Targeting BDNF/TrkB pathways for preventing or suppressing epilepsy.

Traumatic brain injury (TBI) and status epilepticus (SE) have both been linked to development of human epilepsy. Although distinct etiologies, current research has suggested the convergence of molecular mechanisms underlying epileptogenesis following these insults. One such mechanism involves the neurotrophin brain-derived neurotrophic factor (BDNF) and its high-affinity receptor, tropomyosin related kinase B (TrkB). In this review, we focus on currently available data regarding the pathophysiologic role of BDNF/TrkB signaling in epilepsy development. We specifically examine the axonal injury and SE epilepsy models, two animal models that recapitulate many aspects of TBI- and SE-induced epilepsy in humans respectively. Thereafter, we discuss aspiring strategies for targeting BDNF/TrkB signaling so as to prevent epilepsy following an insult or suppress its expression once developed. This article is part of the special issue entitled 'New Epilepsy Therapies for the 21st Century - From Antiseizure Drugs to Prevention, Modification and Cure of Epilepsy'.

Regression of Epileptogenesis by Inhibiting Tropomyosin Kinase B Signaling following a Seizure.

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 promoting 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.

TrkB-Shc Signaling Protects against Hippocampal Injury Following Status Epilepticus.

Temporal lobe epilepsy (TLE) is a common and commonly devastating form of human epilepsy for which only symptomatic therapy is available. One cause of TLE is an episode of de novo prolonged seizures [status epilepticus (SE)]. Understanding the molecular signaling mechanisms by which SE transforms a brain from normal to epileptic may reveal novel targets for preventive and disease-modifying therapies. SE-induced activation of the BDNF receptor tyrosine kinase, TrkB, is one signaling pathway by which SE induces TLE. Although activation of TrkB signaling promotes development of epilepsy in this context, it also reduces SE-induced neuronal death. This led us to hypothesize that distinct signaling pathways downstream of TrkB mediate the desirable (neuroprotective) and undesirable (epileptogenesis) consequences. We subsequently demonstrated that TrkB-mediated activation of phospholipase Cγ1 is required for epileptogenesis. Here we tested the hypothesis that the TrkB-Shc-Akt signaling pathway mediates the neuroprotective consequences of TrkB activation following SE. We studied measures of molecular signaling and cell death in a model of SE in mice of both sexes, including wild-type and TrkBShc/Shc mutant mice in which a point mutation (Y515F) of TrkB prevents the binding of Shc to activated TrkB kinase. Genetic disruption of TrkB-Shc signaling had no effect on severity of SE yet partially inhibited activation of the prosurvival adaptor protein Akt. Importantly, genetic disruption of TrkB-Shc signaling exacerbated hippocampal neuronal death induced by SE. We conclude that therapies targeting TrkB signaling for preventing epilepsy should spare TrkB-Shc-Akt signaling and thereby preserve the neuroprotective benefits.SIGNIFICANCE STATEMENT Temporal lobe epilepsy (TLE) is a common and devastating form of human epilepsy that lacks preventive therapies. Understanding the molecular signaling mechanisms underlying the development of TLE may identify novel therapeutic targets. BDNF signaling thru TrkB receptor tyrosine kinase is one molecular mechanism promoting TLE. We previously discovered that TrkB-mediated activation of phospholipase Cγ1 promotes epileptogenesis. Here we reveal that TrkB-mediated activation of Akt protects against hippocampal neuronal death in vivo following status epilepticus. These findings strengthen the evidence that desirable and undesirable consequences of status epilepticus-induced TrkB activation are mediated by distinct signaling pathways downstream of this receptor. These results provide a strong rationale for a novel therapeutic strategy selectively targeting individual signaling pathways downstream of TrkB for preventing epilepsy.

Vagal nerve stimulation modifies neuronal activity and the proteome of excitatory synapses of amygdala/piriform cortex.

Vagal Nerve Stimulation (VNS) Therapy® is a United States Food and Drug Administration approved neurotherapeutic for medically refractory partial epilepsy and treatment-resistant depression. The molecular mechanisms underlying its beneficial effects are unclear. We hypothesized that one mechanism involves neuronal activity-dependent modifications of central nervous system excitatory synapses. To begin to test this hypothesis, we asked whether VNS modifies the activity of neurons in amygdala and hippocampus. Neuronal recordings from adult, freely moving rats revealed that activity in both amygdala and hippocampus was modified by VNS immediately after its application, and changes were detected following 1 week of stimulation. To investigate whether VNS modifies the proteome of excitatory synapses, we established a label-free, quantitative liquid chromatography-tandem mass spectrometry workflow that enables global analysis of the constituents of the postsynaptic density (PSD) proteome. PSD proteins were biochemically purified from amygdala/piriform cortex of VNS- or dummy-treated rats following 1-week stimulation, and individual PSD protein levels were quantified by liquid chromatography-tandem mass spectrometry analysis. We identified 1899 unique peptides corresponding to 425 proteins in PSD fractions, of which expression levels of 22 proteins were differentially regulated by VNS with changes greater than 150%. Changes in a subset of these proteins, including significantly increased expression of neurexin-1α, cadherin 13 and voltage-dependent calcium channel α2δ1, the primary target of the antiepileptic drug gabapentin, and decreased expression of voltage-dependent calcium channel γ3, were confirmed by western blot analysis of PSD samples. These results demonstrate that VNS modulates excitatory synapses through regulating a subset of the PSD proteome. Our study reveals molecular targets of VNS and point to possible mechanisms underlying its beneficial effects, including activity-dependent formation of excitatory synapses.

A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.

The BDNF receptor tyrosine kinase, TrkB, underlies nervous system function in both health and disease. Excessive activation of TrkB caused by status epilepticus promotes development of temporal lobe epilepsy (TLE), revealing TrkB as a therapeutic target for prevention of TLE. To circumvent undesirable consequences of global inhibition of TrkB signaling, we implemented a novel strategy aimed at selective inhibition of the TrkB-activated signaling pathway responsible for TLE. Our studies of a mouse model reveal that phospholipase Cγ1 (PLCγ1) is the dominant signaling effector by which excessive activation of TrkB promotes epilepsy. We designed a novel peptide (pY816) that uncouples TrkB from PLCγ1. Treatment with pY816 following status epilepticus inhibited TLE and prevented anxiety-like disorder yet preserved neuroprotective effects of endogenous TrkB signaling. We provide proof-of-concept evidence for a novel strategy targeting receptor tyrosine signaling and identify a therapeutic with promise for prevention of TLE caused by status epilepticus in humans.

Brain-derived neurotrophic factor but not vesicular zinc promotes TrkB activation within mossy fibers of mouse hippocampus in vivo.

The neurotrophin receptor, TrkB receptor tyrosine kinase, is critical to central nervous system (CNS) function in health and disease. Elucidating the ligands mediating TrkB activation in vivo will provide insights into its diverse roles in the CNS. The canonical ligand for TrkB is brain-derived neurotrophic factor (BDNF). A diversity of stimuli also can activate TrkB in the absence of BDNF, a mechanism termed transactivation. Zinc, a divalent cation packaged in synaptic vesicles along with glutamate in axons of mammalian cortical neurons, can transactivate TrkB in neurons and heterologous cells in vitro. Yet the contributions of BDNF and zinc to TrkB activation in vivo are unknown. To address these questions, we conducted immunohistochemical (IHC) studies of the hippocampal mossy fiber axons and boutons using an antibody selective for pY816 of TrkB, a surrogate measure of TrkB activation. We found that conditional deletion of BDNF resulted in a reduction of pY816 in axons and synaptic boutons of hippocampal mossy fibers, thereby implicating BDNF in activation of TrkB in vivo. Unexpectedly, pY816 immunoreactivity was increased in axons but not synaptic boutons of mossy fibers in ZnT3 knockout mice that lack vesicular zinc. Marked increases of BDNF content were evident within the hippocampus of ZnT3 knockout mice and genetic elimination of BDNF reduced pY816 immunoreactivity in these mice, implicating BDNF in enhanced TrkB activation mediated by vesicular zinc depletion. These findings support the conclusion that BDNF but not vesicular zinc activates TrkB in hippocampal mossy fiber axons under physiological conditions.

Neuroprotective effects of reactive oxygen species mediated by BDNF-independent activation of TrkB.

Reactive oxygen species (ROS) have diverse biological consequences in the mammalian CNS, but the molecular targets mediating these pleiotropic effects are incompletely understood. Like ROS, the neurotrophin receptor, TrkB receptor tyrosine kinase, has diverse effects in the developing and mature mammalian brain. Our discovery that zinc can transactivate TrkB, together with the finding that ROS can trigger zinc release from cytosolic zinc binding proteins, led us to hypothesize that ROS can transactivate TrkB in CNS neurons by a zinc-dependent mechanism. We found that both exogenous H(2)O(2) and endogenous ROS activate TrkB signaling by a Src family kinase-dependent but brain-derived neurotrophic factor-independent mechanism in cultured rat cortical neurons. Exogenous H(2)O(2) enhances cytosolic zinc content in a metallothionein-3 (MT-3)-requiring manner. Both exogenous H(2)O(2) and endogenous ROS mediated transactivation of TrkB requires intracellular zinc and MT-3. The ROS-triggered transactivation of TrkB exerts neuroprotective effects, because inhibition of TrkB kinase activity or uncoupling Shc signaling from TrkB exacerbates neuronal cell death induced by H(2)O(2). Thus, we propose a molecular signaling event whereby ROS induce release of zinc from cytosolic MT-3, the increased cytosolic zinc transactivates TrkB, and the enhanced Shc signaling downstream from TrkB promotes prosurvival effects. We suggest that such neuroprotective effects mediated by ROS are operative in diverse acute and chronic neurological disorders.

RNA aptamer-based functional ligands of the neurotrophin receptor, TrkB.

Many cell surface signaling receptors, such as the neurotrophin receptor, TrkB, have emerged as potential therapeutic targets for diverse diseases. Reduced activation of TrkB in particular is thought to contribute to neurodegenerative diseases. Unfortunately, development of therapeutic reagents that selectively activate particular cell surface receptors such as TrkB has proven challenging. Like many cell surface signaling receptors, TrkB is internalized upon activation; in this proof-of-concept study, we exploited this fact to isolate a pool of nuclease-stabilized RNA aptamers enriched for TrkB agonists. One of the selected aptamers, C4-3, was characterized with recombinant protein-binding assays, cell-based signaling and functional assays, and, in vivo in a seizure model in mice. C4-3 binds the extracellular domain of TrkB with high affinity (K(D) ∼2 nM) and exhibits potent TrkB partial agonistic activity and neuroprotective effects in cultured cortical neurons. In mice, C4-3 activates TrkB upon infusion into the hippocampus; systemic administration of C4-3 potentiates kainic acid-induced seizure development. We conclude that C4-3 is a potentially useful therapeutic agent for neurodegenerative diseases in which reduced TrkB activation has been implicated. We anticipate that the cell-based aptamer selection approach used here will be broadly applicable to the identification of aptamer-based agonists for a variety of cell-surface signaling receptors.

Molecular signaling mechanisms underlying epileptogenesis.

Epilepsy, a disorder of recurrent seizures, is a common and frequently devastating neurological condition. Available therapy is only symptomatic and often ineffective. Understanding epileptogenesis, the process by which a normal brain becomes epileptic, may help identify molecular targets for drugs that could prevent epilepsy. A number of acquired and genetic causes of this disorder have been identified, and various in vivo and in vitro models of epileptogenesis have been established. Here, we review current insights into the molecular signaling mechanisms underlying epileptogenesis, focusing on limbic epileptogenesis. Study of different models reveals that activation of various receptors on the surface of neurons can promote epileptogenesis; these receptors include ionotropic and metabotropic glutamate receptors as well as the TrkB neurotrophin receptor. These receptors are all found in the membrane of a discrete signaling domain within a particular type of cortical neuron--the dendritic spine of principal neurons. Activation of any of these receptors results in an increase Ca2+ concentration within the spine. Various Ca2+-regulated enzymes found in spines have been implicated in epileptogenesis; these include the nonreceptor protein tyrosine kinases Src and Fyn and a serine-threonine kinase [Ca2+-calmodulin-dependent protein kinase II (CaMKII)] and phosphatase (calcineurin). Cross-talk between astrocytes and neurons promotes increased dendritic Ca2+ and synchronous firing of neurons, a hallmark of epileptiform activity. The hypothesis is proposed that limbic epilepsy is a maladaptive consequence of homeostatic responses to increases of Ca2+ concentration within dendritic spines induced by abnormal neuronal activity.