677-B-100]

A theoretical model of dentinogenesis: dentin and dentinal tubule formation

Dentinogenesis, odontoblast dentin formation, includes dentinal growth, mineralization and dentinal tubule formation. Odontoblasts synthesize collagen resulting in collagen apposition contributing to dentinogenesis. Furthermore, within the tubule, they express non-collagenous proteins, such as dentin phosphoprotein (DPP), associated with hydroxyapatite crystal formation and growth. The aim of this work was to determine patterns of growth and dentin formation and quantification of its mineralization. Findings from our work are relevant to endodontics for future regenerative treatment. We formulated a 3D domain mathematical model, which recreates the events that lead to dentinal tubule mineralization. As reference we used collagen apposition and DPP activity. We obtained a model depicting predentin's mineralization distribution during dentin development. Furthermore, we verified different DPP diffusion coefficients to test the model's sensitivity. We present a model to shed light on the process of dentin and dentinal tubule formation, and its relation to diffusion and mineralization processes

^α<i>N</i>-Acetyl β-Endorphin Is an Endogenous Ligand of σ1Rs That Regulates Mu-Opioid Receptor Signaling by Exchanging G Proteins for σ2Rs in σ1R Oligomers

The opioid peptide β-endorphin coexists in the pituitary and brain in its αN-acetylated form, which does not bind to opioid receptors. We now report that these neuropeptides exhibited opposite effects in in vivo paradigms, in which ligands of the sigma type 1 receptor (σ1R) displayed positive effects. Thus, αN-acetyl β-Endorphin reduced vascular infarct caused by permanent unilateral middle cerebral artery occlusion and diminished the incidence of N-methyl-D-aspartate acid-promoted convulsive syndrome and mechanical allodynia caused by unilateral chronic constriction of the sciatic nerve. Moreover, αN-acetyl β-Endorphin reduced the analgesia of morphine, β-Endorphin and clonidine but enhanced that of DAMGO. All these effects were counteracted by β-Endorphin and absent in σ1R−/− mice. We observed that σ1Rs negatively regulate mu-opioid receptor (MOR)-mediated morphine analgesia by binding and sequestering G proteins. In this scenario, β-Endorphin promoted the exchange of σ2Rs by G proteins at σ1R oligomers and increased the regulation of G proteins by MORs. The opposite was observed for the αN-acetyl derivative, as σ1R oligomerization decreased and σ2R binding was favored, which displaced G proteins; thus, MOR-regulated transduction was reduced. Our findings suggest that the pharmacological β-Endorphin-specific epsilon receptor is a σ1R-regulated MOR and that β-Endorphin and αN-acetyl β-Endorphin are endogenous ligands of σ1R

Human HINT1 Mutant Proteins that Cause Axonal Motor Neuropathy Exhibit Anomalous Interactions with Partner Proteins

The 14 kDa histidine triad nucleotide-binding protein 1 (HINT1) is critical to maintain the normal function of motor neurons. Thus, a series of human HINT1 mutants cause autosomal recessive axonal neuropathy with neuromyotonia. HINT1 establishes a series of regulatory interactions with signaling proteins, some of which are enriched in motor neurons, such as the type 1 sigma receptor or intracellular domain (ICD) of transmembrane teneurin 1, both of which are also implicated in motor disturbances. In a previous study, we reported the capacity of HINT1 to remove the small ubiquitin-like modifier (SUMO) from a series of substrates and the influence of HINT1 mutants on this activity. We now report how human HINT1 mutations affect the interaction of HINT1 with the regulator of its SUMOylase activity, calcium-activated calmodulin, and its substrate SUMO. Moreover, HINT1 mutants exhibited anomalous interactions with G protein coupled receptors, such as the mu-opioid, and with glutamate N-methyl-D-aspartate receptors as well. Additionally, these HINT1 mutants showed impaired associations with transcriptional regulators such as the regulator of G protein signaling Z2 protein and the cleaved N-terminal ICD of teneurin 1. Thus, the altered enzymatic activity of human HINT1 mutants and their anomalous interactions with partner proteins may disrupt signaling pathways essential to the normal function of human motor neurons.This work was supported by Plan Nacional I+D+i [grant number RT-2018-093677-B-100]

Schizophrenia and depression, two poles of endocannabinoid system deregulation

Abstract The activity of certain G protein-coupled receptors (GPCRs) and of glutamate N-Methyl-D-aspartate receptors (NMDARs) is altered in both schizophrenia and depression. Using postmortem prefrontal cortex samples from subjects with schizophrenia or depression, we observed a series of opposite changes in the expression of signaling proteins that have been implicated in the cross-talk between GPCRs and NMDARs. Thus, the levels of HINT1 proteins and NMDAR NR1 subunits carrying the C1 cytosolic segment were increased in depressives and decreased in schizophrenics, respect to matched controls. The differences in NR1 C1 subunits were compensated for via altered expression of NR1 subunits lacking the C1 segment; thus, the total number of NR1 subunits was comparable among the three groups. GPCRs influence the function of NR1 C1-containing NMDARs via PKC/Src, and thus, the association of mu-opioid and dopamine 2 receptors with NR1 C1 subunits was augmented in depressives and decreased in schizophrenics. However, the association of cannabinoid 1 receptors (CB1Rs) with NR1 C1 remained nearly constant. Endocannabinoids, via CB1Rs, control the presence of NR1 C1 subunits in the neural membrane. Thus, an altered endocannabinoid system may contribute to the pathophysiology of schizophrenia and depression by modifying the HINT1-NR1 C1/GPCR ratio, thereby altering GPCR-NMDAR cross-regulation

The ALS-Related o1R E102Q Mutant Eludes Ligand Control and Exhibits Anomalous Response to Calcium

Sigma receptor type 1 (o1R) is a transmembrane protein expressed throughout the central nervous system and in certain peripheral tissues. The human o1R E102Q mutation causes juvenile amyotrophic lateral sclerosis (ALS), likely by inducing a series of alterations in calcium efflux from the endoplasmic reticulum (ER) to mitochondria that affects calcium homeostasis and cellular survival. Here, we report the influence of calcium on o1R E102Q associations with glutamate N-methyl-D-aspartate receptors (NMDARs), binding immunoglobulin protein (BiP), and transient receptor potential calcium channels A1, V1, and M8. The mutant protein inhibited the binding of calmodulin to these calcium channels and interacted less with BiP than wild-type o1R, thereby contributing to calcium homeostasis dysfunction. Mutant o1R, but not wild-type o1R, strongly bound to histidine triad nucleotide binding protein 1, which regulates neuromuscular synaptic organization and target selection through teneurin 1. While ligands regulated the association of o1R wild-type with NMDARs and BiP, they failed to modulate the interaction between these proteins and the o1R E102Q mutant. Thus, the o1R E102Q mutant exhibited an anomalous response to cytosolic calcium levels, altered affinity for target proteins, and a loss of response to regulatory ligands. We believe that these modifications may contribute to the onset of juvenile ALS