下垂体アデニル酸シクラーゼ活性化ポリペプチド（PACAP）は、1980年代、TRH、GHRH、CRHなど向下垂体性視床下部ホルモンが次々と発見された後、さらに未知の視床下部因子を探索する過程において発見された。ラット下垂体細胞のcyclic AMP産生刺激活性を指標として、羊視床下部抽出物より、1989年に38アミノ酸からなるPACAP38が、その後 そのN末端 27 残基からなる PACAP27が単離同定された。それらの構造は血管作動性腸管ペプチド(VIP)に68％の相同性を示し、セクレチン・グルカゴンファミリーに属する。その受容体には、PACAP特異的なPAC1Rと、VIPと共有するサブタイプのVPAC1RとVPAC2Rがあり、いずれもGタンパク共役型受容体である。PACAP38は、脳内に広く分布し、視床下部に最も高濃度に存在するが、ほぼ全ての下垂体ホルモンの分泌を促進することが発見当初に明らかにされたものの、未だ視床下部における生理機能の全容は解明されていない。しかしながら神経栄養因子としての機能に着目し、脳卒中など虚血性脳疾患や神経変性疾患の治療へアゴニストとしての臨床応用を目指す一方で、神経伝達物質として痛覚伝達における機能を基盤として、アンタゴニストによる難治性疼痛治療への臨床応用を目指す研究が進められるようになって来た。

COVID-19発症を~95%という高い効率で阻止するワクチンが驚異のスピードで開発されたのは科学がもたらした福音である。しかし、次々と出現する変異株はワクチン効果に対する懸念の域を超える。B型肝炎に対する予防・治療がその典型であるが、ウイルス感染症のコントロールにはワクチンと抗ウイルス薬の両者が必要とされる。しかし、COVID-19発症者の10-20%が中等・重症の呼吸器症状を発症、高齢者や合併症を有する症例では死亡率が高いが、他は軽症か無症状で自然経過、基礎的知見が蓄積される前に投薬された「候補薬」が希望的に「効いた」と間違えて判断されてきた。加えて、「re-purposing (別の目的に再利用)」等の根拠のない期待がその傾向を助長した。そうした混乱から、COVID-19治療薬開発は大きく遅滞したが、初期の「つまづき」は徐々に克服されつつある。しかし、治療効果があると報告された化合物は数例に過ぎない。本講演ではHIV/AIDSに対する治療薬開発のアプローチを基礎としたB型肝炎とCOVID-19治療薬開発の新展開について述べる。

睡眠覚醒調節の根本的な原理、つまり「眠気」（睡眠圧）の脳内での実体とはいったい何なのか、またそもそもなぜ睡眠が必要なのか等、睡眠学の基本課題は全く明らかになっていない。私たちはこのブラックボックスの本質に迫るべく、ランダムな突然変異を誘発したマウスを8,000匹以上作成し、脳波測定により睡眠覚醒異常を示す少数のマウスを選別して原因遺伝子変異を同定するという探索的な研究を行なってきた。このフォワード・ジェネティクス研究の進展により、睡眠覚醒制御メカニズムの中核を担うと考えられる複数の遺伝子の同定に成功し、現在その機能解析を進めている。フォワード・ジェネティクスによって同定されたSleepy変異マウスと断眠マウスの解析から、シナプス蛋白質の累積的リン酸化状態が睡眠圧の本態の一部である可能性が提示された。本講演では、筑波大学WPI-IIISの私どものラボにおける睡眠覚醒の謎への探索的アプローチを紹介する。

近年、細胞内の標的タンパク質を分解する技術が開発され、創薬研究が活発に行われている。分子糊（Molecular Glue）として作用するレナリドミドや、PROTAC（Proteolysis Targeting Chimera）、SNIPER（Specific and Nongenetic IAP-dependent Protein Eraser）に代表されるキメラ化合物がよく知られているが、これらの化合物は標的タンパク質を分解してしまうため、小分子阻害剤とは異なる薬理学的特性を示す。PROTAC/SNIPERは標的タンパク質に結合するリガンドとE3ユビキチンリガーゼに結合するリガンドを繋いだキメラ化合物であり、細胞内で標的タンパク質を強制的にユビキチン化してプロテアソームによる分解を引き起こす。標的リガンドを置換することによって様々なタンパク質を狙って分解する化合物を合理的に設計できるため、創薬の新しいプラットフォーム技術になると期待されている。本講演では我々が開発したSNIPER化合物、タンパク質分解医薬品開発の現状等について紹介する。

生体内での神経回路の発達には、シナプスの形成、除去、再構築が正確に制御されていることが重要である。マウスの大脳皮質では、シナプスの動態に2つの段階があることが、2光子イメージングによって確認された。第1期（生後20日まで）では、シナプスのターンオーバーが高く維持され、第2期（生後3週間以降）では、シナプス動態が強く抑制され、大脳皮質の神経ネットワークとしての成熟が起こる。このようなシナプス動態の変遷は、神経発達障害や精神疾患の病態生理の背景にあると考えられているが、その正確なメカニズムはまだ明らかになっていない。私たちの研究室では、（１）神経回路やシナプス形成過程の多様なメカニズム、（２）シナプスの動的変化の過程での構造・機能連関、（３）脳疾患とシナプス機能障害の関係、に焦点を当てて研究を行っている。最近では、スパインシナプスの超微細構造を定量的に解析する方法や、スパイン内部の分子ダイナミクスを測定する方法など、神経回路を研究するための新しいツールを開発している。さらに、これらのツールは脳疾患の研究にも応用可能である。本講演では、これらの研究を紹介するとともに、精神疾患の病態をシナプス障害として理解することの妥当性と展望について議論したい。

独立行政法人医薬品医療機器総合機構（PMDA）は、健康被害救済業務、承認審査業務及び安全対策業務の３つの業務を柱とした機関である。

平成16年に発足以来、レギュラトリーサイエンスの考え方に基づく適正な承認審査や安全対策の評価を行い、より安全で有効な医薬品・医療機器等をいち早く国民に届けられるよう様々な対策・組織強化を図ってきた。例えば、医療イノベーション推進に向けた迅速な承認審査制度や相談制度の構築、レギュラトリーサイエンスセンターの設置、さらに世界的に猛威を振るっている新型コロナウイルス感染症に関する取り組みについて紹介する。

さらに、薬事規制は国内に留まらず世界との調和・協力が欠かせない時代となっている。PMDAは薬事規制当局の長官レベルの国際会合であるICMRA（International Coalition of Medicines Regulatory Authorities）に参画し、新型コロナウイルス感染症関連製品を含め、薬事規制の国際的なコンセンサスの構築に貢献している。

今般の薬事規制の国際動向や製品開発の現状と課題について紹介する。

腫瘍溶解性ウイルス（OV）は、革新的がん治療技術の有力候補と期待されている。我々はまず、次世代OVのm-CRA（多因子増殖制御型アデノウイルス）プラットフォーム技術を独自開発した。本技術で発明したSurvivin反応性m-CRAは、全がん種に強力な治療効果、高度がん特異性（安全性）、既存治療が無効の「がん幹細胞」に治療効果増強という、革新的作用を示した。治療遺伝子未搭載のSurv.m-CRA-1を、ICH準拠の非臨床開発（GMP製剤製造、GLP安全性試験、規制対応）後、骨軟部腫瘍へのFirst-In-Human医師主導治験第Ⅰ相を完遂し、高い安全性と著明な治療効果を実証した。現在は骨腫瘍への早期承認を目指した第II相試験、膵癌への第Ⅰ／II相試験を実施中である。さらに全身性抗腫瘍免疫を効率よく誘導する免疫遺伝子搭載のSurv.m-CRA-2を非臨床開発中で、さらなる新技術開発も行なっている。この内容を中心に、難治性疾患への増殖因子遺伝子治療、多能性幹細胞・再生医療へのウイルスベクター技術も簡単に紹介したい。

近年、さまざまな分野でP2受容体（P2Rs）の重要性が高まっているが、今回は痛みの研究に焦点を当ててお話しする。慢性的な痛みは、末梢組織の炎症や神経損傷の後にしばしば発生する。特に神経障害性疼痛は、臨床的に有効な薬剤がほとんどないため、重大な臨床的問題となっている。そこで、一次求心性ニューロンのP2X3RとP2X2/3R、脊髄ミクログリアのP2X4R、P2X7R、P2Y12Rを介したATPシグナル伝達の慢性疼痛における役割に関して議論する。さらに、多くの発見により、P2Rsを標的とする新薬の探索が大幅に加速し、これまでにいくつかの化合物が世界で開発されてきたので紹介する。Gefapixantは、難治性の慢性咳嗽として臨床試験を終了した唯一のP2X3R拮抗薬であり承認申請されており、現在は子宮内膜症に関連する痛みの臨床試験が行われている。NP-1815-PXおよびNC-2600は、最近、日本で新規のP2X4Rアンタゴニストとして同定された。NC-2600は、深刻な副作用なしに第I相試験を終了した。このような状況を考えると私たちが痛みに対する新薬を手に入れるも間近と思われる。

Drug addiction can be viewed as a stable form of drug-induced neural plasticity, whereby long-lasting changes in gene expression mediate some of the stable behavioral abnormalities that define an addicted state. Our laboratory has focused on transcriptional pathways in addiction, deduced from large RNA-sequencing datasets of RNAs that show altered expression in brain reward regions of mice as a consequence of drug self-administration, withdrawal, and relapse. Activation or induction of certain transcription factors represent homeostatic adaptations that oppose drug action and mediate aspects of drug tolerance and dependence. In contrast, induction of other transcription factors exerts the opposite effect and contributes to sensitized responses to drug exposure. We are also characterizing a range of chromatin mechanisms that act in concert with these transcription factors to control gene expression. These studies are identifying many of the molecular targets of drug self-administration in brain reward regions and the biochemical pathways most prominently affected. Parallel work has focused on homologous regions in the brains of addicted humans examined postmortem. These advances can now be mined to develop improved diagnostic tests and treatments for addictive disorders.

Funded by the National Institute on Drug Abuse

Over the past four decades, no class of drugs has had more impact on cardiovascular health than the HMC-CoA reductase inhibitors or statins. Developed as potent lipid-lowering agents, statins were shown to reduce mortality and morbidity of patients who are at risk for cardiovascular disease. However, retrospective analyses of some of these clinical trials have uncovered some aspects of their clinical benefits that may be additional to their lipid-lowering effects. In cell culture and animal studies, these effects alter the expression of endothelial nitric oxide synthase, the stability of atherosclerotic plaques, the production of pro-inflammatory cytokines and reactive oxygen species, the reactivity of platelets, and the development of cardiac hypertrophy and fibrosis. Such cholesterol-independent or "pleiotropic" effects of statins generated intense interest as to their potential mechanism and created debate over their relative contribution to cardiovascular risk reduction. One potential mechanism for statin pleiotropy is through inhibition of isoprenoid synthesis and protein prenylation. In particular, the prenylation of Rho GTPases such as Rho, Rac, and Cdc42 is critical to their cellular localization and function. Thus, inhibition of Rho and its downstream effector, Rho kinase, by statins may constitute an important pleiotropic mechanism that could be exploited therapeutically for non-lipid conditions beyond cardiovascular disease.

トランスポーターは生体内の物質分布に寄与する膜タンパク質であり、それを標的とする薬物により多様な代謝制御を実現することができる。がん細胞をはじめとする病態形成に関わる細胞においては、代謝リプログラミングによる代謝系の変化が細胞生存や機能維持に必須の役割を果たすことが多い。特に代謝改変に寄与する栄養素のトランスポーターは、細胞内代謝に介入して病態進展を阻止する薬物の標的となり得る。演者らは、がん細胞に発現亢進するアミノ酸トランスポーターLAT1 (SLC7A5)を見出し、それがmTORC1を中心とするアミノ酸シグナル系を介してがん細胞特有の機能の維持に寄与することを明らかにしてきた。構造活性相関解析に基づいて創製した阻害薬は、抗腫瘍効果を示し、また血管内皮細胞や免疫系細胞など病態形成の過程でLAT1を発現するがん細胞以外の細胞の機能制御にも有用であることが明らかになった。さらに、最近クライオ電顕によりLAT1の構造が解け、基質認識と阻害薬の作用の機序も示された。本講演では、LAT1阻害薬によって見えてきた代謝リプログラミングにおけるトランスポーターの役割を含め、LAT1の創薬標的としての意義について議論したい。

がん幹細胞は、がん組織の起源となり、その維持を担う細胞である。臨床的に見てがん幹細胞の最も重要な特徴は、種々の治療に抵抗性を示し再発や転移の原因になることである。しかし、その治療抵抗性のメカニズムは起源細胞やそれを取り巻く環境によって多彩であり単純ではない。がん幹細胞が樹立する機構としては、体性幹細胞にドライバー変異が導入され、がん幹細胞に変化するもの（幹細胞起源型）、慢性的な炎症を背景として前駆細胞から脱分化してがん幹細胞に変化するもの（前駆細胞起源型）の2つが考えられている。がん幹細胞の治療抵抗性機構はその成立機構の違いを反映し、通常のがん細胞に比べて巧みな生存戦略が内在されている。幹細胞型では分化制御のメカニズムが、前駆細胞起源型では活性酸素の抑制や細胞死抑制メカニズムが治療耐性に関与することを私たちは明らかにしてきた。本講演ではこれらの治療抵抗性機構を基礎研究データに基づいて解説し、ドラッグリポジショニングに基づく新たな治療戦略を提案したい。

腎不全による人工透析患者数は33万人、その医療費は年間1.5兆円を越えている。腎臓を作るにはネフロン前駆細胞と尿管芽の二つが必須であり、前者からは糸球体と尿細管が、後者からは集合管・尿管が形成される。我々は腎臓の正しい起源を同定することによって、ヒトiPS細胞からネフロン前駆細胞を経由して糸球体および尿細管を誘導できることを報告した (Cell Stem Cell 2014)。ヒトiPS細胞由来のネフロン前駆細胞をマウスに移植すると、ヒト糸球体がマウス血管と接続して糸球体上皮の成熟が進む。これらの技術を応用することによって、先天性ネフローゼ症候群の患者由来iPS細胞からネフロンオルガノイドを誘導して糸球体の異常を再現した (Stem Cell Rep 2018)。さらに、もう一つの腎臓前駆組織である尿管芽の誘導にも成功し、分岐する集合管の周囲にネフロンが配置された腎臓本来の構造をマウスでは再構築できることを示した (Cell Stem Cell 2017)。また多発性嚢胞腎患者由来のiPS 細胞から作ったヒト尿管芽オルガノイドを用いて嚢胞形成を再現した (J Am Soc Nephrol 2020)。一方で、間質前駆細胞という３つ目の前駆細胞の重要性が判明したため、これを誘導することで高次構造をもったヒト腎臓の作製を目指している。

近年、麻酔薬ケタミンがうつ病の画期的な治療薬として注目されている。ケタミンは治療抵抗性うつ病患者に投与して数時間後に抗うつ効果を示し、その効果は1週間以上持続する。さらに、ケタミンはうつ病患者の自殺願望、希死念慮も劇的に改善し、自殺予防という点からも注目されている。ケタミンは、不斉炭素を有しているので、二つの光学異性体を有する。わが国で使用されている麻酔薬ケタミンはラセミ体である。米国Johnson & Johnson社は、NMDA受容体への親和性が強いエスケタミンを開発し、2019年に治療抵抗性うつ病の治療薬として、米国と欧州で承認された。一方、演者らはNMDA受容体への親和性が弱いアールケタミンの方が、エスケタミンより抗うつ作用が強く、副作用が少ないことを発見した。現在、米国企業がアールケタミンの第二相臨床治験を海外で実施中であり、わが国では大塚製薬株式会社が第一相臨床治験を準備中である。本教育講演では、千葉大学で開発した新規抗うつ薬アールケタミンの最新知見について議論したい。

細胞性粘菌が産生する分化誘導因子（DIF）は、多種類のがん細胞の増殖と運動（遊走と浸潤）、内皮細胞への接着を阻害し、がんの成長と転移を抑制することが明らかとなっている。その作用機序については長らく不明のままであったが、最近の研究により、AMP活性化プロテインキナーゼ（AMPK）の活性化によるmTORC1の活性阻害がDIFの早期シグナルであることが明らかとなってきた。mTORC1が阻害されるとS6キナーゼが抑制され、下流のSTAT3やSnailの抑制を介して細胞増殖や細胞運動が抑制される。また、DIF結合タンパク質の解析から、DIFによりAMPKが活性化されるのは、DIFが2型リンゴ酸デヒドロゲナーゼ（MDH2）に結合し、これを阻害することによることが示唆される。DIF合成酵素は細胞性粘菌以外の生物では発見されていないが、標的分子のMDH2はほとんど全ての生物で保存されている。DIFの効果が哺乳類にも及ぶのはこのためであろう。植物アルカロイドなど毒を基原とする薬は多いが、DIFのような他の生物のシグナル分子から作られた薬は稀である。この講演では、DIFの臨床応用の可能性についても考えてみたい。

IT技術の医療への導入は、医療の質の向上、画期的な診断技術の開発、精密医療の実用化を加速している。その中で、2020年にはニコチン依存症治療アプリ及びCOチェッカーが日本で初めてプログラム医療機器として承認された。デジタル治療という新しいカテゴリーの医療が始まろうとしている。デジタル治療に用いられるアプリは、一般にデジタル薬と呼ばれている。これらのアプリは認知行動療法に関わるものが多く、行動変容アプリとも呼ばれる。デジタル薬の開発は米国のベンチャー企業が先行しており、日本の製薬企業、ベンチャー企業がそれを追っている状況にある。新規のデジタル医薬品の承認のためには、医薬品医療機器総合機構（PMDA）による審査を経ることになることから、PMDAにプログラム医療機器審査部門が新設され、審査体制の強化が図られている。今後、デジタル医薬品の開発が進み、多くの製品が臨床現場で使用されるようになると予想される、しかし、その作用機序や医薬品とデジタル薬の併用の際の相互作用が不明であるなどの課題も多い。今後、薬理学的なアプローチによるデジタル薬の作用の解析研究が進展することが望まれる。

シナプス可塑性は、学習や記憶のメカニズムと考えられ、その破綻は精神神経疾患や神経認知障害を引き起こす。 そのためその分子機構は、発見以来、研究の焦点となってきた。私は特にシナプス表面の受容体を制御するシナプス後部のシグナル伝達過程に興味を持ち、シナプス可塑性の際に様々な受容体と細胞内成分の活性依存的な輸送が行われることを発見しました。これは細胞内Ca²⁺ 濃度の上昇が引き金となり、様々なシグナル伝達カスケードが引き起こされるが、中でも、CaMKIIは、シナプス可塑性において極めて重要なシグナル伝達分子と考えられている。しかし、その特徴的な12量体構造やシナプスに多量に存在する理由については謎に包まれていた。私は、CaMKIIが、Ca²⁺/カルモジュリンに依存的に、基質タンパク質と液–液相分離を起こし、架橋することを見出した。これにより、シナプス表面の受容体の分布が調節され、そのナノドメインが形成される。このように、CaMKIIと他のシナプスタンパク質とのCa²⁺依存的な液–液相分離は、シナプス可塑性の間にシナプス伝達を持続的に調節するメカニズムである。

The communication between the brain, immune and vascular systems is a key contributor to the onset and progression of neurological diseases. We discovered the coagulation factor fibrinogen as a blood-derived driver for neuroinflammation and inhibitor of repair in a wide range of neurologic diseases, such as multiple sclerosis, Alzheimer's disease and brain trauma. We showed that fibrinogen is necessary and sufficient for neurodegeneration and a new culprit for microglia-mediated oxidative stress-dependent spine elimination and cognitive impairment. By developing Tox-Seq, we reported the oxidative stress innate immune cell atlas in neuroinflammation. We developed cutting-edge imaging tools to study brain network synchronization and the neurovascular interface. We discovered a first-in-class fibrin-targeting immunotherapy to selectively target inflammatory functions of fibrin without interference with clotting with potent therapeutic effects in autoimmune- and amyloid-driven neurotoxicity. High throughput drug screens identified small molecule compounds to block fibrin-induced activation of microglia with therapeutic effects in neuroinflammatory disease. These findings could be a common thread for the understanding of the etiology, progression, and new treatments for neurologic diseases with neuroimmune and cerebrovascular dysfunction. As fibrin is a global activator of toxic innate immune responses in the brain and periphery, these studies could provide the basis for the development of a new class of therapeutics for autoimmune and inflammatory diseases¹.

1. Akassoglou, K. The immunology of blood: Connecting the dots at the neurovascular interface. Nat Immunol 2020, 21:710-712

パーキンソン病や線維筋痛症といった難治性神経疾患は、様々な神経の複合的変容により発症し、時間軸に従って重症化すると考えられる。ヒト iPS 細胞を用いた疾患メカニズム研究の発展により、一つの iPS 細胞から異なる神経サブタイプへと分化誘導を行い、疾患による細胞変容を抽出することにより、統合的な細胞脆弱連関の形成が推測できるようになった。一方、ヒト iPS 細胞研究より得られた情報を元に、テーラーメード型疾患モデルマウスの作製が可能となり、リバーストランスレーショナルニューロサイエンスリサーチを展開することにより、病態表現系の in vivo解析が可能となった。このような細胞特異的遺伝子改変疾患モデル動物を活用し、薬理学的な治療アプローチを展開することで、将来的に、病気の発症や難治化を最大限にコントロールできるテーラーメード薬物療法の提案が期待される。こうした取り組みから、薬理学分野の発展に貢献できるよう、疾患 iPS 細胞由来分化誘導細胞の統合的解析を応用したリバーストランスレーショナルニューロサイエンスリサーチを進めるために邁進していきたい。

我々はアルツハイマー病の病理変化の一つであるタウ蛋白質を主成分とする神経原線維変化を画像化するためのポジトロン放出核種標識薬剤（PETプローブ）として[¹⁸F]THK-5351を開発し、臨床応用を実施してきた。臨床評価の結果から、同プローブには神経原線維変化とは関係のない標的への結合（オフターゲット結合）があることが確認され、それがモノアミン酸化酵素B（MAO-B）への結合であることを新たに見出した。アストロサイトに高発現するMAO-Bは多様な神経疾患で観察されるアストログリオーシスのバイオマーカーとして有用であると判断されたため、タウプローブとしては"標的外"の分子であったMAO-Bを"標的"とみなし、その結合性を高めるという開発戦略で新たなPETプローブ[ ¹⁸ F]SMBT-1を開発した。このような神経病理像を画像化するPETプローブの基礎開発からPET画像の妥当性の検証に至る一連のトランスサイトーシス・リサーチとリバーストランスレーショナル・リサーチが新たなバイオマーカーの創出につながった。

神経回路は発生初期には主に遺伝的プログラムによって形成されるが、生後は外的因子の影響を受け再編成される。我々は外的因子として社会や環境から受けるストレスに着目し、ストレスによる神経回路の変化とその機能的意義を解析してきた。長期的（慢性）ストレスは内側前頭前野のドパミン応答を抑制してうつ様行動を促す。この行動変化に伴い、長期的ストレスでは内側前頭前野で錐体神経細胞の樹状突起が委縮する。一方で、短期的（急性）ストレスでは内側前頭前野のドパミン応答とドパミンD1受容体シグナルを介してうつ様行動が抑制されること、ドパミンD1受容体依存的に錐体神経細胞の樹状突起の増生が誘導されることを見出した。このストレス抵抗性の増強に付随して、短期的ストレスは内側前頭前野のドパミンD1受容体を介して拡張扁桃体の神経活動を高める。また、ケタミンの即効性抗うつ作用が短期的ストレスと類似した神経回路を介することも示してきた。本講演では、ストレス・うつ病における神経回路の可塑性について、最新の知見と今後の展望を紹介したい。

The number of human patients with chronic kidney disease (CKD) is increasing with the aging of society. Similarly, the incidence of CKD in animals, especially dogs and cats, is also increasing with the aging of individuals.

In order to achieve "One Health" for humans and animals, it is important to study useful targets for the diagnosis and treatment of CKD in these mammals. However, the species specific-differences of renal pathogenesis complicate it; dogs tend to show renal glomerular lesions as humans, but most cats manifest tubulointerstitial lesions. Therefore, there is a need to develop therapeutic and diagnostic methods based on the elucidation of species-specific renal pathogenesis, and we are conducting comprehensive research targeting human as well as model animals and companion animals.

In particular, we continue to focus on the unique role of "urine" in various kidney diseases. Our previous studies have shown that urine from model or companion animals with CKD contains candidate disease markers such as cytokines and non-coding RNAs, which are involved in their renal pathogenesis. In addition, as for a unique recent topic, we found that urine is a biological fluid that alters the local immune status of urogenital tissues. Briefly, tertiary lymphoid structures in the renal pelvis, called "urinary tract-associated lymphoid structures (UTALSs)", were formed in humans and mice with chronic nephritis, regardless of infections. In the development of UTALSs, aging and altered immune status as wells as urine played a role in UTALS formation. In a nephritis mouse model, urine leaked from the lumen of the renal pelvis into its parenchyma. Based on obtained findings, we consider that altered urine-urothelium barrier-based UTALS formation may represent a novel mechanism underlying the pathogenesis of chronic nephritis, regardless of urinary tract infection.

Further studies focusing on urinary biomarker candidates and unique roles of urine in renal pathogenesis would be crucial for clinical development and understanding the mechanism of kidney disease progression, to realize One Health in the field of human and animal nephrology.

Recently, the use of neonicotinoid pesticides (NNs) has been increasing in place of organophosphorus pesticides, because NNs have very low toxicity to humans, and their toxicity is considered negligible in daily life, as long as it is below a certain standard. In Japan, however, food residue limits for fruits, vegetables, and tea leaves are set several to several dozen times higher than in other countries.

In this study, to clarify the exposure to NNs in Japan, we collected urine samples from newborns and infants and measured the amount of NNs in the urine to assess the exposure.

On the other hand, NNs have been reported to have effects on experimental animals even at lower exposure levels than NOAEL. For example, Hirano et al. reported that NNs induced anxiety behavior in mice at a dose level of 5 mg/kg, which is lower than the NOAEL of 9.7 mg/kg for clothianidin. Therefore, we sought to develop sensitive biomarkers to conduct appropriate toxicity assessment. Monoamine neurotransmitters (MAs) such as dopamine (DA) and serotonin (5-HT) regulate brain functions such as behavior, memory, and learning, and their abnormalities are said to be involved in the development of neurological diseases such as depression and Parkinson's disease. In order to clarify the distribution and concentration of MA in the brain, we developed a comprehensive, highly sensitive, and accurate analytical method using mass spectrometry, and attempted to clarify the effects of NN administration on MA in the brain.

As a result of quantification of MAs in various brain regions, no change in DA concentration in the striatum was observed after IMI administration, but decreases in 3-MT and DA in the olfactory bulb, and decreases in 5-HT and histamine in the striatum were observed.

Considering the fact that NNs disrupt neurotransmitters in the brain and that infants are exposed to multiple NNs, the exposure level of infants and newborns is not negligible, and it is necessary to elucidate the toxicity mechanism in more detail.

In our laboratory, we have applied lipidomics technology to elucidate the mechanisms of disease progression, and to find novel bioactive lipid, to search diagnostic biomarker for various types of diseases. We usually search for target molecules and obtained proof of concept using highly reproducible animal experimental models, and based on the results, we apply them to human clinical practice. For example, the urinary biomarkers (tetranor-PGDM) for food allergies that we discovered using animal models have been proved to be disease specific and symptom predictable in human clinical studies. In this symposium, I would like to introduce the biomarkers and physiologically active lipids of food allergies, atopic dermatitis, and allergic rhinitis that we have discovered, and their applications to humans.

Membrane transport proteins, such as transporters, pumps and channels, cannot fully exert their transport activity simply by being translated as polypeptide chains and inserted into membranes, but demonstrate the physiological transport activity only when the proteins are properly folded and localize to membrane domains where the proteins should function. Heterodimeric Amino acid Transporters (HATs) family consists of a light chain, which has a transport function, and a heavy chain, which is essential for the localization to the plasma membrane. HATs play important roles in various physiological processes. The rBAT (heavy chain)-b^0,+AT (light chain) complex, a member of the HAT family, transports cystine and basic and neutral amino acids. Mutations in either b^0,+AT or rBAT lead to cystinuria (renal cystine stones). Since the identification of the rBAT and b^0,+AT complex in the late '90s, hundreds of pathological mutations have been found. The studies of mutations improved the understanding of the biosynthesis and transport mechanisms of the complex. However, the pathological mechanisms of mutations outside the predicted substrate binding sites remain largely unknown. By combining biochemical analysis and cryo-EM structures, we solved the structure and function of rBAT-b^0,+AT and examined their biogenesis. As a result, we found that b^0,+AT recognizes the substrates at different amino acid residues depending on the type of substrate. Furthermore, we identified a Ca²⁺binding site in rBAT, and showed that Ca²⁺-mediated super-dimerization (dimer x dimer) is a key for the formation of higher-order structures of the complex and its localization to the plasma membrane. Accordingly, we elucidated the pathogenesis of HAT by mutations related to biosynthesis, which is different from mutations in the substrate-binding site. This study provides an understanding of the HAT biogenesis and serves as a guide to develop a new therapeutic approach. At molecular basis, the study lights up a novel role of Ca²⁺ on membrane protein biogenesis.

Voltage-gated K⁺ channels are tetramer, and four α (main) subunits form a single K⁺ channel. Each α subunit possesses a single voltage sensor domain consisting of four transmembrane segments (S1-S4) and contributes voltage-dependent gating. The fourth transmembrane segment (S4) is a central part of voltage-sensing. Membrane depolarization induces upward movement of the S4 segments, which leads to the channel opening. Most of the voltage-gated K⁺ channels have auxiliary subunits. These subunits could modulate gating properties and even change the affinity of agonist/antagonist. Therefore, it is essential to understand the structures of the macromolecular complex and how the auxiliary subunits modify the voltage-dependent gating. KCNQ1 channel is one of the most well-studied ion channels for gating modulation because its auxiliary subunit KCNE drastically changes the gating properties. For example, KCNE3 makes the KCNQ1 channel constitutively open. Recent cryo-EM structures clearly show KCNE3 interacts with the voltage sensor domain via the S1 segment of KCNQ1. By introducing mutations on putative interaction sites, we identified that the tight interaction between the S1 segment and KCNE3 is required to stabilize the channel's open state. This KCNQ1-KCNE3 complex could be an excellent model to study how auxiliary subunits modulate the gating behavior of voltage-gated ion channels.

Changes in cytosolic Ca²⁺ are involved in various biological responses such as neurotransmission, muscle contraction, and hormone secretion. Therefore, Ca²⁺ channels and their downstream molecules make supramolecular complexes to activate specific signal pathways. We have shown that caveolin (cav)-1, an essential component of caveolae, and junctophilin (JP)-2, a protein that bridges the plasma membrane and sarcoplasmic reticulum (SR), accumulate Cav1.2 voltage dependent Ca²⁺ channels, large conductance Ca²⁺-activated K⁺ (BK) channels and ryanodine receptors within caveolae-SR junctions. This molecular complex supports an effective conversion of Ca²⁺ signals to membrane hyperpolarization by the activation of BK channels and thus, control vascular tone. In addition, we have recently revealed that the activation of a complex of Cav1.2, Ca²⁺/calmodulin-dependent kinase kinase (CaMKK)-2 and CaMK1a localized in caveolae causes transcription of pro-inflammatory genes, which promotes macrophage recruitments to the adventitia and vascular remodeling. In summary, the caveola-based supramolecular complexes convert Ca²⁺ signals to the changes in membrane potential and gene transcription, which is involved in the regulation of vascular tone and the adaptation to increased circumferential stretch, respectively.

In skeletal muscle excitation-contraction coupling, depolarization of transverse tubule membrane causes conformational change in dihydropyridine receptor (DHPR), which in turn opens type 1 ryanodine receptor (RyR1) to release Ca²⁺ from sarcoplasmic reticulum. This 'depolarization-induced Ca²⁺ release' (DICR) occurs through a supramolecular complex composed of several proteins including RyR1, Cav1.1, β1a, Stac3, and junctophilin. However, it remains so far unclear about molecular mechanism of DICR, especially conformational change in RyR1. Recently, Perni et al. reported successful reconstitution of DICR by co-expressing these essential components in tsA201 cells (PNAS, 114: 13822-13827, 2017). In this study, we developed a high-throughput platform of reconstituted DICR in HEK293 cells. Baculovirus infection of essential components into HEK293 cells expressing RyR1 greatly increased their transduction efficiency, and fluorescent ER Ca²⁺ indicator (R-CEPIA1er) quantitatively measured Ca²⁺ release without contaminant of Ca²⁺ influx. We demonstrated [K⁺]_o-dependent Ca²⁺ release by chemical depolarization in the baculovirus-infected cells, indicating a successful reconstitution of DICR. Our high-throughput platform will accelerate elucidation of molecular mechanism of DICR.

Sepsis is one of the leading cause of death worldwide. Recently, several studies suggested that free-hemoglobin and heme derived from hemolysis are important factors which may be associated with severity of septic patients including COVID-19. In other words, hemolysis-derived products enhance the inflammatory responses as damage-associated molecular patterns (DAMPs) in both intravascular and extravascular space. In addition, hemoglobin has vasoconstrictive activity by depleting nitric oxide, whereas heme or Fe²⁺ produce reactive oxygen species (ROS) through Fenton reaction leading to tissue injury. At present, we have no therapeutic options against sepsis-related hemolysis in clinical settings, however, there are might be two therapeutic strategies in this regard. One is supplemental therapy of depleted scavenging proteins such as haptoglobin and hemopexin, the other is activation of the internal scavenging system including macrophage-CD163 pathway. These novel targets against sepsis are also critical for the next pandemic. We will show you our recent data aiming at anti-hemolytic therapy.

Histidine-rich glycoprotein (HRG) is a plasma glycoprotein and exists in blood at a concentration of approximately 100 µg/ml. Although many previous reports already revealed that HRG has a wide range of activities against coagulation-fibrinolysis, immune and blood vascular system, we newly identified that HRG ameliorates a septic condition due to its inhibitory activity against immunothrombus formation starting from neutrophil-endothelial cell interaction. Additionally, we demonstrated that HRG regulates the translocation of HMGB1, damage-associated molecular pattern (DAMP) protein, from nuclei to cytosol and neutralizes the toxicity of heme, a kind of DAMP, and LPS, a kind of pathogen-associated molecular pattern (PAMP). Based on the above, HRG was thought to contribute the amelioration of a septic condition, caused by its regulatory activities against not only blood cell functions but DAMPs/PAMPs actions. Because DAMPs and PAMPs were associated with the exacerbation of not sepsis alone but a variety of inflammatory diseases, HRG, which regulates those reactions, has the potential of therapeutic drug for many kinds of diseases.

In recent years, it has been elucidated that a series of inflammatory factors called DAMPs (damage-associated molecular patterns) are involved in the onset and exacerbation of lifestyle-related diseases and age-related diseases. It is known that DAMPs molecules are released extracellularly in response to cell death or stress, and stimulate some pattern recognition receptors in immune cells to induce the inflammatory responses. However, at present, few reports have been made on the existence form of DAMPs molecules and the factors capable of interacting with DAMPs to cause functional changes. In this symposium, we discuss the extracellular existence form of DAMPs, the regulation of their activities by complex formation and their pathophysiological roles.

It was well known in the end of 1980s that NADPH oxidase generates superoxide as respiratory burst in phagocytes (later named as NOX2), and that its genetic mutations could be causative for chronic granulomatous disease. Since the finding of NOX1 in 1999, a catalytic subunit highly expressed in colon epithelium, seven isoforms of NOX/DUOX family (NOX1 to NOX5 and DUOX1/2) were identified. Novel cytosolic components or maturation factors have also been identified, and isoform-specific activation mechanisms have been clarified. Using genetically modified mice, reactive oxygen species derived from NOX/NADPH oxidase have been found to be involved in various physiological functions and pathogenesis in multiple tissues in the body. An age ago, reagents that were used as inhibitors of NADPH oxidase were non-specific ones like diphenyleneiodonium, a broad-spectrum flavoprotein inhibitor, 4-(2-aminoethyl)benzenesulfonyl fluoride, an inhibitor of serine proteases, or an antioxidant apocynin. In the past two decades, selective NOX inhibitors have been developed. Among them, Setanaxib (GKT137831), a NOX1/4 inhibitor, is under Phase II clinical trial in patients with primary biliary cholangitis (liver fibrosis), Type 1 Diabetes and Kidney Disease (kidney fibrosis), and idiopathic pulmonary fibrosis, suggesting its potential as a new antifibrotic agent. Thus, NOX/NADPH oxidases are expected as novel targets of pharmacotherapeutics.

The NOX family NADPH oxidases deliberately generate reactive oxygen species (ROS) such as superoxide. The human genome contains seven members of the family: NOX1 to NOX5 and DUOX1/DUOX2. These enzymes are membrane-integrated flavocytochromes that transfer electrons from NADPH to molecular oxygen for ROS production. The founder of the family is the phagocyte oxidase, presently known as NOX2, which is dormant in resting cells but produces superoxide, a precursor of microbicidal oxidants, during phagocytosis of invading microbes. The significance of NOX2 is evident from the fact that recurrent and life-threatening infections occur in patients with chronic granulomatous disease (CGD) because of a hereditary defect of the Nox2-encoding gene. The thyroid oxidase DUOX2 is required for thyroid hormone synthesis and its genetic inactivation causes congenital hypothyroidism. Recent studies have revealed that NOX-produced ROS also have various regulatory functions: NOX2 defect causes not only severe infections but also noninfectious granuloma formation, indicating its role in immune system regulation; NOX1 and DUOX2 are highly expressed in gastrointestinal epithelial cells, and their gene variations are associated with inflammatory bowel disease (IBD); and NOX4 is cytoprotective against heart damage.

Reactive oxygen species (ROS) have been implicated in the development of psychiatric disorders. We presently report NOX1/NADPH oxidase regulates the emotional behavior demonstrated in chronic pain model as well as maternal immune activation (MIA) model. There was no difference in the mechanical allodynia developed by spared nerve injury between Nox1 deficient mice (NOX1-KO) and wild type mice (WT). Increased anxiety- and depressive-like behaviors in WT by chronic pain were markedly ameliorated in mice deficient in NOX1-KO. Similarly, the impaired recognition by chronic pain demonstrated in WT was significantly suppressed in NOX1-KO. These affective and cognitive impairment were ameliorated in mice selectively suppressed the expression of hippocampal NOX1 mRNA. In MIA model of gestational polyinosinic-polycytidylic acid, increased serum levels of IL-6 were observed in both genotypes, however, impairment of social preference and defects in motor coordination were observed in WT but not in NOX1-KO. MIA up-regulated NOX1 mRNA in the cerebral cortex and cerebellum of the fetus but not in the offspring. The dropout of Purkinje cells in lobule VII of MIA-affected offspring was significantly ameliorated in NOX1-KO. Taken together, NOX1 may therefore play a key role in the development of behaviors related to the psychiatric disorders.

Hearing loss (HL) is one of the most common sensory impairments. Acquired HLs, such as age-related (ARHL), noise-induced, and drug-induced HL, are classified based on the underlying mechanism. The most common form of sensorineural HL (SNHL) is ARHL, affecting 25~40% of individuals over 65 years of age. Noise is one of the most common occupational hazards. In 2019, WHO warned that half of young people risk damaging their hearing through excessive use of audio devices. However, treatment options for SNHL rely on medical instruments, with no reliable pharmacological interventions.

Nox family is one of the main sources of reactive oxygen species (ROS). Recently, we have reported transgenic mice expressing NOX4 exhibit hearing vulnerability after noise exposure, demonstrating ROS contribute to SNHL. Nox3, activated by a Rho-family GTPase Rac, was discovered as an inner ear specific Nox; however, Nox3-expressing cells were ambiguous. We generated Nox3-Cre;tdTomato mice, in which tdTomato fluorescence is regulated by the Nox3 promoter-driven Cre. Nox3-expressing cells in cochleae included outer hair cells (OHCs) and supporting cells (SCs), and they increased with aging, noise, and cisplatin. Moreover, increased Nox3 expression in OHCs and SCs played essential roles in ROS-related SNHL through OHC apoptosis. Thus, ROS are promising targets for therapeutics development for acquired SNHLs. I will discuss our hearing research focused on ROS and Rho-family GTPases.

Discovery of receptor activator of NF-κB ligand (RANKL) gave a great impact on identification of the mechanisms regulating osteoclast differentiation and function, establishment of research field bridging bone and the immune system (osteoimmunology), and development of a fully human anti-RANKL neutralizing antibody (denosumab). Soluble RANKL and anti-RANKL antibody have been used in vitro and in vivo as valuable research tools. We have reported a novel and rapid bone loss model by administration of glutathione-S transferase (GST)-RANKL to mice, and continuous inhibition of RANKL for several weeks by a single injection of a denosumab-like anti-mouse RANKL neutralizing monoclonal antibody (OYC1) to mice. It is known that RANKL is a multifunction protein that plays a variety of roles such as regulation of bone metabolism. For example, exploration of its pivotal role in immunology is a recent topic of increasing interest. In this presentation I will talk about the discovery of RANKL and the current and future impact of its research.

In the late 1980s, it was found that receptor activator of nuclear factor-κB ligand (RANKL) is expressed by osteoblasts has a crucial role in osteoblastogenesis, which had been a dogma for long. Later studies, however, have revealed that osteocytes functions as the major source of RANKL in many biological processes, including unloading and unloading onto the bone. Osteocytes are known as a mechanosensor in the bone tissue and to respond to mechanical stimuli by expressing various molecules, such as RANKL, Sclerostin (Scl) and insulin–like growth factor (IGF)-1. These cells employ cell surface molecules and intracellular signaling molecules as mechanosensors and mechanotransducers. Mechanosensors detects the flow of tissue fluid and the strain of bone matrix. These sensors then mediate intracellular signals using mechanotransducers, resulting in the regulation of the expression of effector molecules. Although many molecules have found as such sensors and transducers, there remains room for investigation. In this session, I would like to overview of the recent findings on mechanobiology of the bone, focusing on osteocyte RANKL.

Periodontitis, one of the most common infectious diseases in humans, is a unique "osteoimmune" disease in which antibacterial immune response causes alveolar bone destruction. Recently, it was revealed that the osteoclastic bone damage that occurs during periodontitis is dependent on the receptor activator of NF-kB ligand (RANKL) produced by osteoblastic cells and periodontal ligament cells (Tsukasaki et al., Nature Commun 2018, Nature Rev Immunol 2019). To understand the pathogenesis of and develop future therapeutic strategies for periodontal bone loss, it is vitally important to clarify precise molecular mechanisms underlying osteoclastic bone erosion. Recently, we unveiled the importance of the tight regulation of RANKL activity by the local OPG production in bone and immune systems by generating OPG-floxed mice (Tsukasaki et al., Cell Rep 2020), and deciphered the stepwise cell fate decision pathways during osteoclastogenesis at single-cell resolution (Tsukasaki et al., Nature Metabolism 2020). In this talk, I will summarize the recent progress in the fields of periodontology and RANKL biology.

In bone tissue, RANKL acts as a positive regulator of bone turnover through both RANKL forward signaling, which induces osteoclast maturation, and RANKL reverse signaling, which promotes early osteoblast differentiation. Inhibition of osteoclast maturation by blocking RANKL forward signaling inevitably reduces the supply of osteoclast-derived coupling factors, leading to the suppression of bone formation. If we can design an agent that blocks RANKL forward signaling while simultaneously activating RANKL reverse signaling, the influence of reduced supply of osteoclast-derived coupling factors can be mitigated, which may be useful in the treatment of bone destructive diseases. Our previous study has shown that the cross-linking of RANKL molecules on the cell surface triggers the activation reverse signaling. Therefore, we screened various structures of IgG Fc-fusion protein constructs with multivalent binding sites for RANKL by arranging anti-RANKL single-chain antibody variable regions (scFv). Consequently, it was found that a structure in which the domain order inside the scFv was VH-VL order was advantageous for its ability to activate RANKL reverse signaling. Finally, the optimal structure was selected, and its pharmacological activity was evaluated using the ovariectomized mouse model. Results confirmed that the activation of RANKL reverse signaling could mitigate the decrease in bone formation associated with the suppression of osteoclast maturation.

The production of angiotensin II (Ang II) in the brain plays important roles as neurotransmitter and neuropeptide. Central Ang II is involved in regulating various physiological processes, such as blood pressure and water homeostasis, via Ang II type 1 (AT1) receptors. We have demonstrated that Ang II induces frequent urination via AT1 receptors in the brain even at doses that does not seem to affect the blood pressure in animal experiment. Intracerebroventricular administration of Ang II was also found to reduce the bladder capacity without affecting the maximum voiding pressure, post voiding residual urine volume or voiding efficiency. Additionally, the activation of AT1 receptor downstream signal pathway (phospholipase C/protein kinase C/NADPH oxidase/superoxide anion) and suppression of GABAergic nervous system in the brain are involved in the mechanism underlying the central Ang II-inducted frequent urination. AT1 receptor antagonists have been widely used to treat hypertension. We demonstrated that peripherally administered AT1 receptor antagonist telmisartan, which can penetrate blood brain barrier, exerted an inhibitory effect on central Ang II-inducted frequent urination. At this symposium, we would present and discuss the possible drug therapy targeting AT1 receptors in the brain against frequent urination on the results obtained from our recent research work.

Atrial fibrillation reduces the quality of life and increases mortality from cardiovascular disease. The prevention of AF is of major health importance because the incidence, prevalence, and lifetime risk of AF are increasing globally. However, the number of studies directed at the primary or secondary prevention of AF is limited. Recent evidence suggests the association of inflammation with the incidence and progression of AF. NLRP3 inflammasome is central to the innate immune system. Although several studies suggest the association of NLRP3 with AF, the precise mechanism linking activated NLRP3 to AF remains to be elucidated. Gut-derived metabolites, such as lipopolysaccharides and trimethylamine N-oxide (TMAO), have been shown to play a role in AF susceptibility. Lipopolysaccharides are derived from gut microbiota and may enter the circulation through the gut mucosa. TMAO is produced in the liver by oxidation of trimethylamine, which is derived from dietary choline and L-carnitine. Although these gut-derived metabolites have been shown to increase AF susceptibility, it is unclear as to how lipopolysaccharides and TMAO increase the risk of AF and whether lowering circulating levels of lipopolysaccharides and TMAO lead to the prevention of AF. This presentation will discuss the association of inflammation with AF and the potential of drug discovery to prevent AF by focusing on the involvement of gut-derived metabolites in inflammation.

The roles of the nitric oxide synthases system (NOSs) in inter-organ communication of cardiovascular diseases remain elusive. In this symposium, we introduce our three studies in which we examined this issue in our mice lacking all three NOS isoforms (triple n/i/eNOSs^-/- mice) (PNAS 2005).

There is no experimentally useful model that develops myocardial infarction (MI). We previously reported that our triple NOSs^-/- mice spontaneously develop MI. However, it takes a long time (1 year) to develop MI (Circulation 2008). We then revealed that 2/3-nephrectomized triple NOSs^-/- mice suddenly die due to early onset of MI with high incidence, succeeding in an experimentally useful model of developing MI. These results suggest the protective role of NOSs in reno-cardiac communication (JMCC 2014).

We studied the role of bone marrow (BM) NOSs in vascular lesion formation. Constrictive vascular remodeling and neointimal formation at 2 weeks after carotid artery ligation were markedly accelerated in wild-type (WT) mice transplanted with triple NOSs^-/- BM as compared with those with WT BM. These results suggest the protective role of NOSs in BM-vascular communication (Nitric Oxide 2011).

We investigated the role of NOSs in pulmonary hypertension (PH). The extents of PH at 3 weeks after hypoxic exposure were markedly exacerbated in WT mice transplanted with triple NOSs^-/- BM as compared with those with WT BM, suggesting the protective role of NOSs in BM-lung communication (AJRCCM 2018).

These lines of evidence indicate the protective role of NOSs in inter-organ communication.

The gastrointestinal tract plays an important role in maintaining of our various physiological functions. Although the gastrointestinal tract is inside of our body, it is the "outside world". In fact, large number of bacteria coexist and are involved in maintaining of mucosal function. Unbalanced diet, several drugs, decreased blood flow, stress may cause the damage of this balance. Once the balance is destroyed, dysfunction of mucosa and increased permeability are induced. Such excessively increased permeability of intestinal mucosa is defined as "Leaky gut syndrome (LGS)", and various large-molecular weighted substances are transferred into the blood circulation. The LGS is considered to be a trigger for various systemic diseases such as non-alcoholic steatohepatitis (NASH), inflammatory bowel disease, cardiovascular diseases, allergy, chronic kidney disease, etc.

In this presentation, we show the outline of various systemic diseases caused by LGS, especially focused on multi-organ relationships such as "Gut-liver axis" and "Oral-gut axis". In addition, we show the effect of improving LGS status on various systemic diseases and the possibility of LGS as a target for new drug discovery.

Pluripotent stem cells can be used for both regenerative medicine and in vitro modeling for diseases and drug screening. For regenerative medicine, pluripotent stem cells need to differentiate into a specific cell type that is required for transplantation therapy. Cell products for transplantation therapy have lot-to-lot variations in quality, which affect their efficacy and side effects as well as the cost of the products. Quality evaluation of cell products requires non-invasive methods although well-used methods, such as expression analysis of genes and proteins, deconstruct cell products. To evaluate the quality of cell products in a non-deconstructive way, we developed a machine learning model that predicts the function of cell products using label-free images of cell morphology. Next, for in vitro modeling, we sought to recapitulate the interaction of brain regions by fusing brain-region-specific organoids. Imaging and viral vectors allowed for the evaluation of in vitro interaction of brain regions. In this talk, we will introduce our recent work with multidisciplinary approaches.

The ultimate goals of neuroscience are to understand human brain structures and functions, and to utilize them to overcome neurological disorders. The research on the human brain had been limited to non-invasive MRI or PET studies, pathological analysis with postmortem brains or in silico genomic analyses. Although animal or cellular models had been developed for investigating neurological disorders as alternatives to living human brain tissues, many of them did not represent correct human pathological phenotypes. In these circumstances, emergence of iPSCs and organoid culture systems provides us a novel way to investigate the development and dysfunction of the human brain tissues in vitro. We developed self-organizing 3D organoid culture of iPSCs for construction of human brain tissues. Combining 3D brain organoid cultures of human PSCs, 4D imaging and image analysis, we establish efficient methods to faithfully recapitulate and to quantitatively analyze the ontogenetic formation of human brain tissues. We induce degeneration of the formed tissues to construct human neurological disease models. We further explore the ways to prevent or restore the degeneration for clinical treatment and drug discovery.

The platform based on the techniques for iPSC generation and self-organizing 3D culture will become a powerful tool for investigating human brain development and neurological diseases.

The brain is a well-organized organ consisting of telencephalon, diencephalon, mesencephalon, and metencephalon. Each brain region has specific functions and interacts with other regions by neuronal axons and synaptic connections. Thus, region-specific neuronal differentiation from human pluripotent stem cells would help for better understanding human brain development and the pathogenesis of various neurological diseases, and for use in drug development, disease modeling and regenerative therapies.

Recently, we analyzed gene expression profile of human developing ventral midbrain by single-cell RNA-sequencing and established new protocol for generation of midbrain dopaminergic (DA) neurons from human embryonic stem cells by modification of crucial morphogenic factors and transcription factors. In addition, we generated brain organoid that is co-cultured with human induced pluripotent stem cells (hiPSCs)-derived neurons and microglial progenitor cells. We also generated multiregional assembloid that mimics the neural network formation of nigrostriatal pathway and the pathogenesis of Parkinson's disease (PD). using region-specific differentiation technology. Furthermore, we will introduce new therapeutic advantage of medicine for promoting synaptic formation of grafted hiPSC-derived DA neurons towards more beneficial cell transplantation therapy for PD.

To overcome coronavirus disease 2019 (COVID-19), it is essential to develop a therapeutic drug. For that purpose, an excellent in vitro model that can be used for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) research is needed. It is desired to use a physiologically relevant model rather than a cancer cell line such as Vero cell, which is widely used in SARS-CoV-2 research. We have conducted SARS-CoV-2 research using (1) organoids and (2) organ-on-a-chips. (1) We are trying to clarify the mechanism of SARS-CoV-2 infection using airway organoids. Among various airway epithelial cells, ciliated cells can be easily infected. On the other hand, basal cells, which are stem cells in the airway, cannot be infected with SARS-CoV-2. We confirmed that basal cells that survive after the viral infection can repair damaged airways. (2) Using an organ-on-a-chip that can reproduce the epithelial-endothelial barrier, we are investigating the mechanism by which SARS-CoV-2 destroys the biological barrier. SARS-CoV-2 infects epithelial cells but not endothelial cells. We confirmed that infected epithelial cells secrete interferons (IFN), and then enhance the vascular permeability. Currently, we are trying to develop a therapeutic drug using our organoids and organ-on-a-chip.

The skin forms a barrier between the body and the environment, preventing water loss and penetration of exogenous substances, and upholds homeostasis. The prevention of exogenous substance penetration a priori indicates that a passage of either toxic or medicinal compounds through the skin represents very important problem for topical drug development and application. The topical drugs are developed to deliver an active molecule either to a certain compartment in the skin or through the skin to peripheral blood circulation. In both cases, the pharmacological activity is determined by drug concentration, either in skin or in blood. 

The efficiency and efficacy of topical drug delivery to peripheral blood circulation is judged by the blood concentration of a drug. Moreover, the blood concentration-time course can be predicted using skin permeation parameters obtained from the in vitro skin permeation experiments. Thus, skin permeation experiments are very important to assess systemic exposure of chemicals. Synthetic membranes and three-dimensional cultured human skin models have been utilized for in vitro skin permeation experiments as alternative membranes, especially in the development of cosmetic products. Furthermore, recently, in silico perdiction of absorbed drug after topical application have been developed. In this presentation, usefulness of estimation of skin absorption with synthetic menbrane as well as in silico model would be introduced. I hope this presentation would be helpful for estimation of skin absorption of chemicals to assess systemic exposure.

A well-functioning immune system is essential for maintaining the integrity of an organism. Immune cells are an integral part of other systems including the respiratory, dermal, gastrointestinal, neurological, cardiovascular, reproductive, hepatic, musculoskeletal system, and endocrine systems. As a consequence, exposure to immunotoxic compounds can have serious adverse health consequences affecting responses to both communicable and non-communicable diseases. It is therefore important to understand the immunotoxic potential of xenobiotics and the risk they pose to humans.

Developing novel test methods to evaluating the immunotoxic potential of xenobiotics, Japan has developed five Adverse Outcome Pathways (AOPs) for immunotoxicity in the Organisation for Economic Co-operation and Development (OECD) project.  Based on the AOPs, Japan is on-going a Detailed Review Paper (DRP) for In vitro tests addressing immunotoxicity with a focus on immunosuppression. The DRP aims to present and discuss the application and interpretation of in vitro immunotoxicity assays, mainly covering immunosuppression, and to define a tiered approach to testing and assessment. 　After accepting the above documents, Japan is planning to develop test guidelines for in vitro immunotoxicity to OECD.