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PN-III-P4-ID-PCE-2016-0475

Molecular targets responsible for the pain associated with neurovisceral porphyrias

Project director: Prof. Dr. Alexandru Babes, University of Bucharest

Project Executive Summary

Porphyrias comprise a family of genetic diseases due to loss-of-function mutations in the genes coding for enzymes involved in heme biosynthesis. Some of these diseases manifest as painful neurovisceral attacks. Accumulation of heme precursors appears to be involved in the pathology: aminolevulnic acid and porphobolinogen are massively increased in the plasma during acute attacks of porphyria. This proposal addresses the cellular and molecular mechanisms responsible for pain-sensing in neurovisceral porphyrias. Using an in vitro approach and a combination of functional (calcium imaging, patch clamp) and molecular (qRT-PCR, immunocytochemsitry) techniques, we aim to identify molecular targets involved in this pathological pain signaling. Our hypothesis is that accumulation of heme precursors leads to enhanced generation of reactive oxygen species (ROS), followed by opening of ROS-sensing ion channels and nociceptor activation. Our aim is to identify these ion channels, focusing on known ROS-modulated channels expressed in peripheral nociceptors: Transient receptor potential family members TRPA1, TRPV1 and TRPM8, as well as the TTX-resistant voltage-gated sodium channel Nav1.8. We shall also try to validate our hypothesis by measuring ROS production and identifying the predominant reactive species generated upon prolonged treatment of cultured neurons with heme precursors. Acute porphyria attacks are associated with mitochondrial dysfunction and can also be triggered by severe caloric deprivation. We shall explore the interaction between these factors and excessive levels of heme precursors, trying to pinpoint the mechanisms involved in the activation of pain-sensing primary afferent neurons. We believe that the outcome of this project will lead to a better understanding of the aetiology of neurovisceral porphyrias, but also to effective therapeutic strategies to alleviate the pain perceived by porphyria patients.

 

 

Tinte moleculare responsabile de durerea asociata cu porfiriile neuroviscerale

Director proiect: Prof. Dr. Alexandru Babes, Universitatea din Bucuresti

Rezumatul proiectului

Porfiriile sunt o familie de boli genetice datorate unor mutaţii ale genelor ce codificǎ enzimele implicate în biosinteza hemului. Unele dintre aceste boli se manifestǎ prin atacuri neuroviscerale dureroase. Acumularea precursorilor hemului pare sǎ fie implicatǎ  în patologia acestor boli: acidul aminolevulinic şi porfobilinogenul sunt masiv crescute în plasma sangvinǎ în atacurile de porfirie acutǎ. Proiectul nostru adreseazǎ mecanismele celulare şi moleculare responsabile de durerea din porfiriile neuroviscerale. Folosind o abordare in vitro şi o combinaţie de tehnici funcţionale (imagisticǎ de calciu, patch clamp) şi moleculare (RT-PCR, imunocitochimie), urmǎrim sǎ identificǎm ţinte moleculare implicate în aceastǎ patologie dureroasǎ. Ipoteza noastrǎ este cǎ acumularea de precursori ai hemului genereazǎ specii reactive de oxigen (SRO), ducând la activarea unor canale ionice sensibile la SRO şi la stimularea nociceptorilor. Scopul nostru este sǎ identificǎm aceste canale, axându-ne pe canale ionice modulate de SRO exprimate în nociceptori: membrii familiei Transient Receptor Potential TRPA1, TRPV1 şi TRPM8, precum şi canalul de sodiu dependent de voltaj rezistent la TTX Nav1.8. Vom încerca sǎ validǎm ipoteza noastrǎ masurând producerea de ROS şi identificând specia reactivǎ predominantǎ generatǎ în urma unui tratament prelungit al neuronilor în culturǎ cu precursori ai hemului. Atacurile acute de porfirie sunt asociate cu disfuncţie mitocondrialǎ şi  pot fi declanşate de deprivare caloricǎ severǎ. Vom explora interacţiunea dintre aceşti factori şi nivelul crescut de precursori ai hemului, încercând sǎ surprindem mecanismul implicat în activarea nociceptorilor primari. Avem convingerea cǎ rezultatul acestui proiect va duce la o mai bunǎ  înţelegere a etiologiei porfiriilor neuroviscerale, dar şi la identificarea unor strategii terapeutice pentru ameliorarea durerii pacienţilor care suferǎ de aceste boli.