Characterization of schizophrenia liability genes in models of human microglial synaptic pruning

人类小胶质细胞突触修剪模型中精神分裂症易感基因的表征

基本信息

批准号：
10736092
负责人：
ROY H. Perlis
金额：
$ 60.9万
依托单位：
MASSACHUSETTS GENERAL HOSPITAL
依托单位国家：
美国
项目类别：
财政年份：
2023
资助国家：
美国
起止时间：
2023-07-12 至 2028-03-31
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10736092
关键词：
Address Alleles Applied Genetic Engineering Autopsy Biological Biological Assay Biological Models Biology Brain Brain region CRISPR screen Cell model Cells Chronic Clinical Clustered Regularly Interspaced Short Palindromic Repeats Complement Complement 4b Complement component C4a Data Dendritic Spines Deposition Development Disease Engineering Functional disorder Gene Expression Genes Genetic Engineering Genetic Variation Genomics Heritability Hippocampus Human Immune In Vitro Individual Induced pluripotent stem cell derived neurons Investigation Knock-out Link Maps Measurable Mediating Mental disorders Mentally Disabled Persons Methods MicroRNAs Microglia Modeling Mus Neurons PF4 Gene Patients Peripheral Blood Mononuclear Cell Phagocytosis Phenotype Pluripotent Stem Cells Prefrontal Cortex Process Proteins Protocols documentation Publishing Research Research Personnel Risk Rodent Model Role Schizophrenia Slice Specificity Structure Synapses Synaptosomes System Variant Work biobank density disorder risk effective therapy gene function genetic manipulation genome wide association study genome-wide human genomics human model induced pluripotent stem cell neuroimaging novel novel therapeutics peripheral blood retinogeniculate risk variant schizophrenia risk synaptic pruning synaptogenesis targeted treatment therapeutic development therapy development transcriptomics uptake

项目摘要

Schizophrenia is a chronic, disabling, and strongly heritable illness. Existing treatments do not restore function for many patients, nor do they modify the disease process, and development of novel therapeutics is hindered by a lack of biological targets. Postmortem studies demonstrate reduced cortical dendritic spine density among individuals with schizophrenia, consistent with clinical structural neuroimaging studies. Convergent lines of evidence from rodent models and human genomics suggest that these abnormalities may arise from microgliamediated pruning dysfunction. The investigators have developed, validated and published patient-specific models of microglia-mediated pruning by generating reprogrammed microglia-like cells from patients and healthy controls, and assaying them with isolated and highly purified synapses (synaptosomes) from induced pluripotent stem cell (iPSC)-derived neuronal cultures. In their published studies, they have demonstrated robust evidence of dysfunction in synaptic engulfment attributable to abnormalities in both microglia and neurons from individuals with schizophrenia. To date, efforts to link these abnormalities to disease risk loci have largely been limited to the complement C4 locus, and focused almost entirely on neurons. The proposed investigation will draw on the patient-derived cellular biobank created by the investigators to apply genetic engineering methods to address the role of microglia in synaptic pruning directly. Specifically, it will investigate the contributions on synaptic engulfment of both microglial expressed schizophrenia liability genes and putative upstream regulators of neuronal complement synaptic expression through engineered induced microglia and neuronal cultures, respectively. For Aim 1, the investigators propose to perform CRISPR screening applied to induced microglia-like cells, applying high scale in vitro synaptic pruning assays to discover novel disease-associated gene functions implicated in schizophrenia. In Aim 2, the investigators will further validate loci from the screen in Aim 1 in CRISPR-engineered iPSC-derived microglia models to confirm roles in synaptic engulfment and determine other effects on microglia development and function. To complement these aims, in Aim 3, the investigators will generate engineered iPSC-derived neuronal cultures manipulating the expression of human complement C4 alleles and modulatory genes. They will then use synaptosomes from these lines for functional in-vitro microglialmediated engulfment assays to assess effects of manipulation of these genes in neurons. Taken together, these aims will characterize the effects of schizophrenia liability loci on human synaptic pruning, in terms of microglia function as well as neuronal complement expression, using validated, scalable patient-derived model systems. They will prioritize targets for treatment development and inform the biology of schizophrenia risk.

精神分裂症是一种慢性、致残且具有强烈遗传性的疾病。现有治疗无法恢复功能对于许多患者来说，它们也不能改变疾病过程，新疗法的开发受到阻碍由于缺乏生物靶标。尸检研究表明，皮质树突棘密度降低患有精神分裂症的个体，与临床结构神经影像学研究一致。的收敛线来自啮齿动物模型和人类基因组学的证据表明，这些异常可能源于小胶质细胞介导的修剪功能障碍。研究人员开发、验证并发表了小胶质细胞介导的患者特异性模型通过从患者和健康对照中产生重新编程的小胶质细胞样细胞并对其进行分析来进行修剪含有来自诱导多能干细胞 (iPSC) 的分离且高度纯化的突触（突触体）神经元培养物。在他们发表的研究中，他们证明了突触功能障碍的有力证据吞噬可归因于精神分裂症患者小胶质细胞和神经元的异常。到迄今为止，将这些异常与疾病风险位点联系起来的努力很大程度上仅限于补体 C4 位点，几乎完全集中在神经元上。拟议的研究将利用患者来源的细胞研究人员创建的生物库应用基因工程方法来解决小胶质细胞在直接进行突触修剪。具体来说，它将研究两者对突触吞噬的贡献小胶质细胞表达精神分裂症易感基因和神经元补体的假定上游调节因子分别通过工程诱导的小胶质细胞和神经元培养物进行突触表达。对于目标 1，研究人员建议对诱导的小胶质细胞样细胞进行 CRISPR 筛选，应用大规模体外突触修剪测定来发现新的疾病相关基因功能与精神分裂症有关。在目标 2 中，研究人员将进一步验证目标 1 中筛选的基因座 CRISPR 工程化的 iPSC 衍生的小胶质细胞模型，以确认突触吞噬中的作用并确定其他对小胶质细胞发育和功能的影响。为了补充这些目标，在目标 3 中，研究人员将产生操纵人类补体 C4 表达的工程化 iPSC 衍生神经元培养物等位基因和调节基因。然后他们将使用这些品系的突触体进行功能性体外研究小胶质细胞介导的吞噬测定来评估神经元中这些基因的操作效果。总而言之，这些目标将描述精神分裂症易感位点对人类突触的影响使用经过验证的、可扩展的方法，在小胶质细胞功能和神经元补体表达方面进行修剪源自患者的模型系统。他们将优先考虑治疗开发的目标并告知生物学精神分裂症风险。