Research [New Techniques]
Hayashi Group
| Project Title | Visualization of deep brain synaptic circuit under physiological and pathological conditions |
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A neuronal assembly is a group of neurons that fire together, which is wired together by synaptic plasticity mechanism. The neuronal assembly is considered as a high-order unit of neuronal information processing but its analysis has been hampered by the lack of appropriate methods to detect neuronal activity from multiple neurons at the same time. We will investigate the operation principle of the neuronal assembly during hippocampal learning. For this purpose, we will place a transgenic mouse expressing Ca2+-sensitive fluorescent protein under a two-photon microscopy and observe hippocampal neuronal activity while the animal performs a learning paradigm in virtual reality. We will first study the physiological behavior of neuronal assembly and then in collaboration with other groups, we will study the dynamics of the neuronal assembly under pathological conditions. Our final goal is to establish neural circuit pathology from functional aspect.
Inoue Group
| Project Title | Understanding the neuronal dysfunction/neurocircuit pathology based on iPSC technology |
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In neurodegenerative diseases, specific populations of neuronal cells degenerate and die, reflecting selective vulnerability. It is not easy to diagnose the patients correctly even with the combination of several clinical findings and laboratory tests. The development of new therapies must be based on correct diagnosis. Before the birth of iPSC technology, it was impossible to perform biopsy of neuronal cells from patients, but we have now, as a starting point in this research program, begun to analyze the neurons of patients via patient-specific iPSCs (Imamura, K., Inoue H. Research on neurodegenerative diseases using induced pluripotent stem cells. Psychogeriatrics, 12,115-119 (2012)).
The neurodegeneration process consists of neuronal dysfunction, accumulation of abnormal proteins, neuronal death, and exacerbation by non-neuronal cells. In this program, we are trying to model the synaptic and neurocircuit pathology in neurodegeneration using patient cells. New imaging technology including high content analysis would allow precise observation and understanding of neurodegeneration mechanisms (Egawa N, et al., Drug Screening for ALS Using Patient-Specific Induced Pluripotent Stem Cells. Sci Transl Med, 4, 145ra104 (2012)).
On the other hand, it remains unknown which site in neuronal circuits might be a nascent lesion, whether neurodegeneration might occur in each neuron independently or in a co-ordinated manner, or even whether partial rescue of neurons in the circuits might be effective. We might be able to tackle these issues. However, we should also find solutions for technical matters including differentiation efficiency, clonal variation of differentiation/maturation, and recapitulation of aging process/environmental determinants (Kitaoka S, Kondoh H, Inoue H. Induced Pluripotent Stem Cell Technology for the Study of Neurodegenerative Diseases. Induced Stem Cells, chapter V, Nova Science Publishers Inc, New York, p129-142 (2011)). In this research program, we will address technology development and buttress theories with more concrete evidence.
Publicly Invited Research (H25-26)
Hirano Group
| Project Title | Effects of β-amyloid on location and movement of glutamate receptors around postsynaptic membrane |
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We have developed a method to form postsynaptic-like membrane (PSLM) directly on the glass surface in a neuronal culture preparation and to observe the fluorescence signal attached to glutamate receptor with total internal reflection fluorescence microscopy. This method has enabled us to record the location and movement of glutamate receptors with a high signal to noise ratio around PSLM. Using this method we aim to elucidate pathological effects of β-amyloid on both AMPA-type and NMDA-type ionotropic glutamate receptors around the postsynaptic membrane.
Seiji Okada Group
| Project Title | Investigation of synaptic regeneration with transcriptome analysis of the engrafted neural stem/progenitor cells |
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The transplantation of neural stem/progenitor cells (NSPCs) is an exciting therapeutic option for replacing damaged neural cells and inducing functional recovery. However, no direct analysis has ever been performed on their in vivo profile after transplantation. By combing flow-cytometoric isolation and RNA-sequencing, we have recently shown that NSPCs are highly vulnerable to environmental factors and that the graft environment greatly affects engrafted NSPCs. In this study, we utilize this method and selectively isolate the engrafted NSPCs from the injured spinal cord to investigate whether they are integrated in and contribute to the functional neuronal circuits. We also plan to examine the molecular changes of the integrated NSPCs at each phase of the synaptic regeneration with transcriptome analysis.
Muramatsu Group
| Project Title | Novel models of neurodegenerative diseases created by intravascular delivery of AAV vectors |
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Vectors derived from adeno-associated viruses (AAVs) are very efficient for transduction of neurons and glial cells in the mammalian central nervous system. Using AAV vectors, we have been developing gene therapy for neurodegenerative diseases, including Parkinson's disease, amyotrophic lateral sclerosis, and Alzheimer's disease. In the current project, we plan to apply AAV vectors to create novel models of these diseases in order to study the complex features of the diseases. Intravascular delivery of AAV vectors enables us to introduce genes related to the pathogenesis of the diseases into the neurons and glial cells in broad areas of the adult brain. In contrast to ES cell-based conventional transgenic animals, AAV vector-mediated models are devoid of developmental effects. We also plan to produce primate models and use in vivo imaging to elucidate the synaptic pathology of these diseases.
Yohei Okada Group
| Project Title | The analysis of neuromuscular pathology using disease specific human iPSCs |
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The pathogenetic analysis of human neurological disorders is sometimes quite difficult because of the lack of appropriate disease models. To overcome these problems, we have been establishing disease specific human iPSCs from patients of neurological disorders. By differentiating these patient specific human iPSCs into neural cells, we can develop novel in vitro disease models which adequately recapitulate the pathogenesis of the diseases. In this study, taking advantage of hiPSCs which could differentiate into varieties of human somatic cells, we derive both motor neurons and skeletal muscles from disease specific iPSCs, and examine neuromuscular pathology of neurological disorders.
Publicly Invited Research (H23-24)
Hirano Group
| Project Title | Development of new experimental methods for the functional analyses of synaptic proteins |
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In this project, two experimental methods to study location, movement, interaction and function of synaptic proteins will be developed. One is to form postsynaptic-like membrane directly on the glass surface and to observe the fluorescence signal attached to synaptic proteins with total internal reflection fluorescence microscopy. This method would enable one to observe the location and movement of synaptic proteins such as receptors or postsynaptic scaffold proteins with near single-molecular resolution. Another is to express combinations of synaptic proteins in non-neuronal cultured cells and to co-culture them with neurons prepared from mouse central nervous system. The formation of excitatory or inhibitory synapse-like structure will be formed between non-neuronal cells and neurons, and their structural and functional properties will be studied. This study is aimed to elucidate essential proteins involved in particular properties of synapses.
Muramatsu Group
| Project Title | Novel models of neurodegenerative diseases created by intravascular delivery of AAV vectors |
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Vectors derived from adeno-associated viruses (AAVs) are very efficient for transduction of neurons and glial cells in the mammalian central nervous system. Using AAV vectors, we have been developing gene therapy for neurodegenerative diseases, including Parkinson’s disease, amyotrophic lateral sclerosis, and Alzheimer’s disease. In the current project, we plan to apply AAV vectors to create novel models of these diseases in order to study the complex features of the diseases. Intravascular delivery of AAV vectors enables us to introduce genes related to the pathogenesis of the diseases into the neurons and glial cells in broad areas of the adult brain. In contrast to ES cell-based conventional transgenic animals, AAV vector-mediated models are devoid of developmental effects. We also plan to produce primate models and use in vivo imaging to elucidate the synaptic pathology of these diseases.
Okada Group
| Project Title | The analysis of pathogenesis of Parkinson's disease by patient-derived disease specific human iPS cells |
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The pathogenetic analysis of human neurological disorders is sometimes quite difficult because of the lack of appropriate disease models. To overcome these problems, we have been establishing disease specific human iPS cells from patients of neurological disorders. By differentiating these patient specific human iPS cells into neural cells, we can develop novel in vitro disease models which adequately recapitulate the pathogenesis of the diseases. In this study, we establish human iPS cells from fibroblasts derived from Parkinson's disease patients, and differentiate them into neural cells. Using these neural cells, we investigate how neuronal cell death occurs in the brain of Parkinson's disease patients and clarify the underlying mechanisms.
Yamamoto Group
| Project Title | Pathophysiological examination for pediatric neurodevelopmental disorders using disease-specific induced pluripotent stem cells |
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Pediatric neurodevelopmental disorders include autistic spectrum disorders, severe psychomotor developmental delay, intractable epilepsy, and so on. Recent advance of molecular and cytogenetic analyses, including microarray-based comparative genomic hybridization and exome sequence, enabled us to identify rare pathogenic variants in the patients with pediatric neurodevelopmental disorders. In this study, disease-specific induced pluripotent stem (iPS) cells are going to be established from the skin fibroblasts derived from the patients whose genetic mutations had been identified in the previous our studies. Morphological abnormalities and pathophysiological findings would be examined by neuronal differentiation of the generated disease-specific iPS cells.
Iwata Group
| Project Title | Investigation of a possible interaction between neuron and glial cells on the pathogenesis of Alzheimer’s disease |
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Neprilysin (NEP) is a major peptidase responsible for in vivo degradation of amyloid-β peptide (Aβ) that is implicated in the pathogenesis of Alzheimer’s disease (AD), and plays a protective role in Aβ–induced synaptotoxicity. Recent studies revealed that declined activity of NEP exacerbates brain inflammation and metabolic stabilization of Aβ by up-regulating glial glutaminyl cyclase (QC). In this research, we will introduce NEP gene and QC siRNA using viral vectors driving neuronal and glial expression into the brains of NEP-knockout mice and AD model mice by i.v. administration, respectively, analyze a possible interaction between neurons and glial cells in vivo and examine the gene therapeutic effects on inflammation and synaptic and cognitive functions.
