Anne E. Anderson - US grants

The major epileptic syndromes with onset in infancy or later in life are characterized by modulation of epileptiform activity in synchrony with sleep-wake cycling. Most commonly epileptiform activity is augmented, at times drastically, during non-rapid eye movement (NREM) sleep. Preliminary results using immature rats with tetanus toxin induced epilepsy reveal similar NREM augmentation of epileptiform activity. The goal of the proposed studies is to improve our understanding of the mechanisms underlying modulation of epileptiform activity during sleep-wake cycling in developing brain. NREM sleep is characterized by increased activity in brainstem monoaminergic (noradrenergic and serotonergic) systems. The noradrenergic brainstem nuclei (Locus ceruleus) have widespread cortical projections making it possible for this system to modulate cortical neuronal activity. There is experimental evidence that beta-adrenergic receptor activation increases epileptiform activity in adult rat hippocampal slices. Studies have shown that this occurs in infant rats as well but is more dramatic. Intracellular neurophysiologic studies in adult hippocampal slice preparations have shown that activation of the beta- adrenergic receptor suppresses the slow after hyperpolarization (sAHP) that normally follows the paroxysmal depolarization shift (PDS). Blockade of this potential shortens the post PDS refractory period and thus it is thought to lead to the increases in interictal spike frequency observed experimentally. The beta-adrenergic receptor is a G-protein linked receptor. Ligand binding to the beta-adrenergic receptor mediates G protein activation of adenylyl cyclase. As a result, intracellular cyclic adenosine monophosphate (cAMP) levels increase. This in turn activates cAMP dependent protein kinase (PKA). Activation of PKA has been shown to phosphorylate a protein closely associated with a slow calcium activated potassium channel in hippocampal neurons. Protein phosphorylation leads to reduction in potassium conductance through this channel and a blockade of the sAHP. Based on these ideas, we hypothesize that beta-adrenergic receptor activation and this second messenger cascade mediate NREM sleep augmentation of epileptiform activity. Through the use of a tetanus toxin induced epilepsy model in immature rats, the proposed experiments will further define sleep modulation of epileptiform activity by manipulating the beta-adrenergic system in vivo. Experiments are also proposed to record intracellular and extracellular changes in epileptiform activity in hippocampal slices from these animals following pharmacological manipulation of the beta-adrenergic system. By further defining the role of beta-adrenergic modulation of epileptiform activity in immature brain, we hope to better understand the basic processes contributing to seizures in early life. Thus, these studies will hopefully lead to new avenues of treatment of the epilepsies of infancy and childhood.

DESCRIPTION (Adapted from the Applicant's Abstract): Epilepsy is a common neurological disorder. Basic research in the field of epilepsy has focused on understanding the cellular and molecular mechanisms that underlie the disorder. The goal of this proposal is to evaluate the role that the mitogen-activated protein kinase (MAPK) signaling cascade plays in epilepsy. We have shown that MAPK regulates K channel activity and synaptic plasticity. Furthermore MAPK activation leads to long-lasting changes in the hippocampus through regulation of gene transcription. Recent studies have demonstrated MAPK activation in animal models of epilepsy, although the downstream targets of MAPK in epilepsy are unknown. We propose that the MAPK cascade plays a critical role in the genesis of the acute and chronic phases of epilepsy through regulation of K channel activity and gene transcription. Regulation of K channel activity could impact membrane excitability, and recent studies have shown that humans and genetic mouse models with K channel mutations have an epilepsy phenotype. MAPK regulation of transcription factors such as cyclic AMP response element binding protein (CREB) could contribute to the chronic changes seen in epilepsy (i.e. hippocampal sclerosis). Our preliminary results show hippocampal MAPK activation, an increase in NLAPK phosphorylation of a dendritic K channel subunit, Kv4.2, and increases in CREB phosphorylation in the kainate model of epilepsy. To further support a role for the MAPK cascade in epilepsy we have pilot studies showing that inhibition of the MAPK cascade blocks the expression of kainate-induced limbic motor seizures. In this proposal we wish to test the hypotheses that: 1) the MAPK cascade is activated in hippocampus following kainate-induced status epilepticus and is necessary for kainate-induced epileptogenesis; 2) the K channel subunit, Kv4.2, is an effector of MAPK in the kainate model of epilepsy; and 3) the transcription factor, CREB, is an effector of MAPK in the kainate model of epilepsy. By further defining the role of the MAPK signaling cascade in epilepsy we hope to gain insight into the basic mechanisms contributing to this disorder. Thus these studies may lead to the development of new treatments for epilepsy.

DESCRIPTION (Adapted from the Applicant's Abstract): Epilepsy is a common neurological disorder. Basic research in the field of epilepsy has focused on understanding the cellular and molecular mechanisms that underlie the disorder. The goal of this proposal is to evaluate the role that the mitogen-activated protein kinase (MAPK) signaling cascade plays in epilepsy. We have shown that MAPK regulates K channel activity and synaptic plasticity. Furthermore MAPK activation leads to long-lasting changes in the hippocampus through regulation of gene transcription. Recent studies have demonstrated MAPK activation in animal models of epilepsy, although the downstream targets of MAPK in epilepsy are unknown. We propose that the MAPK cascade plays a critical role in the genesis of the acute and chronic phases of epilepsy through regulation of K channel activity and gene transcription. Regulation of K channel activity could impact membrane excitability, and recent studies have shown that humans and genetic mouse models with K channel mutations have an epilepsy phenotype. MAPK regulation of transcription factors such as cyclic AMP response element binding protein (CREB) could contribute to the chronic changes seen in epilepsy (i.e. hippocampal sclerosis). Our preliminary results show hippocampal MAPK activation, an increase in NLAPK phosphorylation of a dendritic K channel subunit, Kv4.2, and increases in CREB phosphorylation in the kainate model of epilepsy. To further support a role for the MAPK cascade in epilepsy we have pilot studies showing that inhibition of the MAPK cascade blocks the expression of kainate-induced limbic motor seizures. In this proposal we wish to test the hypotheses that: 1) the MAPK cascade is activated in hippocampus following kainate-induced status epilepticus and is necessary for kainate-induced epileptogenesis; 2) the K channel subunit, Kv4.2, is an effector of MAPK in the kainate model of epilepsy; and 3) the transcription factor, CREB, is an effector of MAPK in the kainate model of epilepsy. By further defining the role of the MAPK signaling cascade in epilepsy we hope to gain insight into the basic mechanisms contributing to this disorder. Thus these studies may lead to the development of new treatments for epilepsy.

The proposed studies focus on molecular mechanisms of signaling pathway modulation of excitability in neurons of immature hippocampus. Signaling cascades activate protein kinases, which biochemically modify substrate proteins via phosphorylation. Kinase phosphorylation of ion channel subunits is a well- characterized means of regulating channel function in neurons and therefore, membrane excitability. Despite these insights, the molecular mechanisms underlying the regulation of the membrane properties of immature neurons remain unclear. We propose that an important locus for mediating neuromodulation of excitability of neurons from immature hippocampus is through regulation of potassium (K+) channel activity by phosphorylation. This proposal focuses on Ca2+-activated K+ channels known as the small conductanceSK channels. These subunits contribute to a component of the current known as the afterhyperpolarization (AHP). The AHP follows a single action potential or a series of action potentials. Therefore, the AHP playsa critical role in shaping the electrical responsiveness of hippocampal neurons beginning early in life, and modulation of this current can dramatically affect neuronal excitability. Given that K* channels are critical to the regulation of neuronal excitability, developmental differences in K+ channel expression and kinase regulation may play a role in normal plasticity and pathological processes such as epilepsy in the immature brain. For these studies we will focus on SK2 and the current that it underlies, the apamin-sensitive AHP. The central hypothesis of this proposal is that the cAMP-dependent protein kinase (PKA) pathway regulates SK2 channel function through direct phosphorylation and that this post-translational mechanism as well as the developmental regulation of the expression of SK2 channels and the underlying mAHP contributes to the regulation of neuronal excitability in developing hippocampus. As part of our studies we will investigate the possibility that there are developmental differences in the expression and regulation of SK2 channel subunits and the apamin-sensitive AHP that may underlie the well-characterized observation that immature brain exhibits periods of increased excitability. This feature likely contributes to normal plasticity in developing CNS as well as pathology, such as increased seizure susceptibility in immature brain.

DESCRIPTION (provided by applicant): Prolonged, continuous seizure activity (status epilepticus) is associated with significant mortality and morbidity. The studies outlined here focus on understanding the cellular mechanisms that are involved in status epilepticus. We hypothesize that activity-dependent alterations in ion channel regulation contribute to increased network excitability and possibly the potentiation of status epilepticus. In this proposal we will focus on understanding the mechanisms underlying the regulation of a particular ion channel, Kv4.2 during status epilepticus. Kv4.2 channels are critical regulators of postsynaptic excitability in the hippocampus, which is a seizure prone region of the brain. These channels are localized to the dendrites of hippocampal neurons where they are major contributors to the transient A-type K+ current. In this region where the neurons receive synaptic input, the voltage-dependent activation of Kv4.2 channels provides a critical mechanism for regulating postsynaptic excitability. Post-translational modifications are involved in the regulation of Kv4.2 channels. Recently, we have identified aberrant regulation of Kv4.2 channels in hippocampus acutely following status epilepticus, the net sum of which is predicted to lead to decreases in the A-type K+ current in the dendrites and thereby increase postsynaptic excitability. In these studies, we will evaluate candidate mechanisms involved in the remodeling of Kv4.2 channels in the postsynaptic membrane following convulsant stimulation. The aims of the proposal are: 1) to evaluate whether there are alterations in Kv4.2 channel expression and localization in hippocampus during status epilepticus;2) to investigate whether alterations in the half-life and trafficking of Kv4.2 is a candidate mechanism for these changes and 3) to evaluate whether post-translational mechanisms contribute to this effect. We will use a combination of biochemical, molecular, imaging, and physiology techniques to evaluate Kv4.2 expression and mechanisms of regulation following convulsant stimulation in models in vivo and in vitro. Our hope is that the findings from these studies will provide novel insights into the mechanisms involved in the regulation of Kv4.2 during status epilepticus and that these studies will provide insights into the development of new interventions for the treatment of status epilepticus. PUBLIC HEALTH RELEVANCE: Prolonged, continuous seizure activity (status epilepticus) is associated with significant mortality and morbidity. The studies outlined here focus on understanding the mechanisms that are involved in activity-dependent alterations in ion channels during status epilepticus. We hypothesize that these mechanisms contribute to increased network excitability and potentially the potentiation of status epilepticus. Our hope is that the findings from these studies will provide insights into the development of new interventions for the treatment of status epilepticus.

DESCRIPTION (provided by applicant): Sudden unexpected death in epilepsy (SUDEP) is characterized by sudden unexpected, nontraumatic, and nondrowning deaths in epileptic patients. SUDEP is the most common cause of mortality in individuals with epilepsy. There are reports of the incidence of SUDEP being as high as 1 in 200 in patients with severe epilepsy. However, this is likely to be an underestimate as the actual number of SUDEP cases is thought to be underreported. Among the candidate mechanisms for SUDEP, cardiac etiologies are a strong possibility. Sudden cardiac death resulting from cardiac arrhythmia such as ventricular tachycardia and fibrillation is associated with alterations in cardiac ion channels responsible for the regulation of the heart rate and rhythm. These types of changes also occur in association with cardiac pathology, which is considered a risk factor for arrhythmia. Imbalance in the autonomic nervous system, specifically sympathetic nervous system activity is considered an important player in the development of ventricular tachyarrhythmias. A number of clinical studies support the concept that autonomic alterations exist in individuals with epilepsy and that this may be more pronounced in the group with SUDEP. Thus, cardiac arrhythmogenic events, possibly related to autonomic dysregulation and cardiac ion channel remodeling represent a strong candidate mechanism underlying SUDEP. In the studies outlined we will utilize the pilocarpine model of acquired epilepsy that recapitulates a number of the features of chronic epilepsy in humans, including unprovoked seizures, tachycardia, and an increased risk of sudden death. Our pilot studies in these animals reveal that there is cardiac arrhythmogenesis and ion channel remodeling. Through an interdisciplinary collaboration between investigators from the fields of epilepsy and cardiac electrophysiology we will test the hypothesis that epilepsy results in cardiac molecular remodeling and an associated propensity for arrhythmogenesis. We propose that aberrant autonomic nervous system function is a candidate mediator for these changes.

DESCRIPTION (provided by applicant): Prolonged continuous seizure activity (status epilepticus, SE) is associated with an increased risk for developing epilepsy with hippocampal sclerosis and comorbidities such as long-term cognitive impairments. The molecular mechanisms underlying these changes following SE are not well-understood. In experimental models, SE triggers immediate and long-lasting dysregulation of the mammalian target of rapamycin (mTOR) pathway, which has been implicated in epileptogenesis. Under physiological conditions mTOR modulates dendritic morphology and ion channels, synaptic plasticity, and memory, but the role of mTOR in behavioral deficits following SE has not been evaluated. Previously it has been shown that excessive activation of the mTOR pathway in transgenic rodents is associated with behavioral deficits, seizures, mossy fiber sprouting, and dendritic abnormalities, which are reversed following treatment with the mTOR inhibitor, rapamycin. Based on these studies, we hypothesize that mTOR pathway dysregulation contributes to the morphological and molecular alterations in hippocampal dendrites and the associated hippocampal-dependent learning and memory deficits that occur following SE. We have pilot data demonstrating that treatment with rapamycin improves dendritic morphology and ion channel dysregulation and rescues hippocampal- dependent spatial learning and memory deficits early following SE. Our pilot data also suggest that the rescue of the behavioral phenotype is not sustained as the effect does not last into the period of chronic epilepsy. Thus, transient rapamycin therapy is not sufficient to block long-term alterations in behavior following SE, suggesting that chronic treatment with rapamycin or more potent mTOR inhibitor therapy may be required. In the studies proposed here we will further evaluate changes in the downstream signaling of mTORC1 and 2 following SE. Furthermore, we will investigate whether translation rates are altered following SE. To this end, we will employ polysome profiling and monitor the effectiveness of PP242 compared to rapamycin. We will use inhibitor regimens that include early and late treatments following SE. We will use biochemistry, dendritic morphological assessments, behavior, and electroencephalography as outcome measures. Since these inhibitors are already in use clinically or are being extensively studied and developed in oncology, there is the potential to rapidly translate to treatments for epilepsy and comorbidities in humans. The aims of this proposal are as follows: 1) To further characterize aberrant mTOR signaling and evaluate dysregulation of mTORC1- dependent mRNA translation following SE; 2) To evaluate whether mTOR pathway dysregulation contributes to dendritic structural and molecular alterations following SE; 3) To evaluate whether mTOR pathway dysregulation contributes to learning and memory deficits that occur following SE; and 4) To compare the effect of rapamycin versus PP242 on epileptiform activity.

Project Summary/Abstract The overall goal of the proposed studies is disruption in sympathetic nervous system (SNS) tone during epileptogenesis that contributes to cardiac alterations and potentially sudden death and test whether the cardiac phenotype can be rescued with central modulation of SNS signaling. In sudden unexpected death in epilepsy (SUDEP) affecting humans, the underlying mechanisms are not fully understood but include cardiac and respiratory dysfunction/failure. The focus of this proposal is on cardiac alterations and the role of abnormal SNS tone underlying these phenotype changes in a mouse model of acquired epilepsy with altered SNS tone, cardiac changes, and SUDEP. Detrimental cardiac effects following experimental SE that persist during the development of epilepsy have been shown. Furthermore, pretreatment with a systemic ?1-adrenergic receptor antagonist, blocked some acute changes following SE and stimulation-induced arrhythmias in chronic epilepsy, supporting increased SNS drive in SE and epilepsy. Alterations in SNS drive have not been fully characterized in epileptogenesis, and whether modulation of SNS tone is protective against chronic cardiac changes in epilepsy and associated sudden death has not been evaluated. Mechanisms underlying increased SNS tone following SE are unclear. SNS activity is regulated at many levels in the central nervous system (CNS) with final convergence at the rostral medulla (RM) neurons, which are critical modulators of SNS tone. Models of sympathoexcitation exhibit elevated angiotensin II (ANG II) levels in the RM, which binds to angiotensin type 1 (AT1) and type 2 (AT2) receptors. Activation of excitatory AT1 receptors in the RM is implicated in the maintaining elevated SNS tone, while AT2 receptor activation has an inhibitory effect. In sympathoexcitatory models with similar cardiac alterations as seen post-SE, in RM there are increased AT1 and decreased AT2 receptos. Increased ANG II levels in the brainstem with increased SNS tone has been shown in epilepsy. We hypothesize that persistently increased SNS tone contributes to cardiac dysfunction following SE with altered ANG II signaling as a candidate mechanism. We propose that alterations in ANG II signaling following SE are centrally driven. The aims of the proposed studies will test this hypothesis.