Small noncoding RNA Dysregulation is Implicated in Manganism in a Rat Model of Methylcyclopentadienyl Manganese Tricarbonyl-Induced Unrepaired Striatum Damage

Excessive accumulation of manganese in brain can cause Parkinsonian-like symptoms, known as Manganism. Methylcyclopentadienyl Manganese Tricarbonyl (MMT), a gasoline antiknock additive, is one of environmental exposures of manganese, which can lead to manganism in Rats. Though some researches showed that small non-coding RNAs (sncRNAs) were differently expressed in Parkinson's disease (PD) patients, it was still unclear whether and how sncRNAs dysfunction appeared in Manganism. Unfartunately, transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) are highly modified, including 3' terminal modification such as 3'-phosphate and 2',3'-cyclic phosphate that block the adapter ligation process, and RNA methylations such as m1A, m3C, m1G and m22G that interfere with reverse transcription. Thus, sncRNAs could escape from traditional small RNA-seq. Here, we present the differential expression of sncRNAs in MMT-induced unrepaired striatum in rats compared with control group, using PANDORA-seq, which could discover the highly modified sncRNAs. By removing sncRNAs modification, we found that 599 sncRNAs were differently expressed in Striatum of MMT-treated rats compared with control group, and 1155 sncRNAs in Mn-treated vs control. Further function analysis for predicted targets of these DE-sncRNAs shown that dysregulation of sncRNAs was implicated Manganism in rats. Overall design: The rats were treated as previously described(Zhu et al., 2022). Briefly, After the animals had acclimated for one week, eighteen rats containing 9 males and 9 females (220 ± 20 g, 8-weeks-old) were divided randomly into three groups: Control, MMT and positive control (MnCl2) of rats (with 3 rats in each groups/sex). The MMT group were treated with 4 mg/kg MMT (about 1 mg Mn/kg) with corn oil via the intragastrical (i.g.) route once a day, six days per week, for 8 weeks. The control group were treated with corn oil (1 ml/kg administration volume) in the meantime. The rats in the MnCl2 group were given 200 mg/kg MnCl2·4H2O (i.g.) with physiological saline (10 ml/kg administration volume). Animals were weighed daily.

大脑中锰元素过度蓄积可引发类帕金森症状，即锰中毒（Manganism）。甲基环戊二烯三羰基锰（Methylcyclopentadienyl Manganese Tricarbonyl，MMT）作为汽油抗爆添加剂，是锰的环境暴露来源之一，可诱导大鼠发生锰中毒。尽管已有研究表明帕金森病（Parkinson's disease, PD）患者体内存在非编码小RNA（small non-coding RNAs, sncRNAs）的差异表达，但目前仍不清楚非编码小RNA的功能异常是否以及如何出现在锰中毒进程中。遗憾的是，转运RNA衍生小RNA（transfer RNA-derived small RNAs, tsRNAs）与核糖体RNA衍生小RNA（ribosomal RNA-derived small RNAs, rsRNAs）存在高度修饰，包括会阻断接头连接过程的3'端修饰（如3'-磷酸、2',3'-环磷酸），以及会干扰反转录过程的RNA甲基化修饰（如m1A、m3C、m1G、m22G）。因此，传统小RNA测序（small RNA-seq）无法有效捕获这类非编码小RNA。本研究借助可识别高度修饰非编码小RNA的PANDORA-seq技术，对比了MMT诱导的大鼠纹状体与对照组中sncRNAs的差异表达情况。通过去除非编码小RNA的修饰后，我们发现：MMT处理组大鼠纹状体中共存在599个差异表达的sncRNAs，而氯化锰（MnCl2）处理组与对照组相比则有1155个差异表达sncRNAs。对这些差异表达sncRNAs的预测靶标进行功能富集分析后显示，非编码小RNA的表达失调与大鼠锰中毒密切相关。实验整体设计：实验大鼠的造模流程参照此前发表的研究（Zhu等，2022）。具体操作如下：大鼠适应环境1周后，选取18只8周龄、体重为220±20g的大鼠（雌雄各9只），随机分为三组：对照组、MMT处理组与阳性对照组（MnCl2处理组），每组每性别各3只大鼠。MMT处理组以玉米油为溶媒，经灌胃（intragastrical, i.g.）途径给予4mg/kg MMT（相当于1mg Mn/kg体重），每日1次，每周给药6天，持续8周。对照组同期以相同给药体积（1ml/kg）给予玉米油。MnCl2处理组则以生理盐水为溶媒，经灌胃给予200mg/kg MnCl2·4H2O（给药体积为10ml/kg）。实验期间每日称量大鼠体重。