Project title: Imaging Ras Signaling in Neurons
Investigator’s name : Ryohei Yasuda, PhD
Title: Postdoctoral researcher, Cold Spring Harbor Laboratory.
(Assistant Professor at the Duke University Medical Center from 7/1/2005)
Phone: 516-367-6929, Fax: 516-367-8866
E-mail: yasuda@cshl.edu
Address: Cold Spring Harbor Laboratory, 1 Bungtown Road, Cold Spring Harbor, NY 11724
Category: Track B (Cellular / Molecular Imaging)
Sponsored research officer:
Dean / President :
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Section I: Hypothesis
Synaptic plasticity is considered a cellular basis for learning and memory. Activation of a small GTPase protein Ras is one of the key steps for several forms of synaptic plasticity such as rapid synaptic potentiation 1 / depression 2 and formation of new synapses 3 . Ras activation also leads to gene transcription important for maintenance of synaptic plasticity 4 as well as neuronal differentiation, survival and death 5 . This diverse spectrum of cellular responses by
Ras activation raises the question of how Ras signaling couples certain upstream stimuli to specific downstream effect. Precise regulation of temporal and spatial localization of Ras activation must be an important factor to achieve the specificity. To image directly Ras activity pattern, we have been developing a fluorescence resonance energy transfer (FRET)-based Ras fluorescence sensor. The combination of fluorescence lifetime measurements to measure FRET and two-photon laser scanning microscopy (2 photon fluorescence lifetime microscopy or 2PFLIM) allows for the quantitative examination of Ras dynamics at the single synapse level in highly light-scattering brain tissue. Using this technique, we will visualize spatial-temporal pattern of Ras activity in response to various electrical and pharmacological stimuli. The measurements will improve our understanding of Ras signaling in learning and memory, and hopefully give insights into how we can treat Ras-related mental disorders such as autism
6 , X-linked Mental Retardation 7 and Neurofibromatosis type 1 (NF1) 8 .
Section II: Aim of the proposed research project
Specific aim 1: Optimization and characterization of Ras imaging technology a.
Development and optimization of 2PFLIM. b.
Optimization of signal-to-nose ratio of FRET-based Ras sensor using HEK293 cells. c.
Characterization and verification of the Ras sensor to accurately report endogenous Ras activation in neurons in cultured hippocampal slices.
Specific aim 2: Imaging spatial-temporal patterns of Ras activation at the single synapse level in cultured hippocampal slices. a.
Imaging spatial-temporal patterns of Ras activation in response to physiological stimuli such as synaptic stimulation and back-propagating action potentials in neurons at the single synapse level. b.
Observation of difference in Ras dynamics upon induction of long-term potentiation (LTP) and long-term depression (LTD) of synaptic transmission. c.
Stimulating glutamate receptors on a single dendritic spine using 2-photon glutamate uncaging and observe to what extent Ras activation is restricted in the stimulated spine. d.
Deducing a model to explain observed spatial temporal patterns of Ras signaling.
Section IIII: Research Significance and Potential clinical applications of the research
Learning and memory are based upon changes in the activity dependent regulation of the synaptic strength and the formation / retraction of synapses. The formation of synaptic plasticity is initiated by opening of NMDA-type glutamate receptors (NMDARs) and calcium channels, which increases calcium concentration in postsynaptic compartments called dendritic spines. A small GTPase protein Ras is one of the key molecules that relay the calcium to synaptic plasticity 4 . Ras activity is controlled by its activators, guanine exchange factors (GEFs), and inactivators,
GTPase activating proteins (GAPs). Although several calcium-dependent GEFs and GAPs are identified, their exact roles in synaptic plasticity are unknown. Ras activation leads to phosphorylation of extracellular signal-related kinase (ERK), which branches out to many downstream effects. First, Ras-ERK pathway increases the number of
AMPA-sensitive glutamate receptors into synapses 1 , which is one of the mechanisms of rapid potentiation of synaptic transmission. Also, the signaling activates local protein synthesis important for several forms of long lasting synaptic plasticity 2 4 . Furthermore, downstream of Ras-ERK signaling leads to gene transcription in the nucleus, which is essential for maintenance of synaptic plasticity as well as neuronal differentiation, survival and death 4 SUG. Consequently, Ras-ERK pathway is required for many forms of synaptic plasticity including longterm potentiation 1 , depression 2 and spine-genesis 3 . Finally several behavioral paradigms, such as the Morris water maze and fear conditioning requires Ras-ERK pathway 4 8 9 .
The extensive branching of Ras-ERK pathway, which often leads to opposite cell responses, raises the question of how the pathway achieves the specificity, such that certain upstream stimuli lead to specific downstream effect. One parameter would be temporal dynamics of the pathway, like in case of calcium signaling where high and
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short calcium transient potentates synapses while low prolonged calcium depresses. Also, considering the highly polarized and compartmentalized morphology of neurons, spatial localization of Ras activity should be another important determinant for leading specific downstream effects. Therefore, an examination of spatial-temporal aspects of Ras activity at the single synapse level will improve our understanding of Ras in synaptic plasticity.
Consistent with essential roles of Ras pathway in synaptic plasticity and learning and memory, there are important links between learning and cognitive deficits and mutations in Ras pathway. For example, autism is associated with mutations in a gene encoding Ras 6 , and one of genes causing X-linked mental retardation encodes a downstream kinase of Ras-ERK pathway 7 . Furthermore, neurofibromatosis type 1, which is often associated with learning disability, is caused by mutations in a single gene encoding neurofibromin, a multidomain molecule with
GAP activity. Learning disabilities of a NF1 mouse model is caused by failure of synaptic plasticity due to excessive activity of Ras, and importantly it is reversed by decreasing Ras activity genetically and pharmacologically 8 .
Considering the multi-functionality of Ras, fairly subtle perturbation of spatial-temporal dynamics of Ras activation may cause failure in synaptic plasticity and thus learning deficits. Therefore, understanding mechanisms of controlling Ras activity patterns will hopefully suggest how we can treat these diseases.
Section IV: Methods
Development and optimization of imaging methodology
Optical imaging is the only method by which signaling can be measured at the single synapse level in living neurons. Signaling dynamics can be visualized using FRET-based sensors of protein activity. FRET occurs when two fluorophores are in close proximity (~5 nm), such that the excited donor fluorophore transfers energy to the acceptor fluorophore. With FRET, the donor fluorescence is quenched, and the acceptor fluorescence increases.
Therefore, FRET can be a readout of interactions between proteins tagged with fluorophores. FRET is frequently measured as a ratio of acceptor fluorescence to donor fluorescence. With the ratiometric method, however, quantification of FRET is difficult due to the dependence on fluorophore concentration and the influence of wavelength-dependent light scattering. Instead, we quantify FRET using measurements of fluorescence lifetime, which is defined as the time between excitation and photon emission 10 . The fluorescence lifetime decreases in proportion to FRET efficiency independent of the fluorophore concentration or the wavelength-dependent light scattering. To visualize Ras activity in living neurons in highly scattered brain tissue at the single synapse level, we combined fluorescence lifetime measurements with 2-photon laser scanning microscopy (2PFLIM; Figure 1). We will further optimize software and optical and electrical components to improve signal-to-noise ratio and reduce the time taken for generating a single image (currently ~ 1 min).
FRET-based Ras sensor
To visualize Ras activity, Ras is tagged by green fluorescence protein (GFP) and its binding partner Ras Binding Domain of Raf
(RBD) by red fluorescence protein (RFP; Figure 1A). When Ras-
GFP is activated, RBD-RFP binds to Ras-GFP, causing FRET between GFP and RFP. Thus, we can image Ras activity by quantifying FRET. The efficiency of FRET depends on several factors such as the distance between the two fluorescence proteins, the fluorophore orientation and the fluorescence spectral overlap 10 .
Thus, the signal-to-noise ratio can be optimized by tuning linkers connecting the fluorescence proteins and target proteins and location of the fluorophores. We test and optimize the signal-to-noise ratio of the sensor in a well-characterized system: growth factor dependent
Ras activation in HEK293 cells. The Ras sensor in HEK293 cells show a dramatic decrease of fluorescence lifetime by a growth factor application, reporting Ras activation. The sensor will be optimized further in neuron so that the sensor reports endogenous Ras activity.
We transfect the Ras sensor in neurons in cultured hippocampal slices using gene-gun and measure the activity in response to KCl application, which causes depolarization of neurons to open calcium cannels on spines and dendrites (Figure 1B). We will also measure, using biochemical assay, the endogenous Ras activity and check if the time course and pharmacological properties of endogenous Ras activity agree with the
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fluorescence lifetime measurements. This method has already caught one problem of the sensor: binding between
RBD and active Ras is so strong that the signal of the sensor does not reverse after a washout of KCl. By introducing a point mutation to RBD to decrease the affinity between Ras and RBD, reversible signal was obtained. We will further test whether the overexpression of the sensor alters neuronal properties by using electrophysiological measurements. If significant overexpression effects exist, we will further introduce mutations to minimize the interference with endogenous signaling.
Imaging Ras activity in response to physiological stimulation
We will image Ras dynamics in response to physiological electrical stimulations, backpropagating action potentials
(BAPs) and synaptic stimulations, using 2PFLIM. These two stimulations evoke calcium transients with different spatial-temporal patterns. BAPs cause opening of calcium channels on dendrites and spines to produce global calcium transients 12 . Because calcium transients are proportional to frequency of AP pulses, it is straightforward to correlate the amplitude of calcium transients and Ras activations. In contrast, synaptic stimulations open NMDARs to produce calcium transients largely restricted in the stimulated spines 12 . Repetitive synaptic stimulations with high and low frequency will cause long-term potentiation and depression, respectively. We will observe how Ras activation dynamics differs in response to these two types of stimulations. Furthermore, to visualize Ras activation by single-synapse stimulations, two-photon uncaging of glutamate will be applied 11 . Caged glutamate becomes active upon photolysis, and the extent of glutamate uncaging can be limited to activation of a single dendritic spine by controlling the focal volume using 2-photon excitation. This will give clear answer to what extent Ras activity spreads from stimulated spines into parent dendrites, nearby spines and even nucleus.
Modeling to explain spatial temporal pattern of Ras activation
Spatial-temporal dynamics of Ras activity is controlled by activity of calcium-dependent and independent
GAPs/GEFs. Also, it is shaped by diffusion of Ras, which we will measure with fluorescence recovery after photo bleaching. By tuning these parameters, we will deduce a biophysical model to explain the observed Ras dynamics in spines and dendrites. The model will help understand how Ras signaling can achieve both the multiple functionality and the specificity.
Section V: Qualification of the primary investigator for undertaking the proposed research
The primary investigator of the proposed research, Ryohei Yasuda, have been developing a 2PFLIM and worked on producing FRET-based Ras sensors with Dr. Karel Svoboda at Cold Spring Harbor Lab. We will setup a lab at the
Duke University Medical Center and continue working on Ras signaling in neurons using similar system. The main tool in the proposed research, 2PFLIM will be built using startup fund from the Medical Center. Tissue culture room, machining service and molecular biology facilities (DNA sequencing etc.
) in the Medical Center will be used for the research.
1 Zhu, J.J., et al., R. Cell 110 , 443–455 (2002).
2 Gallagher, S.M., Daly, C.A., Bear, M.F. & Huber, K.M., J Neurosci 24 , 4859-64 (2004).
3 Wu, G.-Y., Disseroth, K. & Tsien, R.W. Nat Neurosci., 4, 151-158 (2001).
4 Thomas, G.M. & Huganir, R.L. Nat Rev Neurosci 5 , 173-183 (2004).
5 Cheung, E.C.C. & Slack, R.S., Sci. STKE pe45, (2002).
6 Comings, D.E., Wu, S., Chiu, C., Muhleman, D., & Sverd, J. Psychiatry Res.
63 , 25–32 (1996).
7 Trivier, E. et al.
, Nature 384, 567-670 (1996).
8 Costa, R.M., et al.
Nature 415 , 526–530 (2002).
9 Brambilla, R., et al. Nature 390 , 281–286 (1997).
10 Bastiaens, P.I., & Pepperkok, R., Trends Biochem. Sci.
25, 631-637 (2000).
11 Matsuzaki, M. et al., Nat Neurosci . 4, 1086-1092 (2001).
12 Yasuda, R. et al., Sci. STKE pl5, 10 (2004).
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Appendix A: List of active grants and pending proposals
Grant: Career Award at the Scientific Interface
Source: Burroghs Wellcome Fund
Title: Visualization of biochemical signaling in single dendritic spines
Period: July 1, 2005 – June 30, 2008
Total amount: $360,000
Abstract: Calcium dependent signaling in postsynaptic compartments underlies synaptic plasticity and ultimately learning and memory. To elucidate mechanisms of postsynaptic signaling, we develop 2-photon fluorescence lifetime imaging microscope that will provide dynamic and spatial information of enzymatic activities and proteinprotein interactions. This technique will allow us to determine where and when enzymes are activated, what kind of protein-protein interactions underlie Ca 2+ dependent signaling, and how signals spread from a single synapse to the rest of the neuron.
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Appendix B: CV
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