A conserved MTMR lipid phosphatase increasingly suppresses autophagy in brain neurons during aging.

Ageing is driven by the progressive, lifelong accumulation of cellular damage. Autophagy (cellular self-eating) functions as a major cell clearance mechanism to degrade such damages, and its capacity declines with age. Despite its physiological and medical significance, it remains largely unknown why autophagy becomes incapable of effectively eliminating harmful cellular materials in many cells at advanced ages. Here we show that age-associated defects in autophagic degradation occur at both the early and late stages of the process. Furthermore, in the fruit fly Drosophila melanogaster, the myotubularin-related (MTMR) lipid phosphatase egg-derived tyrosine phosphatase (EDTP) known as an autophagy repressor gradually accumulates in brain neurons during the adult lifespan. The age-related increase in EDTP activity is associated with a growing DNA N6-adenine methylation at EDTP locus. MTMR14, the human counterpart of EDTP, also tends to accumulate with age in brain neurons. Thus, EDTP, and presumably MTMR14, promotes brain ageing by increasingly suppressing autophagy throughout adulthood. We propose that EDTP and MTMR14 phosphatases operate as endogenous pro-ageing factors setting the rate at which neurons age largely independently of environmental factors, and that autophagy is influenced by DNA N6-methyladenine levels in insects.

Functional indications for transposase domestications - Characterization of the human piggyBac transposase derived (PGBD) activities.

Transposable elements are widespread in all living organisms. In addition to self-reproduction, they are a major source of genetic variation that drives genome evolution but our knowledge of the functions of human genes derived from transposases is limited. There are examples of transposon-derived, domesticated human genes that lost (SETMAR) or retained (THAP9) their transposase activity, however, several remnants in the human genome have not been thoroughly investigated yet. These include the five human piggyBac-derived sequences (PGBD1-5) which share ancestry with the Trichoplusia ni originated piggyBac (PB) transposase. Since PB is widely used in gene delivery applications, the potential activities of endogenous PGBDs are important to address. However, previous data is controversial, especially with the claimed transposition activity of PGBD5, it awaits further investigations. Here, we aimed to systematically analyze all five human PGBD proteins from several aspects, including phylogenetic conservation, potential transposase activity, expression pattern and their regulation in different stress conditions. Among PGBDs, PGBD5 is under the highest purifying selection, and exhibits the most cell type specific expression pattern. In a two-component vector system, none of the human PGBDs could mobilize either the insect PB transposon or the endogenous human PB-like MER75 and MER85 elements with intact terminal sequences. When cells were exposed to various stress conditions, including hypoxia, oxidative or UV stress, the expression profiles of all PGBDs showed different, often cell type specific responses; however, the pattern of PGBD5 in most cases had the opposite tendency than that of the other piggyBac-derived elements. Taken together, our results indicate that human PGBD elements did not retain their mobilizing activity, but their cell type specific, and cellular stress related expression profiles point toward distinct domesticated functions that require further characterization.

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.

Canine Cognitive Dysfunction (CCD) scores correlate with amyloid beta 42 levels in dog brain tissue.

Alzheimer's disease (AD) is a significant burden for human health that is increasing in prevalence as the global population ages. There is growing recognition that current preclinical models of AD are insufficient to recapitulate key aspects of the disease. Laboratory models for AD include mice, which do not naturally develop AD-like pathology during aging, and laboratory Beagle dogs, which do not share the human environment. In contrast, the companion dog shares the human environment and presents a genetically heterogeneous population of animals that might spontaneously develop age-associated AD-like pathology and cognitive dysfunction. Here, we quantitatively measured amyloid beta (Aß42 or Abeta-42) levels in three areas of the companion dog brain (prefrontal cortex, temporal cortex, hippocampus/entorhinal cortex) and cerebrospinal fluid (CSF) using a newly developed Luminex assay. We found significant positive correlations between Aß42 and age in all three brain regions. Brain Aß42 abundance in all three brain regions was also correlated with Canine Cognitive Dysfunction Scale score in a multivariate analysis. This latter effect remained significant when correcting for age, except in the temporal cortex. There was no correlation between Aß42 in CSF and cognitive scores; however, we found a significant positive correlation between Aß42 in CSF and body weight, as well as a significant negative correlation between Aß42 in CSF and age. Our results support the suitability of the companion dog as a model for AD and illustrate the utility of veterinary biobanking to make biospecimens available to researchers for analysis.

Man's best friend in life and death: scientific perspectives and challenges of dog brain banking.

Biobanking refers to the systematic collection, storage, and distribution of pre- or post-mortem biological samples derived from volunteer donors. The demand for high-quality human specimens is clearly demonstrated by the number of newly emerging biobanking facilities and large international collaborative networks. Several animal species are relevant today in medical research; therefore, similar initiatives in comparative physiology could be fruitful. Dogs, in particular, are gaining increasing attention in translational research on complex phenomena, like aging, cancer, and neurodegenerative diseases. Therefore, biobanks gathering and storing dog biological materials together with related data could play a vital role in translational and veterinary research projects. To achieve these aims, a canine biobank should meet the same standards in sample quality and data management as human biobanks and should rely on well-designed collaborative networks between different professionals and dog owners. While efforts to create dog biobanks could face similar financial and technical challenges as their human counterparts, they can widen the spectrum of successful collaborative initiatives towards a better picture of dogs' physiology, disease, evolution, and translational potential. In this review, we provide an overview about the current state of dog biobanking and introduce the "Canine Brain and Tissue Bank" (CBTB)-a new, large-scale collaborative endeavor in the field.

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.

Gut Microbiome Composition is Associated with Age and Memory Performance in Pet Dogs.

Gut microbiota can crucially influence behavior and neurodevelopment. Dogs show unique similarities to humans in their physiology and may naturally develop dementia-like cognitive decline. We assessed 29 pet dogs' cognitive performance in a memory test and analyzed the bacterial 16S rRNA gene from fecal samples collected right after the behavioral tests. The major phyla identified in the dog microbiomes were Bacteroidetes, Firmicutes, and Fusobacteria, each represented by >20% of the total bacterial community. Fewer Fusobacteria were found in older dogs and better memory performance was associated with a lower proportion of Actinobacteria. Our preliminary findings support the existence of links between gut microbiota, age, and cognitive performance in pet dogs.

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.

Genetic Pathways of Aging and Their Relevance in the Dog as a Natural Model of Human Aging.

Aging research has experienced a burst of scientific efforts in the last decades as the growing ratio of elderly people has begun to pose an increased burden on the healthcare and pension systems of developed countries. Although many breakthroughs have been reported in understanding the cellular mechanisms of aging, the intrinsic and extrinsic factors that contribute to senescence on higher biological levels are still barely understood. The dog, Canis familiaris, has already served as a valuable model of human physiology and disease. The possible role the dog could play in aging research is still an open question, although utilization of dogs may hold great promises as they naturally develop age-related cognitive decline, with behavioral and histological characteristics very similar to those of humans. In this regard, family dogs may possess unmatched potentials as models for investigations on the complex interactions between environmental, behavioral, and genetic factors that determine the course of aging. In this review, we summarize the known genetic pathways in aging and their relevance in dogs, putting emphasis on the yet barely described nature of certain aging pathways in canines. Reasons for highlighting the dog as a future aging and gerontology model are also discussed, ranging from its unique evolutionary path shared with humans, its social skills, and the fact that family dogs live together with their owners, and are being exposed to the same environmental effects.

The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/Dynamin.

Phagocytosis of various targets, such as apoptotic cells or opsonized pathogens, by macrophages is coordinated by a complex signaling network initiated by distinct phagocytic receptors. Despite the different initial signaling pathways, each pathway ends up regulating the actin cytoskeletal network, phagosome formation and closure, and phagosome maturation leading to degradation of the engulfed particle. Herein, we describe a new phagocytic function for the nucleoside diphosphate kinase 1 (NDK-1), the nematode counterpart of the first identified metastasis inhibitor NM23-H1 (nonmetastatic clone number 23) nonmetastatic clone number 23 or nonmetastatic isoform 1 (NME1). We reveal by coimmunoprecipitation, Duolink proximity ligation assay, and mass spectrometry that NDK-1/NME1 works in a complex with DYN-1/Dynamin (Caenorhabditis elegans/human homolog proteins), which is essential for engulfment and phagosome maturation. Time-lapse microscopy shows that NDK-1 is expressed on phagosomal surfaces during cell corpse clearance in the same time window as DYN-1. Silencing of NM23-M1 in mouse bone marrow-derived macrophages resulted in decreased phagocytosis of apoptotic thymocytes. In human macrophages, NM23-H1 and Dynamin are corecruited at sites of phagosome formation in F-actin-rich cups. In addition, NM23-H1 was required for efficient phagocytosis. Together, our data demonstrate that NDK-1/NME1 is an evolutionarily conserved element of successful phagocytosis.-Farkas, Z., Petric, M., Liu, X., Herit, F., Rajnavölgyi, É., Szondy, Z., Budai, Z., Orbán, T. I., Sándor, S., Mehta, A., Bajtay, Z., Kovács, T., Jung, S. Y., Afaq Shakir, M., Qin, J., Zhou, Z., Niedergang, F., Boissan, M., Takács-Vellai, K. The nucleoside diphosphate kinase NDK-1/NME1 promotes phagocytosis in concert with DYN-1/dynamin.

Functional characterization of the ABCG2 5' non-coding exon variants: Stem cell specificity, translation efficiency and the influence of drug selection.

ABCG2 is a multidrug transporter with wide substrate specificity, and is believed to protect several cell types from various xenobiotics and endobiotics. This "guardian" function is important in numerous cell types and tissue barriers but becomes disadvantageous by being responsible for the multidrug resistance phenotype in certain tumor cells. ABCG2 regulation at the protein level has already been extensively studied, however, regulation at the mRNA level, especially the functional role of the various 5' untranslated exon variants (5' UTRs) has been elusive. In the present work, we describe a comprehensive characterization of four ABCG2 mRNA variants with different exon 1 sequences, investigate drug inducibility, stem cell specificity, mRNA stability, and translation efficiency. Although certain variants (E1B and E1C) are considered as "constitutive" mRNA isoforms, we show that chemotoxic drugs significantly alter the expression pattern of distinct ABCG2 mRNA isoforms. When examining human embryonic stem cell lines, we provide evidence that variant E1A has an expression pattern coupled to undifferentiated stem cell stage, as its transcript level is regulated parallel to mRNAs of Oct4 and Nanog pluripotency marker genes. When characterizing the four exon 1 variants we found no significant differences in terms of mRNA stabilities and half-lives of the isoforms. In contrast, variant E1U showed markedly lower translation efficiency both at the total protein level or regarding the functional presence in the plasma membrane. Taken together, these results indicate that the different 5' UTR variants play an important role in cell type specific regulation and fine tuning of ABCG2 expression.

Excision efficiency is not strongly coupled to transgenic rate: cell type-dependent transposition efficiency of sleeping beauty and piggyBac DNA transposons.

The Sleeping Beauty (SB) and piggyBac (PB) DNA transposons represent an emerging new gene delivery technology, potentially suitable for human gene therapy applications. Previous studies pointed to important differences between these transposon systems, depending on the cell types examined and the methodologies applied. However, efficiencies cannot always be compared because of differences in applications. In addition, "overproduction inhibition," a phenomenon believed to be a characteristic of DNA transposons, can remarkably reduce the overall transgenic rate, emphasizing the importance of transposase dose applied. Therefore, because of lack of comprehensive analysis, researchers are forced to optimize the technology for their own "in-house" platforms. In this study, we investigated the transposition of several SB (SB11, SB32, SB100X) and PB (mPB and hyPB) variants in various cell types at three levels: comparing the excision efficiency of the reaction by real-time PCR, testing the overall transgenic rate by detecting cells with stable integrations, and determining the average copy number when using different transposon systems and conditions. We concluded that high excision activity is not always followed by a higher transgenic rate, as exemplified by the hyperactive transposases, indicating that the excision and the integration steps of transposition are not strongly coupled as previously thought. In general, all levels of transposition show remarkable differences depending on the transposase used and cell lines examined, being the least efficient in human embryonic stem cells (hESCs). In spite of the comparably low activity in those special cell types, the hyperactive SB100X and hyPB systems could be used in hESCs with similar transgenic efficiency and with reasonably low (2-3) transgene copy numbers, indicating their potential applicability for gene therapy purposes in the future.