Fight Aging! Newsletter, June 15th 2020

Historically, the phenomena of heterochronic parabiosis and blood exchange remained unconfirmed with respect to the key assumption as to whether the addition of young factors is needed for rejuvenation, and if premature aging of young mice stemmed from the introduction of old blood factors or a simple dilution of young factors. To answer these questions in a well-controlled experimental set-up, we took advantage of our recently developed small animal blood exchange model.

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.

Aging, defined as irreversible deterioration of physiological processes of organisms over time, is characterized by nine hallmarks: cellular senescence, mitochondrial dysfunction, deregulated nutrient sensing, epigenetic alterations, telomere attrition, genomic instability, altered intercellular communication, and stem cell exhaustion. Among these, cellular senescence has recently been focused on as one of the key factors in the complex aging process as it is interlinked with other hallmarks. Senescent cells are accumulated in tissues of vertebrates with age. Interestingly, removal of senescent cells in animals results in the delayed onset of age-associated diseases.

Senescence is characterized by a stable cell-cycle arrest in the G1 phase and an inflammatory response called senescence-associated secretory phenotype (SASP), which modifies the microenvironment around senescent cells. Components of the SASP include growth factors, pro-inflammatory cytokines, chemokines, and extracellular matrix remodeling enzymes. SASP contributes to inflammaging, a term that describes low-grade, controlled, asymptomatic, chronic, and systemic inflammation associated with aging processes. Evidence points out that inflammaging may ultimately lead to age-related diseases. Thus, interventions that suppress SASP and inflammaging processes may hold potential to alleviate various chronic diseases.

It has been elusive that circulating mediators are responsible for rejuvenating multiple tissues of old organisms by parabiosis of young organisms. Very recently, it was demonstrated that extracellular vesicles (EVs) from young mice plasma extend the lifespan of old mice by delaying aging through exosomal nicotinamide phosphoribosyl transferase (eNAMPT). Another study also reported that exosomes from young mice could transfer miR-126b-5p to tissue of old mice, and reverse the expression of aging-associated molecules. Another report revealed that EVs derived from serum of young mice attenuated inflammaging in old mice by partially rejuvenating aged T-cell immunotolerance. Implantation of hypothalamic stem cells or progenitor cells, which were genetically engineered to survive from aging-related hypothalamic inflammation, was reported to induce retardation of aging and extension of lifespan in mid-aged mice.

More importantly, growing evidence suggests that cellular senescence can be alleviated or reversed by EVs or exosomes derived from stem cells. For example, human exosomes reduced the high glucose-induced premature senescence of endothelial progenitor cells (EPCs) and enhanced wound healing in diabetic rats. Taken together, mesenchymal stem cell derived exosomes confer anti-senescence effects through their unique miRNA, lnRNA, and enzyme contents. By inducing proliferation and reducing SASP in senescent cells, they hold great potential to reduce senescent cells in tissues. Since removal of senescent cells from tissues was reported to create a pro-regenerative environment and tissue homeostasis, application of mesenchymal stem cell derived exosomes to remove the senescent cells may be a preferable approach to induce the regeneration or rejuvenation of tissues.

Substantial evidence has demonstrated that the accumulation of senescent cells can drive many age-associated phenotypes and pathologies. For example, senescent cells accumulate in adipose tissue of patients with diabetes and age-related metabolic dysfunction, in osteoarthritic joints, in the aorta in vascular hyporeactivity and atherosclerosis, and in the lungs in idiopathic pulmonary fibrosis. While selective elimination of these senescent cells confers notable benefits in some tissues, recent studies have also described beneficial roles for senescent cells, raising the question of the differential roles of these cells in various tissues.

Senescence-ablator mouse models have pioneered the field of in vivo senescence studies. With the use of one such model, known as the INK-ATTAC mouse, that is based on a 2,617 base pair fragment of the p16Ink4a gene promoter, it has been proposed that removal of p16-expressing cells results in life extension in mice. The concern, however, is whether the reporter construct with a part of the p16 genomic sequence fully resembles endogenous p16 gene expression, especially with aging. This is further supported by the fact that some p16-expressing cells are not efficiently removed by the INK-ATTAC system in several tissues, including the liver, colon, and T lymphocytes. Thus, it is unclear whether there are important senescent cell types in tissues where the INK-ATTAC system does not work and what impact their removal has on health span.

Here, we generated two knock-in mouse models targeting the best-characterized marker of senescence, p16Ink4a. Using a genetic lineage tracing approach, we found that age-induced p16High senescence is a slow process that manifests around 10-12 months of age. The majority of p16High cells were vascular endothelial cells mostly in liver sinusoids (LSECs), and to lesser extent macrophages and adipocytes. In turn, continuous or acute elimination of p16High senescent cells disrupted blood-tissue barriers with subsequent liver and perivascular tissue fibrosis and health deterioration. Our data show that senescent LSECs are not replaced after removal and have important structural and functional roles in the aging organism. In turn, delaying senescence or replacement of senescent LSECs could represent a powerful tool in slowing down aging.

Regulation of cellular homeostasis is pivotal for the survival of a cell. Highly conserved mechanisms have evolved which enable cells to cope with various environmental stresses that often disrupt cellular homeostasis. Sequestration of nontranslating mRNAs into stress granules (SGs) is one such mechanism that attenuates protein synthesis during stress. Stress granules are cytosolic assemblies consisting of nontranslating mRNAs, small 40S ribosomes, mRNA-associated translation initiation complexes, and RNA-binding proteins.

It has been suggested that the SGs function as triage sites redirecting mRNAs to either translation, sequestration, or degradation. Therefore, SG assembly represents a key role in protein and RNA homeostasis under adverse conditions and is a tightly regulated process. Not surprisingly, dysregulation of SG dynamic has recently been linked to various diseases, such as cancer, inflammatory, neurodegenerative, and neuromuscular diseases.

It has been previously demonstrated that SG formation enhances cell survival and stress resistance. However, the physiological role of SGs in organismal aging and longevity regulation remains unclear. In this study, we used markers to monitor the formation of SGs in Caenorhabditis elegans. We found that, in addition to acute heat stress, SG formation could also be triggered by dietary changes, such as starvation and dietary restriction (DR). We found that HSF-1 is required for the SG formation in response to acute heat shock and starvation but not DR, whereas the AMPK-eEF2K signaling is required for starvation and DR-induced SG formation but not heat shock. Moreover, our data suggest that this AMPK-eEF2K pathway-mediated SG formation is required for lifespan extension by DR, but dispensable for the longevity by reduced insulin/IGF-1 signaling.

Glial cells play several key roles in the central nervous system, including supplying oxygen to neurons and forming myelin - the protective, fatty substance that protects the nerve cells' axons. In multiple sclerosis (MS), glial cells called oligodendrocytes are attacked by the immune system, causing a breakdown of myelin that disrupts the signals between nerve cells and results in a loss of motor and sensory functions.

Researchers are developing a method for regenerating myelin with progenitor glial cells. When they transplanted the cells into mouse models of MS, the cells transformed into new oligodendrocytes and restored myelin. Now, a company that was spun out last year, Oscine Therapeutics, is preparing the cell therapy for human clinical trials in MS and other glial diseases.

In the mouse study, researchers showed that after transplantation, the human glial progenitor cells migrated to damaged areas of the brain. After they created new oligodendrocytes, myelation was restored, as was motor function. Much of the regenerative medicine research in MS is focused on restoring myelin, and several different approaches are under investigation. Last year, researchers reported that when they implanted stem cells with the surface protein CD34 into mouse models of MS, the cells grew into myelin-forming glial cells. Other experimental approaches to regenerating myelin include using microRNAs and reprogrammed skin cells.

Aging clocks dissociate biological from chronological age. The estimation of biological age is important for identifying gerontogenes and assessing environmental, nutritional, or therapeutic impacts on the aging process. Recently, methylation markers were shown to allow estimation of biological age based on age-dependent somatic epigenetic alterations. However, DNA methylation is absent in some species such as Caenorhabditis elegans and it remains unclear whether and how the epigenetic clocks affect gene expression. Aging clocks based on transcriptomes have suffered from considerable variation in the data and relatively low accuracy.

Here, we devised an approach that uses temporal scaling and binarization of C. elegans transcriptomes to define a gene set that predicts biological age with an accuracy that is close to the theoretical limit. Our model accurately predicts the longevity effects of diverse strains, treatments, and conditions. The involved genes support a role of specific transcription factors as well as innate immunity and neuronal signaling in the regulation of the aging process. We show that this transcriptome clock can also be applied to human age prediction with high accuracy. This transcriptome aging clock could therefore find wide application in genetic, environmental, and therapeutic interventions in the aging process.

Christian Schafmeister's academic research is now taking its first steps towards commercialisation in the form of his new start-up - ThirdLaw Technologies. The company seeks to harness the power of 'spiroligomers' to rapidly build new small molecules. "We are starting up a company to develop an artificial immune system. We're making very large libraries of artificial molecules that could bind to protein surfaces. Proteins are these long chains of amino acids that fold into a three dimensional shape and do something amazing - I wanted to build molecules like that. But the problem with proteins and every other approach to this is trying to figure out how this long flexible molecule will fold into a three dimensional shape."

Schafmeister came up with the idea for spiroligomers - using building blocks that are like amino acids but, instead of connecting through one bond, which allows rotational flexibility, his building blocks connect through two bonds. "So you make building blocks that are rings, and you connect them through rings, and so you build ladder molecules,. And you could programme the shape of those ladder molecules by controlling their stereochemistry, the shapes of the rings, and how you put them together."

By achieving this, Schafmeister is able to create molecules that present as reactive groups like the side chains of amino acids, and hold them in a particular three dimensional constellation. This enables spiroligomers to do things like bind protein surfaces, or point them inwards to create pockets that enable catalysis and speed-up select chemical reactions. "The clearest aging-related application for spiroligomers is developing catalysts that can cleave the glucosepane crosslinks. That's a clear goal and something that I think is unique to what we're doing. We could do it in the next year - but it's just a question of resources."

Astrocytes, star-shaped cells that make up more than half the cells in the central nervous system, belong to a category of brain cells called glia which provide vital support for neurons in the brain. Astrocytes aid in metabolic processes, regulate connectivity of brain circuits, participate in inflammatory signaling, and help regulate blood flow across the blood-brain barrier, among other duties. They are a crucial component of brain function but are often overlooked in research and drug development, although recent mounting evidence implicates them in many neurological diseases.

"We observed in mice that astrocytes in inflammatory environments take on a reactive state, actually attacking neurons rather than supporting them. We found evidence of reactive astrocytes in the brains of patients with neurodegenerative diseases, but without a human stem cell model, it wasn't possible to figure out how they were created and what they are doing in patient brains."

Researchers used a new human stem cell model to determine if the outcome observed in mice could also be happening in humans. They exposed healthy stem-cell-derived astrocytes to inflammation - essentially mimicking the environment of the brain in neurodegenerative diseases - collected their byproducts, and then exposed these secretions to healthy neurons. "What we saw in the dish confirmed observations in mice: the neurons began to die. Observing this 'rogue astrocyte' phenomenon in a human model of disease suggests that it could be happening in actual patients and opens the door for new therapeutics that intervene in this process."

The team also saw that stem-cell-derived astrocytes exposed to inflammation lost their typical astrocyte functions: they did not support neuronal maturation or firing very well, and they didn't uptake as much glutamate. They also changed their morphology, losing their characteristic 'long arms' and taking on a more constricted star-like shape. "Along with secreting a toxin that kills neurons, we also saw that stem-cell-derived astrocytes in disease-like environments simply do not perform their typical jobs as well, and that could lead to neuronal dysfunction. For example, since they do not take up glutamate properly, too much glutamate is likely left around the neurons, which could cause a neuron to atrophy, and that's something we can potentially target in new therapies."

Using skin cells from human volunteers, researchers have created fully functional mini livers, which they then transplanted into rats. In this proof-of-concept experiment, the lab-made organs survived for four days inside their animal hosts. These mini livers secrete bile acids and urea, just like a normal liver, except they're made-to-order in the lab using patient cells. And, although liver maturation takes up to two years in a natural environment, the researchers did it in under a month.

As an ultimate test, the researchers transplanted their lab-grown mini livers into five rats, who were bred to resist organ rejection. Four days after the transplant, researchers investigated how well the implanted organs were faring. In all cases, blood flow problems had developed within and around the graft, but the transplanted mini livers worked - the rats had human liver proteins in their blood serum.

Researchers are optimistic that this research is not merely a stepping-stone on the path toward growing replacement organs in a lab, but also a useful tool in its own right. "The long-term goal is to create organs that can replace organ donation, but in the near future, I see this as a bridge to transplant. For instance, in acute liver failure, you might just need hepatic boost for a while instead of a whole new liver."

Reperfusion therapy, the gold standard for stroke treatment, helps restore blood flow after a stroke caused by a clot, preventing loss of brain tissue. However, only about 10% of stroke patients qualify, in part because of reperfusion therapy's narrow treatment window. New research suggests that this therapy could be both safer and more effective for both motor and cognitive recovery if administered with a specialized compound that blocks the immune response. Reducing the immune response in the brain could be a strategy for improving cognitive recovery. It could also extend the treatment window for therapy, allowing stroke specialists to help many more stroke patients.

"With reperfusion therapy, we're restoring the blood flow, which is necessary to save the tissue, but there is an ongoing inflammatory response by the immune system that is not targeted by reperfusion." This could explain why, though mechanical reperfusion has a success rate of 90% in returning blood flow to the brain, only about 40% of treated patients recover enough motor and reasoning skills within three months to tend to their daily needs independently. Even those who do recover motor function can still struggle with cognitive challenges months later.

During a stroke, the oxygen and energy supply to the brain is cut off by a clot, causing brain tissue to become stressed and die rapidly. Just as it is with a cut to the knee, the immune system is activated to heal the wound, which includes clearing the dead tissue. A family of special immune proteins called complement proteins help to guide and promote this immune response in the damaged areas. These complement proteins flag both dead tissue and stressed brain cells for removal. The stressed brain cells are not yet dead, only damaged by lack of oxygen and energy, and thus salvageable tissue is destroyed by the immune system.

Researchers developed a complement protein blocker, named B4Crry, which acts only at the site of stroke injury. This compound blinds the complement proteins to the signals of stressed brain cells, saving the stressed tissue and reducing overall brain damage. In a mouse model of stroke, reperfusion therapy alone did improve recovery of coordinated movements such as walking. With the addition of B4Crry to treatment, coordinated movement improved even faster, with greater recovery seen as early as three days after the stroke. The improvements to learning and memory were even greater than those seen with motor functions. Reperfusion therapy alone was equal to no treatment at all for learning and memory recovery after stroke. However, when B4Crry was added to their treatments, mice had greatly improved cognitive recovery, making three times fewer errors on a learning and memory task.

After stroke, brain immune cells called microglia began eating the connections between stressed brain cells. Immune system complement proteins were marking these connections for destruction because they displayed the stressed cell signal. Without these connections, brain cells cannot communicate efficiently, and overall brain function decreases. B4Crry concealed the cells' stress signals from the complement proteins and thereby saved the connections between neurons. Preserving connectivity improved learning and memory brain function after stroke.

The main cause of blindness in industrialized countries is the degeneration of photoreceptors, including age-related macular degeneration and retinitis pigmentosa. During the progression of degenerative photoreceptor diseases, light-sensitive and light-insensitive photoreceptor regions in the retina coexist. For example, macular degeneration patients lose vision in the central portion of their retina but retain peripheral eyesight.

Scientists have now succeeded in developing a new therapeutic approach to restore light sensitivity in degenerating retina without negatively affecting remaining vision. They were inspired by species found in nature, such as bats and snakes, that can localize near-infrared light emitted by the bodies of their preys. This is done by using heat-sensitive ion channels which are able to detect the heat of the near-infrared light. This enables the bats and snakes to superimpose thermal and visual images in the brain and thus react to their environment with greater precision.

To equip retinal photoreceptors with near-infrared sensitivity, the researchers devised a three-component system. The first component contains engineered DNA that ensures that the gene coding for the heat-sensitive channel is only expressed in photoreceptors. The second component is a gold nanorod, a small particle, that efficiently absorbs near-infrared light. The third component is an antibody that ensures strong binding between the heat-sensitive channel expressed in photoreceptors and the gold nanorods that locally capture near-infrared light and locally release heat.

The researchers first tested their system in engineered mice with retinal degeneration, confirming that near-infrared light effectively excites photoreceptors and that this signal is transmitted to retinal ganglion cells, the latter representing the output of the retina towards higher visual centers in the brain. Next, they showed that stimulating the mouse eye with near-infrared light is also picked up by neurons in a brain area that is important for conscious vision, the primary visual cortex. They also designed a behavioral test in which untreated blind mice were not able to use near-infrared stimulation to learn a simple task whereas blind mice treated with the three-component system could perform the task related to near-infrared stimulus.

The matrix metalloproteinase (MMP) group of proteins controls a large variety of key physiological and pathological processes, including tissue remodelling, DNA replication, cell-cycle progression, neurodegeneration, and cancer. MMP-2 is constitutively expressed in several tissues and is tightly associated with inflammatory states such as osteoarthritis. MMP-9 is implicated in lipid metabolism and its activity contributes to endothelial dysfunction.

In order to gain insight into the pathophysiology of ageing and to identify new markers of longevity, we analysed the activity levels of MMP-2 and MMP-9 in association with some relevant haematochemical parameters in a Sicilian population, including long-living individuals (LLIs, ≥95 years old). A cohort of 154 healthy subjects (72 men and 82 women) of different ages (age range 20-112) was recruited. The cohort was divided into five subgroups: the first group with subjects less than 40 years old, the second group ranging from 40 to 64 years old, the third group ranging from 65 to 89 years old, the fourth group ranging from 90 to 94 years old, and the fifth group with subjects more than 95 years old.

A relationship was observed between LLIs and MMP-2, but not between LLIs and MMP-9. However, in the LLI group, MMP-2 and MMP-9 values were significantly correlated. Furthermore, in LLIs, we found a positive correlation of MMP-2 with the antioxidant catabolite uric acid and a negative correlation with the inflammatory marker C-reactive protein. Finally, in LLIs MMP-9 values correlated directly both with cholesterol and with low-density lipoproteins. On the whole, our data suggest that the observed increase of MMP-2 in LLIs might play a positive role in the attainment of longevity. This is the first study that shows that serum activity of MMP-2 is increased in LLIs as compared to younger subjects.

Long-lived mutant mice, such as Ames dwarf, Snell dwarf, and GKO mice, have increased percent body fat and abnormal fat distribution, with preservation of subcutaneous and relatively less visceral fat compared to controls, raising the idea that altered function of adipose tissue within these mice may contribute to their insulin sensitivity, longevity, and disease resistance. To delineate the effects of GH on specific tissues, we evaluated adipose tissue in mice with global disruption of GHR (GKO mice), as well as mice with disruption of GHR in liver (LKO), muscle (MKO), or fat (FKO).

Based on the structure and function of adipocytes and their surrounding stroma, adipose tissue is divided into two categories, white adipose tissue (WAT) and brown adipose tissue (BAT). Its function is to store excess energy in the form of triglycerides for future use. BAT is responsible for dissipating energy in the form of heat through non-shivering thermogenesis. Adipose tissue also influences the activity of macrophages, T cells, B cells, mast cells, dendritic cells, and neutrophils. The inflammatory response of adipose tissue is mainly regulated by macrophages. M1 macrophages produce pro-inflammatory cytokines. In contrast, M2 macrophages are anti-inflammatory and help to maintain tissue homeostasis. In principle, delay or reversal of M1/M2 macrophage polarization might contribute to the insulin sensitivity, disease resistance, and longevity of Ames, Snell, or GKO mice, but no data on this point are yet available.

We report here that white (WAT) and brown (BAT) fat have elevated UCP1 in both kinds of mice, and that adipocytes in WAT depots turn beige/brown. These imply increased thermogenesis and are expected to lead to improved glucose control. Both kinds of long-lived mice show lower levels of inflammatory M1 macrophages and higher levels of anti-inflammatory M2 macrophages in BAT and WAT, with correspondingly lower levels of inflammatory cytokines. Experiments with mice with tissue-specific disruption of GHR showed that these adipocyte and macrophage changes were not due to hepatic IGF1 production nor to direct growth hormone (GH) effects on adipocytes, but instead reflect GH effects on muscle. Muscles deprived of GH signals, either globally (GKO) or in muscle only (MKO), produce higher levels of circulating irisin and its precursor FNDC5. The data thus suggest that the changes in adipose tissue differentiation and inflammatory status seen in long-lived mutant mice reflect interruption of GH-dependent irisin inhibition, with consequential effects on metabolism and thermogenesis.

One of the most universal hallmarks of aging is the decline in protein homeostasis. Studies in a variety of organisms have uncovered age-dependent accumulation of misfolded and damaged proteins, which may impair cell function and homeostasis, leading to the development of age-related diseases. Misfolded, aggregated and damaged proteins can be removed by the proteasome or cleared through the autophagy-lysosome pathway. As the key organelle for cellular degradation, lysosomes exhibit age-related changes. In order to understand whether and how lysosomes alter with age and contribute to lifespan regulation, we characterized multiple properties of lysosomes during the aging process in C. elegans.

We uncovered age-dependent alterations in lysosomal morphology, motility, acidity, and degradation activity, all of which indicate a decline in lysosome function with age. The age-associated lysosomal changes are suppressed in the long-lived mutants daf-2, eat-2, and isp-1, which extend lifespan by inhibiting insulin/IGF-1 signaling, reducing food intake and impairing mitochondrial function, respectively. We found that 43 lysosome genes exhibit reduced expression with age, including genes encoding subunits of the proton pump V-ATPase and cathepsin proteases. The expression of lysosome genes is upregulated in the long-lived mutants, and this upregulation requires the functions of DAF-16/FOXO and SKN-1/NRF2 transcription factors. Impairing lysosome function affects clearance of aggregate-prone proteins and disrupts lifespan extension in daf-2, eat-2, and isp-1 worms.

Our data indicate that lysosome function is modulated by multiple longevity pathways and is important for lifespan extension. Further studies are required to understand whether lysosomes make tissue-specific contributions to aging and lifespan extension.