Loss of alkyladenine DNA glycosylase alters gene expression in developing mouse brain and leads to reduced anxiety and increased exploratory behavior

Neurodevelopment is a tightly coordinated process, in which genome is exposed to spectra of endogenous agents at different stages of differentiation. Increasing body of evidence suggests that DNA damage is an important feature of developing brain, tightly linked to gene expression programs and neuronal activity. Some of the most frequent DNA damage includes changes to DNA bases, recognized by DNA glycosylases and repaired through base excision repair (BER). The only mammalian DNA glycosylase able to remove frequent alkylated DNA based is alkyladenine DNA glycosylase (Aag, aka Mpg). Recently we showed that, besides participation in DNA repair, AAG affects expression of neurodevelopmental genes in human cells. Aag was further proposed to act as reader of epigenetic marks, including 5-hydroxymethylcytosine (5hmC), in the mouse brain. Despite the evidence of potential Aag involvement in the key brain processes, the impact of Aag loss on developing brain remains unknown. Here, by using Aag knockout (Aag-/-) mice, we show that Aag absence leads to reduced DNA damage levels, evident in lowered number of gammaH2AX foci in P5 hippocampi. This is accompanied by changes in 5hmC signal intensity in different hippocampal regions. Analysis of gene expression in hippocampus and prefrontal cortex, at multiple developmental stages, indicates that lack of Aag results in altered gene expression, primarily of genes involved in regulation of response to stress. One of the most prominent genes deregulated in Aag-dependent manner, at all tested developmental stages, is aldehyde dehydrogenase 2 (Aldh2). In line with the changes in hippocampal DNA damage levels and the gene expression, adult Aag-/- mice exhibit altered behavior, evident in decreased anxiety in the Elevated Zero Maze and increased alternations in the Elevated T Maze tests. Taken together these results suggests that Aag has functions in modulation of genome dynamics during brain development, important for animal behavior. To characterize brain development in Aag-/- versus WT, mice at postnatal day five (P5), juvenile mice at six weeks (6W) and adult mice from four to six months (6M) were evaluated. Hippocampus and pre-frontal cortex were isolated from P5, 6W and 6M male mice (4 animals per time point) for further RNA extraction. Total RNA from hippocampus and pre-frontal cortex was then compared between the KO (Aag-/-) and WT genotypes in addition to comparisons at the 3 different time points.神经发育是一个高度协同调控的过程，在此过程中基因组在细胞分化的不同阶段会暴露于一系列内源性因子。日益增多的实验证据表明，DNA损伤是发育中大脑的重要特征，且与基因表达程序及神经元活动紧密相关。最常见的DNA损伤类型包括DNA碱基改变，这类损伤可被DNA糖苷酶（DNA glycosylases）识别，并通过碱基切除修复（base excision repair，BER）通路完成修复。目前哺乳动物中唯一能够清除常见烷基化DNA碱基的DNA糖苷酶为烷基腺嘌呤DNA糖苷酶（alkyladenine DNA glycosylase，简称Aag，又称Mpg）。此前本团队的研究表明，除参与DNA修复外，AAG还可调控人类细胞中神经发育相关基因的表达。另有研究提出，AAG在小鼠大脑中可作为表观遗传标记的识别因子，包括5-羟甲基胞嘧啶（5-hydroxymethylcytosine，5hmC）。尽管已有证据表明AAG可能参与大脑的关键生理过程，但AAG缺失对发育中大脑的影响仍不明确。本研究通过使用烷基腺嘌呤DNA糖苷酶敲除（Aag-/-）小鼠，发现AAG缺失会导致DNA损伤水平降低，具体表现为出生后第5天（P5）小鼠海马体中的γH2AX焦点数量减少。同时，不同海马脑区的5hmC信号强度也发生了改变。我们对多个发育阶段小鼠的海马体及前额叶皮层进行了基因表达分析，结果显示AAG缺失会导致基因表达谱发生改变，主要富集于参与应激响应调控的基因。在所有检测的发育阶段中，乙醛脱氢酶2（aldehyde dehydrogenase 2，Aldh2）是受AAG调控最为显著的差异表达基因之一。结合海马体DNA损伤水平及基因表达的变化，成年Aag-/-小鼠表现出行为学改变：在高架零迷宫（Elevated Zero Maze）实验中焦虑水平降低，在高架T迷宫（Elevated T Maze）实验中自发交替行为增加。综上，本研究结果表明，AAG在大脑发育过程中参与调控基因组动态平衡，这对动物的正常行为至关重要。为表征Aag-/-小鼠与野生型（wild type，WT）小鼠的大脑发育差异，本研究对三个发育阶段的小鼠进行了评估：出生后第5天（P5）的幼鼠、6周龄（6W）的幼年小鼠，以及4至6月龄（6M）的成年小鼠。我们从每个时间点的4只雄性小鼠体内分离海马体与前额叶皮层，用于后续的RNA提取。此外，我们还在3个不同时间点下，分别对敲除型（KO，Aag-/-）与野生型（WT）小鼠的海马体及前额叶皮层总RNA进行了比较分析。