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Panx1 Channels Promote Both Anti- and Pro-Seizure-Like Activities in The bioRxiv preprint doi: https://doi.org/10.1101/2021.06.03.446992; this version posted June 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. PWF et al. 2021 1 2 3 4 5 6 7 8 Panx1 channels promote both anti- and pro-seizure-like 9 activities in the zebrafish via p2rx7 receptors and ATP signaling 10 11 12 13 14 15 16 1,2,4* 1,2 1,2 1,2 17 Paige Whyte-Fagundes , Daria Taskina , Nickie Safarian , Christiane Zoidl , Peter L 18 Carlen3,4, Logan W Donaldson1, Georg R Zoidl1,2,4* 19 20 21 22 1 Department of Biology, York University; Toronto, Ontario, M3J1P3; Canada 23 2 Center of Vision Research (CVR), York University; Toronto, Ontario, M3J1P3; Canada 24 3 Department of Medicine, Physiology and BME, University of Toronto, 399 Bathurst St., 5w442, 25 Toronto, ON M5T 2S8, Canada 26 4 Department, Krembil Research Institute, University Health Network, 135 Nassau Street, 27 Toronto, ON M5T 1M8, Canada 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 *Corresponding authors: [email protected], [email protected] 45 46 Keywords: Pannexin1, seizures, epilepsy, electrophysiology, behavior, TALENs, Zebrafish, ATP 47 bioRxiv preprint doi: https://doi.org/10.1101/2021.06.03.446992; this version posted June 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 2 48 Abstract: 49 The molecular mechanisms of excitation-inhibition imbalances promoting seizure generation in 50 epilepsy patients are not fully understood. Experimental evidence suggests that Pannexin1 (Panx1), 51 an ATP release channel, modulates excitability of the brain. In this report, we have performed 52 behavioral and molecular phenotyping experiments on zebrafish larvae bearing genetic or 53 pharmacological knockouts of panx1a or panx1b channels, each highly homologous to human 54 PANX1. When Panx1a function is lost or both channels are under pharmacological blockage, 55 treatment with pentylenetetrazol to induce seizures causes reduced ictal-like events and seizure- 56 like locomotion. These observations were extended by transcriptome profiling, where a spectrum 57 of distinct metabolic and cell signaling states correlates with the loss of panx1a. The pro- and 58 anticonvulsant activities of both Panx1 channels affects ATP release and involves the purinergic 59 receptor p2rx7. We propose that Panx1 zebrafish models offer opportunities to explore the 60 molecular and physiological basis of seizures and assist anticonvulsant drug discovery. 61 62 63 64 Introduction 65 A widely accepted view in epilepsy research is that neuronal hyperexcitability during 66 epileptic seizures is caused by an imbalance of excitatory and inhibitory activities. This view places 67 the imbalance of neuronal transmitters glutamate and GABA release first, but evidence is 68 accumulating for altered ATP- and adenosine-mediated signaling between neurons and glial cells 69 contributing to heightened states of excitability and epileptic seizures 1. ATP release and signaling 70 can be both excitatory and inhibitory, but adenosine strongly inhibits electrical activity. Five groups 71 of ATP-release channels with expression in the nervous system are known: pannexin-1 (Panx1) 2, 72 connexin (Cx) hemichannels 3, calcium homeostasis modulator 1 (CALHM1) 4, volume-regulated 73 anion channels (VRACs, also known as volume-sensitive outwardly rectifying (VSOR) anion bioRxiv preprint doi: https://doi.org/10.1101/2021.06.03.446992; this version posted June 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 3 74 channels) 5, and maxi-anion channels (MACs) 6. Panx1 is recognized as a pro-convulsant channel 75 after behavioral and electrophysiological markers of excitability are ameliorated in distinct models 76 of epilepsy once Panx1 is inhibited pharmacologically or by global deletion in mice 7-10. Other 77 evidence for pro-convulsant actions of Panx1 derived from increased expression of human and 78 rodent Panx1 found in epileptic tissue 8,11-13. However, the simplistic view of inhibiting mammalian 79 Panx1 and causing anti-convulsant effects is challenged. The targeted deletion of mouse Panx1 in 80 astrocytes potentiates, while the absence of Panx1 in neurons attenuates seizure manifestation 14. 81 Furthermore, the contribution of Panx1 to seizures is also brain region dependent 15, raising 82 questions about the underlying molecular and cellular mechanisms. 83 Zebrafish have two panx1 ohnologues, panx1a and panx1b, with distinct expression 84 localizations and biophysical properties 16-18. Like mammalian Panx1 19-21, the zebrafish panx1a is 85 broadly expressed in all tissues tested 16,17, whereas the expression of panx1b is highest in the 86 nervous system 17. We had suggested that both pannexins fulfill different functions in vivo based 87 on differences in the unitary conductance of Panx1a (≈380pS) and Panx1b (480-500pS) channels, 88 the complexity of subconductance stages, and the cumulative open and closed times 17. Here, we 89 interrogated Panx1 channels genetically and pharmacologically, and induced seizure-like activities 90 in the zebrafish using pentylenetetrazole (PTZ) 22 to suppress inhibitory networks by blocking 91 gamma aminobutyric acid (GABA)-A receptors. The zebrafish responses to PTZ were 92 physiologically and behaviorally comparable to mammals 22,23. 93 Gene-edited zebrafish lacking panx1a or panx1b 24,25 revealed opposite seizure-like 94 phenotypes and PTZ-related morbidity. Targeted ablation of panx1b potentiates, while the absence 95 of panx1a attenuates seizure manifestations according to recordings of in vivo local field potentials 96 (ivLFP) and locomotor behavior. A deletion of both fish panx1 genes, in a double knockout fish 97 (DKO), causes a moderate phenotype with reduced seizures. In line with these observations are bioRxiv preprint doi: https://doi.org/10.1101/2021.06.03.446992; this version posted June 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 4 98 significant changes to extracellular ATP levels and biological processes demonstrating that the 99 propensity of developing seizure like activities is correlated with altered regulation of energy 100 metabolism, cell death, and the cellular transportome. The acute pharmacological blocking of both 101 Panx1 channels using Probenecid (Pb) abolished PTZ-induced seizures. The molecular, 102 electrophysiological, and behavioral changes are overlapping, but not identical to genetic 103 interference. Likewise, pharmacological blocking of the Panx1 interaction partner P2rx7 26 reduces 104 seizure-like activities like Pb treatment. Finally, structural modeling and comparing the pores of 105 the two Panx1 ohnologues and the human PANX1 channel supports our experimental data 106 implicating the Panx1a protein as a driver of pro-convulsant activities. 107 In summary, our analysis of genetic and pharmacological models in the zebrafish 108 establishes Panx1a channels as the pro-convulsant Panx1 channel. The different propensities of 109 Panx1a and Panx1b channels in developing seizure-like activities opens opportunities for 110 comparative studies into seizure mechanisms and drug discovery by targeting shared properties of 111 zebrafish Panx1a and human PANX1. 112 113 114 Results: 115 Panx1 genotypes determine evoked seizure-like events. 116 Seven days post fertilization (7dpf) larvae were anesthetized, and agar embedded before an 117 electrode was placed into the optic tectum (OT). Seizure-like events (SLE) were recorded after 118 topical application of 15 mM PTZ, as described 27 (Fig. 1a). Characteristic SLEs induced by PTZ 119 demonstrate an initial action potential burst followed by short paroxysmal bursts, representative of 120 a seizure pattern, which was absent at baseline (Fig. 1b). The number of panx1a-/- larvae exhibiting 121 SLEs was reduced by 88% compared to PTZ treated Tubingen Longfin (TL) controls; only one bioRxiv preprint doi: https://doi.org/10.1101/2021.06.03.446992; this version posted June 4, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. 5 122 panx1a-/- larva responded to PTZ treatment (n = 8). All panx1b-/- larvae exhibited SLEs (n = 9). 123 33% of the DKO larvae seized (panx1a-/-: panx1b-/-; DKO; n = 12), which was significantly 124 reduced compared to controls (n = 7) and indistinguishable from panx1a-/- larvae (p=0.2) (Fig. 1c). 125 The average number of SLEs per hour was similar for DKOs and panx1a-/- larvae (p = 0.58), 126 with less than one event on average (DKO: 0.58 ± 0.29; panx1a-/-: 0.38 ± 0.38 events/hour,). Even 127 though all panx1b-/- fish had SLEs, the average number of events per hour were significantly less 128 compared to controls (panx1b-/-: 36.78 ± 2.67; TL: 49.86 ± 3.86 events/hour) (Fig. 1d). The 129 duration of events in all genotypes were comparable to TL controls (TL: 5.5s ± 0.4s; DKO: 4.1s ± 130 0.2s, p = 0.07; panx1a-/-: 4.2s, p = n/a; panx1b-/-: 4.8s ± 0.4s, p = 0.09) (Fig. 1e). 131 The latency until the first SLE showed differences amongst genotypes. TL larvae exhibited 132 the shortest latent period (12.9 ± 1.3min). Panx1b-/- fish presented a delay until the first SLE, with 133 events starting at 21 ± 1.2min. DKO (51.1 ± 4.5min) and panx1a-/- responded last to PTZ treatment 134 (47.3min) (Fig. 1f). 135 The fractional percentage of seizing time within the first hour revealed additional genotype 136 specific differences. SLEs in panx1b-/- larvae were reduced compared to TL controls (panx1b -/-: 137 4.8% ± 0.3%; TL: 7.6% ± 0.8%). However, both DKO and panx1a-/- fish displayed minimal seizing 138 time (<0.1%).
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