|dc.description.abstracteng||The transient receptor potential A1 (TRPA1) channel is essential for vertebrate pain. TRPA1 plays a fundamental role as a primary detector of noxious stimuli of physical and chemical nature, and is critically involved in different pain states. Even though TRPA1 activation modalities have been studied extensively, the network of protein interactions regulating TRPA1 (the so-called TRPA1 interactome) is only poorly understood. Considering the crucial role of TRPA1 in pain signaling, it is mandatory to shed light on the elusive molecular machinery regulating TRPA1 channels in sensory neurons. This project was therefore aimed at getting insights into the mechanisms of TRPA1 regulation by identifying TRPA1-protein complexes and characterizing their biological meaning in the context of nociception.
A mass spectrometry-based proteomics approach led to the discovery of the physical association of Annexin A2 (AnxA2) with native TRPA1 in mouse sensory neurons. AnxA2 is enriched in a subpopulation of sensory neurons and coexpressed with TRPA1. Furthermore, we observed an increase of TRPA1 membrane levels in cultured sensory neurons from AnxA2-deficient mice. Functional studies suggest that AnxA2 limits the availability and consequently the activity of TRPA1 channels at the plasma membrane of sensory neurons. Moreover, our in vitro observations were reflected in enhanced nocifensive responses of AnxA2 knock-out mice specifically upon TRPA1 activation in vivo. TRPA1 channels have been shown to contribute to hypersensitivity to cold stimuli during inflammation. We therefore investigated the possible impact of AnxA2 on TRPA1 function also in this context employing the well-established complete Freund´s adjuvant (CFA) model of persistent inflammation in mice. AnxA2-deficient mice showed enhanced hypersensitivity on a cold plate upon CFA injection, whereas the hypersensitivity to heat and mechanical stimulation were not affected. In conclusion, we characterized AnxA2 as a novel TRPA1-associated protein which specifically regulates TRPA1 channels in vitro and in vivo.
These findings pave the way for a more thorough investigation of the dynamic changes in TRPA1-associated protein complexes in different conditions. In this context we submitted mice to the established Complete Freund’s Adjuvant (CFA)-model of inflammatory pain and isolated TRPA1-protein complexes from sensory neurons. Samples were submitted to state-of-the-art quantitative mass spectrometry analysis to compare complexes between CFA and control mice and therefore identify dynamic changes in TRPA1 interactome. This work revealed a dramatic alteration of TRPA1-associated protein complexes in the context of inflammatory pain, and the functional characterization of selected candidates that will follow this study might uncover new players in the development and maintenance of inflammatory pain.
In a separate set of experiments we focused our attention on a protein called 4-Nitrophenylphosphatase domain and non-neuronal SNAP25-like protein homolog 1 (NIPSNAP1), which function is largely still unknown. NIPSNAP1 showed to be expressed in nociceptive neurons of mouse dorsal root ganglia and functional studies suggested that it can modulate TRPA1 expression and/or function upon overexpression. A role of NIPSNAP1 in nociceptive transmission has been recently uncovered by the identification of its physical association with Nocistatin, a neuropeptide involved in pain signaling. In this context we investigated Nocistatin and found that it enhances TRPA1-mediated cellular calcium responses in DRG neurons. Mechanistically, this effect does not seem to be mediated by a modulation of TRPA1 plasma membrane expression, and NIPSNAP1 knock-down did not affect it. Future studies will contribute to characterize the underlying mechanisms and also the potential relevance for TRPA1-mediated nociception in vivo.||de