The physiopathological roles of orexins/hypocretins are supported by preclinical and clinical studies. Orexin A and B (hypocretins 1 and 2) and their two receptors (OX1R and OX2R) affect arousal, sleep/wake regulation, reward and stress. The absence of orexin producing cells in the lateral hypothalamus (LH) and of orexin in the CSF, results in narcolepsy with cataplexy (narcolepsy type I). Orexin KO or double OX1R / OX2R KO mice display narcolepsy/cataplexy, whereas OX2R KO and OX1R KO mice have moderate or no sleep phenotype. These features triggered drug discovery programmes for the treatment of insomnia and other disorders. Dual OX1R / OX2R antagonists (DORAs) are close to or marketed. Suvorexant (Belsomra®) and Lemborexant (Dayvigo®) are the first registered orexin hypnotics. DORAs promote sleep primarily by increasing REM (rapid eye movement) sleep, with little effect on NREM (non-rapid eye movement), especially slow wave sleep (SWS), although the primary target mediating sleep by DORAs and other OXR antagonists is the OX2R. We shall briefly review preclinical and clinical data of OXR antagonists and agonists in various neuropsychiatric diseases. REM sleep enhancement by DORAs provides opportunities to treat specific neurological disorders, whereas OX2R antagonists such as Seltorexant (JNJ-54717793), have broader applications, by promoting balanced sleep and antidepressant effects, whereas OX1R antagonists may be indicated in panic/anxiety or addiction. The development of OXR agonists is in very early stages. In preclinical models, OXR agonists produce arousal and have anti narcolepsy/cataplexy effects as expected. In the clinic, only peptide agonists have been tested so far, with very limited success. In summary, OXR antagonists and agonists represent an exciting new class of neuropsychiatric treatments.

L-3,4-ジヒドロキシフェニルアラニン（L-ドーパ）はドパミンの前駆体である。長らくそれ自体は活性がなく単なるドパミンの前駆体として位置付けられてきた。私たちは、神経刺激によってL-ドーパがカルシウム依存性に遊離されることを見出した。さらに、ドパミンへの変換を阻害した条件下において、ドパミンやグルタミン酸とは異なる薬理学的性質を示す応答を示すことを見出し、以来、L-ドーパが神経伝達物質として作動するかを検証してきた。最近になり、L-ドーパを認識する受容体候補分子、GPR143が同定された。GPR143は、L-ドーパの作用を媒介することを示す知見が得られている一方、その詳細なメカニズムは未解明である。GPR143遺伝子欠損マウスの表現型の解析途上、血管平滑筋a1-アドレナリン受容体 (a1AR)とGPR143とが複合体を形成し、a1ARを介するシグナル伝達を正に修飾することを見出した。GPR143欠損マウスは、モノアミン神経伝達に関与する他の複数の薬物応答の異常を示すなど、多様な表現型を示すことも明らかとなった。GPR143 は、L-ドーパの対象疾患であるパーキンソン病患者剖検脳において、レビー小体に局在することを見出した。L-ドーパの作用機序ならびにパーキンソン病の病態との関連性が今後の重要な検討課題である。

生体内では多様な輸送担体（トランスポーター;TP）が生体に必要な物質を積極的に取り込み、異物を積極的に排泄する機能を担っている。TPの分子認識に基づいた創薬を行うことにより、薬効を維持したまま副作用に関連する組織・細胞への移行を抑える理想的な動態特性を持つ薬の開発も可能になると期待され、医薬品開発の面からもTP研究への関心が高まっている。TPは、薬物代謝酵素と同様に、分子多様性、遺伝子多型、臓器特異性、広範な基質認識性という特性を持つことがわかってきた。医薬品開発においては、in vitroからin vivoへの外挿法などを確立していくことで、医薬品開発過程での遺伝子発現系の利用、薬物間相互作用の評価、遺伝子多型による個人間変動の解析などTP研究を実用段階へと進めていく必要がある。当日はその実例を示したいと思う。

近年、米国FDAが中心となり、生理学的薬物速度論モデル（PBPK model）による薬物動態予測を、臨床試験の必要性の判断、投与量の設定に活かそうという動きがある。この実例についても触れながらの講演にしたい。

近年、あらゆる分野において爆発的に増大し続けるビッグデータから知識発見や新たな価値を創造する科学技術として、人工知能（AI）が注目されている。創薬・生命科学分野においても、基礎研究や臨床現場で日々生まれる多種多様かつ膨大なデータを用いた様々なAI研究がなされており、これによる医薬品開発の劇的効率化が期待されている。

演者は、これまでに人工知能・機械学習技術の創薬応用に着手してきており、創薬標的生体分子の探索や薬剤のスクリーニング・最適化を行うAIの研究開発を行ってきた。また、2016年11月に、アカデミアと製薬企業・IT企業が連携し、創薬AI開発を目指す「ライフ・インテリジェンス・コンソーシアム（LINC）」を立ち上げた。LINCでは、医薬品開発の上流から下流（病態解明、創薬標的探索、薬剤設計、前臨床、臨床、市販後）にいたる約30種類のAI技術の開発を行ってきた。

本講演では、演者が取り組む創薬AIの具体的な研究事例を紹介するとともに、AIがもたらす医薬品産業へのインパクトについて議論したい。

The cognitive control of action reflects our ability to extract and encode causal relationships from the environment to guide choice between different courses of action. To investigate this process at both a psychological and a neural level we have adopted a model of cognitive control in rodents in which the influence of predictive learning on action selection is assessed in an instrumental choice situation: the Pavlovian-instrumental transfer paradigm. Over the last several years we have systematically investigated the core neural systems and cellular circuits that mediate transfer effects of various kinds and, in this presentation, I will describe: the behavioural background to this research project; the psychological theories that best account for these kinds of effect; and our most recent findings at a neural level, focusing on neurochemistry of amygdala-striatal interactions and specific modulatory processes within the striatum that enhance motor output via a pallido-thalamic feedback circuit. As a consequence of this latter research, we have uncovered what appears to be a form of GPCR-based ‘memory' process in the nucleus accumbens that encodes specific Pavlovian associations for later use in the control of choice between actions.

多能性幹細胞や、生検サンプルを活用したプライマリ細胞などを活用し、ヒト器官に類似した組織体を生み出すオルガノイド研究が隆盛を極めている。即ち、ヒューマン・オルガノイドは解剖学的・機能的に生体内に存在する器官に近い特徴を示すことから、これまで研究対象とすることが困難であったヒトにおけるさまざまな生命現象に迫ることが可能となった。

　これまでに、われわれは、ヒト多能性幹細胞を活用したオルガノイド研究において、さまざまな疾患表現型の再現に必須となる「複雑性」を付与する技術開発を継続推進してきた（Nature, 2017: Nature, 2019: Cell Metabolism, 2019: Nature Medicine, 2020）。本講演では、ヒト肝臓を対象としたオルガノイドを事例に、「どう創るか？」から「どう使うか？」へと力点が移りつつ有る第4世代のOrganoid4.0研究が指向する未来を考察したい。すなわち、いままで研究対象とすることが困難であった高度なヒト臓器モデルの樹立を通じ薬学・医学への実質的還元を目指す新潮流、オルガノイド医学（Organoid Medicine）研究の最前線について具体事例を交えて紹介する。

ほ乳類の腸管には数百の腸内細菌（マイクロバイオータ）が存在し、宿主の生理機能に深く影響を及ぼしている。従ってマイクロバイオータに人為的に介入することが出来れば、複数の疾患に対する新たな治療戦略となり得る。我々は、消化管の恒常性維持機構を理解すると共に、個々の腸内細菌種が免疫システムにどのように影響を与えているかを還元化して把握して行く独自の研究手法を確立してきた。この方法によってこれまでに、制御性T細胞、Th17細胞、Th1細胞、CD8 T細胞を特異的に誘導する腸内細菌種の同定に成功した。これらの成果は、免疫系が関わる様々な疾患（感染症・アレルギー・自己免疫・がんなど）に対する新しい治療法開発に繋がる可能性がある。

記憶形成時の可塑的刺激によって活性化された神経細胞集団にて、シナプス入力は核内でのCREB依存的転写誘導を引き起こす。シナプス入力は、シナプス局所における神経可塑性シグナルを引き起こすのみならず、長期記憶・長期可塑性を制御するシナプスと核を結ぶ活動依存的神経可塑性メカニズムをも活性化することを我々はまず見いだした。シナプスと核の間の情報伝達の解読により、シナプスから核に至る複数のCREB経路と、その下流の標的遺伝子であるArcの制御に基づく逆シナプスタギング機構を明らかにした。一方、これらの発見により、シナプス可塑性とArc転写可塑性の間の両方向的機能連関を担うゲノム・蛋白の構造モジュール情報同定が実現した。この結果、速い活動電位動態を記録可能な次世代Ca²⁺インディケーター群XCaMP、ならびに活性化神経細胞集団を標識する合成プロモーターE-SAREの開発が可能となった。このようなCa²⁺シグナル伝達の分子基盤解明がもたらした神経回路計測・標識技術開発の革新が、現在数多くのin vivo 脳機能制御機構解明に寄与している。

High mobility group box-1 (HMGB1) は、核内に局在するクロマチンDNA結合タンパクである。HMGB1はストレス刺激や細胞障害により、細胞質を経由して細胞外へ分泌・放出されると、神経突起伸展活性や起炎性活性を発揮する。このような局在、動態、活性の特徴は、既存の活性物質群とは全く異なっている。演者は、HMGB1に対する特異的単クローン抗体を作製し、抗体を用いて脳虚血、脳出血、脳外傷、てんかん等の中枢疾患におけるターゲットバリデーションと治療効果の評価を行ってきた。その結果、抗体治療は、これらの傷害モデルで共通して観察されるBBB破綻を強く抑制し、随伴する脳内炎症を軽減することで難治性脳疾患に治療効果を発揮することを明らかにした。HMGB1結合因子として、血漿タンパクHRGを見出し、敗血症病態において血漿HRG低下を起点とする病態形成の機序について新しい考え方と、HRG補充療法を具体的に提案した。HRGの新しい受容体としてCLEC1Aを同定し、HMGB1動員のHRGによる制御を血管内皮細胞で解明した。ターゲット分子同定から開始するトランスレーショナルリサーチと創薬は、薬理学の一つの方向性を示す。

先進国における高齢化の進展や医療財政の悪化の一方で、ゲノム医療やAI をはじめとする新たなテクノロジーの台頭、更には患者や国民の健康及び医療に対するニーズの多様化など、製薬業界を取り巻く環境は大きく変化している。

製薬協では、製薬業界が人々の健康寿命延伸に貢献するためには何を成すべきかといった観点から2019年に「政策提言２０１９」をとりまとめた。我々は従来取り組んできた疾患治療のための医薬品の創出にとどまらず、予防・先制医療の実現をも見据えた研究開発に取り組む必要がある。そのためには、健康医療ビッグデータ、AI、ゲノム医療といった革新的なテクノロジーを取り入れたイノベーション創出が必須であり、オープンイノベーションの重要性が増している。

そのオープンイノベーションの形態も環境変化により変遷し、従前の１対１での共同研究から多対多の連携、さらには、産学官の広範なステークホルダーにより構築されたエコシステムにおける取り組みが求められている。

ここでは、製薬業界で取り組んできたオープンイノベーションの事例を紹介するとともに、今後のオープンイノベーションの方向性に関して議論したい。

急性骨髄性白血病 (Acute Myeloid Leukemia: AML)は未熟な造血幹細胞または前駆細胞のクローン性増殖を特徴とする血液がんの一つである。AMLにおいて最も高頻度に認められる遺伝子変異としてFms-like tyrosine kinase 3 (FLT3)遺伝子の変異（遺伝子内縦列重複変異とチロシンキナーゼドメイン変異）が知られている。変異型FLT3はリガンド非依存的に恒常的活性化しており、異常増殖の原因になっている。ギルテリチニブはALK阻害剤研究の中で合成された化合物であり、その創薬研究の過程でFLT3阻害作用が見出されたことからFLT3変異陽性AML患者を適応疾患として研究開発された。本薬は国際共同第III相試験によってFLT3変異陽性患者に対する治療効果が立証され、一昨年、日本及び米国において「再発又は難治性のFLT3遺伝子変異陽性の急性骨髄性白血病」を適応として世界で初めて承認された。本発表ではAMLの病態、AMLにおけるFLT3の役割、ギルテリチニブの非臨床データなどについて紹介する。

G protein-coupled receptors (GPCRs) are the largest family of cell surface drug targets. Consequently, there is high interest in understanding the structure of members of this receptor superfamily and the molecular detail of how ligands and transducer proteins interact with them. Our laboratory has been applying single particle cryo-EM to determination of active GPCR structures, using minimally modified receptors. Our work has been principally focused on the class B GPCR subfamily that bind large peptide hormones and are well established clinical targets for the treatment of major disease, including migraine, irritable bowel syndrome, diabetes, obesity and neurodegeneration. Combining novel structural information with molecular pharmacology, biophysical studies and molecular dynamics simulations, we are gaining substantial insights into how ligands and accessory proteins interact with this receptor family to facilitate G protein coupling and downstream signaling.

高齢化社会をむかえパーキンソン病の増加が社会問題であるが、根本的治療薬は未開発でありその開発が期待されている。私たちはパーキンソン病の発症機構の解明と治療薬の開発において、ドパミン神経選択的な変性メカニズムの解明、ドパミン機能を制御する新たな分子機構の発見、新たな創薬標的を用いた治療薬の薬理学的研究に取り組んできた。具体的に、ドパミン生合成の律速酵素であるチロシン水酸化酵素が中脳ドパミン神経で消失する新たな分子機構、消失したドパミン生合成酵素を回復するための新たな創薬標的の探索とドパミン作動性機能を制御する新規シグナルネットワーク、さらにパーキンソン病の原因タンパク質αシヌクレインが中脳ドパミン神経に取り込まれ伝播する新たなメカニズムを明らかにし、パーキンソン病の予防・治療応用が期待される。これらの研究成果から、パーキンソン病を含むレビー小体疾患の克服とその創薬研究、今後の展望について紹介する。

本演者らは、二光子励起顕微鏡を用いて、動物個体を生かしたままで生体骨・関節組織内部をリアルタイムで可視化するライブイメージング系を確立し、骨表面上での生きた破骨細胞の"動き"や"機能"、骨芽細胞との"相互作用"を可視化することに成功した。本技術を駆使して、様々な骨疾患治療薬が実際にどのような薬理作用を発揮するのか、特に生きた組織・細胞レベルでの作用機序について検証するシステムを独自に確立した。その結果、骨粗鬆症治療で汎用されている活性型ビタミンD製剤や副甲状腺ホルモン製剤が、生体内で骨量を増加させる仕組みが明らかとなった。さらに、関節リウマチ治療において臨床応用されている生物学的製剤3剤が、それぞれ異なる作用機序で破骨細胞による骨破壊を抑制し得ることが分かった。本研究で確立した骨・関節組織の生体イメージング技術は、骨疾患の病態解明や新規治療薬の開発において強力な手段となることが強く期待される。

記憶の形成後、長時間の経過や神経細胞の脱落などにより、記憶は想起できなくなる。しかし、ふとした瞬間に自発的に想起できることがあるように、記憶痕跡は脳内に残っていると考えられる。脳内ヒスタミン神経は結節乳頭核から脳の広域へ投射し、認知機能や覚醒、食欲に関与する。私たちの研究グループは、これまでマウスの行動解析、神経活動の操作や神経活動イメージング、臨床研究を融合させて研究を進めてきた。そして脳内ヒスタミン神経系を活性化させることにより、一見忘れたように思える記憶でも、想起を回復できるようになることを明らかにした。さらに詳細な神経回路機構の解析により、ヒスタミンは学習時に活動した嗅周皮質ニューロンの再活性化を促進させることを見出した。このような記憶の想起を回復させる神経回路機構の解明は、アルツハイマー病などの認知機能障害の治療薬開発の一助となることが期待される。本講演では記憶の想起を中心とした記憶・学習の神経回路機構について、in vivoでの神経活動操作やイメージングを含めた最新の知見と今後の展望を紹介したい。

The expansion of COVID-19 in the world has not ended yet, and the situation in Japan is still unpredictable. Under these circumstances, the development of SARS-CoV-2 treatments such as vaccines and medicines is still underway. Since April of this year, we have been conducting research on the drug screening for SARS-CoV-2 using the supercomputer "Fugaku". In my talk, I would like to talk about the impact of the supercomputer "Fugaku" on drug discovery, by exemplifying the in silico drug screening for SARS-CoV-2.

The clinical manifestations of COVID-19 vary from mild or almost asymptomatic to severe ones that require intensive cares such as mechanical ventilation and ECMO in the ICU. In humans with such complex genetic or environmental factors, multi-layered omics networks on the viral and the host side are involved in the formation of various pathologies across all organs. Precision medicine, which provides appropriate medical cares to individual patients based on the omics information including genomic one, is a key to control COVID-19 in humans with diverse biological backgrounds. We construct the multi-layer omics data generation / analysis platform, in which time-series multi-layered omics data of the virus and host, which are linked to medical information, are stored. Those our generating and public data, could be useful for clarifying the complex biological network of COVID-19, and the prediction of disease progression, complications, and effects of interventions for individual patients.

A new pandemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has emerged and its severe and prognostic symptoms have become a social problem. SARS-CoV-2 infection is mainly initiated by binding of its surface Spike protein to angiotensin converting enzyme 2 (ACE2) on the host cell membrane followed by internalization via endocytosis-mediated pathway. We eventually found that ACE2 expression levels were upregulated by the exposure to epidemiologically reported risk factors for COVID-19 severity; cigarette side-stream smoke, anti-cancer drug, diabetes and obesity, through formation of protein complex between transient receptor potential canonical (TRPC) 3 and NADPH oxidase (Nox) 2 on the rodent myocardial plasma membrane. We thus used 20 already approved drugs that has previously reported to have potency to attenuate the formation of TRPC3-Nox2 protein complex and evaluated the inhibitory effect on Spike protein-induced ACE2-GFP internalization in HEK293 cells. We found that several TRPC3-Nox2 complex inhibitors also have potency to inhibit the Spike protein-induced ACE2 internalization, and finally identified one drug that has the most potency to prevent SARS-CoV-2-induced infection and proliferation in TMPRRS2-expressing VeroE6 cells, and fluorescence-labeled S protein-induced pseudo infection in human iPS cardiomyocytes. These results provide a new concept that the inhibition of TRPC3-Nox2 complex formation will be a novel strategy to improve COVID-19 aggravation and sequelae by suppressing ACE2-mediated infection pathway.

The pandemic of COVID-19 presents an urgent need for effective treatments. To date, there are limited therapies that are proven to be effective against COVID-19. Several potential candidates or repurposed drugs are under investigation, including drugs that inhibit SARS-CoV-2 replication and block infection via its receptor ACE2. The most promising therapy is remdesivir, which is approved by US Food and Drug Administration (FDA) for emergency use in adults and children hospitalized with severe suspected COVID-19. In addition, because SARS-CoV-2 may induce long COVID-19 symptoms, such as heart failure and myocarditis, safety issues of anti-COVID-19 drugs including cardiotoxicity are important for patients. Here we show currently available pharmacological strategies, that are based on our understanding of COVID-19. In particular, we have used iPSC-based models to investigate the efficacy and safety of drug candidates for COVID-19. We found several drug candidates which inhibit SARS-CoV-2 replication. These drugs have not shown any cardiotoxicities using iPSC-cardiomyocytes. In the symposium, we would like to discuss our challenge and future perspectives against COVID-19 drugs.

Although depression has a very high lifetime incidence rate of 10%, the remission rate of existing antidepressants is low, and the discovery of new drug targets is urgently needed. On the other hand, large-scale genome-wide association study analysis revealed Sirt1 as a genomic region associated with depression, and because Sirt1 is a factor involved in the regulation of mitochondrial activity, we focused on mitochondria in the present study to examine their relevance to depression. Mice showed depressed- and anxiety-like behaviors when subjected to chronic restraint stress. These mouse brain mitochondrial specimens had significantly reduced oxygen consumption rates and expression of electron transfer chain proteins. Furthermore, mitochondria-specific unfolded protein stress responses (UPRmt) was significantly correlated with the depression-like behavior of mice. Antibiotics that inhibit protein synthesis have been reported to induce UPRmt. We found that doxycycline had antidepressant- and anxiolytic-like effects in mice when it was chronically applied to them. These results indicate that UPRmt may be involved in depressive- and anxiety-like behaviors in mice.

Acetylcholine is crucial for regulation of synaptic transmission in the hippocampus and the cortex, the center of learning and memory. Released acetylcholine from cholinergic terminals is thought to act on muscarinic and nicotinic acetylcholine receptors on plasma membrane, and is subsequently hydrolyzed by cholinesterase. Previous experiments by our group have shown that muscarinic acetylcholine receptor subtype M1 (M1-mAChR) in the brain is highly distributed on intracellular organelles of neurons, such as endoplasmic reticulum and Golgi apparatus, and activates signaling cascades distinct from those of plasma membrane receptor. The intracellular M1-mAChRs is activated by endogenous acetylcholine following its uptake via a putative transport system without degradation, which facilitates long-term potentiation in the hippocampal CA1 synapses. In addition, the cholinergic synaptic regulation through intracellular M1-mAChR in the hippocampus disappeared after chronic physical stress. In this symposium, we demonstrate the function of intracellular M1-mAChR in the brain and discuss about possible contribution to stress and neuropsychiatric disorder.

The ovary is innervated by autonomic nerves in addition to being under the control of hormones. In this symposium, we will introduce our studies on regulation of ovarian estradiol secretion and ovarian blood flow by sympathetic adrenergic innervation in relation to physical stress.

In rats, sympathetic nerves reach the ovary by two routes: the superior ovarian nerve (SON) in the suspensory ligament and ovarian nerve plexus (ONP) along the ovarian artery. Of the two pathways, stimulation of the SON, but not the ONP, reduces estradiol secretion via activation of alpha2-adrenoceptors, whereas stimulation of the either nerves reduces ovarian blood flow via activation of alpha1-adrenoceptors. Reflex activation of ovarian sympathetic nerves by noxious physical stress (noxious cutaneous stimulation of a hindpaw) causes ovarian vasoconstriction and inhibition of ovarian estradiol secretion. The ovarian vasoconstrictive response is produced via reflex activation of the SON and ONP, whereas inhibition of ovarian estradiol secretion occurs as a result of reflex activation of the SON only. These results suggest that reflex activation of sympathetic nerves to the ovary by stressful physical stimulation, for example, noxious stimulation, may be involved in rapid inhibition of ovarian function in emergencies. Rapid and direct regulation of ovarian function by the autonomic nerves may be an important adaptation of female reproductive function to either internal or external environmental changes.

The sympatho-adrenomedullary (SA) system is one of the body's principal responses to stress stimuli. In contrast, dysregulation of the system in response to excessive stress can contribute to the development of various diseases including essential hypertension and other cardiovascular events, gastrointestinal diseases and certain disorders of immune function. Therefore, to establish "fundamental" approaches for treatment of these stress-related diseases, we have to elucidate regulatory mechanisms that control the SA outflow focusing on the brain, which plays a key role in responses to stress. We have investigated a relationship between brain 2-arachodonoylglycerol (2-AG) and central regulation mechanisms for the SA outflow. 2-AG has been recognized as an endogenous ligand for cannabinoid CB receptors (endocannabinoid), which plays an inhibitory role in synaptic neurotransmission via presynaptic CB₁ receptors. On the other hand, 2-AG is hydrolyzed by monoacylglycerol lipase to arachidonic acid, a precursor of prostanoids. In this symposium, we will introduce our data indicating bidirectional roles of brain 2-AG as an endocannabinoid and as a precursor of prostanoids in central regulation of the SA outflow.

Purinergic receptors are nucleoside receptors, adenosine receptors (A1, A2_{A, B}, A3), and nucleotide receptors, P2X and P2Y receptors. Burnstock proposed calling the former the P1 receptor and the latter the P2 receptor(1978), and further subdividing the P2 receptor into ion channel-typed P2X and G protein-coupled P2Y(1985). P2X and P2Y are collectively called ATP receptors. Adenosine-3'-phosphate (ATP) is an agonist of P2X receptors. P2X molecules form a trimer acting as a single non-selective cation channel (Na⁺, K⁺, Ca²⁺). Seven types from P2X1 to P2X7 have been reported. All subunit molecules form homomer receptors, and some P2X molecules can form heteromers (P2X1 with P2X5, P2X2 with P2X3 or P2X6, and P2X4 with P2X6 or P2X7). Various subtypes are expressed in all tissues in the body, and utilize the difference in sensitivity (1 million times difference from nM to mM) to play an important individual physiological functions.

It's already been 61 years since Holton pointed out the relationship between ATP and pain (1959). Today, ATP is involved in a variety of nociceptive transmission through ATP receptor subtypes that are variously expressed in dorsal root ganglion (DRG) neurons, dorsal root ganglion neurons, spinal microglia, and the upper central nervous system.

Currently, drug discovery targeting ATP receptors is being carried out all over the world.

In this memorial lecture, I will review the long drug development history and belated US FDA approval of the adenosine A2A receptor (A2AR) antagonist istradefylline for treatment of Parkinson's disease (PD) and its implications. The A2AR has been long considered as the leading drug target for PD treatment for its highly concentrated expression in the striatopallidal neurons and its colocalization and functional antagonistic interact ion with dopamine D2 receptors in this neuron type. Since the early 1990s, mounting experimental findings have demonstrated the motor enhancement effect of A2AR antagonists, alone or synergizing with L-DOPA in rodents and non-human primates. Since 2001, more than 25 clinical trials were conducted to evaluate the safety and clinical efficacy of A2AR antagonists in PD patients. After more than two decades of preclinical and clinical studies, on August 27, 2019, the U.S. Food and Drug Administration (FDA) approved the adenosine A2A receptor antagonist Nourianz (istradefylline) developed by Kyowa Hakko-Kirin Inc., Japan, as an add-on treatment to levodopa in Parkinson's disease (PD) with "OFF" episodes. This milestone achievement represent is the first A2AR therapeutic drug and the first non-dopaminergic drug for PD treatment approved by US FDA in the last two decades. This approval provides an important lessons to be remembered, namely the selection of defined patients subpopulations who are most responsive to the tested drug is critical for a successful demonstration of the clinical efficacy of the drug. Importantly, this approval paves the way to foster entirely novel therapeutic opportunities for adenosine A2A receptor antagonists, such as neuroprotection or reversal of mood and cognitive deficits in PD and other neuropsychiatric diseases. As a pioneer, Professor Geoff Burnstock established the purinergic signaling field against all the odds more than 50 years ago and thus set up the stage for us to pursuit and achieve many milestone like this in near future.

Molecular identification of purine receptors, particularly the P2X receptor channels of several types, in the late 90's lead researchers to identify their roles in the brain because many subtypes were found there. Except for the first identification of the fast excitatory synaptic transmission mediated by ATP in the medial habenula of rats (Edwards et al., 1992), such demonstration in the mammalian brain remained highly limited, mostly due to the extremely rapid breakdown of ATP to adenosine. In the brain slice preparation, we found that ATP (presumably released from astrocytes) triggers Ca-dependent action potential-independent release by activating presynaptic P2X2/3 receptors, which is followed by reduction of action potential-dependent release through activating presynaptic adenosine A1 receptors (Kato & Shigetomi, 2001; Shigetomi & Kato, 2004). We proposed that spatially limited colocalization of P2X and A1 receptors and ectonucleotidase enables ATP to exert dual functions and dubbed this system "purine signaling complex in the brain synapses," which later found in many brain structures (Kawamura et al., 2004) and also in non-mammalian species (Wakisaka et al., 2017).

The molecular pathogenesis of depression is often explained by the catecholaminetheory. However, there are many cases of so-called refractory depression thatare resistant to existing noradrenergic and serotonin activating drugs, so thetrue molecular pathogenesis of depression needs to be elucidated. Asdemonstrated by Prof Burnstock, ATP is not a merely energy currency but is animportant intercellular signaling molecule that mediates information betweenneurons and glial cells in the central nervous system. Animal studies havereported that "ATP" is the most universally altered intercellular signalingmolecule in depressive symptoms, and that a decrease in this extracellular ATPcauses depressive symptoms (Nat Med 2013). However, its molecular and regulatorymechanisms remain unclear. In the present study, we tested this ATP hypothesisand elucidated the molecular mechanism of this ATP hypothesis using fluoxetine(FLX), a typical SSRI antidepressant, which increased extracellular ATP (ATPo)in the hippocampal brain in a dose-dependent manner. This ATPo increase wasdependent on astrocytes rather than neurons, and the molecular mechanism of thisincrease was found to be an enhancement of the ATP exocytosis. We found thatreleased ATP acts as ATP and degraded adenosine on P2Y11 and A2b receptors,respectively, to enhance the cAMP-CREB system and that this signal enhancementleads to BDNF expression. Very interestingly, the sequence of responses fromATP/adenosine to BDNF production was induced in astrocytes, but not in neurons.We further discuss the importance of astrocytes as a prime target fordepression.

We investigated the roles of nitric oxide synthases (NOSs) in the pathogenesis of lung diseases by using our triple n/i/eNOSs^-/- mice. Bleomycin treatment resulted in pulmonary fibrosis (PF) in wild-type (WT), single nNOS^-/-, iNOS^-/-, and eNOS^-/-, and triple n/i/eNOSs^-/- mice as compared with saline treatment. PF was exacerbated only in triple NOSs^-/- mice, but not in any single NOS^-/- mice, as compared with WT mice, suggesting a protective role of NOSs in the development of PF（Respir Res 2014). Hypoxic exposure led to pulmonary hypertension (PH) in all WT, single NOS^-/-, and triple NOSs^-/- mice as compared with normoxic exposure. PH was aggravated in triple NOSs^-/- mice and, to a lesser extent, in single eNOS^-/- mice as compared with WT mice, again suggesting a protective role of NOSs in the development of PH (AJRCCM 2018). In contrast, ovalbumin-induced bronchial asthmatic changes (BA) were markedly mitigated in triple NOSs^-/- than in WT mice, suggesting an opposing injurious role of NOSs in the development of BA（Lung 2016). We recently found that spontaneous development of pulmonary emphysematous changes (PE) was noted only in triple n/i/eNOSs^-/- mice, suggesting a preventive role of NOSs in the development of PE. Taken together, our findings demonstrate diversity of the roles of NOSs in the pathogenesis of lung diseases.

Pulmonary arterial hypertension (PAH) is a chronic and life-threatening disease characterised by progressive vascular remodelling that leads to increased pulmonary vascular resistance, right ventricular heart failure and death. PAH is defined by >25 mmHg increase in pulmonary arterial blood pressure and a pulmonary capillary wedge pressure of 15 mmHg. If left untreated PAH is fatal; it has a survival rate of just 34 % after 5 years. Current therapies for PAH include stimulating the nitric oxide (NO)–soluble guanylate cyclase (sGC)–cyclic guanosine monophosphate (cGMP) axis, improving the prostacyclin pathway or inhibiting the endothelin pathway. Although the causal relationship remains unproven, the NO–sGC–cGMP axis is ultimately a critical factor in the development of PAH because the condition is associated with endothelial dysfunction, impaired NO synthesis and insufficient stimulation of the NO–sGC–cGMP pathway. NO activates sGC, resulting in the synthesis of cGMP, which is a key mediator of pulmonary arterial vasodilatation that may also inhibit vascular smooth muscle proliferation and platelet aggregation. Dysregulation of the NO–sGC–cGMP axis results in pulmonary vascular inflammation, thrombosis and constriction, and ultimately leads to PAH. Therapeutic options targeting the NO–sGC–cGMP axis include phosphodiesterase type 5 (PDE5) inhibitors, such as sildenafil and tadalafil, and the sGC stimulator riociguat. In this session, I would like to introduce the importance of NO-cGMP-PKG Axis in PAH and discuss the similarities and differences between PDE5 inhibitors and sGC stimulator and considers which is better for the treatment of PAH.

Coronavirus is a type of single-stranded RNA virus with a lipid bilayer called an envelope on the outside, and it looks like a corona under an electron microscope. It cannot self-regenerate, attaches to and enters human cells via ACE2 receptors, and proliferates within the human cells. There have been some coronaviral diseases such as SARS, MERS and "normal" human coronaviruses (HCoV) that cause common colds.

The "new coronavirus" was named as SARS coronavirus-2 (SARS-CoV-2), and new coronavirus infectious disease caused by SARS-CoV-2 was named as COVID-19 short for "corona-virus disease discovered in 2019". SARS-CoV-2 infection spreads before the symptom onset, and prophylactic actions such as wearing masks and social or physical distancing are very important and critical.

Treatment is mainly general respiratory management with various therapeutic agents including approved or nonapproved antiviral drugs for treating new coronavirus infection such as remdesivir, favipiravir in combination with corticosteroids (dexamethasone) and other expected agents for treating COVID-19 such as inhaled corticosteroid (ciclesonide), ivermectin, anticoagulants, antimicrobials and the treatment of comorbid illnesses. Therapeutic methods and agents are selected at the hospital based on the latest information such as treatment results and side effects of each regimen.

In this symposium, I would like to talk about current knowledge of the diagnosis and management of COVID-19 in Japan.

Acute bacterial or viral infections responsible for pneumonia or sepsis cause severe inflammatory damage to the lungs, leading to the development of acute lung injury (ALI) in critically ill patients. ALI is characterized by high permeability pulmonary edema as well as refractory hypoxemia and diffuse pulmonary infiltrates. Our previous study has shown that the inducible NO synthase inhibition or the histamine H₁-receptor blockade significantly but incompletely inhibited LPS-induced lung vascular permeability in mice, suggesting that NO and histamine may also be partly responsible for mediating increased lung vascular leak following LPS challenge. The involvement of histamine in lung vascular leak has also been demonstrated using histidine decarboxylase gene knockout mice. Furthermore, treatment with the VEGF-neutralizing monoclonal antibody bevacizumab significantly reduced the LPS lung hyperpermeability response, suggesting active participation of VEGF in non-cardiogenic lung edema associated with LPS-induced ALI. We thus assume that several vascular permeability molecules, including NO, histamine, and VEGF, can be excessively produced and thereby actually contribute to the development of pulmonary edema in ALI.

Men with benign prostatic hyperplasia (BPH) often experience symptoms of overactive bladder (OAB), and bladder outlet obstruction (BOO) is one of cause of BPH. It has been suggested that bladder myogenic microcontractions or micromotions may partly contribute to the development of urgency (bladder sensory (afferent) hypersensitivity) in OAB related to BOO. We have investigated the direct effects of drugs (anticholinergics, β3-adrenoceptor agonists, α1-adrenoceptor antagonists, PDE type5 inhibitors, etc.) on the bladder afferent function in rodents. In our results, almost all drugs may act on the bladder afferent function, and some of drug (e.g. mirabegron) inhibits the afferent activities through the suppression of the bladder myogenic microcontractions in normal or pathophysiological conditions.

Chronic prostatitis/chronic pelvic pain syndrome (CP/CPPS) causes long-standing pain and/or storage symptoms including storage symptoms, such as urgency and frequency. It has been reported that CP/CPPS is possibly overlapping with symptoms in BPH.

In this symposium, I will introduce our recent studies using pathophysiological animal models with BOO or CP/CPPS.

Erection is initiated by the production and release of nitric oxide (NO) via NO synthase upon sexual stimulation. NO relaxes the penile corpus cavernosum smooth muscle, which increases the inflow of blood into the penile corpus cavernosum and compresses the veins, thereby preventing blood loss. This increases the intracavernous pressure in the penile corpus cavernosum to induce erection. Phosphodiesterase 5 (PDE5) inhibitors, which target the NO/cyclic guanosine monophosphate (cGMP) pathway, are currently used to treat erectile dysfunction (ED). PDE5 inhibitors are useful drugs in the general population with ED; however, these inhibitors are not necessarily useful for ED due to diabetes complications or neurogenic conditions after radical prostatectomy. One reason for this low effectiveness is inadequate production of NO in these conditions. Therefore, NO replacement may be an effective ED treatment, but it has been difficult to achieve to date due to systemic side effects.

To overcome this limitation, we focused on the development of light-controlled NO donors, which can produce NO in response to light, thus producing NO only in localized, light-exposed areas. Light-controlled NO donors can also control the onset and end of drug effects because they can produce NO only for the duration of light exposure. We modified the compound and recently succeeded in developing a red light-responsive NO donor termed "NORD-1" with high tissue permeability.

In this symposium, we introduce the results of an in vivo study on the enhancement of erectile response with NORD-1 and the effectiveness of the ED model.

Mammalian sperm activate their motility upon ejaculation, but sperm motility needs further change to whiplash-like motility to penetrate and fertilize the egg. This specialized motility is called "hyperactivation". It was reported that extracellular Na⁺ is involved in regulation of hyperactivation. Na⁺/K⁺ ATPase (NKA) plays a major role in the regulation of cellular Na⁺ homeostasis. The catalytic subunit of NKA is an α subunit, and there are α1 and α4 subunits in sperm. The sensitivity of these two α subunits to ouabain, a specific NKA inhibitor, is quite different, that is, 10^-6 M ouabain inhibits NKA α4 while >10^-5 M ouabain inhibits both the α1 and α4. Therefore, this difference of sensitivity enables us to distinguish the physiological role of NKA α subunits on sperm function by pharmacological approach. In this symposium, I will introduce my latest study which showed, by utilizing this pharmacological property of ouabain, that the NKA α1 subunits is necessary for the maintenance of motility while the α4 subunit is necessary for the hyperactivation-associated change in flagellar movement. These results suggest that NKA α4 subunit is an attractive target for the male contraception.

Spermatogonial stem cell (SSCs) proliferate and differentiate to sperm in seminiferous tubules. SSCs are under well-organized control of germ cell niche composed of Sertoli cells, Leydig cells, peritubular myoid cells (PMCs) and vasculature. Thus, Sertoli cell dysfunction causes spermatogenic failure. Recently we discovered benzalkonium Chloride (BC) ablates Sertoli cells selectively. We tried manipulating mouse germ cell niche by seminiferous transplantation into BC treated testis. FACS sorted donor Sertoli cells colonize and support recipient spermatogenesis. When we transplanted whole testicular cells into Sertoli-depleted testis, donor SSCs, Sertoli, Leydig and PMCs colonize and reconstruct donor testis in recipient testis. BC has been used clinically for decades and ablates mouse and canine Sertoli cells, suggesting that this novel method of drug-induced Sertoli cell elimination followed by replacement may be useful for male infertility patients with Sertoli cell disorder. Using this novel technique, we can reorganize mouse germ cell niche. This might achieve human testis reconstruction in mouse testis and can be a strategy of fertility preservation for young cancer patients.

In 2020, the number of universities teaching nursing in Japan reached 274. In recent years, increasing social demand has been seen for nurses who can cope with Japan's declining population, advances in medical care, and community-based integrated care systems. Therefore, the expectations of recipients of a bachelor's degree in nursing education have been rising. In 2017, the Ministry of Education, Culture, Sports, Science and Technology of Japan published the "Model Core Curriculum for Nursing Education in Japan" and established learning goals that each university can refer to when organizing the curriculum. In October 2020, the "Regulation for Enforcement of the Act on Public Health Nurses, Midwives, and Nurses" was revised and the total number of credits was increased, and one unit was added to enhance the foundation of clinical judgment when applying pharmacological knowledge. The revision of this regulation provides a good opportunity for universities to revise their own curriculums. It is important both to consider carefully how a curriculum is organized at the basic education stage within a limited period and to create one that attracts students.

Elderly patients who take multiple types of medication have been increasing. Such patient often taking supplements and traditional Chinese medicine. A number of medication errors by nurses have been reported in Japan.

The model core curriculum for nursing education, which was established in 2017, claimed the importance of pharmacology education in nursing education. Produce the desired pharmacology education in science of nursing. We would like to have discussions, for pharmacology education in science of nursing the purpose of produce of nursing practice ability.

I have experienced for clinical practice in the intensive care unit for more than 10 years and am working as a professor in nursing school. In this symposium, I will talk about pharmacology education for nursing student and clinical nurses based on my experience.

The point of our opinion is pharmacology in nursing curriculum at this time is just pharmacology, not but pharmacology for nursing or nurses. Off course, pharmacology is pharmacology, however, its education should be changed depending on medical profession and it should contribute to knowledge in terms practice. It is time for transition from pharmacology education for pharmacology to pharmacology education for specifically nursing students and clinical nurses.

Two active learning programs of practical pharmacotherapy for nursing students have been performed in school of nursing, University of Miyazaki; (1) pharmacotherapy role-play for interprofessional education (IPE) and (2) practical excise for Kampo medicine.

Pharmacotherapy role-play for IPE was performed as joint lecture both medical students and nursing students. This pharmacotherapy role-play is named Case & Communication based approach (C&C approach), since it is studied through communication between physicians, nurses and patients based on cases presented beforehand. In the practical excise for Kampo medicine, nursing students studied Kampo medicines and tried to taste 9 frequently used Kampo medicines. These active-learning programs in nursing pharmacology education may be effective for better understanding of pharmacotherapy, patient's feeling, and improvement of students' awareness and motivation as a nurse.

In this symposium, we would like to show the detail of these programs and possibility of contribution to nursing education.

COVID-19 therapeutics currently under development can be broadly divided into two categories. The first is an antiviral drug with in vitro antiviral activity that inhibits the growth of the virus in infected organs in the body. Second, they do not act directly on the virus, but act on the body to ameliorate cytokine storms and acute respiratory distress syndrome (ARDS) caused by an overactive defense response to infection. For an entirely new drug, the safety of the drug has to be confirmed in pre-clinical animal studies, and the safety and dosing of the drug in humans must be established in Phase I and Phase II studies. However, if the drug has already been administered to humans through drug repositioning, the initial steps in development can be shortened. Antiviral drugs include remdesivir, favipiravir, ciclesonide, nelfinavir, nafamostat and ivermectin. On the other hand, bioactive agents include dexamethasone, tocilizumab, baricitinib, and eritran. These drugs and other candidates and others are summarized and briefly presented in this section.

Countries around the world are currently fighting the coronavirus disease 2019 (COVID-19) pandemic, which is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). SARS-CoV-2 is a betacoronavirus, belonging to the same genus as severe acute respiratory syndrome (SARS)-CoV and Middle East respiratory syndrome (MERS)-CoV. Currently, there are no proven antiviral therapies for COVID-19. Numerous clinical trials have been initiated to identify an effective treatment. One leading candidate is remdesivir (GS-5734), a broad-spectrum antiviral that was initially developed for the treatment of Ebola virus, has been provisionally approved for COVID-19. Remdesivir is a nucleotide analogue that delays replication of viral RNA. It comes in the form of a prodrug which is metabolized to the active form (remdesivir triphosphate) once it enters cells. In vitro and preclinical studies have shown that it has antiviral activity against SARS-CoV-2. There have been several studies of remdesivir in patients with COVID-19. These have included a compassionate use program, placebo-controlled trials and dosing trials. The results present here.

Eritoran (E5564) is Eisai's in-house discovered and developed investigational TLR4 (Toll-Like Receptor 4) antagonist created with natural product organic synthesis technology. It is a structural analogue of Lipid A, which is an activator of endotoxins of bacteria. It has been previously observed to be safe in 14 clinical studies including a large Phase 3 randomized trial in severe sepsis. We are participating in the international network REMAP-CAP-COVID (Randomized, Embedded, Multi-factorial, Adaptive Platform-Community Acquired Pneumonia COVID) which aims for novel coronavirus medicine development through drug repurposing, and began an international collaborative clinical trial in October 2020 which is designated for confirmed novel coronavirus patients who are hospitalized and are in a progressing disease state. It is hoped that through suppressing the most upstream TLR4 activity which controls production of multiple cytokines, the cytokine storm in patients can be suppressed and pneumonia can thus be prevented from becoming severe.

Although months have passed since WHO declared COVID-19 a global pandemic, only a limited number of clinically effective drugs are available, and the development of drugs to treat COVID-19 has become an urgent issue worldwide. The pace of new research on COVID-19 is extremely high and it is impossible to read every report. In order to tackle these problems, we leveraged our artificial intelligence (AI) system, Concept Encoder, to accelerate the process of drug repositioning. The Concept Encoder is a patented AI system based on natural language processing technology and by deep learning papers on COVID-19, the system identified a large group of genes implicated in COVID-19 pathogenesis. The AI system then generated a molecular linkage map for COVID-19, connecting the genes by deep learning the molecular relationship. By thoroughly reviewing the resulting map and list of the genes with rankings, we found potential key players for disease progression and existing drugs that might improve COVID-19 survival. Here, we focus on potential targets and discuss the perspective of our approach.

Excessive or chronic social stress induces emotional and cognitive disturbancesand is a risk for mental illness. Reduced neuronal activity in the medialprefrontal cortex (mPFC) underlies these behavioral abnormalities. However, thesubcellular origin and process of this neuronal change remain elusive. Here weexamined ultrastructural alterations of mPFC neurons after social defeat stressin mice by serial electron microscopy and expansion microscopy. Social stresscaused the loss of dendritic branches as well as morphological abnormality ofsubcellular structures, including mitochondria in remaining dendrites withvaricosity-like membrane deformation. Social stress induced synaptic shrinkageselectively at mitochondria-containing synapses. Furthermore, multi-omics andfunctional analyses revealed that social stress deteriorated mitochondrialfunctions with decreased mitochondrial proteins at synapses. Pharmacologicalmanipulation targeting mitochondria attenuated the synaptic shrinkage anddepression-related behaviors. Together, these findings illustrate the importanceof synaptic subcellular organelle in the synaptic pathology underlying stressand depression.

The processing of stress responses involves brain-wide communication among cortical and subcortical regions. Although considerable effort has been made to discover these brain regions, hypothesis-free and unbiased approaches to identify previously unknown neural elements have been limited until recently due to technical limitations. Here, we performed whole-brain activation mapping and machine learning-based analyses to identify previously uncharacterized brain regions and neuronal ensembles implicated in stress, and found that the claustrum (CLA) most prominently contributes to discrimination in stressed brains. Activity-dependent genetic labeling in TRAP2 mice subjected to social defeat stress revealed reciprocal connection of the CLA with fos-tagged neurons in brain-wide areas, including the basolateral amygdala and medial prefrontal cortex. Chemogenetic reactivation of the fos-tagged CLA neuronal ensemble reproduced anxiety-like behaviors and brain-wide neuronal activation, while its silencing attenuated anxiety-like behaviors induced by acute social defeat stress and increased resistance to chronic social defeat stress. The CLA thus controls stress-induced emotional responses bidirectionally through brain-wide networks, and its inactivation can be a preventative measure that increases stress resilience.

Stress experiences induce diverse psychiatric symptoms. While stress experiences themselves are terminated within short time periods at a time scale of seconds or minutes, the stress-induced mood changes subsequently last for days or even a lifetime. This fact means that physiological activity not only during onsite stress episodes but also during post-stress periods may be crucial to facilitate stress-induced psychiatric changes. We hypothesized that this effect is mediated by memory processing mechanisms in the brain. A well-established theory of memory suggests that learned memory traces need to be consolidated into neuronal circuits for a long-term memory storage. Especially, the ventral part of the HC (vHC) is especially considered a crucial brain region for emotion- and sociality-related information processing and for memory consolidation. We tested this idea, mice were subjected to social defeat (SD) stress. Defeated mice showed deficits in social interactions. This reduction was suppressed by vHC inactivation, suggesting that vHC neuronal activity after stress experiences is especially crucial to induce social behavior deficit. To further examine detailed hippocampal activity patterns, we recorded spike patterns of vHC CA1 neurons using tetrode assemblies. Stress-encoding neurons in the vHC were continuously reactivated for several hours after SD experiences, which likely underlies the long-lasting effects of SD stress on social behavior. To further examine the causal of vHC activity in stress-induced behavioral changes, closed-loop feedback stimulation was applied to the vHC when synchronized reactivations of neuronal population in the vHC were detected. This manipulation abolished the stress-induced reductions in social interaction deficits, suggesting the importance of synchronized reactivation of vHC neurons for subsequent psychiatric responses. The vHC-specific activity patterns may serve as a biomarker and a therapeutic target in stress-induced mood disorders.

Although stressful events predispose individuals to psychiatric disorders, such as depression, not all people who encounter a stressful life experience become depressed, suggesting that gene-environment interactions (GxE) determine depression risk. Accumulating evidence has implicated that stress-induced aberrant synaptic and structural plasticity may be key underlying mechanisms of stress susceptibility. Recent evidence has provided key insights into the biological significance of epigenetic regulation of gene expression in synaptic plasticity and behavioral response to chronic stress. The medial prefrontal cortex (mPFC) is vulnerable to damage from a variety of psychosocial stressors and aberrant structural and functional changes in this brain structure have been implicated in depression. However, little is known about the role of epigenetic mechanisms within the mPFC in chronic stress-induced aberrant neuronal plasticity and depression-like behavior. In this symposium, I will focus on causal and mechanistic evidence implicating altered functions and connectivity of the mPFC circuits in the establishment and the maintenance of stress resilience and susceptibility. I also touch upon recent findings suggesting a role for epigenetic mechanisms in these processes and briefly discuss promising avenues of future investigation.

It has already become a powerful tool for generating neural cells from human induced-pluripotent stem cells (iPSCs) to mimic neurophysiological functions, for instance, by constructing brain organoids. In particular, recent studies using disease-specific iPSCs cells have also been required to utilize a large number of samples, including sporadic diseases. In addition, it becomes important to validate pathological conditions and drug screening in a rapid manner. However, required long period culture, highly cellular heterogeneity and highly clonal diversity make analysis of brain organoids difficult. Therefore, an application of alternative rapid and accurate method has been needed, so target-specific neuronal induction by transient expression of bHLH-type proneural factors and other transcription factors in iPSCs can be very useful.

We have improved this tool based on using TetO-Neurog2 or TetO-Ascl1 system and developed a platform for stable generation and functional analysis of target neuronal cells. In this symposium, we will introduce our new platform and report the results of the studies on Alzheimer's disease and various psychiatric and neurodevelopmental disorders. We believe that our platform will enable us to selectively generate numerous subtype-specific neuronal cells and provide a rapid pathophysiological or drug screening system.

Microglia have been highlighted to understand the underlying pathophysiology of various neuropsychiatric disorders. Postmortem brain analysis and PET imaging analysis are two major methods to estimate microglial activation in humans, however only a limited aspect of microglial activation can be measured by these methods. Dynamic analysis using fresh microglia in human brain is an ideal method, however technological and ethical considerations have limited the ability to conduct research using fresh brain microglia.

To overcome this limitation, we have originally developed a technique to create directly induced microglia-like (iMG) cells from fresh human peripheral blood monocytes adding GM-CSF and IL-34 for 2 weeks, instead of brain biopsy (Sci Rep 2014). Using the iMG cells, dynamic morphological and molecular-level analyses such as real-time cell differentiation, phagocytosis and cytokine releases are applicable.

We have used the iMG cells as surrogate cells of human brain microglia, and revealed previously-unknown dynamic pathophysiology of microglia in patients with Nasu-Hakola disease (Sci Rep 2014), fibromyalgia (Sci Rep 2017) and rapid-cycling bipolar disorder (Front Immunology 2017).

In addition, we have recently shown microglia-related pathophysiology using plasma such as human metabolome analysis focusing on the tryptophan-kynurenine pathway and neuron-related exosome analysis.

We believe that these indirect methods using human bloods shed new light on clarifying dynamic molecular pathologies of microglia in a variety of neuropsychiatric disorders.