Abstract: BACKGROUND: The pathology of Alzheimer’s disease is primarily characterized by abnormal amyloid-β deposition and tau neurofibrillary tangles. Medications currently available can only control certain symptoms. Despite ongoing efforts to develop new treatments, such as targeted immunotherapy against amyloid-β and beta-secretase inhibitors, clinical trials have not yet shown success.
OBJECTIVE: To investigate the methylation changes of Fbln1 gene in the hippocampus of Alzheimer’s disease PSEN1/PSEN2 double gene knockout mice (dKO) without abnormal amyloid-β deposition and APP/PS1 double transgenic mice (DTG) with abnormal amyloid-β deposition, and to explore the mechanisms and potential targets related to Alzheimer’s disease without abnormal amyloid-β deposition.
METHODS: Nine female 7-month-old (early-stage Alzheimer’s disease) and 12-month-old (mid-stage Alzheimer’s disease) dKO mice were selected, along with age-matched wild-type mice as controls. The Fbln1 gene with abnormal methylation changes in the hippocampal tissue of these mice were identified using reduced representation bisulfite sequencing. The altered methylation status of the Fbln1 gene in the hippocampal tissue of mid-stage dKO mice was verified using bisulfite single-gene methylation sequencing. The mRNA and protein expression levels of the Fbln1 gene in early- and mid-stage dKO and 12-month-old DTG mice were detected using reverse transcription-polymerase chain reaction and Western blot methods, respectively. Finally, the expression levels of Fbln1 and amyloid-β in dKO and DTG mice were compared, with age-matched female wild-type mice serving as controls.
RESULTS AND CONCLUSION: (1) The significant hypomethylation of Fbln1 gene in the hippocampus of mid-stage Alzheimer’s disease dKO mice was detected by reduced representation bisulfite sequencing (P < 0.05). Although early-stage dKO mice also exhibited hypomethylation, there was no significant difference compared with wild-type mice (P > 0.05). The abnormal hypomethylation of the Fbln1 gene in the hippocampal tissue of mid-stage Alzheimer’s disease dKO mice was further validated by Bisulfite single-gene methylation sequencing. (2) The mRNA and protein expression levels of the Fbln1 gene in early-stage Alzheimer’s disease dKO mice were not significantly different from those in age-matched wild-type mice (P > 0.05). The mRNA levels and protein expression of Fbln1 in mid-stage Alzheimer’s disease dKO and DTG mice were significantly higher than those in age-matched wild-type mice (dKO: t = 5.336, 8.985, P < 0.01; DTG: t = 4.151, 8.392, P < 0.01), but there was no difference in the mRNA and protein expression levels of Fbln1 between the two mid-stage Alzheimer’s disease animal models (P > 0.05). (3) There was no difference in the protein expression levels of Fbln1 and amyloid-β between mid-stage Alzheimer’s disease dKO mice (P > 0.05), while there was a significant difference in the protein expression levels of Fbln1 and amyloid-β between mid-stage DTG mice (t = 6.348, P < 0.01), indicating that the Fbln1 gene plays a role not only in the DTG mouse model with amyloid-β deposition but also in dKO mice without amyloid-β deposition and there may be additional Fbln1 gene-related mechanisms involved in the development of Alzheimer’s disease. (4) Changes in Fbln1 gene methylation may be involved in age-dependent neurodegeneration in dKO mice and may simultaneously participate in both the amyloid-β abnormal deposition and non-amyloid-β deposition mechanisms in Alzheimer’s disease, providing new insights and evidence for exploring non-amyloid-β deposition-related mechanisms and new potential targets. Fbln1, an aging-related factor and new target, offers wide-ranging application prospects.

Key words: Fbln1 gene, methylation, Alzheimer’s disease, dKO mice, DTG mice, amyloid-β