Mechanisms of ANO1 channel activation in sensory neurons

ANO1 (TMEM16A) is a Ca2+ activated Cl- channel (CaCC) expressed in peripheral somatosensory neurons responding to painful (noxious) stimuli. Previously, our lab has been able to demonstrate specific coupling of ANO1 to inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca2+ release from the endoplasmic reticulum (ER) via G-protein coupled receptor (GPCR) activation. This phenomenon underscores excitatory and noxious effects of some mediators of inflammatory pain, such as pro-algesic and vasoactive neuropeptide bradykinin. To further investigate mechanisms of ANO1 activation in somatosensory neurons, I developed a dual imaging approach, which involved transfecting dorsal root ganglion (DRG) neurons with a halide sensitive EYFP mutant (H148Q/I152L) and simultaneous Ca2+ imaging to monitor CaCC activity. This methodology was successfully used to demonstrate robust coupling of CaCC activity to IP3R activation produced by bradykinin. Blockade of ANO1 using a selective inhibitor (T16A-inhA01) abolished CaCC activity induced by bradykinin application. In contrast to the ER-induced Ca2+ release, Ca2+ influx produced by depolarisation-induced activation of voltage gated Ca2+ channels (VGCCs) was relatively ineffective in activating ANO1, which is in good agreement with previous studies. TRPV1 activation by capsaicin was able to induce robust CaCC activity. Given the ability of TRPV1 to activate PLC isoforms and produce IP3, I further tested the mechanism by which ANO1 is activated by TRPV1. Depletion of the ER Ca2+ stores severely reduced both, the capsaicin-induced Ca2+ signals and the concurrent CaCC activation. Intriguingly, under extracellular Ca2+ free conditions capsaicin was still able to induce [Ca2+]i elevation, further illustrating the ability of TRPV1 to induce intracellular Ca2+ release. Finally, monitoring of ER specific-Ca2+ dynamics concurrently with CaCC activity unambiguously confirmed the ability of TRPV1 to produce ER-Ca2+ mobilisation. Importantly, IP3R blockade with xestospongin C reduced CaCC activity after TRPV1 activation. Collectively, these experiments suggest that a significant fraction of Ca2+ required for activation of ANO1 downstream of TRPV1 is indeed delivered through IP3R activation. Using ‘in-situ proteomics’ and super-resolution microscopy I investigated multi-protein complexes in ER-plasma membrane (ER-PM) junctions of DRG neurons involving ANO1, TRPV1 and IP3R1. I found using proximity ligation assay that all 3 proteins were within 40nm of each other; however there was a greater number of ANO1 and TRPV1 complexes compared to TRPV1/ANO1 and TRPV1/IP3R1 complexes. Two-colour stochastic optical reconstruction microscopy (STORM) was able to confirm these findings and demonstrate that there is indeed a greater percentage of complexes involving ANO1 and TRPV1. Preliminary triple-colour STORM suggested the presence of ANO1, TRPV1 and IP3R1-containing protein complexes. Finally, I used total internal reflection microscopy (TIRF) to monitor the dynamics of the ER-PM junctions following the activation of bradykinin receptors or TRPV1. Application of bradykinin and capsaicin elicited increased intensity proximity of the ER to PM (as evaluated by the TIRF signal of fluorescently-labelled ER), which is suggestive of the ER moving to the PM by internal store mobilisation and highlighting the importance of ER-PM junctions. In sum, the experiments described in this thesis have discovered and characterised a novel mode of ANO1 activation in pain-sensing neurons: TRPV1-mediated ER Ca2+ release in ER-PM junctional signalling complex. These findings describe a hitherto unknown signalling mechanism potentially contributing to inflammatory pain

Snf2h Primes UL Neuron Production

Alterations in the homeostasis of either cortical progenitor pool, namely the apically located radial glial (RG) cells or the basal intermediate progenitors (IPCs) can severely impair cortical neuron production. Such changes are reflected by microcephaly and are often associated with cognitive defects. Genes encoding epigenetic regulators are a frequent cause of intellectual disability and many have been shown to regulate progenitor cell growth, including our inactivation of the Smarca1 gene encoding Snf2l, which is one of two ISWI mammalian orthologs. Loss of the Snf2l protein resulted in dysregulation of Foxg1 and IPC proliferation leading to macrocephaly. Here we show that inactivation of the closely related Smarca5 gene encoding the Snf2h chromatin remodeler is necessary for embryonic IPC expansion and subsequent specification of callosal projection neurons. Telencephalon-specific Smarca5 cKO embryos have impaired cell cycle kinetics and increased cell death, resulting in fewer Tbr2+ and FoxG1+ IPCs by mid-neurogenesis. These deficits give rise to adult mice with a dramatic reduction in Satb2C upper layer neurons, and partial agenesis of the corpus callosum. Mice survive into adulthood but molecularly display reduced expression of the clustered protocadherin genes that may further contribute to altered dendritic arborization and a hyperactive behavioral phenotype. Our studies provide novel insight into the developmental function of Snf2h-dependent chromatin remodeling processes during brain development

Antinociceptive Activity of Methanolic Leaf Extract of Parthenium hysterophorus L.

The present study was aimed to evaluate the antinociceptive property of Parthenium hysterophorus L. The central antinociceptive activity was analyzed by hot plate and tail immersion method; whereas acetic acid-induced writhing test and formalin induced licking tests were carried out for peripheral antinociceptive activity. The acute toxicity study revealed that methanol extract of the plant was moderately toxic at a dose of 500 mg/kg body weight. In hot plate method and tail immersion test the methanol extract exhibited significant analgesic activity ( < 0.001) at a dose of 2.5 and 5 mg/kg revealed its central antinociceptive activity. The significant activity (p<0.01) in acetic acid induced writhing test and formalin induced licking test implies the peripheral antinociceptive property of the extract at both doses. These findings justify that P. hysterophorus L. can be a valuable natural antinociceptive source which seemed to provide potential phytotherapeutics against various ailments