Blanc SVSE 4 - Blanc - SVSE 4 - Neurosciences

Micro-RNAs as new candidates for chronic pain regulation – MirPain

Identifying the causal role of miRNA in the sensitization to chronic pain

We aim to demosntrate the role of miRNAin controlling the hyperexcitability of spinal neurons in conditions of chronic neuropathic pain. We also strive to identify candidates miRNA that are likely to represent new therapeutic targets, exerting an upstream control on the expression of key players in the pathophysiology of chronic pain.

Evidence for the role of miRNA in animal models of pain.

The incidence of chronic pain is estimated to 20-25% worldwide, thus making a real public health problem. However, few patients with chronic pain obtain complete relief from the drugs that are currently available, and more than half report poor relief. Long-lasting modification in pain transmission pathways develop as a result of global changes in gene expression2. However, it is largely unknown how nerve injury brings about such global changes in gene expression to induce chronic pain. <br />MicroRNAs (miRNAs) are a new class of non-coding nucleic acids able to regulate protein synthesis. The intial objectives of the project were to demonstrate the specific role of some miRNAs in the control of neuronal excitability that underlies pain sensitization in the spinal cord. The secondary objective was to assess the miRNA therapeutic potential within the frame of a preclinical study in animla models of neuropathic pain. Our project aims to combine a set of complementary technics to investigate the effects of miRNAs on the expression and the function of receptors and membrane channels in spinal dorsal horn neurons. <br />For this purpose, our project will pioneer the experimental study of miRNA functional role in controlling the excitability of spinal neurons, and more particularly of projection neurons that integrate nociceptive inputs to the spinal cord. The project will also test on animal models the behavioural consequences of treatments aiming to modulate the availability and/or the amount of candidate miRNAs.

The proposal relies on a large array of complementary technics, ranging from in vitro approaches on cell cultures, to in vivo behavioural analysis in animal models of chronic pain. Molecular biology methods will allow to generate gentic contructs required for the expression of recombinant proteins, and for the study of miRNAs. Cell cultures provide the opportunity to test these contructs and to analyse the effects of these recombinant proteins on cell physiology.Electrophysiology approaches make possible functional studies on different types of biological preparations, from cell cultures to spinal neurons recorded in anesthetized animals. Finally, sensory testing will give an assesment of pain behaviour

At mid-term, the results remain partial. As for the task 3, we master now all tools necessary to miRNA study in a subpopulation of spinal neurons that project nociceptive information to the brain. In particular, we can inject a genetic contrsuct-carrying virus in a site of brain projection, thalamus or parabrachial area, then let this virus migrate toward cell bodies of origin. In these neurons, the virus will be expressed and will modify the miRNA content.
The activities regarding the task 2 are largely completed. We have thus demonstrated the causal role of miR-103 in spinal neurons hyperexcitability.In normal conditions, miR-103 represses Cav1.2 calcium channel expression. In neuropathic conditions, this block is relieved and the calcium channel is upregulated. We had already demonstrated that such an overexpression is responsible for an exaggerated activation of spinal neurons, and in particular of projection neurons. Here, we demonstrate that miR-103 down-regulation in pain conditions leads to similar effects.Moreover, miR-103 effects are specific of the Cav1.2 subtype and the miRNA does not affect other calcium channel, such as the Cav1.3 subtype though it is structurally very close. Finally, changes in miR-103 expression have major consequences on the pain behaviour of animal models tested in this study.
The results already obtained have supported several invitations for our team in national and international conferences for 2 years. Owing to this work, we are also member of an international consortium that obtained an important european grant within the frame of a call of proposal dedicated to chronic pain.

Our study paves the ground for a future use of miRNA as therapeutic tools in the treatment of chronic pain.

We have published an important article in EMBO Journal about the role of miR-103 in the control of Cav1.2 calcium channel expression and of pain behaviour. Our preclinical data demonstrate the causal role of miRNA in chronic pain. This article represents

In physiological conditions, pain has a protective role. In contrast, chronic pain relies on maladaptive plasticity that induces neuronal sensitization in dorsal spinal networks. Such long-lasting modification develops as a result of global changes in gene expression. However, it is largely unknown how nerve injury brings about such changes in gene expression to induce chronic pain.
The present project aims to investigate how the translational control of key membrane channels participates in spinal neuron hyperexcitability in chronic pain. We will focus on microRNAs (miRNAs) that repress translation by binding mRNAs. In the present project, we will provide evidence for a causal role of miRNAs in long-lasting neuronal hyperexcitability underlying chronic pain in a rat model of neuropathy (Sciatic Nerve Ligation). We will focus on two complementary sets of mechanisms contributing to neuronal excitability, synaptic plasticity and intrinsic neuronal plasticity.
The project comprises 3 main tasks and will involve 3 partners: Marc Landry (miRNA and intrinsic properties); Olivier Thoumine (AMPA receptors and synaptic plasticity); and Yves De Koninck (deletion of miRNAs in spinal projection neurons).
Task 1 (O Thoumine): At the synaptic level, glutamatergic AMPARs relay nociceptive inputs. Changes in their subunit synthesis and trafficking play a critical role in chronic pain. Our preliminary data have used bioinformatics screening to select miRNA candidates for regulating AMPAR subunits synthesis. We aim to identify which of them are involved in changes of AMPAR expression in chronic pain. We narrowed our screen by showing that homeostatic scaling, a form of AMPAR-dependent synaptic plasticity, correlates with down-regulation of few candidates. We will now explore which candidates bind to AMPAR mRNA by using a Luciferase assay. The impact of selected miRNAs will be tested on AMPAR subunits expression and function at synapses in primary cultures. Then, intrathecal injections of these miRNAs, or their inhibitors, will be performed in Sham and neuropathic rats, and pain behaviour will be measured to evaluate the functional effects of miRNAs on pain thresholds.
Task 2 (M Landry): We already demonstrated the critical role of Cav1.2 L-type calcium channel in regulating intrinsic firing properties, and gene expression in spinal neurons. Our preliminary data identified one miRNA candidate, miR103 that is likely to regulate all three subunits (?1, ?2?1, ?1) of Cav1.2, thus providing a model for integrative regulation of a given macromolecular complex. We showed that miR103 is down-regulated in the spinal cord of neuropathic rats, and that miR103 intrathecal injections reduce pain sensitization. Our objective is to decipher the mode of action of miR103, and to search whether its antinociceptive effect is mediated by its regulatory role on the three Cav1.2 subunits. We will first confirmed miR103 binding to all three mRNAs. We will then use immunohistochemistry and qRT-PCR to investigate miR103-mediated repression of Cav1.2 subunits synthesis after transfection of spinal neuron cultures with miR103 or its inhibitor. Calcium imaging in transfected cultures will document the functional regulation of Cav1.2 by miR103. The effects of miR103 on Cav1.2 expression in vivo will be assessed after intrathecal injection of the miRNA, or its inhibitor, in Sham and neuropathic rats. Possible effects on other calcium channels (Cav2.2 and Cav3.2), will be also considered.
Task 3 (Y De Koninck): We will target modifications in the expression of miRNAs in spinal projection neurons. To do so, we will inject in the thalamus viral constructs expressing miRNA or their inhibitors, which will be retrogradely transported into spino-thalamic projection neurons. We will monitor the effects on pain behavior.
Our findings will be the first demonstrating the functional implication of miRNAs in chronic pain by integrative regulation of several targets.

Project coordinator

Monsieur Marc LANDRY (CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION AQUITAINE LIMOUSIN) – marc.landry@u-bordeaux2.fr

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IINS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION AQUITAINE LIMOUSIN
IINS CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE - DELEGATION AQUITAINE LIMOUSIN

Help of the ANR 344,000 euros
Beginning and duration of the scientific project: December 2011 - 36 Months

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