Timothy Andrew Benke - US grants

Little is understood about the mechanisms developmental epilepsy, i.e., the progression from episodic seizures in early-life to chronic epilepsy in adulthood. This progression likely causes childhood learning impairment that could be prevented by a better understanding of the process. A mechanistic connection exists between the expression of an in vitro correlate of learning, long-term depression (LTD), and the consequences of epileptogenesis: LTD induces a near complete removal of a specific GluR subunit (GluR2) from synapses by an N-ethyl-maleimide-sensitive fusion protein (NSF) -dependent mechanism while epileptiform discharges in vitro can produce LTD and epileptogenesis leads to impaired LTD. I hypothesize that these two processes, the expression of LTD and epileptogenesis, saturate the same NSF-GIuR2 interaction that decreases the number of synaptic GIuR2 receptors and results in chronic LTD impairment. First, the developmental progression of LTD in hippocampal slices from control and epileptic rats will be studied using field-recording techniques. Developmental epileptogenesis will be induced with the infra-hippocampal tetanus toxin injection method. It is hypothesized that epileptic rats will develop a progressive impairment in the expression of hippocampal LTD. Second, the developmental interaction of GluRs with NSF will be studied using whole-cell patch-clamp recordings of GluR mediated synaptic currents along with pharmacological agents that modulate the G1uR2-NSF interaction in slice preparations from epileptic and control rats at different ages. It is hypothesized that epileptogenesis will saturate and thus occlude further pharmacological disruption of the GluR2-NSF interaction. Lastly, the impairment of LTD caused by developmental epileptogenesis could alternatively be the result of alterations in the single-channel and/or kinetic properties of GluRs. It is hypothesized that G1uR single- channel conductance and kinetics in epileptic rats, measured by whole- dendrite patch-clamp recordings of GluR mediated synaptic currents and non-stationary noise analysis, will be similar to values measured in control rats.

DESCRIPTION (provided by applicant): Clinical evidence demonstrates that seizures in infants and children cause long-term learning disabilities. However in laboratory studies, the proposed mechanisms for this are inconsistent, partly due to different experimental paradigms. Synaptic properties mediating learning and memory (long-term potentiation (LTP) and long-term depression (LTD)) change with development. These processes involve alterations in glutamate receptor (GluR) subunits that are triggered by specific NMDA receptor (NR) subunits. Our behavioral data demonstrate that a single episode of early-life seizures (ELS) in rats causes later learning disability. This occurs without alteration of neuronal morphology and connections. In vitro studies show alterations in hippocampal LTP and LTD that are consistent with abnormal regulation of glutamate receptors. Contrary to prior biochemical studies suggesting isolated down-regulation of GluR2, our electrophysiological data suggests synaptic GluR1 is transiently up-regulated shortly after ELS but later held in internal pools. Total expression of NR2A becomes permanently decreased. We hypothesize that the transient up-regulation of GluR1 consolidates an abnormal developmental trajectory. This trajectory leads to further abnormal regulation of GluR1 and NR2A that subsequently mediates the abnormal LTP and LTD and the learning disability that we observe following ELS. Abnormal expression of sub-synaptic machinery regulates these alterations in GluR1 and NR2A and hard-wires the long term effects. Currently, there are no therapeutic interventions clinically available for this common, debilitating and costly condition. Thorough characterization of both what makes immature excitatory synapses vulnerable to early-life seizures and the resulting long lasting changes will provide valuable insight into this deficiency as well as the neurobiology of developing synapses. To test our hypothesis and resolve contrary observations we propose three Specific Aims involving in vitro electrophysiological and biochemical studies in rats: Specific Aim 1 (SA1): Expression of homomeric GluR1 receptors early in development makes CA1 hippocampal synapses vulnerable to ELS. Specifc Aim 2 (SA2): ELS causes a rapid, persistent alteration of expression of GluR1 and NR2A receptors that is contrary to normal development. Specific Aim 3 (SA3): The persistent alteration of expression of GluR1 and NR2A receptors following ELS is mediated by altered expression of sub-synaptic machinery. These Aims will provide the evidence necessary to support future R01-funded mechanistic studies that address pharmacological interventions that could prevent the effects of ELS on learning impairment.

DESCRIPTION (provided by applicant): We seek to ultimately understand 3 basic questions: (1) Do early-life seizures (ELS) trigger intellectual disability (ID) with an autistic phenotype? (2) What signaling programs triggered by ELS potentially underlie this phenotype? (3) What are the rational, long-term pharmacological treatments to improve the abnormalities in synaptic plasticity and the in vivo phenotype? Our preliminary studies provide electrophysiological, pharmacological and biochemical evidence to suggest that ELS induces a chronic phenotype similar to other genetic forms of ID and autism such as Fragile X (FRAX) with FMRP dysfunction and Tuberous Sclerosis (TSC) with mTOR dysfunction. We propose three specific aims, utilizing electrophysiological, biochemical, immuno-cytochemistry and behavioral studies. Studies will use adult rats following a single kainate-induced ELS at post-natal day (P) 7. These studies will further investigate the mechanisms underlying altered mGluR-LTD measured electrophysiologically and test the hypothesis that ELS leads to a phenotype similar to other genetic forms of autism. Comparisons will be made to age-matched saline injected controls. Specific Aim 1: Determine the mechanisms underlying enhanced mGluR-dependent LTD observed following ELS. This will test the hypothesis that mGluR-dependent LTD is altered following ELS similar to that mediated by genetically disrupted FMRP expression and can be modified pharmacologically in a similar fashion. Specific Aim 2: Characterize the signaling pathways associated with S6 kinase (SK1) hyperactivation following ELS. This will test the hypothesis that signaling pathways are altered following ELS in a similar fashion to that mediated by disrupted FMRP expression and hyperactive mTOR, as in FRAX and TSC, respectively. Specific Aim 3: Further characterize the behavioral and electrographic in vivo phenotype following ELS. This will test the hypothesis that behavioral modalities beyond abnormal fear conditioning are induced by ELS that are consistent with an autism-like phenotype. By determining the pharmacological modulators of enhanced LTD following ELS, we will determine if this alteration in plasticity shares key features with that associated with FRAX and TSC. The pharmacological interventions advanced in FRAX and TSC are mirrored by our studies proposed here. Our studies will inform whether other forms of ID and autism potentially may share their pharmacological sensitivity and determine other potential therapeutic targets. These studies will advance the novel hypothesis that FMRP dysfunction can be seen in ID and/or autism outside of FRAX. This could impact treatment strategies for all causes of ID and/or autism. Our findings will support our hypothesis that seizures, as an external environmental factor, directly influence the development of ID and/or an autistic phenotype. These hypotheses cannot be tested in a straightforward fashion in patients. The use of our novel animal model of ELS triggering ID with an autistic phenotype allows these elusive clinical questions to be addressed more directly.

DESCRIPTION (provided by applicant): In response to RFA-NS-11-008, we are proposing to develop the Rocky Mountain Network for Neuroscience Clinical Studies (RMNNCS). This program will reside at the University Of Colorado School Of Medicine within the Departments of Adult and Child Neurology, and will be executed between three institutions: University of Colorado School of Medicine, University of Colorado Hospital and The Children's Hospital. The Program Director will be Dr. Timothy Vollmer, M.D., Professor of Neurology, with Co-Program Director Dr. Paul Levisohn M.D., Associate Professor of Child Neurology. Co-Investigators will include numerous faculty members with experience in human research in adult and child neurology neuroimaging, and neurosurgery. Co-investigators include specialists in the fields of Cerebrovascular Disease, Movement Disorders, Epilepsy, Behavioral/Cognitive Neurology, Neurogenetics, Neuroimmunology, Neuroopthalmology and Neuromuscular Disease in both adult and pediatric patients. In conjunction with the RMNNCS, we propose to develop the Rocky Mountain Alliance for Neuroscience Clinical Studies (RMANCS). This will be collaboration between the RMNNCS, community-based neurologists and community-based patient advocacy groups focused on neurological disease from throughout the Rocky Mountain region. This will further enhance RMNNCS' ability to reach rare patient populations for NEXT studies and lay the foundation for identifying new clinical research concepts for the NEXT Centers of Excellence.

DESCRIPTION (provided by applicant): Advancing the care of children with nervous system disorders is critically dependent on training the next generation of academic pediatric neurologists to be both outstanding clinicians and successful independent basic and translational researchers who can work effectively in multidisciplinary research teams. The Colorado Neurosciences Academic Development Award (NSADA K12) program based at the University of Colorado Anschutz Medical Campus and Children's Hospital Colorado will leverage the combined resources of an outstanding neuroscience research community, our extensive university infrastructure for research training and one of the best children's hospitals in the country to provide exceptional basic-translational pediatric neuroscience research training for the next generation of academic pediatric neurologists. The overall goal is to provide our Scholars the scientific and professional skills needed for productive academic research careers as independent investigators and leaders who will make significant impacts in Child Neurology research. We will meet this goal with two aims: 1) provide the highest quality, personally designed training and career development opportunities focused in basic and translational science research; 2) provide training experiences that will educate the Scholars in multidisciplinary, integrated, team science required for translation of basic discoveries into opportunities to help solve and prevent major unmet problems in Child Neurology. To accomplish these aims, we have engaged outstanding research faculty in basic and translational science that have highly successful research and research training programs with proven track records of excellence to provide our scholars with optimal research training and career development. During this 5 year program we will recruit 3 Scholars from graduating pediatric neurology fellows and junior faculty (Instructors or Assistant Professors) who have strong academic records, are motivated and committed to establishing independent research careers, and have demonstrated evidence of research potential, and provide each of them with 3 years of uninterrupted research time. We will create a leadership and training program structure that will establish: a) criteria and processes for recruitment of scholars, research mentors, and individual mentorship teams; b) required research and career development training; c) evaluation of all aspects of the program. The Scholars will be exposed to a broad array of research and career development opportunities, and will learn the mechanisms involved in multidisciplinary, collaborative team science. We are committed to preparing the next generation of independent Pediatric Neurology/Neuroscience investigators to discover mechanisms of disease and effectively translate these discoveries into improvements in care for children with neurological disease.

Core C ? Behavioral and In Vivo Neurophysiology Core (BINC) Summary The Behavioral and In Vivo Neurophysiology Core (BINC) will provide appropriate facilities and educational and consulting services that allow Rocky Mountain Neurological Disorders Center Core (RMNDC) investigators to perform behavioral and in vivo neurophysiological phenotyping of rodent models. Behavioral phenotyping, through structured established and novel tasks, is necessary for all aspects of basic and translational neuroscience research. Behavioral testing allows detailed characterization of motor, sensory, emotional, social and cognitive output of brain circuits that can be correlated to investigator initiated manipulations. Similarly, video monitoring coupled with electroencephalography (EEG) /local field potentials (LFP) characteristics can be used to determine disease status, to understand the mechanisms of co-morbidity that can occur following an injury and to track the evolution of disease pathology after an initial insult. The ability to link findings from the subcellular level (e.g. Core B) to the output of brain circuits (e.g. Core A) through behavioral and neurophysiological characterization is both exciting and highly relevant to the mission of NINDS.

ABSTRACT: ?Exploratory determination of the role of L-type calcium channels in mediating abnormal plasticity and behavior after early life seizures? Pediatric epileptic encephalopathy (P-EE) syndromes, characterized by frequent early-life seizures (ELS), are associated with catastrophic neurocognitive impairment including autism spectrum disorder and intellectual disability (ASD/ID). P-EE can be associated with injury or genetic causes. Currently, clinical opinions by leaders vary widely in the field, with some believing that ELS adds insult to injury and favoring the aggressive treatment of ELS. For most of our patients, ?the cow is already out of the barn?: ELS has already happened and there are no therapies to address the long-term consequences of ELS itself. Therefore, there is an urgent need to investigate these issues with rodent models: 1) What are the mechanisms? 2) Can this be treated? We hypothesize that hyperactive L-type calcium channels (LTCCs) play a substantial role to mediate abnormal plasticity and behavior in P-EE. Studies in CA1 hippocampal slices in vitro will pharmacologically compare mechanistic changes in synaptic plasticity (SA1) mediated by LTCCs that have developed chronically (P60+) after ELS. Behavioral studies (SA2) will determine the neurocognitive benefit of an LTCC antagonist after ELS. Our rationale is that these studies will segue to mechanistically inspired, novel therapeutics for neurocognitive deficits associated with ELS where none exist that could provide significant societal benefit. Specific Aim 1 (SA1): Mechanistically link KA-ELS-mediated LTCC dysfunction to disruption of key LTCC protein-protein interactions that impact mGluR-LTD. Specific Aim 2 (SA2): Determine whether chronic LTCC antagonism ameliorates neurocognitive impairment after KA-ELS. These exploratory studies will lay the groundwork to segue into comprehensive studies investigating the mechanistic roles of LTCC in mediating neurocognitive deficits in P-EE with ELS. By understanding mechanistically how LTCC modulate plasticity after ELS (SA1) and determining whether broad-spectrum LTCC inhibition improves neurocognition (SA2), we can then advance LTCC-related neurotherapeutic strategies for application to genetic models of P-EE with ELS, to be characterized in future studies. These studies are essential, as genetic or other protein replacement strategies in P-EE may be insufficient to correct the long-term consequences of ELS.

Pathological mutations in cyclin-dependent kinase-like 5 (CDKL5) result in CDKL5 deficiency disorder (CDD, OMIM 300203, 300672), a relatively common genetic cause of early-life epilepsy. Recent analysis of genetic variants in CDD have indicated that CDKL5 kinase function is central to disease pathology. In other words, loss of kinase function seems equivalent to loss of the protein. Big gaps are present in rodent models of CDD. A key feature is missing in these models: severe epilepsy. Specific inhibitors of CDKL5 are essential for addressing the role of CDKL5 without confounding chronic effects of genetic CDKL5 deletion. A key waypoint in the path to address this question is knowledge of specific CDKL5 substrates. Our collaborator, Dr. Sila Ultanir, has determined that microtubule end-binding protein 2 (EB2) is a specific CDKL5 substrate and has developed a phoshospecific antibody. With this tool in hand, our immediate goal in this pilot project is to use this read-out of CDKL5 activity to identify sensitive and specific CDKL5 inhibitors in brain tissues. Our collaborators, Dr. Axtman and Ms. Wells, have identified additional compounds. We will use these inhibitors to address our hypothesis that the acute blockade of CDKL5 activity has unique effects on hippocampal signaling and function compared to chronic knock-out through 2 aims: 1) Identify sensitive and specific inhibitors of CDKL5 and 2) Determine effect of acute inhibition of CDKL5 on hippocampal synaptic function and plasticity. Two developmental time points will be studied. Overall Impact: Our goal in this pilot project is to identify a CDKL5 inhibitor. This will allow us to bypass the gaps in rodent models of human CDD. In future studies, we hypothesize that specific CDKL5 kinase inhibitors will be necessary to fully probe and understand CDKL5 neuronal function throughout development. Since CDD is a strong candidate for gene therapy, an important clinical question is whether gene replacement of CDKL5 later in life improves symptoms. Our future experiments are necessary to determine whether such therapy may have an optimal developmental therapeutic window. Further, it may reveal that some symptoms of CDD do not depend on CDKL5 function later in life, as these were triggered developmentally, and will require alternative interventions beyond gene replacement.

Pathogenic variants in the Cyclin-dependent kinase like 5 (CDKL5) gene cause CDKL5 deficiency disorder (CDD, MIM 300672, 105830) a severe developmental and epileptic encephalopathy (DEE) associated with cognitive and motor dysfunction and cortical visual impairment. Recent data suggest CDD is one of the most common genetic causes of DEE. Work in CDD animal models has demonstrated the ability for disease modification and symptom reversal: worldwide efforts are now underway to develop therapeutic strategies (including gene therapy) to treat and potentially cure CDD. While there are four active clinical trials, none assesses the full spectrum of this DEE to address true disease modification. While capability for disease modifying therapies is accelerating, there is a critical barrier for clinical trial readiness that may result in failure of these therapies, not due to lack of efficacy but due to lack of validated outcome measures. CDD has been associated historically with Rett syndrome but there are many clear distinctions and CDD has emerged as an independent disorder. Some Clinical Outcome Measures (COMs) can be adapted from Rett syndrome COMs, whereas others need to be developed specifically for CDD. Our research network is uniquely positioned to develop clinical trial readiness for CDD by pairing exceptional experience in the development and validation of outcome measures with an extensive network of CDD experts and clinical trialists. Our goals are to 1) refine and validate appropriate quantitative COMs and biomarkers and 2) conduct a multi-site clinical trial readiness study to ensure that they can be successfully implemented. We will test the hypothesis that CDD specific COMs can be refined to accurately and reproducibly track meaningful changes in clinical trials: Aim 1: Generate and validate a suite of COMs and biomarkers necessary to comprehensively assess disease modification in CDD. Aim 2: Conduct a multi-site clinical trial readiness study to assess implementation, longitudinal stability, and collect baseline COMs and EEG/evoked potential data. Overall Impact: These outcome measures will establish clinical trial readiness for CDD and generate historic baseline outcome data, ensuring optimal testing of potential new therapeutics including gene therapy. Furthermore, these measures will be adaptable to other DEEs by enabling choices of outcome measures beyond existing NINDS supported measurement tools (NeuroQoL, PROMIS, Toolbox) that are not designed for the severity of the DEE populations.