【作者】 王旭；

【导师】 王苏；

【作者基本信息】 东南大学 ， 生物学， 2024， 硕士

【摘要】 亨廷顿病(Huntington’s Disease,HD)是由Huntingtin(HTT)基因上第一个外显子处CAG三核苷酸序列重复异常扩增引起的一种遗传性的神经退行性疾病。CAG重复序列可以编码出聚谷氨酰胺链,过长的聚谷氨酰胺链影响了亨廷顿蛋白的正常结构和功能,形成不溶性的突变型亨廷顿蛋白。突变型亨廷顿蛋白的积累会干扰细胞内正常的代谢过程,导致HD患者中纹状体和皮质神经元损伤和死亡,最终影响了患者的运动能力、认知功能和精神健康。但近期研究发现,突变型亨廷顿蛋白在胚胎发育时期就已经表达并影响了神经系统的正常发生,表明HD不仅是一种神经退行性疾病,也涉及神经发育的过程。自HD致病基因发现以来,关于其病理机制的研究已经取得了显著进展,但目前仍缺乏有效的治疗方法。早期对于HD研究依赖于动物模型和死后人脑样本,这些方法存在一定的局限性,如不同物种间差异性和临床样本的稀缺。随着诱导多能干细胞技术的发展,研究人员现在能够利用患者特定的诱导多能干细胞来建立脑类器官模型,这是一种包含多种脑细胞类型的三维体外模型,能够在体外环境模拟人脑的结构和功能。我们利用源自HD患者的诱导多能干细胞,成功构建了三种体外疾病模型:人类神经祖细胞(neural progenitor cells,NPC),巨噬细胞前体(primitive macrophage progenitors,PMP)以及脑类器官模型。通过采用转录组测序技术,我们发现了在HD患者的早期神经发育阶段,NPC和PMP中的PAR复合体相关基因下调,可能导致PAR复合体组装异常,这一异常导致了HD患者来源的NPC(HD NPC)与HD患者来源的PMP(HD PMP)的细胞极性出现缺陷。HD NPC的细胞极性异常影响了其分化及定位功能,进而影响了正常脑结构的形成。而HD PMP的细胞极性异常则降低了其迁移和吞噬功能,进一步加剧了细胞的炎症反应,导致了早期的神经炎症的发生。同时,我们为了更好的理解HD的发病过程,我们建立了含有小胶质细胞类器官,发现其可以在一定程度上重现HD的发育异常。并且我们通过对成熟阶段脑类器官的转录组测序分析发现,对照组PMP(Control PMP)与HD NPC共培养,可以促进HD NPC的增殖与分化,并减轻其炎症反应。我们这些发现证实了突变型亨廷顿蛋白的积累会影响HD PMP对神经发育的调控作用,最终导致神经发育异常。这些结果为理解HD早期神经发育异常的机制提供了新的理论基础,并为在胚胎期或新生期采用基因治疗HD提供了新的思路。更多还原

【Abstract】 Huntington’s Disease(HD)is a genetic neurodegenerative disorder caused by the abnormal expansion of CAG trinucleotide repeats in the first exon of the HTT gene.This expansion leads to the production of an elongated polyglutamine chain in the huntingtin protein,disrupting its normal structure and function and resulting in the formation of insoluble mutant huntingtin protein.The accumulation of this mutant protein interferes with normal cellular metabolic processes,leading to damage and death of neurons in the striatum and cortex,impacting patients’motor skills,cognitive functions,and mental health.Recent studies suggest that mutant huntingtin protein is expressed and impacts normal neural development during embryonic stages,indicating that HD involves neurodevelopmental processes in addition to being a neurodegenerative disease.Since the identification of the gene causing HD,substantial progress has been made in understanding its pathological mechanisms,but effective treatments are still lacking.Initially,HD research relied heavily on animal models and post-mortem human brain samples,which have limitations like interspecies differences and the scarcity of clinical samples.The development of iPSCs technology has enabled researchers to create patient-specific brain organoid models.These 3D in vitro models contain various brain cell types and simulate human brain structure and function outside the body.In our study,we utilized iPSCs derived from patients with HD to establish three in vitro disease models:human neural progenitor cells(NPCs),primitive macrophage progenitors(PMPs),and brain organoid models.By applying transcriptome sequencing technology,we found that PAR complex-related genes were down-regulated in NPC and PMP during the early neurodevelopmental stages of HD patients,possibly leading to abnormal PAR complex assembly,leading to defects in cellular polarity in HD-derived NPCs(HD NPCs)and HD-derived PMPs(HD PMPs).The polarity defects in HD NPCs affect their differentiation and localization abilities,impacting the development of normal brain structures.The cellular polarity abnormalities in HD PMPs result in reduced migration and phagocytosis capabilities,exacerbating cellular inflammatory responses and triggering early neuroinflammation.Meanwhile,in order to better understand the pathogenesis of HD,organoids containing microglia were established and found to reproduce the developmental abnormalities of HD to some extent.Moreover,through transcriptome sequencing analysis of mature brain organoids,we found that co-culture of Control PMP(Control PMP)with HD NPC could promote the proliferation and differentiation of HD NPC and reduce its inflammatory response.Our findings demonstrate that the accumulation of mutant huntingtin protein affects the regulatory role of HD PMPs in neurodevelopment,ultimately leading to neurodevelopmental abnormalities.These results provide a novel theoretical basis for understanding the mechanisms behind early neurodevelopmental abnormalities in HD and suggest new avenues for gene therapy in HD during fetal or neonatal stages.更多还原