Pinson Marisa R, Chung Dae D, Mahnke Amanda H, Salem Nihal A, Osorio Daniel, Nair Vijay, Payne Elizabeth A, Del Real Jonathan J, Cai James J, Miranda Rajesh C
Department of Neuroscience and Experimental Therapeutics, Texas A&M University Health Science Center, Bryan, Texas, USA.
Women's Health in Neuroscience Program, Texas A&M University Health Science Center, Bryan, Texas, USA.
Alcohol Clin Exp Res. 2022 Apr;46(4):556-569. doi: 10.1111/acer.14796. Epub 2022 Mar 2.
We previously showed that ethanol did not kill fetal neural stem cells (NSCs), but that their numbers nevertheless are decreased due to aberrant maturation and loss of self-renewal. To identify mechanisms that mediate this loss of NSCs, we focused on a family of Gag-like proteins (GLPs), derived from retroviral gene remnants within mammalian genomes. GLPs are important for fetal development, though their role in brain development is virtually unexplored. Moreover, GLPs may be transferred between cells in extracellular vesicles (EVs) and thereby transfer environmental adaptations between cells. We hypothesized that GLPs may mediate some effects of ethanol in NSCs.
Sex-segregated male and female fetal murine cortical NSCs, cultured ex vivo as nonadherent neurospheres, were exposed to a dose range of ethanol and to mitogen-withdrawal-induced differentiation. We used siRNAs to assess the effects of NSC-expressed GLP knockdown on growth, survival, and maturation and in silico GLP knockout, in an in vivo single-cell RNA-sequencing dataset, to identify GLP-mediated developmental pathways that were also ethanol-sensitive.
PEG10 isoform-1, isoform-2, and PNMA2 were identified as dominant GLP species in both NSCs and their EVs. Ethanol-exposed NSCs exhibited significantly elevated PEG10 isoform-2 and PNMA2 protein during differentiation. Both PEG10 and PNMA2 were mediated apoptosis resistance and additionally, PEG10 promoted neuronal and astrocyte lineage maturation. Neither GLP influenced metabolism nor cell cycle in NSCs. Virtual PEG10 and PNMA2 knockout identified gene transcription regulation and ubiquitin-ligation processes as candidate mediators of GLP-linked prenatal alcohol effects.
Collectively, GLPs present in NSCs and their EVs may confer apoptosis resistance within the NSC niche and contribute to the abnormal maturation induced by ethanol.
我们之前发现乙醇不会杀死胎儿神经干细胞（NSCs），但其数量会因异常成熟和自我更新能力丧失而减少。为了确定介导NSCs这种损失的机制，我们聚焦于一类源自哺乳动物基因组中逆转录病毒基因残余的类Gag蛋白（GLPs）。GLPs对胎儿发育很重要，但其在大脑发育中的作用几乎未被探索。此外，GLPs可能在细胞外囊泡（EVs）中在细胞间转移，从而在细胞间传递环境适应性。我们假设GLPs可能介导乙醇对NSCs的一些影响。
将离体培养为非贴壁神经球的性别分离的雄性和雌性胎儿小鼠皮质NSCs暴露于一系列乙醇剂量以及有丝分裂原撤去诱导的分化环境中。我们使用小干扰RNA（siRNAs）评估NSC表达的GLP敲低对生长、存活和成熟的影响，并在体内单细胞RNA测序数据集中进行GLP的虚拟敲除，以鉴定也是乙醇敏感的GLP介导的发育途径。
PEG10亚型-1、亚型-2和PNMA2被确定为NSCs及其EVs中的主要GLP种类。乙醇暴露的NSCs在分化过程中PEG10亚型-2和PNMA2蛋白显著升高。PEG10和PNMA2均介导抗凋亡作用，此外，PEG10促进神经元和星形胶质细胞谱系成熟。两种GLP均不影响NSCs的代谢和细胞周期。虚拟PEG10和PNMA2敲除确定基因转录调控和泛素连接过程是GLP相关产前酒精影响的候选介导因素。
总体而言，NSCs及其EVs中存在的GLPs可能赋予NSC生态位内的抗凋亡能力，并导致乙醇诱导的异常成熟。
