Gary W. Mathern - US grants

Human hippocampal epilepsy is one of the most difficult epileptic syndromes to control. These intractable and often severe seizures are associated with electrophysiologic hyperexcitability, severe Ammon's horn neuron loss, and evidence of aberrant axonal plasticity, termed hippocampal sclerosis (HS). The etiology of HS is unknown, and it is likewise unclear if HS is the pathophysiologic substrate of chronic epilepsy or the pathologic consequence of multiple seizures. A unique developmental feature of the hippocampus is the postnatal neurogenesis, migration and axon formation of granule cells (GC). The GCs are the parent neurons for mossy fibers (MF), one of the principal aberrantly sprouted axon systems in HS. We recently have had the unique opportunity to study surgically resected hippocampi from epileptic children to discern the evolution of pathologic changes as they relate to seizures and postnatal GC maturation. Preliminary results show evidence of aberrant MF sprouting at the time of postnatal GC development. The size of the MF fibers and boutons increased in older seizure patients, suggesting maturation of these axons. Seizure associated regio inferior neuron loss was found in all epileptic children, but greater neuron loss was found after age 2 years, when the GCs had nearly completed neurogenesis. This proposal is designed to further study hippocampi from epileptic children and will determine the following specific aims. l) Determine the progressive pathologic changes in the hippocampus that might suggest an etiology of adult HS. Our two hypotheses are that adult HS is either the consequence of progressive seizure related pathologic changes or it is a result of some severe perinatal insult that is associated with structural damage. 2) Determine the time course of postnatal hippocampal development. We will look for the in vivo expression of proteins associated with neurogenesis, migration, and axon growth cone formation. Our hypothesis is that postnatal hilar cells and GCs will differentially express these proteins compared with the remaining hippocampus which forms prenatally. 3) Determine the postnatal differential expression of neurotrophic factors (NTF) in the developing hippocampus. Our hypothesis is that mRNA NTF expression is altered in epileptic hippocampi in a manner that promotes and maintains aberrant sprouting during postnatal axon development. Age- matched autopsy hippocampi without evidence of brain pathology will serve as controls. Parallel developmental studies will be performed in rat pups to determine if postnatal hippocampal maturation is different in another mammalian species. These studies on epileptic children form a data base that will be expanded upon in future human and animal experiments to study the cellular mechanisms and pathophysiology of HS. A CIDA is also a training grant. The most significant component of the training will be in acquiring research skills that will complement my existing clinical knowledge and training in Neurosurgery. The research program contains a didactic exposure to basic Neuroscience graduate courses, hands on technical and theoretical exposure to molecular neurobiology, teaching, and close supervision by senior neuroscientists. The goal is sufficient research training and exposure to become an independent clinical investigator familiar with basic research applied to clinical epilepsy related problems.

In the mesial temporal lobe epilepsy syndrome (MTLE), 65% of surgical specimens show hippocampal sclerosis which consists of severe neuronal loss, and aberrant axon sprouting. We hypothesized that it is the reorganized inhibition on surviving neurons. Also, recent human studies indicate that sclerosis is associated with changes in: 1) NMDA and AMPA mRNA subunits; and 2) GABA and glutamate and transporters. Unfortunately, one problem with human studies is that the pathology is final and static, and it is difficult to discern the "cause" of the epilepsy from the "consequence" of repeated seizures without time course studies in animal models. This research project is designed to understand the pathophysiology of human hippocampal sclerosis by studying time course changes in animal models of spontaneous limbic epilepsy. We will: 1) Determine, using in situ hybridization techniques and immunocytochemistry, the time course and evolution of NMDA R1-2a-d and AMPA GluR1-3 receptor subunits, associated with spontaneous seizures in three chronic models of limbic epilepsy (intra-hippocampal kainate, systemic pilocarpine, and self-sustained limbic status epilepticus); and 2) in the same rat models, determine the time course and evolution of glutamate (EAAC1; GLT-1, and GLAST) and GABA transporters (GAT-1; and GAT4). The chronic animal models will be compared to kindled animals and our recent human studies, and the results correlated to hippocampal neuron loss and signals of aberrant axon sprouting. We hypothesize that compare to controls and kindled rats, animals with spontaneous limbic epilepsy will show increased hippocampal AMPA and NMDA subunits; 2) decreased astroglial glutamate and GABA transporters; and 3) increased in neuronal EAAC1 transporters. An increase in AMPA and NMDA receptors could lead to neuronal hyperexcitability, changes in GABA and glutamate transporters would raise or decrease intra- synaptic and extracellular GABA and glutamate levels, and these factors interacting with aberrant axon sprouting could generate mesial limbic seizures. These Specific Aims will be accomplished by performing multiple histologic techniques on the same rats that have been intensively monitored for spontaneous seizures, and the resulting data will provide an important understanding of the molecular pathophysiology of hippocampal epilepsy, suggest pathogenic mechanisms of limbic seizures, and provide insights into possible ways of controlling the chronic seizures.

Resective neurosurgery is a frequent treatment for children with therapy-resistant epilepsy. About half of surgical cases have cortical dysplasia (CD), consisting of cortical dyslamination, heterotopic neurons, and dysmorphic cytomegalic neurons and balloon cells. The others have non-CD pathologies such as infarcts and Rasmussen syndrome. The goals of this research project are to examine the electrophysiologic and anatomic properties of cells in pediatric CD tissue to discern mechanisms that lead to epileptogenesis. In the previous funding period, we found that cytomegalic neurons displayed increased voltage-gated calcium currents, and cytomegalic neurons and about 30% of pyramidal neurons showed decreased Mg++ sensitive NMDA receptors similar to immature cortex. Balloon cells did not display active membrane properties or synaptic activity. In Preliminary studies, we also found that severe CD has other immature features. These include more GABA than glutamate spontaneous synaptic currents and terminals, greater layer 1 evoked GABA-mediated currents, GABA-A receptors with depolarized reversal potentials, longer GABA-A receptor decay time constants, and more interneurons than expected including recently discovered cytomegalic GABA neurons. These findings lead us to hypothesize that severe CD consists of a proportion of cells that retain immature GABA signaling properties interacting with normal mature-like pyramidal cells to produce "pro-epileptic" conditions and seizures. This renewal will address this hypothesis by determining in severe CD if: 1) Synaptic signaling is similar to immature cortex with GABA (not glutamate) as the predominant neurotransmitter;2) GABAA receptors on cytomegalic and some pyramidal neurons show immature characteristics, such as depolarized reversal potentials;3) Enhancement of GABA function from GABA altering medications are excitatory and "pro-epileptic";and 4) Interneurons display immature characteristics associated with spontaneous rhythmic "pacemaker" GABA activity on pyramidal neurons. The results of these experiments will discern developmental pathologic mechanisms of epileptogenesis associated with CD that cannot be obtained from animal CD models, and begin translational research studies that will provide insight into rational pharmacological treatments for pediatric CD patients with epilepsy.

DESCRIPTION (provided by applicant): In post-hemispherectomy children for seizure control, this proposal will examine motor and gait functions pre- and post- Body Weight Support Treadmill (BWST) training, and correlate changes from BWST training with functional MRI (fMRI) cortical activation from voluntary ankle movements. Our goals are to understand functional plasticity in the developing human brain by determining if age at injury/surgery, from specific pathologies, predicts baseline motor/gait functions and response to BWST training that can be used to design rehabilitation programs, using fMRI, that improve quality-of-life (QOL) for brain injured children. We hypothesize that injury/surgery from birth to 2 years maintains the developmental regulated ipsilateral corticospinal tract (ICS) that innervates ipsilateral muscles from primary motor/sensory (M1S1) cortex. By comparison, later injury/surgery, where the ICS tract has partially developmental regressed, will result in poorer functional recovery, especially for distal ankle/foot functions affecting walking. Based on Preliminary Data obtained from a Pilot Study we propose to determine if: 1) Age at injury/surgery related to pathology predicts motor and gait deficits pre- and post-hemispherectomy for proximal and distal muscles of the upper and lower limbs; 2) BWST training improves motor function and walking measures, if the changes are better in children with an early age at lesion/surgery, and if training induced walking and motor improvements are associated with better QOL measures; and 3) post-BWST gains of motor and walking function correlate with and increase in fMRI activation of M1S1 and cingulate cortex with ipsilateral and contralateral foot dorsiflexion consistent with cortical plasticity involving preservation of the ICS tract. The results of this research will have a conceptual impact regarding concepts of cortical plasticity in the developing human brain: and has practical application for neurorehabilitation in children post-hemispherectomy and for those with other forms of early developmental cerebral injury, such as trauma, hemorrhage, and tumors.

DESCRIPTION (provided by applicant): Epilepsy is a frequent neurological disorder, and 25% to 30% have therapy-resistant epilepsy. Of those with uncontrolled epilepsy, some may be candidates for epilepsy neurosurgery if the EEG can be localized to a single site. Scalp EEG has limitations with regard to source localization because of spatial non-uniformity of the brain's electrical conductivity. The goals of this project are to improve our understanding of the brain micro-structure that effect EEG source localization by integrating different research techniques in epilepsy surgery patients. In Preliminary Studies, we demonstrate that cortical electrical conductivities are directionally specific and depend on the type of pathology. Also, there is a relationship between measures of brain electrical conductivity and brain water diffusivity measured by Diffusion Tensor Imaging (DTI) MRI. These findings support our goal of measuring brain electrical conductivity and DTI, and relating these to altered brain structure as steps toward improving methods of EEG source localization. We will accomplish our goals by: 1) Measuring the electrical conductivity of cortical gray matter and subcortical white matter parallel and perpendicular to the pial surface;2) validate that ex vivo measures of brain electrical conductivity replicate in vivo conditions by performing in vivo and ex vivo experiments in animal models of normal and abnormal brains;3) use presurgical DTI to determine the water diffusion tensor and the associated principal diffusivities (eigenvalues), principal directions (eigenvectors) and FA from the gyri and cortical sites on which the electrical conductivity measures are performed;3) determine histopathologic measures of cortical disorganization in sites adjacent to those used for the electrical conductivity and DTI measurements;and 4) model EEG interictal discharges from scalp EEG and MEG/MSI using models that incorporate measures of isotrophic brain from electrical conductivity and DTI and determine if these new methods more closely determine EEG sources as determined by ECoG. The results of these experiments will provide the necessary Preliminary data for an R01 application to develop methods of intracranial EEG source localization that will be individual patients. PUBLIC HEALTH RELEVANCE: One-third of epilepsy patients have seizures that are not controlled by drugs, and are at risk for seizure- induced death and mental retardation. Surgery is an option to treat uncontrolled seizures, but less than half are candidates because of limitations of EEG and MRI techniques. This grant will improve these tools by studying if electrical signals move through the brain differently in epilepsy patients, whether electrical signal changes can be estimated using new MRI techniques, and if the electrical and MRI changes are from disorganization of the cerebral cortex.

DESCRIPTION (provided by applicant): Molecular characterization of hemimegalencephaly Abstract Somatic mutations, in which a fraction of the cells in the body have a deleterious mutation, is well recognized in cancer but only recently appreciated in neurological disease. In the setting of a somatic mutation in a population of progenitor cells, all daughter cells inherit te mutation and are able to express the resultant phenotype as a function of the differentiation program. We recently identified the first de novo somatic mutations in the developing brain in the condition hemimegalencephaly, (HME) a catastrophic focal epilepsy condition associated with a malformation of cerebral cortical development (MCD). HME is one of the most severe MCD syndromes, characterized by massive hamartomatous overgrowth of either of the two cerebral hemispheres. Cerebral hemispherectomy is a frequent treatment for the refractory epilepsy, allowing sampling of diseased tissue. By comparing DNA from diseased brain with DNA from blood/saliva, we identified de novo somatic mutations in PIK3CA, AKT3 or MTOR, part of the mTOR pathway. Mutations were present in 8-40% of sequenced alleles in various brain regions sampled during surgery, and some in codons known to activate the protein. However, the pilot study was based on a limited sample size. The goal of this application is to expand upon our initial findings, and elucidate the genetic, developmental, signaling and cell biological mechanisms of HME, particularly in the context of mammalian cortex development. We will combine next-generation sequencing of diseased brain from HME patients with advanced bioinformatics, complete clinical correlated neuroanatomy, and mouse modeling to help advance our understanding of the mechanism of this important disease. We will: 1] Test for de novo somatic mutations in a larger retrospectively and prospectively collected cohort of HME patients. 2] Correlate genetic disease burden with clinical, imaging, and histopathological findings (phenotype). 3] Test how these de novo mutations alter progenitor cell functions in the developing cerebral cortex. The goal of the experiments is to determine how mutations in these genes lead to disrupted cortical development, why these lesions are epileptogenic, and whether repurposing approved medications might benefit patients, with relevance to other focal dysplasias and focal epilepsies.

PROJECT SUMMARY In children with drug-resistant epilepsy, resective brain surgery may be the only option to obtain seizure freedom but leads to permanent deficits when the zone of resection involves eloquent brain. There is a clear need to find alternative ways to treat children with intractable epilepsy. Controlling brain inflammation may be one important therapeutic strategy. We and others have found activated T cells and other peripheral immune cells in brain tissue removed to control seizures even in cases where an underlying immune disorder is not suspected. We have also identified specific T cell subsets in the blood of patients undergoing resective surgery that are indicative of an active immune response. We hypothesize that recurrent seizures could cause a ?sterile? infection in which activated T cells and other nonresident immune cells are recruited to sites of seizure-induced inflammation. Adjunctive treatment with drugs that block the recruitment of pro-inflammatory peripheral lymphoid and myeloid cells may therefore be therapeutically beneficial. To better understand the involvement of cellular immunity in intractable pediatric epilepsy we will use mass cytometry and a panel of antibodies to distinguish between T cell, innate lymphoid, and myeloid subsets in CD45+ immune cells isolated from resected brain tissue, and blood collected at the time of surgery from a cohort of pediatric epilepsy surgery patients and following their recovery from surgery at 12 weeks, when the effects of the surgery on clinical status are assessed. We will also measure the diversity of ?? and ?? T cells in resected brain tissue and peripheral blood by sequencing the third complementarity regions (CDR3) of rearranged T cell receptor (TCR) ? and ? genes. Showing that there are only a limited number of different ?? and ?? T cell clones in the brain tissue would support the idea of a specific adaptive response. We hypothesize that removal of the epileptogenic area of the brain with a commensurate improvement in seizure status would resolve the sterile infection, and result in a reduction in the frequency of T cell subsets in the blood associated with an active immune response. A decrease in the frequency T cell clones in the peripheral circulation that were originally found in the brain would also be expected.