Paraoxonase 2 Deficiency in Mice Alters Motor Behavior and Causes Region-Specific Transcript Changes in the Brain

Araoxonase 2 (PON2) is an intracellular antioxidant enzyme shown to play an important role in mitigating oxidative stress in the brain. Oxidative stress is a common mechanism of toxicity for neurotoxicants and is increasingly implicated in the etiology of multiple neurological diseases. While PON2 deficiency increases oxidative stress in the brain in-vitro, little is known about its effects on behavior in-vivo and what global transcript changes occur from PON2 deficiency. Therefore, we sought to characterize the effects of PON2 deficiency on behavior in mice, with an emphasis on locomotion, and evaluate transcriptional changes with RNA-Seq. Behavioral endpoints included home-cage behavior (Noldus PhenoTyper), motor coordination (Rotarod) and various gait metrics (Noldus CatWalk). Home-cage behavior analysis showed PON2 deficient mice had increased activity at night compared to wildtype controls and spent more time in the center of the cage, displaying a possible anxiolytic phenotype. PON2 deficient mice had significantly shorter latency to fall when tested on the rotarod, suggesting impaired motor coordination. Minimal gait alterations were observed, with decreased girdle support posture noted as the only significant change with PON2 deficiency. Finally, A subset of samples were utilized for RNA-Seq analysis, looking at three discrete brain regions: cerebral cortex, striatum and cerebellum. Highly regional- and sex-specific changes in RNA expression were found when comparing PON2 deficient and wildtype mice, suggesting PON2 may play distinct regional roles in the brain in a sex-specific manner. Taken together, these findings demonstrates that PON2 deficiency significantly alters the brain on both a biochemical and phenotypic level, with a specific impact on motor function. This data has implications for future gene-environment toxicological studies and warrants further investigation of PON2 in the brain. Male and female wildtype (WT) and PON2 deficient (PON2-def) mice (Ng et al., 2006) on a C57BL/6J background were used for this study. Seven to ten animals were used per sex per group. Mice were bred from PON2 deficient heterozygous crosses with resulting WT and PON2-def homozygous littermates used in rolling cohorts. Behavioral testing began at 3-4 months of age. Mice were housed in a specific pathogen-free facility on a 14-hour light/ 10-hour dark cycle with ad libitum access to food and water. All procedures were conducted in accordance with the National Institute of Health Guide for the Use and Care of Laboratory Animals and were approved by the University of Washington Institutional Animal Care and Use Committee. Mice were sacrificed at 3-4 months of age upon conclusion of behavioral testing. Cerebral cortex, striatum and cerebellum were fresh dissected (Spijker 2011) and flash frozen in liquid nitrogen. Tissue was pulverized with a pre-chilled mortar and pestle into a fine powder, stored at 80°C and aliquoted into appropriate buffers for downstream testing.对氧磷酶2（Araoxonase 2，PON2）是一种细胞内抗氧化酶，已被证实可在大脑中发挥重要的抗氧化应激作用。氧化应激是神经毒物产生毒性的常见机制，且越来越多的研究表明其与多种神经系统疾病的病因学密切相关。尽管已有研究证实PON2缺失会在体外培养条件下加剧大脑的氧化应激，但目前对于PON2缺失在活体动物中对行为学的影响，以及PON2缺失所引发的全转录组变化仍知之甚少。因此，本研究旨在表征PON2缺失对小鼠行为学的影响，重点关注运动功能，并通过RNA测序（RNA-Seq）评估其转录组变化。本研究的行为学检测终点包括笼养行为（使用Noldus PhenoTyper系统）、运动协调能力（转棒实验，Rotarod）以及多种步态参数（Noldus CatWalk系统）。笼养行为分析结果显示，与野生型（WT）对照组相比，PON2缺失型小鼠夜间活动量显著增加，且在笼内中心区域停留的时间更长，表现出潜在的抗焦虑表型。转棒实验中，PON2缺失型小鼠的跌落潜伏期显著缩短，提示其运动协调能力受损。步态分析仅观察到轻微改变，仅发现肢体带支撑姿势减少这一与PON2缺失相关的显著变化。最后，我们选取部分样本进行RNA-Seq分析，检测了三个离散的大脑区域：大脑皮层、纹状体和小脑。对比PON2缺失型与野生型小鼠后，发现RNA表达存在显著的区域特异性和性别特异性变化，提示PON2在大脑中可能以性别依赖的方式发挥不同的区域性功能。综上，本研究结果表明，PON2缺失会在生化和表型层面显著改变大脑功能，尤其对运动功能产生特异性影响。该数据可为未来的基因-环境毒理学研究提供参考，同时也值得进一步深入探索PON2在大脑中的作用机制。本研究使用的实验动物为C57BL/6J背景的野生型（WT）及PON2缺失型（PON2-def）小鼠（Ng等，2006），其中雄性和雌性小鼠各分为两组。每个性别每组使用7至10只动物。小鼠由PON2缺失型杂合子交配繁育而来，以同窝产生的WT及PON2-def纯合子小鼠作为实验队列。行为学测试于小鼠3-4月龄时开始。小鼠饲养于无特定病原体（SPF）级别的动物设施中，光照周期为14小时光照/10小时黑暗，自由采食饮水。所有实验操作均遵循"National Institute of Health Guide for the Use and Care of Laboratory Animals"，并经华盛顿大学实验动物护理与使用委员会批准。行为学测试结束后，于3-4月龄时对小鼠实施安乐死。取新鲜解剖的大脑皮层、纹状体和小脑（Spijker，2011），经液氮快速冷冻后，使用预冷的研钵和杵将组织研磨为细粉，储存于-80℃冰箱，随后分装至适配下游实验的缓冲液中。