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

• 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
• 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

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的、致残的、具有强烈遗传性的疾病。现有的治疗不能恢复功能 对于许多患者，它们也不能改变疾病过程，并且阻碍了新疗法的开发 缺乏生物学目标。死后研究表明，皮质树突棘密度降低， 精神分裂症患者，与临床结构神经影像研究一致。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。 研究人员已经开发、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组中产生重编程的小胶质细胞样细胞进行修剪， 用来自诱导多能干细胞（iPSC）衍生的分离和高度纯化的突触（突触体）， 神经元培养在他们发表的研究中，他们已经证明了突触功能障碍的有力证据， 吞噬归因于精神分裂症患者的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基因座联系起来的努力主要限于补体C4基因座， 几乎完全集中在神经元上。拟议的调查将利用患者来源的细胞 研究人员创建了一个生物库，应用基因工程方法来解决小胶质细胞在 突触修剪直接。具体来说，它将研究突触吞噬的贡献， 小胶质细胞表达精神分裂症易感基因和神经元补体上游调控因子 分别通过工程化诱导的小胶质细胞和神经元培养物的突触表达。 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试验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iPSC衍生的小胶质细胞模型，以确认在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 产生操纵人补体C4表达的工程化iPSC衍生的神经元培养物 等位基因和调节基因。然后，他们将使用来自这些细胞系的突触体进行功能性体外研究。 小胶质细胞介导的吞噬试验，以评估这些基因在神经元中的操作效果。 总之，这些目标将描述精神分裂症易感基因座对人类突触的影响。 修剪，在小胶质细胞功能以及神经元补体表达方面，使用经验证的，可扩展的 患者衍生模型系统。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。