RESEARCH ARTICLE Regression of Epileptogenesis by Inhibiting Tropomyosin Kinase B Signaling following a Seizure Kamesh Krishnamurthy, MD, PhD ,1 Yang Zhong Huang, PhD,1 Stephen C. Harward, MD, PhD,2 Keshov K. Sharma, BS,1 Dylan L. Tamayo,1 and James O. McNamara, MD1,3,4 Objective: Temporal lobe epilepsy (TLE) is a devastating disease in which seizures persist in 35% of patients despite optimal use of antiseizure drugs. Clinical and preclinical evidence implicates seizures themselves as one factor promot- ing epilepsy progression. What is the molecular consequence of a seizure that promotes progression? Evidence from preclinical studies led us to hypothesize that activation of tropomyosin kinase B (TrkB)–phospholipase-C-gamma-1 (PLCγ1) signaling induced by a seizure promotes epileptogenesis. Methods: To examine the effects of inhibiting TrkB signaling on epileptogenesis following an isolated seizure, we implemented a modified kindling model in which we induced a seizure through amygdala stimulation and then used either a chemical–genetic strategy or pharmacologic methods to disrupt signaling for 2 days following the seizure. The severity of a subsequent seizure was assessed by behavioral and electrographic measures. Results: Transient inhibition of TrkB-PLCγ1 signaling initiated after an isolated seizure limited progression of epi- leptogenesis, evidenced by the reduced severity and duration of subsequent seizures. Unexpectedly, transient inhibi- tion of TrkB-PLCγ1 signaling initiated following a seizure also reverted a subset of animals to an earlier state of epileptogenesis. Remarkably, inhibition of TrkB-PLCγ1 signaling in the absence of a recent seizure did not reduce severity of subsequent seizures. Interpretation: These results suggest a novel strategy for limiting progression or potentially ameliorating severity of TLE whereby transient inhibition of TrkB-PLCγ1 signaling is initiated following a seizure. ANN NEUROL 2019;86:939–950 espite the introduction of a panoply of new antiseizure in 1881 that “seizures beget seizures.”9 In support of this idea, Ddrugs in the past quarter century, there has been no mea- longitudinal observations of a cohort of patients with newly surable improvement in the proportion of patients with newly diagnosed epilepsy revealed that individuals at low risk of diagnosed epilepsy rendered free of seizures.1 Approximately recurrent seizures exhibited a progressive increase in risk with one-third of such patients experience recurrent seizures despite increasing number of seizures.5 Direct evidence that an iso- treatment by skilled clinicians with recently introduced thera- lated seizure could promote both development and progres- peutics.1 These failures underscore the need to elucidate the sion of epilepsy emerged from preclinical observations.10 mechanisms underlying the development and/or progression Repeatedly evoking brief, localized seizures induced a pro- of epilepsy, a process termed epileptogenesis.2 gressive increase in duration and severity of subsequent Of the various forms of epilepsy, temporal lobe epilepsy evoked seizures, a model termed kindling.10,11 Evoking many (TLE) is both common and commonly debilitating.3,4 Nota- (eg, 70–80) such seizures culminated in recurrent seizures bly, TLE is frequently progressive, with worsening of clinical occurring without stimulation, often associated with fatal- course,5 comorbidities,6 and structural lesions.7,8 One factor ity.12,13 Furthermore, the frequency and severity of seizures that might contribute to this progression is the occurrence of progressively increases long after the onset of epilepsy in seizures themselves, as first posited by Gowers, who proposed diverse models including hypoxia–ischemia, status epilepticus View this article online at wileyonlinelibrary.com. DOI: 10.1002/ana.25602 Received Apr 15, 2019, and in revised form Sep 11, 2019. Accepted for publication Sep 12, 2019. Address correspondence to Dr McNamara, Departments of Neurobiology, Duke University, Durham, NC 27708. E-mail: [email protected] From the Departments of 1Neurobiology, 2 Neurosurgery, 3 Pharmacology & Cancer Biology, and 4 Neurology, Duke University, Durham, NC © 2019 American Neurological Association 939 ANNALS of Neurology (SE), and genetic channelopathies; the occurrence of seizures a 1-second train of 1-millisecond biphasic-rectangular pulses at 60Hz in all of these models is thought to contribute to the beginning at 20μA. Additional stimulations were given in 20μA – progression.14 16 increments at 1-minute intervals until an electrographic seizure was Understanding the molecular consequences by which detected. Animals then received 2 stimulations per day at the EST, spaced at least 6 hours apart, with the behavioral seizure scores classi- a seizure promotes progression of epilepsy could provide fied according to a modification of the Racine scale for mice: 0, nor- novel targets and strategies to limit subsequent worsening mal activity; 1, arrest and rigid posture; 2, head nodding; of disease. One molecular consequence of an isolated sei- 3, unilateral forelimb clonus; 4, rearing with bilateral forelimb clo- zure is the increased activation of the brain-derived neuro- nus; 5, rearing and falling; 6, tonic–clonic seizures with violent run- trophic factor receptor, tropomyosin kinase B (TrkB), and ning and/or jumping.23 The criterion for “kindled” was the its signaling effector, phospholipase-C-gamma-1 (PLCγ1), occurrence of 3 consecutive seizures of class 4 or greater, with limb spanning a couple of days as evident in biochemical and clonus/tonus lasting at least 12 seconds. Once “kindled,” subsequent histochemical measures.17,18 Transient inhibition of TrkB- evoked seizures were characterized by abrupt onset concomitantly of PLCγ1 signaling following prolonged seizures prevents electrographic seizure activity in the stimulated amygdala and behav- subsequent development of epilepsy.19,20 We therefore ioral seizures culminating in class 4, 5, or 6. Electrographic seizure hypothesized that transiently inhibiting TrkB-PLCγ1sig- activity was typically followed by postictal depression of EEG activity naling by treatment for 2 days following an isolated seizure in the stimulated amygdala during which animals exhibited immobil- ity and occasional orofacial clonus; this sequence was followed by would inhibit progression of epilepsy. We used the kindling abrupt return of exploratory activity in the cage. model to test our hypothesis because of the convenience For each seizure, 4 variables were quantified by offline ana- afforded over timing of a seizure. lyses of video-EEG recordings by a blinded, trained observer: (1) the maximum behavioral seizure class, (2) the duration of Materials and Methods electrographic seizure activity, (3) the duration of overt seizure activ- Animals ity defined as clonic and/or tonic behaviors of class 4 or greater, and All animal procedures were approved by the institutional animal care (4) the total time elapsed between seizure onset and abrupt return and use committee at Duke University and conform to the US Pub- of exploratory activity in the cage. For variables 2 through 4, data lic Health Service’s Policy on Humane Care and Use of Laboratory are presented normalized to the seizure evoked prior to treatment, Animals, as well as the National Institutes of Health and Duke Uni- thereby enabling each animal to serve as its own control. versity institutional guidelines for the care and use of experimental Design of individual experiments is presented in panel A of “ ” animals. Animals were maintained on a 12-hour light/dark cycle with each figure. All seizures following kindling (Fig 1A), whether des- food and water available ad libitum. Wild-type (WT) adult 8- to ignated seizure 1 or 2, were evoked with a stimulation intensity 12-week-old C57BL/6 male mice were obtained from Charles River determined by assessing the EST a second time, administering stim- F616A μ μ (Wilmington, MA). TrkB mice were originally obtained from ulations in 20 A increments every 1 minute, beginning at 20 A, Dr David Ginty21 andbackcrossedtotheC57BL/6lineforatleast until an electrographic seizure (duration >5 seconds) was evoked. – 7 generations. This knockin mouse harbors a point mutation on the Notably, in experiments presented in Figures 1A E, 2, 3, and 5, TrkB allele, substituting an alanine for phenylalanine within the experimental design mandated that the behavioral pattern accompa- adenosine triphosphate binding pocket of the TrkB kinase domain. nying electrographic seizure 1 was class 4 or greater. This mutation renders TrkB protein uniquely susceptible to kinase inhibition by small molecule derivatives of the general kinase inhibi- Treatments tor PP1, including 1-(1,1-dimethylethyl)-3-(1- naphthalenylmethyl)- Prior to each administration, a solution of 100mM 1NMPP1 was 1H-pyrazolo[3,4-d]pyrimidin-4-amine (1NMPP1). Importantly, dissolved in a solubilization buffer (vehicle) containing 0.9% NaCl 1NMPP1 does not have any detectable effect in WT mice, and there and 2.5% Tween-20 to a concentration of 1.67mg/ml and dosed F616A are no differences in TrkB kinase activity in the TrkB compared at 16.6μg/g intraperitoneal (IP) every 12 hours for a total of to WT mice in the absence of this compound.19,21 Both male and 5 treatments. Vehicle injection served as a control. In addition to female adult (8–12 weeks old) homozygous mice were used. treatments by IP injection, either 1NMPP1 (25μM) or vehicle was included in drinking water for the 2 days of