Shona H. Wood

Genome-Environment Interactions That Modulate Aging: Powerful Targets for Drug Discovery

Aging is the major biomedical challenge of this century. The percentage of elderly people, and consequently the incidence of age-related diseases such as heart disease, cancer, and neurodegenerative diseases, is projected to increase considerably in the coming decades. Findings from model organisms have revealed that aging is a surprisingly plastic process that can be manipulated by both genetic and environmental factors. Here we review a broad range of findings in model organisms, from environmental to genetic manipulations of aging, with a focus on those with underlying gene-environment interactions with potential for drug discovery and development. One well-studied dietary manipulation of aging is caloric restriction, which consists of restricting the food intake of organisms without triggering malnutrition and has been shown to retard aging in model organisms. Caloric restriction is already being used as a paradigm for developing compounds that mimic its life-extension effects and might therefore have therapeutic value. The potential for further advances in this field is immense; hundreds of genes in several pathways have recently emerged as regulators of aging and caloric restriction in model organisms. Some of these genes, such as IGF1R and FOXO3, have also been associated with human longevity in genetic association studies. The parallel emergence of network approaches offers prospects to develop multitarget drugs and combinatorial therapies. Understanding how the environment modulates aging-related genes may lead to human applications and disease therapies through diet, lifestyle, or pharmacological interventions. Unlocking the capacity to manipulate human aging would result in unprecedented health benefits.

Whole transcriptome sequencing of the aging rat brain reveals dynamic RNA changes in the dark matter of the genome

Brain aging frequently underlies cognitive decline and is a major risk factor for neurodegenerative conditions. The exact molecular mechanisms underlying brain aging, however, remain unknown. Whole transcriptome sequencing provides unparalleled depth and sensitivity in gene expression profiling. It also allows non-coding RNA and splice variant detection/comparison across phenotypes. Using RNA-seq to sequence the cerebral cortex transcriptome in 6-, 12- and 28-month-old rats, age-related changes were studied. Protein-coding genes related to MHC II presentation and serotonin biosynthesis were differentially expressed (DE) in aging. Relative to protein-coding genes, more non-coding genes were DE over the three age-groups. RNA-seq quantifies not only levels of whole genes but also of their individual transcripts. Over the three age-groups, 136 transcripts were DE, 37 of which were so-called dark matter transcripts that do not map to known exons. Fourteen of these transcripts were identified as novel putative long non-coding RNAs. Evidence of isoform switching and changes in usage were found. Promoter and coding sequence usage were also altered, hinting of possible changes to mitochondrial transport within neurons. Therefore, in addition to changes in the expression of protein-coding genes, changes in transcript expression, isoform usage, and non-coding RNAs occur with age. This study demonstrates dynamic changes in RNA with age at various genomic levels, which may reflect changes in regulation of transcriptional networks and provides non-coding RNA gene candidates for further studies.

Clocks for all seasons: unwinding the roles and mechanisms of circadian and interval timers in the hypothalamus and pituitary.

Adaptation to the environment is essential for survival, in all wild animal species seasonal variation in temperature and food availability needs to be anticipated. This has led to the evolution of deep-rooted physiological cycles, driven by internal clocks, which can track seasonal time with remarkable precision. Evidence has now accumulated that a seasonal change in thyroid hormone (TH) availability within the brain is a crucial element. This is mediated by local control of TH-metabolising enzymes within specialised ependymal cells lining the third ventricle of the hypothalamus. Within these cells, deiodinase type 2 enzyme is activated in response to summer day lengths, converting metabolically inactive thyroxine (T4) to tri-iodothyronine (T3). The availability of TH in the hypothalamus appears to be an important factor in driving the physiological changes that occur with season. Remarkably, in both birds and mammals, the pars tuberalis (PT) of the pituitary gland plays an essential role. A specialised endocrine thyrotroph cell (TSH-expressing) is regulated by the changing day-length signal, leading to activation of TSH by long days. This acts on adjacent TSH-receptors expressed in the hypothalamic ependymal cells, causing local regulation of deiodinase enzymes and conversion of TH to the metabolically active T3. In mammals, the PT is regulated by the nocturnal melatonin signal. Summer-like melatonin signals activate a PT-expressed clock-regulated transcription regulator (EYA3), which in turn drives the expression of the TSHβ sub-unit, leading to a sustained increase in TSH expression. In this manner, a local pituitary timer, driven by melatonin, initiates a cascade of molecular events, led by EYA3, which translates to seasonal changes of neuroendocrine activity in the hypothalamus. There are remarkable parallels between this PT circuit and the photoperiodic timing system used in plants, and while plants use different molecular signals (constans vs EYA3) it appears that widely divergent organisms probably obey a common set of design principles.

Gene expression in canine atopic dermatitis and correlation with clinical severity scores

BACKGROUND Canine atopic dermatitis (cAD) is a common condition in dogs that may be a naturally occurring model for human atopic dermatitis (hAD). Despite this, comparative research is limited, particularly into the genetic background of cAD. OBJECTIVES 1. Measure candidate gene expression in cAD skin using quantitative real time PCR (qPCR). 2. Correlate gene expression to clinical cAD scores (Canine Atopic Dermatitis Extent and Severity Index[CADESI]-03 and intradermal allergen test [IDT]). METHODS mRNA was extracted from biopsies of non-lesional and lesional skin from atopic dogs, and healthy skin from non-atopic dogs. Gene expression was quantified using qPCR, and compared between non-lesional atopic, lesional atopic and healthy skin. Gene expression in atopic skin was correlated with clinical severity (CADESI-03) and the number of positive reactions on an IDT. RESULTS Of the 20 quantified genes, 11 demonstrated statistically significant altered mRNA expression between atopic and healthy skin; dipeptidyl-peptidase-4 (DPP4), phosphatidylinositol-3,4,5-trisphosphate-5-phosphatase-2 (INPPL1), serine protease inhibitor kazal type-5 (SPINK5), sphingosine-1-phosphate lyase-1 (SGPL1), peroxisome proliferator-activated receptor gamma (PPARgamma), S100 calcium-binding protein A8 (S100A8), Plakophilin-2 (PKP2), Periostin (POSTN), Cullin4A, TNF-alpha and metalloproteinase inhibitor-1 (TIMP-1). Three genes correlated with CADESI-03: serum amyloid A 1 (SAA-1), S100A8, and PKP2; and four with IDT results: mast cell protease I (CMA1), SAA-1, S100A8 and SPINK5. CONCLUSION Genes with altered expression included those relevant to skin barrier formation and immune function, suggesting both are relevant in the pathogenesis of AD. Many of these genes reflect the proposed pathogenesis in hAD, supporting the use of dogs as a model for hAD. Furthermore, these genes may be considered suitable targets for future genetic and protein function studies in human and canine AD.

Serotonin: from top to bottom

Serotonin is a monoamine neurotransmitter, which is phylogenetically conserved in a wide range of species from nematodes to humans. In mammals, age-related changes in serotonin systems are known risk factors of age-related diseases, such as diabetes, faecal incontinence and cardiovascular diseases. A decline in serotonin function with aging would be consistent with observations of age-related changes in behaviours, such as sleep, sexual behaviour and mood all of which are linked to serotonergic function. Despite this little is known about serotonin in relation to aging. This review aims to give a comprehensive analysis of the distribution, function and interactions of serotonin in the brain; gastrointestinal tract; skeletal; vascular and immune systems. It also aims to demonstrate how the function of serotonin is linked to aging and disease pathology in these systems. The regulation of serotonin via microRNAs is also discussed, as are possible applications of serotonergic drugs in aging research and age-related diseases. Furthermore, this review demonstrates that serotonin is potentially involved in whole organism aging through its links with multiple organs, the immune system and microRNA regulation. Methods to investigate these links are discussed.

Despite identifying some shared gene associations with human atopic dermatitis the use of multiple dog breeds from various locations limits detection of gene associations in canine atopic dermatitis

Canine atopic dermatitis (cAD) is a common, severe pruritic and inflammatory skin disease and is a major veterinary welfare issue. This study genotyped 97 single nucleotide polymorphisms (SNPs) in 25 candidate genes in 659 dogs across eight breeds from three locations (UK, USA and Japan). These genes were selected from hAD literature, and previous cAD gene expression experiments. The aim of this study was to identify any shared gene associations between cAD and hAD. Only one SNP within the TSLP-receptor was associated with all eight breeds (corrected p=0.037). Five SNPs within Filaggrin, DPP4, MS4A2, and INPPL1 were associated with cAD, but only in certain breeds from different locations. Though these associations are broadly similar to hAD the variability of results across the breeds and locations demonstrates that a candidate gene approach using mixed breeds from different locations is not appropriate. This study therefore suggests that further candidate gene studies in cAD should be breed and location specific to increase the likelihood of finding associations with the disease.

Gene (mRNA) expression in canine atopic dermatitis: microarray analysis

Genes potentially involved in the pathology of canine atopic dermatitis (AD) were identified using gene expression microarrays. Total RNA extracted from skin biopsies was hybridized to an Agilent Technologies custom-designed 22K canine array. The arrays were analysed using Genedata Analyst software. Data were corrected for multiple hypothesis testing and tested for significance using the National Institute on Aging array analysis tool. For comparison, data were divided into separate groups: lesional atopic (n = 16), nonlesional atopic (n = 17) and healthy controls (n = 9). Fifty-four genes were differentially expressed at a significance level of 0.05 in canine AD compared to healthy controls. Sixteen genes were differentially expressed in both nonlesional and lesional atopic skin, 26 genes only in nonlesional skin and 12 only in lesional skin. These genes were associated with innate immune and inflammatory responses, cell cycle, apoptosis, barrier formation and transcriptional regulation. The most dysregulated gene in lesional skin was S100A8, which showed an almost 23-fold increase in expression. This is a pro-inflammatory cytokine located in the epidermal differentiation complex. Microarray analysis is a novel technique in canine AD. Significant changes in gene expression were identified in atopic skin. These were relevant to skin barrier formation and the immune response, suggesting that they both participate in AD. Gene expression restricted to lesional skin may be involved in inflammatory changes, whereas those shared or restricted to nonlesional skin may reflect the atopic phenotype. Investigating gene polymorphisms in the targets identified in this study will help improve our understanding of the genetic basis of this disease.

GeneFriends: An online co-expression analysis tool to identify novel gene targets for aging and complex diseases

BackgroundAlthough many diseases have been well characterized at the molecular level, the underlying mechanisms are often unknown. Nearly half of all human genes remain poorly studied, yet these genes may contribute to a number of disease processes. Genes involved in common biological processes and diseases are often co-expressed. Using known disease-associated genes in a co-expression analysis may help identify and prioritize novel candidate genes for further study.ResultsWe have created an online tool, called GeneFriends, which identifies co-expressed genes in over 1,000 mouse microarray datasets. GeneFriends can be used to assign putative functions to poorly studied genes. Using a seed list of disease-associated genes and a guilt-by-association method, GeneFriends allows users to quickly identify novel genes and transcription factors associated with a disease or process. We tested GeneFriends using seed lists for aging, cancer, and mitochondrial complex I disease. We identified several candidate genes that have previously been predicted as relevant targets. Some of the genes identified are already being tested in clinical trials, indicating the effectiveness of this approach. Co-expressed transcription factors were investigated, identifying C/ebp genes as candidate regulators of aging. Furthermore, several novel candidate genes, that may be suitable for experimental or clinical follow-up, were identified. Two of the novel candidates of unknown function that were co-expressed with cancer-associated genes were selected for experimental validation. Knock-down of their human homologs (C1ORF112 and C12ORF48) in HeLa cells slowed growth, indicating that these genes of unknown function, identified by GeneFriends, may be involved in cancer.ConclusionsGeneFriends is a resource for biologists to identify and prioritize novel candidate genes involved in biological processes and complex diseases. It is an intuitive online resource that will help drive experimentation. GeneFriends is available online at: http://genefriends.org/.

Genome-wide association analysis of canine atopic dermatitis and identification of disease related SNPs.

In humans, genome-wide association studies (GWAS) have been shown to be an effective and thorough approach for identifying polymorphisms associated with disease phenotypes. Here, we describe the first study to perform a genome-wide association study in canine atopic dermatitis (cAD) using the Illumina Canine SNP20 array, containing 22,362 single-nucleotide polymorphisms (SNPs). The aim of the study was to identify SNPs associated with cAD using affected and unaffected Golden Retrievers. Further validation studies were performed for potentially associated SNPs using Sequenom genotyping of larger numbers of cases and controls across eight breeds (Boxer, German Shepherd Dog, Labrador, Golden Retriever, Shiba Inu, Shih Tzu, Pit Bull, and West Highland White Terriers). Using meta-analysis, two SNPs were associated with cAD in all breeds tested. RS22114085 was identified as a susceptibility locus (p?=?0.00014, odds ratio?=?2) and RS23472497 as a protective locus (p?=?0.0015, odds ratio?=?0.6). Both of these SNPs were located in intergenic regions, and their effects have been demonstrated to be independent of each other, highlighting that further fine mapping and resequencing is required of these areas. Further, 12 SNPs were validated by Sequenom genotyping as associated with cAD, but these were not associated with all breeds. This study suggests that GWAS will be a useful approach for identifying genetic risk factors for cAD. Given the clinical heterogeneity within this condition and the likelihood that the relative genetic effect sizes are small, greater sample sizes and further studies will be required.

Binary Switching of Calendar Cells in the Pituitary Defines the Phase of the Circannual Cycle in Mammals.

Summary Persistent free-running circannual (approximately year-long) rhythms have evolved in animals to regulate hormone cycles, drive metabolic rhythms (including hibernation), and time annual reproduction. Recent studies have defined the photoperiodic input to this rhythm, wherein melatonin acts on thyrotroph cells of the pituitary pars tuberalis (PT), leading to seasonal changes in the control of thyroid hormone metabolism in the hypothalamus. However, seasonal rhythms persist in constant conditions in many species in the absence of a changing photoperiod signal, leading to the generation of circannual cycles. It is not known which cells, tissues, and pathways generate these remarkable long-term rhythmic processes. We show that individual PT thyrotrophs can be in one of two binary states reflecting either a long (EYA3+) or short (CHGA+) photoperiod, with the relative proportion in each state defining the phase of the circannual cycle. We also show that a morphogenic cycle driven by the PT leads to extensive re-modeling of the PT and hypothalamus over the circannual cycle. We propose that the PT may employ a recapitulated developmental pathway to drive changes in morphology of tissues and cells. Our data are consistent with the hypothesis that the circannual timer may reside within the PT thyrotroph and is encoded by a binary switch timing mechanism, which may regulate the generation of circannual neuroendocrine rhythms, leading to dynamic re-modeling of the hypothalamic interface. In summary, the PT-ventral hypothalamus now appears to be a prime structure involved in long-term rhythm generation.