N6-Methyladenine Progressively Accumulates in Mitochondrial DNA during Aging.

N6-methyladenine (6mA) in the DNA is a conserved epigenetic mark with various cellular, physiological and developmental functions. Although the presence of 6mA was discovered a few years ago in the nuclear genome of distantly related animal taxa and just recently in mammalian mitochondrial DNA (mtDNA), accumulating evidence at present seriously questions the presence of N6-adenine methylation in these genetic systems, attributing it to methodological errors. In this paper, we present a reliable, PCR-based method to determine accurately the relative 6mA levels in the mtDNA of Caenorhabditis elegans, Drosophila melanogaster and dogs, and show that these levels gradually increase with age. Furthermore, daf-2(-)-mutant worms, which are defective for insulin/IGF-1 (insulin-like growth factor) signaling and live twice as long as the wild type, display a half rate at which 6mA progressively accumulates in the mtDNA as compared to normal values. Together, these results suggest a fundamental role for mtDNA N6-adenine methylation in aging and reveal an efficient diagnostic technique to determine age using DNA.

Poly(A) RNA sequencing reveals age-related differences in the prefrontal cortex of dogs.

Dogs may possess a unique translational potential to investigate neural aging and dementia because they are prone to age-related cognitive decline, including an Alzheimer's disease-like pathological condition. Yet very little is known about the molecular mechanisms underlying canine cognitive decline. The goal of the current study was to explore the transcriptomic differences between young and old dogs' frontal cortex, which is a brain region often affected by various forms of age-related dementia in humans. RNA isolates from the frontal cortical brain area of 13 pet dogs, which represented 7 different breeds and crossbreds, were analyzed. The dogs were euthanized for medical reasons, and their bodies had been donated by their owners for scientific purposes. The poly(A) tail RNA subfraction of the total transcriptome was targeted in the sequencing analysis. Cluster analyses, differential gene expression analyses, and gene ontology analyses were carried out to assess which genes and genetic regulatory mechanisms were mostly affected by aging. Age was the most prominent factor in the clustering of the animals, indicating the presence of distinct gene expression patterns related to aging in a genetically variable population. A total of 3436 genes were found to be differentially expressed between the age groups, many of which were linked to neural function, immune system, and protein synthesis. These findings are in accordance with previous human brain aging RNA sequencing studies. Some genes were found to behave more similarly to humans than to rodents, further supporting the applicability of dogs in translational aging research.

CDKN2A Gene Expression as a Potential Aging Biomarker in Dogs.

Describing evolutionary conserved physiological or molecular patterns, which can reliably mark the age of both model organisms and humans or predict the onset of age-related pathologies has become a priority in aging research. The age-related gene-expression changes of the Cyclin Dependent Kinase Inhibitor 2A (CDKN2A) gene have been well-documented in humans and rodents. However, data is lacking from other relevant species, including dogs. Therefore, we quantified the CDKN2A mRNA abundance in dogs of different ages, in four tissue types: the frontal cortex of the brain, temporal muscle, skin, and blood. We found a significant, positive correlation between CDKN2A relative expression values and age in the brain, muscle, and blood; however, no correlation was detected in the skin. The strongest correlation was detected in the brain tissue (CDKN2A/GAPDH: r = 0.757, p < 0.001), similarly to human findings, while the muscle and blood showed weaker, but significant correlation. Our results suggest that CDKN2A might be a potential blood-borne biomarker of aging in dogs, although the validation and optimization will require further, more focused research. Our current results also clearly demonstrate that the role of CDKN2A in aging is conserved in dogs, regarding both tissue specificity and a pivotal role of CDKN2A in brain aging.

A Preliminary Study to Investigate the Genetic Background of Longevity Based on Whole-Genome Sequence Data of Two Methuselah Dogs.

Aging is the largest risk factor in many diseases and mortality alike. As the elderly population is expected to increase at an accelerating rate in the future, these phenomena will pose a growing socio-economic burden on societies. To successfully cope with this challenge, a deeper understanding of aging is crucial. In many aspects, the companion dog is an increasingly popular model organism to study aging, with the promise of producing results that are more applicable to humans than the findings that come from the studies of classical model organisms. In this preliminary study we used the whole-genome sequence of two extremely old dogs - age: 22 and 27 years (or 90-135% more, than the average lifespan of dogs) - in order to make the first steps to understand the genetic background of extreme longevity in dogs. We identified more than ∼80 1000 novel SNPs in the two dogs (7500 of which overlapped between them) when compared to three publicly available canine SNP databases, which included SNP information from850 dogs. Most novel mutations (∼52000 SNPs) were identified at non-coding regions, while 4.6% of the remaining SNPs (n∼1600) were at exons, including 670 missense variants - 76 of which overlapped between the two animals - across 472 genes. Based on their gene ontologies, these genes were related - among others - to gene transcription/translation and its regulation, to immune response and the nervous system in general. We also detected 12 loss-of-function mutations, although their actual effect is unclear. Several genetic pathways were also identified, which pathways may be tempting candidates to be investigated in large sample sizes in order to confirm their relevance in extreme longevity in dogs (and possibly, in humans). We hypothesize a possible link between extreme longevity and the regulation of gene transcription/translation, which hypothesis should be further investigated in the future. This phenomenon could define an interesting direction for future research aiming to better understand longevity. The presented preliminary results highlight the utility of the companion dog in the study of the genetic background of longevity and aging.

Comparison of Behavior and Genetic Structure in Populations of Family and Kenneled Beagles.

In dogs, the social and spatial restriction associated with living in a kennel environment could lead to chronic stress and the development of abnormal behaviors ("kennel-dog syndrome"). However, little is known about how kenneled dogs differ from their conspecifics living as pets in human families. In the current study, using a test battery exposing the dogs to novel stimuli, we compared the behavior of three groups of beagles: (1) kenneled dogs living in a restricted environment with limited human contact (N = 78), (2) family dogs living in human families as pets (N = 37), and (3) adopted dogs born in the kenneled population but raised in human families (N = 13). We found one factor comprising most of the test behaviors, labeled as Responsiveness. Family dogs and adopted dogs scored higher in Responsiveness than kenneled dogs. However, 23% of the kenneled dogs were comparable to family and adopted dogs based on a cluster analysis, indicating a similar (positive) reaction to novel stimuli, while 77% of the kenneled dogs were unresponsive (mostly immobile) in at least part of the test. To assess if the behavioral difference between the family and kenneled dogs could be due to genetic divergence of these two populations and/or to lower genetic diversity of the kenneled dogs, we analyzed their genetic structure using 11 microsatellite markers. We found no significant difference between the populations in their genetic diversity (i.e., heterozygosity, level of inbreeding), nor any evidence that the family and kenneled populations originated from different genetic pools. Thus, the behavior difference between the groups more likely reflects a G × E interaction, that is, the influence of specific genetic variants manifesting under specific environmental conditions (kennel life). Nevertheless, some kenneled individuals were (genetically) more resistant to social and environmental deprivation. Selecting for such animals could strongly improve the welfare of kenneled dog populations. Moreover, exploring the genetic background of their higher resilience could also help to better understand the genetics behind stress- and fear-related behaviors.