Manoj Patel - US grants

DESCRIPTION (provided by applicant): Epilepsy, a neurological disorder, is a major public health issue affecting over 2 million Americans. Each year over 100,000 new cases of epilepsy are diagnosed. Current treatment for patients suffering from epileptic seizures involves the suppression of these seizures with antiepileptic drugs (AEDs). Since sodium channels control cellular excitability, they have become important targets for the suppression of seizures. In fact, many of the clinically used AEDs target sodium channels as a major mechanism of their action. However, the continued introduction of new AEDs has not helped the estimated 750,000 Americans that are resistant to current drugs. In addition, many achieve seizure control but only at the cost of significant toxic side effects. One potential reason for this unacceptable statistic is the use of screens that are not representative of the pathological conditions that exist in a diseased "epileptic" neuron. For example, studies have shown that in both animal and human limbic epilepsy, alterations in sodium channel isoform expression as well as their activity/gating occurs. These changes have been associated with the hyper-excitability of neurons involved in limbic epilepsy and also the altered pharmacology that has been well documented in temporal lobe epilepsy, reducing the efficacy of the sodium channel blockers in suppressing epileptic seizures. These observations emphasize the important need to develop screening assays that represent the "epileptic" condition and not the control "non-epileptic" condition. A direct consequence of developing such a screen would be the identification of candidate therapeutics effective in therapy resistant patients. In this exploratory/developmental translational proposal we plan to develop an "epileptic" screening profile using neurons isolated from animals with chronic limbic epilepsy (CLE). We will develop an electrophysiology screening profile using clinically used AEDs, known to target sodium channels as a mechanism of action, and a series of selective sodium channel blockers that have a different efficacy profile against sodium channel isoforms. We will validate our screening assay by assessing anticonvulsant activity in CLE rats, a model of pharmacoresistant epilepsy with spontaneous seizures. This proposal will allow us to ascertain two important features 1) it will allow us to develop and validate an electrophysiology screening assay that will permit the preliminary screening of candidate therapeutics 2) it will provide preliminary data on the efficacy of candidate therapeutics leading to the identification of candidate therapeutics that can be evaluated through further preclinical testing. Specifically we plan to: 1) Develop an electrophysiology screening profile for candidate therapeutics using "epileptic" brain slices - identification of candidate therapeutics. 2) Pre-clinical testing of candidate therapeutics for anticonvulsant activity in an animal model of chronic limbic epilepsy - validation of the electrophysiology screen. Public Health Relevance: Epilepsy, a neurological disorder, is a major public health issue affecting over 2 million Americans. Approximately 750,000 Americans experience seizures that cannot be suppressed by currently available drugs. The patients continue to experience un-controlled, life threatening, epileptic seizures. In this exploratory/developmental proposal, we will develop a drug testing screen that will identify drugs that are effective in epileptic neurons. These drugs could be effective in patients that are "pharmaco-resistant" to currently available drugs.

DESCRIPTION (provided by applicant): Seizures with a focal onset, such as temporal lobe epilepsy (TLE), are a set of debilitating disorders that affect millions of people worldwide. Despite the size of this problem, medical treatment of TLE fails in most cases. For over 40 years, the only option left for these patients has been surgery to remove the temporal lobe. Therefore, the development of novel treatments that can prevent focal seizures will have a significant impact on the quality of life of many epilepsy patients. The hypothesis guiding this R21 grant is that silencing hyperactive neurons in the Entorhinal Cortex (EC) will prevent seizures. Substantial evidence in both animal models and human patients incriminate the role of EC neurons in TLE seizures. Preliminary data show that in rodent models of TLE, EC neurons have increased firing due to increased after-depolarizing currents and increased expression of Na channel subunits. The hypothesis is that knocking down the expression of the Na channel beta-4 subunit using short-hairpinned RNA (shRNA) will reduce seizures. Recombinant adeno-associated virus (rAAV) has emerged as the top choice for human gene therapy. An important safety feature is the ability to regulate gene therapy. This will be accomplished using a newly developed tetracycline regulator cassette that controls expression of the shRNA and requires drug for activation (doxycycline-ON). The overall goal is to develop a delivery system for the shRNA that can be used in the clinic and to test its efficacy in animal models of TLE.

DESCRIPTION (provided by applicant): Epilepsy is a significant neurological disorder characterized by recurrent spontaneous seizures. It is estimated that over 2.3 million Americans have epilepsy with 200,000 new cases of epilepsy being diagnosed each year. Epilepsy is a factor in the deaths of between 25,000 to 50,000 patients each year and is estimated to cost the US $12.5 billion each year. Epilepsy therefore, is a major economic and personal burden for the American public. Unfortunately, antiepileptic drugs (AEDs) are ineffective in approximately 30% of patients. Too often treatment is associated with adverse side effects which may be the result of the AEDs affecting their targets in regions outside the seizure onset zone. In order to develop more effective treatments with fewer side effects there has been a concerted effort to understand the underlying mechanisms by which neurons become hyperexcitable in epilepsy. In chronic epilepsy molecular and cellular changes occur within the seizure onset zone, making it capable of generating spontaneous seizures. It has become clear that these changes have an altered pharmacology so that the development of new therapies that are more specific for the causes of epilepsy will be greatly aided by identifying important changes that are unique to the seizure onset zone. In this proposal we will examine the changes in sodium (Na) channels in epileptogenesis. Na channels play a critical role in controlling neuronal excitability, and so changes in Na channel thresholds and firing patterns would have significant effects on system excitability. Alterations in Na channel behavior, as a result of Na channel mutations, are known to be responsible for a number of inherited forms of generalized epilepsy. Our central hypothesis is that alterations in the expression and physiology of Na channels that make neurons more excitable are found broadly in the limbic system seizure onset zone. To help support the hypothesis that these changes contribute to the development of epilepsy it is necessary to show that the changes occur before the onset of spontaneous seizures and are thus not a consequence of the seizures. Our proposal will focus on medial entorhinal cortex (mEC) and subiculum neurons using an animal model of temporal lobe epilepsy (TLE), a common form of adult pharmaco-resistant epilepsy. We provide preliminary data demonstrating that mEC layer II neurons are intrinsically hyperexcitable in epileptic animals and that Na channel physiology is also altered. We show that changes in neuronal excitability and Na channel behavior occur before the appearance of spontaneous seizures. These findings support our central hypothesis that changes in Na channel expression and physiology contribute to the development of epilepsy. PUBLIC HEALTH RELEVANCE: Epilepsy is a neurological disorder and is a major public health issue affecting over 2 million Americans. Temporal lobe epilepsy (TLE) is the most common form of adult epilepsy that is difficult to treat pharmacologically. The limbic system is known to be heavily implicated in seizure generation in TLE. In this proposal we will determine the changes in sodium channel activity within the limbic system during the development of epileptic seizures in an animal model of TLE. Since Na channels are the target for many clinically used anticonvulsant drugs a better understanding of the importance of Na channels in epileptogenesis could provide a target for therapy development.

De novo mutations of SCN8A, the gene that encodes for the sodium (Na) channel isoform Nav1.6, are known to cause early infantile epileptic encephalopathy 13 (EIEE13). To date, more than 150 SCN8A mutations have been identified. Patients experience a variety of seizure types and motor features that can lead to wheelchair dependence. Intellectual disability varies from mild to severe and becomes progressively worst with seizure onset. Sudden unexpected death in epilepsy (SUDEP) occurs in approximately 10% of patients and increases significantly if seizures are not controlled. Unfortunately, a majority of patients have drug refractory epilepsy or a mixed response to anti-epileptic drugs (AEDs). Very little is known about the pathogenesis of SCN8A epileptic encephalopathy or treatment options for patients. In this proposal we will use a highly novel and innovative knock-in mouse model, developed by the Meisler lab, carrying the human SCN8A encephalopathy mutation p.Asn1768Asp (N1768D). The mutation was identified in a child who presented with refractory epilepsy at the age of 6 months, intellectual disability, ataxia and SUDEP at 15 years of age. The mouse model exhibits many of the pathological phenotypes seen in patients, including spontaneous seizures and sudden death. In homozygous mutant mice (D/D), seizures begin at 3 weeks of age and progress to death within 24 hours. Heterozygous mutant mice (D/+) have later seizure onset starting at 8 weeks of age and progression to death within one to two months. The availability of this mouse model provides a unique opportunity to fully investigate the pathogenesis of this devastating human epileptic encephalopathy and to also test new and selective therapies. In this proposal we will investigate when alterations in Na channel physiology and membrane excitability begin to appear in our model of epileptic encephalopathy, testing both excitatory and inhibitory neurons within brain regions known to be involved in seizure activity. In Aim 1 we will determine the pathogenesis of these alterations at specific time points before and after seizure onset using mutant mice. In Aim 2, we will silence Nav1.6 using virally delivered, dox-inducible, Nav1.6 shRNA, targeting either excitatory or inhibitory neurons and determine the effects on Na channel activity, neuronal excitability and seizure activity in mutant mice. We will determine if we can delay the onset of seizures by targeting at a time point before the onset of seizures and also if we can modulate seizure activity in mice with spontaneous seizures. In Aim 3, we will test whether our Nav1.6 subtype selective compound (MV1505) can reduce seizure activity in heterozygous (D/+) mice and delay SUDEP. We will also evaluate two clinically used anticonvulsants (phenytoin and lacosamide). These studies will significantly impact our current understanding of the physiological consequences of increased Nav1.6 activity in SCN8A epileptic encephalopathy. They will also provide important insight into the selective targeting of Nav1.6 for therapy.

Epilepsy is a major economic and personal burden for the American public, affecting over 3 million Americans (1-2% of the population) with over 200,000 new cases diagnosed each year. There is no cure for epilepsy. Seizures can only be suppressed using antiepileptic drugs. Unfortunately, these drugs are ineffective in approximately 30% of patients and are often associated with adverse side effects. T-type calcium channels (T-channels) play an important role in controlling neuronal excitability. T- channels open near the resting membrane potential of many neurons, allowing them to act as pacemaker currents that trigger sodium dependent action potential. Increases in T-channel expression and activity have been reported in animal models of temporal lobe epilepsy (TLE), contributing to neuronal hyperexcitability. In absence epilepsy, T-channel activity has been linked to thalamocortical network oscillations that give rise to spike wave discharges (SWD). Despite growing evidence for a role of T-channels in both TLE and absence epilepsy, little is known about the mechanisms by which T-channel activity and expression levels are increased, facilitating increases in neuronal excitability and seizure susceptibility. We recently discovered a novel T-channel modulator, the Ca2+ channel and chemotaxis receptor domain containing 1 (CACHD1) protein. CACHD1 is structurally similar to ?2? subunits, the major target of gabapentinoids. CACHD1 is highly expressed in both human and rodent hippocampal and thalamic brain regions with overlapping expression patterns to all three T- channel subtypes. CACHD1 promotes cell surface expression levels of T-channels and increases peak current densities, leading to an increase in neuronal excitability and increased seizure susceptibility. Knockout of CACHD1 prevents ?-butyrolactone (GBL) induced absence seizures and delays the onset of kindled seizures and reduces seizure durations. In view of these findings, CACHD1 could facilitate increases in neuronal excitability associated with TLE and absence epilepsy, making it a novel target for therapy. In this proposal we will test our central hypothesis that CACHD1 increases neuronal excitability via increases in T-channel function, facilitating the onset and severity of both absence epilepsy and TLE. On completion of these studies we will have advanced our current understanding for the role of CACHD1 in the development of absence epilepsy and TLE, providing a novel target for therapy development.