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Microglia and Neuropathic Pain: The role of calcium activated chloride channels

Final Report Summary - CACCINNP (Microglia and Neuropathic Pain: The role of calcium activated chloride channels)

Neuropathic pain is a major worldwide health problem that is poorly understood and often untreatable. The overall prevalence of neuropathic pain ranges between 0.9% and 8% of the population and its costs to society are substantial. Microgliosis in the spinal cord resulting from injury of peripheral sensory neurons is a key factor in the pathogenesis of neuropathic pain. Although microglia have been demonstrated to contribute to the pathology of chronic pain, they also exert protective effects by clearance of apoptotic neurons at the injury site. An understanding of the mechanisms that underlie neuronal-glial communication may allow these pathological and protective functions of microglia to be separated. Among a plethora of candidates involved in neuron-glia communication, we focused on a membrane protein called Anoctamin-6 that was recently shown to regulate membrane asymmetry and chloride transport. In this project we have investigated the role of this protein in neuropathic pain. Given the impact of chloride regulation on the pathogenesis of neuropathic pain, our research was firstly aimed at the development of tools for ex vivo monitoring of intracellular chloride changes. To address these issues, we have taken advantage of a new genetically encoded indicator termed Cl-Sensor, that contains a triple mutation in YFP, which renders it very sensitive to Cl (Markova et al., 2008;Waseem et al., 2010). We have created two mouse lines that express Cl-Sensor either under the control of the Thy1 mini promoter, or via Cre-mediated recombination from the Rosa26 locus. Together with the characterization of these lines we demonstrated that they allow for robust ratiometric monitoring of [Cl]i across different tissues. Transgenic mice expressing Cl-Sensor under the control of cell-type specific promoters provide a novel tool for the functional characterization of intracellular Cl distribution in defined subsets of neurons in various experimental models (Batti et al., 2013). Anoctamin-6 is an uncharacterized protein that belongs to a family of putative chloride channels. Recently, Suzuki et al., proposed an essential role for Anoctamin-6 for Ca2+-dependent phospholipids scrambling (Suzuki et al., 2010). We therefore aimed to characterize the dual role of this transmembrane protein. Using an electrophysiological approach combined with our recently developed ratiometric tools we identified Anoctamin6 as a chloride channels. The analysis was carried out in a heterologous system transiently transfected with Anoctamin-6 cDNA. The same system was then assessed for the putative scrambling activity. Using FACS analysis and time-lapse microscopy we assessed tools for the identification of the Ca2+ dependent phosphatidylserine (PS) exposure on the cell membrane. Site direct mutagenesis showed two point mutations on the transmembrane protein associated with a constitutive activation and inactivation, respectively of the scramblase. We have further generated conditional Anoctamin-6 KO mice to investigate the role of Anoctamin-6 in myeloid cells. The excision of exon 13 of Anoctamin-6 gene resembles the human mutation found on Scott Syndrome patients (Suzuki et al., 2010). The conditional KO animal, driven by LysMcre, showed a robust inactivation of Anoctamin-6 gene. KO macrophages primed with IFN-γ showed a diminished scramblase activity. Using a polarity-sensitive biosensor for PS and time lapse microscopy we observed transient exposure on the cell membrane of WT macrophages during the phagocytic occurrence. This event that was not present on KO macrophages. Change in anionic phospholipids is associated with several processes of phagocytosis (Yeung et al., 2008) we focused our attention on possible role for Anoctamin-6 in those mechanisms. A reduced phagocytic rate was observed for macrophages lacking Anoctamin-6 and this was likely due to a reduced capacity of volume changes and formation of late endosomes. Together those results suggest a dual function for Anoctamin-6 as a chloride channel and as a scramblase in a heterologous system and myeloid cells. We identified a role for Anoctamin-6 during phagocytosis, which is likely associated with the change in membrane asymmetry during the phagocytic event. We are currently exploring the function of Anoctamin-6 in the partial sciatic nerve ligation (PNL) mouse model of neuropathic pain. We observed an increase in microgliosis in neuropathic tissue and increased LysMCre expressing cells in the ipsilateral side of lumbar spinal cord. In this work we developed tools that should greatly facilitate non-invasive monitoring of [Cl]i and allow for the analysis of Cl transients in pathological conditions. We also assigned a dual function to an as yet uncharacterized channel and determined its role in phagocytosis, a common event occurring in the injury site of neuropathic tissue. Ongoing pain behavioral analysis will shortly address the potential contribution of Anoctamin-6 to the pathogenesis of neuropathic pain.
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Batti, L., Mukhtarov, M., Audero, E., Ivanov, A., Paolicelli, O., Zurborg, S., Gross, C., Bregestovski, P., and Heppenstall, P.A. (2013). Transgenic mouse lines for non-invasive ratiometric monitoring of intracellular chloride. Front Mol Neurosci 6, 11. Markova, O., Mukhtarov, M., Real, E., Jacob, Y., and Bregestovski, P. (2008). Genetically encoded chloride indicator with improved sensitivity. J Neurosci Methods 170, 67-76. Suzuki, J., Umeda, M., Sims, P.J. and Nagata, S. (2010). Calcium-dependent phospholipid scrambling by TMEM16F. Nature 468, 834-U135. Waseem, T., Mukhtarov, M., Buldakova, S., Medina, I., and Bregestovski, P. (2010). Genetically encoded Cl-Sensor as a tool for monitoring of Cl-dependent processes in small neuronal compartments. J Neurosci Methods 193, 14-23. Yeung, T., Gilbert, G.E. Shi, J., Silvius, J., Kapus, A., and Grinstein, S. (2008). Membrane phosphatidylserine regulates surface charge and protein localization. Science 319, 210-213.