The process of wound healing involves a complex network of signaling pathways working to promote rapid cell migration and wound closure. Activation of purinergic receptors by secreted nucleotides plays a major role in calcium mobilization and the subsequent calcium-dependent signaling that is essential for proper healing. The role of the purinergic receptor P2X7 in wound healing is still relatively unknown. We demonstrate that P2X7 expression increases at the leading edge of corneal epithelium after injury in an organ culture model, and that this change occurs despite an overall decrease in P2X7 expression throughout the epithelium. Inhibition of P2X7 prevents this change in localization after injury and impairs wound healing. In cell culture, P2X7 inhibition attenuates the amplitude and duration of injuryinduced calcium mobilization in cells at the leading edge. Immunofluorescence analysis of scratchwounded cells reveals that P2X7 inhibition results in an overall decrease in the number of focal adhesions along with a concentration of focal adhesions at the wound margin. Live cell imaging of green fluorescent proteinelabeled actin and talin shows that P2X7 inhibition alters actin cytoskeletal rearrangements and focal adhesion dynamics after injury. Together, these data demonstrate that P2X7 plays a critical role in mediating calcium signaling and coordinating cytoskeletal rearrangement at the leading edge, both of which processes are early signaling events necessary for proper epithelial wound healing. (Am J Pathol 2016, 186: 285e296; http://dx

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GABAA receptors mediate rapid responses to the neurotransmitter GABA and are robust regulators of the brain and spinal cord neural networks that control locomotor behaviors, such as walking and swimming. In developing zebrafish, gross pharmacological blockade of these receptors causes hyperactive swimming, which is also a feature of many zebrafish epilepsy models. Although GABAA receptors are important to control locomotor behavior, the large number of subunits and homeostatic compensatory mechanisms have challenged efforts to determine subunit-selective roles. To address this issue, we mutated each of the eight zebrafish GABAA α subunit genes individually and in pairs using a CRISPR-Cas9 somatic inactivation approach, then we examined the swimming behavior of the mutants at two developmental stages, 48 and 96 hours post fertilization (hpf). We found that disrupting the expression of specific pairs of subunits resulted in different abnormalities in swimming behavior at 48 hpf. Mutation of α4 and α5 selectively resulted in longer duration swimming episodes, mutations in α3 and α4 selectively caused excess, large-amplitude body flexions (C-bends), and mutation of α3 and α5 resulted in increases in both of these measures of hyperactivity. At 96 hpf, hyperactive phenotypes were nearly absent, suggesting that homeostatic compensation was able to overcome the disruption of even multiple subunits. Taken together, our results identify subunit-selective roles for GABAA α3, α4, and α5 in regulating locomotion. Given that these subunits exhibit spatially restricted expression patterns, these results provide a foundation to identify neurons and GABAergic networks that control discrete aspects of locomotor behavior.

Amyotrophic lateral sclerosis (ALS) is an incurable neurodegenerative disease characterized by the death of upper and lower motor neurons. While causative genes have been identified, 90% of ALS cases are not inherited and are hypothesized to result from the accumulation of genetic and environmental risk factors. While no specific causative environmental toxin has been identified, previous work has indicated that the presence of the organochlorine pesticide cis-chlordane in the blood is highly correlated with ALS incidence. Never before tested on the motor system, here, we show that cis-chlordane is especially toxic to motor neurons in vitro- and in vivo-independent of its known antagonism of the GABAA receptor. We find that human stem-cell-derived motor neurons are more sensitive to cis-chlordane than other cell types and their action potential dynamics are altered. Utilizing zebrafish larvae, we show that cis-chlordane induces motor neuron and neuromuscular junction degeneration and subsequent motor deficits in a touch-evoked escape response. Together, our work points to cis-chlordane as a potential sporadic ALS exacerbating environmental pollutant.

GABAA receptors mediate rapid responses to the neurotransmitter GABA and are robust regulators of the brain and spinal cord neural networks that control locomotor behaviors, such as walking and swimming. In developing zebrafish, gross pharmacological blockade of these receptors causes hyperactive swimming, which has been embraced as an epilepsy model. Although GABAA receptors are important to control locomotor behavior, the large number of subunits and homeostatic compensatory mechanisms have challenged efforts to determine subunit-selective roles. To address this issue, we mutated each of the eight zebrafish GABAA α subunit genes individually and in pairs using a CRISPR-Cas9 somatic inactivation approach, then we examined the swimming behavior of the mutants at two developmental stages. We found that disrupting the expression of specific pairs of subunits resulted in different abnormalities in swimming behavior at the first development stage. Mutation of α4 and α5 selectively resulted in longer duration swimming episodes, mutations in α3 and α4 selectively caused excess, large-amplitude body flexions (C-bends), and mutation of α3 and α5 resulted in increases in both of these measures of hyperactivity. At the later stage of development, hyperactive phenotypes were nearly absent, suggesting that homeostatic compensation was able to overcome the disruption of even multiple subunits. Taken together, our results identify subunit-selective roles for GABAA α3, α4, and α5 in regulating locomotion. Given that these subunits exhibit spatially restricted expression patterns, these results provide a foundation to identify neurons and GABAergic networks that control discrete aspects of locomotor behavior.