Alzheimer's disease (AD) is a major health concern with limited effective treatments. The incomplete understanding of AD pathogenesis hinders the development of clinical solutions. Recent research highlights the long-term impact of neurogenic brain regions on age-related neurodegeneration. Three major neurogenic regions – the hippocampal dentate gyrus, the subventricular zone, and the mediobasal hypothalamus – are involved in adult neurogenesis related to limbic system functions. The hypothalamus has a regulatory role in systemic aging and physiology, influenced by neuropeptides like GnRH and OXT. However, comparative studies of the hypothalamus with other limbic system components in aging are lacking. This study uses DNA methylation epigenetic analysis to compare the hypothalamus, hippocampus, and olfactory bulb (OB) in aging mice, aiming to identify age-related DNA methylation patterns and explore the potential for developing hypothalamic peptide-based therapies for AD.

Existing literature establishes a strong link between aging and DNA methylation. Studies have profiled genome-wide methylation changes associated with aging rates across various human tissues and cell types. Research also shows the importance of DNA methylation in the epigenetics of aging, impacting cellular senescence and influencing biological age. Previous work from the authors' lab has implicated the hypothalamus in the regulation of systemic aging and physiology through the actions of GnRH and OXT. This study builds upon this foundation by directly comparing the methylation patterns of the hypothalamus with other key limbic system structures to identify unique signatures associated with aging and to explore therapeutic targets.

The study used male C57BL/6 mice at 2 and 12 months of age (representing young and middle age) and 5xFAD mice (an AD model) to investigate age-related DNA methylation. Hypothalamus, hippocampus, and OB were isolated, and DNA was extracted. Bisulfite oligonucleotide-captured sequencing (BOCS) was employed to profile CG-rich genomic regions. This approach covers promoters, CG islands, shores, and shelves. Average methylation levels of CG and CH sites were calculated for each tissue. Differentially methylated cytosines (DMCs) and regions (DMRs) were identified. Pathway analysis was performed to identify affected molecular pathways. Deconvolution analysis was used to account for cellular heterogeneity. For the therapeutic study, 9-month-old 5xFAD mice received 2 months of nasal administration of OXT and GnRH individually or in combination. Neurobehavioral assays (open field, grip strength, Y-maze, novel object recognition, social interaction, Morris water maze) and amyloid-beta (Aβ) immunostaining were performed to assess the effects of the treatment. Quantitative RT-PCR was used to measure mRNA levels of OXT, GnRH1, and Adcy family members. NGS BSP was used to assess the effect of the treatment on Adcy promoter methylation.

The hypothalamus showed distinctly higher DNA methylation levels in young mice compared to the hippocampus and OB. This difference decreased with age. DMCs and DMRs analysis revealed that age-related DNA methylation changes are common across the three brain regions, particularly affecting genes involved in hypothalamic regulatory pathways, including OXT and GnRH pathways. In middle-aged mice, transcription of OXT and GnRH genes decreased in the hypothalamus. Similarly, transcription of these genes was also downregulated in the hypothalamus of 5xFAD mice compared to wild-type controls. OXT-GnRH combinational therapy significantly improved various neurological functions and dramatically reduced Aβ plaque deposition in the 5xFAD AD mouse model compared to individual peptide treatments. While neither treatment significantly changed average Adcy promoter methylation, DMC analysis identified several short sequences with altered methylation levels following OXT-GnRH treatment.

The findings support the hypothesis that the hypothalamus plays a crucial role in age-related DNA methylation and that age-related changes in hypothalamic DNA methylation affect multiple brain regions. The significant effects of OXT-GnRH combinational therapy suggest that targeting these neuropeptide pathways may be a promising therapeutic strategy for AD. The superior efficacy of combinational therapy compared to individual peptide treatments may be attributed to synergistic effects and reduced off-target effects at lower doses. This study provides a strong rationale for further investigation of OXT-GnRH combinational therapy for AD in clinical settings. The observed changes in Adcy gene expression in the 5xFAD model and the subtle methylation alterations in Adcy promoter regions following OXT-GnRH treatment warrant further exploration of the molecular mechanisms underlying the therapeutic effects.

This study demonstrates that the hypothalamus exhibits unique age-related DNA methylation patterns, particularly affecting OXT and GnRH pathways. OXT-GnRH combinational therapy is highly effective against AD-like pathologies in a male mouse model, suggesting a promising therapeutic strategy. Future research should investigate the mechanism of action, optimize treatment parameters, evaluate female models, and explore the applicability to other aging-related diseases.

The study used only male mice, limiting the generalizability of the results. The sample size for some experiments was relatively small, and further studies with larger sample sizes are warranted. The study focused on an AD-like model, and future research should examine the applicability to other neurodegenerative diseases. While the study established proof of principle for OXT-GnRH combinational therapy, further optimization of dosage, duration, and assessment of off-target effects are necessary before clinical translation.