Browsing by Author "McNamara, James O"
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Item Open Access A Peptide Selectively Uncoupling BDNF Receptor TrkB from Phospholipase C gamma 1 Prevents Epilepsy and Anxiety-like Disorder(2015) Gu, BinTemporal lobe epilepsy is a common and devastating disorder that features recurrent seizures and is often associated with pathologic anxiety and hippocampal sclerosis. An episode of prolonged seizures (status epilepticus) is thought to promote development of human temporal lobe epilepsy years later. A chemical-genetic approach established proof of concept that transiently inhibiting the receptor tyrosine kinase, TrkB, following status epilepticus prevented epilepsy, anxiety-like behavior and hippocampal damage in a mouse model, providing rationale for developing a therapeutic targeting TrkB signaling. To circumvent the undesirable consequence that global inhibition of TrkB exacerbates neuronal degeneration following status epilepticus, we sought to identify both the TrkB-activated signaling pathway mediating these pathologies and a compound that uncouples TrkB from the responsible signaling effector. To accomplish these goals, we used genetically modified mice and a model of seizures and epilepsy induced by a chemoconvulsant. Genetic inhibition of TrkB-mediated phospholipase C gamma 1 (PLC gamma 1) signaling suppressed seizures induced by a chemoconvulsant, leading to design of a peptide (pY816) that inhibited the interaction of TrkB with PLC gamma 1. We demonstrate that pY816 selectively inhibits TrkB-mediated activation of PLC gamma 1 both in vitro and in vivo. Treatment with pY816 prior to administration of a chemoconvulsant suppressed seizures in a dose- and time-dependent manner. Treatment with pY816 initiated after chemoconvulsant-evoked status epilepticus and continued for just three days suppressed seizure-induction of epilepsy, anxiety-like behavior and hippocampal damage assessed months later. This study elucidates the signaling pathway by which TrkB activation produces diverse neuronal activity-driven pathologies and demonstrates therapeutic benefits of an inhibitor of this pathway in an animal model in vivo. A strategy of uncoupling a receptor tyrosine kinase from a signaling effector may prove useful in diverse diseases in which excessive receptor tyrosine kinase signaling contributes.
Item Open Access A Peptide Uncoupling BDNF Receptor TrkB from Phospholipase Cγ1 Prevents Epilepsy Induced by Status Epilepticus.(Neuron, 2015-11-04) Gu, Bin; Huang, Yang Zhong; He, Xiao-Ping; Joshi, Rasesh B; Jang, Wonjo; McNamara, James OThe 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.Item Open Access A Three-Molecule Model of Structural Plasticity: the Role of the Rho family GTPases in Local Biochemical Computation in Dendrites(2015) Hedrick, Nathan GrayIt has long been appreciated that the process of learning might invoke a physical change in the brain, establishing a lasting trace of experience. Recent evidence has revealed that this change manifests, at least in part, by the formation of new connections between neurons, as well as the modification of preexisting ones. This so-called structural plasticity of neural circuits – their ability to physically change in response to experience – has remained fixed as a primary point of focus in the field of neuroscience.
A large portion of this effort has been directed towards the study of dendritic spines, small protrusions emanating from neuronal dendrites that constitute the majority of recipient sites of excitatory neuronal connections. The unique, mushroom-like morphology of these tiny structures has earned them considerable attention, with even the earliest observers suggesting that their unique shape affords important functional advantages that would not be possible if synapses were to directly contact dendrites. Importantly, dendritic spines can be formed, eliminated, or structurally modified in response to both neural activity as well as learning, suggesting that their organization reflects the experience of the neural network. As such, elucidating how these structures undergo such rearrangements is of critical importance to understanding both learning and memory.
As dendritic spines are principally composed of the cytoskeletal protein actin, their formation, elimination, and modification requires biochemical signaling networks that can remodel the actin cytoskeleton. As a result, significant effort has been placed into identifying and characterizing such signaling networks and how they are controlled during synaptic activity and learning. Such efforts have highlighted Rho family GTPases – binary signaling proteins central in controlling the dynamics of the actin cytoskeleton – as attractive targets for understanding how the structural modification of spines might be controlled by synaptic activity. While much has been revealed regarding the importance of the Rho GTPases for these processes, the specific spatial and temporal features of their signals that impart such structural changes remains unclear.
The central hypotheses of the following research dissertation are as follows: first, that synaptic activity rapidly initiates Rho GTPase signaling within single dendritic spines, serving as the core mechanism of dendritic spine structural plasticity. Next, that each of the Rho GTPases subsequently expresses a spatially distinct pattern of activation, with some signals remaining highly localized, and some becoming diffuse across a region of the nearby dendrite. The diffusive signals modify the plasticity induction threshold of nearby dendritic spines, and the spatially restricted signals serve to keep the expression of plasticity specific to those spines that receive synaptic input. This combination of differentially spatially regulated signals thus equips the neuronal dendrite with the ability to perform local biochemical computations, potentially establishing an organizational preference for the arrangement of dendritic spines along a dendrite. Finally, the consequences of the differential signal patterns also help to explain several seemingly disparate properties of one of the primary upstream activators of these proteins: brain-derived neurotrophic factor (BDNF).
The first section of this dissertation describes the characterization of the activity patterns of one of the Rho family GTPases, Rac1. Using a novel Förster Resonance Energy Transfer (FRET)- based biosensor in combination with two-photon fluorescence lifetime imaging (2pFLIM) and single-spine stimulation by two-photon glutamate uncaging, the activation profile and kinetics of Rac1 during synaptic stimulation were characterized. These experiments revealed that Rac1 conveys signals to both activated spines as well as nearby, unstimulated spines that are in close proximity to the target spine. Despite the diffusion of this structural signal, however, the structural modification associated with synaptic stimulation remained restricted to the stimulated spine. Thus, Rac1 activation is not sufficient to enlarge spines, but nonetheless likely confers some heretofore-unknown function to nearby synapses.
The next set of experiments set out to detail the upstream molecular mechanisms controlling Rac1 activation. First, it was found that Rac1 activation during sLTP depends on calcium through NMDA receptors and subsequent activation of CaMKII, suggesting that Rac1 activation in this context agrees with substantial evidence linking NMDAR-CaMKII signaling to LTP in the hippocampus. Next, in light of recent evidence linking structural plasticity to another potential upstream signaling complex, BDNF-TrkB, we explored the possibility that BDNF-TrkB signaling functioned in structural plasticity via Rac1 activation. To this end, we first explored the release kinetics of BDNF and the activation kinetics of TrkB using novel biosensors in conjunction with 2p glutamate uncaging. It was found that release of BDNF from single dendritic spines during sLTP induction activates TrkB on that same spine in an autocrine manner, and that this autocrine system was necessary for both sLTP and Rac1 activation. It was also found that BDNF-TrkB signaling controls the activity of another Rho GTPase, Cdc42, suggesting that this autocrine loop conveys both synapse-specific signals (through Cdc42) and heterosynaptic signals (through Rac1).
The next set of experiments detail one the potential consequences of heterosynaptic Rac1 signaling. The spread of Rac1 activity out of the stimulated spine was found to be necessary for lowering the plasticity threshold at nearby spines, a process known as synaptic crosstalk. This was also true for the Rho family GTPase, RhoA, which shows a similar diffusive activity pattern. Conversely, the activity of Cdc42, a Rho GTPase protein whose activity is highly restricted to stimulated spines, was required only for input-specific plasticity induction. Thus, the spreading of a subset of Rho GTPase signaling into nearby spines modifies the plasticity induction threshold of these spines, increasing the likelihood that synaptic activity at these sites will induce structural plasticity. Importantly, these data suggest that the autocrine BDNF-TrkB loop described above simultaneously exerts control over both homo- and heterosynaptic structural plasticity.
The final set of experiments reveals that the spreading of GTPase activity from stimulated spines helps to overcome the high activation thresholds of these proteins to facilitate nearby plasticity. Both Rac1 and RhoA, the activity of which spread into nearby spines, showed high activation thresholds, making weak stimuli incapable of activating them. Thus, signal spreading from a strongly stimulated spine can lower the plasticity threshold at nearby spines in part by supplementing the activation of high-threshold Rho GTPases at these sites. In contrast, the highly compartmentalized Rho GTPase Cdc42 showed a very low activation threshold, and thus did not require signal spreading to achieve high levels of activity to even a weak stimulus. As a result, synaptic crosstalk elicits cooperativity of nearby synaptic events by first priming a local region of the dendrite with several (but not all) of the factors required for structural plasticity, which then allows even weak inputs to achieve plasticity by means of localized Cdc42 activation.
Taken together, these data reveal a molecular pattern whereby BDNF-dependent structural plasticity can simultaneously maintain input-specificity while also relaying heterosynaptic signals along a local stretch of dendrite via coordination of differential spatial signaling profiles of the Rho GTPase proteins. The combination of this division of spatial signaling patterns and different activation thresholds reveals a unique heterosynaptic coincidence detection mechanism that allows for cooperative expression of structural plasticity when spines are close together, which in turn provides a putative mechanism for how neurons arrange structural modifications during learning.
Item Open Access BDNF-TrkB Signaling in Single-Spine Structural Plasticity(2016) Harward, Stephen CannadaMultiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.
Item Open Access Disease Modification of Epilepsy by Disruption of TrkB Signaling(2019) Krishnamurthy, KameshEpilepsy is the most common acquired neurological disorder and is characterized by spontaneous, recurrent seizures. Of the various forms of epilepsy, Temporal Lobe Epilepsy (TLE) has received intense clinical and research interest. Current therapeutic options for TLE are anti-convulsive and purely symptomatic. Improved treatments are needed that either (1) prevent epileptogenesis or (2) ameliorate existing disease. Studies suggest that TLE may be induced by a preceding episode of prolonged seizure activity (status epilepticus, or SE). Our lab previously utilized a chemical-genetic strategy to establish proof of concept that transient inhibition of the receptor tyrosine kinase TrkB following SE prevented TLE. Subsequent studies identified the downstream effector of TrkB activation by demonstrating that transient administration of a peptide (“pY816”) uncoupling TrkB from the enzyme PLCγ1 also prevented SE-induced TLE.
TLE is analogous to associative memory formation in that both involve activity-determined plasticity. Associative memories can be rendered labile following re-exposure to the inciting stimulus; during this labile period inhibition of molecular mechanisms necessary for initial learning inhibits reconsolidation and results in memory “erasure”. Given the proposed parallels between epileptogenesis and memory formation as well as the central role of TrkB-PLCγ1 signaling in the development of epilepsy, I sought to test whether the occurrence of a seizure introduces a period of lability and whether inhibition of TrkB-PLCγ1 signaling prevents subsequent reconsolidation. I demonstrate in the kindling model of TLE that the combination of an evoked seizure and chemical-genetic inhibition of TrkB kinase, but not inhibition of TrkB kinase alone, reduces the severity of subsequent evoked seizures. Combination of an evoked seizure and pY816 (but not pY816 alone) produces the same effect. These results suggest that seizures induce a period of lability in a model of TLE and perturbation of TrkB-PLCγ1 signaling inhibits reconsolidation of pathologic plasticity.
In specimens from patients who underwent surgical resection for medically refractory TLE there is a striking increase in expression of the ligand for TrkB, BDNF. In a second study, I demonstrate that this increase, as well as an increase in TrkB-PLCγ1 signaling, is also seen in a TLE model exhibiting spontaneous seizures. Given the result that TrkB-PLCγ1 inhibition prevents reconsolidation, I asked what effect treatment with pY816 has in an animal model after spontaneous seizures emerged. I demonstrate that pY816 induced a remission in seizures that persists after treatment termination.
These studies elucidate a signaling pathway (TrkB-PLCγ1) underlying epilepsy progression and persistence, connect TLE to other disorder of pathologic plasticity like PTSD and neuropathic pain, and open the door to a novel therapeutic approach for treating patients with existing epilepsy.
Item Open Access Locales and Mechanisms of TrkB Activation Within Hippocampus(2014) Helgager, Jeffrey JamesUnderstanding the mechanisms of limbic epileptogenesis in cellular and molecular terms may provide novel therapeutic targets for its prevention. The neurotrophin receptor tropomyosin-related kinase B (TrkB) is thought to be critical for limbic epileptogenesis. Enhanced activation of TrkB, revealed by immunodetection of enhanced phosphorylated TrkB (pTrkB), a surrogate measure of its activation, has been identified within the hippocampus in multiple animal models. Knowledge of the cellular locale of activated TrkB is necessary to elucidate its functional consequences. Using an antibody selective to pTrkB in conjunction with confocal microscopy and cellular markers, we determined the cellular and subcellular locale of enhanced pTrkB induced by status epilepticus (SE) evoked by infusion of kainic acid into the amygdala of adult mice. SE induced enhanced pTrkB immunoreactivity in two distinct populations of principal neurons within the hippocampus--the dentate granule cells and CA1 pyramidal cells. Enhanced immunoreactivity within granule cells was found within mossy fiber axons and giant synaptic boutons. By contrast, enhanced immunoreactivity was found within apical dendritic shafts and spines of CA1 pyramidal cells. A common feature of this enhanced pTrkB at these cellular locales is its localization to excitatory synapses between excitatory neurons, presynaptically in the granule cells and postsynaptically in CA1 pyramidal cells. Long-term potentiation (LTP) is one cellular consequence of TrkB activation at these excitatory synapses that may promote epileptogenesis.
The importance of TrkB in diverse neuronal processes, as well as its involvement in various disorders of the nervous system, underscores the importance of understanding how it is activated. The canonical neurotrophin ligand which activates TrkB is brain derived neurotrophic factor (BDNF). Zinc, however, has also been demonstrated to activate this receptor through a mechanism whereby it does not directly interact with it, known as transactivation. Presynaptic vesicles of mossy fiber boutons of stratum lucidum are particularly enriched in zinc, where it is co-released with glutamate in an activity dependent fashion, and incorporated into these vesicles by the zinc transporter, ZnT3. Given the presence of large quantities of zinc within stratum lucidum, we hypothesized that this metal may contribute to TrkB transactivation at this locale. To this end, we examined the contributions of both BDNF and synaptic vesicular zinc to TrkB activation in stratum lucidum of mouse hippocampus under physiological conditions. Utilization of mice which are genetic knockouts for BDNF and/or ZnT3 allowed us to examine TrkB activation in the absence of one or both of these ligands. This was done using an antibody for pTrkB in conjunction with confocal microscopy, assaying immunoreactivity at the cellular and synaptic locales within stratum lucidum where pTrkB was previously found to be enriched. Our results suggest that BDNF contributes to TrkB activation within stratum lucidum. Interestingly, ZnT3 mice displayed an increase in BDNF protein and TrkB activation, demonstrating that synaptic zinc regulates BDNF and TrkB signaling at this locale.
Item Open Access Mechanisms of specificity in neuronal activity-regulated gene transcription.(2012) Lyons, Michelle RenéeIn the nervous system, activity-regulated gene transcription is one of the fundamental processes responsible for orchestrating proper brain development–a process that in humans takes over 20 years. Moreover, activity-dependent regulation of gene expression continues to be important for normal brain function throughout life; for example, some forms of synaptic plasticity important for learning and memory are known to rely on alterations in gene transcription elicited by sensory input. In the last two decades, increasingly comprehensive studies have described complex patterns of gene transcription induced and/or repressed following different kinds of stimuli that act in concert to effect changes in neuronal and synaptic physiology. A key theme to emerge from these studies is that of specificity, meaning that different kinds of stimuli up- and down regulate distinct sets of genes. The importance of such signaling specificity in synapse-to-nucleus communication becomes readily apparent in studies examining the physiological effects of the loss of one or more forms of transcriptional specificity – often, such genetic manipulations result in aberrant synapse formation, neuronal cell death, and/or cognitive impairment in mutant mice. The two primary loci at which mechanisms of signaling specificity typically act are 1) at the synapse – in the form of calcium channel number, localization, and subunit composition – and 2) in the nucleus – in the form of transcription factor expression, localization, and post-translational modification. My dissertation research has focused on the mechanisms of specificity that govern the activity-regulated transcription of the gene encoding Brain-derived Neurotrophic Factor(Bdnf). BDNF is a secreted protein that has numerous important functions in nervous system development and plasticity, including neuronal survival, neurite outgrowth, synapse formation, and long-term potentiation. Due to Bdnf’s complex transcriptional regulation by various forms of neural stimuli, it is well positioned to function as a transducer through which altered neural activity states can lead to changes in neuronal physiology and synaptic function. In this dissertation, I show that different families of transcription factors, and even different isoforms or splice variants within a single family, can specifically regulate Bdnf transcription in an age- and stimulus-dependent manner. Additionally, I characterize another mechanism of synapse-to-nucleus signaling specificity that is dependent upon NMDA-type glutamate receptor subunit composition, and provide evidence that the effect this signaling pathway has on gene transcription is important for normal GABAergic synapse formation. Taken together, my dissertation research sheds light on several novel signaling mechanisms that could lend specificity to the activity-dependent transcription of Bdnf exon IV. My data indicate that distinct neuronal stimuli can differentially regulate the Calcium-Response Element CaRE1 within Bdnf promoter IV through activation of two distinct transcription factors: Calcium-Response Factor (CaRF) and Myocyte Enhancer Factor 2 (MEF2). Furthermore, individual members of the MEF2 family of transcription factors differentially regulate the expression of Bdnf, and different MEF2C splice variants are unequally responsive to L-type voltage-gated calcium channel activation. Additionally, I show here for the first time that the NMDA-type glutamate receptor subunit NR3A (also known as GluN3A) is capable of exerting an effect on NMDA receptor-dependent Bdnf exon IV transcription, and that changes in the expression levels of NR3A may function to regulate the threshold for activation of synaptic plasticity-inducing transcriptional programs during brain development. Finally, I provide evidence that the transcription factor CaRF might function in the regulation of homeostatic programs of gene transcription in an age- and stimulus-specific manner. Together, these data describe multiple novel mechanisms of specificity in neuronal activity-regulated gene transcription, some of which function at the synapse, others of which function in the nucleus.Item Open Access Regression of Epileptogenesis by Inhibiting Tropomyosin Kinase B Signaling following a Seizure.(Annals of neurology, 2019-12) Krishnamurthy, Kamesh; Huang, Yang Zhong; Harward, Stephen C; Sharma, Keshov K; Tamayo, Dylan L; McNamara, James OOBJECTIVE: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.Item Open Access Transient Inhibition of TrkB Kinase after Status Epilepticus Prevents Development of Temporal Lobe Epilepsy(Neuron, 2013) Liu, Gumei; Gu, Bin; He, Xiao-Ping; Joshi, Rasesh B; Wackerle, Harold D; Rodriguiz, Ramona Marie; Wetsel, William C; McNamara, James OTemporal lobe epilepsy is the most common and often devastating form of human epilepsy. The molecular mechanism underlying the development of temporal lobe epilepsy remains largely unknown. Emerging evidence suggests that activation of the BDNF receptor TrkB promotes epileptogenesis caused by status epilepticus. We investigated a mouse model in which a brief episode of status epilepticus results in chronic recurrent seizures, anxiety-like behavior, and destruction of hippocampal neurons. We used a chemical-genetic approach to selectively inhibit activation of TrkB. We demonstrate that inhibition of TrkB commencing after status epilepticus and continued for 2weeks prevents recurrent seizures, ameliorates anxiety-like behavior, and limits loss of hippocampal neurons when tested weeks to months later. That transient inhibition commencing after status epilepticus can prevent these long-lasting devastating consequences establishes TrkB signaling as an attractive target for developing preventive treatments of epilepsy in humansItem Open Access TrkB and Epileptogenesis(2008-04-09) Kotloski, RobertDiscovering the cellular and molecular mechanisms underlying the pathophysiology underlying the development of epilepsy is key to the creation of improved treatments. The neurotrophins and their receptors, in particular BDNF and TrkB, are likely candidates to be involved in the process by which a normal brain becomes epileptic (epileptogenesis). The work presented in the dissertation has investigated the hypothesis that TrkB is a central factor in epileptogenesis in multiple animal models of epilepsy.
Conditional deletion of TrkB in the Syn-Cre TrkB-/- mouse prevented nearly all epileptogenesis in the kindling model, despite the ability to have a tonic-clonic seizure. Reduction of TrkB de novo in mature Act-CreER TrkB-/- mice also delayed epileptogenesis in the kindling model. Additionally, Syn-Cre TrkB+/- and Act-CreER TrkB-/- mice had impaired persistence of the hyperexcitable state following kindling. It remained unclear from these findings whether reduction of TrkB during and/or following induction of kindling was responsible for the impaired persistence. The inducible Act-CreER TrkBflox/flox mice were used to reduce TrkB only after the fully kindled state had been reached and demonstrated that loss of TrkB after completion of kindling impairs persistence of the hyperexcitable state.
Status epilepticus is a medical emergency defined by prolonged continuous seizure activity. Conditional deletion of TrkB in the Syn-Cre TrkB-/- mice prevents sustained seizure activity evident in wild type mice following pilocarpine injection. Furthermore, the Syn-Cre TrkB-/- mice may also retain greater sensitivity to diazepam following status epilepticus than control mice. Together with biochemical evidence of TrkB activation during status epilepticus, these findings suggest that TrkB activation is required for persistence of status epilepticus.
In conclusion, the findings in this dissertation demonstrate TrkB to be a molecular mechanism critical for: 1) epileptogenesis in the kindling model; 2) persistence of hyperexcitability in the kindling model; 3) persistence of limbic status epilepticus in a chemoconvulsant model. These discoveries provide the basis for developing novel therapeutic approaches to three distinct and devastating aspects of the limbic epilepsy in humans. These aspects are: 1) preventing progression of limbic epilepsy to a medically refractory state; 2) reversal of medically refractory limbic epilepsy; 3) medically refractory status epilepticus.
Item Open Access TrkB-Shc Signaling Protects against Hippocampal Injury Following Status Epilepticus.(The Journal of neuroscience : the official journal of the Society for Neuroscience, 2019-06) Huang, Yang Zhong; He, Xiao-Ping; Krishnamurthy, Kamesh; McNamara, James OTemporal 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.