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Unravelling mammalian mechanosensor diversity by functional genomics

Periodic Reporting for period 2 - MECHANOGENOMICS (Unravelling mammalian mechanosensor diversity by functional genomics)

Reporting period: 2018-03-01 to 2019-08-31

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 somatosensory system are mechanosensitive ion channels, which identification constitutes one of the most important challenges in the field of sensory transduction. Our goal is to identify molecular components of these mechanosensitive channels and to characterize their roles in touch, mechanical pain sensing and proprioception.
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 is to target initial steps in the pain pathway. In this context, mechanosensitive channels are the molecular players which initiate the pain response by detecting noxious stimuli. Therefore, this work will provide new therapeutics targets for treating touch and proprioceptive disorders and mechanical pain.
At least three distinct type of mechanosensitive currents based on biophysical properties, mainly adaptation to sustained stimulation, are expressed in dorsal root ganglion (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 (mechano patch-seq). Two rounds of this approach led us to generate the specific expression profile of 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 evaluate the overall quality of these data by looking at expected enriched genes in given populations based on current knowledge. Indeed, Piezo2 is enriched in the population of neurons expressing rapidly adapting mechanosensitive currents, and many nociceptive markers are enriched in the neuronal population expressing slowly adapting mechanosensitive currents defined in the literature as nociceptive neurons. Therefore, we generated lists of candidate genes by selecting and prioritizing those enriched in populations of interest.
We next tested these candidates one by one by performing siRNA knock-down in DRG neurons primary culture and patch-clamp recordings of mechanosensitive currents. We tested over 150 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 performed at least two independent siRNA electroporation and recorded 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). However, two candidate genes induce a significant decrease of maximal amplitude of intermediately- or slowly-adapting mechanosensitive currents. While still testing candidate genes using this approach, we are further characterizing the two identified genes. These genes code for transmembrane proteins of unknown function. Especially, we are performing behavior experiments on transgenic animals to characterize the physiological roles of these two genes on mechanosensation in vivo.
Combining patch-clamp recordings of mechanosensitive currents and single-cell transcriptome sequencing to generate the specific expression profile of distinct populations of mouse mechanosensitive neurons was a technical challenge that we successfully achieved. Using these data to identify gene candidates specifically expressed in neuronal subpopulations, we expect to identify molecular component(s) of specific mechanosensitive channels using a siRNA screening approach. Identification of such a component is 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.
The identification of components and/or specific modulators of somatosensory mechanosensitive channels is an important challenge for the study of nociception. Their identification would be transformative as a scientific finding, and have many clinical implications in acute, inflammatory and chronic pain. Furthermore, the conversion of mechanical force into biochemical signaling is crucial to physiology of many additional cell types. Although this research is aimed for advancement of pain research, it could potentially impact a variety of other biological systems that could share mechanically-gated ion channels with the somatosensory system.