LINE-1 CpG methylation in canine blood and cortical DNA tracks epigenetic changes with age

Aging is accompanied by progressive epigenetic alterations, including changes in DNA methylation affecting both gene-associated regions and repetitive elements such as LINE-1 retrotransposons. Reduced epigenetic repression of transposable elements has been implicated in age-related genomic instability. Circulating cell-free DNA (cfDNA) provides a minimally invasive substrate for monitoring systemic molecular changes; however, age-related LINE-1 methylation dynamics in cfDNA remain poorly characterized in non-human species. Dogs represent a valuable translational model for aging research due to their shared environment with humans and shorter lifespan. We investigated age-associated LINE-1 CpG methylation in plasma-derived cfDNA and in prefrontal cortex genomic DNA from dogs. Sequence analysis of 264 potentially active canine LINE-1 elements identified a highly conserved central region spanning ORF1 and ORF2, with the targeted CpG site located within a locally low-variability segment, supporting multi-copy assessment. Methylation was quantified using methylation-sensitive restriction endonuclease digestion followed by real-time quantitative PCR (MSRED-PCR), with bisulfite conversion-based PCR (BSC-PCR) used for technical validation. In clinically healthy dogs, LINE-1 methylation in blood cfDNA showed a strong negative association with chronological age, best described by a nonlinear (logarithmic) model. In prefrontal cortex samples, older dogs exhibited significantly lower LINE-1 methylation compared to younger individuals. Across tissues, aging was consistently associated with LINE-1 hypomethylation. These findings indicate that age-related LINE-1 hypomethylation is detectable in both brain tissue and circulating cfDNA. While based on cross-sectional cohorts, the results suggest that LINE-1 methylation in cfDNA may represent a candidate minimally invasive marker of chronological aging in dogs.