The Nature and Function of Genomic Imprinting in Monoaminergic Neurons

 描述(由申请人提供)：在受影响的个体中，许多影响神经精神障碍风险的变异是杂合的。基因组印记是一种可遗传的表观遗传机制，导致某些基因的母系或父系等位基因优先表达，但通过沉默健康的等位基因，导致只表达突变的等位基因，从而恶化杂合突变的影响。规范印记 包括使父母的一个等位基因完全沉默。作者最近发现，在大脑中有数百个基因表现出母亲或父亲的等位基因偏见。他们把这称为非规范的印记。使用一种新的方法，作者有证据表明，在组织水平上检测到的非规范印记效应涉及大脑中神经元亚群的等位基因沉默。他们进一步发现，非规范印记显著影响杂合突变对后代行为的影响，这取决于突变的父母来源。因此，这些结果表明在大脑中存在高度细胞类型的特异性印记效应。目前，还不清楚哪些神经元群体参与其中，特定的行为如何受到影响，以及特定神经元类型中是否存在额外的基因印记。非规范印迹被发现影响酪氨酸羟基酶(Th)和多巴脱羧酶(DDC)的表达，这两个基因是合成单胺类神经递质多巴胺、去甲肾上腺素和5-羟色胺的必需基因。焦虑症、抑郁症、双相情感障碍、精神分裂症和其他精神疾病都会导致单胺系统功能障碍。这项拟议的研究将检验这一假设，即非规范印记导致单胺能神经元亚群中的等位基因沉默效应，以调节行为和生理的重要方面。在目标1中，将使用分别表达母系和父系等位基因的EGFP和tdTomato报告基因的转基因小鼠，在男性和女性的大脑中定义解剖学上定义的富含Th和DDC印记效应的单胺能神经元亚群。在目标2中，将使用一系列行为分析来定义影响单胺能神经元的非规范印记效应的功能(S)，以比较Th和DDC的母系和父系遗传缺失突变小鼠。最后，在目标3中，将使用单细胞RNAseq对从转基因F1杂交后代中纯化的EGFP+神经元的单胺类神经元的印记效应进行全面的描述。在C57BL/6J x CastEiJ遗传背景上，DDC：：EGFP转基因报告鼠(AIM 1)的相互交配将产生杂交后代。F1代的多态位点将在RNAseq数据中显示等位基因的表达。总体而言，这项研究将确定大脑中单胺能神经元印记效应的细胞性质和功能，并将非规范印记确立为影响单胺能回路功能和等位基因结构的重要表观遗传现象。

 DESCRIPTION (provided by applicant): Many variants that influence the risk for neuropsychiatric disorders are heterozygous in the affected individual. Genomic imprinting, which is a heritable epigenetic mechanism that results in preferential expression of the maternal or paternal allele for some genes, can worsen the effects of heterozygous mutations by silencing the healthy allele, leading to expression of only the mutant allele. Canonical imprinting involves complete silencing of one parent's allele. The authors recently discovered hundreds of genes that exhibit a maternal or paternal allele bias in the brain. They refer to this as noncanonical imprinting. Using a novel method, the authors have evidence that noncanonical imprinting effects detected at the tissue level involve allele-silencing in subpopulations of neurons in the brain. They further found that noncanonical imprinting significantly influences the impact of heterozygous mutations on offspring behavior depending on the parental origin of the mutation. Thus, the results indicate the existence of highly cell-type specific imprinting effects n the brain. Currently, it is unknown which neuronal populations are involved, how specific behaviors are impacted, and whether additional genes are imprinted in specific neuron types. Noncanonical imprinting was found to influence the expression of tyrosine hydroxylase (Th) and dopa decarboxylase (Ddc), two genes essential for the synthesis of the monoamine neurotransmitters dopamine, noradrenaline and serotonin. Dysfunction of the monoaminergic systems occurs in anxiety disorders, major depression, bipolar, schizophrenia and other mental disorders. The proposed study will test the hypothesis that noncanonical imprinting causes allele- silencing effects in subpopulations of monoaminergic neurons to regulate important aspects of behavior and physiology. In aim 1, anatomically-defined subpopulations of monoaminergic neurons that are enriched for Th and Ddc imprinting effects will be defined in the male and female brain using transgenic mice that express egfp and tdTomato reporters from the maternal and paternal alleles, respectively. In aim 2, the function(s) of noncanonical imprinting effects influencing monoaminergic neurons will be defined using a battery of behavioral assays to compare mutant mice with maternally versus paternally inherited deletions in Th and Ddc. Finally, in aim 3, imprinting effects in monoaminergic neurons will be comprehensively profiled using single-cell RNASeq on EGFP+ neurons purified from transgenic F1 hybrid offspring. Hybrid offspring will be generated from reciprocal matings of Ddc::egfp transgenic reporter mice (Aim 1) on the C57BL/6J x CastEiJ genetic backgrounds. Polymorphic sites in the F1 hybrids will reveal allelic expression in the RNASeq data. Overall, the study will define the cellular nature and function of imprinting effects in monoaminergic neurons in the brain and establish noncanonical imprinting as an important epigenetic phenomenon that influences the function and allelic architecture of monoaminergic circuits.