1995 — 1999 |
Lynch, David Robinson |
P30Activity Code Description: To support shared resources and facilities for categorical research by a number of investigators from different disciplines who provide a multidisciplinary approach to a joint research effort or from the same discipline who focus on a common research problem. The core grant is integrated with the center's component projects or program projects, though funded independently from them. This support, by providing more accessible resources, is expected to assure a greater productivity than from the separate projects and program projects. |
Nmda Receptors--Biochemical Definition and Pharmacological Characterization @ Children's Hospital of Philadelphia
NMDA receptors; neuropharmacology; developmental neurobiology; neurochemistry; central nervous system; dizocilpine; retinoate; stoichiometry; cell differentiation; neurons; biological models; radiotracer; laboratory rabbit; immunoprecipitation; western blottings; transfection;
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0.909 |
1995 — 1999 |
Lynch, David Robinson |
K08Activity Code Description: To provide the opportunity for promising medical scientists with demonstrated aptitude to develop into independent investigators, or for faculty members to pursue research aspects of categorical areas applicable to the awarding unit, and aid in filling the academic faculty gap in these shortage areas within health profession's institutions of the country. |
Biochemical Approach to Nmda Receptors &Excitotoxicity @ Children's Hospital of Philadelphia
The proposed program of research, clinical activity, and academic exposure should provide the primary investigator with a strong foundation for research in basic neurology and movement disorders. Research on mechanisms of glutamatergic neurotransmission and excitotoxicity will form the core of the proposed program. Glutamate induced excitotoxicity through the N- methyl-D-aspartate (NMDA) receptor has been implicated in many chronic and acute neurologic disorders including hypoxic-ischemic damage, Huntington's disease, secondary damage from head trauma, glutaric acidemia, hyperammonemia and other central nervous system disorders. The exact pathologic character of excitotoxic damage varies depending on many factors including developmental stage and brain region. One possible explanation for such variations in excitotoxicity is variability in NMDA receptor structure and pharmacology. Understanding the biochemical and pharmacological basis of heterogeneity in NMDA receptors may elucidate the mechanistic reasons for variability in excitotoxic mechanisms. A series of cDNA's have been cloned which are proposed to encode NMDA receptor subunit proteins. These diverse cDNA's suggest possible mechanisms for production of NMDA receptor subtypes. This proposal will use in vitro model systems to examine the biochemical and pharmacological heterogeneity of NMDA receptors composed of different subunits. NMDA receptor assembly will be modeled in transfected cells and then in cells with a neuronal phenotype. Biochemistry and pharmacology of the NMDA receptors in these systems will be examined. These findings will be extended to determine the role of specific NMDA receptor subunits in excitotoxicity and thus provide a rational basis for design of pharmacological agents acting at NMDA receptor subtypes.
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0.909 |
1996 — 1999 |
Lynch, David Robinson |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Pcp and the Nmda Receptor @ Children's Hospital of Philadelphia
Phencyclidine (PCP) is a widely abused drug (e.g. angel dust) which has a variety of actions in the central nervous system. Many of the behavioral effects induced by PCP are thought to result from the blockade of excitatory neurotransmission through the NMDA receptor. The activity of this receptor is critical for many normal brain functions including learning and memory. However, not all compounds that block the NMDA receptor have the same behavioral effects as PCP. Dextromethorphan is another non-competitive NMDA receptor antagonist that is thought to bind to the same site as PCP, but this drug has very different behavioral effects. The two major goals of this project are to understand the molecular interactions between the NMDA receptor and non-competitive antagonists like PCP and to determine the subunit composition and stoichiometry of the NMDA receptor. Several specific questions will be addressed during the course of the project including: l) are there subtypes of NMDA receptors which interact differently with PCP, 2) How are subtypes of NMDA receptors assembled from various subunits, 3) what is the stoichiometry of the NMDA receptor, 4) Which regions of the NMDA receptor bind PCP 5) Which amino acids in the NMDA receptor are most important for drug binding, 6) do amino acids from multiple NMDA receptor subunits interact with PCP, 7) do all non-competitive agonists of the NMDA receptor bind to the same subtypes of NMDA receptor, 8) is it feasible to develop drugs to block PCP binding which will not block the NMDA receptor channel.
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0.909 |
1999 — 2002 |
Lynch, David Robinson |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Nmda Receptors, Protein Kinase C, and Excitotoxicity @ Children's Hospital of Philadelphia
Excitotoxicity, a major pathophysiological mechanism of many neurological disorders, is mediated by a specific type of glutamate receptor, the n-methyl-D-aspartate (NMDA) receptor. Understanding NMDA receptor activation is crucial for developing therapeutic strategies which block excitotoxicity. The NMDA receptor is present in brain as many different subtypes, which differ in composition by the type of subunits which are present. The endogenous enzyme protein kinase C modulates NMDA receptor function heterogeneously in the brain, perhaps reflecting varying composition of NMDA receptors. In heterologous expression systems, the two types of NMDA receptor subunits (NR 1 and NR2) subunit contribute differently to modulation by PKC. In the present study, we will determine the mechanisms by which such modulation occurs and whether findings in heterologous systems explain the diversity of effects of PKC found in the brain. In the first aim, we will use biochemical and molecular biological approaches to determine the exact site or sites on the NR2A subunit which are phosphorylated by PKC. This will begin with phosphorylation experiments in vitro. Site directed mutations will allow determination of whether these sites are causally linked with NR 2A-dependent potentiation of NMDA receptors by PKC. In the second aim we will ascertain the cellular mechanism which mediates NR1 dependent attenuation of NMDA receptors by PKC. Specific experiments will examine the effects of agents which alter calmodulin, actin, and intermediate filaments, all of which interact with NR 1 in subtype specific manners. In our third aim we will determine whether the cell specific effects of PKC on native NMDA receptors reflect cell specific differences in the type of NMDA receptors which are made. These experiments will use measurements of the effect of PKC on NMDA receptors of single cells, and use RNA amplification techniques to determine which NMDA receptors mRNAs are made in those specific cells. The type of NMDA receptor mRNAs made will be correlated quantitatively with the cellular response. In our final aim we will use a model of excitotoxicity to determine the role which PKC plays in potentiation or inhibition of NMDA receptor mediated excitotoxicity. This systematic approach should provide critical information on the complete mechanisms of control of NMDA receptors by PKC and their significance in physiologic and pathophysiologic processes.
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0.909 |
2003 — 2006 |
Lynch, David Robinson |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Calpain Mediated Cleavage of Nr2 in Excitotoxicity @ Childrens Hospital of Philadelphia
[unreadable] DESCRIPTION (provided by applicant): The glutamate receptor called the N-methyl-D-aspartate (NMDA) receptor is a crucial mediator of many forms of excitotoxicity, a pathophysiological mechanism of neurologic disease. The NMDA receptor is constructed from combinations of NR1 and NR2 subunits, with each subunit conveying distinct properties. Different subtypes may play distinct roles in excitotoxicity, possibly based on the variable features of their intracellular domains. These domains link the receptor to intracellular signaling processes, and are potential sites for proteolytic modification by the enzyme calpain. Such modifications could play a crucial role in control of the subtype specific properties of NMDA receptors. Previously we have shown that the NR2A subunit is a selective substrate for calpain while the NR1 subunit is not cleaved by calpain. This cleavage may create receptors with novel properties, which could be regulated in a subunit selective manner during excitotoxicity and hypoxia. In this proposal, we will assess the effect of calpain cleavage on the physiological properties and localization of different NMDA receptor subtypes and the role of these events in excitotoxicity. We will initially determine the sites of cleavage by calpain in each NR2 subunit in vitro, in heterologous systems and in neurons. Following these experiments, we will define the effect of calpain cleavage on physiological properties of the NMDA receptor, and determine whether cleavage by calpain alters NMDA receptor localization in heterologous expression systems and in neurons. In the final Aim, we will investigate whether calpain cleavage of NMDA receptors occurs during models of excitotoxicity and hypoxia, and whether calpain cleavage of NMDA receptors alters the severity of cellular damage. In addition, we will ascertain whether calpain cleavage plays a role in a specific model of epilepsy, the kindling model. Overall, the present I proposal will define how calpain proteolysis of the NMDA receptor modulates receptor properties, allowing us to determine the role of this process in neuronal function and disease processes. [unreadable] [unreadable]
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0.91 |
2007 — 2011 |
Lynch, David Robinson |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Regulation of Nmda Receptors in Excitotoxicity by Calpain and Fyn @ Children's Hosp of Philadelphia
DESCRIPTION (provided by applicant): Glutamate, the major excitatory transmitter in the central nervous system is crucial for synaptic transmission and plasticity as well as the pathophysiological process termed "excitotoxicity." Excitotoxicity usually requires activation of a specific glutamate receptor, the N-methyl-D-aspartate (NMDA) receptor. Therapeutic applications of NMDA receptor antagonists in humans though have been unsuccessful due to adverse effects. Better understanding of the modulation of NMDA receptor activity, localization, and turnover may provide novel ways to control excitotoxicity while minimizing deleterious effects of NMDA receptor blockade. NMDA receptor localization and function is controlled by many events including cleavage of the C-terminal of the NR2B subunit by calpain and phosphorylation of NR2B by Src family tyrosine kinases (SFK) (1). Our preliminary data demonstrate that phosphorylation of NR2B by Fyn controls NR2B cleavage by calpain and activation of downstream signaling events including activation of p38 MAK kinase. Activation of Fyn in this paradigm depends on NMDA receptor activation, allowing Y1336 phosphorylation to be part of positive and negative feedback mechanisms controlling neuronal responses. In this proposal, we will dissect the molecular mechanism of the interactions of calpain, Fyn and MAGUK proteins in the control of NMDA receptor properties, and investigate their importance in NMDA receptor physiology and models of excitotoxic mechanisms of human diseases. In aim 1, we will examine the ability of NMDA receptor-activated Fyn to phosphorylate distinct sites on the NMDA receptor and other substrates. This will allow us to understand the mechanism by which NMDA receptors directly or indirectly activate SFK and how such activity is directed to distinct sites on the NMDA receptor. Aim 2 will define the structural determinants and mechanisms that mediate the interactions of calpain (and its subtypes) with NMDA receptors and SFK. In Aim 3, we will assess examine electrophysiological properties of calpain-cleaved NMDA receptors and whether cleavage by calpain alters activation of downstream MAP kinases in order to link further calpain mediated cleavage of NR2B with physiological and pathophysiological events. The final aim will investigate whether the interactions of calpain and SFK alter pathophysiological events in an in vitro model of excitotoxicity. Better understanding of the mechanisms by which calpain modulates NMDA receptors and how MAGUK proteins and SFK alter this process should allow a rational basis for use of agents modulating these events in neurological disorders. PUBLIC HEALTH RELEVANCE: The proposal addresses the mechanisms of degradation of the N-methyl-D-aspartate receptor. Through understanding of this process, the proposal may facilitate therapies for preventing damage in neurologic disorders such as stroke.
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0.913 |
2009 — 2010 |
Lynch, David Robinson |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Defining the Epitope in Antinmda Receptor Encephalitis @ Children's Hosp of Philadelphia
DESCRIPTION (provided by applicant): Glutamate, the major excitatory transmitter in the central nervous system, is crucial not only for synaptic transmission but also for long-term neuronal changes such as synaptic plasticity and control of gene expression. However, excessive release of glutamate (as occurs in pathological situations) can result in cell death and NMDA receptor hypofunction has been implicated in schizophrenia. Recently, in collaboration with Dr. Josep Dalmau, we have found that antibodies to the NMDA receptor mediate an immune encephalitis associated with personality and behavioral changes, acute psychosis, and short-term memory deficits. Individuals with this syndrome make antibodies that selectively react with the hippocampus, an area involved in memory, and the antigen is the NR1 subunit of the NMDAR. Using molecular biological approaches we have defined the epitope to be within the first 380 amino acids on NR1. Furthermore, creation of the epitope is blocked by tunicamycin and disruption of a specific N- linked glycosylation/deamidation site in NR1 removes immunoreactivity, suggesting that site specific N-linked glycosylation and deamidation are specifically involved in the production of the epitope. We will characterize the features of this epitope in order to better understand the role of these 2 crucial biochemical processes in NMDAR properties. We will ascertain the distribution of deamidated and differentially glycosylated NMDA receptors in the brain and comparing this to the unique pattern of patients'related anti-NMDAR immunoreactivity that is found in anti NMDA receptor encephalitis. In addition, we will immunolabel NMDAR with patient serum and assess whether labeled NMDAR are glycosylated or deamidated at specific sites. In our second aim, we introduce NMDA receptors into cell lines lacking specific glycosylation enzymes. We will then assess the effect on the glycosylation pattern of NMDAR, on their cellular trafficking, and their physiological properties. These will be compared to the unique properties of NMDAR in specific brain regions and cell types. Together, these aims will supply new data on the mechanisms involved in antiNMDAR encephalitis, provide new understanding of the role of glycosylation and deamidation in neuronal chemistry, and devise new strategies for studying the biochemistry of NMDA. PUBLIC HEALTH RELEVANCE: The proposal addresses the mechanisms by which antibodies are generated in the disorder known as anti-N-methyl-D-aspartate Receptor encephalitis. Through understanding of this process, the proposal may facilitate therapies for preventing damage in this disorder and a basic understanding of other neurologic disorders such as stroke.
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0.913 |
2011 — 2012 |
Lynch, David Robinson |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Nicotinic-Glutamatergic Interactions in Axonal Development @ Children's Hosp of Philadelphia
DESCRIPTION (provided by applicant): A variety of neurological and psychiatric disorders are associated with alterations in synaptic plasticity, including Alzheimer's disease, schizophrenia, and Down syndrome. Each of these conditions has also been suggested to involve pharmacological modulation by multiple neurotransmitter receptors, including both the N-methyl D-aspartate receptor (NMDA) and cholinergic nicotinic receptors, particularly the a7 receptor. Understanding the role of such receptors in synaptic alterations and development may thus be crucial for the development of novel therapeutic approaches. The cellular basis for neural plasticity that underlies learning and memory involves a combination of functional and structural alterations in neurons and synapses. While much attention has focused on N- methyl-D-aspartate receptor (NMDAR)-dependent postsynaptic mechanisms involved in long-term change, presynaptic mechanisms are also crucial to such these processes. As seen in postsynaptic mechanisms of synaptic modification, presynaptic glutamatergic receptors including presynaptic NMDA receptors have been proposed to be involved in the regulation of axonal branching and bouton formation. Our preliminary data demonstrate that the axonal a7 nicotinic acetylcholine receptors (nAChR) modulate the location and size of glutamatergic presynaptic boutons and presynaptic NMDAR-mediated glutamatergic transmission in cortical cultures, suggesting that the axonal a7 nAChR and presynaptic NMDARs may be intrinsic factors and signaling mechanisms that mediate synaptogenesis and structural plasticity of glutamatergic axons. In the present proposal, we will extend this work through detailed assessment of these events in models of development and in neonatal brain. This will also allow us to ascertain the role of nAChR-NMDAR interactions in synaptogenesis and structural plasticity, and the temporal periods for such events. We will begin with complete definition of the pharmacological mechanism underlying the presynaptic interactions of a7 and NMDAR. We will then assess the development course of these events to define the critical period during which such interactions occur using neuronal cultures, hippocampal slice cultures and neonatal animals. Collectively these experiments will allow us to identify the mechanisms that mediate the presynaptic interactions of nicotinic and glutamatergic systems, and provide a basis for interpreting these results in the contexts of specific disorders. This will allow new investigations directly targeting these diseases.
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0.913 |
2012 — 2013 |
Lynch, David Robinson |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Defining the Epitope in Anti-Ampa Receptor Encephalitis @ Children's Hosp of Philadelphia
DESCRIPTION (provided by applicant): Glutamate, the major excitatory transmitter in the central nervous system, is crucial not only for synaptic transmission but also for long-term neuronal changes such as synaptic plasticity and control of gene expression. Many of the actions of glutamate are exerted through a specific receptor, the N-methyl-D- aspartate receptor (NMDAR), which controls the trafficking of a second glutamate receptor, the a-amino-3- hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPAR). Recently, in collaboration with Dr. Josep Dalmau, we identified two distinct encephalitic syndromes, associated directly with antibodies to NMDAR in one syndrome and with antibodies to the AMPAR in the other. In antiAMPAR encephalitis, subjects present with symptomatology (amnesia, seizures) analogous to that predicted to occur with AMPAR hypofunction. However, the syndrome can be treated in some individuals by tumor removal or plasmapheresis, suggesting that it is mediated directly by antibodies. Patients with this syndrome make antibodies to the extracellular portion of the AMPAR subunits GluR1 and/or GluR2. Our new preliminary data have identified structural determinants in the amino terminal domain (ATD) of GluR1/2 that are necessary for immunoreactivity to patients' antiAMPAR antibodies. We have also used fusion proteins from these regions to demonstrate diversity in the immune response and to identify individuals with possible antiAMPAR encephalitis who tested negatively by previous approaches. In this proposal we will continue to create new tools for investigation of patients' antiAMPAR antibodies in order to assess whether this syndrome is dramatically under diagnosed, and to link the pathophysiology to specific AMPAR portions First, we will test for the presence of antiAMPAR antibodies and antibodies to specific structural determinants on the AMPAR in samples from a large cohort of individuals with potential encephalitis. We will then correlate the results with clinical features of the subjects and determine whether pathophysiological mechanisms in anti AMPAR encephalitis are mediated through specific epitopes. Cumulatively, completion of these aims will lead to improved understanding of the pathophysiological mechanisms in this disorder, develop tools for further exploration of the mechanisms, and potentially allow creation of novel therapeutic approaches for antiAMPAR encephalitis. PUBLIC HEALTH RELEVANCE: The proposal addresses the mechanisms by which antibodies cause neurological dysfunction in the disorder known as anti-AMPA receptor encephalitis. Through understanding of this process, the proposal may facilitate therapies for preventing damage in this disorder and other forms of encephalitis. It will also provide tools to investigate mechanisms of memory loss in people.
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0.913 |
2014 — 2015 |
Lynch, David Robinson |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Metabolic Approaches to Friedreich Ataxia @ Children's Hosp of Philadelphia
DESCRIPTION (provided by applicant): FRDA is an inherited mitochondrial disease resulting from a triplet nucleotide expansion in the first intron of the gene for frataxin, a protein involve in iron sulfur cluster (ISC) formation. Patients with FRDA have decreased levels of frataxin and secondarily ISC-containing proteins, resulting in the degeneration of dorsal root ganglion cells, the dentate nucleus of the cerebellum, selected other neurons, cardiomyocytes, and ¿-islet cells. Clinically, these changes manifest in a characteristic ataxia, dysarthria, hypertrophic cardiomyopathy and diabetes mellitus. Although increased reactive oxygen species (ROS) formation and oxidative stress have been proposed as pathogenic mechanisms of FRDA based on results from cellular models, treatments aimed at these have been minimally successful. In addition, as frataxin is a ubiquitously expressed protein, the mechanism behind the exquisite tissue selectivity in FRDA is unexplained. An alternative hypothesis to ROS production for the pathophysiology of FRDA is that insufficient bioenergetic capacity with downstream metabolic dysfunction plays a crucial role in the disease. In addition to their role the in the electron transport chain, iron-sulfur containing enzymes are also involved in the Krebs cycle and fatty acid breakdown. Consequently, the metabolic functions of mitochondria might be crucially compromised in FRDA, leading to tissue selective metabolic dysfunction. In order initially to define such abnormalities, we have used rigorous stable isotope dilution and isotopic tracer liquid chromatography-mass spectrometry techniques to examine metabolism in FRDA. Using Stable Isotope Labeling by Essential nutrients in Cell culture (SILEC), a novel stable isotope dilution LC-MS method for assessing metabolic changes reflected revealed by intracellular levels of short chain acyl-coenzyme (CoA) thioesters, we have found that decreased frataxin expression results in changes of intracellular levels of specific CoA thioesters. In addition, we have identified a decreased metabolism through fatty acid pathways from palmitate to Acetyl CoA in platelets and fibroblasts. Such observations may be useful as biomarkers of disease activity, and potentially for developing therapeutic approaches. In this proposal, we will expand the understanding fatty acid metabolism in FRDA by examining other metabolism other substrates, in different cellular models, and with a broader scope of FRDA patients. We will examine metabolism of other substrates, as well as the tissue specificity of metabolic changes. Such measurements will develop biomarkers that can be used in clinical studies, including therapeutic trials, in FRDA. This will be tested in the final aim. Overall this approach will have immediate impact on ongoing trials in FRDA and potentially revise current pathophysiological concepts of the disorder and related diseases.
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0.913 |
2015 — 2016 |
Lynch, David Robinson |
R13Activity Code Description: To support recipient sponsored and directed international, national or regional meetings, conferences and workshops. |
Ataxia Investigators Meeting 2016: From Basic Science to Clinical Therapeutics @ Children's Hosp of Philadelphia
? DESCRIPTION (provided by applicant): Ataxia, a disabling and frequently fatal neurological disorder, results from a wide variety of genetic and acquired etiologies. The 6th Ataxia Investigators' Meeting, AIM 2016: From Basic Science to Clinical Therapeutics will assemble an international roster of investigators to address the diverse causes of ataxia, to define better the pathogenic basis of ataxia, and to explore routes to therapy. The conference will focus on the most recent scientific advances and emerging integrative approaches toward therapy, with the following objectives: 1) Identify common disease mechanisms, 2) Explore therapeutic strategies, 3) Help establish the future leaders of ataxia research, and 4) Bring trainees (graduate students and postdocs) into contact with ataxia patients and families. AIM 2016 will represent a critical mechanism to facilitate collaboration and discussion on ataxia research and therapeutic approaches, which is of particularly great importance now that the field is entering the phase of meaningful, multi-center clinical trials both in the United States and Europe. The AIM 2016 meeting, affiliated and overlapping with the annual meeting of the largest ataxia foundation in the country occurring at the same hotel, will maximize the impact of this meeting for scientists and patients alike.
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0.913 |
2016 — 2017 |
Lynch, David Robinson |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Anti-Nmda Receptor Antibodies From Patients With Limbic Encephalitis @ Children's Hosp of Philadelphia
Project Summary: Anti-NMDA receptor encephalitis is a potentially lethal encephalitis attributed to autoantibodies against the N- methyl-D-aspartate receptor (NMDAR). Patients with anti-NMDAR encephalitis present with psychiatric and cognitive symptoms, and then progresses to severe neurological dysfunction, including seizures, abnormal movements, impaired consciousness, and autonomic instability, frequently requiring mechanical ventilation. In spite of the severity of the symptoms, substantial recovery is possible for patients treated with immunosuppressive therapies that reduce titers of anti-NMDAR antibodies. Despite the strong correlation between patient anti-NMDAR antibodies and the clinical syndrome, the pathogenic role of anti-NMDAR antibodies in the disease has not been definitively established. Likewise, the actions of anti-NMDAR antibodies on neurons are incompletely understood. The primary obstacle to definitive study is the lack of pure anti-NMDAR antibodies cloned from patients. Prior studies have been restricted to the limited amounts of polyclonal antibodies obtainable from patient sera and CSF. Monoclonal anti-NMDAR antibodies obtained from patients would, for the first time, allow demonstration that the pathophysiological events believed to underlie anti-NMDAR encephalitis are attributable to anti- NMDAR antibodies alone. The antibodies would enable study of how they affect glutamatergic signaling in the brain, facilitate the development of animal models for anti-NMDAR encephalitis, and permit structural modeling of the antibody-NMDAR complex, which could then direct the design of targeted therapies. Our hypothesis is that, in anti-NMDAR encephalitis, antibodies to the extracellular region of the NMDAR lead to internalization of receptors on neurons, leading the NMDAR hypofunction. Our primary objective is to test this hypothesis by cloning antibodies from patients with anti-NMDAR encephalitis and using them to understand the properties of this disease. We will collect B-cells from patients with anti-NMDAR encephalitis, clone their antibodies, and characterize their NMDAR binding properties and effects on receptor function. These experiments will establish whether anti-NMDAR mAbs have features consistent with a primary role in disease pathogenesis. They will lay a foundation for definitive experiments in animal models as well as structural studies to delineate the molecular structure of disease-related immune complexes. 1
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0.913 |
2017 — 2021 |
Lynch, David Robinson |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Natural History of Friedreich Ataxia in Children @ Children's Hosp of Philadelphia
Abstract Friedreich?s ataxia (FRDA) is the most common form of hereditary ataxia, affecting approximately 1 in every 50,000 people in the United States and Europe. Symptoms typically begin between the ages of 5 and 15 years and worsen over time. The pathophysiology of FRDA reflects the deficiency of the protein frataxin. Reduced frataxin levels impair the function of mitochondrial iron-sulfur-cluster-containing enzymes and ability to produce ATP. Recently, amelioration of frataxin deficiency by gene therapy in mouse models of FRDA has produced impressive benefit in reversing the phenotype, providing an evidenced-based approach for treatment of FRDA patients. Mitigation of mitochondrial dysfunction also represents a valid therapeutic approach. However, if attempts at these therapies were made today, they would be limited by the inability to assess the human biology of FRDA in detail, as well as the inability to target therapies to the most biologically responsive individuals, children. To achieve this goal, we will study the natural history of FRDA in children, to understand the course of disease activity in this age group. In the first aim, we will assess potential measures of disease progression in the youngest subjects with FRDA (n=100 at 3 sites) These will include specific revisions and modifications of timed walks (in order to identify a test more suitable for use in young individuals, and an automated measure of upper extremity coordination (the CCFS) that is useful in older FRDA subjects. In aim 2 we will assess biochemical measures of frataxin deficiency and downstream metabolic function, and understand their utility in serial monitoring.in children with FRDA. Peripheral samples (blood, buccal cells, isolated platelets) will be obtained from a large heterogeneous cohort of subjects with FRDA (n=100 at 3 sites). We will then assay the primary biomarker of disease severity, frataxin level, in the samples with a newly devised mass spectrometry-based assay to understand how such levels reflect disease status. In parallel, we will examine mitochondrial-derived alterations in metabolic pathways in platelets to examine events downstream from frataxin deficiency. Finally we will examine physiological tests (motor evoked potentials, Cr-CEST of muscle) that can link clinical parameters with biochemical measurements. Cumulatively these aims will define the utility of such approaches in clinical measurement of FRDA in children, and validate such approaches as the definitive measures needed for design of informative trials in children with FRDA.
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0.913 |
2019 — 2020 |
Lynch, David Robinson |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Frataxin Measurement For Clinical Trial Readiness in Friedreich Ataxia @ Children's Hosp of Philadelphia
Friedreich?s ataxia (FRDA) is the most common form of hereditary ataxia, affecting approximately 1 in every 50,000 people in the United States and Europe. Symptoms typically begin between the ages of 5 and 15 years and worsen over time. The pathophysiology of FRDA reflects the deficiency of the protein frataxin. Reduced frataxin levels impair the function of mitochondrial iron- sulfur-cluster-containing enzymes and ability to produce ATP. Recently, amelioration of frataxin deficiency by gene therapy in mouse models of FRDA has produced impressive benefit in reversing the phenotype, providing an evidenced-based approach for treatment of FRDA patients. However, if attempts at reversal of frataxin deficiency were made today, they would be limited by the inability to assess frataxin levels in detail, as well as the inability to target therapies to the most biologically responsive individuals. To achieve this goal, we will validate the rigorous measurement of frataxin in larger cohorts to understand how it reflects disease activity. In the first 2 aims, we will test improved measures of frataxin deficiency and downstream metabolic function, and understand their utility in therapeutic monitoring. Peripheral samples (blood, buccal cells, isolated platelets, muscle) will be obtained from a large heterogeneous cohort of subjects with FRDA (n=200, n=20 for muscle). We will then assay the primary biomarker of disease severity, frataxin level, in the samples with a newly devised mass spectrometry based assay in order to understand the sensitivity of the assay and how such levels reflect disease status. In the second aim, we will examine mitochondrial-derived alterations in metabolic pathways in platelets and muscle to examine events downstream from frataxin deficiency. In Aim 3, we will reevaluate long term natural history data from already established clinical instruments in the context of frataxin levels. Such data is available for a large >500 person cohort including the 200 who provide samples for the present study, and can be used to understand the relationship between frataxin levels and clinical status. Cumulatively these aims will define the utility of frataxin levels in clinical measurement of FRDA, and validate such approaches as definitive measures needed for design of informative trials of frataxin restoration.
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0.913 |
2020 — 2021 |
Blair, Ian Alexander Hwang, Wei-Ting (co-PI) [⬀] Lynch, David Robinson |
U01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Analytical Validation of Frataxin Proteoforms in Blood as Biomarkers of Friedreich's Ataxia @ University of Pennsylvania
ABSTRACT: ANALYTICAL VALIDATION OF FRATAXIN PROTEOFORMS IN BLOOD AS BIOMARKERS OF FRIEDREICH?S ATAXIA Friedreich?s ataxia (FA) is an autosomal recessive disease caused by an intronic GAA triplet expansion in the FXN gene, leading to reduced expression of the mitochondrial protein frataxin. FA is estimated to affect 1 in 50,000 with a mean age of death in the fourth decade of life. There are no approved treatments for FA, although experimental approaches, which involve up-regulation or replacement of frataxin protein, are being tested. Frataxin is undetectable in serum or plasma, and whole blood could not be used because frataxin was thought to be present in long-lived erythrocytes. An assay for analyzing frataxin in platelets, which have a half- life of 10 days, which would allow therapeutic interventions to be tested, was developed by the Blair and Lynch labs. The assay is based on stable isotope dilution immunopurification two-dimensional nano-ultra high performance liquid chromatography/parallel reaction monitoring/high resolution mass spectrometry (nano- UPLC-MS/HRMS) and is monitoring three tryptic peptides from frataxin. The assay had 100 % sensitivity and specificity for discriminating between controls and FA cases but analyzing platelets on a routine basis is very challenging in many clinical settings. We have now discovered that in erythrocyte is in fact a novel proteoform of frataxin (isoform E) with 135-amino acids (76-210) and an N-terminally acetylated methionine residue. It arises through an alternative splice-site form that is used for the canonical full-length form of frataxin (1-210). There is three times as much isoform E in erythrocytes (26.7 ± 6.4 ng/mL) from the blood of healthy volunteers (n=10) when compared with the mature mitochondrial frataxin present in other blood cells (7.1 ± 1.0 ng/mL). We recently found that both isoform E (8.5 ± 1.1 ng/mL) and mature mitochondrial frataxin (2.1 ± 1.1 ng/mL) are both reduced by > 70 % in blood from FA patients (n=29) when compared with healthy control subjects. Isoform E lacks a mitochondrial targeting sequence and so it is distributed to both cytosol and the nucleus when expressed in cultured cells. The ability to specifically quantify extra-mitochondrial and mitochondrial isoforms of frataxin in whole blood would make it possible to follow the progress of the disease and monitor the efficacy of therapeutic interventions. This will require the development of rigorously validated assays that address the pre-analytical and analytical issues that are relevant to the use of blood as a biological matrix for biomarker analysis rather than the more common use of serum or plasma. The present proposal addresses these issues under following specific aims: Aim 1: To conduct pre-analytical validation for collection and storage of mature frataxin and isoform E protein in whole blood. Aim 2: To conduct analytical validation of the method for quantifying mature mitochondrial frataxin and frataxin isoform E in whole blood. Aim 3: Validation of the mature frataxin and frataxin isoform E proteoform analysis in whole blood from controls, FA carriers, and FA cases from multiple sites.
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