Yasunori Hayashi - US grants

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by a selective loss of motoneurons. It affects 500,000 individuals worldwide annually and remains untreatable. To develop an effective treatment for ALS, a precise understanding of its pathogenesis is indispensable. One approach toward this goal involves identifying the gene(s) responsible for familial forms of ALS. Such an approach has uncovered mutations in the gene encoding Cu/Zn cytosolic superoxide dismutase (SOD1); these mutations only account for 20%-25% of familial cases. Despite intense efforts, the pathogenesis of the remainder of familial cases and all of sporadic cases are practically unknown. We have recently identified a new NMDA type glutamate receptor, NR3B. Interestingly, it is expressed almost exclusively in the motoneurons known to be affected in ALS and negatively regulates the function of NMDA receptor to limit Ca2+ influx. Because it is well documented that an overactivation of NMDA receptor causes the cell death associated with various neurological disorders, the identification of NR3B lead us to propose the "NR3B hypothesis" of the pathogenesis of ALS. This hypothesis proposes that defects in NR3B function may be neurotoxic in motoneurons through loss of the negative regulation of motoneuronal NMDA-mediated Ca2+ currents. Loss of NR3B function may increase the NMDA receptor current and Ca2+ influx in these cells, thereby predisposing motoneurons to excitotoxicity. This mechanism may cause ALS by itself or may predispose carrier to ALS when it is combined other genetic or epigenetic factors. In this proposal, we will test this NR3B hypothesis by genetic analysis of the NR3B gene in humans and modifications of NR3B gene dose in ALS mice. We will first analyzing DNA from ALS patients for mutations in the NR3B gene. We will first screen ALS families without a known genetic defect for genetic linkage to the NR3B gene (Aim 1). We will then perform mutational analysis of the full NR3B gene in linked cases and in a series of individuals with both familial and sporadic ALS, as well as controls (Aim 2). Thereafter (Aim 3) we will test the hypothesis that polymorphisms in the NR3B gene are associated with an increased risk of developing ALS or with some aspect of the clinical course of ALS (e.g. onset age, life span after onset, disease distribution). In related studies in mice, we will determine whether variations in the dose of the NR3B gene alter the phenotype of transgenic SOD1-G93A mice (Aim 4). In our view, this is a significant translational and explorative investigation that promises to relate a newly discovered, novel motoneuronal glutamate receptor subunit, NR3B, with a fatal human motor neuron disease; the findings have potential implications for the development of new approaches to therapy in ALS.

[unreadable] DESCRIPTION (provided by applicant): Synaptic plasticity has been suggested to be a cellular counterpart for learning and memory. However, it is practically impossible to visually monitor synapses actually undergoing synaptic plasticity at a given memory paradigm in a given neuronal network. This project proposal, written in response to the program announcement "Developing Novel Genetic Methods for Mapping Functional Neuronal Circuits and Synaptic Change", describes the development of a technology for visualizing synapses that are undergoing synaptic plasticity in neurons in living animal. This goal will be accomplished by a combination of two technologies: fluorescence resonance energy transfer (FRET) and two-photon laser scanning microscopy. We will first develop a FRET-based construct for optically measuring the CaMKII activity and actin polymerization/depolymerization equilibrium, which will allow non-destructive optical detection of CaMKII activation in intact neurons. Since the enzymatic activity of CaMKII is constitutively enhanced after the induction of NMDA receptor-mediated synaptic plasticity and this activity is required for maintaining synaptic potentiation, we expect that the activation of CaMKII will be a good indicator of synaptic plasticity. In contrast, in a work which we published, we found actin polymerization/depolymerization equilibrium can be detected with FRET and that it follows LTP and LTD respectively. To detect FRET at the synaptic structure, we will take advantage of a two-photon microscope. We will then test the feasibility of our strategy by expressing the construct in neurons and induce synaptic plasticity by either high-speed ionophoretic stimulation of individual synapses or local electrical stimulation or combined with detection of structural plasticity. Finally, we will generate a transgenic animal expressing this construct. The barrel cortex of the resultant animal will be observed with the two-photon microscope. Paradigms known to induce synaptic plasticity in this structure such as sensory deprivation will be tested to see whether synaptic plasticity is reflected by FRET. In summary, our technology will provide a unique system for detecting NMDAR-mediated synaptic plasticity with spatial resolution at the single synapse level on a sub-second time scale. This technique, in combination with technologies currently under development in other laboratories, such as in vivo two-photon imaging in freely moving animals and deep-structure two-photon microscope imaging with a relay lens, will pro- vide a versatile system for monitoring synaptic plasticity that cannot be achieved with existing experimental systems. In the future, this technique could be applied to higher mammals, such as macaque monkeys, after viral or transgenic introduction of our reporter construct. It may be possible to have monkeys perform a task and observe synaptic plasticity in the brain during learning of the task. [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Synaptic plasticity has been suggested to be a cellular counterpart for learning and memory. However, it is practically impossible to visually monitor synapses actually undergoing synaptic plasticity at a given memory paradigm in a given neuronal network. This project proposal, written in response to the program announcement "Developing Novel Genetic Methods for Mapping Functional Neuronal Circuits and Synaptic Change", describes the development of a technology for visualizing synapses that are undergoing synaptic plasticity in neurons in living animal. This goal will be accomplished by a combination of two technologies: fluorescence resonance energy transfer (FRET) and two-photon laser scanning microscopy. We will first develop a FRET-based construct for optically measuring the CaMKII activity and actin polymerization/depolymerization equilibrium, which will allow non-destructive optical detection of CaMKII activation in intact neurons. Since the enzymatic activity of CaMKII is constitutively enhanced after the induction of NMDA receptor-mediated synaptic plasticity and this activity is required for maintaining synaptic potentiation, we expect that the activation of CaMKII will be a good indicator of synaptic plasticity. In contrast, in a work which we published, we found actin polymerization/depolymerization equilibrium can be detected with FRET and that it follows LTP and LTD respectively. To detect FRET at the synaptic structure, we will take advantage of a two-photon microscope. We will then test the feasibility of our strategy by expressing the construct in neurons and induce synaptic plasticity by either high-speed ionophoretic stimulation of individual synapses or local electrical stimulation or combined with detection of structural plasticity. Finally, we will generate a transgenic animal expressing this construct. The barrel cortex of the resultant animal will be observed with the two-photon microscope. Paradigms known to induce synaptic plasticity in this structure such as sensory deprivation will be tested to see whether synaptic plasticity is reflected by FRET. In summary, our technology will provide a unique system for detecting NMDAR-mediated synaptic plasticity with spatial resolution at the single synapse level on a sub-second time scale. This technique, in combination with technologies currently under development in other laboratories, such as in vivo two-photon imaging in freely moving animals and deep-structure two-photon microscope imaging with a relay lens, will pro- vide a versatile system for monitoring synaptic plasticity that cannot be achieved with existing experimental systems. In the future, this technique could be applied to higher mammals, such as macaque monkeys, after viral or transgenic introduction of our reporter construct. It may be possible to have monkeys perform a task and observe synaptic plasticity in the brain during learning of the task. [unreadable] [unreadable]

Blood Coagulation Factor IV; CRISP; Ca++ element; Calcium; Coagulation Factor IV; Computer Retrieval of Information on Scientific Projects Database; FRET; Factor IV; Fluorescence Resonance Energy Transfer; Funding; Grant; Institution; Investigators; Ionophores; Learning; Monitor; NIH; National Institutes of Health; National Institutes of Health (U.S.); Proteins; Research; Research Personnel; Research Resources; Researchers; Resources; Source; System; System, LOINC Axis 4; United States National Institutes of Health; fluorophore; gene product; memory process; mutant