Ryohei Yasuda - US grants

The fascinating capabilities of neural networks arise from the interplay between the intrinsic properties of neurons and their interconnections. Synapses are the first place where the neuron receives information. In order to understand information processing and storage in the brain, it is important to first understand the properties of synapses that define their response to stimulation and how these properties are altered during development or by experience. Recent modeling work by the PI has shown that the microscopic details of glutamate receptor activation have important implications for synaptic plasticity. The studies conducted in the present project will extend this research in several important directions. First, a combination of experiment (focal glutamate uncaging using two-photon microscopy) and modeling (Monte Carlo models of stochastic receptor activation) will be used to assess how the differences in the biophysical properties of the NMDA subtype of glutamate receptor shape the synaptic response. Second, a large scale computational model of signal transduction networks involved in synaptic plasticity will be developed to parse out the differential contributions of different glutamate receptor subtypes in long-term modifications of synaptic strength. The models will be based on detailed anatomical and physiological data, and the results will be compared with experiments. Results of the proposed research will shed light on some of the complex features of signaling events that drive bidirectional modification of synaptic strength. The investigators include a theorist and experimental neuroscientist and the project thus serves as a model for how distinct approaches can be brought together into a cohesive effort to address a general problem in neuroscience. The results of this project are also expected to generate interest from a diverse community of researchers ranging from mathematics to molecular, cellular and systems neuroscience. The research is part of an initiative at Duke to promote multidisciplinary approaches to multi-scale modeling, computation and analysis. The funding will also support the PI in the development of a course on theoretical neuroscience at Duke for students from neurobiology, biomedical engineering, and mathematics.

[unreadable] DESCRIPTION (provided by applicant): The small GTPase protein Ras is important for many neuronal processes essential to the regulation of synaptic connections such as strengthening of synaptic transmission, formation of new synapses and regulation of cell excitability. Ras is also important for protein synthesis and gene transcription required for long-term maintenance of synaptic plasticity. Consistent with essential roles of Ras signaling in synaptic plasticity, failures in Ras signaling are associated with diseases causing cognitive impairments and learning deficits such as autism, X-linked mental retardation and neurofibromatosis 1. Although the importance of Ras signaling in synaptic plasticity is well recognized, it is not clear how Ras decodes and relays calcium dynamics to regulate its diverse downstream effects. In neurons, Ras signaling is involved in signaling events spanning different compartments, including spines, dendrites and the nucleus. Thus, the spatiotemporal dynamics of Ras signaling are likely to be important in determining its downstream effects. To study Ras signaling mechanism in neurons, we have recently developed a fluorescence technique that allows us to image Ras activity with single synapse resolution in living neurons deep in brain tissue, using this technique, the objective of this proposal is to understand the mechanisms of spatiotemporal regulation of Ras signaling. Our hypothesis is that the spatiotemporal pattern of Ras signaling is shaped by 1) Ras activation controlled by calcium-dependent signaling networks involving multiple kinases and feedback loops, and 2) spatial spreading of Ras activation due to the diffusion and trafficking of Ras and Ras regulators. To test this hypothesis, we will image Ras activity in spines and dendrites in response to activation of glutamate receptors on a single spine using 2-photon glutamate uncaging. Our preliminary data suggested that Ras activation occurs at the stimulated spine, subsequently spreading into its parent dendrite and nearby spines. The specific aims of this proposal are to 1) identify upstream signaling that activates Ras in individual spines, 2) determine the mechanisms and roles of the spatial regulation of Ras in dendrites, and 3) elucidate mechanisms underlying differential activation of the Ras GTPase family. This work will advance our understanding of how Ras couples calcium with synaptic plasticity, and ultimately with learning and memory. Moreover, our study will provide insights into the molecular mechanisms underlying Ras-related mental disorders. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Dendritic spines are small (0.1 - 0.01 femtolitter) excitatory postsynaptic compartments emanating from the dendritic surface. Ca2+ influx into spines activates signaling networks consisting of hundreds of species of proteins that induce diverse forms of synaptic plasticity. Rho GTPase proteins, particularly Rac1, RhoA and Cdc42, are critical components of these signaling networks, and their activation plays an important role in regulating the morphology and function of dendritic spines. Consistent with the important role of Rho signaling in spine morphology and function, mutations in Rho signaling pathways are associated with many forms of mental retardation and autism. In this study, we will develop a technique to measure Rho signaling in single dendritic spines while they undergo morphological and functional plasticity in brain slices. To do so, we will combine 2-photon fluorescence lifetime imaging microscopy (2pFLIM) with fluorescent resonance energy transfer-based Rho activity sensors extensively optimized for 2pFLIM. Our preliminary data demonstrates that the activity of Cdc42 is restricted to spines undergoing synaptic plasticity, while Rac1 and RhoA activation spreads along dendrites over ~10 5m and invades neighboring spines. These results suggest that each Rho signaling pathway functions on a different length scale. We will study the mechanisms and roles of the spatial spreading of Rho GTPase proteins by measuring and perturbing the spatiotemporal dynamics of Rho signaling. The specific aims of this project are to 1) establish techniques to image Rho signaling in individual spines, 2) elucidate the mechanisms and roles of spatiotemporal dynamics of Rho during synaptic plasticity, and 3) identify signaling pathways connecting calcium with Rho GTPase activation and synaptic plasticity. This study will illuminate the molecular mechanisms of morphological and functional plasticity of dendritic spines, and will provide insights into mental diseases caused by mutations in Rho signaling pathways. PUBLIC HEALTH RELEVANCE: The shape and function of synapses are regulated by signaling mediated by Rho proteins. Many forms of mental retardation and autism are caused by abnormal Rho signaling. This project will develop a novel technique to measure the activity of Rho proteins in single synapses to elucidate the mechanisms linking the activity of Rho proteins and the morphology and function of synapses. This will facilitate understanding of mental retardation and autism.

DESCRIPTION (provided by applicant): Signaling mechanisms at synapses requires a precise number and arrangement of receptors, ion channels, and adhesion molecules. AMPA-type ionotropic glutamate receptors are the major mediators of fast excitatory synaptic transmission in the brain, and alterations in the number of AMPA receptors at the synapse is a critical feature of synapse formation, maturation, and synaptic plasticity. Although previous studies have helped define the functional significance of AMPA receptor trafficking during NMDA receptor-dependent synaptic potentiation at diverse glutamatergic synapses, the protein machinery that transports AMPA receptors through intracellular compartments and the mechanisms by which this machinery senses NMDA receptor- dependent Ca2+ influx remain poorly understood. To address these important questions, my laboratory has initiated a program of cell biological and physiological studies to analyze the intracellular signaling pathways that activate endosomal transport of AMPA receptors in dendrites and dendritic spines - the primary postsynaptic compartment in the mammalian brain. We have identified a key actin-based motor protein, myosin Vb (MyoVb), which resides in dendritic spines, traffics AMPA receptors, and responds to elevated intracellular Ca2+ by associating with AMPA receptor- containing recycling endosomes (REs) through the Rab11/Rab11-FIP2 endosomal adaptor complex. Further, we have recently found that activation of MyoVb by NMDA receptor-induced Ca2+ influx leads to the physical translocation of REs into dendritic spines and subsequent local spine exocytosis of AMPA receptors and other membrane material for synaptic potentiation and spine growth during long-term potentiation (LTP). Taking advantage of these preliminary data and our ability to monitor and manipulate endosomal transport in dendrites and spines, we propose to define the underlying molecular and cellular mechanisms that activate, regulate, and terminate AMPA receptor transport by the MyoVb/Rab11/Rab11-FIP2 complex. This work will provide insight into fundamental mechanisms that underlie neuronal signaling and synaptic plasticity. Moreover, because endosomal sorting and transport of AMPA receptors regulates plasticity in diverse neural circuits in a wide range of pathologic insults and in response to therapeutic agents relevant to numerous neurologic and psychiatric diseases, these studies hold promise for the development of novel therapeutic strategies. PUBLIC HEALTH RELEVANCE: The proposed research will uncover molecular mechanisms that regulate brain cell communication at synapses. Abnormal function of synapses contributes to epilepsy, memory decline, depression, autism, schizophrenia, and addiction. By helping to understand how nerve cell synapses are adjusted during brain development and modified as we learn, the proposed research will define novel targets and therapeutic strategies for these devastating neurological and psychiatric disorders, which currently have a profound negative impact on public health.

DESCRIPTION (provided by applicant): In the central nervous system, most excitatory synapses terminate on dendritic spines, small postsynaptic compartments emanating from the dendritic surface. Ca2+ influx into spines activates a signaling network required for diverse forms of synaptic plasticity. In particular, the family of ~150 small GTPase proteins is important for many aspects of synaptic plasticity, including regulation of the actin cytoskeleton, membrane trafficking, vesicular transport and gene transcription. In this study, we will develop a technique to monitor the activity of more than 60 small GTPase proteins in single dendritic spines in brain slices. To do so, we will develop scalable designs and optimization schemes to make fluorescence resonance energy transfer (FRET)-based sensors reporting small GTPase activity with high sensitivity. To quantitatively image FRET signal with high sensitivity and resolution in light scattering brain tissue, we will use 2-photon fluorescence lifetime imaging microscopy (2pFLIM). Our preliminary data demonstrates that our design can be applied to many small GTPase proteins. Using these sensors, we will screen small GTPase proteins activated by NMDA receptors, and image their activity in single dendritic spines undergoing structural and functional plasticity. Our specific aims are 1) to develop and test sensors for small GTPase proteins, 2) to screen small GTPase proteins for those activated by Ca2+ through NMDA receptors, 3) to measure the spatiotemporal dynamics of selected small GTPase proteins in single dendritic spines. This study will provide insights into how the activity of small GTPase proteins is coordinated in spines to produce structural and functional plasticity of dendritic spines, and will illuminate the molecular mechanisms of synaptic plasticity and ultimately learning and memory. PUBLIC HEALTH RELEVANCE: Signaling mediated by small GTPase proteins is important for synaptic plasticity and ultimately learning and memory. This project will develop a novel technique to measure the activity of small GTPase proteins in single synapses to elucidate the mechanisms by which small GTPase signaling regulates synaptic function. This will facilitate understanding the molecular mechanisms of mental diseases related to abnormal small GTPase signaling such as mental retardation, autism, schizophrenia and Alzheimer's disease.

? DESCRIPTION (provided by applicant): Synaptic plasticity, a cellular basis of learning and memory, is mediated by a complex biochemical signaling network consists of hundreds of signaling proteins. In particular, Ca2+- dependent signaling in dendritic spines, tiny postsynaptic compartments emanating from dendritic surface, plays a key role in the induction of long-term synaptic plasticity. In order to understand the operational principles of this network and the mechanisms underlying synaptic plasticity, the activity of hundreds of proteins under many manipulations needs to be measured in single dendritic spines during synaptic plasticity. The activity of proteins in spines has been measured using advanced fluorescence resonance energy transfer (FRET)-based techniques. However, thus far only a small fraction of the entire network has been measured. Our understanding of signaling networks is limited by this scarcity of measurements for signaling components. Thus, the goal of this project is to establish a high-throughput system for the development and optimization of signaling sensors, and a fully automated system for imaging signal transduction during plasticity in single dendritic spines. Using this high-throughput imaging system, we aim to improve the overall efficiency of data output by orders of magnitude, producing large data sets that could be further analyzed for information flow in the signaling network and connectivity between network elements. Thus, this project is expected to lead to a dramatic advance in our understanding of intracellular signaling in neurons and provide key insights into the mechanisms underlying synaptic plasticity and ultimately learning and memory.

Project Summary/Abstract Synaptic plasticity is thought to be a basis of learning and memory of the brain. Signaling mechanisms underlying synaptic and behavioral plasticity have been extensively studied with the aid of pharmacological and genetic manipulation of signaling. However, it has been difficult to assess the spatiotemporal aspects of signaling activity particularly. The goal of this proposal is to develop a new technique based on genetically encoded light- inducible kinase inhibitors to resolve the spatiotemporal dynamics of signaling required for synaptic and behavioral plasticity in vivo. Our preliminary data using a newly- developed photo-inducible CaMKII inhibitor demonstrates that ~60 s of CaMKII activity is sufficient to induce structural plasticity of dendritic spines. We aim to 1) develop photo- inducible inhibitors for kinases including CaMKII, PKC and PKA, 2) determine the timing of kinase activity required for synaptic plasticity in slices, and 3) identify the spatiotemporal window of kinase activity required for learning-related dendritic spine turnover in vivo and animal?s performance improvement after learning. The new tool will provide new insights into the action of kinases in vivo during synaptic and behavioral plasticity.

Abstract Dendritic spines are mushroom-shaped postsynaptic compartments that host intracellular signaling cascades important for synaptic plasticity and, thereby, learning and memory. Signaling events in spines involve a network composed of hundreds of proteins interacting with each other extensively. Synaptic plasticity is typically induced by Ca2+ elevation in spines, which activates a variety of signaling pathways. This leads to changes in the actin cytoskeleton and membrane dynamics, which in turn causes structural and functional changes of the spine. Recent studies have demonstrated that the activities of these proteins have a variety of spatiotemporal patterns, which orchestrate signaling activity in different subcellular compartments at different time scales. To better understand the operational principles of this network and the mechanisms underlying plasticity, we will develop tools to optically measure and manipulate signaling activity in neurons in both brain slices and in awake, behaving animals during plasticity. In particular, we aim to develop innovative technology to image and measure endogenous proteins by combining advanced imaging techniques, new optogenetic tools and genome-editing technology. Using these tools, we will determine time windows of signaling activity mediated by endogenous proteins, and elucidate how intracellular signaling mediates synaptic, circuit and behavioral plasticity. This will thus lead to a better understanding of how information is processed at different time scales and provide new insights into the molecular mechanisms underlying learning and memory.