锌指蛋白ZFP488调控新生儿脑白质发育和损伤后修复的机制研究

The perinatal brain injury is a leading cause of childhood mortality and lifelong disability. Periventricular leukomalacia (PVL) is the principal form of brain injury in the premature infant. Cerebral palsy and cognitive impairment are usually related to periventricular white matter damage. Despite recent improvements in neonatal care, no effective treatment for perinatal brain lesions is available. Myelin abnormalities and loss of pre-oligodendrocyte cell processes occurring in PVL without a loss of oligodendrocyte cell density suggested the necessity to analyze in the greater depth the potential factors critical for oligodendrocyte differentiation. Molecular mechanisms underlying oligodendrocyte myelination and remyelination are poorly understood. Transcriptional regulation of oligodendrocyte precursor formation from neural progenitor cells is relatively well characterized in the developing CNS, however, the molecular mechanisms governing oligodendrocyte myelinogenesis remain elusive. Recent studied indicate that Olig1/2 play a critical role in oligodendrocyte differentiation and myelination as well as remyelination. Myelin assembly by oligodendrocytes in the postnatal CNS of Olig1 null (Olig1∆KO) mice is severely compromised. Our preliminary search for Olig1 downstream genes identifies ZFP488 gene. Nuclear zinc-finger transcriptional regulator ZFP488 is an oligodendrocyte-specific transcription modulator that cooperates with the bHLH transcription factor Olig2 to promote oligodendrocyte differentiation. RNAi knockdown of ZFP488 leads to down-regulation of myelin gene expression in an oligodendroglial cell line. Thus, ZFP488 acts as a transcriptional co-regulator for oligodendrocyte maturation and myelination. In this study, we will investigate the oligodendrocyte differentiation and myelination in developing GFP-ZFP488 knockout mice. Secondly, we will explore the role of ZFP488 in oligodendrocyte differentiation and (re)myelination in two different mice models, neonatal hypoxic-ischemic brain injury model and adulthood spinal cord injury model and figure out whether the effect of ZFP488 on oligodendrocyte differentiation is related to Notch signaling pathway. Finally, we will isolate the OPC from neonatal rat and analyze the possible downstream target genes of ZFP488 and the signaling pathways by ChIP sequencing and RNA sequencing. The goal of this proposal is to define the biological function of ZFP488 in oligodendrocyte myelination through molecular, biochemical and genetic approaches. Understanding the molecular and genetic basis of oligodendrocyte myelination should provide potential gene therapy protocols to reverse the CNS damage in demyelinating diseases such as periventricular leukomalacia and multiple sclerosis.

发育期脑白质损伤是一系列与脑发育相关的损伤病变，由环境作用和基因遗传变异两方面引起，可遗留严重神经系统后遗症。新生儿缺氧缺血性脑病是最常见的发育脑白质损伤之一，其病因与少突胶质细胞发育及相关调控基因有关。少突胶质细胞发育受一系列细胞内外因子调控，目前其分化成熟的调控机制尚待完善。本课题组前期研究发现锌指蛋白ZFP488协同转录因子Olig2促进少突胶质细胞分化，其表达降低可引起髓鞘基因表达下调。本课题旨在探究ZFP488调控少突胶质细胞分化成熟和髓鞘形成的分子机制。我们拟通过建立ZFP488基因敲除小鼠，观察ZFP488在正常发育期和病理状态下对少突胶质细胞分化发育、髓鞘形成和再生的影响；分析其与Notch信号通路关系；并通过ChIP-seq和RNA-seq分析并验证其下游目的基因，从而阐明ZFP488基因在少突胶质细胞分化成熟、髓鞘形成以及损伤后髓鞘再生中的转录调控作用和相关分子机制。

脑白质损伤是常见的早产儿神经系统损伤之一，其主要病理表现为中枢神经系统（CNS）低髓鞘化/脱髓鞘，导致远期神经功能障碍甚至脑瘫的发生，目前尚无有效的临床治疗手段。哺乳动物中最大的转录因子家族锌指蛋白通过调控基因的表达在细胞分化过程中发挥重要作用。既往研究发现，经典锌指蛋白ZFP488是少突胶质细胞谱系基因Olig1的下游因子，可与Olig2结合促进鸡胚中的少突胶质细胞分化。在斑马鱼中，过表达小鼠ZFP488可以挽救Znf16l突变引起的髓鞘形成障碍。此外，神经干细胞中ZFP488过表达可促进CNS髓鞘损伤后再生。但ZFP488在哺乳动物髓鞘发育和损伤后修复过程中的作用仍未明确。本项目通过构建Zfp488基因敲除小鼠，观察发育过程中ZFP488缺失对少突胶质细胞分化和髓鞘形成的影响；通过构建LPC诱导的小鼠脑白质脱髓鞘模型，观察ZFP488缺失对髓鞘损伤后修复过程的影响；通过RNA-seq和CUT&tag测序分析，探讨ZFP488调控少突胶质细胞发育分化和髓鞘形成的分子机制。研究发现，ZFP488表达于CNS中的少突胶质细胞谱系，且主要表达于成熟少突胶质细胞中；ZFP488缺失导致发育过程中少突胶质细胞分化和髓鞘发育延迟，但对髓鞘的最终形成无显著影响。在LPC诱导的脑白质脱髓鞘损伤模型中，ZFP488促进损伤区域的髓鞘再生。RNA-seq和CUT&tag分析结果证实，ZFP488发挥转录因子作用，通过直接调控促髓鞘形成相关基因Tenm4和Opalin的表达，正向调控少突胶质细胞分化和髓鞘形成相关信号通路，从而影响CNS中髓鞘形成。本项目的顺利开展揭示了ZFP488在哺乳动物髓鞘形成和损伤后修复过程中的作用及相关机制，为中枢神经系统低/脱髓鞘疾病的治疗提供了新的针对性思路，为ZFP488基因突变导致的中枢神经系统髓鞘发育异常疾病的分子诊断和预后分析提供了实验依据。