Hee Jung Chung - US grants

[unreadable] DESCRIPTION (provided by applicant): G-protein coupled inwardly rectifying potassium channel (GIRK) regulates neuronal excitability by mediating postsynaptic inhibitory effects of various transmitter in brain. Recent studies showing the presence of GIRK in dendritic spines suggest that excitatory neurotransmitter receptors may regulate GIRK function. Indeed, neuronal activity increased GIRK surface expression in dendrites and spines of hippocampal neurons in a protein synthesis and a protein phosphatase (PPI)-dependent manner. The objectives of this proposal are to investigate the molecular mechanisms and physiological consequence of activity-induced GIRK surface expression. Live-imaging and surface immunostaining will be performed to investigate how protein synthesis, post translational surface trafficking of GIRK and PP1 activity contribute to activity-induced GIRK surface expression (Specific Aim A-C). To study the functional significance of activity-induced GIRK surface expression (Specific Aim D), whole-cell patch clamp recording of GABAb-mediated GIRK current before and after neuronal activity will be performed. These studies will reveal novel mechanisms for modulating inhibitory synaptic function [unreadable] [unreadable]

? DESCRIPTION (provided by applicant): Epilepsy is caused by excessive neuronal excitability characterized by seizures, which are abnormal and uncontrolled discharges of action potentials. This common brain disorder is often caused by mutations in ion channels that form the basis for neuronal intrinsic excitability. Voltage-gated Kv7/KCNQ K+ channels potently inhibit repetitive and burst firing of action potentials. Kv7 activators are used as anti-epileptic drugs whereas mutations in Kv7.2 and Kv7.3 subunits cause epilepsy in humans. My work has shown that some epilepsy mutations impair polarized enrichment of Kv7 channels at the axonal surface, the subcellular domain governing action potential initiation and conduction, suggesting that their correct axonal targeting is critical for their physiologic function. However, the underlying mechanisms remain poorly understood. The goal of this proposal is to understand how axonal targeting of Kv7 channels is achieved and regulated by epilepsy mutations and neuronal activity, and how basal and regulated targeting impacts intrinsic excitability. This proposal is significant because dissecting these unexplored mechanisms will increase our understanding of the role of axonal Kv7 channels in regulating excitability, and foster the development of new therapeutic strategies for epilepsy that could enhance axonal surface density of Kv7. Given that chronic blockade of neuronal activity in hippocampus leads to temporal lobe epilepsy, characterizing the regulation of Kv7 axonal targeting by chronic activity blockade of hippocampal neurons may provide mechanistic insights into plasticity of intrinsic excitability and epileptogenesis. My work has shown that Kv7 enrichment at the axonal surface involves a region in the Kv7.2 C- terminal tail upstream of ankyrin-G binding domain. This axon-targeting domain contains phosphorylation sites and interacts with calmodulin, syntaxin 1A, AKAP79/150 and PIP2. In our preliminary studies, we discover that Kv7 axonal targeting is regulated by calmodulin-mediated exit from the endoplasmic reticulum, dynamin-mediated endocytosis, and phosphorylation of Kv7.2. We further show that homeostatic increase in excitability induced by chronic activity blockade accompanies reduction in Kv7 current, Kv7.3, and AKAP150. Based on these data, our hypothesis is that Kv7.2 interacting molecules and phosphorylation mediate basal and activity-regulated Kv7 axonal targeting by linking Kv7 to the core traffic machinery for polarized targeting. To test this hypothesis, Aim 1 will determine the traffic pathways that mediate Kv7 axonal targeting. Aim 2 will identify Kv7.2 interacting proteins and phosphorylation that bind to the core traffic machinery for polarized targeting. Aim 3 will characterize the regulation of Kv7 axonal targeting by chronic activity blockade. To execute these aims, we will use live imaging, immunostaining, binding assays, and electrophysiology in dissociated and organotypic hippocampal cultures, which preserve the functional and morphological polarity of neurons. We will also utilize epilepsy mutations, knock-out mice, and in vivo alterations of neuronal activity.

PROJECT SUMMARY / ABSTRACT Alzheimer's disease (AD) afflicts more than 5 million Americans, yet no known drug is able to prevent or stop the disease. Before AD fully develops with insoluble amyloid-? plaque deposits and neurodegeneration, there is a progressive cognitive decline associated with the impairment of synaptic plasticity that underlies learning and memory. This abnormal synaptic plasticity is likely caused by soluble amyloid-? oligomers affecting the synaptic levels of AMPA and NMDA receptors, two glutamatergic receptors that mediate induction and expression of synaptic plasticity. However, the underlying detailed mechanisms are not known and are exceptionally challenging to study due to the complex behavior of these receptors and the small nanometer-scale dimensions of the synaptic domains in which they reside. The goal of this proposal is to understand the molecular details of abnormal synaptic plasticity present in early AD by developing small nanoparticle-based optical probes and new microscopy techniques to analyze the position and dynamics of AMPA and NMDA receptors in normal and AD brains. This goal will be accomplished through the individual and collective efforts of three principle investigators, Paul Selvin (microscopy), Andrew Smith (quantum dots) and Hee Jung Chung (neurobiology). They have previously worked as a team to publish two manuscripts on generating small quantum dots (sQD) (< 10 nm diameter) that can enter the neuronal synapse and accurately follow the receptor number and dynamic placement in dissociated cultured neurons. To achieve this goal, Aim 1 will optimize super-resolution imaging techniques for sQDs in dissociated hippocampal culture and thick hippocampal slices with intact circuitry, specifically focusing on 1- and 2-photon excitation with FIONA and PALM/STORM microscopy. This will allow < 20 nm resolution in all three dimensions. Aim 2 will develop a novel set of sQDs that are smaller, stable, and monovalent with minimal non-specific interaction with tissue. Aim 3 will apply sQDs and super-resolution optical methods to perform single-molecule imaging of glutamate receptors during synaptic plasticity in hippocampal culture and acute slices from wild-type and AD transgenic model mice. Because of our on-going successful collaboration, we are able to work with the AD model immediately, while new microscopy and quantum dots are being generated. This research will increase our understanding of the early pathogenesis of AD and therefore foster the development of new therapeutic strategies that could specifically inhibit the progression of cognitive decline of this disease.

Abstract The ability to measure the molecular mechanisms of neuronal communication at the nanometer spatial scale will have enormous impact in both basic bioscience and in future clinical neuroscience. In particular, AMPA- and NMDA-type glutamate receptors (AMPARs/NMDARs, known as iGluRs) are involved in neuron-to-neuron communication across synapses, where these receptors contribute to learning and memory, and when dysregulated, to neurodegenerative diseases including Alzheimer's, Parkinson's and complications from strokes. A critical mechanistic event is the transport of iGluRs into and out of synapses (or parts of synapses) in a dynamic process called synaptic plasticity. A revolution is underway because of the recent ability to resolve these events at the nanometer-scale using fluorescence super-resolution microscopy (FSRM). However significant inherent problems with this technology have led to confounding results and misinformation. The biggest problem has been with the fluorescent probes used to image receptors: conventional organic fluorescent probes last only a few seconds; commercial (and big) quantum dots (bQDs), despite their exceptional brightness and photostability, are over 20 nm in diameter and are too large to fit inside the synaptic cleft where iGluRs are active. We recently overcame this problem through an R21, which enabled us to develop small quantum dots (sQDs) that are <10 nm in diameter. They specifically label iGluRs in the synaptic cleft, which is just ~20-30 nm wide. The sQDs do this with tremendous brightness and stability, resulting in FSRM images in 3-dimensions with 100 ms time-resolution for greater than 2 minutes of continuous excitation. In contrast, bQD-labeled AMPARs are predominantly stuck in the extra-synaptic space because steric hindrance prevents them from going inside. We have recently extended these findings with a newer sQD that is completely stable, and with small organic fluorophores that we now show are stable enough, on live neurons (which previously had been too photolabile for such measurements.) Our findings, some of which have been published in 3 papers resulting from our R21 grant, may have tremendous implications for basic science and health: the surface mobility and trafficking of iGluRs, which depend on the ease of diffusion inside and outside of synapses, regulates synaptic efficacy. Here we wish to understand the distribution and dynamics of iGluRs, both within the synapses and between synapses, using our new sQDs and other new photoactivatable fluorescent proteins and some organic fluorophores. For this, a number of new advances in optics, probe design, and care with receptor monovalency are necessary. After these technical problems are solved (which will be useful to answer many different biological questions), we will validate the biology that we have observed, and to apply these to proof-of-principle experiments involved in two key biological questions: 1) In what way do receptors move into and around the synapse during homeostatic and synaptic plasticity? 2) Do endocytosed receptors communicate with each other between synapses on the same neuron?

Significance: The ability to measure the molecular mechanisms of neuronal communication at the nanometer spatial scale will have enormous impact on basic bioscience and likely to future clinical neuroscience. In particular, AMPA- and NMDA-type glutamate receptors (known as iGluRs) are dynamically involved in neuron- to-neuron communication across the thin (?30 nm) synapse; when dysregulated, neurodegenerative diseases result, such as Alzheimer?s and Parkinson?s diseases. We?and others?have tracked these events with nanometric resolution using super-resolution fluorescence microscopy (SRFM). Using small probes?quantum dots (?12 nm diameter) and other photostable fluorophores, developed in our lab in the preceding grant?we came up with some surprises. We find that a large fraction of the AMPARs reside in the synapse where their mobility is restricted; during long-term-potentiation (LTP, a molecular underpinning of memory formation), we?ve quantified their numbers and find during their maintenance phase that their lateral diffusion is rare; NMDARs have extra-synaptic nanodomains which may keep their numbers from rising during LTP. But are these, and other results, correct? To validate these preliminary results, we will measure the placement and diffusion of the iGluRs, primarily AMPARs, using three different SRFM techniques, each one having its own advantages and disadvantages. We will also determine the 3D-orientation of the synapse, the effect of probe size and type, the details of LTP activation, and quantitatively determine the number of iGluRs at each synapse. The results between the three techniques will be compared. Innovation: Each SRFM technique has new aspects, particularly with respect to neuroscience. First, we will improve the PALM/STORM technique (one type of SRFM) to test the distribution and dynamics of iGluRs more accurately. We will use new probes?nanobodies and scFv?s?against post-synaptic proteins and iGluRs, and test new sQDs and new cross-linking reagents against iGluRs. We will also determine the orientation and position of the synaptic zone by labeling neuroligin and various presynaptic proteins, such as Bassoon and RIM1/2, first under basal conditions and then with chemical LTP (cLTP). Second, we will use and develop PAINT, another form of SRFM, which has recently been shown to have a 100× increase in speed with excellent spatial resolution??5 nanometers in 0.2 sec. We will show that quantitative-PAINT can be applied to fixed neurons and can be used to measure cLTP on an individual synapse. And for the first time, we will apply PAINT to a living neuron under physiological conditions to measure AMPAR dynamics. With PAINT, we will be able to test how many iGluRs there are per synapse, whether they are synaptic or extra-synaptic, and how the number of iGluRs change with cLTP. Third, we will utilize a fluorogenic activating protein (FAP) with iGluRs and show that the number of receptors can be measured in living neurons with nanometric resolution, no background, and potentially fast response to cLTP. This method will therefore provide another test of iGluR structure & dynamics.