Multi-omic rejuvenation and lifespan extension on exposure to youthful circulation

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• Published: 27 July 2023

• Bohan Zhang,

• David E. Lee,

• Alexandre Trapp,

• Alexander Tyshkovskiy,

• Ake T. Lu,

• Akshay Bareja,

• Csaba Kerepesi,

• Lauren K. McKay,

• Anastasia V. Shindyapina,

• Sergey E. Dmitriev,

• Gurpreet S. Baht,

• Steve Horvath,

• Vadim N. Gladyshev &

• James P. White

Nature Aging volume 3, pages948–964 (2023)Cite this article

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ABSTRACT Heterochronic parabiosis (HPB) is known for its functional rejuvenation effects across several mouse tissues. However, its impact on biological age and long-term health is unknown. Here we performed extended (3-month) HPB, followed by a 2-month detachment period of anastomosed pairs. Old detached mice exhibited improved physiological parameters and lived longer than control isochronic mice. HPB drastically reduced the epigenetic age of blood and liver based on several clock models using two independent platforms. Remarkably, this rejuvenation effect persisted even after 2 months of detachment. Transcriptomic and epigenomic profiles of anastomosed mice showed an intermediate phenotype between old and young, suggesting a global multi-omic rejuvenation effect. In addition, old HPB mice showed gene expression changes opposite to aging but akin to several lifespan-extending interventions. Altogether, we reveal that long-term HPB results in lasting epigenetic and transcriptome remodeling, culminating in the extension of lifespan and healthspan.

INTRODUCTION

Aging is the primary risk factor for chronic diseases (Brett and Rando, 2014; Lopez-Otin et al., 2013). It brings accumulation of damage at many levels of biological organization and a pervasive and destructive decline of organ function, resulting in inevitable mortality. Although many attempts have been made to extend lifespan and ameliorate specific aging phenotypes through interventions, aging itself has generally been regarded as an irreversible process. Currently, there is no clear evidence that any intervention can rewind the biological age of a whole organism. With recent developments of advanced aging biomarkers based on DNA methylation (i.e., methylation clocks) (Horvath, 2013; Meer et al., 2018; Olova et al., 2019; Petkovich et al., 2017), the concept of aging as an irreversible process has been challenged via precise measurements of biological age. With their convincing assessment of the attenuated aging effect of various longevity interventions, including caloric restriction (CR), genetic models and reprogramming, methylation clocks have been generally recognized as a robust readout of organismal biological age. Notably, DNA methylation clocks have successfully predicted the reversal of biological age by several interventions, including reprogramming factor expression and treatment with drugs or blood components (Fahy et al., 2019; Horvath et al., 2020; Lu et al., 2020; Rando and Chang, 2012; Sarkar et al., 2020). Clocks have been also recently used to reveal and describe a natural rejuvenation event occurring during early embryonic development (Kerepesi et al., 2021; Trapp et al., 2021). However, in the case of interventions, it remains generally enigmatic whether the predicted reversal of biological age is sustained, correlated with longer lifespan, and manifests in improved physiological function. The heterochronic parabiosis (HPB) model has been used to study circulating factors that regulate the aging process since the 1950s (Lunsford et al., 1963; McCay et al., 1956; Pope et al., 1956). More recent work has established the model as a proof of concept that youthful circulation can restore old tissue functions (Baht et al., 2015; Conboy et al., 2005). Indeed, the effects of HPB on the amelioration of aging phenotypes are evident across tissues including muscle (Conboy et al., 2005), liver (Conboy et al., 2005), heart (Loffredo et al., 2013), brain (Ruckh et al., 2012; Villeda et al., 2014) and bone (Baht et al., 2015; Vi et al., 2018). Remarkably, these effects are observed typically after only 4-5 weeks of parabiosis. Similar results are observed with acute heterochronic blood exchange (non-parabiosis), showing beneficial effects of “young blood” on muscle, liver and brain of old recipients (Rebo et al., 2016). Heterochronic young blood plasma transfer also improves pathology of age-related diseases, such as in a model of Alzheimer’s disease in mice (Middeldorp et al., 2016). Although HPB leads to diverse effects on old cells and tissues, our understanding of the molecular mechanisms involved remains limited. Likewise, whether the immediate effects of blood/plasma exchange are sustained after the procedure is still unknown. Lastly, due to the previous absence of a precise quantification method, it remains unclear whether HPB can slow or rewind the biological aging of organisms. A previously reported (Conboy et al., 2013; Wright et al., 2001), but seldomly used HPB procedure is the detachment of mice following parabiosis. This technique has previously been used to verify cell engraftment using different tracer techniques (Donskoy and Goldschneider, 1992). A recent investigation revealed the persistence of aged hematopoietic stem cells in the young bone marrow niche resulting from HPB months after a surgical separation of parabionts (Ho et al., 2021); however, to our knowledge there have been no studies to investigate longevity or long-term effects on healthspan. Although detachment involves performing a second surgery on the animals, it permits physiological and longevity measurements, which are difficult to obtain in the case of anastomosed mice. Here, we report the results of a long-term HPB study followed by a detachment period. We found that old mice detached from young mice showed an extended lifespan and improvements across several dimensions of aging biology. By comprehensive epigenetic clock and RNA-seq analyses, we observed a robust reduction in biological age of old mice following 3-month HPB, sustained even after a detachment period of two months. Notably, this rejuvenation effect was significantly stronger than that observed upon short-term HPB. We find that transcriptomic and epigenetic profiles of long-term HPB are intermediate between young and old isochronic pairs, and that HPB positively associates with the effects of common lifespan-extending interventions and counteracts aging-related gene expression changes. Our findings suggest the presence of profound and persisting molecular rejuvenation effects following exposure to young circulation, leading to extended lifespan and healthspan.

RESULTS

Long-term parabiosis followed by detachment extends lifespan and healthspan in mice We used a long-term (3-month) parabiosis (either heterochronic or isochronic) period in mice, followed by detachment of the parabionts. Young mice started parabiosis at 3 months and old mice at 20 months of age (Figure 1A). Transcriptomic and epigenetic profiling of the liver and blood was conducted to assess molecular changes resulting from HPB (Figure 1B). For longevity and healthspan experiments, mice were detached and allowed 1 month of recovery after parabiosis prior to physiological data collection (Figure S1A). After separation from respective parabiosis pairs, some mice were allowed to live freely until their natural death, in order to examine the effect of parabiosis on lifespan. We observed a significant 6-week extension in median lifespan and a 2-week extension in maximum lifespan in old mice detached from heterochronic pairs as compared to their isochronic controls (Figure 1C). This was accompanied by an initial reduction in body weight coupled with better preservation of body weight in the final months of life (Figure 1D). The initial drop in body weight appeared to be primarily caused by a reduction in fat mass, while the preservation of body weight was due to the maintenance of both lean and fat mass later in life (Figure 1D). These changes in body composition were independent of changes in food consumption (Figure S1B). In addition to improvements in body composition, old heterochronic mice showed higher voluntary cage activity than isochronic controls (Figure S1C). While it is possible that old heterochronic mice received some small training effect due to attachment to young, more active partners, mouse physical capacity decreases rapidly following aerobic exercise training, returning to pretraining levels by 4 weeks (Brace et al., 2016). This detraining would be apparent by the second month following detachment, if the training were the sole contributor to the increased physical activity; however, the observed increases in cage activity persist for at least 7-9 months (Figure 1D, Figure S1C).

Epigenetic age of old mice is reversed by parabiosis with a sustained effect after detachment.

For epigenetic analyses, we subjected mice to the same prolonged attachment, and harvested tissues either immediately after the 3-month parabiosis procedure, or 2 months after detachment (Figure S1A). We first subjected the blood and liver of the animals to Reduced Representation Bisulfite Sequencing (RRBS)(Meissner et al., 2005) (Figure 2A, Table S1). Since blood samples taken immediately after detachment contained a mixture of old and young blood (Figure S2), we initially chose to quantify methylation changes in blood after 2-month detachment to investigate whether epigenomic remodeling upon exposure to young circulation persists without young blood contamination. Additionally, we took liver as an example of a solid tissue to quantify the indirect effects on the methylome happening immediately after parabiosis and following 2-month detachment. To quantify the biological age of the animals, we applied four RRBS-based epigenetic clocks to 6 different groups of mice: young isochronic, old isochronic, and old heterochronic mice, as well as detached variants of all these three groups. Clocks used included two recently developed multi-tissue clocks (Meer et al., 2018; Thompson et al., 2018), a blood-specific clock (Petkovich et al., 2017), and the first single-cell clock framework (Trapp et al., 2021), which was modified to accommodate and profile epigenetic age in bulk data. Application of these clocks to blood RRBS data revealed a profound epigenetic age decrease (19-28%) when comparing old isochronic and heterochronic pairs. Remarkably, this effect was sustained even after two months of detachment (reduction in age by 16-32%) (Figure 2B). Application of these same four clocks to liver data showed comparable immediate (5-26%) and sustained (10-26%) epigenetic age reduction as observed in blood (Figure 2C). Together, these results indicate that long-term HPB rejuvenates the epigenome compared to isochronic pairs in both blood and liver. Moreover, these unbiased rejuvenation signals in solid organs (such as the liver) further suggest that long-term HPB acts in a systemic manner, leading to global epigenomic remodeling and organism-wide age reversal. (Con't...)