tRFAla-AGC-3-M8 attenuates neuroinflammation and neuronal damage in Alzheimer's disease via the EphA7-ERK1/2-p70S6K signaling pathway

Background: 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.

Methods: 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_25-35 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^Ala-AGC-3-M8-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.

Results: 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_1/2 and p70S6K in AD. This upregulation occurred following the downregulation of tRF^Ala-AGC-3-M8 due to Aβ stimulation and was confirmed via in vitro experiments. By inhibiting EphA7 expression and increasing tRF^Ala-AGC-3-M8 expression, we suppressed the ERK_1/2-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_25-35 (see Graphical Abstract).

Conclusions: This study clarifies the specific role of tRF^Ala-AGC-3-M8 in AD pathology and offers a promising target for therapeutic interventions.

Alzheimer’s disease; ERK1/2; EphA7; Microglia; Neuroinflammation; p70S6K; tRFAla-AGC-3-M8; tsRNA.