The role of retrotransposable elements in ageing and age-associated diseases.

The genomes of virtually all organisms contain repetitive sequences that are generated by the activity of transposable elements (transposons). Transposons are mobile genetic elements that can move from one genomic location to another; in this process, they amplify and increase their presence in genomes, sometimes to very high copy numbers. In this Review we discuss new evidence and ideas that the activity of retrotransposons, a major subgroup of transposons overall, influences and even promotes the process of ageing and age-related diseases in complex metazoan organisms, including humans. Retrotransposons have been coevolving with their host genomes since the dawn of life. This relationship has been largely competitive, and transposons have earned epithets such as 'junk DNA' and 'molecular parasites'. Much of our knowledge of the evolution of retrotransposons reflects their activity in the germline and is evident from genome sequence data. Recent research has provided a wealth of information on the activity of retrotransposons in somatic tissues during an individual lifespan, the molecular mechanisms that underlie this activity, and the manner in which these processes intersect with our own physiology, health and well-being.

Analysis of Active Transport by Fluorescence Recovery after Photobleaching.

Fluorescence recovery after photobleaching (FRAP) is a well-established experimental technique to study binding and diffusion of molecules in cells. Although a large number of analytical and numerical models have been developed to extract binding and diffusion rates from FRAP recovery curves, active transport of molecules is typically not included in the existing models that are used to estimate these rates. Here we present a validated numerical method for estimating diffusion, binding/unbinding rates, and active transport velocities using FRAP data that captures intracellular dynamics through partial differential equation models. We apply these methods to transport and localization of mRNA molecules in Xenopus laevis oocytes, where active transport processes are essential to generate developmental polarity. By providing estimates of the effective velocities and diffusion, as well as expected run times and lengths, this approach can help quantify dynamical properties of localizing and nonlocalizing RNA. Our results confirm the distinct transport dynamics in different regions of the cytoplasm, and suggest that RNA movement in both the animal and vegetal directions may influence the timescale of RNA localization in Xenopus oocytes. We also show that model initial conditions extracted from FRAP postbleach intensities prevent underestimation of diffusion, which can arise from the instantaneous bleaching assumption. The numerical and modeling approach presented here to estimate parameters using FRAP recovery data is a broadly applicable tool for systems where intracellular transport is a key molecular mechanism.

Using in vivo imaging to measure RNA mobility in Xenopus laevis oocytes.

RNA localization in the Xenopus oocyte is responsible for the establishment of polarity during oogenesis as well as the specification of germ layers during embryogenesis. However, the inability to monitor mRNA localization in live vertebrate oocytes has posed a major barrier to understanding the mechanisms driving directional transport. Here we describe a method for imaging MS2 tagged RNA in live Xenopus oocytes to study the dynamics of RNA localization. We also focus on methods for implementing and analyzing FRAP data. This protocol is optimized for imaging of the RNAs in stage II oocytes but it can be adapted to study dynamics of other molecules during oogenesis. Using this approach, mobility can be measured in different regions of the oocyte, enabling the direct observation of molecular dynamics throughout the oocyte.

Directional transport is mediated by a Dynein-dependent step in an RNA localization pathway.

Cytoplasmic RNA localization is a key biological strategy for establishing polarity in a variety of organisms and cell types. However, the mechanisms that control directionality during asymmetric RNA transport are not yet clear. To gain insight into this crucial process, we have analyzed the molecular machinery directing polarized transport of RNA to the vegetal cortex in Xenopus oocytes. Using a novel approach to measure directionality of mRNA transport in live oocytes, we observe discrete domains of unidirectional and bidirectional transport that are required for vegetal RNA transport. While kinesin-1 appears to promote bidirectional transport along a microtubule array with mixed polarity, dynein acts first to direct unidirectional transport of RNA towards the vegetal cortex. Thus, vegetal RNA transport occurs through a multistep pathway with a dynein-dependent directional cue. This provides a new framework for understanding the mechanistic basis of cell and developmental polarity.

Death by transposition - the enemy within?

Here we present and develop the hypothesis that the derepression of endogenous retrotransposable elements (RTEs) - "genomic parasites" - is an important and hitherto under-unexplored molecular aging process that can potentially occur in most tissues. We further envision that the activation and continued presence of retrotransposition contribute to age-associated tissue degeneration and pathology. Chromatin is a complex and dynamic structure that needs to be maintained in a functional state throughout our lifetime. Studies of diverse species have revealed that chromatin undergoes extensive rearrangements during aging. Cellular senescence, an important component of mammalian aging, has recently been associated with decreased heterochromatinization of normally silenced regions of the genome. These changes lead to the expression of RTEs, culminating in their transposition. RTEs are common in all kingdoms of life, and comprise close to 50% of mammalian genomes. They are tightly controlled, as their activity is highly destabilizing and mutagenic to their resident genomes.

Drug repurposing for neurodegenerative diseases using Zebrafish behavioral profiles.

Drug repurposing can accelerate drug development while reducing the cost and risk of toxicity typically associated with de novo drug design. Several disorders lacking pharmacological solutions and exhibiting poor results in clinical trials - such as Alzheimer's disease (AD) - could benefit from a cost-effective approach to finding new therapeutics. We previously developed a neural network model, Z-LaP Tracker, capable of quantifying behaviors in zebrafish larvae relevant to cognitive function, including activity, reactivity, swimming patterns, and optomotor response in the presence of visual and acoustic stimuli. Using this model, we performed a high-throughput screening of FDA-approved drugs to identify compounds that affect zebrafish larval behavior in a manner consistent with the distinct behavior induced by calcineurin inhibitors. Cyclosporine (CsA) and other calcineurin inhibitors have garnered interest for their potential role in the prevention of AD. We generated behavioral profiles suitable for cluster analysis, through which we identified 64 candidate therapeutics for neurodegenerative disorders.

Combining supervised and unsupervised analyses to quantify behavioral phenotypes and validate therapeutic efficacy in a triple transgenic mouse model of Alzheimer's disease.

Behavioral testing is an essential tool for evaluating cognitive function and dysfunction in preclinical research models. This is of special importance in the study of neurological disorders such as Alzheimer's disease. However, the reproducibility of classic behavioral assays is frequently compromised by interstudy variation, leading to ambiguous conclusions about the behavioral markers characterizing the disease. Here, we identify age- and genotype-driven differences between 3xTg-AD and non-transgenic control mice using a low-cost, highly customizable behavioral assay that requires little human intervention. Through behavioral phenotyping combining both supervised and unsupervised behavioral classification methods, we are able to validate the preventative effects of the immunosuppressant cyclosporine A in a rodent model of Alzheimer's disease, as well as the partially ameliorating effects of candidate drugs nebivolol and cabozantinib.

primiReference: a reference for analysis of primary-microRNA expression in single-nucleus sequencing data.

Single-nucleus RNA-sequencing technology has revolutionized understanding of nuanced changes in gene expression between cell types within tissues. Unfortunately, our understanding of regulatory RNAs, such as microRNAs (miRNAs), is limited through both single-cell and single-nucleus techniques due to the short length of miRNAs in the cytoplasm and the incomplete reference of longer primary miRNA (pri-miRNA) transcripts in the nucleus. We build a custom reference to align and count pri-miRNA sequences in single-nucleus data. Using young and aged subventricular zone (SVZ) nuclei, we show differential expression of pri-miRNAs targeting genes involved in neural stem cells (NSC) differentiation in the aged SVZ. Furthermore, using wild-type and 5XFAD mouse model cortex nuclei, to validate the use of primiReference, we find cell-type-specific expression of pri-miRNAs known to be involved in Alzheimer's disease (AD). pri-miRNAs likely contribute to NSC dysregulation with age and AD pathology. primiReference is paramount in capturing a global profile of gene expression and regulation in single-nucleus data and can provide key insights into cell-type-specific expression of pri-miRNAs, paving the way for future studies of regulation and pathway dysregulation. By looking at pri-miRNA abundance and transcriptional differences, regulation of gene expression by miRNAs in disease and aging can be further explored.

A cluster of X-linked miRNAs are de-repressed with age in mouse liver and target growth hormone signaling.

Growth hormone (GH) signaling influences lifespan in a wide variety of mammalian species. We previously reported that a cluster of miRNAs located on the X-chromosome are de-repressed with age in male mouse liver, and a subset, the mir-465 family, can directly attenuate expression of the growth hormone receptor (GHR) in vitro leading to a reduction in GH signaling. Here we show that this cluster of miRNAs is also upregulated in the liver with age in females, and that calorie restriction and the Ames dwarf genotype, both known to delay aging, attenuate the upregulation of the miRNA cluster. Upregulation of mir-465 in vivo leads to a reduction in GHR mRNA in the liver and an attenuation of GH signaling, indicated by a reduction in GHR, IGF-1, IGFBP3, and ALS mRNA expression. There is a corresponding reduction in IGF-1 protein levels in the liver and plasma. These results suggest that the age-associated upregulation of the X-chromosomal cluster of miRNAs could influence lifespan.

Regulation of the somatotropic axis by MYC-mediated miRNA repression.

The transcription factor MYC is overexpressed in many human cancers and has a significant causal role in tumor incidence and progression. In contrast, Myc +/- heterozygous mice, which have decreased MYC expression, exhibit a 10-20% increase in lifespan and a decreased incidence or progression of several age-related diseases. Myc heterozygous mice were also reported to have decreased mTOR and IGF1 signaling, two pathways whose reduced activity is associated with longevity in diverse species. Given MYC's downstream role in these pathways, the downregulation of mTOR and IGF1 signaling in Myc heterozygotes suggests the presence of feedback loops within this regulatory network. In this communication we provide further evidence that the reduction of Myc expression in Myc +/- heterozygous mice provokes a female-specific decrease in circulating IGF1 as well as a reduction of IGF1 protein in the liver. In particular, reduced Myc expression led to upregulation of miRNAs that target the Igf1 transcript, thereby inhibiting its translation and leading to decreased IGF1 protein levels. Using Argonaute (AGO)-CLIP-sequencing we found enrichment of AGO binding in the Igf1 transcript at the target sites of let-7, miR-122, and miR-29 in female, but not male Myc heterozygotes. Upregulation of the liver-specific miR-122 in primary hepatocytes in culture and in vivo in mice resulted in significant downregulation of IGF1 protein, but not mRNA. Reduced levels of IGF1 increased GH production in the pituitary through a well-documented negative-feedback relationship. In line with this, we found that IGF1 levels in bone (where miR-122 is not expressed) were unchanged, consistent with the decreased incidence of osteoporosis in female Myc heterozygotes, despite decreased circulating IGF1.

Finding Drug Repurposing Candidates for Neurodegenerative Diseases using Zebrafish Behavioral Profiles.

Drug repurposing can accelerate drug development while reducing the cost and risk of toxicity typically associated with de novo drug design. Several disorders lacking pharmacological solutions and exhibiting poor results in clinical trials - such as Alzheimer's disease (AD) - could benefit from a cost-effective approach to finding new therapeutics. We previously developed a neural network model, Z-LaP Tracker, capable of quantifying behaviors in zebrafish larvae relevant to cognitive function, including activity, reactivity, swimming patterns, and optomotor response in the presence of visual and acoustic stimuli. Using this model, we performed a high-throughput screening of FDA-approved drugs to identify compounds that affect zebrafish larval behavior in a manner consistent with the distinct behavior induced by calcineurin inhibitors. Cyclosporine (CsA) and other calcineurin inhibitors have garnered interest for their potential role in the prevention of AD. We generated behavioral profiles suitable for cluster analysis, through which we identified 64 candidate therapeutics for neurodegenerative disorders.

An 8-cage imaging system for automated analyses of mouse behavior.

The analysis of mouse behavior is used in biomedical research to study brain function in health and disease. Well-established rapid assays allow for high-throughput analyses of behavior but have several drawbacks, including measurements of daytime behaviors in nocturnal animals, effects of animal handling, and the lack of an acclimation period in the testing apparatus. We developed a novel 8-cage imaging system, with animated visual stimuli, for automated analyses of mouse behavior in 22-h overnight recordings. Software for image analysis was developed in two open-source programs, ImageJ and DeepLabCut. The imaging system was tested using 4-5 month-old female wild-type mice and 3xTg-AD mice, a widely-used model to study Alzheimer's disease (AD). The overnight recordings provided measurements of multiple behaviors including acclimation to the novel cage environment, day and nighttime activity, stretch-attend postures, location in various cage areas, and habituation to animated visual stimuli. The behavioral profiles were different in wild-type and 3xTg-AD mice. AD-model mice displayed reduced acclimation to the novel cage environment, were hyperactive during the first hour of darkness, and spent less time at home in comparison to wild-type mice. We propose that the imaging system may be used to study various neurological and neurodegenerative disorders, including Alzheimer's disease.

Timing and cell specificity of senescence drives postnatal lung development and injury.

Senescence causes age-related diseases and stress-related injury. Paradoxically, it is also essential for organismal development. Whether senescence contributes to lung development or injury in early life remains unclear. Here, we show that lung senescence occurred at birth and decreased throughout the saccular stage in mice. Reducing senescent cells at this stage disrupted lung development. In mice (<12 h old) exposed to hyperoxia during the saccular stage followed by air recovery until adulthood, lung senescence increased particularly in type II cells and secondary crest myofibroblasts. This peaked during the alveolar stage and was mediated by the p53/p21 pathway. Decreasing senescent cells during the alveolar stage attenuated hyperoxia-induced alveolar and vascular simplification. Conclusively, early programmed senescence orchestrates postnatal lung development whereas later hyperoxia-induced senescence causes lung injury through different mechanisms. This defines the ontogeny of lung senescence and provides an optimal therapeutic window for mitigating neonatal hyperoxic lung injury by inhibiting senescence.

A zebrafish model for calcineurin-dependent brain function.

Small-molecule modulators of calcineurin signaling have been proposed as potential therapeutics in Down syndrome and Alzheimer's disease. Models predict that in Down syndrome, suppressed calcineurin-NFAT signaling may be mitigated by proINDY, which activates NFAT, the nuclear factor of activated T-cells. Conversely, elevated calcineurin signaling in Alzheimer's disease may be suppressed with the calcineurin inhibitors cyclosporine and tacrolimus. Such small-molecule treatments may have both beneficial and adverse effects. The current study examines the effects of proINDY, cyclosporine and tacrolimus on behavior, using zebrafish larvae as a model system. To suppress calcineurin signaling, larvae were treated with cyclosporine and tacrolimus. We found that these calcineurin inhibitors induced hyperactivity, suppressed visually-guided behaviors, acoustic hyperexcitability and reduced habituation to acoustic stimuli. To activate calcineurin-NFAT signaling, larvae were treated with proINDY. ProINDY treatment reduced activity and stimulated visually-guided behaviors, opposite to the behavioral changes induced by calcineurin inhibitors. The opposing effects suggest that activity and visually-guided behaviors are regulated by the calcineurin-NFAT signaling pathway. A central role of calcineurin-NFAT signaling is further supported by co-treatments of calcineurin inhibitors and proINDY, which had therapeutic effects on activity and visually-guided behaviors. However, these co-treatments adversely increased excitability, suggesting that some behaviors are regulated by other calcineurin signaling pathways. Overall, the developed methodologies provide an efficient high-throughput platform for the evaluation of modulators of calcineurin signaling that restore neural function, while avoiding adverse side effects, in a complex neural system.

Novel use of FDA-approved drugs identified by cluster analysis of behavioral profiles.

Repurposing FDA-approved drugs is an efficient and cost-effective approach in the development of therapeutics for a broad range of diseases. However, prediction of function can be challenging, especially in the brain. We screened a small-molecule library with FDA-approved drugs for effects on behavior. The studies were carried out using zebrafish larvae, imaged in a 384-well format. We found that various drugs affect activity, habituation, startle responses, excitability, and optomotor responses. The changes in behavior were organized in behavioral profiles, which were examined by hierarchical cluster analysis. One of the identified clusters includes the calcineurin inhibitors cyclosporine (CsA) and tacrolimus (FK506), which are immunosuppressants and potential therapeutics in the prevention of Alzheimer's disease. The calcineurin inhibitors form a functional cluster with seemingly unrelated drugs, including bromocriptine, tetrabenazine, rosiglitazone, nebivolol, sorafenib, cabozantinib, tamoxifen, meclizine, and salmeterol. We propose that drugs with 'CsA-type' behavioral profiles are promising candidates for the prevention and treatment of Alzheimer's disease.

Sex-specific aging in animals: Perspective and future directions.

Sex differences in aging occur in many animal species, and they include sex differences in lifespan, in the onset and progression of age-associated decline, and in physiological and molecular markers of aging. Sex differences in aging vary greatly across the animal kingdom. For example, there are species with longer-lived females, species where males live longer, and species lacking sex differences in lifespan. The underlying causes of sex differences in aging remain mostly unknown. Currently, we do not understand the molecular drivers of sex differences in aging, or whether they are related to the accepted hallmarks or pillars of aging or linked to other well-characterized processes. In particular, understanding the role of sex-determination mechanisms and sex differences in aging is relatively understudied. Here, we take a comparative, interdisciplinary approach to explore various hypotheses about how sex differences in aging arise. We discuss genomic, morphological, and environmental differences between the sexes and how these relate to sex differences in aging. Finally, we present some suggestions for future research in this area and provide recommendations for promising experimental designs.

The mir-465 family is upregulated with age and attenuates growth hormone signaling in mouse liver.

We analyzed the small RNA transcriptome from 5-month-old, 24-month-old, and 36-month-old mouse liver and found 56 miRNAs that changed their expression profile with age. Among these is a cluster of 18 miRNAs that are upregulated between 50- and 1,000-fold at 24 and 36 months of age. This cluster is located in a 60-kb region of the X-chromosome that is devoid of other coding sequences and is part of a lamin-associated domain. Potential targets of the miRNAs in the cluster suggest they may regulate several pathways altered in aging, including the PI3K-Akt pathway. Total transcriptome analyses indicate that expression of several potential genes in the PI3K-Akt pathway that may be targeted by the mir-465 family (mmu-mir-465a, mmu-mir-465b, and mmu-mir-465c) is downregulated with age. Transfection of the liver cell line AML12 with mir-465 family members leads to a reduction of three of these potential targets at the mRNA level: a 40% reduction of the growth hormone receptor (GHR), and a 25% reduction in Kitl and PPP2R3C. Further investigation of the GHR 3'UTR revealed that the mir-465 family directly targets the GHR mRNA. Cells transfected with mir-465 showed a reduction of JAK2 and STAT5 phosphorylation upon growth hormone (GH) stimulation, resulting in a reduction in insulin-like growth factor 1 (IGF-1) and IGF-1-binding protein 3 expression. With age, GH signaling falls and there is a reduction in circulating IGF-1. Our data suggest that an increase in expression of the mir-465 family with age may contribute to the reduction in the GH signaling.

Early embryonic exposure to polychlorinated biphenyls disrupts heat-shock protein 70 cognate expression in zebrafish.

Polychlorinated biphenyls (PCBs) are persistent environmental contaminants that have documented neurological effects in children exposed in utero. To better define neuronally linked molecular targets during early development, zebrafish embryos were exposed to Aroclor 1254, a mixture of PCB congeners that are common environmental contaminants. Microarray analysis of the zebrafish genome revealed consistent significant changes in 38 genes. Of these genes, 55% (21) are neuronally related. One gene that showed a consistent 50% reduction in expression in PCB-treated embryos was heat-shock protein 70 cognate (Hsc70). The reduction in Hsc70 expression was confirmed by real-time polymerase chain reaction (PCR), revealing a consistent 30% reduction in expression in PCB-treated embryos. Early embryonic exposure to PCBs also induced structural changes in the ventro-rostral cluster as detected by immunocytochemistry. In addition, there was a significant reduction in dorso-rostral neurite outgrowth emanating from the RoL1 cell cluster following PCB exposure. The serotonergic neurons in the developing diencephalon showed a 34% reduction in fluorescence when labeled with a serotonin antibody following PCB exposure, corresponding to a reduction in serotonin concentration in the neurons. The total size of the labeled neurons was not significantly different between treated and control embryos, indicating that the development of the neurons was not affected, only the production of serotonin within the neurons. The structural and biochemical changes in the developing central nervous system following early embryonic exposure to Aroclor 1254 may lead to alterations in the function of the affected regions.

Reorganization of chromosome architecture in replicative cellular senescence.

Replicative cellular senescence is a fundamental biological process characterized by an irreversible arrest of proliferation. Senescent cells accumulate a variety of epigenetic changes, but the three-dimensional (3D) organization of their chromatin is not known. We applied a combination of whole-genome chromosome conformation capture (Hi-C), fluorescence in situ hybridization, and in silico modeling methods to characterize the 3D architecture of interphase chromosomes in proliferating, quiescent, and senescent cells. Although the overall organization of the chromatin into active (A) and repressive (B) compartments and topologically associated domains (TADs) is conserved between the three conditions, a subset of TADs switches between compartments. On a global level, the Hi-C interaction matrices of senescent cells are characterized by a relative loss of long-range and gain of short-range interactions within chromosomes. Direct measurements of distances between genetic loci, chromosome volumes, and chromatin accessibility suggest that the Hi-C interaction changes are caused by a significant reduction of the volumes occupied by individual chromosome arms. In contrast, centromeres oppose this overall compaction trend and increase in volume. The structural model arising from our study provides a unique high-resolution view of the complex chromosomal architecture in senescent cells.

A mixture of environmental contaminants increases cAMP-dependent protein kinase in Spisula embryos.

Using the surf clam embryo, we investigated the effects of the combination of bromoform, chloroform, and tetrachloroethylene, three pollutants found in high concentrations in the municipal water supply in Brick, New Jersey. Exposure produced an increase in an isoform of the regulatory subunit (RII) of cAMP-dependent protein kinase, demonstrated by confocal microscopy and western blotting. Embryos showed an increase in RII where the primordial gill and ciliated velar epithelium are innervated. This increase correlated with increased ciliary activity, indicating a corresponding rise in the catalytic subunit. Treatment resulted in decreased threonine phosphorylation of actin. There was no effect on neurotransmitters or receptors of the serotonergic-dopaminergic nervous system. These effects occurred only with the ternary mixture. No significant effect was seen with individual or paired components. This is the first report showing that bromoform, chloroform, and tetrachloroethylene act synergistically to alter a key regulator of neuronal development.