Non-Intrinsic, Systemic Mechanisms of Cellular Senescence.

Cellular senescence is believed to contribute to aging and disease through the activity of secreted factors that promote inflammation, remodel the extracellular matrix, and adversely modify the behavior of non-senescent cells. While the markers and properties of senescent cells are still under investigation, it is postulated that cellular senescence manifests in vivo as the consequence of cellular damage that accumulates and becomes exacerbated with time. Yet, the notions that senescence has a solely intrinsic and time-dependent nature are questioned by the rapid induction of senescence in young mice and young cells in vitro by exposure to blood from aged animals. Here, we review some of the research on the systemically present factors that increase with age and may contribute to extrinsically induced senescence or "bystander senescence". These include proteins, reactive oxygen species, lipids, and nucleic acids, which may be present in individual soluble form, in vesicles, and in non-membranous multi-component macromolecules.

Fail-tests of DNA methylation clocks, and development of a noise barometer for measuring epigenetic pressure of aging and disease.

This study shows that Elastic Net (EN) DNA methylation (DNAme) clocks have low accuracy of predictions for individuals of the same age and a low resolution between healthy and disease cohorts; caveats inherent in applying linear model to non-linear processes. We found that change in methylation of cytosines with age is, interestingly, not the determinant for their selection into the clocks. Moreover, an EN clock's selected cytosines change when non-clock cytosines are removed from the training data; as expected from optimization in a machine learning (ML) context, but inconsistently with the identification of health markers in a biological context. To address these limitations, we moved from predictions to measurement of biological age, focusing on the cytosines that on average remain invariable in their methylation through lifespan, postulated to be homeostatically vital. We established that dysregulation of such cytosines, measured as the sums of standard deviations of their methylation values, quantifies biological noise, which in our hypothesis is a biomarker of aging and disease. We term this approach a "noise barometer" - the pressure of aging and disease on an organism. These noise-detecting cytosines are particularly important as sums of SD on the entire 450K DNAme array data yield a random pattern through chronology. Testing how many cytosines of the 450K arrays become noisier with age, we found that the paradigm of DNAme noise as a biomarker of aging and disease remarkably manifests in ~1/4 of the total. In that large set even the cytosines that have on average constant methylation through age show increased SDs and can be used as noise detectors of the barometer.

Small-animal blood exchange is an emerging approach for systemic aging research.

We describe a small-animal blood exchange approach developed for aging research as an alternative to heterochronic parabiosis or plasma injections. In parabiosis, animals are surgically coupled, which has several disadvantages, including difficulty controlling experimental procedure, the effects of shared organs, environmental enrichment from jointly exploring the housing enclosure, involuntary exercise and an imprecise onset of blood sharing. Likewise, in plasma injections, the added volumes need to be small, and there is little flexibility in changing the relative contributions of ectopic to endogenous blood components. These factors complicate the conclusions and interpretations, including the identification of key mechanisms and molecular or cellular determinants. Our approach, where blood is exchanged between animals without them being surgically coupled, is less invasive than parabiosis. The percentage of exchanged blood or other exchanged fluids is known and precise. The age of plasma and cells can be mixed and matched at all desired relative contributions to the endogenous systemic milieu, and the onset of the effects can be accurately delineated. In this protocol, we describe the preparatory and animal surgery steps required for small-animal blood exchange in mice and compare this process with parabiosis and plasma injections. We also provide the design, hardware and software for the blood exchange device and compare automated and manual exchange methods. Lastly, we report mathematical modeling of the dilution of blood factors. The fluid exchange takes ~30 min when performed by a well-trained biomedical scientist; the entire process takes ~2 h.

Old plasma dilution reduces human biological age: a clinical study.

This work extrapolates to humans the previous animal studies on blood heterochronicity and establishes a novel direct measurement of biological age. Our results support the hypothesis that, similar to mice, human aging is driven by age-imposed systemic molecular excess, the attenuation of which reverses biological age, defined in our work as a deregulation (noise) of 10 novel protein biomarkers. The results on biological age are strongly supported by the data, which demonstrates that rounds of therapeutic plasma exchange (TPE) promote a global shift to a younger systemic proteome, including youthfully restored pro-regenerative, anticancer, and apoptotic regulators and a youthful profile of myeloid/lymphoid markers in circulating cells, which have reduced cellular senescence and lower DNA damage. Mechanistically, the circulatory regulators of the JAK-STAT, MAPK, TGF-beta, NF-κB, and Toll-like receptor signaling pathways become more youthfully balanced through normalization of TLR4, which we define as a nodal point of this molecular rejuvenation. The significance of our findings is confirmed through big-data gene expression studies.

Systemic induction of senescence in young mice after single heterochronic blood exchange.

ABSTACT: Ageing is the largest risk factor for many chronic diseases. Studies of heterochronic parabiosis, substantiated by blood exchange and old plasma dilution, show that old-age-related factors are systemically propagated and have pro-geronic effects in young mice. However, the underlying mechanisms how bloodborne factors promote ageing remain largely unknown. Here, using heterochronic blood exchange in male mice, we show that aged mouse blood induces cell and tissue senescence in young animals after one single exchange. This induction of senescence is abrogated if old animals are treated with senolytic drugs before blood exchange, therefore attenuating the pro-geronic influence of old blood on young mice. Hence, cellular senescence is neither simply a response to stress and damage that increases with age, nor a chronological cell-intrinsic phenomenon. Instead, senescence quickly and robustly spreads to young mice from old blood. Clearing senescence cells that accumulate with age rejuvenates old circulating blood and improves the health of multiple tissues.

Autologous treatment for ALS with implication for broad neuroprotection.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is characterized by a progressive loss of motor neurons (MNs), leading to paralysis, respiratory failure and death within 2-5 years of diagnosis. The exact mechanisms of sporadic ALS, which comprises 90% of all cases, remain unknown. In familial ALS, mutations in superoxide dismutase (SOD1) cause 10% of cases. METHODS: ALS patient-derived human-induced pluripotent stem cells (ALS hiPSCs, harboring the SOD1AV4 mutation), were differentiated to MNs (ALS-MNs). The neuroprotective effects of conditioned medium (CM) of hESCs (H9), wt hiPSCs (WTC-11) and the ALS iPSCs, on MN apoptosis and viability, formation and maintenance of neurites, mitochondrial activity and expression of inflammatory genes, were examined. For in vivo studies, 200 µl of CM from the ALS iPSCs (CS07 and CS053) was injected subcutaneously into the ALS model mice (transgenic for the human SOD1G93A mutation). Animal agility and strength, muscle innervation and mass, neurological score, onset of paralysis and lifespan of the ALS mice were assayed. After observing significant disease-modifying effects, the CM was characterized biochemically by fractionation, comparative proteomics, and epigenetic screens for the dependence on pluripotency. CM of fibroblasts that were differentiated from the wt hiPSCs lacked any neuroprotective activity and was used as a negative control throughout the studies. RESULTS: The secretome of PSCs including the ALS patient iPSCs was neuroprotective in the H2O2 model. In the model with pathogenic SOD1 mutation, ALS iPSC-CM attenuated all examined hallmarks of ALS pathology, rescued human ALS-MNs from denervation and death, restored mitochondrial health, and reduced the expression of inflammatory genes. The ALS iPSC-CM also improved neuro-muscular health and function, and delayed paralysis and morbidity in ALS mice. Compared side by side, cyclosporine (CsA), a mitochondrial membrane blocker that prevents the leakage of mitochondrial DNA, failed to avert the death of ALS-MNs, although CsA and ALS iPSC-CM equally stabilized MN mitochondria and attenuated inflammatory genes. Biochemical characterization, comparative proteomics, and epigenetic screen all suggested that it was the interactome of several key proteins from different fractions of PSC-CM that delivered the multifaceted neuroprotection. CONCLUSIONS: This work introduces and mechanistically characterizes a new biologic for treating ALS and other complex neurodegenerative diseases.

Mechanisms and Minimization of False Discovery of Metabolic Bioorthogonal Noncanonical Amino Acid Proteomics.

Metabolic proteomics has been widely used to characterize dynamic protein networks in many areas of biomedicine, including in the arena of tissue aging and rejuvenation. Bioorthogonal noncanonical amino acid tagging (BONCAT) is based on mutant methionine-tRNA synthases (MetRS) that incorporates metabolic tags, for example, azidonorleucine [ANL], into newly synthesized proteins. BONCAT revolutionizes metabolic proteomics, because mutant MetRS transgene allows one to identify cell type-specific proteomes in mixed biological environments. This is not possible with other methods, such as stable isotope labeling with amino acids in cell culture, isobaric tags for relative and absolute quantitation and tandem mass tags. At the same time, an inherent weakness of BONCAT is that after click chemistry-based enrichment, all identified proteins are assumed to have been metabolically tagged, but there is no confirmation in mass spectrometry data that only tagged proteins are detected. As we show here, such assumption is incorrect and accurate negative controls uncover a surprisingly high degree of false positives in BONCAT proteomics. We show not only how to reveal the false discovery and thus improve the accuracy of the analyses and conclusions but also approaches for avoiding it through minimizing nonspecific detection of biotin, biotin-independent direct detection of metabolic tags, and improvement of signal to noise ratio through machine learning algorithms.

Erythrocytes, a New Contributor to Age-Associated Loss of Blood-Brain Barrier Integrity.

Blood exchanges between young and old partners demonstrate old blood has a detrimental effect on brain health of young animals. Previous studies primarily investigate soluble blood factors, such as transforming growth factor-beta, on the brain and the blood-brain barrier (BBB). However, the role of blood cellular components, particularly erythrocytes, has not been defined. Erythrocyte morphology and rigidity change as mammals age, altering their transport within the capillary bed. This impacts downstream biological events, such as the release of reactive oxygen species and hemoglobin, potentially compromising the BBB. Here, a micro electrical BBB (µE-BBB), with cocultured endothelial and astrocytic cells, and a built-in trans-endothelial electrical resistance (TEER) system is described to monitor the effect of capillary shear stress on erythrocytes derived from young and old mice and people and the subsequent effects of these cells on BBB integrity. This is monitored by the passage of fluorescein isothiocyanate-dextran and real-time profiling of TEER across the BBB after old and young erythrocyte exposure. Compared to young erythrocytes, old erythrocytes induce an increased permeability by 42% and diminished TEER by 2.9% of the µE-BBB. These results suggest that changes in circulating erythrocytes are a biomarker of aging in the context of BBB integrity.

Attenuation of age-elevated blood factors by repositioning plasmapheresis: A novel perspective and approach.

Aging is associated with the impairment of stem cell activation, leading to the functional decline of tissues and increasing the risk for age-associated diseases. The old, damaged or unrepaired tissues disturb distant tissue homeostasis by secreting factors into the circulation, which may not only serve as biomarkers for specific age-associated pathologies but also induce a variety of degenerative phenotypes. In this review, we summarize and discuss systemic determinants that perpetuate age-related tissue dysfunction. We further elaborate on the effects of attenuating these circulating factors by highlighting recent advances which utilize plasmapheresis in a pre-clinical or clinical setting. Overall, we postulate that repositioning therapeutic plasma exchange (TPE) to dilute the systemic factors, which become deleterious at their age-elevated levels, could be a rapidly effective rejuvenation therapy that recalibrates crucial signaling pathways to a youthful state.

Discrimination of single-point mutations in unamplified genomic DNA via Cas9 immobilized on a graphene field-effect transistor.

Simple and fast methods for the detection of target genes with single-nucleotide specificity could open up genetic research and diagnostics beyond laboratory settings. We recently reported a biosensor for the electronic detection of unamplified target genes using liquid-gated graphene field-effect transistors employing an RNA-guided catalytically deactivated CRISPR-associated protein 9 (Cas9) anchored to a graphene monolayer. Here, using unamplified genomic samples from patients and by measuring multiple types of electrical response, we show that the biosensors can discriminate within one hour between wild-type and homozygous mutant alleles differing by a single nucleotide. We also show that biosensors using a guide RNA-Cas9 orthologue complex targeting genes within the protospacer-adjacent motif discriminated between homozygous and heterozygous DNA samples from patients with sickle cell disease, and that the biosensors can also be used to rapidly screen for guide RNA-Cas9 complexes that maximize gene-targeting efficiency.

Rapid and Electronic Identification and Quantification of Age-Specific Circulating Exosomes via Biologically Activated Graphene Transistors.

Increasing access to modern clinical practices concomitantly extends lifespan, ironically revealing new classes of degenerative and inflammatory diseases of later years. Here, an electronic graphene field-effect transistor (gFET) is reported, termed EV-chip, for label-free, rapid identification and quantification of exosomes (EV) associated with aging through specific surface markers, CD63 and CD151. Studies suggest that blood-derived exosomes carry specific biomolecules that can be used toward diagnostic applications of age and health. However, to observe improvements in patient outcomes, earlier detection at the point-of-care (POC) is required. Unfortunately, conventional techniques and other electronic-based platforms for exosome sensing are burdensome and inept for the POC distinction of aged blood factors. It is shown that EV-chip can quantitatively detect purified exosomes from plasma, with a limit of detection (LOD) of 2 × 104 particles mL-1 and a limit of quantification (LOQ) of 6 × 104 particles mL-1 . The sensitivity and compact electronics of the EV-chip improves upon previously published electronic biosensors, making it ideal for a physician's office or a simple biological laboratory. The sensitivity, selectivity, and portability of the EV-chip demonstrate the potential of the biosensor as a powerful point-of-care diagnostic and prognostic tool for age-related diseases.