Hearing is essential for echolocating bats that rely extensively on their auditory systems to forage, navigate, and avoid obstacles. The evolution of echolocation in bats has been correlated with adaptations at all levels of auditory processing to enable active acoustic sensing of complex and dynamic environments. Consequently, echolocating species are exposed to intense self-generated sounds. Further, many species form high-density aggregations, where sonar sounds emitted by other individuals may be potentially damaging to the cochlea. The critical role of hearing in the fitness and survival of echolocating bats suggests that the evolution of this active sensing system may have introduced selective pressures to protect the auditory system from damage over a lifetime of exposure to sound.

The aging auditory system in most mammals shows a progressive loss of hearing sensitivity that begins with high-frequency deficits and extends to low frequencies over time. Although the etiology of age-related hearing loss (ARHL) is highly variable, depending on genetic, epigenetic, and environmental factors, its onset is generally correlated with senescent changes to the peripheral structures of the auditory system, including loss of inner and outer hair cells, loss of ribbon synapses and retraction of auditory nerve fibers (i.e., cochlear synaptopathy), and deterioration of the stria vascularis. The molecular mechanisms underlying ARHL are hypothesized to result from inter-related metabolic and physiological changes over the lifespan that lead to the accumulation of reactive oxygen species and increase susceptibility to cellular dysfunction.

We hypothesize that echolocation imposes selective pressures to preserve hearing function across the lifespan, especially in species that require echolocation-based active sensing for prey capture. Although bats are not immune to hearing loss, and indeed, some species appear vulnerable to ARHL, recent evidence indicates that species differences in echolocation behaviors may correlate with differential susceptibility to hearing loss. For example, echolocating bat species have shown evidence for resistance to noise-induced cochlear hair cell damage whereas non-echolocating visually dominant species were susceptible to acoustic overexposure and showed levels of hair cell loss comparable to that observed in mice.

In this study, we used DNA methylation to estimate the ages of wild-caught big brown bats (Eptesicus fuscus) and measured hearing sensitivity in young and aging bats using auditory brainstem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs). We found no evidence for hearing deficits in aging bats, demonstrated by comparable thresholds and similar ABR wave and DPOAE amplitudes across age groups. We additionally found no significant histological evidence for cochlear aging, with similar hair cell counts, afferent, and efferent innervation patterns in young and aging bats. Here we demonstrate that big brown bats show minimal evidence for age-related loss of peripheral hearing sensitivity and therefore represent informative models for investigating mechanisms that may preserve hearing function over a long lifetime.

Translation of academic findings into practical applications within industry and clinical context is of the utmost importance within the field of longevity. The hallmarks of ageing have played a key role in paving the way for providing mechanisms for therapeutics to target and modify them for lowering biological age and enhancing longevity, attracting interest from academia, industry, and investors. Several speakers presented various data on the pharmacological and nutraceutical interventions currently under investigation in clinical trials, or in the pipeline.

Metformin and rapamycin, and its analogues (rapalogs), are among the most extensively studied longevity compounds, targeting AMPK and mTOR, respectively, as discussed by multiple speakers. Repurposing existing drugs has shown promise in combating ageing. These compounds exhibit a range of beneficial effects, including immunomodulation and alteration of cellular metabolism mechanisms, in both in vitro and in vivo models of healthy ageing. However, for their prescription solely for longevity purposes, further research is warranted to determine appropriate dosages and potential impacts on other bodily systems.

In addition to repurposing existing drugs, there are ongoing efforts to identify novel compounds and devise effective therapeutic regimes using machine learning and network pharmacology. Longevity and ageing research has necessitated a shift in disease research from reductionist to systems theory. Joao Pedro Magalhaes (University of Birmingham) discussed his research and the emergence of network pharmacology and in silico models, incorporating various disciplines such as systems biology, genomics, and proteomics, among others. By examining drug-network interactions through omics data analysis and network database retrieval, network pharmacology provides comprehensive insights into drug mechanisms and efficacy, making it particularly suitable for investigating longevity therapeutics.

A growing array of supplements, such as NAD+ and its precursors for cellular energy, calorie restriction mimetics for fasting-like effects, and other bioactives and nutraceuticals, are being explored for their potential anti-ageing benefits. These compounds target specific pathways or provide broad antioxidant and anti-inflammatory properties, which can alleviate age-related damage and promote overall health. Despite being promoted by numerous commercial entities, clinicians may exhibit hesitancy due to less stringent regulatory processes.

At the cellular level, senolytics are a class of drugs that aim to interfere with senescent cells. Several speakers outlined the process of cellular senescence and its contribution to unhealthy ageing via mechanisms including disrupting tissue functionality and limiting the regenerative potential of adult stem cells. The accumulation of senescent cells leads to an increase in biological age and increases the risk of disease. Senolytics hold promise in eliminating senescent cells, and in model organisms, they have shown potential to extend lifespan, enhance healthspan, and treat or even reverse age-related diseases.

Advancing to the systemic level, dysbiosis and chronic inflammation, recently recognised hallmarks of ageing, are interconnected and responsive to dietary interventions, as elucidated by Richard Siow (King's College London). Bioactive compounds in whole foods, potentially acting synergistically to mitigate age-related changes and enhance vitality pathways, are currently being investigated by commercial entities to isolate and validate them in vivo. Siow also emphasised the significant impact of dietary choices on rates of functional decline. With the emergence of longevity nutrition frontiers such as nutrigenomics, personalised nutritional regimens tailored to individuals' genetic profiles are now feasible, enhancing the effectiveness of interventions.

To promote longevity and lower biological age, pharmaceuticals, nutraceutical supplements, and other interventions should be considered as part of a comprehensive anti-ageing regimen that incorporates other complementary protocols. These may include adopting a healthful diet, engaging in regular exercise and resistance training, practising intermittent fasting, managing stress effectively, and optimising sleep patterns.

Renal transmembrane protein αKlotho has several important functions. On the one hand, it serves as a co-receptor for phosphaturic hormone fibroblast growth factor 23 (FGF23), which is mainly produced in bone. On the other hand, enzymatic cleavage of transmembrane αKlotho results in an extracellular form, called soluble Klotho (sKL), which exerts endocrine and paracrine effects in several tissues and organs. FGF23 exerts further effects in other organs including heart and is correlated with outcomes in kidney and cardiovascular disease.

The joint action of FGF23 and αKlotho in the kidney is pivotal for phosphate and vitamin D metabolism. Lack of either FGF23 or αKlotho results in massive phosphate and active vitamin D excess in mice, causing a phenotype of rapid aging with a plethora of aging-associated diseases that are reminiscent of human aging and affect almost all tissues and organs. Conversely, overexpression of αKlotho has powerful antiaging effects, expanding the life span by about 30% in mice. αKlotho has also been demonstrated to be highly beneficial in several acute and chronic disorders. Further putatively health-promoting effects of αKlotho may include the reduction in oxidative stress or anti-inflammatory effects to name a few.

AMP-dependent kinase (AMPK) is basically expressed in all cell types and consists of three subunits, α, β, γ. Physiologically, it is activated by increase in cellular AMP concentration, indicating lack of ATP and hence energy deficiency. In rough summary, AMPK reduces cellular processes consuming energy and induces pathways providing energy. Higher AMPK activity is associated with some remarkable health benefits. These may include the protection of the heart during ischemia, the reduction of microvascular disease in diabetes, or nephroprotection in insulin resistance. These beneficial effects are largely attributed to improvements in cell metabolism or stimulation of autophagy.

Given that both αKlotho and AMPK have beneficial effects in similar organs, we studied whether AMPK regulates αKlotho gene expression in Madin-Darby canine kidney cells, normal rat kidney 52E cells, and human kidney 2 cells. We measured αKlotho expression upon pharmacological manipulation or siRNA-mediated knockdown of AMPKα. AMPK activator 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) enhanced αKlotho expression, an effect reduced in the presence of AMPK inhibitor compound C or siRNA targeting AMPK catalytic subunits. Similarly, AMPK activators metformin and phenformin upregulated αKlotho transcripts. Taken together, our results suggest that AMPK is a powerful inducer of αKlotho and could thereby contribute to the development of future therapeutic interventions.

As it says on the first slide here, we have achieved significant reductions in the burden of atherosclerotic plaque in mice and we intend to continue the way we started. With respect to the current standard of care for atherosclerosis, the big problem here, the thing that I'm going to beat you over the head with for five minutes or so, is that present treatments cannot greatly, rapidly, or reliably reduce the existing burden of plaque. But we can, and that is our value proposition.

Atherosclerosis is probably going to kill you if nobody does something about it. The rupture of fatty plaque that grows in blood vessels kills 27% of us directly via heart attack or stroke - and that is in a world in which everybody who can use statins is using statins. Further, that doesn't account for the people who are killed less directly by the reduced blood flow to the heart that leads to heart failure. In short, atherosclerosis is not a good thing and is the greatest single cause of of human mortality, greater even than than cancer. The more plaque you have, the bigger the problem: looking at this great data from a 2004 Dutch study, your mortality risk is five times greater if you have five or six plaques evident in imaging than somebody who doesn't have those plaques. You will notice the mortality risk is a very large range, by the way, as the type of plaque matters greatly; how much fat is in it, how much cholesterol is stuck in that plaque, how likely is it to rupture. Either way if your cardiologist can see five plaques in your major arteries you are not in a good place. You want to do something about it, but today what can you do about it? Not an awful lot.

Let me call your attention to two meta-analysis studies as good examples - there are any number of others we could look at - in which researchers looked over several dozen studies and thousands of patients. If you look at the numbers on the right, the percent atheroma volume is the percentage of the volume of the artery that's obstructed by the plaque. We are looking for percentage change in that in that number. You want it lower, but if after 18 months of statin therapy the outcome is a range of minus 5% to plus 3%, well, that is not a great form of treatment. The standard mean difference is even worse, as it is essentially zero! So if you take statins for 18 months then your existing plaque is still going to be there at the end of the day. But the plaque is the story! It is the most important part of this, because there are large studies that show that if you can produce a 1% reduction in your percent atheroma volume - just 1% - then the the reduction in cardiovascular event risk is much bigger, on the order of 20%. But that takes 18 months or thereabouts and only a subset of patients actually achieve that outcome no matter how low their blood cholesterol. There are people taking combinations of the modern PCSK9 inhibitors and statins and despite very low LDL cholesterol levels they have existing plaque and it's going to stay there.

Why am I pointing out this 1% figure? That leads us to the next slide, to make the point that in mice we can reduce plaque cross-sectional area in the aortic root by 17% over only 6 weeks of treatment. We look at a cross-sectional area at random in the body of the aortic root plaque to get that 17% figure, and if one assumes it will hold all the way through the slices of the plaque, then this is a 17% volume reduction, not just cross-sectional area. This is a big deal! The importance here is that we can reliably achieve this in mice, and we hope to be able to do the same in humans. You might ask how we achieve this outcome in mice, and the short answer is that use a LNP-mRNA gene therapy to make cells clear out localize deposits of excess free cholesterol - and I'm sure you you understand all the words in isolation but the context would probably help a little more. Excess free cholesterol is a feature of aging, and unfortunately it is also a feature of obesity - which is one of the reasons why a lot of the consequences of obesity appear similar to those of aging. Cholesterol is transported around the the body, it isn't really made or destroyed locally, and that complex transport system breaks down in ways that give rise to localized excesses of cholesterol. That localized excess overwhelms the cell's ability to make free cholesterol safe by esterifying it or incorporating it into cell membranes or attaching it to transport particles, and that free cholesterol is toxic. It is toxic in the liver particularly because the liver is the center of cholesterol metabolism; your liver function is greatly diminished and harmed by this this excess free cholesterol.

Now unfortunately this free cholesterol is undruggable. There is no "break down the cholesterol" mechanism operating throughout the human body, or even in liver cells, that you can adjust with a small molecule. You can't bind and sequester enough free cholesterol with something like a cyclodextrin to get rid of these excesses without killing the patient first - by removing too much cholesterol from cell membranes. In particular LDL cholesterol in the bloodstream, which is the target of lipid lowering therapies such as statins, has a tenuous relationship at best with a localized excess of cholesterol that might occur at some place in your body. You can lower LDL cholesterol in the bloodstream as much as you like and it won't really do that much to a localized excess of cholesterol.

That said there are human proteins that can act in conjunction to degrade excess free cholesterol. These proteins are just not expressed together in near all cells in our body. We took the best of these proteins, turned them into an optimized fusion protein, encoded that fusion protein in messenger RNA, and put the messenger RNA into a lipid nanoparticle that is targeted to the liver. We introduce the therapy via intravenous injection, it travels to the liver, it expresses our fusion protein in liver cells, and that clears out the excess free cholesterol in the liver. The liver is thereby restored to homeostasis and the result is systemic benefits throughout the body via removal of this age-related and obesity-related contribution to disease and dysfunction. It is not just atherosclerosis that is affected, but many other conditions as well.

We are heading in the direction of our our first human trials and our focus is on the rare genetic disease of homozygous familial hypercholesterolemia - that I will refer to HoFH from now on for the obvious reason that it is hard to say repeatedly. HoFH patients have enormous blood cholesterol levels, and over a lifetime that means that they exhibit accelerated atherosclerosis. Absent any sort of intervention these people die in their 30s - they are not not in good shape. There are very few HoFH patients, and this rare disease status means that the barriers are lower for FDA approval, and potentially fast track approval. This slide shows our timeline to clinical trials; note that I'm including our non-human primate studies in because I'm going to make a point about those in a moment. By late 2025 we are going to have efficacy data in a high fat diet model in cynomolgus macaques, but that runs in parallel with getting ready for the first clinical trial for HoFH in early 2026. Of course questions of time are essentially questions of money - all of this depends on raising a series A round this year, enabling licensing, GMP manufacture with a CDMO, IND-enabling studies, and all the rest of it. Everything is lined up, it just needs the funding and off we go. This timeline is 18 months to the IND submission, and you might feel that this is a long time - but this is in fact very fast in the world of biotech, as I'm sure many of you are aware.

But coming back to the non-human primate studies, here is why I wanted to mention our work there. It is an interesting aside there's a company called Verve Therapeutics, you might have heard of them because they recently ran into trouble with one of their clinical trials, but back in 2021 they were riding high because they had just gone public at a something approaching a 1 billion valuation. At that time, the only data they had was in non-human primates. They had not conducted human trials at that point, and our therapy is objectively better than theirs - which is just a better a better statin, in brief. It lowers LDL cholesterol, and thus cannot possibly reverse plaque, just as is the case for all of the other LDL lowering technologies. As I said, we will be in this position about 15 months from now with a much better therapy, which is hopefully food for thought for those who might be thinking about investing in us.

I should say that while you know that atherosclerosis is why we are running this program, the starting point, it is not where we will will stop. This is a first-in-class therapy that's going to spawn half an industry's worth of further effort, research groups and companies tackling the many age-related conditions that are aggravated by free cholesterol pathology. This really should be a hallmark of aging, and I'm sure that as the hallmarks expand somebody will add it. We have demonstrated that we can reverse the liver fibrosis of metabolic dysfunction-associated steatohepatitis (MASH). We have also demonstrated over the course of that preclinical work that this form of therapy is likely relevant to type 2 diabetes as well, as we have demonstrated improved glucose tolerance and insulin levels following clearance of excess free cholesterol from the liver. Further, there are numerous neurodegenerative conditions in which the lipid metabolism of the brain is relevant. Patients and models exhibit cells with lipid droplets in the brain; there is clearly something going wrong in there, and we believe that excess cholesterol is probably relevant. Similarly I can point out a range of evidence in the cancer field, in immunology, in a number of other rare diseases, in which cholesterol metabolism runs off the rails in some part of the body, and where a therapy like ours could treat these conditions. This work will obviously be undertaken by an industry that will come after us, not by us - atherosclerosis is a very big problem in and of itself, but there is an even bigger pipeline here.

In the remaining 10 minutes I'm going to take you through a very brief tour of our results in atherosclerotic mice. This is the high level only, we have much more data, but the most important data is of course what happens to the plaque. We'll start with a really great picture of serum samples from a time series study. These LDLR-knockout mice are the model for HoFH, the human patients also have loss of function in the LDLR gene, meaning the liver can't take up cholesterol so the blood is filled with gunk: lots of cholesterol, lots of triglycerides. As the picture shows, when you pull serum from these mice it is actually opaque because it has so much so much stuff in there, unlike normal serum which is clear. In this time series study we injected the mice once with our therapy, and then sacrificed them at various time points afterwards. You can see that by 96 hours, a single injection of our of our therapy has essentially reversed the problem in the serum. The serum is back to being clear; we've removed the gunk, and that contributes to an impressive effect on the plaque.

Next up is the data showing 17% aortic root plaque reversal that I mentioned earlier. Just to fill in the details here, we took LDLR knockout mice and placed them on the a high fat diet for 16 weeks to make them severely atherosclerotic, and then gave them six weeks of weekly injections with our LNP-mRNA therapy. We used a very broad range of doses, from 0.25 mg/kg to 1.50 mg/kg. Those who know the mRNA space will know you you won't go far wrong by picking something between 0.5 mg/kg and 1.0 mg/kg - many therapies seem to resolve to that dose in the end, and perhaps there is some universal reason for that. In any case, we picked a range of doses and they all worked to reduce plaque, and when averaging across the groups we see the 17% reduction in plaque cross-sectional area. By the way, there is nothing stopping us from extending these study timelines - waiting a few weeks and introducing another six weeks of therapy with these mice. We haven't done that yet but in principle we should observe a greater regression of plaque.

Looking at further data, we took the mice and put them on a treadmill at study close, prior to sacrifice. Bear in mind that these are not mice that will win any prizes for exercise capacity because they are fat and sedentary, but all of the treated mice, at whatever dose, regained cardiovascular function to a very sizable degree versus the mice who were still impeded by the severe atherosclerosis that they were suffering. I want you to note that this is a very broad therapeutic window, with no side effects and yet benefits were observed at all doses tried.

Now we'll move over to the APOE knockout mice which are another accepted model for atherosclerosis in the general population. We conducted a study in which we compared our therapy with a statin. It starts the same way as before, put the mice on a lengthy high fat diet and then provide them with six weeks of treatment. In this case it was a single LNP-mRNA dose at 1.0 mg/kg alongside a reasonable 5.0 mg/kg daily dose of atorvastatin, a dosing level that will not provoke liver problems in the mice but should still provide a visible effect size on lipid metabolism. As the data shows, our therapy is not only much better than statins, but also synergizes with statins. It is important to note that all other treatments are complementary to ours, be that the Cyclarity approach, or statins, or PCSK9 inhibitors - they can all be used in conjunction. It is an enormous industry, there is room for everybody. Again, this six week treatment could be repeated. In principle you can keep going. You could take a break and conduct the treatment again for 6 weeks, and we would expect larger results on plaque composition and size.

An important point is we're not just removing lipids from the plaques and thereby stabilizing them, but we're also increasing the collagen content of the plaques. The lipids are effectively being replaced by collagen, and thus the result is a more fibrotic plaque which is stable and safer. Further, we're not replacing lipids with calcium - the plaque is not becoming calcified. I'm not going to present data on that topic, but I will say that in these models we do see a reversal of calcification in these plaques. You should probably take that with the grain of salt given that LDLR and APOE knockout models are not specifically models of calcification - if you put 100 of these mice on a lengthy high fat diet, you will maybe see 10 or 20 that have that exhibit a large degree of calcification in their plaques, not really enough to robustly draw conclusions.

I'll finish up by mentioning our team I think most of you know Bill Cherman and I, the co-founders. We come from a mixed investment and patient advocacy background in the longevity community. Morad Topors is a tremendously talented researcher who is here today so you should take the chance to talk to him - he has a very good background in cardiometabolic disease and is probably the world's leading expert at this point on clearing cholesterol for therapeutic effect. Bobby Khan, our CMO, is a very reputable, very well-known cardiovascular physician who has put drugs through the FDA and gives us good advice on our forthcoming forthcoming clinical program. Of course I really should mention that none of this would be possible without our very talented lab team, who in this picture are standing outside one of the two restaurants in Syracuse, New York that you have to go to if you go to Syracuse, New York. Now you know! If you're there go to this restaurant. Collectively this is now the world brain trust on clearing cholesterol for therapeutic effect, as that this program is only being conducted by us - nobody else has this, nobody else owns this, nobody else is working on this. We are the first-in-class approach to reversing cardiovascular disease.

Recently researchers have found that amino acid homeostasis is disrupted in the serum and brain of patients with Alzheimer's disease (AD). Moreover, alterations in the levels of different amino acids in the physiological range have been linked to various pathological conditions, including neurological disorders. Longitudinal studies using mouse models of AD have also demonstrated abnormal essential amino acid levels. These findings suggest that dietary intervention may affect the progression of AD by regulating amino acids metabolism.

Methionine is a widely-used sulfur-containing amino acid that serves as a precursor for substances such as spermine, spermidine, and ethylene. It plays a pivotal role in various aspects of growth and development, including cell division, differentiation, apoptosis, homeostasis, and gene expression. Studies have shown that the methionine cycle is involved in the pathogenesis of AD. Methionine serves as a crucial methyl donor in certain methyltransferase reactions, providing methyl groups to various compounds. High methionine diet has been proven to induce AD-like symptoms. As a dietary intervention, methionine restriction has been reported to alleviate AD, but the molecular mechanisms remain unclear. Therefore, it is of great significance to explore the specific mechanisms, by which methionine is involved in the pathogenesis of AD.

This study utilized the data from BXD recombinant inbred (RI) mice to establish a correlation between the AD phenotype in mice and methionine level. Gene enrichment analysis indicated that the genes associated with the concentration of methionine in the midbrain are involved in the dopaminergic synaptic signaling pathway. Protein interaction network analysis revealed that glycogen synthase kinase 3 beta (GSK-3β) was a key regulator of the dopaminergic synaptic pathway and its expression level was significantly correlated with the AD phenotype. Finally, in vitro experiments demonstrated that methionine deprivation could reduce the expression of amyloid-β and phosphorylated tau, suggesting that lowering methionine levels in humans may be a preventive or therapeutic strategy for AD. In conclusion, our findings support that methionine is a high risk factor for AD. These findings predict potential regulatory network, theoretically supporting methionine restriction to prevent AD.

Antisense oligonucleotides (ASOs) are compounds that can be engineered to induce the target mRNA-specific degradation, which in turn decreases the levels of its corresponding protein. Even though they are not the only drugs that can control the expression of α-synuclein, ASOs have a crucial advantage over other approaches. "Currently, antibody drugs and vaccines targeting α-synuclein are under development, but their effects may not prevent the disease from progressing inside cells. In contrast, nucleic acid drugs like ASOs that specifically control intracellular levels of normal α-synuclein could offer higher safety and efficacy by both retaining the natural physiological functions of the protein while inhibiting the spread of pathogenic α-synuclein."

Considering that Parkinson's disease often emerges and then spreads out from specific regions in the brain, the researchers tested whether administering ASOs locally at diseased sites could be a sound treatment or preventive strategy. To this end, they injected ASOs directly into either the left or right striatum of mice brain and analyzed the spread of α-synuclein pathologies throughout various brain regions using the presence of Lewy body-like and Lewy neurite-like pathologies as indicators.

When ASOs were injected into the left striatum two weeks before inoculation of the mouse brain with disease-causing fibrils at the same site, a significant decrease of over 90% in Lewy pathology-like neuronal inclusion was observed. Compared to the control group, this pre-treatment effectively prevented the spread of abnormal fibrils-induced aggregate towards multiple regions of the brain. Even when ASOs were administered at the same time or even after inoculation with fibrillar α-synuclein, there was a notable inhibitory effect in the left striatum and other areas of the brain.

Folate, or vitamin B9, is an essential dietary component used in the body to form red blood cells, as well as DNA, RNA, and proteins. It is especially vital for children, young adults and pregnant women because of its role in growth processes. Researchers wanted to explore its impact in lesser studied age groups. To simulate the effects in older adults, the researchers cut folate from the diets of animal models at an age corresponding roughly to human middle-age. A comparison group was raised the same but continued a typical diet inclusive of folate.

The researchers found the female folate-limited models were able to transition quicker between carbohydrate metabolism and fat metabolism across night and day compared to females on a typical diet. "When you sleep, your metabolism burns fat. And when you're awake and active, you're typically burning carbohydrates for quicker energy. As you get older, it takes longer to switch between these fat-burning and carbohydrate-burning states, but this metabolic plasticity seems to be better maintained in animal models on a folate-limited diet." The males on folate-limited diets had an overall increase in their metabolic rate during active periods, potentially helping them to maintain energy levels and physical activity. The folate-limited group maintained their weight and body fat into old age as opposed to the control group.

The research team began this work a few years ago by using methotrexate to reduce folate intake in yeast cells, then in the worm C. elegans. In both cases, cutting folate led the models to live longer. Looking forward, the team's next step will be to repeat the experiment in more genetically diverse models, simulating the genetic diversity of humans. The researchers will also expand their study of novel compounds to limit folate intake, which could later transition to clinical trials.

In 1998, the U.S. mandated that staple foods, particularly grains, be "enriched" or "fortified" with folic acid and other B vitamins following the refinement process. While helpful for some age groups, it might do more harm than good for older adults. As a result, this research opens a new avenue for developing drugs to limit dietary folate uptake for individuals who don't need as much, rather than cutting foods that contain folate or folic acid.

Reactivation of retroelements in the human genome has been linked to aging. However, whether the epigenetic state of specific retroelements can predict chronological age remains unknown. We provide evidence that locus-specific retroelement DNA methylation can be used to create retroelement-based epigenetic clocks that accurately measure chronological age in the immune system, across human tissues, and pan-mammalian species.

We also developed a highly accurate retroelement epigenetic clock compatible with EPICv.2.0 data that was constructed from CpGs that did not overlap with existing first- and second-generation epigenetic clocks, suggesting a unique signal for epigenetic clocks not previously captured. We found retroelement-based epigenetic clocks were reversed during transient epigenetic reprogramming, accelerated in people living with HIV-1, and responsive to antiretroviral therapy. Our findings highlight the utility of retroelement-based biomarkers of aging and support a renewed emphasis on the role of retroelements in geroscience.

True sex differences exist in aging. Women live longer than men around the world; they also show resilience to cognitive decline and higher baseline function in typical aging in many populations, when dementia and its subsequent development are carefully excluded. Since cognition is a key manifestation of brain function eroded by aging, understanding sex differences and their causes are high value areas of investigation.

Whether female mice show better cognition in typical aging - and whether sex chromosomes or gonads influence sex difference in cognitive aging - remain largely unknown. To examine this, we utilized genetic mouse models of sex biology. The Four Core Genotype (FCG) model tests whether gonads or sex chromosomes contribute to a sex difference. The XY* model tests whether the X or Y chromosome contributes. We tested for cognitive aging and any accompanying sex differences in young and aged mice using the two-trial Y maze paradigm which measures spatial and working memory, a target of aging. As expected, aging decreased cognition in both sexes. While no sex differences were observed in young mice, female sex attenuated age-induced cognitive decline.

Sepsis-associated acute kidney injury (S-AKI) is among the most serious and common complications of sepsis. Although S-AKI has attracted widespread attention, the detailed pathophysiological mechanisms of S-AKI remain complicated and poorly understood. Extensive studies have reported various abnormal physiological processes in S-AKI, including impaired energy metabolism, excess oxidative stress, apoptosis and necrosis of renal tubular epithelial cells, impaired renal microcirculation, activation of inflammatory cells, and inflammatory storms. Moreover, because the kidneys are among the most energy-demanding organs in the body, energy metabolism is crucial for proper renal function.

Mitochondrial damage and dysfunction are highly involved in tubular cell injury or death in acute kidney injury. Therefore, maintaining the structural and functional integrity of the mitochondria may prevent tubular cell apoptosis, thereby facilitating renal recovery from acute kidney injury. Furthermore, the macrophage polarization state and production of inflammatory mediators markedly affect the progression of S-AKI. For example, the release of pro-inflammatory cytokines or excessive oxidative stress from M1 macrophages can exacerbate renal injury. Moreover, M2 macrophages releasing anti-inflammatory factors and growth factors have protective effects against kidney damage. Accordingly, renal mitochondria and macrophage polarization may be promising targets for the prevention and mitigation of S-AKI.

Cordyceps sinensis (CS) is a fungus with a long history of use in traditional Chinese medicine, owing to its anti-aging and anti-cancer properties. The active ingredients of CS include cordycepin, polysaccharides, sterols, and phenolic compounds. Because of its broad physiological effects, including antioxidant, anti-fibrotic, and anti-inflammatory activity, CS has been generally used for renal protection. In the present study, we first evaluated the protective and therapeutic effects of CS against LPS-induced AKI in mice via assays including histopathological staining, serum renal function indexes, and inflammatory cytokine analyses. We performed transcriptomic and proteomic assays on kidney tissues, revealing the molecular targets and pathways through which CS ameliorates S-AKI. We confirmed that CS protects the kidneys against S-AKI by synergistically reprogramming mitochondrial energy metabolism and macrophage polarization.

Epigenetic clocks are age predictors that use machine-learning models trained on DNA CpG methylation values to predict chronological or biological age. Increases in predicted epigenetic age relative to chronological age (epigenetic age acceleration) are connected to aging-associated pathologies, and changes in epigenetic age are linked to canonical aging hallmarks. However, epigenetic clocks rely on training data from bulk tissues whose cellular composition changes with age.

Here, we found that human naive CD8+ T cells, which decrease in frequency during aging, exhibit an epigenetic age 15-20 years younger than effector memory CD8+ T cells from the same individual. Importantly, homogenous naive T cells isolated from individuals of different ages show a progressive increase in epigenetic age, indicating that current epigenetic clocks measure two independent variables, aging and immune cell composition. To isolate the age-associated cell intrinsic changes, we created an epigenetic clock, the IntrinClock, that did not change among 10 immune cell types tested. IntrinClock shows a robust predicted epigenetic age increase in a model of replicative senescence in vitro and age reversal during OSKM-mediated reprogramming.

Long-lived individuals have been extensively studied as a model to investigate the role of the gut microbiota in aging, but their gut fungi remain almost unexplored. Here, we recruited a community-dwelling cohort of 251 participants (24-108 years, including 47 centenarians) from Guangxi in China to characterize the gut mycobiome signatures. We found gut mycobiome markedly varied during aging and determined aging as a predominant factor driving these variations. For long-lived individuals, core taxa, including Penicillium and Aspergillus, were maintained and Candida enterotype was enriched when compared with old counterparts.

Individuals with this enterotype were more likely to possess Bacteroides enterotype enriched in young and centenarians. Moreover, the drivers from Candida enterotype were positively linked with the bacteria components dominated in Bacteroides enterotype. We also identified potentially beneficial yeasts-enriched features to differentiate long-lived individuals from others. Our findings suggest that the gut mycobiome develops with aging, and long-lived individuals possess unique fungal signatures.

Age-associated neurodegenerative disorders present a significant challenge due to progressive neuronal decline and limited treatments. In aged mice, partial reprogramming, characterized by pulsed expression of reprogramming factors, has shown promise in improving function in various tissues, but its impact on the aging brain remains poorly understood. Here we investigated the impact of in vivo partial reprogramming on mature neurons in the dentate gyrus of young and aged mice.

Using two different approaches - a neuron-specific transgenic reprogrammable mouse model and neuron-specific targeted lentiviral delivery of OSKM reprogramming factors - we demonstrated that in vivo partial reprogramming of mature neurons in the dentate gyrus, a neurogenic niche in the adult mouse brain, can influence animal behavior, and ameliorate age-related decline in memory and learning. These findings underscore the potential of in vivo partial reprogramming as an important therapeutic intervention to rejuvenate the neurogenic niche and ameliorate cognitive decline associated with aging or neurodegeneration.

The molecule, DDL-920, works differently from recent FDA-approved drugs for Alzheimer's disease such as lecanemab and aducanumab, which remove harmful plaque that accumulates in the brains of Alzheimer's disease patients. While removing this plaque has been shown to slow the rate of cognitive decline, it does not restore the memory and cognitive impairments. "They leave behind a brain that is maybe without plaque, but all the pathological alterations in the circuits and the mechanisms in the neurons are not corrected."

Similar to a traffic signal, the brain fires off electric signals at different rhythms to start and stop various functions. Gamma oscillations are some of the highest-frequency rhythms and have been shown to orchestrate brain circuits underlying cognitive processes and working memory - the type of memory used to remember a phone number. Patients with early Alzheimer's disease symptoms such as mild cognitive impairment have been shown to have reduced gamma oscillations.

Researchers thought that perhaps there was a way to trigger these electrical rhythms from inside cells using a molecule. Specifically, they needed a compound to target certain fast-firing neurons, known as the paravalbumin interneurons, that are critical in generating gamma oscillations and therefore memory and cognitive functions. However, certain chemical receptors in these neurons that respond to the chemical messenger known as GABA work like brake pedals to reduce the gamma oscillations entrained by these neurons. Researchers identified the compound DDL-920 to antagonize these receptors, allowing the neurons to sustain more powerful gamma oscillations.

Alzheimer's disease model mice and wild-type mice underwent baseline cognitive testing in a Barnes maze - a circular platform surrounded by visual clues and containing one escape hole. The maze is used to measure how well rodents can learn and remember the location of the escape hole. After the initial tests, researchers orally administered DDL-920 to the Alzheimer's model mice twice daily for two weeks. Following treatment, the Alzheimer's disease model mice were able to recall the escape hole in the maze at similar rates as the wild-type mice. Additionally, the treated mice did not display any abnormal behavior, hyperactivity, or other visible side effects over the two-week period.

The primary assumption of aging clocks is that the deviation ∆ of predicted age from the chronological age C represents an accelerated or decelerated aging, that is, an increase or decrease in the biological age B. Since biological age cannot be measured directly, the epigenetic age estimated by the clocks is therefore considered a proxy measure of the biological age. However, before aging clocks could be integrated into clinical practice, these models should provide an estimate of uncertainty for their own predictions.

Uncertainty manifests itself in three ways: (i) Model choice uncertainty, part of a broader category known as epistemic uncertainty, represents how well a proposed model reflects the actual underlying process. (ii) Out-of-distribution (OOD) uncertainty, another type of epistemic uncertainty, emerges when the testing data are not represented in the training data distribution, leading to a high risk of model prediction failure (iii) Aleatoric uncertainty originates from data variations that cannot be reduced to zero by the model.

From the clinical perspective, epistemic uncertainty must be estimated to make reliable conclusions about whether to trust a model or not. Specifically, epistemic uncertainty resulting from the dataset shift should be scrutinized, considering the prevalence of batch effects in biological data. Dataset shift describes the case of OOD sampling where the testing population is under-represented within the training distribution. However, most popular DNA methylation aging clocks fail to meet this criterion because they are typically built using algorithms from the penalized multivariate linear regression (MLR) family. Such algorithms do not yield information on any of the uncertainties, except for the error between chronological and predicted ages in the training data.

In this work, we question the applicability of existing aging clock methodology for measuring rejuvenation by specifically examining prediction uncertainty. We present an analytical framework to consider rejuvenation predictions from the uncertainty perspective. Our analysis reveals that the DNA methylation profiles across reprogramming are poorly represented in the aging data used to train clock models, thus introducing high epistemic uncertainty in age estimations. Moreover, predictions of different published clocks are inconsistent, with some even suggesting zero or negative rejuvenation. While not questioning the possibility of age reversal, we show that the high clock uncertainty challenges the reliability of rejuvenation effects observed during in vitro reprogramming before pluripotency and throughout embryogenesis. Conversely, our method reveals a significant age increase after in vivo reprogramming. We recommend including uncertainty estimation in future aging clock models to avoid the risk of misinterpreting the results of biological age prediction.