Geoffrey T. Swanson - US grants

DESCRIPTION (provided by applicant): Kainate receptors (KARs) are a family of glutamate receptors whose activation leads to seizures in the hippocampus. KARs also modulate synaptic transmission and mediate long-term plasticity at a number of brain synapses. In CA3 pyramidal cells in the hippocampus, KARs are localized to one particular excitatory synapse-those formed by mossy fiber inputs from granule cells. KARs at mossy fiber synapses underlie an postsynaptic current (KA-EPSC) and play a role in the induction of long- and short-term potentiation of synaptic strength. The objective of these experiments is to determine the mechanisms by which KAR activation leads to altered synaptic strength and action potential firing patterns in hippocampal CA3 pyramidal neurons. These questions will be examined with a physiological approach that utilizes selective pharmacological compounds as well as gene-targeted mice lacking one or more KAR subunits. In Specific Aim 1, the cellular mechanisms that shape mossy fiber KA-EPSCs will be examined by analyzing their properties while altering synaptic strength or activating second messenger systems. These experiments will further understanding of the distinct physiological roles that mossy fiber KARs subserve, which will be integral for elucidating the role of KAR5 in hippocampal circuitry. In Specific Aim 2, we will determine the critical receptor subunits and physiological processes that lead from activation of mossy fiber KARs to synchronized neuronal firing patterns. We will test if changes in firing properties arise from non-ionic mechanisms, perhaps G-protein-mediated, or a particularly close association between KARs and ion channels involved in action potential initiation. In Specific Aim 3 we will establish the role of KARs involved in synaptic plasticity at excitatory synapses on CA3 pyramidal neurons. KARs were recently implicated in the induction of long-term potentiation (LTP) at mossy fiber synapses. We will extend our studies by examining the function of presynaptic KARs and determining the subunits underlying LTP using gene-targeted mice that lack one or more KAR subunits. The role of KARs in other forms of mossy fiber plasticity will also be investigated.

[unreadable] DESCRIPTION (provided by applicant): Natural source compounds that differentiate between similar subtypes of receptor proteins are critical tools in modern neuroscience and often serve as valuable lead molecules for therapeutic applications. This has been particularly true with respect to lonotropic glutamate receptors, the receptors that underlie fast excitatory neurotransmission in the central nervous system; pharmacologically active glutamate receptor compounds have been isolated from a number of diverse organisms. Most recently, dysiherbaine (DH), a novel ligand for kainate (KA) and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) type glutamate receptors, was isolated from the marine sponge Dysidea herbacea. DH was shown to be a potent convulsant with an unusually high affinity for a subset of KA receptors; this compound, as well as its natural and synthetic analogues, represents a novel set of tools for investigating biophysical and physiological properties of excitatory amino acid receptors. The diversity and complexity of glutamate receptor expression, as well as the central role of these proteins in brain function, underscore the importance of isolation and characterization of new pharmacological tools with unique selectivity profiles. This project will characterize a new set of pharmacological tools - DH and its analogues - and use these compounds to examine functional aspects of glutamate receptor activity at the biophysical and neuronal level.In the first specific aim, the biological activity of novel analogues of DH will be determined. In the second specific aim, we will use DH as a tool for understanding structure-function relationships of kainate receptors. Kainate receptor subunits exhibit a high degree of variability in their responses to DH, which affords an opportunity to use the marine toxin as a key probe for uncovering important functional domains of the receptor proteins. The third specific aim will be focused on determining the subunitcomposition of neuronal kainate receptors using a combined pharmacological and genetic approach, with our most important tools consisting of gene-targeted mice and subunit-selective marine toxins.

DESCRIPTION (provided by applicant): The objective of these experiments is to determine the signals that control the intracellular trafficking of kainate receptors. Kainate receptors (KARs) comprise a family of ionotropic glutamate receptors whose role in brain physiology and pathology is poorly understood. Little is known regarding the signals that regulate the assembly, transport, and membrane expression of KARs. Physiological characterization of recombinant KARs demonstrated that "low-affinity" subunits, encoded by the GIuR5, GluR6 and GluR7 genes, formed functional glutamate receptors. In contrast, the "high-affinity" subunits KA-1 and KA-2 did not form functional homomeric ion channels, but rather modified the physiological properties of the channel-forming subunits. What is the basis for this difference in functionality between low- and high-affinity KAR subunits? Recent results provide some clues and form the foundation for this project application. Recent results provide some clues and form the foundation for this project application. While characterizing a newly generated anti-KA-1 antibody we found that much, if not all, of KA-1 subunit protein expressed in mammalian cells is retained in the endoplasmic reticulum (ER) and does not reach the cell surface as a homomeric receptor. This pattern of ER retention suggested the possibility that KA-1 and KA2 subunits may either contain ER retention signals or lack ER export motifs in their protein sequence. Strikingly, an examination of the carboxy-terminal sequences revealed that only three kainate receptor subunits contained RXR sequences-KA-1, KA-2 and the weakly expressed GluR7b receptor. The correlation between lack of function and presence of RXR sequence make these motifs likely candidates for KAR ER retention signals. In this project application we will examine the role of RXR motifs in determining the trafficking of KARs. First, using a combination of immunohistochemical and physiological techniques we will test the hypothesis that RXR motifs in the carboxyterminal domains of the KAR subunits act as endoplasmic reticulum retention signals. Second, we will determine if GIuR5 and GluR6 subunits contain carboxy-terminal signals that act as masking domains for retention motifs on KA-1 and KA-2 subunits. Third, a potential role phosphorylation in modulation of trafficking will be examined. These experiments are important for understanding what cellular mechanisms control the subunit composition of KARs and how functional expression of aberrant receptor complexes may alter excitatory neurotransmission.

DESCRIPTION (provided by applicant): In this project we will test the hypothesis that pharmacological targeting of kainate receptors, a family of ionotropic glutamate receptors, represents a viable therapeutic strategy in an animal model of multiple sclerosis (MS). Glutamate receptor activation contributes to excitotoxic degeneration of oligodendrocytes that occurs in diseases of aberrant myelination, such as MS. Oligodendrocytes and their progenitor cells express all three types of ionotropic glutamate receptors (AMPA, kainate, and NMDA subfamilies), with each appearing to play a distinct role in mediating degeneration in models of glial excitotoxicity. Kainate receptor activation, in particular, produces toxicity through at least two distinct mechanisms, calcium-mediated excitotoxicity and priming of oligodendrocytes to complement attack, suggesting that these receptors could play a prominent role in oligodendrocyte death prevalent in MS. Broad-spectrum AMPA/kainate receptor antagonists are efficacious in animal models of MS, ameliorating disease in mice with experimental autoimmune encephalomyelitis (EAE), and reducing physiological measures of ischemic damage in cortical oligodendrocytes when combined with non-competitive NMDA receptor antagonists. Inhibition of AMPA and NMDA receptors, however, has proven clinically problematic. In contrast, subtype-selective antagonists of kainate receptors are well tolerated in animal models and preclinical studies, but these have not been tested in animal models of MS. Furthermore, it is unclear what subtype of kainate receptors are critical for oligodendrocyte excitotoxicity. This is a particularly important question because the subunit composition of native kainate receptors will determine their sensitivity to selective antagonists. In this exploratory study, we will test our central hypothesis first by defining the contribution of specific receptor subunits to spinal oligodendrocyte excitotoxicity using gene-targeted mice and selective pharmacological tools. Secondly, we will test if progression of experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, is attenuated by kainate receptor antagonists or in kainate receptor knockout mice. These experiments will enable us to definitively assess whether targeting of kainate receptors represents an approach with therapeutic potential in treatment of MS. PUBLIC HEALTH RELEVANCE In this project we will test the hypothesis that inhibition of kainate receptors represents a viable strategy for slowing disease progression in an animal model of multiple sclerosis. Kainate receptors comprise a family of proteins responding to the excitatory neurotransmitter L-glutamate;these receptors in part mediate excitotoxic death of oligodendrocytes, which are glial cells in the brain required for normal neuronal function. To test our hypothesis we will utilize newly developed selective pharmacological agents and gene-targeted mice lacking kainate receptor subunits.

DESCRIPTION (provided by applicant): Kainate receptors (KARs) are a family of ionotropic glutamate receptors whose primary function is to help maintain the critical balance between excitatory and inhibitory processes in the central nervous system. Because KARs are primarily serve modulatory roles, rather than underlying integral components of basic excitatory transmission, they may prove more approachable drug targets than AMPA or NMDA receptors. KARs subserve their modulatory role in part by virtue of a remarkably heterogeneous distribution in different neuronal populations. The neuron-specific cellular mechanisms that direct polarized distribution and synaptic (or extrasynaptic) targeting of KARs remain largely unknown. Our long-term objective is to elucidate the molecular processes that control constitutive and regulated KAR localization in neurons. We have discovered that Epb4.1 proteins alter neuronal KAR localization and function through interactions with conserved regions of receptor subunit carboxy-terminal domains. In this project, we will identify the site(s) and subunit specificity of this association, how 4.1 proteins control localization of KARs in neurons, and determine how posttranslational modifications such as palmitoylation and phosphorylation modulate the interaction between 4.1 proteins and KAR subunits. Understanding the molecular and cellular bases for these processes is an important objective, because KARs represent new therapeutic targets for a number of neuropathologies, including chronic pain, anxiety and epilepsy.

DESCRIPTION (provided by applicant): The purpose of this project is to understand the role of animal-derived lectins in nervous system activity. Investigation into the neuronal activities of galectins, a family of mammalian soluble galactoside-binding proteins, thus far has focused primarily on how they impact neurogenesis or act as metastatic factors for brain cancer cells. However, galectins also are known to be secreted by glia and neurons in the mature nervous system, and therefore are likely to have a physiological role in brain function. We hypothesize that galectins impact neuronal function through short-term morphological changes in dendritic spines, alterations in synaptic plasticity, and reductions in neuronal viability. These activities re mediated by engagement of intracellular signaling cascades that include ERK/MAPK. Integral proteins expressed on neuronal plasma membranes typically contain the glycan N acetyllactosamine, a common disaccharide constituent of complex oligosaccharides, and therefore could serve as targets for galectin binding and cross-linking of signaling molecules, leading to their activation and initiation of intracellular enzymatic cascades. In addition to thei actions on neurons, galectins are useful tools for understanding the relevance of N-glycans to ionotropic glutamate receptor (iGluR) function due to their allosteric modulatory actions. Accordingly, the proposed project will elucidate how galectins impact neuronal function and viability and use these proteins as glycan-specific tools in structure-function studies with recombinant iGluRs. In Specific Aim 1, we will examine galectin activity on neurotransmission and synaptic plasticity in the mouse hippocampus. Experiments in Specific Aim 2 will test the hypothesis that galectins alter neuronal structural plasticity through intracellular kinase cascades. Finally, in Specific Aim 3, we will determine the spectrum of galectin actions on iGluRs and their molecular basis. We will probe the physical characteristics of galectins that optimize their functional action on iGluRs. These studies will yield insight into the as-yet unexplored relevance of galectins to neuronal function, which also will be relevant to understanding their importance to glioma-induced alterations in neuronal excitability.

DESCRIPTION (provided by applicant): Mossy cells in the hilar region are important but relatively understudied contributors to integration of cortical input to the hippocampus. Signaling mechanisms that impact mossy cell excitability and synaptic function are relevant to physiological network function, such as encoding of patterns of input, and play a role in pathological adaptations, as occur in epilepsy. Kainate receptors play important modulatory roles in network excitability elsewhere in the hippocampus through diverse functional activities that are neuron- and synapse-specific. How kainate receptors might contribute to excitatory signaling in hilar mossy cells and between mossy cells and either hilar interneurons or dentate granule cells is entirely unknown and is the focus of this project. We will determine if kainate receptors contribute to either efferent or afferent signaling in mossy cells. This objective is relevant to models of temporal lobe epilepsy because (i) mossy cells are central to the two predominant models of circuit hyperexcitability in chronic forms of temporal lobe epilepsy, and (ii) aberrant kainate receptor function was recently shown to contribute to seizures in rodent seizure models. In Specific Aim 1, we will determine the role and composition of kainate receptors in mossy cells, focusing particularly on postsynaptic kainate receptor function at granule cell - mossy cell synapses. The subunit composition of mossy cell kainate receptors will be determined using a combination of pharmacological tools and gene-targeted mice. In Specific Aim 2, we will examine potential contributions by kainate receptors to signaling between mossy cells and dentate granule cells or hilar interneurons. The architecture of mossy cell projections has made studying these synapses challenging. We will develop a new mouse model in which channelrhodopsin is selectively expressed in mossy cells and subsequently photostimulate inputs to granule cells or hilar interneurons independent of either perforant path or CA3 collaterals. Pre- and postsynaptic function, mechanisms of short- and long-term synaptic plasticity, and the role of kainate receptors at these synapses will be determined for the first time. These studies will elucidate the physiological role played by kainate receptors in hilar circuits and lay the framework for understanding their pathological role in network hyperexcitability in seizure states.

DESCRIPTION (provided by applicant): Beta-arrestins are cytosolic proteins that interact with the carboxy-terminal tails of seven-transmembrane receptors (i.e., GPCRs) and play a role in desensitization, internalization, and scaffolding other proteins that initiate intracellular signalng cascades independent of G protein activation. Because the latter non-canonical signaling mode is a relatively new discovery, the extent to which beta-arrestins transduce G protein-independent signaling downstream of most types of GPCRs is unknown. Metabotropic glutamate receptors (mGluRs) represent one such family of receptors with a largely uncharacterized relationship to beta-arrestins. In this project, we will test the hypothesis that group I mGluRs (mGluR1 and mGluR5) have the potential to activate b-arrestin-dependent signaling pathways, and that signaling through non-G protein-mediated mechanisms in part underlie mGluR-dependent forms of synaptic plasticity in the hippocampus. In Specific Aim 1, we will determine which group I mGluRs associate with b-arrestin-1 and -2 in the mammalian brain and examine how an mGluR1-dependent form of hippocampal plasticity at mossy fiber - CA3 pyramidal neuron synapses is altered by association with one or both b-arrestins. In Specific Aim 2, we will elucidate which signaling cascades underlie mGluR1-dependent plasticity at mossy fiber synapses. In Specific Aim 3, we will test the role of b-arrestins in a group I mGluR-dependent plasticity at Schaffer collateral - CA1 pyramidal neuron synapses. These experiments have the potential to reveal an unexpected contribution by novel signaling pathways, those downstream of mGluRs but independent of canonical G protein processes, in synaptic plasticity in the hippocampus. The existence of beta-arrestin-dependent signaling would also support the potential development of biased ligands for mGluRs. The outcomes of this study could therefore yield insight into new strategies for therapeutic targeting of group I mGluRs, which in recent years has been pursued for a number of neuropathologies.

SUMMARY Kainate receptor signaling is required for the appropriate development of the central nervous system. Children with de novo loss-of-function or missense mutations exhibit intellectual disability and other severe developmental phenotypes. Why this occurs is unknown, in part because we do not have a clear understanding of how aberrant kainate receptor function disrupts neural development. The objectives of this project are to (i) gain insight into normal neurodevelopmental roles played by kainate receptors and (ii) to determine the nature of circuit and behavioral disruptions when kainate receptor signaling is aberrant or completely lost in mouse models. We will pursue these objectives using comparative studies in mice that model known genetic variants causative for human disorders. These include a new mouse line generated in the Swanson laboratory, GluK2(A657T), that models a human de novo missense mutation in the Grik2 gene that causes intellectual disability (ID) and ataxia, as well as mice which model Grik2 haploinsufficiency which is associated with developmental delay and ID in human populations. The Contractor, Swanson and Savas laboratories will use these mice to test the hypotheses that kainate receptors establish an appropriate balance between excitation and inhibition in developing hippocampal circuits, are required for correct development of synapses, and regulate intrinsic excitability in the CNS. In the first Specific Aim, we will determine how missense or loss-of-function mutations in Grik2 alter synaptic connectivity, function, morphology and expression of the synaptic and non-synaptic proteome in brain regions associated with altered behaviors. In the second Specific Aim, we determine how intrinsic excitability is altered in kainate receptor mutant mice. In the third Specific Aim, we will carry out behavioral studies on kainate receptor mutant mice to determine the expanse of cognitive, social, habitual, and motor dysfunction, which will also inform the physiological studies in Aims 1 and 2. We anticipate these studies will reveal some of the underlying circuit disruptions that give rise to human cognitive and motor phenotypes. We therefore aim to develop a comprehensive and integrated understanding of the importance of kainate receptor signaling to establishment of appropriate neuronal function in the CNS and establish how aberrant signaling leads to maladaptive development and behaviors in mice that are correlates of the core symptoms of human developmental disorders.

PROJECT SUMMARY Diffusely infiltrative glioma is the most common primary brain tumor in adults. Most glioma patients experience at least one seizure during the course of their disease, and over 30% suffer from repeated seizures, known as tumor-associated epilepsy (TAE). Current front-line treatment for TAE is levetiracetam (LEV) (a.k.a. Keppra®), but this fails to control seizures in over 50% of patients. Such patients then require more powerful second-line antiepileptic drugs that often have greater side effects. TAE is more common in World Health Organization (WHO) grade II-III gliomas than in grade IV glioblastomas, but the reason for this is not clear. The vast majority of grade II-III gliomas contain mutations in isocitrate dehydrogenases 1 and 2 (collectively ?IDHmut?), which lead to the production and release of large amounts of D-2-hydroxyglutarate (D2HG). D2HG bears a great deal of structural similarity to glutamate, an excitatory neurotransmitter that binds to N-methyl-D-aspartate receptor (NMDAR) on neurons. Our data show that D2HG increases in vitro neuronal membrane depolarization and neuronal network activity, and that this can be completely blocked by an NMDA receptor (NMDAR) antagonist. We also found that IDHmut glioma increases seizure activity in engrafted mice compared to IDHwt glioma, and that this is greatly reduced by treatment with IDHmut enzyme inhibitor. Finally, we found that IDHmut gliomas are much more likely to cause seizures compared to IDHwt gliomas. This is the first direct evidence of a mechanistic link between IDHmut and seizures; therefore, our hypothesis is that D2HG contributes to an increased incidence of seizures in patients with IDHmut gliomas, and that new targeted therapeutic strategies can decrease seizures in these patients. In Aim 1, we will explore the mechanisms by which D2HG triggers neuronal depolarization and increased neuronal network activity. Our two main hypotheses are: (i) D2HG directly stimulates NMDA receptors; (ii) D2HG inhibits glutamate reuptake transporters that normally prevent the pathologic accumulation of glutamate in the synaptic cleft. We will use patch clamping and multi-electrode arrays to study the effects of D2HG on the electrical activity of cultured mouse cortical neurons, as well as on mouse brain slices. In Aim 2, we will explore the effects of IDHmut glioma on the surrounding nonneoplastic tissue in vivo, focusing on changes that are characteristic of epilepsy, including neuronal loss, NMDAR downregulation, oxidative stress, inflammation, hippocampal damage, and altered mouse behavior. Results will be validated in patient-derived IDHwt and IDHmut gliomas. In Aim 3, we will compare the anti-seizure effects of two next- generation IDHmut inhibitors, AG-120 and AG-881, as well as memantine, an NMDAR antagonist that is already used to treat Alzheimer?s Disease. Each of these drugs will be tested as monotherapy and in combination with LEV. Successful completion of these Aims will establish the D2HG product of IDHmut as an epileptogenic agent, will shed more light on how IDHmut alters the nonneoplastic neural tissues surrounding glioma, and will foster clinical trials to determine the efficacy of IDHmut inhibitors, and memantine, against seizures in these patients.