シンポジウム
54 in vivoゲノム編集が切り開くハイスループット 精神疾患研究〜系統維持に、時間とコストかけていませんか？〜
54 Transformative high-throughput functional neuroscience with in vivo genome editing
座長：相田 知海（McGovern Institute for Brain Research, Massachusetts Institute ofTechnology）・奥山 輝大（東京大学 定量生命科学研究所） |
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| 2022年6月30日 16:10～16:13 沖縄コンベンションセンター 会議場A1 第2会場
1S02e-01
Welcome to in vivo genome editing revolution
*Tomomi Aida(1,2)
1. McGovern Inst, MIT, MA, USA, 2. Stanley Center, Broad Institute of MIT and Harvard. MA, USA
Keyword: CRISPR, mouse, marmoset, macaque
The history of neuroscience has been developed with genetically modified animal models. These animal models have provided us invaluable in vivo opportunities to investigate gene function, model human diseases, and manipulate specific brain regions, cell types, and neural circuits in a spatiotemporally specific manner. The recent development of genome editing technologies such as CRISPR further revolutionized the target scope of genetic manipulation from “traditional” fly, zebrafish, and mouse models, to virtually any species on the earth including non-human primates (NHPs). However, although the generation of genetically modified animal strains became now easier and faster than ever, the requirement of following the sexual maturation of the founders, colony expansion/maintenance, and breeding with other strains are still unchanged. These old-fashioned steps are costly, laborious, and time-consuming, and thus hampering high-throughput in vivo functional analysis of genes as well as the development of basic research and clinical translation using NHPs. In this symposium, we will focus on the cutting-edge “in vivo genome editing” which directly manipulates target gene(s) in the brain of wild-type animals, skipping all the previous steps required for genetically modified animal model production. The delivery of CRISPR by using viral vectors into the brain enables target gene manipulation in a brain region-, cell type- or neural circuit-specific manner. Furthermore, by leveraging the multiplexity of CRISPR guideRNA which defines the target gene in the genome, simultaneous functional interrogation of tens of genes in wild-type animals can be achieved. These innovations make analysis-ready genetically modified animals readily available just within a few weeks, enabling ultra-rapid functional analysis of genes in vivo at scale and thus, transforming neuroscience research. Welcome to in vivo genome editing revolution! | | 2022年6月30日 16:13～16:40 沖縄コンベンションセンター 会議場A1 第2会場
1S02e-02
In vivo Perturb-seq: scaled investigation of gene functions in the developing brain
*Xin Jin(1)
1. Scripps Research
Keyword: in vivo genome editing, single cell genomics, autism spectrum disorder
The thousands of disease risk genes and loci identified through human genetic studies far outstrip our current capacity to systematically study their functions. I will discuss our attempt to develop a scalable genetic screen approach, in vivo Perturb-seq, and apply this method to the functional evaluation of a panel of autism spectrum disorder (ASD) de novo loss-of-function risk genes. We identified recurrent and cell type-specific gene signatures from both neuronal and glial cell classes that are affected by genetic perturbations and pointed at elements of both convergent and divergent cellular effects across many ASD risk genes. In addition, I will also briefly discuss the research directions in my lab, established in July 2021, in applying spatial transcriptomic approaches to study cell intrinsic and extrinsic effects of these disease risk genes. Our lab will use these systematic approaches, connecting genomic technology development with rigorous dissection of molecular mechanisms, to learn new insight about how complex inputs are integrated into the developing brain. | | 2022年6月30日 16:40～16:51 沖縄コンベンションセンター 会議場A1 第2会場
1S02e-03
自閉症モデルShank3変異マウスにおける社会性記憶表象の障害
Disrupted social memory representation in autism-associated Shank3 mutant mice
*奥山 輝大(1)
1. 東京大学 定量生命科学研究所
*Teruhiro Okuyama(1)
1. IQB, Univ of Tokyo, Tokyo, Japan
Keyword: Social memory, ventral CA1, hippocampus, mice
For social animals, it is crucial to remember and recognize different conspecific individuals (i.e., social memory) and exhibit appropriate social behaviors, such as preference behavior or avoidance behavior, to each individual. Since mice naturally tend to spend more time interacting with novel mice, rather than familiar mice (social discrimination behavior), we can quantify the degree of memory of individuals by calculating the total duration of time spent with novel versus familiar individuals. Using the social discrimination behavioral assay, we recently demonstrated that vCA1 pyramidal neurons in the hippocampus store social memory engram. Even when the memory seemed lost after long separation periods, optogenetic activation of the engram can fully restore that social memory. Additionally, an artificial association between social engram encoding the memory of a specific individual with fear or reward events can elicit avoidance from or preference to that individual, respectively. One tiny dissonance in social memory can easily disrupt the appropriate social behavior, even for humans. Social impairments caused by a genetic mutation, especially those related to the familiarization with other individuals, are commonly exhibited by patients diagnosed with autism spectrum disorder (ASD). Patients with ASD have difficulty either with social memory itself or showing normal social communication driven by social memory. In this meeting, we will show our recent findings regarding neural mechanisms underlying the impairments of social memory representation in autism-associated Shank3 mutant mice. | | 2022年6月30日 16:51～17:18 沖縄コンベンションセンター 会議場A1 第2会場
1S02e-04
Anterior thalamic dysfunction underlies cognitive deficits in neuropsychiatric disease models
*Dheeraj Roy(1)
1. Broad Institute of MIT and Harvard
Keyword: Memory, Thalamus, Autism, Schizophrenia
Neuropsychiatric disorders are often accompanied by cognitive impairments/intellectual disability (ID). It is not clear whether there are converging mechanisms underlying these debilitating impairments. We found that many autism and schizophrenia risk genes are expressed in the anterodorsal subdivision (AD) of anterior thalamic nuclei, which has reciprocal connectivity with learning and memory structures. CRISPR-Cas9 knockdown of multiple risk genes selectively in AD thalamus led to memory deficits. While the AD is necessary for contextual memory encoding, the neighboring anteroventral subdivision (AV) regulates memory specificity. These distinct functions of AD and AV are mediated through their projections to retrosplenial cortex, using differential mechanisms. Furthermore, knockdown of autism and schizophrenia risk genes PTCHD1, YWHAG, or HERC1 from AD led to neuronal hyperexcitability, and normalization of hyperexcitability rescued memory deficits in these models. This study identifies converging cellular to circuit mechanisms underlying cognitive deficits in a subset of neuropsychiatric disease models. | | 2022年6月30日 17:18～17:45 沖縄コンベンションセンター 会議場A1 第2会場
1S02e-05
Targeting parafascicular thalamic circuits rescues motor deficits in a mouse model of Parkinson's disease
*Ying Zhang(1), Dheeraj Roy(1), Yi Zhu(2), Yefei Chen(1), Tomomi Aida(1), Guoping Feng(1)
1. MIT, 2. Zhejiang University, Zhejiang, China
Keyword: Parkinson, Motor , Parafascicular thalamus
Although bradykinesia, tremor, and rigidity are the hallmark motor defects in Parkinson’s disease (PD) patients, they also experience motor learning impairments. The neural circuit basis for these different PD symptoms is not well understood. While current treatments are effective for locomotion deficits in PD, therapeutic strategies targeting motor learning are lacking. We found that distinct parafascicular (PF) thalamic subpopulations project to caudate-putamen (CPu), and subthalamic nucleus (STN). While PFCPu circuits is critical for locomotion, inhibition of PFàSTN circuits induced motor learning deficit. We identified nicotinic acetylcholine receptors within each PF circuits capable of modulating both motor and motor learning. Importantly, modulating these receptors with CRISPR/Cas9 rescues different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating locomotion and motor learning deficits in PD.
| | 2022年6月30日 17:45～18:10 沖縄コンベンションセンター 会議場A1 第2会場
1S02e-06
Disease Modeling using Marmosets with Germ Line Modification and In Vivo Genome Editing
*Hideyuki Okano(1,2)
1. Laboratory for Marmoset Neural Architecture, RIKEN Center for Brain Science, Saitama, Japan, 2. Department of Physiology, Keio University School of Medicine, Tokyo, Japan
Keyword: Marmoset, Genome editing, CRISPR/Cas9, Parkinson disease
The common marmoset (Callithrix jacchus) is a small New World primate that has been extensively used as biomedical research models. There is also an increasing interest in common marmoset in Brain Science and as appropriate models for major human brain disorders (Okano, Annu Rev Neurosci, 2021; Okano et al., Neuron, 2016; Grillner et al., Nat Neurosci, 2016).
We have been working with a number of collaborators to build a platform for marmoset brain science research. In 2009, we succeeded in generating the world's first genetically modified (GM) marmoset with germline transmission by lentiviral mediated transgenesis (Sasaki et al., Nature, 2009), and in 2015, we succeeded in determining the detailed sequence of the entire marmoset genome using next-generation sequencing (Sato et al., Sci Rep, 2015). On this basis, we successfully created individual genome-edited marmosets (GM) using lentiviral mediated transgenesis (Sasaki et al., Nature, 2009), and in 2015, we successfully sequenced the entire genome of a marmoset using a next-generation sequencer (Sato et al., Sci Rep, 2015). In 2015, we successfully sequenced the entire genome of the marmoset using next-generation sequencing (Sato et al., Sci Rep, 2015), and on this basis, we successfully created genome-edited marmosets (Sato et al., Cell Stem Cell, 2016).
In the present talk, I wish to mention our recent data of generation of transgenic marmoset models of neurodegenerative diseases, including Parkinson disease (PD) which overexpressed the mutant form of α-synuclein using lentiviral vector. The PD model marmoset showed stage-dependent progression of the disease, such as sleeping disturbance followed by motor deficit. In addition, I will mention a model marmoset of a neurodevelopmental disorder, the Rett syndrome, obtained by genome editing of MECP2 gene. Abnormalities in brain structure and function in these marmoset models may accelerate discovery of disease biomarkers and mechanisms toward translation (Okano et al., Neuron, 2016). | |
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