背景:尽管WD重复结构域45(WDR45)突变与β-螺旋桨蛋白相关的神经变性(BPAN)有关,这种疾病背后的确切分子和细胞机制仍然难以捉摸。本研究旨在阐明WDR45缺乏对神经变性的影响,特别是轴突变性,在中脑多巴胺能(DAergic)系统内。我们希望通过检查病理和分子改变来更好地了解疾病过程,尤其是在DAergic系统中。
方法:为了研究WDR45功能障碍对小鼠行为和DA能神经元的影响,我们开发了一种小鼠模型,其中WDR45在中脑DAer能神经元(WDR45cKO)中被有条件地敲除。通过纵向研究,我们使用开放视野评估小鼠行为的改变,旋转杆,Y-迷宫,和三室社会方法测试。我们利用免疫荧光染色和透射电子显微镜的组合来检查DAer能神经元体细胞和轴突的病理变化。此外,我们对年轻和老年小鼠的纹状体进行了蛋白质组学和脂质组学分析,以确定衰老过程中可能涉及纹状体病理学的分子和过程.更多,采用原发性中脑神经元培养来探索导致轴突变性的分子机制。
结果:我们对WDR45cKO小鼠的研究揭示了一系列缺陷,包括运动功能受损,情绪不稳定,和记忆丧失,与中脑发能神经元的大幅减少相吻合。神经元的损失,我们在背侧和腹侧纹状体观察到大量轴突扩大。这些扩大的特征是广泛破碎的管状内质网(ER)的积累,轴突变性的标志.纹状体的蛋白质组学分析表明,差异表达的蛋白质在代谢过程中富集。碳水化合物代谢和蛋白质分解代谢过程出现较早,和氨基酸,脂质,和三羧酸代谢在衰老过程中增加。值得注意的是,我们观察到溶血磷脂酰胆碱酰基转移酶1(Lpcat1)的表达急剧增加,调节磷脂代谢,特别是在酰基辅酶A存在下溶血磷脂酰胆碱(LPC)向磷脂酰胆碱(PC)的转化中。脂质组学结果一致表明,差异脂质集中在PC和LPC上。通过干扰原代培养的WDR45缺陷的DA能神经元中的Lpcat1表达,可以有效地改善轴突变性,证明Lpcat1及其调节脂质代谢,尤其是PC和LPC代谢,参与控制由WDR45缺陷引起的轴突变性。
结论:在这项研究中,我们发现了WDR45缺乏导致轴突变性的分子机制,这涉及到磷脂代谢之间的复杂关系,自噬,和管状ER。这些发现极大地促进了我们对驱动轴突变性的基本分子机制的理解,并可能为开发针对BPAN和其他神经退行性疾病的新型机械治疗干预提供基础。
BACKGROUND: Although WD repeat domain 45 (WDR45) mutations have been linked to β -propeller protein-associated neurodegeneration (BPAN), the precise molecular and cellular mechanisms behind this disease remain elusive. This study aims to shed light on the impacts of WDR45-deficiency on neurodegeneration, specifically axonal degeneration, within the midbrain dopaminergic (DAergic) system. We hope to better understand the disease process by examining pathological and molecular alterations, especially within the DAergic system.
METHODS: To investigate the impacts of WDR45 dysfunction on mouse behaviors and DAergic neurons, we developed a mouse model in which WDR45 was conditionally knocked out in the midbrain DAergic neurons (WDR45cKO). Through a longitudinal study, we assessed alterations in the mouse behaviors using open field, rotarod, Y-maze, and 3-chamber social approach tests. We utilized a combination of immunofluorescence staining and transmission electron microscopy to examine the pathological changes in DAergic neuron soma and axons. Additionally, we performed proteomic and lipidomic analyses of the striatum from young and aged mice to identify the molecules and processes potentially involved in the striatal pathology during aging. Further more, primary midbrain neuronal culture was employed to explore the molecular mechanisms leading to axonal degeneration.
RESULTS: Our study of WDR45cKO mice revealed a range of deficits, including impaired motor function, emotional instability, and memory loss, coinciding with the profound reduction of midbrain DAergic neurons. The neuronal loss, we observed massive axonal enlargements in the dorsal and ventral striatum. These enlargements were characterized by the accumulation of extensively fragmented tubular endoplasmic reticulum (ER), a hallmark of axonal degeneration. Proteomic analysis of the striatum showed that the differentially expressed proteins were enriched in metabolic processes. The carbohydrate metabolic and protein catabolic processes appeared earlier, and amino acid, lipid, and tricarboxylic acid metabolisms were increased during aging. Of note, we observed a tremendous increase in the expression of lysophosphatidylcholine acyltransferase 1 (
Lpcat1) that regulates phospholipid metabolism, specifically in the conversion of lysophosphatidylcholine (LPC) to phosphatidylcholine (PC) in the presence of acyl-CoA. The lipidomic results consistently suggested that differential lipids were concentrated on PC and LPC. Axonal degeneration was effectively ameliorated by interfering
Lpcat1 expression in primary cultured WDR45-deficient DAergic neurons, proving that
Lpcat1 and its regulated lipid metabolism, especially PC and LPC metabolism, participate in controlling the axonal degeneration induced by WDR45 deficits.
CONCLUSIONS: In this study, we uncovered the molecular mechanisms underlying the contribution of WDR45 deficiency to axonal degeneration, which involves complex relationships between phospholipid metabolism, autophagy, and tubular ER. These findings greatly advance our understanding of the fundamental molecular mechanisms driving axonal degeneration and may provide a foundation for developing novel mechanistically based therapeutic interventions for BPAN and other neurodegenerative diseases.