Targeting the hypothalamus for modeling age-related DNA methylation and developing OXT-GnRH combinational therapy against Alzheimer's disease-like pathologies in male mouse model

Abstract

The hypothalamus plays an important role in aging, but it remains unclear regarding the underlying epigenetics and whether this hypothalamic basis can help address aging-related diseases. Here, by comparing mouse hypothalamus with two other limbic system components, we show that the hypothalamus is characterized by distinctively high-level DNA methylation during young age and by the distinct dynamics of DNA methylation and demethylation when approaching middle age. On the other hand, age-related DNA methylation in these limbic system components commonly and sensitively applies to genes in hypothalamic regulatory pathways, notably oxytocin (OXT) and gonadotropin-releasing hormone (GnRH) pathways. Middle age is associated with transcriptional declines of genes which encode OXT, GnRH and signaling components, which similarly occur in an Alzheimer's disease (AD)-like model. Therapeutically, OXT-GnRH combination is substantially more effective than individual peptides in treating AD-like disorders in male 5×FAD model. In conclusion, the hypothalamus is important for modeling age-related DNA methylation and developing hypothalamic strategies to combat AD.

Fig. 1. Brain regional analysis on DNA methylation and the changes by middle age.

DNA samples were generated from the hypothalamus (HT), hippocampus (HC), and OB of 2-month-old versus 12-month-old male C57BL/6 and were profiled for cytosine methylation of CG-enriched genomic regions via BOCS and analyzed for average levels of CG and CH sites methylation. A, B Average percentage levels of CG site methylation (A) and CH site methylation (B). C–F Average percentage levels of CG site methylation (C, E) and CH site methylation (D, F) in individual chromosomes. The Y chromosome was not included due to its short size. Data are presented as mean values ± SEM. Statistics: Exact p-values are labeled as text for comparisons between two groups (A, B) and across chromosomes (C–F). Significance is indicated by *p < 0.05 and **p < 0.01, with comparisons conducted at the individual chromosome level among three groups (C–F). n = 3 mice per group (A–F), two-way ANOVA with tissue and age as factors, and post-hoc Tukey test (A, B), and Kruskal-Wallis and post-hoc Dunn test (C–F). Source data are provided as a Source Data file.

Fig. 2. Brain regional DNA methylation difference per DMCs and changes by middle age.

Differentially methylated cytosines (DMCs) were analyzed pairwise among the hypothalamus (HT), hippocampus (HC), and olfactory bulb (OB) of 2- and 12-month-old mice, based on q < 0.05 and methylation changes of at least 25%. A–F DMCs between HT and HC (A, B), HT and OB (C, D), and HC and OB (E, F) using Volcano plots (A, C, E) and Venn diagrams (B, D, F). In the Volcano plots, hypermethylation and hypomethylation are shown as green and red dots, respectively. Venn diagrams show the numbers of DMCs, with green arrows pointing upward to indicate hypermethylation and red arrows pointing downward to indicate hypomethylation. The bar graphs display the percentage of tissue-related DMCs that were lost, retained, or gained by middle age, calculated over the total DMCs observed in young mice (B, D, F). All analyses were performed on n = 3 mice per group, statistically via logistic regression using the MethylKit package. Statistical tests were two-sided, with p-values adjusted to q-values using the SLIM method to control for multiple comparisons, with all data (B, D, F) meeting q < 0.05. DMCs are listed as Supplementary Dataset 1. Source data are provided as a Source Data file.

Fig. 3. Age-related DMRs in brain regions and molecular pathway analysis.

DMRs in the hypothalamus (HT), hippocampus (HC), and OB between 2 and 12 months of age were calculated per DNA length of 500 bps, with at least 10% of methylation difference with q < 0.05 (false discovery rate, FDR). DMRs were based on CG sites in the sequences. A, B The total number of DMRs in each brain tissue due to hypermethylation and hypomethylation (A) and percentage in individual chromosomes (B). C Venn Diagram of the numbers of the underlying genes per age-related DMRs that are either common or unique among HT, HC, and OB. D A total of 173 common genes per age-related DMRs among HT, HC, and OB were processed with KEGG pathway analysis and grouped according to functions. The left Y axis and bar graphs indicate the numbers of genes, and the right Y axis and red dots represent −log₁₀(p-values). All analyses were based on n = 3 mice per group, statistically via logistic regression using MethylKit package (two-sided test) with p-values adjusted to q-values using the SLIM method to control for multiple comparisons, with all data meeting FDR values of less than 0.05 (A–C) and Fisher’s Exact test (two-sided) (D). DMRs and genes for Fig. 3A, C are listed in Supplementary Dataset 2. Source data are provided as a Source Data file.

Fig. 4. In-depth pathway analysis according to age-related DMRs of brain regions.

A–C Pathway analysis per age-related DMRs (500 bps per length, CG-based DMR analysis) in the hypothalamus (HT) (A), hippocampus (HC) (B), and OB (C), the list was based on p-values in logarithm in the x-axis. The numbers of involved genes in each pathway were indicated with color gradients. The size of each circle indicated the combined scores, computed by taking the log of the p-value from the Fisher’s exact test and multiplying it by the z-score of deviation from the expected rank. Due to space limit, representative pathways are listed along y-axis, while all pathways and genes for Fig. 4A–C are provided as Supplementary Dataset 3. D Components in OXT signaling pathway and GnRH signaling pathway according to age-related DMRs (500 bps per length, CG-based DMR analysis) in the HT, HC, and OB. The size of each circle indicated the levels of hypermethylation or hypomethylation. All analyses were based on n = 3 mice per group, and statistics were based on Fisher’s Exact test (two-sided) (A–C) and logistic regression using MethylKit package (two-sided test) with p-values adjusted to q-values using the SLIM method to control for multiple comparisons (D). Source data are provided as a Source Data file.

Fig. 5. Gnrh1 and Oxt gene methylation and transcription in middle age or 5xFAD model.

A, B Differentially methylated cytosines (DMCs) were analyzed in BOCS metadata covering Oxt gene (A) and Gnrh1 gene (B) for the hypothalamic samples of mice between 2 months and 12 months of age. The graphs present the DMCs with at least 1% methylation difference, with genomic positions of cytosines labeled on the Y axis. C, D The mRNA levels of Oxt and Gnrh1 measured in hypothalamic tissues from male C57BL/6 mice under middle age (15 months) vs. young age (2 months) (C) and from male 5xFAD mice vs. male littermate wildtype (WT) controls of 8 months of age (D). Statistics: For panels A and B, analyses were performed using logistic regression with the MethylKit package, with q-values < 0.05 indicating significance (two-sided test), and n = 3 mice per group. Data in panels C and D are presented as mean values ± SEM. au arbitrary unit. Statistical significance was assessed using two-tailed unpaired t-test, with p-values reported, and n = 5 mice per group. Source data are provided as a Source Data file.

Fig. 6. Adcy family gene methylation and transcriptional in middle age or 5XFAD model.

A Diagrams depict the promoter regions and subregions of Adcy genes in the hippocampus, showing age-related methylation differences. Comparisons are based on average methylation levels in each promoter region or subregion between middle-aged (12 months) and young (2 months) mice. The promoters are categorized into CG island, distal subregion, and proximal subregion. Red arrows indicate decreased methylation, while green arrows indicate increased methylation in 12-month-old mice compared to 2-month-old mice. B, C The mRNA levels of Adcy genes in hippocampal tissues were measured from male C57BL/6 mice at middle age (15 months) versus young age (2 months) (B), and from male 5xFAD mice versus male littermate wildtype (WT) controls at 8 months of age (C). Data are presented as mean ± SEM. au arbitrary unit. Statistical significance was assessed using a two-tailed unpaired t-test. p-values are reported in the graphs, with n = 3 mice per group for panel A and n = 5 mice per group for panels B, C. Source data are provided as a Source Data file.

Fig. 7. The effects of OXT-GnRH treatment on physiological disorders in 5xFAD model.

Male 5×FAD mice (9 months old) received 2-month OXT (50 ng) and GnRH (5 ng) treatment individually or in combination vs. vehicle control through daily nasal administration and were subsequently examined for a battery of neurobehavioral assays. Results demonstrated body weight, locomotion in open field test, muscle strength in grip test, cognitive function in Y-maze test, novel object recognition in novel object test, social function in sociality test (A), and spatial learning/memory function in Morris Water Maze (MWM) test (B, C) which consisted of 5-day training session followed by probe test while swimming speed in the probe trial was provided as a technical control. Data are presented as mean ± SEM. The p-values are reported based on one-way ANOVA and Tukey post-hoc test between indicated groups (A, C) and two-way ANOVA and Tukey post-hoc test between vehicle and OXT-GnRH combinational treatment at matched time (B), n = 10 mice per group. Source data are provided as a Source Data file.

Fig. 8. The effects of OXT-GnRH treatment on Aβ plaques in 5xFAD model.

Aged male 5xFAD mice received 2-month OXT and GnRH treatment individually or in combination vs. vehicle control, as detailed in Fig. 7. At the end of behavioral tests, subgroups of mice were processed for brain amyloid β (Aβ) immunostaining. A, B Aβ immunostaining in hippocampal subregions CA1, CA3 and dentate gyrus (DG) (A), and Aβ immunostaining in hypothalamic region MBH and entorhinal cortex (B). Images represent 5 mice per group. Red, Aβ; blue, DAPI nuclear staining. Bar, 100 µm. C–G Quantification of Aβ plaques. Data are presented as mean ± SEM. Statistical significance was assessed between the indicated groups, p-values are reported based on one-way ANOVA and Tukey post-hoc test, n = 5 mice per group. Source data are provided as a Source Data file.