Regression of Epileptogenesis by Inhibiting Trkb Signaling Following A

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 epileptogenesis, evidenced by the reduced severity and duration of subsequent seizures. Unexpectedly, transient inhibition 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

(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 treatment (see Kindling Figs 2 and 4). Kindling experiments were performed as previously described.18,22 Peptides were prepared as previously described.20 The sequence A bipolar stimulating and recording electrode was inserted into the of human TrkB amino acids 807 to 820 (LQNLAKASPVpYLDI) right amygdala (1.0mm posterior, 2.9mm lateral to bregma; with the tyrosine at residue 817 phosphorylated (note that this corre- 4.6mm below dura). A skull screw was placed over the left frontal sponds to residue 816 in mouse TrkB protein) was conjugated at the lobe as a ground electrode. After a postoperative recovery period of at N-terminus to the HIV transactivating protein transduction domain least 5 days, animals were connected to a Grass Stimulator and mon- (tat: YGRKKRRQRRR) to facilitate membrane permeability (termed itored by both video and electroencephalogram (EEG). The current “pY816”).TheHIVtatsequenceconjugated to a scrambled peptide required to evoke an electrographic seizure with duration >5 seconds (LVApYQLKIAPNDLS) served as a control (termed “Scr”). Peptides (electrographic seizure threshold [EST]) was determined by applying were synthesized and purified by Tufts Peptide Core Facility,

940 Volume 86, No. 6 Krishnamurthy et al: Targeting TrkB-PLCγ1

FIGURE 1: Time and evoked seizure in kindled animals increased duration of subsequent electrographic and behavioral seizure, with a more robust effect of evoked seizure. (A) Schematic of experimental design for animals receiving evoked seizure 6 days after kindling. Control animals from experiments depicted in Figures 2B–Eand3B–E were pooled for representation. (B–E) Electrographic seizure duration, overt behavioral seizure duration, combined ictal/postictal duration, and seizure score for animals receiving control treatment after an evoked seizure (n = 28). (F) Schematic of experimental design for animals not receiving evoked seizure 6 days after kindling. Control animals from experiments depicted in Figure 4 were pooled for representation. (G–J) Electrographic seizure duration, overt behavioral seizure duration, combined ictal/postictal duration, and seizure score for animals not receiving control treatment after an evoked seizure (n = 24). Data were analyzed by 2-way analysis of variance with repeated measures and post hoc Bonferroni test. *p < 0.05, **p <0.01, ***p <0.001. EEG = electroencephalographic; Kind = kindled; Sz = seizure; Tx = treatment. [Color figure can be viewed at www.annalsofneurology.org] dissolved in sterile phosphate-buffered saline at 2mg/ml, stored at quality of each batch of peptide was assessed by reverse phase high- −80C, thawed just prior to treatment administration, and given at a performance liquid chromatography, verifying that at least 95% elutes dose of 20mg/kg IP, for a total of 5 doses given 12 hours apart. The as a single peak.

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Carbamazepine (Sigma, St Louis, MO) was dissolved in a therapeutic blood levels.24 Treatments were given every 4 hours solubilization buffer of 2% Tween-80 and 70% propylene glycol for 2 days. Solubilization buffer was used as a control. at a concentration of 2mg/ml and administered at a dose of 20mg/kg, a dose of carbamazepine sufficient to produce Statistical Analysis All data analyses were performed by individuals blinded to treatment group and experimental condition. Animals were random- ized to treatment groups following completion of kindling and prior to any data analysis. Sample sizes were chosen based on power analysis. Unless otherwise stated, data are presented as mean Æ standard error of the mean. Unless otherwise stated, comparisons between 2 groups were analyzed using 2-way analysis of variance with repeated measures and post hoc Bonferroni test. A p < 0.05 was considered significant.

Results Progression of Epileptogenesis To examine progression of epileptogenesis, we implemented a variation of the kindling model. To induce “kindling,” adult mice were subjected to repeated brief (1 second) low-intensity stimulations locally within the amygdala twice daily, resulting in evoked seizures of increasing duration and propagation. Ani- mals were termed “kindled” following the third consecutive evoked seizure with score of class 4 or greater. Following a 6-day stimulus-free period, an additional seizure was evoked (seizure 1), and the electrographic and behavioral features were quantified (see Fig 1). Following an additional 8-day stimulation-free period, another seizure was evoked (seizure 2), and the electrographic and behavioral features were again quantified. The increased duration of electrographic and behavioral features of seizure 2 compared to seizure 1 provided a model of progression, enabling us to ask whether and when brief inhibition of TrkB signaling limited epileptogenesis. Notably, the increased duration of the seizure evoked 14 days after the final kindled seizure was due in part to time elapsed, as evident in animals in which no seizure was evoked during the 2-week interval.

Chemical–Genetic Inhibition of TrkB Kinase after an Evoked Seizure Prevents Progression We previously demonstrated that TrkB kinase is activated following an evoked seizure in the kindling model.18 We therefore asked whether initiating inhibition of TrkB signaling immediately following an evoked seizure would prevent

FIGURE 2: Chemical–genetic inhibition of TrkB kinase after an evoked seizure reduces duration and severity of subsequent seizures. (A) Schematic of experimental design. (B–E) Electrographic seizure duration, overt behavioral seizure duration, combined ictal/postictal duration, and seizure score in animals receiving 1NMPP1 (n = 16) or vehicle (Veh; n = 15) following an evoked seizure. Data were analyzed by 2-way analysis of variance with repeated measures and post hoc Bonferroni test. *p < 0.05, ***p < 0.001. EEG = electroencephalographic; Sz = seizure; Tx = treatment. [Color figure can be viewed at www. annalsofneurology.org]

942 Volume 86, No. 6 Krishnamurthy et al: Targeting TrkB-PLCγ1

FIGURE 3: pY816 treatment after an evoked seizure reduces duration and severity of subsequent seizures evoked at either 6 or 14 days after treatment termination. (A) Schematic of experimental design for animals receiving subsequent seizure 6 days after treatment termination. (B–E) Electrographic seizure duration, overt behavioral seizure duration, combined ictal/postictal duration, and seizure score for animals receiving pY816 (n = 17) or Scr (n = 13) after an evoked seizure 6 days following treatment termination. (F) Schematic of experimental design for animals receiving subsequent seizure 14 days after treatment termination. (G–J) Electrographic seizure duration, overt behavioral seizure duration, combined ictal/postictal duration, and seizure score for animals receiving pY816 (n = 13) or Scr (n = 13) after an evoked seizure 14 days following treatment termination. Data were analyzed by 2-way analysis of variance with repeated measures and post hoc Bonferroni test. *p <0.05,**p <0.01,***p < 0.001. EEG = electroencephalographic; Sz = seizure; Tx = treatment. [Color figure can be viewed at www.annalsofneurology.org]

December 2019 943 ANNALS of Neurology progression seen in our kindling model. To accomplish this, temporal control of inhibition of TrkB kinase activity.21 Initi- F616A we employed a chemical–genetic approach with TrkB ating inhibition of TrkB kinase with 1NMPP1 immediately F616A mice, because this provides both molecular specificity and following seizure 1 in TrkB mice prevented the

FIGURE 4: 1NMPP1 or pY816 treatment in the absence of a preceding evoked seizure has no effect on subsequent seizure severity. (A) Schematic of experimental design for 1NMPP1 experiments. (B–E) Electrographic seizure duration, overt behavioral seizure duration, combined ictal/postictal duration, and seizure score for animals receiving 1NMPP1 (n = 10) or vehicle (Veh; n = 10) in the absence of a preceding evoked seizure. (F) Schematic of experimental design for pY816 experiments. (G–J). Electrographic seizure duration, overt behavioral seizure duration, combined ictal/postictal duration, and seizure score for animals receiving pY816 (n = 16) or Scr (n = 14) in the absence of a preceding evoked seizure. Data were analyzed by 2-way analysis of variance with repeated measures and post hoc Bonferroni test. EEG = electroencephalographic; Sz = seizure; Tx = treatment. [Color figure can be viewed at www.annalsofneurology.org]

944 Volume 86, No. 6 Krishnamurthy et al: Targeting TrkB-PLCγ1

approximately 50% increase in duration of electrographic seizure evident in vehicle-treated animals (see Fig 2). Likewise, F616A 1NMPP1 treatment of TrkB mice eliminated the approximately 25% increase in duration of overt seizures and 75% increase in duration of combined ictal and postictal behavior of vehicle-treated animals. A similar trend was evident with respect to behavioral seizure class (p = 0.06) when comparing treatment with 1NMPP1 compared to vehicle. F616A Ascribing the effects of 1NMPP1 in the TrkB mice to inhibition of TrkB kinase requires that 1NMPP1 treatment of WT mice be ineffective. The duration of both electrographic and behavioral features of seizure 2 exhibited striking increases in comparison to seizure 1 when WT animals (n = 9) were treated with 1NMPP1 immediately following seizure 1 (electrographic: 223%; overt behavioral seizure: 184%; combined ictal and postictal behavior: 168%). The F616A ability of 1NMPP1 to limit progression in TrkB mice contrasts sharply with its ineffectiveness in WT animals and implicates inhibition of TrkB kinase as the mechanism F616A underlying the effect of 1NMPP1 in the TrkB mice.

pY816 Treatment after an Evoked Seizure Prevents Progression Our prior studies of SE-induced TLE implicated a causal role of a single signaling pathway downstream of TrkB, namely PLCγ1; this conclusion is based in part on the beneficial effects of pY816, a peptide that uncouples TrkB from PLCγ1.20 Importantly, an evoked seizure in the kindling model induced activation of PLCγ1 as assessed by a surrogate biomarker, the phosphorylation of PLCγ1 tyrosine 783.18 Moreover, treatment with pY816 immediately following repeated seizures inhibits TrkB-induced activation of PLCγ1.20 Together, these observations led us to ask whether treatment with pY816 following a seizure prevented progression in duration and severity of a subsequent seizure. Treatment of WT mice with pY816 immediately following seizure 1 eliminated the approximate 200% increase of duration of electrographic seizure, duration of overt seizure, and combined ictal and postictal behavior of seizure 2 evident in Scr-treated control animals (see Fig 3). pY816 treatment also induced significant reductions of the behavioral seizure class of seizure 2 in comparison to Scr controls.

FIGURE 5: Treatment with carbamazepine (CBZ) immediately γ after an evoked seizure has no effect on subsequent seizure class Inhibition of TrkB-PLC 1 Signaling following an or duration. (A) Experimental design. (B–E) Electrographic seizure Evoked Seizure: Evidence of Persistent duration, overt behavioral seizure duration, combined ictal/ Regression of Epileptogenesis to an Earlier postictal duration, and seizure score for animals receiving CBZ Stage (n = 7) or vehicle (n = 6) following an evoked seizure. The short Experiments described above demonstrate that transient inhi- half-life of CBZ in mice necessitated treatment at 4-hour intervals, γ which led us to treat all animals in a single experiment; as a result, bition of TrkB-PLC 1 signaling following an evoked seizure the latency between the final kindled seizure and postkindling limits progression of epileptogenesis, as evident in comparisons seizure 1 was variable in this group compared to the experiments between treatment groups and controls for seizure 2 compared in Figures 1–3, and 4. Data were analyzed by 2-way analysis of variance with repeated measures and post hoc Bonferroni test. to seizure 1 (see Figs 2 and 3). Unexpectedly, this transient dis- EEG = electroencephalographic; Sz = seizure; Tx = treatment. ruption also induced a regression of epileptogenesis evident in

December 2019 945 ANNALS of Neurology comparisons of seizure 2 with either seizure 1 or the final kin- Treatment with Carbamazepine after an Evoked dled seizure. This effect was particularly notable in a cohort of Seizure Has No Effect on Subsequent Seizure animals treated identically to those described in the preceding Class or Duration γ paragraph with one exception, namely that the interval The beneficial effects of inhibitors of TrkB-PLC 1 signal- between seizures 1 and 2 was extended from 8 to 14 days. In ing administered transiently following an evoked seizure comparison to seizure 1, treatment with pY816 produced a raised the question of whether carbamazepine, a clinically 50% reduction in electrographic seizure duration and ictal effective antiseizure drug, might have similar benefits. To and postictal behavior for seizure 2, as well as a reduction in address this question, a similar experimental design was overt seizure duration by 75% (Table). Furthermore, a signif- employed whereby carbamazepine was administered for icant reduction in behavioral seizure class for seizure 2 com- 2 days following an evoked seizure (see Fig 5). Fifteen to “ ” pared to seizure 1 was noted for the pY816-treated group. 27 days after establishing kindling, seizure 1 was evoked and These effects contrast sharply with the increases of seizure animals were treated with either carbamazepine (n = 7, 20mg/ 2 compared to seizure 1 in the Scr-treated control groups. kg IP at 4-hour intervals for 2 days) or vehicle (n = 6). Comparison of the final kindled seizure with seizure 2 in Fourteen days after the last dose, seizure 2 was evoked. No dif- pY816-treated animals further demonstrated a regression to ferences were detected between carbamazepine- and vehicle- an earlier state of epileptogenesis, as evident by a significant treated animals with respect to duration of electrographic or reduction in electrographic seizure duration, overt seizure behavioral seizures or seizure class. The increased variability in duration, and seizure score (see Table). These results demon- seizure duration and class notwithstanding, these results dem- strate that transient inhibition of TrkB-PLCγ1 signaling fol- onstrate that treatment with a clinically effective anticonvul- lowing a seizure induces persistent regression of sant immediately following an evoked seizure is not sufficient epileptogenesis. to ameliorate seizure-induced progression in duration and sei- Similar effects were evident for treatment with zure class of subsequent seizures. 1NMPP1 or pY816 with an 8-day interval between seizure 1 and seizure 2, although these were less robust (see Figs 2 Discussion F616A and 3, Table). Treatment of TrkB mice with 1NMPP1 We tested the hypothesis that transient inhibition of TrkB- or WT mice with pY816 produced significant reductions in PLCγ1 signaling following a seizure evoked in a kindled duration of overt behavioral seizure of seizure 2 by approxi- animal would limit progression of seizure-induced epi- mately 50% in comparison to seizure 1; these effects contrast leptogenesis. Four principal findings emerged: (1) either sharply with the increases of seizure 2 compared to seizure 1 in chemical–genetic inhibition of TrkB kinase or treatment the vehicle or Scr peptide control groups. Similar effects were with pY816 peptide initiated after an evoked seizure and evident with respect to behavioral seizure class. continued for 2 days prevented the increased duration and severity of subsequent seizures; (2) unexpectedly, transient inhibition of TrkB-PLCγ1 signaling initiated following an Ineffectiveness of pY816 and 1NMPP1 in the evoked seizure also induced a regression to an earlier state Absence of a Preceding Evoked Seizure of epileptogenesis, evident by reduced duration and severity The dramatic impact on epileptogenesis seen with transient of behavioral seizures; (3) treatment with inhibitors of inhibition of TrkB-PLCγ1 signaling following a seizure led us TrkB-PLCγ1 signaling in the absence of a recent seizure to ask whether such inhibition had similar effects in the absence did not reduce severity of subsequent evoked seizures; and of a preceding seizure; restated, we wondered whether the pre- (4) treatment with a clinically effective anticonvulsant (car- ceding seizure activity is necessary for regression of epi- bamazepine) transiently following an evoked seizure did not leptogenesis induced by inhibition of TrkB-PLCγ1signaling. reduce severity of subsequent seizures. These results estab- To address this question, 6 days following the final seizure lish TrkB-PLCγ1 signaling as a molecular mechanism by evoked to establish kindling (denoted here “seizure 1”), which a seizure promotes progression of epileptogenesis. F616A TrkB mice were treated with 1NMPP1 for 2 days and a TLE is a progressive disorder in some patients, as seizure (seizure 2) was evoked 6 days following cessation of evidenced by increasing hippocampal and cortical atrophy 1NMPP1 (see Fig 4). No differences were detected between monitored by magnetic resonance imaging (MRI).7,8 Clini- 1NMPP1- and vehicle-treated mice with respect to duration of cal observations led Gowers to propose that seizures them- electrographic or behavioral seizure or seizure class. The effects selves could promote the worsening of epilepsy.9 Consistent of pY816 in WT mice were investigated using a similar experi- with this notion, the risk of seizure recurrence increased mental design. No differences were detected between pY816 with each subsequent seizure in a cohort of patients studied and Scr peptide-treated mice with respect to duration of by Hauser and Lee.5 The increasing hippocampal atrophy electrographic or behavioral seizure or behavioral seizure class. detected by MRI in TLE patients correlated with increased

946 Volume 86, No. 6 Krishnamurthy et al: Targeting TrkB-PLCγ1

TABLE. Raw Values for EEG Sz Duration, Overt Sz Duration, Ictal and Postictal Behavior, and Sz Score for Final Kindled Sz, Sz 1, and Sz 2 for All Experiments in Figures 2–5 Sz EEG Sz Duration, s Overt Sz Duration, s Ictal & Postictal Duration, s Sz Score

pY816 after an evoked Sz & 14-day interval between Sz 1 & 2 (see Fig 3F–J)

Final kindled Sz Scr: 39.9 Æ 4.8; Scr: 38.2. Æ 4.2; Scr: 80.2 Æ 6.0; Scr: 4.8 Æ 0.22; pY816: 47.2 Æ 7.3a pY816: 42.9 Æ 4.6a pY816: 82.3 Æ 4.8 pY816: 4.8 Æ 0.26b

Sz 1 Scr: 43.6 Æ 4.8; Scr: 43.6 Æ 3.8; Scr: 77.4 Æ 7.0; Scr: 4.6 Æ 0.18; pY816: 41.9 Æ 3.3c pY816: 46.0 Æ 3.6a pY816: 77.6 Æ 6.5 pY816: 5.0 Æ 0.20b

Sz 2 Scr: 48.9 Æ 5.5; Scr: 47.5 Æ 4.1; Scr: 113.0 Æ 9.6; Scr: 5.23 Æ 0.23; pY816: 25.2 Æ 4.1 pY816: 13.6 Æ 5.3 pY816: 61.7 Æ 10.8 pY816: 3.1 Æ 0.37

1NMPP1/Veh after an evoked Sz & 6-day interval between Sz 1 & 2 (see Fig 2)

Final kindled Sz Veh: 47.9 Æ 5.3; Veh: 47.2 Æ 4.6; Veh: 74.9 Æ 4.1; Veh: 4.7 Æ 0.16; 1NMPP1: 43.8 Æ 4.5a 1NMPP1: 44.1 Æ 4.9c 1NMPP1: 84.1 Æ 7.6 1NMPP1: 4.6 Æ 0.18

Sz 1 Veh: 42.6 Æ 4.6; Veh: 45.0 Æ 4.8; Veh: 85.8 Æ 9.8; Veh: 4.5 Æ 0.16; 1NMPP1: 43.8 Æ 4.5a 1NMPP1: 46.8 Æ 4.1a 1NMPP1: 91.0 Æ 7.5 1NMPP1: 4.9 Æ 0.18c

Sz 2 Veh: 56.6 Æ 7.4; Veh: 49.1 Æ 6,8; Veh: 139.8 Æ 16.5; Veh: 4.9 Æ 0.27; 1NMPP1: 29.2 Æ 3.8 1NMPP1: 26.2 Æ 4.4 1NMPP1: 63.5 Æ 10 1NMPP1: 4.0 Æ 0.4

pY816/Scr after an evoked Sz & 6-day interval between Sz 1 & 2 (see Fig 3A–E)

Final kindled Sz Scr: 31.9 Æ 2.8; Scr: 31.8 Æ 3.4; Scr: 67.4 Æ 7.3; Scr: 4.5 Æ 0.21; pY816: 28.2 Æ 2.8 pY816: 31.7 Æ 3.4 pY816: 57.1 Æ 3.4 pY816: 4.5 Æ 0.17

Sz 1 Scr: 36.6 Æ 3.6; Scr: 38.9 Æ 3.0; Scr: 63.4 Æ 6.3; Scr: 4.6 Æ 0.21; pY816: 34.4 Æ 2.9 pY816: 42.9 Æ 3.0 pY816: 66.7 Æ 6.4 pY816: 4.8 Æ 0.18

Sz 2 Scr: 64.5 Æ 9.7; Scr: 58.8 Æ 5.3; Scr: 115.8 Æ 19.8; Scr: 5.4 Æ 0.18; pY816: 27.4 Æ 2.1 pY816: 23.5 Æ 5.3 pY816: 48.9 Æ 5.7 pY816: 3.7 Æ 0.4

1NMPP1/Veh without a preceding evoked Sz (see Fig 4A–E)

Final kindled Sz Veh: 33.9 Æ 3.6; Veh: 40.8 Æ 6.4; Veh: 81.9 Æ 10.5; Veh: 4.5 Æ 0.22; 1NMPP1: 37.5 Æ 5.1 1NMPP1: 40.8 Æ 6.4 1NMPP1: 82.0 Æ 10.3 1NMPP1: 4.8 Æ 0.17

Sz 2 Veh: 46.9 Æ 5.9; Veh: 50.8 Æ 5.1; Veh: 91.0 Æ 9.9; Veh: 4.9 Æ 0.23; 1NMPP1: 44.6 Æ 5.1 1NMPP1: 48.9 Æ 4.7 1NMPP1: 106.1 Æ 12.4 1NMPP1: 5.5 Æ 0.17

pY816/Scr without a preceding evoked Sz (see Fig 4F–J)

Final kindled Sz Scr: 41.1 Æ 3.4; Scr: 41.1 Æ 3.2; Scr: 69.1 Æ 4.1; pY816: Scr: 4.5 Æ 0.17; pY816: 37.9 Æ 3.7 pY816: 41.1 Æ 4.4 65.1 Æ 3.4 pY816: 4.3 Æ 0.14

Sz 2 Scr: 52.5 Æ 6.7; Scr: 56.9 Æ 6.5; Scr: 111.4 Æ 14.0; Scr: 5.1 Æ 0.22; pY816: 39.6 Æ 4.7 pY816: 48.6 Æ 5.0 pY816: 85.9 Æ 10.8 pY816: 5.0 Æ 0.29

CBZ after an evoked Sz (see Fig 5)

Final kindled Sz Veh: 36.3 Æ 6.5; Veh: 35.5 Æ 4.0; Veh: 85.5 Æ 10.1; Veh: 4.3 Æ 0.21; CBZ: 38.0 Æ 4.6 CBZ: 34.3 Æ 3.3 CBZ: 94.4 Æ 12.7 CBZ: 4.7 Æ 0.36

Sz 1 Veh: 44.8 Æ 7.1; Veh: 50.5 Æ 6.2; Veh: 76.9 Æ 5.1; Veh: 4.5 Æ 0.22; CBZ: 39.3 Æ 5.9 CBZ: 49.9 Æ 5.6 CBZ: 53.1 Æ 6.4 CBZ: 5.3 Æ 0.18

Sz 2 Veh: 50.8 Æ 6.6; Veh: 58.5 Æ 5.9; Veh: 122.0 Æ 12.1; Veh: 5.2 Æ 0.3; CBZ: 46.3 Æ 8.9 CBZ: 57.4 Æ 6.3 CBZ: 95.7 Æ 11.7 CBZ: 5.4 Æ 0.20

Statistical analyses were done by 2-way analysis of variance with repeated measures and post hoc Bonferroni test. ap < 0.01. bp < 0.001. cp < 0.05, significant differences in experimental (ie, 1NMPP1 or pY816) animals when compared to Sz 2. CBZ = carbamazepine; EEG = electroencephalographic; Sz = seizure; Veh = vehicle.

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– seizure frequency.7,8,25 Discovery of the kindling model LTP of each of these synapses,36 39 further supporting the provided unambiguous preclinical evidence supportive of putative role of this mechanism. Gowers’ hypothesis.10 In this model, repeated administra- Why is it that inhibition of TrkB-PLCγ1 signaling tion of brief, low-intensity electrical stimuli to a limbic limits epileptogenesis only when introduced following a structure induces progressively enhanced sensitivity to subse- seizure? The suggestion that LTP is a mechanism by which quent stimuli evidenced by increased duration and propaga- enhanced TrkB-PLCγ1 signaling promotes epileptogenesis tion of electrographic and behavioral seizures.11,23 In contrast could provide an explanation. Inhibitors of TrkB-PLCγ1 to the brief localized electrographic seizures without overt signaling prevent high-frequency stimulation–induced LTP behavioral change evoked by initial stimuli, prolonged and of excitatory synapses in in vitro preparations, a scenario – widely propagated electrographic seizures accompanied by akin to limiting seizure-promoted epileptogenesis.36 39 tonic–clonic seizures are evident following 10 to 15 stimula- Importantly, inhibition of TrkB-PLCγ1 signaling has no tions.23 Spontaneous recurrent seizures (epilepsy), often asso- effect on basal transmission of these synapses,36,37 paralleling ciated with sudden unexpected death, emerge after evoking its ineffectiveness when administered to an animal in the many (eg, 70–80) seizures.12,13 Importantly, the seizure itself, absence of a recent seizure. Similar parallels emerge with not the electrical stimulus, is the key variable causing respect to protein synthesis inhibitors in that anisomycin has – epileptogenesis.26 28 Interestingly, spontaneously occurring no effect on basal synaptic transmission yet inhibits subclinical seizures are thought to contribute to increased stimulation-induced LTP of the Schaeffer collateral synapse – severity of subsequent seizures in other models of epilepsy. with CA1 pyramidal cells.40 42 Animals in which spontaneous recurrent seizures arise after The requirement of a recently evoked seizure for the seizure-free latent periods of days to weeks following SE effectiveness of TrkB-PLCγ1 inhibitors exhibits striking exhibit a progressive increase in frequency and severity of sei- parallels to previous studies of fear conditioning and an ani- zures long after onset of the initial seizure14,15,29; recurrent mal model of neuropathic pain. For example, recall of pre- subconvulsive seizures are thought to promote progression of viously encoded classical fear conditioning introduces a the epilepsy in these animals.15 A similar increase in seizure period of lability during which perturbations such as an frequency and severity was observed in rats long after postnatal evoked seizure43 or transient inhibition of protein synthesis hypoxia–ischemia.30,31 Finally, inducing several brief seizures can eliminate the pathologic memory44; as in the present in an asymptomatic mouse carrying a mutation of SCN1A study, introducing these perturbations without prior recall identified in humans with Dravet syndrome causes severe epi- of the encoded memory fails to eliminate the memory. lepsy and cognitive decline; remarkably, inducing similar brief Likewise, reversal of “neuropathic pain” can be accom- seizures in WT mice has no overt deleterious consequences.16 plished by transient exposure to protein synthesis inhibitors Collectively, these findings imply that some molecular administered following re-exposure to the pain stimulus; consequences of a seizure promote the worsening of epilepsy. treatment with the protein synthesis inhibitors in the Our findings implicate TrkB-PLCγ1 signaling as one such absence of a preceding pain stimulus are ineffective.45 Evi- F616A consequence. The effectiveness of 1NMPP1 in TrkB but dence of TrkB activation following the inciting stimuli in not in WT animals provides unambiguous evidence implicat- each of these models46,47 provides a rationale for determin- ing TrkB kinase in seizure-induced worsening. Among signaling whether transient inhibition of TrkB-PLCγ1signaling ing pathways downstream of TrkB kinase, the efficacy of will limit progression or reverse these plasticities. pY816 implicates PLCγ1 in particular.20 These findings raise Whereas limiting progression of epileptogenesis by the question of the cellular consequence of seizure-evoked TrkB-PLCγ1 inhibitors was consistent with our hypothe- TrkB-PLCγ1 signaling that promotes epileptogenesis. One sis, the regression to an earlier stage of epileptogenesis was plausible mechanism is the long-term potentiation (LTP) of unexpected. Two weeks after transient treatment following excitatory synapses between principal neurons. Goddard pro- a seizure, both behavioral and EEG features of seizures posed that repeatedly evoking electrographic seizures induces were significantly reduced in comparison to the final kin- LTP of excitatory synapses at various “nodes” within a spatially dled seizure in pY816- but not Scr-treated controls (see distributed but synaptically connected network of limbic Fig 3F–J). Similar results were evident in both 1NMPP1- sites,32 a proposal supported by findings of Sutula and Stew- and pY816-treated animals tested 8 days following treat- ard.33,34 If causal, then disruption of LTP at any of these loci ment (see Figs 2 and 3A–E). Whether complete reversal may impair propagation and severity of seizure activity. Immu- of epileptogenesis can be achieved with additional trials of nohistochemical evidence of TrkB-PLCγ1 activation at hippo- seizures followed by TrkB-PLCγ1 inhibitors remains to be campal mossy fiber-CA3 and Shaffer collateral-CA1 synapses determined. Likewise, whether transient inhibition of following kindling and SE17,35 suggests that these particular TrkB-PLCγ1 signaling following a spontaneous seizure synapses may be two such “nodes.” TrkB inhibition prevents (as distinct from evoked seizures in the kindling model) in

948 Volume 86, No. 6 Krishnamurthy et al: Targeting TrkB-PLCγ1 other models of TLE will limit progression or induce a Potential Conflicts of Interest remission is a subject of future study. K.K. has a pending patent, “Compositions and Methods One possible confound in the interpretation of the cur- for the Treatment of Epilepsy,” which includes data pres- rent results, that the reduction in seizure class and duration is ented in this article. Y.Z.H. owns equity in and J.O.M. is a an anticonvulsant effect due to retained 1NMPP1 or peptide, founder of LVM Biosciences, a company that develops – is unlikely for several reasons. First, the chemical genetic inhibitors of TrkB signaling for the treatment of epilepsy. strategy for the experiments depicted in Figure 2 utilized the small molecule 1NMPP1, a compound with a half-life in the 48 brain of approximately 30 minutes, which would be cleared References well before seizure 2 evoked 6 days after treatment termina- 1. Chen Z, Brodie MJ, Liew D, Kwan P. Treatment outcomes in patients tion. Second, pY816 exhibits an anticonvulsant effect in the with newly diagnosed epilepsy treated with established and new intra-amygdala kainate model of SE if administered antiepileptic drugs: a 30-year longitudinal cohort study. JAMA Neu- rol 2018;75:279–286. 10 minutes to 24 hours before induction, but no effect if given 72 hours before induction,20 providing indirect evi- 2. Hauser WA. Questioning the effectiveness of newer antiseizure med- ications. JAMA Neurol 2018;75:273–274. dence that the peptide is cleared from the brain within this 3. Téllez-Zenteno JF, Hernández-Ronquillo L. A review of the epidemi- time frame. Finally, the reduction in behavioral and EEG ology of temporal lobe epilepsy. Epilepsy Res Treat. 2012;2012: measures of seizures persisted when the latency between 630853. pY816 treatment and testing was increased from 6 days to 4. Pitkänen A, Sutula, TP. Is epilepsy a progressive disorder? Prospects 2 weeks (see Fig 3F–J). for new therapeutic approaches in temporal-lobe epilepsy. Lancet Neurol 2002;1:173–181. The present findings suggest a novel strategy for manag- 5. Hauser WA, Lee JR. Do seizures beget seizures? Prog Brain Res ing patients with TLE. In contrast to continuous exposure to 2002;135:215–219. standard antiseizure drugs aimed at inhibiting a seizure, this γ 6. Hermann BP, Seidenberg M, Bell B, et al. Comorbid psychiatric strategy would consist of transient inhibition of TrkB-PLC 1 symptoms in temporal lobe epilepsy: association with chronicity of signaling introduced following the occurrence of a seizure. epilepsy and impact on quality of life. Epilepsy Behav 2000;1: 184–190. Engaging the TrkB-PLCγ1 target selectively in a disease (eg, postseizure) context may limit progression and poten- 7. Bernhardt BC, Worsley KJ, Kim H, et al. Longitudinal and cross- sectional analysis of atrophy in pharmacoresistant temporal lobe epi- tially ameliorate the severity of the epilepsy. The improved lepsy. Neurology 2009;72:1747–1754. 49,50 efficacy of automated seizure detection systems would 8. Coan AC, Appenzeller S, Bonilha L, et al. Seizure frequency and lat- facilitate notification of need to introduce treatment in a eralization affect progression of atrophy in temporal lobe epilepsy. – timely manner, thereby enhancing the feasibility of such an Neurology 2009;73:834 842. approach. 9. Gowers WR. Epilepsy and other chronic convulsive diseases. London, UK: Churchill, 1881.

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