Anis Contractor, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Activity driven modification of excitatory synapses is an exquisitely powerful process for the refinement of synaptic connections. Long-term potentiation (LTP) of synaptic transmission in the hippocampus is widely accepted to be integral to the formation and consolidation of memories. Due to the complexity and heterogeneity of the molecules underlying LTP, many of the cellular mechanisms are still not fully established. A complete description of these fundamental processes will allow us to understand the pathology of neurological disorders that result in the disruption of memory storage and retrieval. The mossy fiber synapse provides one of the main excitatory inputs to the CA3 region of the hippocampus. The recurrent network of the CA3 is particularly important in the storage and retrieval of associative memories. The mossy fiber synapse is critical to the modulation of this network and therefore is central to hippocampal function. Mossy fiber plasticity demonstrates some very interesting properties. LTP is expressed in the presynaptic terminal of the synapse, however there is still controversy over the site of induction. Recent findings have demonstrated a role for postsynaptic mechanism in the induction of mossy fiber LTP followed by trans-synaptic signaling to the pre-synapse, mediated by Eph receptor-ephrin interactions. In order to further explore the molecules involved in this unique pathway, this study will make use of mutant mice in which the relevant proteins have been genetically ablated or mutated to disrupt their signaling function. In addition, we will explore the exact pre- and postsynaptic mechanisms by which these molecules exert their action. These studies will provide new insight relevant to synaptic transmission and memory processes in the mammalian brain. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Kainate receptors are a functionally unique sub-family of glutamate-gated ion channels that mediate synaptic transmission, modulate neurotransmitter release and regulate cellular excitability in the central nervous system (CNS). Because of the critical role these receptors play in brain function, they have been linked to several neurological conditions including chronic pain, neuroinflammatory demyelinating diseases and temporal lobe epilepsy (TLE). Despite significant progress in recent years, there remain a number of fundamental questions about the function of kainate receptors that have been elusive because comprehensive pharmacological tools targeting the kainate receptors have been lacking. In these studies we will make use of mice with targeted mutations in the genes that encode the kainate receptors in order to address some of these remaining questions. These animal models provide unique opportunities to describe the basic function of these receptors and clarify their contribution to normal and convulsant activity in the brain. Thus, using in vitro electrophysiological recording techniques in brain slices, we will: 1. Delineate the role of calcium permeable kainate receptors in regulating synaptic transmission and developmental plasticity in the hippocampus and cortex;2. Determine the molecular pathways through which kainate receptors modulate intrinsic conductances, and thus regulate neuronal excitability in the hippocampus;3. Test the contribution of postsynaptic kainate receptors to spike coupling, and thus determine their role in the recurrent CA3 network of the hippocampus;a major focus for the generation of synchronized epileptiform activity. These studies will provide important insight into the molecular mechanisms by which kainate receptors affect synapses and cellular excitability, and will validate them as potential therapeutic targets in human (TLE). PUBLIC HEALTH RELEVANCE: Kainate receptors are glutamate-gated neurotransmitter receptors that are critical to synaptic signaling and cellular excitability in the central nervous system. Pathophysiological activation of these receptors has been linked to several important neurological conditions including chronic pain, neuroinflammatory demyelinating diseases, and temporal lobe epilepsy. The goals of this study are to delineate the actions of kainate receptors at synapses and to comprehensively uncover their roles in modulating neuronal excitability, thus providing further validation of these receptors as potential therapeutic targets.

DESCRIPTION (provided by applicant): Obsessive-compulsive disorder (OCD) is a chronic and debilitating anxiety disorder characterized by persistent intrusive thoughts, obsessions, compulsions, and repetitive habitual actions. It is estimated to affect 1-2% of the population, making it the fourth most common mental illness, yet current treatment options and therapies are limited and many OCD patients are unresponsive to first-line treatment. There is considerable evidence for the involvement of glutamatergic signaling in the cortico-striatal loop having a direct role in the abnormal behavioral inhibition and inappropriate compulsive or habitual actions symptomatic of the disorder. In preliminary experiments we found that mutant mice in which the genes encoding for kainate receptors, a modulatory glutamate receptor, are ablated have abnormal behaviors marked by elevated compulsive grooming and increased anxiety. This compulsive grooming and anxiety phenotype parallels that seen with several genetic mouse mutant strains displaying obsessive, self-injurious grooming behavior that have been proposed as mouse models of OCD. Kainate receptors are abundantly expressed in the striatum; however, their roles in regulating striatal synapses and circuits have not been defined. In this proposal we will take combined electrophysiological, optogenetic, behavioral and biochemical approaches to determine the cellular roles of kainate receptors in striatal circuits and their contribution to perseverative behaviors. Our hypothesis is that kainate receptor signaling in striatal spiny projection neurons (SPNs) plays a role in regulating synaptic transmission and plasticity. Loss of this regulation significantly impairs the output of the striatm and results in the maladaptive habitual and compulsive behavior. Thus, in specific aim 1 we will determine the contribution of kainate receptors to striatal synaptic function and plasticity ad test whether they regulate SPN synapses through a novel signaling mechanism discovered in our preliminary studies. In specific aim 2 we will determine whether interaction between kainate receptors and another known OCD-associated synaptic scaffold regulates synaptic kainate receptor function in the striatum. In specific aim 3 we will determine the circuit basis for compulsive behavior in mice by utilizing conditional genetic and pharmacological approaches to study behavior after specific manipulations. Together these studies will determine the cellular and circuit roles for kainate receptors in the striatum and their potential role in compulsive behaviors. This proposal will break new ground as the first to mechanistically associate the kainate receptor family to OCD, and to potentially provide a novel approachable target for the design of therapeutic strategies for this debilitating neuropsychiatric disorder.

DESCRIPTION (provided by applicant): Spontaneous electrical activity in the neonate is critical to synaptic refinement and epigenetic processes of neural development. Network bursts that contribute to circuit development are a hallmark of the young hippocampus when neurons are more excitable than in the mature CNS. Spontaneous activity of CA3 neurons in the hippocampus is largely dependent on intrinsic conductances that underlie the after hyperpolarization potential (AHP). This conductance can be dynamically regulated by a class of glutamate-gated receptors, kainate receptors (KARs), which have been prominently associated with neurodevelopmental and neuropsychiatric disorders. Neonatal KARs are potentially a key regulator of the AHP in the developing hippocampus~ however it is not known whether certain properties of neonatal KARs, (e.g. their editing status, their linkage to particular signaling pathways, or how they are activated), make them more likely to play this role in neonate versus the mature hippocampus. Here we propose to address these fundamental questions by determining how neonatal KARs produce long-lasting inhibition of AHPs to regulate excitability of hippocampal neurons. Overall these studies will define a role for neonatal KARs in regulating activity in the hippocampus and will address a gap in our knowledge about their specialized role in developing circuits. In Aim 1 we will use in vitro electrophysiological recording to determine whether KARs play a role in regulating the spontaneous activity of CA3 hippocampal neurons. Recordings will be made from KAR knockout mice and mutant mice that express only the mature form of the receptor to determine how these manipulations affect spontaneous bursting of hippocampal neurons. KARs are predominantly extrasynaptic during early development raising the question of how they might be activated. In Aim 2 we hypothesize that extrasynaptic KARs can be activated by ambient glutamate, and this form of tonic signaling is critical to their specialized function in the neonate. Finally, in Aim 3 we will determine how extrasynaptic hippocampal KARs might be activated in the neonate. We will test two specific hypotheses (i) that neonatal KARs are activated by synaptic glutamate (e.g. through spillover) or (ii) by glutamate released from a non-conventional mechanism (e.g. gliotransmission). Together these studies will determine the mechanism by which neonatal KARs regulate excitability in the developing hippocampus. Altered developmental processes in the neonate could ultimately contribute to some of the neurodevelopmental and neuropsychiatric disorders that are associated with KARs such as mental retardation, autism, schizophrenia, bipolar disorder, and epilepsy.

? DESCRIPTION (provided by applicant): Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and also the single most common known cause of autism. FXS results from an expansion of a CGG repeat sequence in the 5' untranslated region of the gene, which causes hypermethylation, transcriptional silencing, and a resultant loss of expression of the Fragile X mental retardation protein (FMRP). FMRP is a polyribosome associated RNA binding protein that regulates the translation of a large number of messenger RNAs, has roles in mRNA transport, as well as regulating the expression of miRNAs. Altered trajectories for the maturation and stability of synapses have been observed in the cortex of the mouse model of FXS (Fmr1 ko). However, the underlying mechanisms of these alterations in cellular and synaptic development are unknown. One important regulator of cortical development is the inhibitory neurotransmitter GABA. During early development GABA responses in many cortical neurons are excitatory, only maturing to their inhibitory, hyperpolarizing type later. Excitatory effects of GABA have been demonstrated to be important for neuronal proliferation, cell migration, and synaptogenesis. In recent studies we discovered that the regulated development of hyperpolarizing GABA responses is delayed in the cortex of Fmr1 ko mice. We propose that this will lead to an alteration in GABA signaling that contributes to a delay in synaptic and cellular maturation in the cortex. The objectives of this proposal are twofold. The first is to determine how the efficacy of GABA signaling is altered during the critical period, and whether there is a causal link between delayed maturation of GABA signaling and delayed maturation of fast spiking interneurons. The second is to determine whether the underlying mechanism for these changes result from alterations in miRNA expression that normally regulate the transporters underlying the reversal potential for GABA. We will achieve these objectives using two specific aims. Aim 1 will test whether a commonly used diuretic bumetanide (which acts centrally to inhibit the juvenile chloride cotransporter, NKCC1) can rectify the changes in GABA responses and delayed maturation of fast spiking interneurons. In Aim 2 using in vitro assays combined with validation in mouse and human derived neurons we will determine whether several candidate miRNAs can regulate expression of the juvenile chloride cotransporter NKCC1, which sets the reversal potential for GABA. These studies will address important knowledge gaps and potentially provide novel insights into developing cures for FXS. Because similar abnormalities of neuron connectivity and synapse maturation are implicated in other childhood brain disorders, it is possible that this research will open new avenues of study in other disorders characterized by autism and intellectual disability.

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.

Adult neurogenesis in the hippocampus occurs in the well-defined neurogenic niche in the subgranular zone of the dentate gyrus. Newborn neurons are continuously generated and mature over the course of weeks to integrate into the hippocampal circuit. Adult-born immature dentate gyrus cells (DGCs) have unique functional properties that give them a privileged role in circuits that define specific behaviors, and which are critical to episodic memory formation and retrieval. In particular these neurons play important roles in an animal?s ability to separate similar patterns and disambiguate overlapping memories, processes that become impaired during normal aging and in neurodegenerative and neuropsychiatric disorders. Therefore, mechanisms that regulate adult-born DGC maturation and integration are important in understanding diverse neurological disorders including Alzheimer?s disease, schizophrenia and post-traumatic stress disorders. Kainate receptors are a class of glutamate receptor whose contributions to heterogeneous synaptic processes are still not fully understood. The premise of these studies is built upon foundational studies in which we discovered that the maturation of adult-born DGCs is more rapid after ablation of kainate receptors. We found that this effect was likely through a disruption of intracellular Cl- gradient because of an effect on a neuronal Cl- transporter. We will fully describe the altered molecular, synaptic, and functional alterations after loss of kainate receptor signaling and will test whether GABA disruption is causal to the altered integration of maturating DGCs into the hippocampal circuit. We will determine how altered maturation of adult-born DGCs affects the animal?s ability to discriminate between similar patterns and temporal overlap of episodic memories and using in vivo microendoscope imaging correlate cellular activity to behavioral measurements in a pattern separation task. The goal of this project is to examine a new mechanism by which glutamate receptors affect adult-born DGC integration by modulating GABA signaling. These studies would define novel processes that regulate adult-born neurons that could underlie the known involvement of kainate receptor signaling in mechanisms of learning and memory.

Synaptic plasticity in the hippocampus is critical to the formation, storage and retrieval of episodic memories. The separate regions of the hippocampus have evolved to play distinct roles in spatial navigation, contextual memories, social memories, and our ability to separate patterns or complete patterns to reconstruct partial memories. In particular the dentate and CA3 regions of the hippocampus are involved in our pattern separation that is vital to the integrity of episodic memories. At the center of this region are the mossy fiber afferents that make conditional detonator synapses onto CA3 pyramidal neurons, which have a distinct form of presynaptic cAMP dependent plasticity. Despite the importance of cAMP plasticity to memory formation and retrieval in the CA3 the exact molecular mechanisms underlying MF LTP have not been uncovered. The premise of this research builds upon our finding that there are at least two downstream cAMP effectors, PKA (protein kinase A) and Epac2 (exchange protein directly activated by cAMP 2), that contribute to cAMP dependent MF LTP. Despite these findings it is still not known how signaling by each of these effectors results in elevated release from MF synapses and, what are the important targets and substrates that are involved in MF LTP. Here we will use a comprehensive approach with leading edge proteomic, biochemical and electrophysiological approaches to determine the signaling partners of the cAMP effectors, and uncover the physiological mechanism of their actions. Thus, in Aim 1 we will take orthogonal approaches to find the interactors and substrates of PKA and Epac2 and validate and verify these by performing high resolution labeling in situ. In Aim 2 we will determine the exact physiological mechanism that underlie increases in release of neurotransmitter at MF synapses using a combined optogenetic-knockout/pharmacological strategy. In the final Aim we will answer the question of how these different but convergent mechanisms are engaged during naturalistic activity patterns, and whether selective disruption of these effectors impairs the ability of mice to separate similar patterns that underlie the formation and retrieval of episodic memories.