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.

Outcomes of living liver donor candidate evaluations in the Living Donor Collective pilot registry.

BACKGROUND: To gather information on long-term outcomes after living donation, the Scientific Registry of Transplant Recipients (SRTR) conducted a pilot on the feasibility of establishing a comprehensive donor candidate registry. METHODS: A convenience sample of 6 US living liver donor programs evaluated 398 consecutive donor candidates in 2018, ending with the March 12, 2020, COVID-19 emergency. RESULTS: For 333/398 (83.7%), the donor or program decided whether to donate; 166/333 (49.8%) were approved, and 167/333 (50.2%) were not or opted out. Approval rates varied by program, from 27.0% to 63.3% (median, 46%; intraquartile range, 37.3-51.1%). Of those approved, 90.4% were white, 57.2% were women, 83.1% were < 50 years, and 85.5% had more than a high school education. Of 167 candidates, 131 (78.4%) were not approved or opted out because of: medical risk (10.7%); chronic liver disease risk (11.5%); psychosocial reasons (5.3%); candidate declined (6.1%); anatomical reasons increasing recipient risk (26.0%); recipient-related reasons (33.6%); finances (1.5%); or other (5.3%). CONCLUSIONS: A comprehensive national registry is feasible and necessary to better understand candidate selection and long-term outcomes. As a result, the US Health Resources and Services Administration asked SRTR to expand the pilot to include all US living donor programs.

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.

Outcomes of Living Kidney Donor Candidate Evaluations in the Living Donor Collective Pilot Registry.

BACKGROUND: Gaps in our knowledge of long-term outcomes affect decision making for potential living kidney donors. METHODS: The Scientific Registry of Transplant Recipients was asked to determine the feasibility of a candidate registry. RESULTS: Ten living kidney donor programs evaluated 2107 consecutive kidney donor candidates; 2099 of 2107 (99.6%) completed evaluations, 1578 of 2099 (75.2%) had a decision, and 790 of 1578 (50.1%) were approved to donate as of March 12, 2020. By logistic regression, candidates most likely to be approved were married or had attended college or technical school; those least likely to be approved had ≥1 of the following characteristics: Black race, history of cigarette smoking, and higher blood pressure, higher triglycerides, or higher urine albumin-to-creatinine ratios. Reasons for 617 candidates not being approved included medical issues other than chronic kidney disease risk (25.3%), chronic kidney disease risk (18.5%), candidate withdrawal (15.2%), recipient reason (13.6%), anatomical risk to the recipient (10.3%), noneconomic psychosocial (10.3%), economic (0.5%), and other reasons (6.4%). CONCLUSIONS: These results suggest that a comprehensive living donor registry is both feasible and necessary to assess long-term outcomes that may inform decision making for future living donor candidates. There may be socioeconomic barriers to donation that require more granular identification so that active measures can address inequities. Some candidates who did not donate may be suitable controls for discerning the appropriateness of acceptance decisions and the long-term outcomes attributable to donation. We anticipate that these issues will be better identified with modifications to the data collection and expansion of the registry to all centers over the next several years.

TMPRSS11a is a novel age-altered, tissue specific regulator of migration and wound healing.

Aging is a gradual biological process characterized by a decrease in cellular and organism functions. Aging-related processes involve changes in the expression and activity of several proteins. Here, we identified the transmembrane protease serine 11a (TMPRSS11a) as a new age-specific protein that plays an important role in skin wound healing. TMPRSS11a levels increased with age in rodent and human skin and gingival samples. Strikingly, overexpression of TMPRSS11a decreased cell migration and spreading, and inducing cellular senescence. Mass spectrometry, bioinformatics, and functional analyses revealed that TMPRSS11a interacts with integrin ß1 through an RGD sequence contained within the C-terminal domain and that this motif was relevant for cell migration. Moreover, TMPRSS11a was associated with cellular senescence, as shown by overexpression and downregulation experiments. In agreement with tissue-specific expression of TMPRSS11a, shRNA-mediated downregulation of this protein improved wound healing in the skin, but not in the skeletal muscle of old mice, where TMPRSS11a is undetectable. Collectively, these findings indicate that TMPRSS11a is a tissue-specific factor relevant for wound healing, which becomes elevated with aging, promoting cellular senescence and inhibiting cell migration and skin repair.

The Microfluidic Toolbox for Analyzing Exosome Biomarkers of Aging.

As the fields of aging and neurological disease expand to liquid biopsies, there is a need to identify informative biomarkers for the diagnosis of neurodegeneration and other age-related disorders such as cancers. A means of high-throughput screening of biomolecules relevant to aging can facilitate this discovery in complex biofluids, such as blood. Exosomes, the smallest of extracellular vesicles, are found in many biofluids and, in recent years, have been found to be excellent candidates as liquid biopsy biomarkers due to their participation in intercellular communication and various pathologies such as cancer metastasis. Recently, exosomes have emerged as novel biomarkers for age-related diseases. Hence, the study of exosomes, their protein and genetic cargo can serve as early biomarkers for age-associated pathologies, especially neurodegenerative diseases. However, a disadvantage of exosome studies includes a lack in standardization of isolating, detecting, and profiling exosomes for downstream analysis. In this review, we will address current techniques for high-throughput isolation and detection of exosomes through various microfluidic and biosensing strategies and how they may be adapted for the detection of biomarkers of age-associated disorders.

Case Report: Therapeutic and immunomodulatory effects of plasmapheresis in long-haul COVID.

Many patients with COVID-19 experience a range of debilitating symptoms months after being infected, a syndrome termed long-haul COVID. A 68-year-old male presented with lung opacity, fatigue, physical and cognitive weaknesses, loss of smell and lymphocytopenia. After rounds of therapeutic plasma exchange (TPE), the patient returned to normal activities and work. Mechanistically in the patient's peripheral blood mononuclear cells (PBMCs), markers of inflammatory macrophages diminished and markers of lymphocytes, including natural killer (NK) cells and cytotoxic CD8 T-cells, increased. Circulating inflammatory proteins diminished, while positive regulators of tissue repair increased. This case study suggests that TPE has the capacity to treat long-haul COVID.

Plasma dilution improves cognition and attenuates neuroinflammation in old mice.

Our recent study has established that young blood factors are not causal, nor necessary, for the systemic rejuvenation of mammalian tissues. Instead, a procedure referred to as neutral blood exchange (NBE) that resets signaling milieu to a pro-regenerative state through dilution of old plasma, enhanced the health and repair of the muscle and liver, and promoted better hippocampal neurogenesis in 2-year-old mice (Mehdipour et al., Aging 12:8790-8819, 2020). Here we expand the rejuvenative phenotypes of NBE, focusing on the brain. Namely, our results demonstrate that old mice perform much better in novel object and novel texture (whisker discrimination) tests after a single NBE, which is accompanied by reduced neuroinflammation (less-activated CD68+ microglia). Evidence against attenuation/dilution of peripheral senescence-associated secretory phenotype (SASP) as the main mechanism behind NBE was that the senolytic ABT 263 had limited effects on neuroinflammation and did not enhance hippocampal neurogenesis in the old mice. Interestingly, peripherally acting ABT 263 and NBE both diminished SA-ßGal signal in the old brain, demonstrating that peripheral senescence propagates to the brain, but NBE was more robustly rejuvenative than ABT 263, suggesting that rejuvenation was not simply by reducing senescence. Explaining the mechanism of the positive effects of NBE on the brain, our comparative proteomics analysis demonstrated that dilution of old blood plasma yields an increase in the determinants of brain maintenance and repair in mice and in people. These findings confirm the paradigm of rejuvenation through dilution of age-elevated systemic factors and extrapolate it to brain health and function.

Author Correction: Application of bio-orthogonal proteome labeling to cell transplantation and heterochronic parabiosis.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Rejuvenation of three germ layers tissues by exchanging old blood plasma with saline-albumin.

Heterochronic blood sharing rejuvenates old tissues, and most of the studies on how this works focus on young plasma, its fractions, and a few youthful systemic candidates. However, it was not formally established that young blood is necessary for this multi-tissue rejuvenation. Here, using our recently developed small animal blood exchange process, we replaced half of the plasma in mice with saline containing 5% albumin (terming it a "neutral" age blood exchange, NBE) thus diluting the plasma factors and replenishing the albumin that would be diminished if only saline was used. Our data demonstrate that a single NBE suffices to meet or exceed the rejuvenative effects of enhancing muscle repair, reducing liver adiposity and fibrosis, and increasing hippocampal neurogenesis in old mice, all the key outcomes seen after blood heterochronicity. Comparative proteomic analysis on serum from NBE, and from a similar human clinical procedure of therapeutic plasma exchange (TPE), revealed a molecular re-setting of the systemic signaling milieu, interestingly, elevating the levels of some proteins, which broadly coordinate tissue maintenance and repair and promote immune responses. Moreover, a single TPE yielded functional blood rejuvenation, abrogating the typical old serum inhibition of progenitor cell proliferation. Ectopically added albumin does not seem to be the sole determinant of such rejuvenation, and levels of albumin do not decrease with age nor are increased by NBE/TPE. A model of action (supported by a large body of published data) is that significant dilution of autoregulatory proteins that crosstalk to multiple signaling pathways (with their own feedback loops) would, through changes in gene expression, have long-lasting molecular and functional effects that are consistent with our observations. This work improves our understanding of the systemic paradigms of multi-tissue rejuvenation and suggest a novel and immediate use of the FDA approved TPE for improving the health and resilience of older people.

Skeletal muscle as an experimental model of choice to study tissue aging and rejuvenation.

Skeletal muscle is among the most age-sensitive tissues in mammal organisms. Significant changes in its resident stem cells (i.e., satellite cells, SCs), differentiated cells (i.e., myofibers), and extracellular matrix cause a decline in tissue homeostasis, function, and regenerative capacity. Based on the conservation of aging across tissues and taking advantage of the relatively well-characterization of the myofibers and associated SCs, skeletal muscle emerged as an experimental system to study the decline in function and maintenance of old tissues and to explore rejuvenation strategies. In this review, we summarize the approaches for understanding the aging process and for assaying the success of rejuvenation that use skeletal muscle as the experimental system of choice. We further discuss (and exemplify with studies of skeletal muscle) how conflicting results might be due to variations in the techniques of stem cell isolation, differences in the assays of functional rejuvenation, or deciding on the numbers of replicates and experimental cohorts.