Celecoxib Ameliorates Seizure Susceptibility in Autosomal Dominant Lateral Temporal Epilepsy

This Accepted Manuscript has not been copyedited and formatted. The final version may differ from this version. Research Articles: Neurobiology of Disease Celecoxib Ameliorates Seizure Susceptibility in Autosomal Dominant Lateral Temporal Epilepsy Lin Zhou1, Liang Zhou1, Li-da Su2, Sheng-Long Cao3, Ya-Jun Xie1, Na Wang4, Chong-Yu Shao1, Ya-Nan Wang1, Jia-Huan Zhou1, John K. Cowell5 and Ying Shen1 1Department of Neurobiology, Key Laboratory of Medical Neurobiology of the Ministry of Health, Zhejiang University School of Medicine, Hangzhou, China. 2Neuroscience Care Unit 3Department of Neurosurgery, Second Affiliated Hospital of Zhejiang University School of Medicine, Hangzhou, China. 4School of Medicine, Zhejiang University City College, Hangzhou, China. 5GRU Cancer Center, Augusta University, Augusta, GA, USA. DOI: 10.1523/JNEUROSCI.3245-17.2018 Received: 14 November 2017 Revised: 24 January 2018 Accepted: 20 February 2018 Published: 28 February 2018 Author contributions: Lin Zhou and Y.S. designed research; Lin Zhou, Liang Zhou, L.-D.S., S.C., Y.-J.X., N.W., C.-Y.S., Y.-N.W., and J.-H.Z. performed research; Lin Zhou, Liang Zhou, N.W., and Y.S. analyzed data; Lin Zhou, j.c., and Y.S. wrote the paper; j.c. contributed unpublished reagents/analytic tools. Conflict of Interest: The authors declare no competing financial interests. This work was supported by grants from the National Natural Science Foundation of China (81625006, 31471024, and 31571051) and the Natural Science Foundation of Zhejiang Province (Z15C090001). The generation of LGI1-null mice was supported by a grant from the National Institutes of Health to JKC (CA046706). We thank Dr. Z. Josh Huang (Cold Spring Harbor Laboratory, New York, NY) for providing the G42 mutant mice, the Core Facilities of Zhejiang University Institute of Neuroscience for technical assistance, Dr. Zhouyan Feng (Zhejiang University School of Biomedical Engineering, Hangzhou, China) for the suggestions in statistics, and Dr. Iain C. Bruce for reading the manuscript. Corresponding author: Ying Shen, 866 Yu Hang Tang Road, Hangzhou 310058, China. Tel: (86)571-88208240. Email: [email protected] Cite as: J. Neurosci ; 10.1523/JNEUROSCI.3245-17.2018 Alerts: Sign up at www.jneurosci.org/cgi/alerts to receive customized email alerts when the fully formatted version of this article is published. Accepted manuscripts are peer-reviewed but have not been through the copyediting, formatting, or proofreading process. Copyright © 2018 the authors 1 Celecoxib Ameliorates Seizure Susceptibility in Autosomal Dominant 2 Lateral Temporal Epilepsy 3 Lin Zhou,1,† Liang Zhou,1,† Li-da Su,2 Sheng-Long Cao,3 Ya-Jun Xie,1 Na Wang,4 4 Chong-Yu Shao,1 Ya-Nan Wang,1 Jia-Huan Zhou,1 John K. Cowell,5 Ying Shen1,* 5 6 1Department of Neurobiology, Key Laboratory of Medical Neurobiology of the Ministry of 7 Health, Zhejiang University School of Medicine, Hangzhou, China. 8 2Neuroscience Care Unit; 3Department of Neurosurgery, Second Affiliated Hospital of 9 Zhejiang University School of Medicine, Hangzhou, China. 10 4School of Medicine, Zhejiang University City College, Hangzhou, China. 11 5GRU Cancer Center, Augusta University, Augusta, GA, USA. 12 †These authors contributed equally to this work. 13 *Corresponding author: Ying Shen, 866 Yu Hang Tang Road, Hangzhou 310058, 14 China. Tel: (86)571-88208240. Email: [email protected]. 15 16 Number of text pages: 49 / Figures: 6 / Tables: 7 17 Number of words for Abstract: 186 / Introduction: 428 / Discussion: 955 18 Abbreviated Title: Celecoxib ameliorates LGI1-related epilepsy 19 20 Acknowledgements 21 This work was supported by grants from the National Natural Science Foundation of 22 China (81625006, 31471024, and 31571051) and the Natural Science Foundation of 23 Zhejiang Province (Z15C090001). The generation of LGI1-null mice was supported by a 24 grant from the National Institutes of Health to JKC (CA046706). We thank Dr. Z. Josh 25 Huang (Cold Spring Harbor Laboratory, New York, NY) for providing the G42 mutant mice, 26 the Core Facilities of Zhejiang University Institute of Neuroscience for technical assistance, 1 27 Dr. Zhouyan Feng (Zhejiang University School of Biomedical Engineering, Hangzhou, 28 China) for the suggestions in statistics, and Dr. Iain C. Bruce for reading the manuscript. 2 29 Abstract 30 31 Autosomal dominant lateral temporal epilepsy (ADLTE) is an inherited syndrome caused 32 by mutations in the leucine-rich glioma inactivated 1 (LGI1) gene. It is known that 33 glutamatergic transmission is altered in LGI1 mutant mice and seizures can be reduced by 34 restoring LGI1 function. Yet, the mechanism underlying ADLTE is unclear. Here, we 35 propose that seizures in male LGI1-/- mice are due to non-synaptic epileptiform activity in 36 cortical neurons. We examined the intrinsic excitability of pyramidal neurons in the 37 temporal cortex of male LGI1-/- mice and found that the voltage-gated K+ channel Kv1.2 38 was significantly downregulated. We also found that cytosolic phospholipase A2 -/- 39 (cPLA2)-cyclooxygenase 2 (Cox2) signaling was enhanced in LGI1 mice. Interestingly, 40 Cox2 inhibition effectively restored the dysregulated Kv1.2 and reduced the intrinsic 41 excitability of pyramidal neurons. Moreover, in vivo injection of celecoxib, an 42 FDA-approved nonsteroidal anti-inflammatory drug, rescued the defective Kv1.2 (a 43 ~1.9-fold increase), thereby alleviating the seizure susceptibility and extending the life of 44 LGI1-/- mice by 5 days. In summary, we conclude that LGI1 deficiency dysregulates 45 cPLA2-Cox2 signaling to cause hyperexcitability of cortical pyramidal neurons, and 46 celecoxib is a potential agent to manage human ADLTE. 47 48 Key words: ADLTE; Cox2; epilepsy; Kv1.2; celecoxib 49 3 50 Significance statement 51 52 Haploinsufficiency of the leucine-rich glioma inactivated 1 (LGI1) gene is the major 53 pathogenic basis for ADLTE, an inherited syndrome with no cure to date. Existing studies 54 suggest that altered glutamatergic transmission in the hippocampus causes this disease, 55 but the data are paradoxical. We demonstrate that the loss of LGI1 decreases Kv1.2 56 expression, enhances intrinsic excitability, and thereby causes epilepsy. Interestingly, we 57 for the first time show that an FDA-approved drug, celecoxib, rescues the Kv1.2 defect and 58 alleviates seizure susceptibility in LGI1-/- mice, as well as improving their survival. Thus, 59 we suggest that celecoxib is a promising drug for the treatment of ADLTE patients. 60 4 61 Introduction 62 63 Autosomal dominant lateral temporal epilepsy (ADLTE, also known as autosomal 64 dominant epilepsy with auditory features) is an inherited syndrome characterized by the 65 onset of temporal cortical seizures in adolescence or early adulthood (Ottman et al., 1995) 66 in the absence of structural abnormalities by magnetic resonance imaging (Michelucci et 67 al., 2003). ADLTE seizures occur at a low frequency (1-2 per month) and usually during 68 sleep (Michelucci et al., 2003; Michelucci et al., 2007). Aphasia and visual hallucinations 69 are reported in a minority of patients, either alone or accompanying auditory symptoms 70 (Michelucci et al., 2003; Michelucci et al., 2007). Haploinsufficiency of the leucine-rich 71 glioma-inactivated 1 (LGI1) gene is the major pathogenic basis for human ADLTE 72 (Kalachikov et al., 2002; Senechal et al., 2005; Fukata et al., 2006). Homozygous LGI1-/- 73 mice display generalized seizures and premature death (Chabrol et al., 2010; Fukata et al., 74 2010; Yu et al., 2010), while heterozygous LGI1+/- mice display increased seizure 75 susceptibility to acoustic stimuli (Chabrol et al., 2007) and the convulsant agent 76 pentylenetetrazole (PTZ) (Fukata et al., 2010). 77 Several functions of LGI1 in the central nervous system have been demonstrated: (i) 78 inhibiting the inactivation of the presynaptic K+ channel Kv1 (Schulte et al., 2006); (ii) 79 regulating the pruning of glutamatergic synapses in the hippocampus (Zhou et al., 2009) 80 through interaction with ADAM (a disintegrin and metalloproteinase) (Fukata et al., 2006; 81 Fukata et al., 2010; Ohkawa et al., 2013); (iii) increasing neuronal growth through an 82 interaction with Nogo receptors (Thomas et al., 2010); and (iv) causing subtle cortical 5 83 dyslamination (Silva et al., 2015) and reduced thickness of the external granule layer with 84 abnormal foliation in the embryonic cerebellum (Xie et al., 2015). Using conditional lines, 85 Boillot et al. demonstrated that glutamatergic neurons contribute to the seizure 86 phenotypes in LGI1-/- mice (Boillot et al., 2014). Similarly, two independent studies have 87 suggested that altered glutamatergic transmission is the pathologic basis for the seizure 88 phenotypes in LGI1-/- mice (Fukata et al., 2010; Yu et al., 2010). 89 However, how LGI1 deficiency leads to seizures remains unclear. In LGI1-/- mice, Yu 90 et al. (2010) showed enhanced glutamate transmission in the hippocampus, but Fukata et 91 al. (2010) suggested that it is reduced. While this discrepancy may be due to knockout 92 strategies, it is possible that hippocampal synaptic transmission is not the decisive factor 93 in ADLTE. In fact, increased epileptic discharges are evident characteristics of ADLTE 94 patients with LGI1 mutations (Ottman et al., 1995; Michelucci et al., 2009). Here, we set 95 out to investigate the intrinsic excitability of pyramidal neurons in the temporal lobe cortex 96 of LGI1-/- mice and the effects of celecoxib. 97 6 98 Materials and Methods 99 100 Animals. The animal experiments were approved by the Animal Experimentation Ethics 101 Committee of Zhejiang University. Wild-type (WT) and LGI1-/- mice were maintained at the 102 Experimental Animal Center of Zhejiang University and kept in temperature-controlled 103 conditions under a 12:12 h light/dark cycle with food and water ad libitum. Male mice were 104 used in all experiments. 105 106 Antibodies and reagents. Antibodies against Kv1.2 (#K14/16), Kv1.4 (#K13/31), Kv1.6 107 (#K19/36), Nav1.1 (#K74/71), Nav1.2 (#K69/3), and Nav1.6 (#K87a/10) were from 108 Neuromab (Davis, CA).

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