核定位人α-突触核蛋白转基因小鼠行为变化的建立与表征。

IF 3 Q2 MEDICINE, RESEARCH & EXPERIMENTAL
Ziou Wang, Mengchen Wei, Shengtao Fan, Zheli Li, Weihu Long, Haiting Wu, Yiwei Zhang, Zhangqiong Huang
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引用次数: 0

摘要

目的:建立表达人核定位α-突触核蛋白(α-syn)的转基因小鼠模型,探讨其对中枢神经系统和行为的影响及其机制。方法:在人SNCA (hSNCA)基因末端添加核定位序列(NLS)。随后,构建了hSNCA-NLS的空载体和哺乳动物慢病毒载体。通过显微注射制备转基因小鼠,通过PCR和western blotting鉴定基因分型和蛋白表达。随后的行为和分子实验只使用雄性小鼠。免疫荧光法在小鼠脑组织中鉴定了人α-syn与细胞核的共定位。采用开放场、旋转棒和o型迷宫试验评估转基因小鼠的行为变化。qPCR和Western blotting检测炎症、内质网应激(endoplasmic reticulum stress, ERS)和细胞凋亡相关基因和蛋白的表达水平。大量RNA测序用于筛选差异表达基因和信号通路。结果:成功构建了表达人α-syn的转基因小鼠模型。人α-syn在小鼠的心、肝、脾、肾、脑中广泛表达,具有明显的核定位。行为评估表明,到2个月大时,小鼠表现出运动功能障碍、星形胶质细胞增殖和神经炎症。6个月时,ERS相关基因(ATF6、PERK和IRE1)的表达升高以及PERK- beclin1 - lc3ii通路的激活表明ERS进展。9个月时,凋亡事件发生,并伴有明显的焦虑样行为。大量RNA测序进一步鉴定出关键的差异表达基因,包括IL-1α、TNF、PERK、BECLIN、GABA、IL-6α、P53、LC3II、NOS和SPAG,表明它们参与了所观察到的病理和行为表型。结论:成功建立了核定位的人α-syn转基因小鼠。这些发现表明,核定位α-syn诱导早期运动缺陷,这可能是由神经炎症介导的,而后期的焦虑样行为可能是由ers诱导的细胞凋亡引起的。该模型为阐明核α-syn在帕金森病中的作用提供了有价值的工具,并支持进一步的机制和治疗研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Establishment and Characterization of Behavioral Changes in the Nuclear Localization Human α-Synuclein Transgenic Mice.

Objectives: This study aimed to establish a transgenic mouse model expressing nucleus-localized human α-synuclein (α-syn) to investigate its impact on the central nervous system and behavior and the underlying mechanisms involved. Methods: A nuclear localization sequence (NLS) was added to the end of the human SNCA (hSNCA) gene. Subsequently, an empty vector and a mammalian lentiviral vector of the hSNCA-NLS were constructed. Transgenic mice were generated via microinjection, with genotyping and protein expression confirmed by PCR and western blotting. Only male mice were used in subsequent behavioral and molecular experiments. Immunofluorescence identified the colocalization of human α-syn with the cell nucleus in mouse brain tissues. Behavioral changes in transgenic mice were assessed using open field, rotarod, and O-maze tests. qPCR and Western blotting detected expression levels of genes and proteins related to inflammation, endoplasmic reticulum stress (ERS), and apoptosis. Bulk RNA sequencing was used to screen for differentially expressed genes and signaling pathways. Results: We successfully constructed a transgenic mouse model expressing human α-syn. Human α-syn was widely expressed in the heart, liver, spleen, kidneys, and brain of the mice, with distinct nuclear localization observed. Behavioral assessments demonstrated that, by 2 months of age, the mice exhibited motor dysfunction alongside astrocyte proliferation and neuroinflammation. At 6 months, the elevated expression of ERS-related genes (ATF6, PERK, and IRE1) and activation of the PERK-Beclin1-LC3II pathway indicated progressive ERS. By 9 months, apoptotic events had occurred, accompanied by significant anxiety-like behaviors. Bulk RNA sequencing further identified key differentially expressed genes, including IL-1α, TNF, PERK, BECLIN, GABA, IL-6α, P53, LC3II, NOS, and SPAG, suggesting their involvement in the observed pathological and behavioral phenotypes. Conclusions: The nuclear localization human α-syn transgenic mice were successfully established. These findings demonstrate that nucleus-localized α-syn induces early motor deficits, which are likely mediated by neuroinflammation, whereas later anxiety-like behaviors may result from ERS-induced apoptosis. This model provides a valuable tool for elucidating the role of nuclear α-syn in Parkinson's disease and supports further mechanistic and therapeutic research.

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