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- Circulating Citrulline Declines with Age, and Supplementation is Anti-Inflammatory in Mice
- Prevalence of Specific Gut Microbial Species Associated with Muscle Mass in Later Life
- An Approach to Manufacture Large Numbers of Mitochondria for Transplantation
- Entropy of DNA Methylation States as the Basis for an Epigenetic Clock
- Reviewing the State of Therapies for Alzheimer's Disease
- Intranasal Administration of the Diterpenoid EOF2 Promotes Generation of New Neurons
- Endothelial Dysfunction of Microvessels in the Aging of the Vasculature
- Elamipretide / SS-31 Improves Muscle Function But Doesn't Affect Epigenetic Age
- LAMP1 as a Cell Surface Marker of Senescent Cells
- The Aging of the Gut Microbiome is Different by Sex and Mitochondrial Haplotype
- CD2AP in Alzheimer's Disease
- Gene Regulatory Networks in the Design of Approaches to Slow Aging
- Hypothesizing that Non-Coding RNAs are a Major Determinant of Species Life Span
- Advanced Glycation Endproducts in Muscle Loss Leading to Sarcopenia
- Tissue Resident Macrophages in the Heart in Cardiovascular Disease
Circulating Citrulline Declines with Age, and Supplementation is Anti-Inflammatory in Mice
https://www.fightaging.org/archives/2025/03/circulating-citrulline-declines-with-age-and-supplementation-is-anti-inflammatory-in-mice/
Reminiscent of recent work on supplementation of the amino acid taurine as a means to modestly slow aging, the authors of today's open access paper examine the role of the amino acid citrulline in age-related changes occurring in mice. As is true of taurine in mice, blood and tissue levels of citrulline decline to varied degrees with age, while long-term oral supplementation with citrulline both restores youthful levels and improves a number of measures of metabolism. This includes a reduction in age-related chronic inflammation that appears mediated by changes in the behavior of the innate immune cells known as macrophages.
Human trials of taurine supplementation have not produced clearly positive outcomes, but the researchers involved may have been looking at the wrong measures in the years prior to the commonplace use of aging clocks. Taurine touches on many areas of metabolism, and a simple self-experiment for taurine supplementation produced a modest reduction in phenotypic age driven by lower levels of circulating neutrophils, another form of innate immune cell. Increased neutrophil count in a blood sample is representative of inflammation.
Citrulline has been used in a sizable number of human clinical trials versus only the few for taurine, and has shown modestly beneficial results for a range of conditions. Since inflammation contributes to the progression of near all age-related conditions, this is much what one might expect see if it is in fact reducing the contribution of macrophages to the inflammatory environment of aged tissues. Given all of this, one might be tempted to run a similar self-experiment as for taurine, using citrulline supplementation instead, and see what results - it would be straightforward to conduct.
Citrulline regulates macrophage metabolism and inflammation to counter aging in mice
Metabolic dysregulation and altered metabolites are widely recognized as key characteristics of aging. Numerous studies have investigated the roles of endogenous metabolites, such as NAD, taurine, spermidine, and others, as drivers of the aging process. In this study, we conducted a comprehensive analysis of metabolic changes in multiple organs of mice at various ages. Our findings provide the first evidence linking citrulline deficiency to aging. We identified multiple antiaging effects of citrulline, including the reduction of cellular senescence, protection against DNA damage, prevention of cell cycle arrest, modulation of macrophage metabolism, and mitigation of inflammaging. Notably, long-term supplementation of citrulline in aged mice demonstrated significant benefits by alleviating age-associated phenotypes and increasing health span. These findings underscore the critical role of citrulline deficiency as a key driver of the aging process and highlight the potential therapeutic intervention of citrulline supplementation to counteract age-related diseases.
To explore the biological mechanism by which citrulline counteracts aging, we demonstrated that citrulline acts as an endogenous metabolite antagonist to inflammation. Macrophages are primary contributors to age-associated inflammation. Our findings unveil that the decline in endogenous citrulline levels impairs the anti-inflammatory function of macrophages, thereby enhancing susceptibility to inflammatory responses during aging. The anti-inflammatory effect of citrulline has been validated in various mouse models, and its efficacy remains intact even in the context of aging. This observation suggests that the age-dependent deficiency of citrulline, acting as an endogenous antagonist to inflammation, triggers inflammaging and accelerates the aging process. In-depth mechanistic investigations have revealed that citrulline supplementation rescues age-associated metabolic alterations in macrophage metabolism. Specifically, we have demonstrated that citrulline modulates the inflammatory responses by regulating the activities of the mTOR-HIF1α-glycolysis signaling pathway in macrophages. Collectively, these results establish that citrulline governs macrophage metabolism and inflammation as a means to counteract aging.
Our study also underscores the remarkable potential of citrulline as an endogenous metabolite inhibitor of the mTOR pathway in the context of inflammation and aging. mTOR serves as a nutrient sensor that regulates cellular metabolism and is linked to cell proliferation, growth, and survival. Extensive research has established the mTOR pathway as a negative regulator of lifespan and aging. Pharmacological inhibition of mTOR using small-molecule compounds such as rapamycin has been shown to effectively extend longevity in various animal models. However, the discovery of an endogenous metabolite that inhibits mTOR to counter aging has remained elusive. Arginine, leucine, and S-adenosylmethionine (SAM; downstream metabolite of methionine) are the known metabolites that are directly sensed by mTOR components. Restriction of methionine or the three branched-chain amino acids - leucine, isoleucine, and valine - extends lifespan in mice, but the roles of these metabolites in regulating aging and mTOR are complicated. Our metabolomics data confirmed that arginine, leucine, SAM, and methionine levels remained unchanged during aging in mice. In our study, we extensively demonstrated that citrulline inhibits the activation of the mTOR pathway in macrophages in both inflammatory and aging contexts. This finding highlights citrulline as a promising endogenous metabolite with the potential to inhibit mTOR signaling.
Prevalence of Specific Gut Microbial Species Associated with Muscle Mass in Later Life
https://www.fightaging.org/archives/2025/03/prevalence-of-specific-gut-microbial-species-associated-with-muscle-mass-in-later-life/
16S rRNA is a gene that varies in sequence distinctively by microbial species, and thus can be cheaply sequenced to catalog the contents of the gut microbiome for a given individual, determining the composition of specific species and their prevalence. Given this capability, researchers have determined that the distribution of microbial populations alters with age in ways that are harmful to health, such as via loss of beneficial metabolite production or increased inflammation. Further, researchers are increasingly correlating specific features of the gut microbiome to specific age-related conditions.
All of this is groundwork for a near future in which the gut microbiome can be tailored to a specific composition of populations to produce a desired outcome. Lasting rejuvenation of the composition of the gut microbiome, reversing age-related changes, is possible today via fecal microbiota transplantation using a young donor. It is possible in principle (though not yet reduced to practice) to achieve other forms of long-lasting adjustment of the composition of the microbiome by culturing and delivering a specific mix of microbes. Present approaches to oral delivery of probiotics do not achieve this goal, however.
Today's open access paper is an example of present research into correlations between gut microbiome composition and aspects of aging, here the focus is on progressive loss of bone density and muscle mass. The interesting question is the degree to which gut microbiome composition is causative versus being a consequence of other factors that drive osteoporosis and sarcopenia, such as age-related immune dysfunction or the reduced intake of protein and calories typical of older people.
The association of gut microbiome composition with musculoskeletal features in middle-aged and older adults: a two-cohort joint study
The role of the gut microbiome in the development of osteoporosis and sarcopenia has received increased interest given its promising potential in improving musculoskeletal health. The gut microbiome is commonly assessed in the stool and comprises a collection of microorganisms from the digestive tract that impact human physiology through different biological processes. Previous research has indicated that the community of commensal microbes residing in the gut may represent a potentially modifiable factor contributing to muscle and skeletal health. For instance, the gut microbiome can affect the inflammatory environment through effects on the T-cell landscape, which influences osteoclastogenesis and bone loss in mice, and through the production of complex polysaccharides (e.g., short chain fatty acids (SCFA)). The gut microbiota also interacts with important diet components associated with musculoskeletal health such as vitamin K, vitamin D, and calcium. Moreover, the gut microbiota can modulate lipopolysaccharide (LPS) production and various metabolites that directly or indirectly (i.e., through the brain and liver) affect host skeletal muscle metabolism potentially playing a role in sarcopenia etiology.
We leveraged information from two large population-based cohorts, the Rotterdam Study (mean age 62.7 ± 5.6 years; n=1,249) and the Framingham Heart Study (mean age 55.2 ± 9.1 years; n=1,227). For individuals included in this study, gut microbiome 16S rRNA sequencing, musculoskeletal phenotyping, lifestyle and socioeconomic data, and medication records were available. Using 16S rRNA sequencing data we investigated the association between the human gut microbiome (alpha diversity, genera, and predicted functional pathways) and appendicular lean mass (ALM), femoral neck bone mineral density (FN-BMD), and trabecular bone score (TBS) using multilinear regression models controlling for multiple confounders, and performed a joint analysis from both cohorts. Sex-stratified analyses were also conducted.
The gut microbiome alpha diversity was not associated with either tested phenotype after accounting for multiple-testing. In the joint analysis, lower abundance of Oscillibacter, Anaerotruncus, Eisenbergiella, and higher abundance of Agathobacter were associated with higher ALM. Lower abundance of Anaerotruncus, Hungatella, and Clostridiales bacterium DTU089 was associated with higher ALM only in females. Moreover, the biotin biosynthesis II pathway was positively associated with ALM in females while no associations were observed in males. We did not observe any robust association of bone traits with gut microbiome features. Overall, our study suggests that the gut microbiome is linked to muscle aging in middle-aged and older adults. However, larger sample sizes are still needed to underpin the specific microbiome features involved.
An Approach to Manufacture Large Numbers of Mitochondria for Transplantation
https://www.fightaging.org/archives/2025/03/an-approach-to-manufacture-large-numbers-of-mitochondria-for-transplantation/
Mitochondria are the power plants of the cell, producing the chemical energy store molecule adenosine triphosphate (ATP) to power cellular biochemistry. Mitochondrial function declines with age, in part due to damage to mitochondria DNA and in part due to changes in nuclear gene expression that affect proteins needed by mitochondria. This is considered an important contribution to age-related degeneration, particularly in tissues such as muscle and brain that have high energy needs.
Cells will take up mitochondria from their surroundings and make use of them. Studies in mice have indicated that transplantation of mitochondria harvested from cell cultures can produce lasting benefits. The process of aging that diminish mitochondrial function take a while to operate, and youthful mitochondria can improve function for an extended period of time. The challenge here is that mice are small and people are large; reliable production of the large numbers of mitochondria needed is the primary hurdle preventing clinical use of this approach in old people.
A number of companies are working on the mitochondrial manufacturing challenge, including cellvie and Mitrix Bio. In today's open access paper, an academic group describes a potential approach to the problem, though this is aimed at local injection into cartilage. The goal of whole-body infusions of replacement mitochondria might require another two orders of magnitude of increased scale, and it remains to be seen if this approach will work at that level.
Organelle-tuning condition robustly fabricates energetic mitochondria for cartilage regeneration
Mitochondria are vital organelles whose impairment leads to numerous metabolic disorders. Mitochondrial transplantation serves as a promising clinical therapy. However, its widespread application is hindered by the limited availability of healthy mitochondria, with the dose required reaching up to 10^9 mitochondria per injection/patient. This necessitates sustainable and tractable approaches for producing high-quality human mitochondria.
In this study, we demonstrated a highly efficient mitochondria-producing strategy by manipulating mitobiogenesis and tuning organelle balance in human mesenchymal stem cells (MSCs). Utilizing an optimized culture medium (mito-condition) developed from our established formula, we achieved an 854-fold increase in mitochondria production compared to normal MSC culture within 15 days. These mitochondria were not only significantly expanded but also exhibited superior function both before and after isolation, with ATP production levels reaching 5.71 times that of normal mitochondria.
Mechanistically, we revealed activation of the AMPK pathway and the establishment of a novel cellular state ideal for mitochondrial fabrication, characterized by enhanced proliferation and mitobiogenesis while suppressing other energy-consuming activities. Furthermore, the in vivo function of these mitochondria was validated in the mitotherapy in a mouse osteoarthritis model, resulting in significant cartilage regeneration over a 12-week period. Overall, this study presented a new strategy for the off-the-shelf fabrication of human mitochondria and provided insights into the molecular mechanisms governing organelle synthesis.
Entropy of DNA Methylation States as the Basis for an Epigenetic Clock
https://www.fightaging.org/archives/2025/03/entropy-of-dna-methylation-states-as-the-basis-for-an-epigenetic-clock/
Entropy is one of those slippery concepts wherein the same word has been adopted by different scientific disciplines to mean subtly different things. I'd recommend a recent article that attempts to explain for the layperson how these this different meanings arose, and that they overlap at the concept of measuring our ignorance of the state of a system, our inability to predict the state of that system. Here we'll talk about entropy as a measure of the randomness of a distribution; the more random the distribution, the less our ability to predict its specifics. The distribution of interest for today is the methylation state (methylated or not methylated) at one or more CpG sites on the genome, across many genome copies in many cells.
DNA structure determines whether or not a given gene sequence is exposed to transcription machinery and RNA is produced. One of the mechanisms determining the shape of DNA is whether or not methyl groups are added at specific locations called CpG sites, named because a a cytosine nucleotide © is followed by a guanine nucleotide (G) with the two linked by a phosphate group (p). This DNA methylation is the basis for epigenetic clocks that assess chronological and biological age, because the methylation status of some CpG sites is characteristic of the damage and dysfunction of aging. While whether or not a CpG site is methylated is a binary outcome, this data is measured across the many, many cells and genomes in a given blood or tissue sample. Current epigenetic clocks take the average of all of those 1s and 0s as the input of that specific CpG site to the clock algorithm.
In today's open access paper, researchers start instead by considering the entropy of the distribution of methylation status at a CpG site across the many measured genomes. For this purpose, entropy is a measure of how noisy or random the data is. The researchers then show that one can construct an epigenetic clock from the entropy values per CpG site that performs as well as clocks built using average values of methylation state. This suggests that aging is not just resulting in a move of some CpG sites towards one status, but also an increase in noise in DNA methylation, a move in both directions, an increase in randomness. Age-related noise in gene transcription is already a topic for discussion in the field, so why not age-related epigenetic noise as well?
DNA methylation entropy is a biomarker for aging
To measure age associated changes in DNA methylation, we collected buccal swabs from 100 individuals ranging from 7.2 to 84 years old. The DNA methylation profiles were generated using targeted bisulfite sequencing. Our target panel contained approximately 3000 regions that were selected to cover age associated CpG sites that were identified in multiple epigenetic clocks. Each probe is 120 base pairs, and therefore captures a region of DNA that is slightly larger than the probe length. We obtained an average coverage of 293 reads per sample across these regions.
We first calculated the mean methylation of each CpG site in each of the 3000 loci across the 100 samples, and then averaged these levels over a region. We also computed the Cellular Heterogeneity-Adjusted cLonal Methylation (CHALM). This approach computes the read level methylation of a region after reads are dichotomized into methylated or unmethylated based on the presence of one or more methylcytosines. We also computed the methylation entropy for each locus using four CpG sites within each region, using the Shannon entropy formula. With four CpG sites, there are 16 possible methylation states, and we computed the probability of each state as well as the entropy of the four CpG sites.
We next generated scatter plots that compared the values of the three metrics across loci. Age-related changes in mean methylation and CHALM were strongly correlated. By contrast, the scatter plots of entropy versus mean methylation or CHALM resulted in more complex patterns with both positive and negative trends. This demonstrates that methylation entropy is measuring different properties of a locus compared to mean methylation and CHALM, and that loci can become both more or less disordered with age, independently of whether the methylation is increasing or decreasing with age.
We next asked whether we could compare the use of these three metrics to construct epigenetic clocks that predict the age of each individual. Selecting only four CpG sites per region to calculate entropy was sufficient to achieve chronological age estimates that were correlated with the actual age. The mean average error was 5.199 years, which was lower than the other mean-based methods that incorporated many more CpG sites. This suggests that the entropy of a locus is potentially a more useful biomarker of aging than the methylation level of individual sites. Though the 3000 loci analyzed may or may not be representative of the whole genome, this suggests that the entropy of an organism's methylation profile is informative of its epigenetic age.
Reviewing the State of Therapies for Alzheimer's Disease
https://www.fightaging.org/archives/2025/03/reviewing-the-state-of-therapies-for-alzheimers-disease/
The history of attempts to treat Alzheimer's disease is littered with costly failures. One can blame the complexity of both the brain and the condition, which resists attempts to pick apart its contributing mechanisms. One can blame the fact that Alzheimer's is a condition that only naturally occurs in humans (and perhaps dolphins and chimpanzees, with limited evidence in both cases). Access to the biochemistry of the living brain in humans in the ways needed for Alzheimer's research is essentially impossible for ethical and practical reasons. Equally, any practical animal model of Alzheimer's disease, such as the many mouse models, is artificial and embodies certain assumptions about which pathology and processes are most important. Treatments that address the artificially created pathology in the model tend to be successful in that model. Then they fail in humans, after great expense, demonstrating that some of the assumptions were incorrect.
Today's open access review paper is a concise tour of the major categories of drug development. It does omit a range of therapies targeting pathological neurofibrillary tangles made of hyperphosphorylated tau protein, a feature of late stage Alzheimer's disease, and a number of more recent approaches such as clearance of senescent cells as a way to reduce inflammation and tissue dysfunction. Overall it is a cautionary tale for anyone who might be feeling enthused about any of these other approaches to the condition. At some point, the right mechanism will be targeted, but which one is it? The classic problem for every age-related condition is that there is no shortage of contributing mechanisms to consider, but without having already developed a therapy that can address one mechanism in isolation, it is next to impossible to determine whether that one mechanism is important and a good target.
Therapeutic agents for Alzheimer's disease: a critical appraisal
Alzheimer's disease (AD), the most common cause of dementia, is a progressive neurodegenerative disorder, characterized by the degeneration of cholinergic neurons in the nucleus basalis, and the presence of extracellular plaques of beta amyloid (Aβ) and intracellular neurofibrillary tangles composed of phosphorylated tau. AD presents with an impairment in early episodic memory, followed by a gradual and progressive deterioration in cognition and behavior.
The characteristic features of the familial form (FAD) were originally described by Alois Alzheimer in 1906. In FAD, Aβ-containing plaques appear at least 20 years before any signs of memory impairment. While prevention of Aβ formation could provide a treatment option for FAD if started early enough, it represents only about 1% of subjects with AD. The rest have the sporadic form of AD (SAD), with an age of onset of more than 65 years. Their brains also have Aβ-containing plaques, but so do those of healthy, older people with no overt signs of dementia. Since no correlation was found between the number of Aβ plaques and the degree of cognitive impairment in individuals with SAD, the original hypothesis was changed and soluble oligomers of Aβ proposed as the cause of neurodegeneration.
During the last decade, the pharmaceutical industry has concentrated its efforts to affect the processes leading to neurodegeneration by developing drugs to decrease Aβ. Mutations in genes and precursors of Aβ are found in the familial form of the disease. This led to the evaluation of seven monoclonal antibodies against Aβ in subjects with AD, two of which were approved for use by the FDA. They caused only a small improvement in cognitive function, probably because they were given to those with much more prevalent sporadic forms of dementia. They also have potentially serious adverse effects.
γ-secretase is a multi-subunit protease that was identified as responsible for the generation of Aβ, and thus considered a prime therapeutic target in AD. This led to the development of γ-secretase inhibitors like semagacestat to inhibit the formation of Aβ. However, a phase 3 trial in patients with mild to moderate AD was prematurely stopped because the drug actually worsened several measures of cognitive function. Like other γ-secretase inhibitors, avagacestat and tarenflurbil, semagacestat caused serious adverse effects, including cancer, skin related disorders, hypersensitivity reactions, increase in infections, and renal failure. β-secretase inhibitors also prevent formation of Aβ from amyloid precursor protein and their adverse effects are less serious than those of γ-secretase inhibitors. However, verubecestat, atabecestat, and lanabecestat all worsened cognitive function in subjects with mild-moderate AD.
Oxidative stress and elevated pro-inflammatory cytokines are present in all subjects with AD and are well correlated with the degree of memory impairment. Drugs that affect these processes include TNFα blocking antibodies and MAPK p38 inhibitors that reduce cognitive impairment when given for other inflammatory conditions. However, their adverse effects and inability to penetrate the brain preclude their use for dementia. Rosiglitazone is used to treat diabetes, a risk factor for AD, but failed in a clinical trial because it was given to subjects that already had dementia. Ladostigil reduces oxidative stress and suppresses the release of pro-inflammatory cytokines from activated microglia without blocking their effects. Chronic oral administration to aging rats prevented the decline in memory and suppressed overexpression of genes adversely affecting synaptic function in relevant brain regions. In a phase 2 trial, ladostigil reduced the decline in short-term memory and in whole brain and hippocampal volumes in human subjects with mild cognitive impairment and had no more adverse effects than placebo.
Intranasal Administration of the Diterpenoid EOF2 Promotes Generation of New Neurons
https://www.fightaging.org/archives/2025/03/intranasal-administration-of-the-diterpenoid-eof2-promotes-generation-of-new-neurons/
Formation of new neurons and their integration into existing neural networks is necessary for repair and change in the brain. Producing a larger supply of neurons could be beneficial, helping to resist the consequences of age-related damage that builds up over time. Here, researchers find a way to influence one source of new neurons using a diterpenoid compound that can be delivered intranasally to find its way into the brain. They demonstrate increased production of neurons in mice using this approach.
Neural stem cells from the subventricular zone (SVZ) neurogenic niche provide neurons that integrate in the olfactory bulb circuitry. However, in response to cortical injuries, the neurogenic activity of the SVZ is significantly altered, leading to increased number of neuroblasts with a modified migration pattern that leads cells towards the site of injury. Despite the increased neurogenesis and migration, many newly generated neurons fail to survive or functionally integrate into the cortical circuitry. Providing the injured area with the adequate signaling molecules may improve both migration and functional integration of newly generated neurons.
Protein kinases (PK) such as PKA or PKC have an important role in neuroblast migration. Previous reports have revealed that the treatment of mouse cortical injuries with a novel PKC activating diterpenoid resulted in neuroblast enrichment and in their differentiation into mature neurons. Within the injury environment and the SVZ, growth factors that promote proliferation and glial differentiation are highly expressed such as transforming growth factor alpha (TGFα) and they need to be counterbalanced with signals that promote differentiation such as neuregulins to allow regeneration and replacement of the lost neurons. Interestingly, evidence shows that in response to diterpenoid EOF2, which activates novel PKC activity and neuregulin release, these signaling cues may be altered to promote the premature differentiation of neuroblasts and their migration toward the injured area suggesting a role for neuregulin 1 (NRG1) and novel PKC in neuronal replacement in cortical injuries.
We have found that EOF2 treatment of adult mice with mechanical cortical injuries facilitates the delivery of neuroblasts into these injuries. The newly generated neurons develop features of fully functional neurons. Our results show that the newly generated neurons receive electrical inputs, fire action potentials, and undergo complete differentiation into neurons recapitulating the stages that distinguish ontogenic differentiation. These neurons develop features representative of neurons belonging the cortical layer in which they are situated. We have also studied that EOF2 facilitates neuregulin release in SVZ cells, a signaling factor that promotes neuronal differentiation. Neuregulin is expressed in microglial cells that reach the injury in response to the damage and its release is increased by EOF2 treatment.
Endothelial Dysfunction of Microvessels in the Aging of the Vasculature
https://www.fightaging.org/archives/2025/03/endothelial-dysfunction-of-microvessels-in-the-aging-of-the-vasculature/
The endothelium is the inner lining of blood vessels. It is important to a range of vital functions performed by the vasculature, from the selective passage of molecules enforced by the blood-brain barrier to the ability of blood vessels to properly contract and dilate. Further, localized damage and inflammation of the endothelium is an early step in the formation of an atherosclerotic plaque. In this paper, researchers focus on the endothelium of the microvasculature, the smallest blood vessels. All of the same issues occur here, and may contribute over time to the decline of microvascular density as the mechanisms needed for maintenance of these vessels, such as aspects of angiogenesis, become impaired.
Cardiovascular disease (CVD) is the main cause of morbidity and mortality in the adult and the elderly, with increasing prevalence worldwide. A growing body of research has focused on the earliest stage of vascular decline - endothelial dysfunction (ED) - which at the microvascular level can anticipate in decades the diagnosis of CVD. This review aims to provide a prospect of the literature regarding the development of ED as an indissociable feature of the aging of the cardiovascular system, highlighting the role of inflammation in the process.
Vascular aging consists of a lifelong continuum, which starts with cell respiration and its inherent production of reactive oxygen species. Molecular imbalance is followed by cellular epigenetic changes, which modulate immune cells, such as macrophage and lymphocyte subtypes. These mechanisms are influenced by lifestyle habits, which affect inflammation hotspots in organism, such as visceral fat and gut microbiota. The process can ultimately lead to an environment committed to the loss of the physiological functions of endothelial cells. In addition, we discuss lifestyle changes targeting the connection between age-related inflammation and vascular dysfunction. Addressing microvascular ED represents a critical endeavor in order to prevent or delay vascular aging and associated diseases.
Elamipretide / SS-31 Improves Muscle Function But Doesn't Affect Epigenetic Age
https://www.fightaging.org/archives/2025/03/elamipretide-ss-31-improves-muscle-function-but-doesnt-affect-epigenetic-age/
Elamipretide was formerly known as SS-31, and is a mitochondrially targeted antioxidant molecule that improves mitochondrial function, and may or may not achieve that result through the antioxidant mechanism. For many of the current stable of small molecules known to improve mitochondrial function, it isn't entirely clear as to whether their known mechanisms of action are actually the important ones. Here, researchers demonstrate that elamipretide improves muscle function in old mice, an expected outcome, but does not affect epigenetic age, which is perhaps surprising. Epigenetic clocks that assess biological age based on changing patterns of epigenetic modifications to DNA are known to have some blind spots, but mitochondrial function should not be one of them, given the importance of mitochondria in the aging process.
Aging-related decreases in cardiac and skeletal muscle function are strongly associated with various comorbidities. Elamipretide (ELAM), a novel mitochondria-targeted peptide, has demonstrated broad therapeutic efficacy in ameliorating disease conditions associated with mitochondrial dysfunction across both clinical and pre-clinical models. Herein, we investigated the impact of 8-week ELAM treatment on pre- and post-measures of C57BL/6J mice frailty, skeletal muscle, and cardiac muscle function, coupled with post-treatment assessments of biological age and affected molecular pathways.
We found that health status, as measured by frailty index, cardiac strain, diastolic function, and skeletal muscle force, is significantly diminished with age, with skeletal muscle force changing in a sex-dependent manner. Conversely, ELAM mitigated frailty accumulation and was able to partially reverse these declines, as evidenced by treatment-induced increases in cardiac strain and muscle fatigue resistance. Despite these improvements, we did not detect statistically significant changes in gene expression or DNA methylation profiles indicative of molecular reorganization or reduced biological age in most ELAM-treated groups. However, pathway analyses revealed that ELAM treatment showed pro-longevity shifts in gene expression, such as upregulation of genes involved in fatty acid metabolism, mitochondrial translation, and oxidative phosphorylation, and downregulation of inflammation.
Together, these results indicate that ELAM treatment is effective at mitigating signs of sarcopenia and cardiac dysfunction in an aging mouse model, but that these functional improvements occur independently of detectable changes in epigenetic and transcriptomic age. Thus, some age-related changes in function may be uncoupled from changes in molecular biological age.
LAMP1 as a Cell Surface Marker of Senescent Cells
https://www.fightaging.org/archives/2025/03/lamp1-as-a-cell-surface-marker-of-senescent-cells/
Cell surface proteins distinctive to senescent cells can be used as a foundation for immunotherapies that target these cells for destruction. Partial clearance of the age-related burden of lingering senescent cells via small molecule drugs that provoke apoptosis has been shown to produce rejuvenation in aged mice and promising initial results in small human trials, but researchers continue to search for approaches that can remove a greater fraction of senescent cells. Here, find an example of work on surface features of senescent cells, in which a cell surface marker is identified and exploited to construct a proof of principle immunotherapy.
One of the most well-documented hallmarks of senescent cells is their increased lysosomal content and activity. This hallmark feeds into multiple other features of senescence, such as changes in morphology, the senescence-associated secretory phenotype (SASP), and metabolic alterations. Indeed, early efforts quantifying lysosomal activity resulted in the discovery of senescence-associated β-galactosidase (SA-β-Gal), one of the most widely used biomarkers of senescence.
Lysosomal Associated Membrane Protein 1 (LAMP1, also known as CD107a) is a master orchestrator of the structural integrity of lysosomes. LAMP1, as a type I transmembrane glycoprotein, is mostly localized in late endosomes and lysosomes. In immune cells, CD107a is also a cell-surface marker of immune activation and cytotoxic degranulation; however, its expression on the plasma membrane is transient and the protein is internalized rapidly. LAMP1 is only briefly found at the cell surface of healthy cells due to the fusion of lysosomes with the plasma membrane, and is thus mostly undetectable.
The ability to identify and characterize senescent cells is crucial for understanding their role in aging and developing targeted interventions. Here, we describe LAMP1 as a cell surface-specific marker of senescence. LAMP1's presence on the cellular membrane is highly increased in human and mouse senescent cells. In mouse tissue, cells expressing LAMP1 on their surface showed features of senescence. Additionally, senescence induction in the lungs of mice using bleomycin caused an increase in LAMP1+ cells. Finally, senescent cells are eliminated using a LAMP1-targeting antibody. These findings describe a biomarker that can be leveraged to further understand and target senescent cells.
The Aging of the Gut Microbiome is Different by Sex and Mitochondrial Haplotype
https://www.fightaging.org/archives/2025/03/the-aging-of-the-gut-microbiome-is-different-by-sex-and-mitochondrial-haplotype/
Researchers here find that age-related changes in the gut microbiome are quite different by sex, and are influenced by the host's mitochondrial haplotype as well. It is now well known that changes occur in the gut microbiome with age, and these changes contribute to age-related degeneration via reduced production of beneficial metabolites and increased inflammation - but there may well be a great deal of variation from individual to individual. The work noted here was conducted in rats, so it remains to be seen as to whether matters are much the same in humans.
We evaluated the impact of sex and mitochondrial-haplotype on the age-related changes in the fecal gut microbiome of the genetically heterogeneous rodent model, the OKC-HETB/W rat. The age-related changes in the microbiome differed markedly between male and female rats. Five microbial species changed significantly with age in male rats compared to nine microbial species in female rats. Only three of these microbes changed with age in both male and female rats. The mitochondrial-haplotype of the rats also affected how aging altered the microbiome.
Interestingly, most of the microbial species that changed significantly with age were mitochondrial-haplotype and sex specific, i.e., changing in one sex and not the other. We also discovered that sex and mitochondrial-haplotype significantly affected the age-related variations in content of fecal short-chain fatty acids and plasma metabolites that influence or are regulated by the microbiome, e.g., tryptophan derived metabolites and bile acids. This study demonstrates that the host's sex plays a significant role in how the gut microbiome evolves with age, even within a genetically diverse background. Importantly, this is the first study to show that the mitochondrial-haplotype of a host impacts the age-related changes in the microbiome.
CD2AP in Alzheimer's Disease
https://www.fightaging.org/archives/2025/03/cd2ap-in-alzheimers-disease/
The age-related dysfunction of the brain is a deep, complicated, and incompletely understood field of study. Even if aging is driven by relatively simple processes of damage, the brain is a very complex organ, and thus the outcome of even simple dysfunctions will be a very complex web of further interacting consequences. Thus there are any number of papers similar to the one noted here, in which researchers focus on one specific protein and what is known of its interactions in the brain. As a rule, discoveries regarding disease-specific mechanisms are usually broadly true, but it is rarely clear (or even easily understood) as to how much these mechanisms contribute to the overall burden of pathology, whether they are in fact useful targets to pursue for the development of therapies, or just minor components of the disease as a whole.
CD2AP is expressed throughout the body. Since the identification of the association between CD2AP and Alzheimer's disease (AD), the function of CD2AP in the brain has been attracting more and more attention. The mRNA data from the Allen Brain Atlas suggest that although CD2AP may be expressed at low levels in neurons, it is relatively enriched in highly plastic brain regions such as the hippocampus, cortex, and cerebellum. In addition, high expression of CD2AP was observed in dendritic endosomes of primary cultured mouse neurons and the absence of CD2AP in neurons was shown to cause synaptic damage. Moreover, we recently discovered that CD2AP was expressed at higher levels in microglia compared to neurons in mice and that CD2AP could regulate microglial activation in response to amyloid-β toxicity.
CD2AP is intricately involved in intracellular protein transport and degradation, vesicle trafficking, cell signaling, and cytoskeleton remodeling. As a risk factor for AD, abnormalities in CD2AP in the nervous system may contribute to the pathogenesis of AD through various mechanisms, including influencing the transport and processing of amyloid precursor protein (APP) and thus amyloid-β generation, participating in Tau-mediated neurotoxicity, disrupting synaptic function and vesicle release, modulating microglial activation, and compromising the integrity of the blood-brain barrier. However, the specific molecular mechanisms by which CD2AP participates in these processes have yet to be fully elucidated.
Gene Regulatory Networks in the Design of Approaches to Slow Aging
https://www.fightaging.org/archives/2025/03/gene-regulatory-networks-in-the-design-of-approaches-to-slow-aging/
Researchers here outline how it is possible to use what is known of gene regulatory networks in order to design better approaches to slow aging. Proteins interact with one another, and feedback loops involving interactions and changes in expression among many proteins determine each aspect of cell behavior. The key realization is that in such a complex system, one has to think about these networks rather than any one individual protein in order to maximize the chance of producing a useful approach to altering cell behavior.
Earlier aging studies focused on individual genes or pathways in isolation and measured lifespan as a static endpoint. As a result, how aging-related genes interact with one another and how these gene regulatory networks (GRNs) operate dynamically to drive aging remain significant unanswered challenges. GRNs consist of nodes, that symbolize genes or regulatory elements, and edges, that depict the interactions or regulatory connections between these nodes. Highly connected nodes at the center of a GRN are the major orchestrators of the response of a cell to stimuli.
The dynamics of these nodes can often be explained by focusing on a few key local interactions, namely subgraphs. Network motifs are recurrent sub-GRNs, typically including up to four nodes, that have characterized behaviors. Network motifs can be as simple as positive autoregulation which ensures the sustained activity of a node. By contrast, mutual inhibition between two nodes can lead to two distinct cell fates where the system stabilizes in one of two states based on initial conditions. The negative feedback loop is a motif that is especially crucial for ensuring homeostasis, and is activated by deviations from a set point that trigger mechanisms to counteract those changes. These motifs are observed in many GRNs and are reinforced by redundant and compensatory pathways to increase the resilience of the system to perturbations.
Decoding the emergent behavior of aging-related GRNs sets the stage for rational design of new interventional strategies to mitigate age-related diseases and promote healthy longevity. However, the intricate nature of aging-related processes cannot be fully understood through traditional reductionist methods. Instead, systems-level approaches designed to analyze the nonlinear dynamics of gene circuits are required. In addition, such network-based approaches can be naturally integrated with synthetic biology to reveal the design principles of prolongevity strategies.
Hypothesizing that Non-Coding RNAs are a Major Determinant of Species Life Span
https://www.fightaging.org/archives/2025/03/hypothesizing-that-non-coding-rnas-are-a-major-determinant-of-species-life-span/
Non-coding RNA sequences in the genome undergo transcription to produce an RNA molecule, but that RNA is not translated into a protein. Nonetheless, non-coding RNAs collectively form just as complex an interacting environment as proteins, important to the function of the cell. Non-coding RNAs remain poorly explored, as much of the work on cell biology to date has focused on proteins. It is unclear if the present catalog of non-coding RNAs is complete, and many of the known entries have unknown functions. Here, the argument is made for non-coding RNAs to collectively be an important determinant of species life span, based on the differences observed between short-lived and long-lived species.
Lifespan is a complex process that interacts with multifactors, yet it is fundamentally an evolutionary process in which genetic factors evolve to cope with lifespan evolution. Thus, it is essential to uncover the genetic factors that contribute to lifespan variations among different species. Current studies have focused on protein-coding genes in the search for longevity determinants, but the results from these studies have not provided sufficient evidence to explain the evolutionary lifespan disparity, even between a small group of species or individuals. The genetic factors contributing to large-scale lifespan gaps between species remain elusive.
When species genomes evolve, they usually acquire more noncoding RNAs (ncRNAs) than proteins. For example, the human genome contains a larger number of ncRNAs than its mouse counterpart, whereas most proteins remain similar. Importantly, these ncRNAs are actively transcribed with their own functional system and they endogenously execute fundamental functions, including lifespan extensions. Therefore, it is reasonable to hypothesize that ncRNAs play a key role in the evolution of the lifespan of an organism.
The present study analyzed multiple large datasets and revealed that ncRNAs indeed work as the primary evolutionary drivers extending animal lifespans and serve as crucial determinants of reproductive systems. Longevity and reproduction are two most important traits of any organism evolution, suggesting that ncRNAs work as the fundamental drivers driving the long evolutionary process and they carry crucial functions in the organism's genome.
Advanced Glycation Endproducts in Muscle Loss Leading to Sarcopenia
https://www.fightaging.org/archives/2025/03/advanced-glycation-endproducts-in-muscle-loss-leading-to-sarcopenia/
Advanced glycation endproducts (AGEs) are an undesirable form of metabolic waste. The formation of long-lived AGEs that cross-link molecules in the extracellular matrix can change the physical properties of tissue, such as by contributing to the stiffening of blood vessel walls that occurs with age. Further, most varieties of AGE, while being only short-lived, can interact with cell receptors to provoke a maladaptive inflammatory response, thereby contributing to the chronic inflammation of aging. Inflammation, in turn, alters cell behavior for the worse throughout the body. Here, researchers provide an overview of how AGEs contribute to the age-related loss of muscle mass that leads to sarcopenia.
By binding with receptor for advanced glycation end products (RAGEs), AGEs can activate a series of intracellular signalling pathways in skeletal muscle cells related to the elevated levels of inflammation and oxidative stress, as well as impaired insulin/insulin-like growth factor-1 (IGF-1) signalling and mitochondrial biogenesis, which lead to reduced protein synthesis, increased protein degradation, intracellular lipid accumulation, changes in muscle fibre type composition and muscle energy metabolism, and a higher rate of apoptosis, finally resulting in muscle atrophy and impaired regeneration abilities.
Through directly targeted glycosylation, AGEs can damage the biological properties and functions of proteins which include the functional and structural proteins of skeletal muscle as well as collagens in the extracellular matrix, resulting in muscle dysfunction such as impaired force production and increased stiffness. Furthermore, AGEs can also indirectly affect skeletal muscle by contributing to neuromuscular junction lesion and vascular disorders.
Tissue Resident Macrophages in the Heart in Cardiovascular Disease
https://www.fightaging.org/archives/2025/03/tissue-resident-macrophages-in-the-heart-in-cardiovascular-disease/
The innate immune cells known as macrophages can be found in tissues throughout the body, where they perform many functions. Macrophages do not just find and destroy pathogens and potentially problematic cells, they also help to coordinate regeneration from injury. They can take on pro-inflammatory or anti-inflammatory states depending on circumstances. Researchers are interested in finding ways to reduce inflammation and promote greater regeneration by manipulating macrophage state and activities, and here the focus is on macrophages resident in the heart, an organ that exhibits relatively little regenerative capacity following injury.
Macrophages are essential factors of the body's innate immune system and mononuclear phagocyte system and are widely present in the structure of the tissues, including the heart. Cardiac macrophages play an integral physiological role to regulate the physiological and pathological processes of the cardiovascular system. Resident macrophages are heterogeneous and plastic, and multiple subsets with different phenotypes and functions are present in the same tissue and are involved in different pathophysiological processes. There is increasing evidence suggesting that cardiac-resident macrophage populations play a critical role in regulating heart development, electrical conduction, and ventricular remodelling processes.
The mechanisms used by cardiac macrophages to influence cardiovascular disease (CVD) vary and include both direct and indirect interactions with other cardiac cells. In particular, the identification of specific targets for cardiac resident macrophages to regulate CVD would be crucial. Due to the development of various exogenous (using delivery of toxic substances, blocking antibodies and small interfering RNAs) and genetic methods (transgenic methods) to broadly and specifically target these macrophage populations, this has provided us with the opportunity to understand the function of various cardiac and pericardial macrophages. Relatively few studies have addressed therapies targeting cardiac resident macrophages in patients with CVD although mechanistic knowledge about cardiac resident macrophages and their contribution to cardiovascular risk have accumulated in recent years.
View the full article at FightAging
