Susan A. Masino - US grants

DESCRIPTION(From applicant's abstract): In his long-standing interests in determining neurobiological mechanisms of adenosine, the principal investigator has constructed a novel hypothesis that release of intracellular adenosine results not only from an increase in intracellular calcium levels but also from an acidification of intracellular milieu, as measured by an decrease in intracellular pH values. This proposal will predominately use the hippocampal slice preparation to explore various aspects of adenosine neurobiology from an electrophysiological point of view. The first aim will explore the role of intracellular pH and calcium in mediating adenosine release. These studies will explore whether changes in intracellular calcium and pH are both necessary and sufficient to produce adenosine release by using a variety of reagents which affect the intracellular milieu. The second specific aim explore whether adenosine plays an important role in regulating the basic excitability of neurons by performing experiments in adenosine A1 receptor-knockout mice. These studies will determine whether hippocampal slices from KO mice are hyperexcitable, and whether KO of adenosine A1 receptors affect the function of other inhibitory receptors.

[unreadable] DESCRIPTION (provided by applicant): Adenosine is found throughout the nervous system, and its interaction with specific adenosine receptors modulates ongoing neuronal activity in many brain regions. As the core molecule of adenosine triphosphate (ATP), and a component of the overall cellular energy charge, adenosine provides a unique cellular link between energy demand and brain function. The specific inhibitory influence of the adenosine A1 receptor subtype reduces activity and helps neurons survive a short-term compromised energy supply, such as during insufficient oxygen or glucose. Its modulation of physiology and protection against pathology make adenosine a coveted therapeutic target for disorders as diverse as pain, Parkinson's disease, stroke, epilepsy, and sleep disorders. Accumulated research shows that adenosine levels are altered significantly by changes in temperature, pH, oxygen and glucose. The objective of this proposal is to elucidate the relationship between adenosine and these physiologically-relevant variables to inform and benefit both basic research and clinical conditions. The specific aims outlined herein quantify the influence of adenosine across the typical range of recording temperatures in the hippocampal slice, and systematically examine the effect(s) of reducing available oxygen and/or glucose using a combination of electrophysiology, pharmacology and genetically modified mice. Beyond the clinical significance, the proposed studies are relevant to understanding features of the model systems we use in neuroscience, and they represent a succinct set of fundamental research experiments useful as a training platform for young scientists. Ultimately, understanding the regulation of endogenous adenosine, its influence on widely used model systems, and its functional consequences during pathological conditions will enable us to explore and enhance its therapeutic possibilities in situations as diverse as stroke, epilepsy and spinal cord injury. [unreadable] [unreadable]

Changes in brain cell energy affect activity throughout the brain, but the exact mechanisms that link brain energy metabolism with brain activity are not well understood. Nevertheless, the relationship between brain energy and brain activity is central to understanding how brain function is regulated and how metabolism influences ongoing behaviors such as sleep, sensory processing, learning and memory, and cognition. ATP and its core molecule adenosine provide a unique link between energy and brain activity, and may play a key role in translating changes in cell energy directly to changes in brain activity.

The investigators will test the hypothesis that ATP and adenosine link brain energy to brain activity both at the cellular level and the behavioral level by performing a combination of electrophysiological recordings of brain activity, measurements of ATP and adenosine using an enzyme-based sensor, and behavioral testing. These experiments will provide new insight into the evolutionarily important relationship between brain energy and brain activity. In addition, the investigators will mentor undergraduate students as research partners both during the academic year as part of their academic courseload and support them in doing research full-time during the summer. Undergraduate students will be involved in all aspects of this interdisciplinary proposal, participate in weekly laboratory meetings and attend and present research at several local, regional and national conferences.

Taken together, this project is an ideal use of both research monies and tax dollars: it offers innovative and important research on the relationship between brain metabolism and brain activity, and provides a comprehensive year-round educational and training environment and individualized mentoring for a diverse set of undergraduate students aimed toward a career in scientific research.

DESCRIPTION (provided by applicant): A high-fat, low-carbohydrate (ketogenic) diet is an effective treatment for pediatric epilepsy. Though clinical use of this diet is well established and growing, its mechanism(s) of action remain putative, and, more importantly, cognitive effects of this diet are poorly understood. Published hypotheses suggest that clinical effects of a ketogenic diet stem from increased brain mitochondrial bioenergetics and/or inhibitory neurotransmitters or neuromodulators, including GABA and adenosine. Enhanced central inhibition would be predicted to influence normal synaptic plasticity and alter cognitive/behavioral outcomes, and the magnitude of diet-induced synaptic and bioenergetic changes may differ with age. In both juvenile and adult rats, we propose to quantify mitochondrial function in a variety of brain areas and determine the regional selectivity of ketogenic diet-related bioenergetic changes (Specific Aim 1), and to investigate synaptic transmission in one selected brain region (the hippocampus) in awake freely-moving rats and determine if synaptic plasticity is affected by the diet (Specific Aim 2). Our hypothesis is that the ketogenic diet will cause regional changes in mitochondrial function in brain areas critical for seizures, and will decrease the magnitude of synaptic plasticity measured in the in vivo hippocampus. Our Preliminary Data support our hypothesis by showing strikingly diminished hippocampal long-term potentiation in rats maintained on the ketogenic diet for three weeks. This proposal is an interdisciplinary collaboration among the Neuroscience Program and the Departments of Engineering and Psychology, and it promotes mutually beneficial interactions between laboratories with electrophysiological and biochemical expertise. The feasibility of these proposed experiments is extremely high: The standard biochemical methodology is demonstrated in our preliminary studies and well-characterized in the literature. The in vivo electrophysiological methodology has been used extensively in our laboratory in multiple species and size ranges, leading to a number of publications over the last decade. The undergraduate participation and training is clear, as trained, supervised students are involved in all steps of all proposed experiments. The clinical relevance includes a better understanding of the regional brain responses to a ketone-based metabolism, and a much-needed characterization of the influence of a current therapy for pediatric epilepsy on hippocampal synaptic plasticity. PUBLIC HEALTH RELEVANCE: The ketogenic diet is a high fat/very-low carbohydrate diet used successfully to treat pediatric epilepsy. Despite its use in children, little is known about how diet therapy affects brain energy, learning or memory. Based on published and preliminary work, we hypothesize that brain energy and activity will change in areas of the brain affected by seizures. Using an animal model (rat) we will test the effects of ketogenic diet therapy on brain energy and on the biological mechanisms that are important for learning and memory.

DESCRIPTION (provided by applicant): Epilepsy affects approximately 1% of the population across all age groups and is one of the most prevalent chronic neurological disorders. Unfortunately, current pharmacological treatments do not control seizures adequately in up to 35% of persons with epilepsy. As an alternative, therapy with a high-fat low-carbohydrate (ketogenic) diet can be highly effective in medically-refractory epilepsy, and the mechanisms underlying its success can offer insight into other neurological disorders with metabolic underpinnings. Despite intense interest, key mechanisms underlying the clinically- established anticonvulsant success of ketogenic diet therapy remain unknown. Adenosine, the core molecule of ATP, is an inhibitory neuromodulator that links cell metabolism directly to neuronal activity. Understanding how to regulate adenosine, an endogenous anticonvulsant and neuroprotectant, offers powerful therapeutic benefits. Akin to a ketogenic diet's success with refractory epilepsy, adenosine is an effective anticonvulsant in models of drug- resistant epilepsy. The central hypothesis is that ketogenic diets increase adenosine A1 receptor activation, and that this increased inhibitory influence of adenosine is critical for the anticonvulsant success of ketogenic diet therapy. To test this hypothesis, bioenergetic, neurochemical, electrophysiological and behavioral techniques will be applied after ketogenic diet therapy in rats and mice with normal or genetically-altered adenosine signaling. Experiments using the most accurate bioenergetic and neurochemical techniques will quantify changes in energy molecules, adenosine, adenosine receptors, and ketone bodies (Aim 1). Changes in adenosine's influence on synaptic transmission and neuronal excitability will be quantified using detailed electrophysiology and electrochemistry (Aim 2). Experiments using behavioral paradigms of genetically-based seizures or modeled epilepsy will quantify changes in seizure frequency and severity (Aim 3). Preliminary and published data from others and us support the hypothesis that ketogenic strategies increase levels and actions of adenosine. The proposed experiments will measure levels of adenosine and test the role of adenosine acting at adenosine A1 receptors in the anticonvulsant success of dietary therapy in vitro and in vivo. Our expertise in bioenergetics, adenosine regulation and epilepsy, coupled with unique and complementary methodological approaches, will yield clear experimental outcomes. The long-term goal of this research is to understand critical mechanisms underlying metabolic strategies and yield new options in the treatment of epilepsy and other conditions such as brain injury and stroke, where adenosine offers therapeutic benefits. PUBLIC HEALTH RELEVANCE: Low carbohydrate ketogenic diets prevent epileptic seizures and protect neurons, but the reason why dietary therapy is successful is unknown. We hypothesize that ketogenic diets increase adenosine, the brain's own seizure-control molecule. Ultimately, understanding the relationship between adenosine and ketogenic diet therapy will facilitate development of an entirely new family of treatments for epilepsy that are easy to administer, effective and well-tolerated.

? DESCRIPTION (provided by applicant): Clinical evidence demonstrates dietary therapy as highly effective and even a cure for epilepsy. Specifically, children treated with a high-fat, lo-carbohydrate ketogenic diet (KD) can achieve and maintain seizure freedom even after weaning off the KD and drugs. Its potential likely extends well beyond epilepsy, with emerging research identifying benefits in other acute and chronic conditions. Yet despite continuous use since the 1920s it is still not possible to predict whether KD therapy will be successful, and its mechanism remains unknown. One of the most striking aspects of KD treatment is its proven efficacy even in medically-refractory seizures - indicating that this diet mobilizes anti-seizure mechanisms distinct from available drug treatments. To gain new insight into the mysteries of KD therapy we will leverage an exclusive opportunity to analyze a singular clinical repository. A team in Stockholm, Sweden, led by Maria Dahlin, MD, PhD, obtained cerebrospinal fluid (CSF) from each of 25 children with refractory epilepsy at two time points: 1) before and 2) at three months of maintenance on KD therapy. Clinical data and outcomes were tracked and an initial neurotransmitter analysis was performed. Due to their extremely unique and precious nature (repeated lumbar punctures in each child) these samples have been frozen and awaiting the right opportunity: novel and plausible hypotheses regarding KD efficacy, and a reliable and comprehensive analysis. Now, after several years of preparation, we propose to test our ongoing hypotheses on purinergic mechanisms of KDs and, simultaneously, perform a complete metabolic profiling of CSF obtained from humans and rats fed control vs. KD. CSF will be analyzed for more than 200 molecules by Metabolon (Durham, NC) using their most sophisticated platform. Our team is working with Metabolon regarding initial metabolomic analysis and pathway mapping of the human samples. We will continue with a detailed metabolomic and statistical analysis, collect rat CSF and blood, perform a parallel metabolic and subsequent statistical analysis on the rat samples, and proceed to detailed comparison, interpretation, hypothesis testing, and target validation. We hypothesize that these studies will 1) determine how a KD changes the metabolic signature of various pathways as well as individual metabolites; 2) determine which changes correlate with KD efficacy in children; 3) determine which baseline characteristics predict efficacy in children; 4) enable comparison between diet-mediated changes in human and rodent CSF fluid. Predicting efficacy could help identify and motivate patients who could achieve the greatest benefits. Furthermore this work will yield targets to pursue regarding key metabolic changes mobilized by a KD. Student training is included in all conceptual and proposed experimental aspects; students have been involved in the planning and initial analysis. This work represents a significant opportunity to benefit numerous disorders by revealing key mechanisms, identifying biomarkers, and validating animal models.

? DESCRIPTION (provided by applicant): The high-fat, low-carbohydrate ketogenic diet (KD) has been used to treat epilepsy successfully for nearly 100 years and it remains an important clinical option for refractory seizures in adults and children. Whereas it declined in popularity with the advent and evolution of pharmacological therapies, clinical use of the ketogenic diet and basic research into its underlying mechanisms experienced a major resurgence in recent years. This renewed interest in the ketogenic diet is due to high profile cases where the diet was remarkable successful, continued inadequacy of available therapies for up to 30% of patients with epilepsy, general popularity of and interest in diet-based approaches, and increasing recognition that metabolic therapies like the KD have clinical relevance and untapped potential extending well beyond epilepsy. Recent clinical and experimental evidence has shown that a KD and similar dietary and metabolic strategies have significant promise in treating diverse neurological and non-neurological disorders. The Fifth Global Symposium on Ketogenic Therapies is part of a successful and evolving series of global symposia. The first in the series, the International Symposium on Dietary Therapies for Epilepsy and Other Neurological Disorders, held in Phoenix, Arizona (2008), was supported by NIH and became the first in this biennial series. The first symposium established a high level of science and participation for subsequent successful global symposia in 2010 (Edinburgh), 2012 (Chicago) and 2014 (Liverpool). It also established a collaborative spirit and common purpose, particularly since dietary therapy is known to be highly effective. Now, the Fifth Global Symposium on Ketogenic Therapies (Banff, 2016) represents another leap forward for the field: while one focus remains on ketogenic therapy for epilepsy, a strong emphasis will be placed on the emerging links between metabolism and epigenetics, and the program will delve into metabolic therapy for brain cancer, autism spectrum disorder and cognitive disorders such as Alzheimer's disease. We also focus on bringing young professionals into the field, and, like previous symposia, offer all attendees a collaborative spirit and motivation to further leverage this successful therapy. There will be ample time for attendee engagement and group discussions, a well-received feature of previous symposia. The faculty and moderators are all internationally recognized experts and the fifth global symposium is unquestionably the definitive global venue for metabolism-based therapies for neurological disorders.

SUMMARY The ketogenic diet (KD) is an established therapy for drug-resistant epilepsy since its origins in 1921 at the Mayo Clinic in Rochester, Minnesota. While scientists continue to study its broad anticonvulsant effects there is mounting experimental evidence for disease-modifying and neuroprotective properties. In this rapidly advancing field there is emerging data on mechanisms and evidence supporting broader use of the KD (and variants) against diverse neurological conditions including cancer, migraine, autism and dementia. Despite efficacy and affordability large regions of the world do not offer KD therapy ? particularly in Southeast Asia, Eastern Europe and Africa. Centers interested in offering KD may elect not to do so because of perceived inadequate resources; physicians and/or dietitians familiar with KD may be absent. Furthermore, the latest research on mechanisms, applications and protocols may not be well disseminated, and a lack of standardized protocols and management recommendations present additional barriers. Without adequate support patients may not consider this treatment due to its restrictiveness. International meetings are needed where scientists, physicians, dietitians, nurses, social workers and students can meet and discuss protocols, research innovation, clinical experience and collaboration in the KD field. The Sixth Global Symposium on Ketogenic Therapies is part of a successful and evolving biennial series. The first, the International Symposium on Dietary Therapies for Epilepsy and Other Neurological Disorders (Phoenix, Arizona, 2008), was NIH-supported and became the first in the series, establishing a high level of science and participation for subsequent successful symposia (Edinburgh, 2010; Chicago, 2012; Liverpool, 2014; Banff, 2016). It also established an enduring collaborative spirit and common purpose. The Sixth Global Symposium on Ketogenic Therapies (Jeju Island, Korea, 2018) represents another leap forward: the focus remains on promoting collaborative interactions among clinical and basic scientists and trainees, and disseminating the latest research to the community and beyond, and a strong emphasis is placed on standardizing and enhancing international dissemination and implementation. To achieve the highest international impact experts from around the world will share their data and experience, and education sessions will be held for attendees from resource-limited regions with a limited number of trained KD experts. We continue the tradition of bringing young professionals, women and people with disabilities into the field, and, like previous symposia, offer all attendees a collaborative spirit and motivation to further leverage metabolic therapies. Ample time is scheduled for attendee engagement and group discussions, a well-received and robust feature of previous symposia. The faculty and moderators are internationally recognized experts and the Sixth Global Symposium is the definitive venue for continued progress in and advocacy for metabolism-based therapies for neurological disorders.

PROJECT SUMMARY Current epilepsy therapies are inadequate: at least 30% of epilepsy patients suffer residual or medically- refractory seizures and/or comorbidities as well as significant side-effects from antiepileptic drugs. Some of these patients are treated successfully with a ketogenic diet (KD), a poorly understood and potentially underutilized metabolic therapy established in 1921. The consistent clinical success of the KD in suppressing seizures in refractory adult and pediatric epilepsies has been verified in multi-center, international and randomized prospective clinical studies. Clinical observations and recent translational work strongly suggest that a KD has antiepileptogenic and disease-modifying properties, and recent work indicates that metabolic therapy may benefit a greatly expanded spectrum of diseases including pain, autism, brain cancer, and Alzheimer?s disease. Nevertheless, therapeutic use of the KD has been limited largely to pediatric refractory epilepsy: there are virtually no data on using a metabolic therapy as a first-line therapy, and thus its true clinical efficacy and ability to prevent epileptogenesis is unknown. Understanding key mechanisms by which the clinical benefits of a KD are exerted is urgent and of the highest biomedical significance because it is anticipated that these mechanisms will lead to the rapid genesis of effective new metabolism-based therapeutics with disease-modifying capabilities for epilepsy. Here we test our OVERALL HYPOTHESIS that epigenetic changes in DNA methylation are mobilized during epileptogenesis and provide a therapeutic target for epilepsy prevention through diet-based metabolic therapy. In Aim 1 we will identify epigenetic epileptogenic mechanisms that are (i) common to etiologically different rodent models of temporal lobe epilepsy (TLE) and (ii) laboratory independent ? thus fulfilling an unmet need in epilepsy research and establishing a high degree of scientific rigor among our team. In Aim 2 we will quantify antiepileptogenesis and test mechanisms mobilized by KD therapy, including increased adenosine as a key downstream antiepileptogenic mechanism. Finally, in Aim 3 we will validate whether candidate epigenetic changes are required for KD-based antiepileptogenic effects and thereby provide mechanistic evidence for a causal relationship among metabolic therapy, epigenetic alterations, and antiepileptogenesis. Our approach represents the first systematic and comprehensive mechanistic analysis of an understudied metabolic treatment that can stop ? and even permanently resolve ? seizures. The expected outcome is the identification and characterization of epigenetic mechanisms through which metabolic therapy interferes with the process of epileptogenesis. In particular we will determine whether the expected antiepileptogenic effects are specific to KD-therapy and dependent on identified epigenetic mechanisms. A thorough mechanistic understanding of the KD may reveal an entirely new class of therapies for epilepsy and its prevention.