シンポジウム（Symposium）

Symposium Interpretation of human brain mechanisms and drug discovery using leading-edge stem cell technologies シンポジウム Interpretation of human brain mechanisms and drug discovery using leading-edge stem cell technologies

7月27日（土）16:30～16:55　第5会場（朱鷺メッセ　3F　302） 3S05e-1 ApoE2, E3 and E4 differentially activate MAP-kinase signaling to regulate amyloid-β secretion and synapse formation paralleling their role in Alzheimer's disease Yu-Wen Alvin Huang（Huang Yu-Wen Alvin） Stanford University School of Medicine Background: Nearly a quarter-century since apolipoprotein E (ApoE) was discovered as the first and to date the single greatest genetic risk factor for late-onset Alzheimer's disease (AD), researchers continue to struggle to comprehend how ApoE functions in the brain and modify AD occurrence. Human ApoE uniquely has three genetic isoforms, ApoE2, ApoE3 and ApoE4, which are encoded by three human-specific allelic variants of APOE gene - ε2 (8%), ε3 (78%) and ε4 (14%) - and differ only in two residues. The most common isoform ApoE3 conveys standard AD risk. The ApoE4 genotypes strikingly contribute to ~50% of AD cases worldwide, and are also associated with age-related cognitive decline. In contrast, ApoE2 is reported to lower AD risk. How the minimal structural differences among human ApoE isoforms influence AD risk is incompletely understood, owing to inherent complexity within the brain as well as the inadequate sensitivity and species-specificity of conventional AD model systems. Methods: Our group recently developed a novel approach to overcome these technical hurdles using iN cells. This methodology incorporated two critical experimental design strategies: 1) excluding glial cells, the major source of ApoE in the brain, to eliminate the background ""noise"" and 2) using human wildtype ES/iPSC-derived iN cells for evolutionarily-consistent genomic context to boost the ""signal"". Results: As a result, we have delineated a signaling pathway whereby ApoE activates a non-canonical MAP kinase cascade which requires dual leucine zipper kinase (DLK) and eventually leads to increased synthesis of amyloid-β (Aβ), the central component of AD pathology. Most importantly, this original methodology is sensitive enough to detect the intrinsic difference among three human ApoE isoforms in activating this pathway, with the potency rank order mirroring their relative risk for AD: ApoE4> ApoE3>ApoE2. We have recently showed that this ApoE signaling also governs synaptic formation in the same isoform-specific manner, explaining in part the intriguing phenomenon of brain hyperexcitability underlying ApoE4 carriers' cognitive anomaly from early, even developmental stage. Conclusion: Collectively, these findings shed light on how ApoE functions in the brain and how ApoE4 contributes to AD pathogenesis.		7月27日（土）16:55～17:20　第5会場（朱鷺メッセ　3F　302） 3S05e-2 Staged Dysregulation of Cortical Development Underlies Psychoses Tomoyo Sawada（澤田知世）^1,2,3，Thomas E Chater（Chater E Thomas）⁴，Yohei Sasagawa（笹川洋平）⁵，Mika Yoshimura（芳村美佳）⁵，Noriko Fujimori（藤森典子）³，Kaori Tanaka（田中かおり）⁵，Yukiko Goda（合田裕紀子）⁴，Itoshi Nikaido（二階堂愛）^5,6，Tadafumi Kato（加藤忠史）³ ¹Lieber Inst. for Brain Development, Baltimore, USA ²Dept Neurology, Johns Hopkins Univ Sch of Med, Balrimore, USA ³理研CBS 精神疾患動態 ⁴理研CBS シナプス可塑性・回路制御 ⁵理研BDR バイオインフォマティクス研究開発ユニット ⁶筑波大グローバル教育院ライフイノベーション学位プログラム生物情報 Neuropsychiatric disorders such as schizophrenia (SCZD) and bipolar disorder (BD) are among the most intractable in brain health. SCZD and BD share some of their clinical features, including psychosis. Given their complex genetic/environmental etiologies and sporadic occurrence, there are few reliable experimental approaches for addressing mechanistic selectivity and the order of events in pathogenesis. Recent genomic analysis and iPS cell-based modeling have identified several candidate cellular pathways in these disorders, including alterations in synaptic function and Wnt signaling. However, understanding the origins of and relationships among these pathways remains difficult without analyzing patient neurons by simulating brain development with a well-controlled genetic background. Here, we describe the use of functional cellular genomics to establish the selectivity and order of developmental mechanisms in a patient with schizoaffective bipolar disorder (SA-B), exhibiting psychotic symptoms. We established multiple iPS cell lines from the patient and, to reconstruct disease pathology and avoid genetic variability, generated parallel lines from the patient's unaffected monozygotic twin. Morphological analysis of iPS cell-derived organoid models of corticogenesis revealed thinning of the SOX2⁺ ventricular zone-like layer in the patient. Additionally, single-cell RNA-seq demonstrated diminished Wnt signaling pathway accompanied by reduced cell proliferation, accelerated neuronal maturation, and enhanced GABAergic specification in organoids from the patient. Time-point profiling of the patient's iPS cell-derived neuronal cells in a monolayer culture system confirmed alterations in Wnt signaling, cell proliferation, cellular maturity, and GABAergic differentiation during early developmental stages. Comparison of twins with psychosis and the unaffected sibling, including two additional monozygotic twin pairs discordant for SCZD, suggested enhanced ventralization during early neurodevelopment as a common pathogenesis of psychoses. Based on a strategy to minimize genetic variability and to use cortical organoid models to simulate brain development, our study proposes that staged dysregulation in early neurodevelopmental processes underlies the clinical expression of psychoses. Furthermore, the present study outlines a robust methodological approach for mapping cellular pathways and their order in the pathogenesis of complex disorders.

7月27日（土）17:20～17:45　第5会場（朱鷺メッセ　3F　302） 3S05e-3 Detecting somatic variations in human brain cells at single cell level Miki Bundo（Bundo Miki）^1,2，Tadafumi Kato（Kato Tadafumi）³，Kazuya Iwamoto（Iwamoto Kazuya）¹ ¹Dept. Mol Brain, Kumamoto Univ, Kumamoto ²PRESTO JST ³Lab. for Mol. Dynamics of Mental Disorders, RIKEN CBS Recent studies have revealed that the genome of human brain cells contains several kinds of somatic variations, such as single nucleotide variants (SNV), copy number variations (CNV) and novel insertions of retrotransposons. These somatic variations arise during embryonic development and may play an important role in the development of psychiatric diseases. We recently reported that the copy number of long interspersed nucleotide element (LINE-1) retrotransposon was higher in the neurons of patients with schizophrenia than in the neurons of healthy controls. As each cell in the brain is thought to have different pattern of variations, single cell analysis should be required to describe the complete somatic variations in the brain. We have developed some techniques for isolating cell nuclei from various types of brain cells and detecting somatic SNV and LINE-1 insertions at single cell level. We assessed somatic variations (SNVs and LINE-1 insertions) using a postmortem brain of a control subject without any psychiatric diseases (Japanese female, 93 years old). We isolated single neuronal nuclei from prefrontal cortex, amplified whole genomic DNA and performed whole genome sequencing. Our bioinformatic analysis detected somatic SNVs from single neuronal nuclei, and we successfully validated some of them by independent amplicon sequencing. Notably, some of SNVs were validated in region and/or cell-type specific manner. We also performed LINE-1 insertion analysis using DNA from single nuclei of several different types of brain cells, including neuron, oligodendrocyte, microglia, and astrocyte. We detected 30-40 novel insertions of LINE-1 at each single nuclei, and found the cell-type dependent novel insertion patterns. We have been conducting a case-control study using postmortem brain samples from patients with schizophrenia and controls to clarify the role of brain somatic variations in the pathogenesis of schizophrenia.		7月27日（土）17:45～18:10　第5会場（朱鷺メッセ　3F　302） 3S05e-4 精神疾患の橋渡し研究にヒト直接誘導神経細胞（iN）は有用か？ Noriaki Sagata（佐方功明），Takahiro A Kato（加藤隆弘）九州大院医精神病態医学 Direct conversion technique to produce human induced-neuronal (iN) cells from human fibroblasts within 2 weeks is expected to discover unknown neuronal phenotypes of neuropsychiatric disorders. In addition to translational studies using human iPS cell-derived neuronal cells (iPS-neurons), we would like to propose the possibility that research using the iN cells is important for psychiatric research and future medical systems. At first, we present unique gene expression profiles in iN cells from patients with neurofibromatosis type 1 (NF1), a single-gene multifaceted disorder with comparatively high co-occurrence of autism spectrum disorder (ASD). Microarray-based transcriptomic analysis on iN cells from male healthy controls (HC) and male NF1 patients revealed that 149 genes expressions were significantly different (110 upregulated and 39 downregulated). Interestingly, 31 of those 149 genes expressions in NF1-iN cells were rescued to HC levels by forskolin application. Thus, we propose that administration of the drug forskolin and/or other AC activators to NF1 patients (especially males) during the early developmental period have a possibility of preventing the occurrences of ASD and neuropsychiatric disorders in later life. Furthermore, in NF1-iN cells at 14 days post infection (DPI), higher expression of FOS mRNA was observed. Interestingly, BCL2 mRNA was higher in NF1-iN cells at DPI 5 (early-period) but not at DPI 14. We revealed that the expression of FOS and BCL2 mRNA, which act as anti-apoptotic in neuronal cells, were higher in developed- and early-stage iN cells of NF1 patients, respectively. Thus, apoptosis of neuronal cells during neurodevelopmental period may be disturbed in NF1 patients. These results have suggested that analysis of early-stage iN cells may reflect characteristics of premature neuronal cells during neurodevelopmental periods, and we thus believe that molecular analysis of not only developed-stage but also early-stage iN cells may explore the novel pathophysiology of neuronal cells in various neurodevelopmental disorders including ASD and schizophrenia. In particular, iN cells can be produced much faster compared to iPS-neurons, thus iN cells can be useful in the future application for assessment and treatment selection in clinical settings. We believe that translational research/clinical application using both iPS-neurons and iN cells would be an ideal approach in the future.

7月27日（土）18:10～18:30　第5会場（朱鷺メッセ　3F　302） 3S05e-5 ヒト興奮性・抑制性神経細胞を用いた神経発達障害の in vitro モデル Mitsuru Ishikawa（石川充），Hideyuki Okano（岡野栄之）慶應義塾大学医学部生理学教室 The importance of human pluripotent stem cells (hPSCs) as a biomedical model for studying human brain function is well-established. However, current protocols for generating neurons from hPSCs remain limited by protracted differentiation timelines and production of heterogeneous neuronal phenotypes. Few studies have been able to produce differentiated neuronal subtypes and to culture functional synapses and circuits. Here, we constructed rapidly inducible neuronal cell lines to generate excitatory neurons or inhibitory neurons using the piggyBac transposon system along with either the tetO-Neurog2 or tetO-Ascl1+Dlx2 system. Moreover, we induced transient expression of other genes in these systems resulting in the rapid generation of functional neurons. As characterized by using morphological analysis, Ca²⁺ imaging, and electrophysiology, differentiated neurons belonging to specific subtypes were generated . Using induced pluripotent stem cells (iPSCs) specific for neurodevelopmental disorders and epilepsy, we established an in vitro epilepsy model and performed functional phenotyping of disease-specific excitatory or inhibitory neurons. This methodology, relying on a robust and scalable starting population of hPSCs, is not only a highly efficient neuronal differentiation protocol but is also readily amenable to scaling for hPSC-based high-throughput disease phenotyping and drug screening.