シンポジウム

シンポジウム 46 脳神経疾患の解明に向けた「シンギュラリティ生物学」の新技術 46 New technologies of "Singularity Biology" for elucidation of neurological diseases. 座長：坂内博子（早稲田大学　理工学術院）・佐原成彦（量子科学技術研究開発機構）

2022年7月2日　9:00～9:22　ラグナガーデンホテル羽衣：中　第9会場 3S09m-01 100万以上の細胞をミクロンレベルの空間分解能でワンショット観察可能なトランススケールスコープAMATERAS　－外れ値科学の創出をめざして－ One-shot imaging of multi-million cells at micrometer spatial resolution using the Trans-Scale-Scope, AMATERAS -Conception of a novel discipline: outlier science- 永井健治(1) 1. 大阪大学 TAKEHARU NAGAI(1) 1. Osaka University Keyword: outlier, trans-scale-scope, Singularity, rare cellular activity The way of scientific analysis generally involves measuring the object of interest by some method, then focusing on the major components that make up the measured data and understanding the characteristics of the observed object from the statistical values such as mean, median, and variance, etc. In such a method, it is common to exclude "outliers" that are far from the mean value from the analysis. Therefore, little knowledge has been accumulated on rare components that show outliers. To focus on rare components (particularly cells) that have been overlooked so far, we have developed AMATERAS, a trans-scale-scope that can simultaneously observe more than one million cells with micrometer-level spatial resolution. In this symposium, examples of observation and analysis using AMATERAS will be introduced from the viewpoint of how singularity is formed by such rare cells, and new concepts in life science research will be outlined.		2022年7月2日　9:22～9:44　ラグナガーデンホテル羽衣：中　第9会場 3S09m-02 てんかん発作を惹起するシンギュラリティ構造の脳オルガノイドを用いた解析 Analysis and control of singularity structures triggering epileptic seizure in human iPSC derived brain organoid 六車恵子(1)、玉田篤史(1) 1. 関西医科大学 Keiko Muguruma(1), Atsushi Tamada(1) 1. Kansai Medical University Keyword: neuronal network, brain organoid, epilepsy, iPSC Epilepsy is a neurological disorder that is characterized by epileptic seizure due to abnormally excessive neuronal activity. We aim to understand and control epileptic seizure as a singular transition of neuronal activity from normal stable state to abnormal hyper-excitable state. To this end, we developed a culture system that recapitulates the seizure by applying iPSC differentiation technology. Several lines of iPSCs were generated from patients with epilepsy, and were differentiated into 3D cerebral cortical organoids that recapitulate human corticogenesis. The organoids were dissociated into neocortical neurons and were further cultured on 2D substrates to reconstitute dense cortical neuronal network. In these 2D cultures, the pattern of neuronal activity was analyzed by performing electrophysiological recording with multi-electrode array. We found that patient-derived cultures, but not healthy control cultures, exhibit a periodic bursting pattern emerging from specific spatial positions. Such pattern of activity could be the recapitulation of the phenotype of epileptic seizure. To further investigate the mechanism that triggers the seizure, precise real-time measurement and quantitative analysis of the neuronal activity is required. Here we have established calcium imaging system of specific cell types with tamoxifen-inducible expression of calcium indicators. The pattern of calcium signals is currently analyzed with a standard microscope and AMATERAS, a transscale scope that enables to detect rare neuronal activity among wide field of view. We are also searching for the structural basis that triggers the bursting at specific foci by imaging the individual neuronal morphology in large-scale neuronal network by utilizing Brainbow stochastic fluorescent labeling. These functional and structural analyses with patient iPSC-derived neuronal cultures will be effective tools to investigate the pathogenic mechanisms of epilepsy and to develop therapeutic treatment or drugs against the disease.

2022年7月2日　9:44～10:06　ラグナガーデンホテル羽衣：中　第9会場 3S09m-03 タウ蛋白質相転移は神経変性疾患のシンギュラリティポイントとなりうる？ Investigating phase transition of tau protein as a singularity point of neurodegenerative diseases 佐原成彦(1) 1. 量子科学技術研究開発機構 Naruhiko Sahara(1) 1. National Institutes for Quantum Science and Technology Keyword: tau protein, tauopathy, animal model, in vivo imaging The microtubule-associated protein tau abnormally aggregates into intracellular, filamentous inclusions (neurofibrillary tangles; NFTs) in brains of individuals with neurodegenerative diseases including Alzheimer’s disease (AD), progressive supranuclear palsy, corticobasal degeneration, argyrophilic grain disease, Pick’s disease, and familial frontotemporal lobar degeneration with underlying tau pathology collectively referred to as tauopathies. NFTs are closely associated with the severity of brain function loss in AD. Thus, making tau protein a target in the treatment of AD has become a major therapeutic strategy. Although the exact pathways have not yet been clarified, it is conceivable that NFT formation passes through dimerization, multimerization, oligomerization, and protofibril formation. In the central nervous system, tau is a highly soluble and natively unfolded protein dominated by a random coil structure in solution. This soluble form became converted into insoluble aggregates during the pathogenesis of tauopathy. We hypothesize that tau protein phase transition induces neurotoxicity and cell death in the brain. Tauopathy mouse models (e.g., rTg4510, PS19 lines) which represent an accumulation of tau pathology and a neurodegeneration are used for searching singularity points of tauopathy. Since in vivo imaging techniques (e.g., positron emission tomography [PET], two-photon microscopy, magnetic resonance imaging) have led to significant breakthroughs for detecting pathological tau accumulations and related neurodegeneration, we have examined to observe a singularity point of tauopathy in live animals (Sahara et al. 2017, Front. Neurology). Moreover, as neuroinflammation was thought to be another hallmark in neurodegenerative disease, temporal change of microglial activation was examined by in vivo PET imaging (Ishikawa et al. 2018 JAD). To narrow down a linkage between tau-induced neurodegeneration and neuroinflammation, we examined homeostatic microglial markers and revealed early reduction of homeostatic microglia in tauopathy mice (Maeda et al. 2021 Brain Comm.). Our data suggest that phenotypic changes of microglia can be one of checkpoint markers of tauopathy. Nevertheless, it is essential to identify both tau protein transition and microglial activation for early diagnosis of tauopathy. Ongoing studies to investigate singularity points of tauopathy will be presented in this symposium.		2022年7月2日　10:06～10:28　ラグナガーデンホテル羽衣：中　第9会場 3S09m-04 マウスおよびマーモセットでα-シヌクレイン病変の伝播をin vivoで可視化できる多用途PETプローブ In vivo visualization of propagating α-synuclein pathologies in mouse and marmoset models by a multimodal PET probe 小野麻衣子(1) 1. 国立研究開発法人量子科学技術研究開発機構 Maiko Ono(1) 1. National Institutes for Quantum Science and Technology Keyword: α-synuclein , propagation, in vivo PET imaging, two-photon microscopy Parkinson’s disease and dementia with Lewy bodies are neurodegenerative diseases of high prevalence, and they are pathologically characterized by the appearance of Lewy bodies and Lewy neurites, which are mainly composed of aggregated α-synuclein. Abnormal α-synuclein is also a major component of glial cytoplasmic inclusions, which are a pathological feature of multiple system atrophy, a neurodegenerative disease presenting with movement and autonomic disorders. Previous studies experimentally demonstrated that α-synuclein fibrils acted as templates for the conversion of normal α-synuclein molecules into misfolded species, leading to the prion-like propagation of the α-synuclein fibrillogenesis throughout the brain via neural circuits. Thus, deposition and propagation of intracellular α-synuclein fibrils is implicated in a group of neurodegenerative disorders, while high-contrast in vivo detection of α-synuclein depositions and propagations has been unsuccessful in animal models and humans. Recently, we have developed a bimodal imaging probe, C05-05, for visualizing α-synuclein inclusions in the brains of living murine and non-human primate models of α-synuclein propagation. In vivo optical and positron emission tomographic (PET) imaging of a mouse model demonstrated visualization of pathological α-synuclein aggregates by C05-05, revealing a dynamic propagation of fibrillogenesis along neural pathways followed by disruptions of these structures. Moreover, longitudinal ¹⁸F-C05-05-PET of a marmoset model captured widespread dissemination of fibrillary pathologies accompanied by neurodegeneration detected by dopamine transporter PET. Collectively, a new neuroimaging platform incorporating C05-05 is implementable for multi-scale analysis of the neurodegenerative α-synuclein fibrillogenesis and pharmacological actions of a drug candidate on this etiological process in animal models.

2022年7月2日　10:28～10:50　ラグナガーデンホテル羽衣：中　第9会場 3S09m-05 Modulation of Tau Liquid-Liquid Phase Separation by Disease-Related Modifications and Interacting Proteins Nicholas M Kanaan(1) 1. Michigan State University, College of Human Medicine, Department of Translational Neuroscience Keyword:* Tau, Alzheimer's disease, Tauopathy, liquid phase separation Formation of membrane-less organelles via liquid-liquid phase separation is a route by which cells can meet the biological requirement for spatiotemporal regulation of cellular components and reactions. Indeed, nucleoli, stress granules, P granules and Cajal bodies are examples of membrane-less organelles involved in multiple cellular processes including gene regulation, ribosome function and regulation of signal transduction Several proteins associated with neurodegenerative diseases undergo phase separation. Tau, a protein known for its involvement in Alzheimer’s disease, frontotemporal dementia and other tauopathies was recently found to undergo liquid-liquid phase separation. We found that tau forms dynamic liquid droplets at physiological levels upon molecular crowding. The formation of tau droplets is enhanced by disease-associated modifications such as the AT8 phospho-epitope or the P301L tau mutation associated with an inherited tauopathy. Extended phase separation of all forms of tau promoted the adoption of known toxic conformations and oligomerization. Tau interacting proteins have differential effects on tau phase separation in vitro. For example, the stress-induced chaperone protein, Hsp70, inhibits phase separation. On the other hand, the calcium-binding protein, EFhd2, appears to modulate the formation of aggregate-like or liquid droplet-like tau structures in a calcium-dependent fashion. Collectively, our findings highlight a potential role for tau phase separation in the formation of neurotoxic tau species, provide insights into the effects that disease-related modifications, and demonstrate how tau interacting partners can modulate tau phase transitions.