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ERC

MECHANOGENOMICS Report Summary

Project ID: 678610
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - MECHANOGENOMICS (Unravelling mammalian mechanosensor diversity by functional genomics)

Reporting period: 2016-09-01 to 2018-02-28

Summary of the context and overall objectives of the project

Mechanosensation refers to our ability to perceive touch, pain and proprioception through our somatosensory system, constituting with vision, olfaction and hearing the crucial senses governing our perception of surrounding environment and our social interactions. Mechanosensation relies on mechanotransduction, the signaling by which external mechanical stimuli are converted into biological signals within the cell. Molecular mechanosensors of the somato-sensory system are mechanically-activated ion channels, which identification constitutes one of the most important challenges in the field of sensory transduction. Our goal is to characterize mechanosensation from molecular to behavior levels.
Patients presenting with chronic pain and sensory malfunction often complain of a heightened perception of pain to noxious mechanical stimuli (mechanical hyperalgesia) and/or pain to innocuous touch (mechanical allodynia). One strategy for developing novel analgesics targets initial steps in the pain pathway, aiming potential treatments directly at the sensory neurons that detect noxious stimuli. Therefore, this work will provide new therapeutics target for treating touch and proprioceptive disorders and mechanical pain.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

At least three distinct type of mechanosensitive currents based on biophysical properties, mainly adaptation to sustained stimulation, are expressed in DRG neurons. If the rapidly adapting currents are mediated by Piezo2 channels, the identity of ion channels sustaining other types of mechanosensitive currents is unknown.
We combined patch-clamp recordings of DRG neuron mechanosensitive currents and single-cell RNA sequencing. Two rounds of this approach led us to generate the specific expression profile of 49 individual neurons belonging to four distinct populations based on the type of mechanosensitive current expressed. Comparison of these expression profiles using two different bioinformatics approaches, FPKM and DESeq2 analysis, led us to generate lists of candidates genes enriched in populations of interest. The overall quality of these data was evaluated and confirmed by looking at expected enriched genes in given populations based on current knowledge. Indeed, Piezo2 is enriched in the population of cells expressing rapidly adapting mechanosensitive currents, and several nociceptive markers are enriched in the population of cells expressing slowly adapting mechanosensitive currents described in the literature as nociceptors.
We next started to test these candidates one by one by performing siRNA knock-down in DRG neurons primary culture and patch-clamp recordings of mechanosensitive currents. We tested 90 candidates so far by comparing for each of them the proportions of neurons expressing the various types of mechanosensitive currents with control scramble siRNA treated DRG neurons. For each candidate, we perform at least two independent siRNA electroporation and record over 30 neurons. Control experiments using Piezo2 siRNA give rise to a statistically significant reduction of the proportion of neurons expressing rapidly adapting mechanosensitive currents, but none of the candidates tested so far reduces significantly the population of interest (intermediately- or slowly- adapting current expressing neurons). We are still testing candidate genes using this approach.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

Combining patch-clamp methodology and single-cell transcriptome sequencing to generate the specific expression profile of distinct populations of mouse mechanosensitive neurons was technically challenging, and our results suggests that this step has been successfully achieved.
We expect to identify molecular component(s) of specific mechanosensitive channels using our siRNA approach. Identification of such a component will be further examined using cloning/expression strategy, and histological staining. Ultimately, generation of transgenic animals and behavioral experiments will lead to characterize the physiological role of identified genes in somatosensory functions.
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