Division of Integrative Neuroscience , New York State Psychiatric Institute and Columbia University , New York , New York 10032 , United States.
Departments of Molecular, Cellular, and Developmental Biology and Psychology and Neuroscience , University of Colorado Boulder , Boulder , Colorado 80309 , United States.
ACS Chem Neurosci. 2019 Jul 17;10(7):3197-3206. doi: 10.1021/acschemneuro.8b00638. Epub 2019 Feb 12.
The serotonin 1A receptor (5-HT1A) system has been extensively implicated in modulating mood and behavior. Notably, 5-HT1A levels in humans display remarkable variation, and differences in receptor levels have been linked with a variety of psychiatric disorders. Further, reduction of receptor levels by 30-50% in mice suggests that changes in receptor levels that model existing human variation are sufficient to drive behavioral alterations. As a result, genetic mechanisms that modulate human 5-HT1A levels may be important for explaining individual differences in mood and behavior, representing a potential source of psychiatric disease risk. One common genetic variant implicated in differential 5-HT1A levels is the G/C single nucleotide polymorphism (SNP) rs6295, located upstream of the human 5-HT1A gene. This SNP differentially binds the transcription factor, NUDR/Deaf1, leading to cell-type specific effects on transcription in vitro. To investigate the direct effects of this SNP in the heterogeneous cellular context of the brain, we generated humanized transgenic mice using a design that maximized the local transcriptional landscape of the human HTR1A gene while also controlling for effects of genomic insertion location. We integrated a 180 kb human bacteria artificial chromosome (BAC) transgene containing G- and C-alleles of rs6295 flanked by FRT or loxP sites. Subsequent deletion of each allele by Cre- or Flp-recombinase resulted in rs6295G and C alleles in the same genomic location. These alleles were bred onto a 5-HT1A null mouse such that the human BAC was the sole source of 5-HT1A in these mice. We generated three separate lines, two of which had detectable human 5-HT1A levels in the brain, although none displayed expression in the raphe. Of these, one line exhibited rs6295-dependent differences in 5-HT1A levels and differences in behavior, even though the overall levels were considerably lower than native expression levels. The line-dependent effect of rs6295 on protein levels and behavior may depend upon differences in background genetic factors or different insertion sites across each line. This work confirms that relatively subtle differences in 5-HT1A levels can contribute to differences in behavior and highlights the challenges of modeling human noncoding genetic variation in mice.
5-羟色胺 1A 受体（5-HT1A）系统在调节情绪和行为方面起着重要作用。值得注意的是，人类的 5-HT1A 水平存在显著差异，受体水平的差异与多种精神疾病有关。此外，在小鼠中，受体水平降低 30-50%表明，模拟现有人类变异的受体水平变化足以导致行为改变。因此，调节人类 5-HT1A 水平的遗传机制可能对解释情绪和行为的个体差异很重要，代表了精神疾病风险的一个潜在来源。一个与 5-HT1A 水平差异相关的常见遗传变异是 G/C 单核苷酸多态性（SNP）rs6295，位于人类 5-HT1A 基因的上游。该 SNP 不同程度地结合转录因子 NUDR/Deaf1，导致体外细胞类型特异性转录效应。为了研究该 SNP 在大脑异质细胞环境中的直接影响，我们使用一种设计生成了人源化转基因小鼠，该设计最大限度地模拟了人类 HTR1A 基因的局部转录景观，同时控制基因组插入位置的影响。我们整合了一个 180kb 的人类细菌人工染色体（BAC）转基因，其中包含 rs6295 的 G 和 C 等位基因，两侧为 FRT 或 loxP 位点。随后，通过 Cre 或 Flp 重组酶删除每个等位基因，导致 rs6295G 和 C 等位基因位于相同的基因组位置。这些等位基因被繁殖到 5-HT1A 缺失小鼠上，使得人类 BAC 成为这些小鼠中 5-HT1A 的唯一来源。我们生成了三个独立的品系，其中两个在大脑中检测到可检测的人类 5-HT1A 水平，尽管没有一个在中缝核中表达。其中，一个品系表现出 rs6295 依赖性的 5-HT1A 水平差异和行为差异，尽管总体水平明显低于天然表达水平。rs6295 对蛋白质水平和行为的品系依赖性影响可能取决于每个品系之间的背景遗传因素或不同插入位置的差异。这项工作证实，5-HT1A 水平的相对细微差异可能导致行为差异，并强调了在小鼠中模拟人类非编码遗传变异的挑战。
