Symposium

Symposium 9 Neuronal function assay of excellence: in vitro technologies using neuronal culture シンポジウム9 ヒト由来神経細胞の機能評価法へ向かって：培養細胞を用いた多彩なアプローチ

SY9-1 Regulation of the maturation of neurons by neurotrophic molecules 神経栄養因子による神経細胞成熟制御 Takei Nobuyuki（武井延之）¹，金村米博²，那波宏之¹ ¹Brain Research Institute, Niigata University ²Institute for Clinical Research, Osaka National Hospital Neuronal differentiation and maturation, both functionally and morphologically, are regulated by neurotrophic molecules. Classically, molecules that promote neurite extension are designated as a neurotrophic factor that induces neural differentiation. Recent studies show that synapse formation, maturation, and plastic changes are also regulated by neurotrophic molecules. Brain-derived neurotrophic factor (BDNF), the most prominent neurotrophic factor in the brain, induces morphological and functional maturation of neurons. It increases presynaptic, exocytosis-associated proteins and enhances stimulation-evoked release of neurotransmitters. BDNF also increases the levels of glutamate receptor proteins and PDZ proteins. Functionally, AMPA-induced current are largely enhanced by BDNF. Contrary, epidermal growth factor (EGF) and its family molecules decrease presynaptic molecules. It also downregulates glutamate receptor levels and PDZ proteins. As a functional index, EGF reduces mEPSC amplitude in cortical neurons. Thus, neuronal maturation is regulated by neurotrophic molecules either positive or negative directions. It is still unclear whether neurotrophic molecules play crucial roles in maturation of human neurons, which show slow development and have long lifespan. Human iPS-derived neural stem cells (NSCs) require EGF, bFGF and LIF for proliferation and maintenance. BDNF is often used for acceleration of differentiation. We analyze and discuss the effect of these molecules on the maturation of once differentiated human neurons.		SY9-2 Neuronal primary cilium as a signaling platform for environmental cue 環境センサー「一次繊毛」機能アッセイ系の構築に向けて Saito Yumiko（斎藤祐見子）¹，三木大輔¹，宮本達雄²，関野祐子³，白尾智明⁴，小林勇喜¹ ¹Laboratory for Behavi Neurosci, Grad Sch of Integrated Arts and Sciences, Hiroshima Univ ²Research Institute for Radiation Biology and Medicine, Hiroshima Univ, Hiroshima, Japan ³Grad Schl of Pharma Sci, Depart. Pharmacology, Univ of Tokyo, Tokyo, Japan ⁴Depart. Neurobio and Behav, Gunma Univ Grad Sch Med Primary cilia are microtubule-based cellular appendages from the centrosomal mother centriole (basal body) in most non-dividing vertebrate cell types. The membrane surrounding the cilium is enriched for specific membrane proteins and lipids that confer the cilium with unique sensory properties as a cellular antenna. Their importance is crucially underscored by a spectrum of human diseases, known as ciliopathies, which include obesity, mental retardation, and perhaps even psychiatric disorders. Further, shortened primary cilia length have recently been observed in several neurodegenerative conditions. Understanding neurological disease mechanisms, rodent neuronal cultures represent powerful tools, yet it is hard to efficiently translate new findings into new medicines to human. Commercially available human iPSC-derived neurons might be useful for drug screening and toxicity studies. However, primary cilia in human iPSC-derived neurons have not been described yet. Here, we assessed the morphological features and functionality of primary cilia in human iPSC-derived cortical neurons, iCell and iCell GlutaNeuron which is a mixture of post-mitotic neuronal subtypes, respectively. To characterize neuronal cilia, the cells were cultured through 25 day and subjected to immunohistochemistry by co-staining with antibodies against various basal body/ciliary markers and against MAP2. Primary cilia were first detected on neurons on Day 4, and their lengths were elongated with cultivation days. Further, by using Day 11-18 neurons, we confirmed the effects of several pharmacological agents that have been previously shown to modulate ciliary dynamics. Our work will help to develop mature iPSC-derived cortical neurons toward meaningful functional assays for drug discovery.

SY9-3 Evaluation of presynaptic functions by fluorescence imaging techniques 蛍光イメージングによるシナプス前部機能評価 Namiki Shigeyuki（並木繁行），坂本寛和，廣瀬謙造 Dept. of Neurobiology, Gad. Sch.of Med, The Univ. of Tokyo Presynaptic function involving neurotransmitter release from presynaptic terminals is a key determinant of neuronal function. Most previous studies addressing presynaptic function have relied on an analysis of the ensemble postsynaptic responses of many synapses, not on direct examination of neurotransmitter release at individual synapses. In this study, we developed an optical method to evaluate the presynaptic functional parameters of a single synapse using a fluorescence glutamate imaging technique with an enhanced glutamate optical sensor (eEOS). Throughout our attempts we established an analytical platform for quantification of presynaptically released glutamate with single-synapse resolution, then we found that multiple quantal release sites of synaptic vesicle for individual synapses enable multivesicular release on the arrival of single action potentials in cultured hippocampal neurons. To clarify the molecular correlates, we combined this approach with super-resolution imaging by stochastic optical reconstruction microscopy (STORM), identifying a nanoscale molecular distribution of the presynaptic protein Munc13-1 as a core component of the quantal release site. Furthermore we also found nanometer-scale molecular assembly for several synaptic proteins at presynaptic terminal. These results suggest that super-resolution imaging of synaptic proteins and glutamate imaging are applicable to the assay of presynaptic activity based on neurotransmitter release and nanometer-scale distribution of synaptic molecules in cultured and differentiated neurons.		SY9-4 Synapse imaging of Alzheimer's disease model neurons: localization change of synaptic proteins アルツハイマー病のシナプス機能異常を視る：シナプスタンパク質の微小局在変化解析 Koganezawa Noriko（小金澤紀子），山川佳苗，山崎博幸，白尾智明 Department of Neurobiology and Behavior, Gunma University Graduate School of Medicine Alzheimer’s disease (AD) is the most common neurodegenerative disease. Pathology of AD includes the extracellular accumulation of amyloid beta peptide, the intracellular accumulation of hyper-phosphorylated tau, the loss of drebrin from dendritic spines, and the loss of neuronal cells mainly in the cerebral cortex and hippocampus. Among them synaptic dysfunction has most correlation with cognitive dysfunction in the AD brains. Drebrin is an actin binding protein and stabilizes actin filaments. Drebrin-decorated stable actin filaments accumulate in dendritic spines and are thought to be crucial for synaptic plasticity. Drebrin decreases its expression level before onset of AD. We therefore hypothesized that loss of drebrin, that is, loss of stable actin filaments from dendritic spines elicits synaptic dysfunction and causes dementia in AD. To examine this hypothesis, we conducted experiments using AD model neurons, which is cultured hippocampal neurons treated by amyloid beta oligomer. We evaluated synaptic status based on drebrin cluster number using high-content imaging analysis. Decrease of drebrin cluster number was observed after amyloid beta oligomer treatment for 24 hours and this might be a result of localization change of drebrin. To further investigate the details of drebrin localization change in AD model neurons, we will conduct experiments using super resolution microscopy. In this talk, the drebrin loss underlying the pathology of AD, analysis of AD model neurons using high-throughput analysis or super resolution microscopy and a possibility of drebrin usage as a marker of synaptic dysfunction in AD will be discussed.

SY9-5 Differentiation method of human iPS cells derived neurons and evaluation of their maturation level ヒトiPS細胞由来神経細胞の分化誘導法とその成熟度評価 Kanemura Yonehiro（金村米博）^1,2,3 ¹Department of Biomedical Research and Innovation, Institute for Clinical Research, Osaka National Hospital, National Hospital Organization, Osaka, Japan ²Department of Neurosurgery, Osaka National Hospital, National Hospital Organization, Osaka, Japan ³Department of Physiology, Keio University School of Medicine, Tokyo, Japan Risk assessment of neurotoxicity caused by chemical substances or pharmaceuticals is very important. At present, neurotoxicity is mainly examined using in vivo tests with experimental animals. However, it is often indicated these neurotoxicity assays using non-human animals does not always predict human neurotoxicity. Cost of tests is high but throughput is generally low. And more, in vivo tests with experimental animals is subject of discussion in animal welfare. Therefore, more effective and reasonable alternatives to animal testing are desired.One hopeful approach is in vitro neurotoxicity tests using human normal cells. There are several reports suggest usefulness of human neuronal cells differentiated from human somatic neural stem/progenitor cells (hNSPCs) or human embryonic stem cells (hESCs). But, application of hNSPCs and hESCs to neurotoxicity tests is also ethically controversial. Recent great breakthroughs in cell reprogramming technology led to the generation of human induced pluripotent stem cells (hiPSCs) from various less ethical and accessible human tissues. Now, in vitro tests using hiPSCs technology represent one of the most promising next-generation assays for drug discovery and toxicity tests. In this session, I will introduce effective and reproducible neuronal differentiation methods of hiPSCs and our resent progress for evaluation strategies of maturation level of hiPSCs derived neurons, and finally discuss future possibility of new in vitro neurotoxicity assay using hiPSC.