Methylation alterations of Fbln1 gene in the hippocampus of PSEN1/PSEN2 double knockout and APP/PS1 transgenic mice

Abstract

BACKGROUND: Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by abnormal deposition of β-amyloid (Aβ) and neurofibrillary tangles of tau protein. Current medications only alleviate some symptoms, and despite extensive efforts to develop new therapies, such as anti-Aβ immunotherapy and β-secretase inhibitors, clinical trials have not been successful. OBJECTIVE: To investigate the methylation changes of the Fbln1 gene in the hippocampus of PSEN1/PSEN2 double knockout (dKO) mice, which lack Aβ deposition, and APP/PS1 double transgenic (DTG) mice, which exhibit Aβ deposition, to explore non-Aβ-related mechanisms and potential targets in AD. METHODS: Hippocampal tissues were collected from female dKO mice at 7 months (early AD) and 12 months (mid-stage AD) of age, with age-matched wild-type (WT) mice as controls. Epigenetic reduced representation bisulfite sequencing (RRBS) was used to screen for aberrantly methylated genes, identifying Fbln1. Bisulfite sequencing PCR (BSP) was performed to validate the methylation status of Fbln1 in mid-stage dKO mice. RT-PCR and western blot were used to measure Fbln1 mRNA and protein expression in early and mid-stage dKO mice, as well as in 12-month-old DTG mice. Finally, the expression levels of Fbln1 and Aβ were compared between dKO and DTG mice, with age-matched WT mice as controls. RESULTS AND CONCLUSION: RRBS showed that Fbln1 was hypomethylated in the hippocampus of mid-stage dKO mice (P < 0.05), while early-stage dKO mice showed a trend of hypomethylation but without statistical significance (P > 0.05). BSP confirmed the abnormal hypomethylation of Fbln1 in mid-stage dKO mice. In early-stage dKO mice, Fbln1 mRNA and protein levels were not significantly different from WT (P > 0.05). In mid-stage dKO mice, Fbln1 mRNA and protein levels were significantly higher than in WT (t=5.336, P < 0.01; t=8.985, P < 0.01). Similarly, mid-stage DTG mice showed significantly higher Fbln1 mRNA and protein levels than WT (t=4.151, P < 0.01; t=8.392, P < 0.01), but there was no significant difference between the two AD models (P > 0.05). In mid-stage dKO mice, there was no significant difference between Fbln1 and Aβ protein levels (P > 0.05), whereas in DTG mice, the difference was significant (t=6.348, P < 0.01), indicating that Fbln1 plays a role in both Aβ-dependent and Aβ-independent mechanisms. These findings suggest that Fbln1 methylation changes may contribute to age-dependent neurodegeneration in dKO mice and may be involved in both Aβ and non-Aβ pathways in AD, providing new insights and potential targets for non-Aβ-related mechanisms. Fbln1, as an aging-related factor, holds promise as a novel target.

1. Introduction

Alzheimer's disease (AD) is the most common age-dependent neurodegenerative disorder, characterized pathologically by abnormal deposition of β-amyloid (Aβ) and neurofibrillary tangles of tau protein. Currently available medications only manage some symptoms and do not effectively halt disease progression [1-2]. Despite extensive efforts to develop new therapies, including anti-Aβ immunotherapy and β-secretase inhibitors, clinical trials have not yielded success [3-4]. Therefore, exploring non-Aβ-related mechanisms and potential targets is of paramount importance for developing novel therapeutic strategies.

Due to the difficulty in obtaining human brain tissue with Aβ deposition, the establishment of AD animal models lacking Aβ deposition is crucial for studying non-Aβ mechanisms. The PSEN1/PSEN2 conditional double knockout (dKO) mouse, which lacks the presenilin genes associated with familial AD, exhibits age-dependent neurodegeneration and AD-like phenotypes without Aβ plaque formation. This model provides a unique opportunity to investigate Aβ-independent pathways in AD.