Edward Perez-Reyes - US grants

Considerable progress on skeletal muscle L-type Ca2+ channels has been made at the pharmacological, biophysical, biochemical, and molecular biological levels. In fact, all the known subunits of the skeletal muscle L-type Ca2+ channel have been purified and cloned (alpha1, alpha2, beta, gamma, delta). However, less is known of the subunit structure of the cardiac channel. I will focus my research on the cardiac Ca2+ channel, since this channel plays a pivotal role in both the physiology and pathophysiology of the cardiovascular system, as well as being the site of action of a number of clinically relevant drugs, the "calcium antagonists." I have discovered that the gene encoding the a, subunit of the cardiac Ca2+ channel is alternatively spliced to produce at least three transcripts. I hypothesize that these transcripts encode L-type Ca2+ channel subtypes that differ in their biophysical properties. The first aim of this project is to characterize the electrophysiological properties of these alpha1 subtypes using the Xenopus laevis oocyte expression system. The second hypothesis I will test is that the other Ca2+ channel subunits modulate the biophysical properties of the alpha1 subunit. I plan to clone the other subunits of the cardiac L-type Ca2+ channel using the skeletal muscle cDNA's as probe, then coexpress them with alpha1. Since the hormonal regulation of cardiac contractility is in part mediated by phosphorylation of the L-type Ca2+ channel, my third aim is to study how these channels are regulated by phosphorylation. Since the submission of my original grant application I have achieved considerable progress on these research goals. I have optimized and partially characterized the expression of a cardiac alpha1 subtype in oocytes using the 2 microelectrode voltage clamp. In addition, I have cloned a new beta subunit that is expressed in heart and brain, and shown that it can modulate the biophysical properties of alpha1. These preliminary studies demonstrate the feasibility of the proposed studies, as well as my ability to carry them out.

Intracellular calcium controls a variety of cellular functions such as contraction, secretion, proliferation, and gene expression. One of the major pathways of calcium influx into cardiac myocytes is through T- and L-type voltage-gated Ca2+ channels. T-type, or low voltage-activated (LVA) channels, open after small depolarizations of the membrane. T-type currents have been found in atrial myocytes and pacemaker cells, where they are involved in determining pacemaker activity. Numerous drugs block L-type channels, however, there are no selective blockers of T-type channels. One of the long-term objectives of this research is to provide an assay system that will allow the development of T-type channel blockers. Calcium channels are multisubunit complexes composed of a large, ion-conducting subunit, alpha1, and several accessory subunits(alpha 2delta, beta and gamma). Six genes encoding alpha1 subunits have been cloned. Cloning and expression of channels has allowed their electrophysiological and pharmacological classification. These studies established that all of these alpha1s are high voltage-activated channels. The aim of this project is to clone the cardiac low voltage-activated T- type Ca2+ channel. Exciting preliminary studies have identified a novel Ca2+ channel gene expressed in heart. The hypothesis is that this gene encodes a T-type channel. The specific aims of this project are to: 1) clone the full-length cDNA, 2) suppress the expression of native T-type channels in rat neonatal ventricular myocytes, 3) determine the tissue specific expression of this gene (Northerns and in situ hybridizations, 4) express the cDNA in heterologous expression systems and measure the biophysical properties of the channel (single channel conductance, voltage-dependence), and 5) determine the pharmacological properties of the expressed current. The research design uses recombinant DNA techniques to clone T-type channels from human heart tissue and express the cloned channels in both Xenopus laevis oocytes and HEK-293 cells. Electrophysiological methods are used to study the expressed channel at both the single channel and whole cell level. The hypothesis will then be tested by comparing the properties of the cloned channel to that observed from T-type channels in isolated cells.

DESCRIPTION (provided by applicant): Low threshold calcium (Ca2+) spikes mediated by T-type Ca2+ channels play a key role in neuronal excitability. These channels open after small fluctuations in the neuronal membrane potential, leading to a further depolarization and the opening of other channels, such as voltage-gated sodium (Na +) channels and high voltage-activated Ca 2+ channels, often leading to bursts of neuronal activity. Over-active burst firing of thalamic neurons is thought to trigger not only absence epileptic seizures, but have also been implicated in a wide range of mental disorders characterized by the presence of thalamocortical dysrhythmias. The discovery of three genes encoding T-type channels has led to many breakthroughs in our understanding of their physiology, and the renewal this grant will extend these studies further into their biophysics, pharmacology, and structure-function of T-type channels. One hypothesis to be tested is that mutations in a T channel gene triggers childhood absence epilepsy (CAE). This channelopathy hypothesis will be tested by introducing these mutations into the channel, and measuring how this affects functional activity. Half of the CAE mutations are clustered in a particular region of the channel, so studies will explore its role in channel function. One goal of these studies is to provide the proof of concept that developing a novel T-type channel blocker will produce an effective antiepileptic drug. Such proof might also come from studies on the mechanism of action of new generation antiepileptic drugs that can treat many types of epilepsy and neuropathic pain. Studies will explore the selectivity of these drugs using patch clamp electrophysiology of cells engineered to express human Na + and Ca2+ channels. Novel compounds have been synthesized that block both channels at lower doses than the parent drug, the antiepileptic phenytoin. Therefore a final goal of this grant will be to use computer modeling to design novel compounds. A fluorescence-based assay has been developed that allows for medium throughput screening for active compounds. Lead compounds will be sent to the NIH Anticonvulsant Drug Development Program for in vivo testing. These studies will test the hypothesis that more potent channel blockers are better antiepileptics and the importance of selectivity.

DESCRIPTION: (Applicant's Abstract) Intracellular calcium controls a variety of cellular functions such as contraction, secretion, proliferation, and gene expression. One of the major pathways of calcium influx into cells is through voltage-activated Ca2+ channels. Low voltage-activated, T-type, Ca2+ channels can open after small depolarizations of the plasma membrane, leading to further depolarization of the membrane (pacemaker activity) and changes in intracellular Ca2+. T channels are also thought to play important roles in burst firing and in oscillatory behavior of neurons. Their ability to inactivate and recover quickly over a narrow voltage range are considered key properties of thalamic neurons, allowing them to show distinct firing patterns that correlate with sleep and wakefulness. Many antiepileptic drugs can block these channels in vitro, leading to the hypothesis that abnormal expression of T channels may be involved in epilepsy. Exciting preliminary studies demonstrate the cloning and expression of a new family of alpha1 subunits that encode T-type Ca2+ channels. Cloning of these channels has opened up new areas of research that should identify the physiology of this important class of ion channel. The specific aims of this project are to: 1) characterize the electrophysiological properties of these cloned T-type channels; 2) characterize their pharmacology, in particular their block by divalent cations, antihypertensives, and antiepileptics; 3) investigate structure-function relationships of T-type channels, focusing on inactivation properties; and 4) investigate the subunit structure of these channels, focusing on how Ca channels are regulated by their beta subunits. The research design uses recombinant DNA techniques to clone and modify T-type channels and express the cloned channels in both Xenopus laevis oocytes and HEK-293 cells. Electrophysiological methods are used to study the expressed channel at both the single channel and whole cell level. These studies should provide significant insights into the functional diversity of voltage-activated Ca channels, their pharmacology, and how their structure determines their function in neuronal signaling.

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Voltage-gated calcium channels are an established drug target. Nevertheless, therapeutically useful drugs only target one of the ten members of the Ca2+ channel family, the cardiovascular L-type channel (Cav1.2). Considerable evidence supports the hypothesis that a small molecule blocker of N-type channels (Cav2.2) would be effective in the treatment of chronic pain. N-type channels in sensory neurons mediate calcium influx into presynaptic terminals, thereby triggering neurotransmitter release onto neurons in the dorsal horn of the spinal cord. Knock-out of the gene encoding the a1 subunits of N-type channels (?12.2) diminishes pain sensitivity and reduces the development of neuropathic pain symptoms after spinal nerve ligation. Finally, ziconotide, a peptide toxin that is highly selective for N-type channels, demonstrated safety and efficacy in clinical trials for the treatment of intractable pain in cancer and AIDS patients after intrathecal infusion. These studies establish the proof-of-concept that an orally available small molecule blocker of N-type channels would be a major therapeutic advance for chronic pain. Two major obstacles have hampered the development of such drugs: one, N-type channels mediate neurotransmitter release at many synapses, so a blocker might have many side effects; and two, the lack of a high throughput assay to screen candidate compounds. Recent studies demonstrate that nociceptive neurons express a specific splice variant isoform of ?12.2. Therefore, a selective and state-dependent blocker of this isoform might produce analgesia without side effects. The goal of this grant is to develop stable cell lines of recombinant N-type channels that will be useful in high throughput screening. The cell lines will be tested for channel expression using whole cell clamp electrophysiology, and their usefulness in a screen will be tested using a fluorescent dye assay to measure calcium influx. A final goal is to test whether the N-type channel variants have unique pharmacological profiles. [unreadable] [unreadable] Chronic pain continues to be a major public health problem, affecting 40 million Americans, with little relief from current drugs. By targeting an important protein in the pain pathway, the research funded by this grant will provide tools that can be used to screen candidate compounds during the development of novel analgesics. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): [unreadable] [unreadable] Voltage-gated calcium channels are an established drug target. Nevertheless, therapeutically useful drugs only target one of the ten members of the Ca2+ channel family, the cardiovascular L-type channel (Cav1.2). Considerable evidence supports the hypothesis that a small molecule blocker of N-type channels (Cav2.2) would be effective in the treatment of chronic pain. N-type channels in sensory neurons mediate calcium influx into presynaptic terminals, thereby triggering neurotransmitter release onto neurons in the dorsal horn of the spinal cord. Knock-out of the gene encoding the a1 subunits of N-type channels (?12.2) diminishes pain sensitivity and reduces the development of neuropathic pain symptoms after spinal nerve ligation. Finally, ziconotide, a peptide toxin that is highly selective for N-type channels, demonstrated safety and efficacy in clinical trials for the treatment of intractable pain in cancer and AIDS patients after intrathecal infusion. These studies establish the proof-of-concept that an orally available small molecule blocker of N-type channels would be a major therapeutic advance for chronic pain. Two major obstacles have hampered the development of such drugs: one, N-type channels mediate neurotransmitter release at many synapses, so a blocker might have many side effects; and two, the lack of a high throughput assay to screen candidate compounds. Recent studies demonstrate that nociceptive neurons express a specific splice variant isoform of ?12.2. Therefore, a selective and state-dependent blocker of this isoform might produce analgesia without side effects. The goal of this grant is to develop stable cell lines of recombinant N-type channels that will be useful in high throughput screening. The cell lines will be tested for channel expression using whole cell clamp electrophysiology, and their usefulness in a screen will be tested using a fluorescent dye assay to measure calcium influx. A final goal is to test whether the N-type channel variants have unique pharmacological profiles. [unreadable] [unreadable] Chronic pain continues to be a major public health problem, affecting 40 million Americans, with little relief from current drugs. By targeting an important protein in the pain pathway, the research funded by this grant will provide tools that can be used to screen candidate compounds during the development of novel analgesics. [unreadable] [unreadable] [unreadable]

Significance -- Epilepsy affects about 2.5 million people in the United States. If not properly controlled by antiepileptic drugs, seizure disorders can lead to lower quality of life and loss of productivity. One class of epilepsy is called idiopathic generalized epilepsy (IGE), because the cause is not known (idiopathic) and the seizures can alter activity throughout the brain (generalized). Although this type of epilepsy can be inherited, there appear to be many genes involved (polygenic). Mutations in the gene, CACNA1H, which encodes the Cav3.2 channel, have been discovered in IGE patients. Approach -- This study will test the hypothesis that these epilepsy variants lead to a gain of function, either by altering Cav3.2 channel activity, or by altering its expression in neurons. Cav3.2 channels are a special class of calcium channel that can open near the resting membrane potential of neurons. This calcium entry can affect neurons in two important ways, one to depolarize the membrane and cause the neuron to fire sodium action potentials, and two, to increase calcium within the neuron, a 2nd messenger involved in the activation of many cellular processes such as dendritic arborization. Which of these roles is affected by epilepsy variants? To provide insights into this question, the present grant will address the following specific aims: [1] to establish the mechanisms by which Cav3.2 channels are trafficked in neurons;and [2] determine whether epilepsy variants of Cav3.2 increase dendritic arborization and whether this can be reversed by novel T-channel antagonists. The approaches used to address these important issues include molecular biology, cell biology, fluorescent microscopy, electrophysiology, and pharmacology. Impact -- These studies are likely to impact the field of neuroscience by uncovering novel roles of T- channels in dendritic arborization and neuronal excitability, and by validating novel antagonists. The studies will likely have a clinical impact: showing that T-channel antagonists reverse the effects of Cav3.2 variants on dendritic development, thereby providing the rationale for their early use to cure epilepsy.

DESCRIPTION (provided by applicant): Pain is a leading health problem in the United States with 1 in 10 Americans suffering from moderate to severe pain. Yet, the treatment of chronic pain remains a clinical challenge, with only half of patients receiving adequate pain relief. Therefore, the development of novel analgesics without abuse potential and side effects would have significant impact on the treatment of pain. The hypothesis guiding these exploratory studies is that chronic pain can be treated by inhibiting ion channels in nociceptors such as the Cav3.2 T-type voltage-gated calcium channel. T-currents are upregulated in animal models of chronic constrictive nerve injury and diabetic neuropathy, and Cav3.2 directed antisense oligonucleotides reverse the mechanical hyperalgesia and allodynia observed in these animals. Importantly, Cav3.2 is important for pain signaling in both rodents and humans, since the clinically relevant analgesic, lipoic acid, blocks human Cav3.2 currents in vitro yet is ineffective in Cav3.2 knockout mice in vivo. These studies will develop short hairpin RNAs (shRNA) that inhibit the expression of mouse and human Cav3.2 channels. In contrast to chemically synthesized siRNA, delivery of DNA-based shRNA can be specifically targeted to neuronal subtypes using gene promoters. The present study will explore the use of the sodium channel promoter Scn10a, whose expression is largely limited to nociceptors. An important feature for clinical use is the ability to regulate gene therapy. This will be accomplished using a newly developed version of the tetracycline repressor, which requires drug for activation (doxycycline-ON). Recombinant adeno-associated virus (rAAV) has emerged as the top choice for human gene therapy. AAV particles can be produced with protein coats that effectively infect sensory neurons and are retrogradely transported to the nucleus (e.g. serotype 8). The goal of these studies is to develop rAAV targeting vectors that direct shRNA- mediated knockdown of Cav3.2, use this to prepare viral particles, and then test for nociceptor- specific expression in rats with neuropathic pain. The research team includes Dr. Edward Perez- Reyes, a T-channel expert, Dr. Guanping Gao, an AAV expert, and Dr. Hui-Lin Pan, a neuropathic pain expert. PUBLIC HEALTH RELEVANCE: Project Narrative Chronic pain affects over 10% of the population, resulting in lowered productivity and quality of life. The present proposal is focused on developing a novel treatments of pain that can provide long-term treatment without abuse potential. Disclaimer: Please note that the following critiques were prepared by the reviewers prior to the Study Section meeting and are provided in an essentially unedited form. While there is opportunity for the reviewers to update or revise their written evaluation, based upon the group's discussion, there is no guarantee that individual critiques have been updated subsequent to the discussion at the meeting. Therefore, the critiques may not fully reflect the final opinions of the individual reviewers at the close of group discussion or the final majority opinion of the group. Thus the Resume and Summary of Discussion is the final word on what the reviewers actually considered critical at the meeting.

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 major neurological disorder with significant economic and human burdens. One of the most common forms is mesial temporal lobe epilepsy (TLE). Unfortunately, medical treatment of TLE fails in many cases, leaving a large unmet clinical need. Therefore, a thorough understanding of the neuronal circuits involved in seizure initiation and propagation will drive the development of novel therapies. A hallmark of mesial temporal lobe epilepsy for many patients is hippocampal sclerosis. The hippocampus is particularly susceptible to excitotoxicity. Key sites of neuronal death are the hilus of the dentat gyrus, where many inhibitory GABAergic interneurons are lost. Recurrent seizures trigger an increase in both neurogenesis and the development of remaining neurons. This is particularly true for the dentate gyrus, whose responses include: birth and migration of new granule cells, appearance of novel basal dendrites, and an increase in their axonal projections including excitatory collaterals back onto neighboring granule cells (mossy fiber sprouting). Which of all these responses is most responsible for the development of spontaneous seizures? This proposal will test the hypothesis that reduced activity of inhibitory interneurons is a key driver f epileptogenesis. This hypothesis was developed to explain the serendipitous finding that expression of a modified leak K+ channel (TREK-M) in rat dentate hilar neurons triggered limbic seizures similar to those that occur in TLE patients. The research will use a novel chemogenetic approach based on adeno-associated viral delivery of TREK-M whose expression is dependent on the action of Cre recombinase and regulated by doxycycline. This allows us to exploit Cre-driver mice that have been developed as a result of the NIH Neuroscience Blueprint. The ability of chemogenetic silencing of GABAergic neurons will be first tested in mice where Cre is expressed in all GABAergic interneurons. The first aim will validate delivery, record seizure activity, and examine the pathology that results, focusing on mossy fiber sprouting. The second aim will begin to dissect which of the many GABAergic subtypes are critical for seizure generation. This innovative chemogenetic approach will likely have a major impact on the field of neuroscience, as it provides a longer time scale to probe complex behaviors than is possible with optogenetics. The studies may also provide the first animal model of spontaneous recurrent seizures without neuronal death, which would mimic human TLE patients who do not show hippocampal sclerosis.

There are 3 million Americans with epilepsy. This research aims to develop first-in-class drug- inducible gene therapies for the most common form of focal epilepsy, temporal lobe epilepsy (TLE), which affects half a million Americans. Despite the size of this problem, medical treatment of TLE fails in 35% of these patients, leaving a large unmet clinical need. For over 40 years, the only option left for some of these patients has been surgical resection of the temporal lobe. Although this reduces seizures, it has many problems. One, it is not a cure, so most patients must continue to take antiepileptic drugs with attendant side effects. Two, many patients are unwilling to take the risk of permanent brain damage, such as impaired learning and memory. TLE is one of the best studied forms of epilepsy and there are excellent animal models of TLE that can be used to test novel therapies. The overarching hypothesis of this grant is that by silencing critical neurons in the epileptic circuit with gene therapy, one can effectively stop seizures cold. This approach could be extended to other types of focal seizures, such as: focal cortical dysplasia and epilepsy after traumatic brain injury. Notably, this treatment could be used on patients for whom surgery is not an option due to fear of neurological deficits, those with multiple seizure foci or when the focus is in a critical, or ?eloquent? region of the cortex. To address this critical unmet need, this grant will develop novel gene therapies based on recombinant adeno-associated virus (AAV). Advances in imaging and EEG source localization already allow doctors to localize seizure foci almost to the cellular level. Imagine a future when, rather than cutting, neurosurgeons inject AAV to disrupt the seizure from spreading to the rest of the brain. In fact, magnetic resonance imaging (MRI)-guided delivery of therapeutics has already been shown to be an effective method for targeting specific brain regions. Using funds from the CURE Foundation, preliminary data was collected that show AAV delivery of a modified leak K+ channel (TREK-M) reduces status epilepticus. Importantly, the gene therapy reduced spontaneous recurring seizures by 90% in a rat model of TLE. Aim 1 will compare injection of TREK-M at different sites and study its effect on spontaneous seizures in a chronic TLE rat model. Aim 2 will test the safety of the gene therapy in control animals and whether TREK-M can correct seizure-induced deficits in learning and memory. Aim 3 is a preclinical study to establish the efficacy of the gene therapy on spontaneous recurring seizures in a rat model of TLE. We predict drug-inducible gene therapy will have a large impact on both clinical and basic research.

Project summary The long-term goal of these studies is to develop novel therapeutics for epilepsy patients whose seizures are not well-controlled by current drugs (pharmacoresistant). Many of these patients have a type of focal epilepsy called temporal lobe epilepsy (TLE). There has been little progress in the development of novel therapies for these patients because of the lack of suitable animal models. Current TLE models show much greater neuronal death and hippocampal sclerosis than observed in patients, and a highly variable occurrence of seizures that precludes drug testing. The proposed studies will validate the usefulness of new mouse model of TLE that overcomes these problems. It was discovered that a mild kindling protocol of a specific strain of mice, VGAT- Cre, led to spontaneous seizures. Kindling refers to the process where repeated electrical stimulations eventually trigger tonic-clonic seizures. However, kindling only leads to spontaneous seizures in VGAT-Cre mice. These mice express Cre recombinase under the control of the vesicular GABA transporter (VGAT), a gene that is specifically expressed in GABAergic inhibitory neurons. Loss, or dysfunction, of these neurons in the hippocampus has been linked to the development of temporal lobe epilepsy. The first aim of this study will optimize kindling protocols to generate mice with an ideal frequency of spontaneous seizures for drug testing. The second aim is to validate that these mice respond to currently available antiseizure drugs. This work will be done by Dr. Wilcox?s group at the University of Utah, who also directs the NIH-sponsored Epilepsy Therapy Screening Program.