Deng Zihao, Li Yudi, Chi Wenjun, Zhang Wanzhou, Li Fangming, Ling Li
Department of Neurology, Shenzhen Hospital, Southern Medical University, Shenzhen, 518000, China.
The First School of Clinical Medicine, Southern Medical University, Guangzhou, 510000, China.
Alzheimers Res Ther. 2025 May 15;17(1):104. doi: 10.1186/s13195-025-01734-6.
Alzheimer's disease (AD) is a chronic, progressive neurodegenerative disorder clinically characterized by memory decline, cognitive dysfunction, language impairment, deterioration of visuospatial skills, and personality changes. Pathologically, AD is marked by the deposition of β-amyloid (Aβ) plaques in the brain, the formation of neurofibrillary tangles, and progressive neuronal loss. Recent research has highlighted transfer RNA (tRNA)-derived small RNAs (tsRNAs) as crucial regulators in various biological processes; however, their roles in the pathophysiology of AD remain largely unexplored. The erythropoietin-producing hepatocellular (Eph) receptor family has recently drawn attention in the study of neurodegenerative diseases due to their role in regulating critical processes, including cell migration, neural development, angiogenesis, and tumor formation. This study aimed to investigate specific tsRNAs associated with AD by performing RNA sequencing on the cortex of APP/PS1 transgenic mice and to explore the relationship between tsRNAs and their target genes within the Eph receptor family, thereby elucidating insights into the specific regulatory functions of these molecules.
Eight-month-old male C57BL/6 and APP/PS1 transgenic mice were used in the study. BV-2 and HT22 cells were cultured and treated with Aβ at concentrations ranging from 0 µM to 40 µM. RNA was extracted from cortical tissues, and tRNA-derived fragments were analyzed after pre-treatment to remove RNA modifications. Differential expression of tRFs and tiRNAs was identified through sequencing, followed by bioinformatics analysis of target genes using TargetScan and miRanda. Transfection of BV-2 and HT22 cells with EphA7-siRNA and tRF-mimic was conducted, and their interaction was validated using dual-luciferase reporter assays. Protein expression levels were assessed by western blotting and immunofluorescence. Statistical analyses were performed using R and GraphPad Prism, with significance set at p < 0.05.
We identified for the first time that EphA7 expression is upregulated in aggregated microglia and neuronal cells in the dentate gyrus region of the hippocampus, with increased phosphorylation of ERK and p70S6K in AD. This upregulation occurred following the downregulation of tRF due to Aβ stimulation and was confirmed via in vitro experiments. By inhibiting EphA7 expression and increasing tRF expression, we suppressed the ERK-p70S6K signaling pathway in BV-2 and HT22 cells. This intervention alleviated neuronal damage and tau hyperphosphorylation in HT22 cells and reduced the M1-type polarization state of BV-2 cells induced by Aβ (see Graphical Abstract).
This study clarifies the specific role of tRF in AD pathology and offers a promising target for therapeutic interventions.
阿尔茨海默病（AD）是一种慢性进行性神经退行性疾病，其临床特征为记忆减退、认知功能障碍、语言障碍、视觉空间技能退化和人格改变。在病理上，AD的特征是大脑中β-淀粉样蛋白（Aβ）斑块沉积、神经原纤维缠结形成以及神经元进行性丢失。最近的研究强调了转运RNA（tRNA）衍生的小RNA（tsRNAs）在各种生物学过程中作为关键调节因子的作用；然而，它们在AD病理生理学中的作用在很大程度上仍未被探索。促红细胞生成素产生肝细胞（Eph）受体家族最近在神经退行性疾病研究中受到关注，因为它们在调节包括细胞迁移、神经发育、血管生成和肿瘤形成等关键过程中发挥作用。本研究旨在通过对APP/PS1转基因小鼠的皮质进行RNA测序，研究与AD相关的特定tsRNAs，并探索tsRNAs与其在Eph受体家族中的靶基因之间的关系，从而阐明这些分子的特定调节功能。
本研究使用8月龄雄性C57BL/6和APP/PS1转基因小鼠。培养BV-2和HT22细胞，并用浓度范围为0μM至40μM的Aβ进行处理。从皮质组织中提取RNA，并在预处理以去除RNA修饰后分析tRNA衍生片段。通过测序鉴定tRFs和tiRNAs的差异表达，随后使用TargetScan和miRanda对靶基因进行生物信息学分析。用EphA7-siRNA和tRF模拟物转染BV-2和HT22细胞，并使用双荧光素酶报告基因检测验证它们的相互作用。通过蛋白质免疫印迹和免疫荧光评估蛋白质表达水平。使用R和GraphPad Prism进行统计分析，显著性设定为p < 0.05。
我们首次发现EphA7在海马齿状回区域的聚集小胶质细胞和神经元细胞中表达上调，在AD中ERK和p70S6K的磷酸化增加。这种上调是在Aβ刺激导致tRF下调后发生的，并通过体外实验得到证实。通过抑制EphA7表达并增加tRF表达，我们在BV-2和HT22细胞中抑制了ERK-p70S6K信号通路。这种干预减轻了HT22细胞中的神经元损伤和tau过度磷酸化，并降低了Aβ诱导的BV-2细胞的M1型极化状态（见图1）。
本研究阐明了tRF在AD病理中的具体作用，并为治疗干预提供了一个有前景的靶点。
