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Effects of 16p11.2 copy number variation on neuronal development and pathology

16p11.2 拷贝数变异对神经元发育和病理学的影响

基本信息

批准号：
10659523
负责人：
MIRJANA MALETIC-SAVATIC
金额：
$ 86.16万
依托单位：
BOSTON CHILDREN'S 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：
2023-03-10 至 2028-02-29
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/10659523
关键词：
16p 16p11.2 3-Dimensional Address Affect Animal Model Animals Architecture Axon Behavior Bioenergetics Bipolar Disorder Brain Brain Diseases CRISPR/Cas technology Cell Line Cell membrane Cell model Cells Ceramides Clinical Coculture Techniques Code Complex Copy Number Polymorphism Cytoskeleton Data Development Disease Energy Metabolism Engineering Environment Enzymes Functional disorder Gene Expression Profiling Generations Genes Genetic Transcription Genotype Glutamates Homeostasis Human Hyperactivity Induced pluripotent stem cell derived neurons Intellectual functioning disability Knowledge Laboratories Lipids Mass Spectrum Analysis Mediating Mental disorders Metabolic Metabolism Methodology Midbrain structure Mitochondria Modeling Molecular Molecular Target Neurites Neurocognitive Neurodevelopmental Disorder Neuronal Differentiation Neuronal Dysfunction Neurons Organoids Pathology Pathway interactions Patients Pattern Penetrance Phenotype Property Prosencephalon Regulation Reporting Risk Factors Role Schizophrenia Signal Transduction Sorting Synapses Time Transcript Transcriptional Regulation Variant autism spectrum disorder brain volume clinical phenotype connectome density dihydroceramide desaturase dopaminergic neuron excitatory neuron genomic locus induced pluripotent stem cell inhibitory neuron insight lipid metabolism lipidome lipidomics metabolome metabolomics migration molecular phenotype multi-electrode arrays multimodal data neurite growth neuron development neuronal cell body neuronal circuitry neuronal excitability neuronal metabolism new therapeutic target patch clamp pleiotropism protein expression synaptic function synaptogenesis three-dimensional modeling tool transcription factor transcriptome transcriptome sequencing two-dimensional

项目摘要

Project Summary/Abstract Copy number variations (CNVs) of the human 16p11.2 genetic locus, containing 29 coding genes, are associated with a number of neurodevelopmental and psychiatric disorders. The deletion (16pdel) and duplication (16pdup) variants of this region have poorly understood pleiotropic effects. Although autism is more common in patients with deletions, and schizophrenia is more common in those with duplications, underlying mechanisms are not clear. Several molecular pathways from the 16p11.2 region modulate neuronal differentiation, migration, axonal development, and synapse formation, as well as energy and lipid metabolism. Studies of 16p-animal models have suggested deficits in the KCTD13-RhoA pathway activation, neuronal migration, axonal development, and behavior. In turn, disruption of ceramide homeostasis due to 16p11.2 CNVs at FAM57B locus altered lipid abundance, cell membrane dynamics, synaptic protein expression, and synaptic transport, suggesting that lipidome dysregulation could contribute to neuronal function and activity. However, the exact molecular mechanisms underlying these neuronal dysfunctions in excitatory versus inhibitory neurons are lacking. Moreover, contradictory results have been reported from different animal and human cell models. To address this gap of knowledge, we developed human iPSC-derived neuronal models of 16p11.2 CNVs and demonstrated that i) KCTD13 regulates RhoA pathway activation, and increased RhoA expression leads to hyperactivity of the 16pdel human cortical neuron networks, and ii) there are significant changes in key mitochondrial and lipid enzyme transcripts, including decrease in FAM57B-mediated ceramide synthase expression, that directly correlate with observed changes in the metabolome and lipidome. These data suggest that 16pdel leads to complex metabolic disruptions and deficient ceramide expression that might contribute to the observed functional neuronal network phenotypes. These data have led us to hypothesize that 16p11.2 CNVs cause dysregulation of ceramide abundance in glutamatergic and GABAergic neurons that in turn promotes deficits in synaptic development and function leading to network disorganization and hyperactivation. Here, we will investigate this hypothesis and the effects of 16p11.2 CNVs on cortical neuron development and function in human iPSC-derived 2-dimensional excitatory-inhibitory neuron co-cultures and human iPSC-derived 3-dimensional forebrain organoids. To reduce variability caused by different genotypic backgrounds, we will study CRISPR-Cas9 induced 16p11.2 CNV iPSC lines in addition to iPSC lines derived from patients and healthy controls. We will utilize state- of-the-art molecular methodologies to uncover mechanisms underlying the synaptic dysfunction of the excitatory- inhibitory neurons in 16p11.2 CNVs, including single cell transcriptional gene expression profiling and lipidome/metabolome profiling. Finally, we will investigate the excitatory-inhibitory network function, connectivity, and oscillation patterns with multi-electrode arrays and patch clamping. We anticipate that this study will uncover new molecular targets related to cortical neuron dysfunction in 16p11.2 CNV disorders.

项目总结/摘要人类16p11.2基因座的拷贝数变异（CNVs），包含29个编码基因，患有多种神经发育和精神疾病缺失（16 pdel）和重复（16 pdup）该区域的变体具有知之甚少的多效性效应。虽然自闭症在患者中更常见缺失，精神分裂症在那些有重复的人中更常见，潜在的机制不是清楚来自16p11.2区域的几种分子途径调节神经元分化、迁移、轴突生长和神经元分化。发育和突触形成，以及能量和脂质代谢。16 p-动物模型的研究已经表明KCTD 13-RhoA通路激活、神经元迁移、轴突发育和行为反过来，由于FAM 57 B基因座16p11.2 CNV导致的神经酰胺稳态破坏改变了脂质代谢，丰度、细胞膜动力学、突触蛋白表达和突触转运，表明脂质体调节异常可能有助于神经元功能和活性。然而，确切的分子缺乏兴奋性神经元与抑制性神经元中这些神经元功能障碍的潜在机制。此外，不同的动物和人类细胞模型报告了相互矛盾的结果。解决基于这一知识空白，我们开发了16p11.2 CNV的人iPSC衍生的神经元模型，并证明了 i）KCTD 13调节RhoA通路活化，并且RhoA表达增加导致细胞内的细胞过度活跃， 16 pdel人类皮层神经元网络，和ii）关键线粒体和脂质酶转录，包括FAM 57 B介导的神经酰胺合酶表达的减少，与观察到的代谢组和脂质组的变化相关。这些数据表明，16 pdel导致复杂的代谢中断和神经酰胺表达不足，可能有助于观察到的功能性神经网络表型这些数据使我们假设16p11.2 CNVs引起调节异常神经酰胺丰富的多巴胺能和GABA能神经元，反过来又促进突触缺陷，发展和功能导致网络解体和过度激活。在这里，我们将调查这一点 16p11.2 CNV对人iPSC衍生的皮质神经元发育和功能的影响 2-三维兴奋-抑制神经元共培养物和人iPSC衍生的三维前脑类有机体为了减少由不同基因型背景引起的变异性，我们将研究CRISPR-Cas9诱导 16p11.2 CNV iPSC系以及源自患者和健康对照的iPSC系。我们将利用国家- 最先进的分子方法来揭示兴奋性突触功能障碍的潜在机制， 16p11.2 CNVs中的抑制性神经元，包括单细胞转录基因表达谱，脂质组/代谢组分析。最后，我们将研究兴奋-抑制网络的功能，连通性，以及多电极阵列和膜片钳的振荡模式。我们预计这项研究将揭示与16p11.2 CNV疾病中皮质神经元功能障碍相关的新分子靶点。