Epigenetics and Longevity Interventions

As of now we’ve gone over what epigenetics is and what it does. We then talked about which mechanisms epigenetics uses to exert control over the various aspects in the cell. Then lastly, we talked about how the epigenome changes with age along with what drives that change and how it can be quantified. Now all that’s left is to reverse it. 

As of the release date of this article, the control we are able to exert over the epigenome in our own cells is limited. We have yet to elucidate the mechanisms that make the epigenome tick let alone manipulate it with ease. Thus, most of the interventions we are able to apply to the epigenome (as of now) are either through indirect consequences and/or by leveraging already existing tools found in nature. Because of the indirect nature of our interventions the line between an epigenetic intervention and another intervention that targets another aspect of aging may be blurred. 

In the future there may be engineered mechanisms that allow precision epigenetic editing, however presently we are stuck with optimizing present systems to do our bidding. 

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This article is part four of a four-part series on epigenetics.

1. Introduction to Epigenetics
2. Epigenetics and Gene Expression
3. Epigenetics and Aging
4. Epigenetics and Longevity Interventions

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The Potential Challenges of Tracking Aging

Now before going on to the interventions of aging we first have to talk about measurement. To say that we have something that can reverse aging, i.e interventions, we first have to have a number that represents aging which goes up or down. The ability to measure aging is perhaps one of the biggest challenges in the field. In the previous article we mentioned this problem and how aging clocks were a proposed solution to this problem. Aging clocks are one of the first solutions that come to mind when measuring aging yet they still remain somewhat unreliable.
Although the pool of evidence supporting their abilities to reflect aging are increasing we are still unable to make definitive statements through the results of aging clocks. For example a recent paper showed that 13 of the 17 most common aging clocks used today oscillated throughout the day.¹ For example, the pan tissue horvath clock we talked about in the last article predicted the epigenetic age of a 52 year old individual 55.3 years at ~11:30 PM which changed to 58.5 years at ~11:30 AM in the same day.

However, some clocks such as DunedinPACE, which NOVOS uses in NOVOS Age, do not show the same oscillation. Suggesting they navigate noise better and capture aging more accurately. Even though discrepancies like this exist, it should be noted that for every inconsistency, there are several areas where aging clocks are accurate. Their ability to reflect age-related changes, such as lifestyle habits, diseases, and interventions, is improving, with certain clocks outperforming others in specific aspects. Nevertheless, these clocks still have a long way to go and must be stress-tested, as shown in the study above, to be used as conclusive evidence for age reversal.

Other than aging clocks there exists other methods to characterize aging. On the cellular level options are limited. The measurements to assess changes in the hallmarks of aging. These include the changes we mentioned in the previous article: expression changes in some genes, erosion of heterochromatin, mitochondrial activity, metabolic profiles etc. But the gold standard for aging measurements are on the organismal level. These are really not very different from the things you can think about: How do they look? How much do they weigh? How far can they run? How strong are they? How well can they remember? And, of course, how long do they live? All of these are things that we know that change with age and ideally the best aging interventions should show improvements in several of these areas. 

Diet & Lifestyle

Aging is influenced by both genetic and environmental factors, with diet and lifestyle changes playing significant roles. Epigenetic mechanisms, such as DNA methylation and histone modifications, mediate these effects. Calorie restriction (CR), which reduces calorie intake without sacrificing nutrients, has been shown to extend lifespan and improve healthspan across species, including humans.²,^3,4 CR helps prevent age-related changes in DNA methylation and histone modifications, effectively reducing biological age.⁵ This is partly achieved through the upregulation of sirtuins, which promote genomic stability and stress resistance.⁶ The composition of the diet also impacts aging through epigenetic modifications. Restricting branched-chain amino acids (BCAAs) slows aging by altering histone acetylation, while nutrients like glucose and fatty acids influence longevity by modulating DNA methylation and histone modifications.^7,8 For example, monounsaturated fatty acids extend lifespan in C. elegans by affecting chromatin remodeling.⁹

Exercise contributes to healthy aging by triggering beneficial epigenetic changes and mitigating age-related hazards. It influences DNA methylation patterns and histone modifications, regulating gene expression in various tissues.^10,11 Late-life exercise, for instance, reduces epigenetic aging in skeletal muscle by decreasing DNA methylation.¹² This effect was demonstrated in a study where older mice, after engaging in weighted wheel running, exhibited an eight-week reduction in their epigenetic age compared to sedentary mice. Lifestyle factors such as sleep, smoking cessation, and emotional stability also impact aging through epigenetic mechanisms. Regular sleep and quitting smoking can decelerate epigenetic aging, while chronic stress accelerates it through harmful epigenetic changes.¹³ 

In conclusion, adopting healthy lifestyle changes—including dietary modifications, regular exercise, and stress management—are promising strategies to combat aging. These interventions work through epigenetic mechanisms, promoting longevity and improving healthspan.

Pharmacology

Pharmacological strategies targeting aging and aging-associated disorders can be divided into two main classes: geroprotective compounds and senolytics. Geroprotective compounds bring beneficial outcomes such as metabolic improvement, inflammation relief, and epigenomic stabilization. 

Extensive efforts have focused on developing small-molecule-based aging interventions, with metformin and rapamycin being the best studied and most effective. Metformin, originally an antidiabetic drug, shows promise in extending lifespan and healthspan.¹⁴ It activates AMP-activated protein kinase (AMPK), leading to improved insulin sensitivity, reduced inflammation, and enhanced mitochondrial function. Metformin also affects epigenetics by regulating enzymes such as histone acetyltransferases (HATs), histone deacetylases (HDACs), and DNA methyltransferases (DNMTs).¹⁵ By modulating these enzymes, metformin alters gene expression patterns to promote a youthful cellular state, reduces oxidative stress, and lowers the risk of age-related diseases like cancer and cardiovascular diseases. Rapamycin, an mTOR inhibitor, mimics the effects of caloric restriction by downregulating the mTOR pathway.¹⁶This inhibition reduces protein synthesis and cell growth, enhancing autophagy, which cleans out damaged cellular components.¹⁷ Rapamycin also influences epigenetics by slowing the accumulation of epigenetic aging signatures, such as DNA methylation changes, in liver cells of mice.¹⁸ Additionally, it improves immune function, reduces inflammation, and delays age-related diseases such as cancer and neurodegenerative disorders.¹⁹ Its ability to target fundamental aging mechanisms and epigenetic changes makes it a promising candidate for extending both lifespan and healthspan.

NAD+ (nicotinamide adenine dinucleotide) is a vital coenzyme involved in numerous metabolic processes, including energy production and cellular repair. NAD+ precursors, such as nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), and nicotinamide (NAM), help maintain NAD+ levels, which are crucial for DNA repair, epigenetic regulation, and cellular health.²⁰ Supplementing with these precursors can prevent NAD+ decline, benefiting aging and related diseases.²⁰ Studies in animals show that NAD+ repletion extends lifespan, improves mitochondrial function, delays cellular aging, and enhances cognitive functions.²¹ While more human studies are needed, boosting NAD+ levels with these supplements shows promise in combating aging and associated disorders.

NMN is among the primary ingredient in NOVOS Boost. Coupled with NOVOS Core ingredients such as fisetin, glycine, pterostilbene, Rhodiola Rosea, calcium alpha-ketoglutarate, glucosamine sulfate, hyaluronic acid, and L-theanine, it supports various aspects of cellular health and epigenetic stability. Fisetin and pterostilbene, for example, activate sirtuins and reduce oxidative stress, while glycine and L-theanine support cellular functions and stress responses.²², ²³, ²⁴, ²⁵ Rhodiola Rosea and micro-dosed lithium have neuroprotective effects, and alpha-ketoglutarate and malate contribute to metabolic processes and mitochondrial function.²⁶, ²⁷, ²⁸, ²⁹ Glucosamine and hyaluronic acid support joint and skin health, crucial for healthy aging.³⁰, ³¹ These supplements, among others present in NOVOS supplements, have many benefits, some unmentioned, targeting the various hallmarks of aging and help promote healthy aging.

Senolytics, which eliminate senescent cells, improve age-related impairments and indirectly affect the epigenome.³² While primarily known for inducing apoptosis in senescent cells, senolytics like dasatinib and quercetin also influence DNA and histone methylation profiles.³³, ³⁴ Although the connection between senolytics and epigenetics is less direct, their role in removing senescent cells can lead to a rejuvenated tissue environment, potentially affecting the epigenetic landscape.

In conclusion, pharmacological strategies that target aging through epigenetic mechanisms show great promise. Geroprotective compounds, by modulating DNA methylation and histone modifications, and senolytics, through their indirect impact on the epigenome, represent promising avenues for promoting longevity and improving healthspan. Further research is needed to fully elucidate how these drugs and supplements interact with epigenetic networks to delay the aging process.

Partial Reprogramming

During a process called differentiation the fertilized zygote goes through a complex and intricate process that results in a human being with a multitude of systems, organs, tissues and cell types. We talked about the metaphor of the waddington epigenetic landscape and how the differentiating cell is represented by a ball rolling down the landscape through a series of hills and valleys to find its final resting place, the terminally differentiated cell type.³⁵ Then through the transfer of the nucleus of a cell that traversed through this journey through the landscape and had already found its final resting place was able to be reprogrammed when it was placed into enucleated egg.³⁶ Then comes reprogramming where the infamous Japanese scientist Shinya Yamanaka discovered that the expression of 4 transcription factors were enough to reprogramming terminally differentiated cells into pluripotent stem cells.³⁷ This discovery ignited a whole new area of research that better characterized the process of these somatic cells becoming stem cells. The main characteristic of stem cells is that they are able to make several different cell types. In the case of pluripotent stem cells which are one of the most potent stem cells they are able to differentiate into all the different cell types present in the adult human body. During reprogramming for somatic cells to be able to gain the ability to make multiple different cell types the first step in their journey to become stem cells is for them to forget what type of cell they were. 

Through the hundreds of papers published on cellular reprogramming scientists began to see signs of rejuvenation during the earlier stages of the reprogramming process. This prompted the first mention of “partially reprogrammed cells” for rejuvenation. A decade after the discovery of cellular reprogramming in 2016 came proof that partial reprogramming was successful in mice.³⁸ Where when the reprogramming process is interrupted before the cells lose their identities on their journey to become stem cells. Partial reprogramming was first shown to be able to extend the lifespan of mice with accelerated aging syndromes. In the same paper it was also shown that partial reprogramming could alleviate some hallmarks of aging in human cells in vitro as well. As of now partial reprogramming still remains an emerging area with some tens of papers published about it. But what specifically does partial reprogramming change in these cells and organisms and how can we be sure that it’s aging? Well, in the first organismal partial reprogramming study we’ve been mentioning in 2016, they looked at the most straightforward and irrefutable aging measurement, which is lifespan. Through their partial reprogramming, they were able to extend the lifespans of mice with an accelerated aging syndrome by up to 50%.

Almost 10 years after this landmark paper, partial reprogramming still remains a new and niche area of research with only some tens of papers published in this area. Among these papers the more impactful and rigorous studies don’t target aging directly but rather focus on the abilities of partial reprogramming to induce regeneration or treat certain diseases. For example one of the best studies using partial reprogramming, published in one of the three apex journals, Nature used partial reprogramming to reverse vision loss caused by nerve damage^.40 They do this the researchers crush the optic nerve which results in vision loss. From the figure below the damage site is marked by the blue stars. The optic nerve is stained with a dye that shows active neurons, meaning they’re active if they’re glowing. On the bottom panel there is the optic nerve from mice that have received the partial reprogramming treatment and the ones that haven’t in the top panel. Upon partial reprogramming you can see the regenerating neurons on the right side of the damage site. This neuronal regeneration is also accompanied by a partial restoration of vision in the mice. In fact, this paper eventually led to the creation of a startup called Life Biosciences, which is focused on treating an eye-related disorder known as NAION. They are conducting pre-clinical studies in non-human primates and have shown promising results, with plans to begin human clinical trials in 2025.⁴¹

Another great example of using partial reprogramming to induce regeneration was done in the heart, published in the journal Science, one of the 3 apex journals. This study showed that transient expression of reprogramming factors (OSKM) in adult cardiomyocytes, which normally have limited regenerative capacity, can induce a proliferative state. This process significantly enhanced cardiac repair and function following myocardial infarction by reducing scar size and promoting the proliferation of existing cardiomyocytes.⁴²

The previous two studies’ main focus was treating a disease through reprogramming induced regeneration. Another study showed that partial reprogramming can make old fibroblasts resemble young fibroblasts in various hallmarks of aging measurements. Partially reprogrammed old human fibroblasts showed improvements in several hallmarks of aging, including enhanced epigenetic marks (H3K9me3, HP1γ, LAP2α), increased proteolytic activity, improved mitochondrial function, and reduced cellular senescence.⁴³

Researchers have also been working to optimize the reprogramming process. In one study, scientists measured gene expression at different stages and experimented with various combinations of reprogramming factors. They discovered that reprogramming temporarily causes cells to lose their identity, which is crucial for rejuvenating their gene expression. Once the reprogramming process is halted, the cells regain their original identities.⁴⁴

Chemical Partial Reprogramming

Up until a couple years ago the reprogramming of human cells were only able to be done through the expression of genes. The expression of the transcription factors led to the remodeling of the epigenome erasing the existing cell identity and eventually producing stem cells. Delivering genes into cells equally and efficiently is a challenging problem to tackle that is bottlenecking not only the area of partial reprogramming but many other therapies as well. In gene therapy treatments this is usually done through engineered viral systems. Although these viral systems are effective delivery systems they are more appropriate for specific targeted areas rather than whole body delivery.⁴⁵

An alternative that has recently emerged to genetic reprogramming is reprogramming with chemicals. Chemical reprogramming involves the use of small molecule inhibitors and activators that are able to target specific epigenetic modifiers. Human chemical reprogramming was finally achieved in 2022 after years of research on the effects of small molecules on reprogramming.⁴⁶ Chemical reprogramming of mouse cells was achieved almost a decade before that.⁴⁷ Using chemicals for partial reprogramming has many benefits. Firstly and most importantly, it solves the delivery problem as small molecules are able to diffuse more easily throughout tissues and can be administered systemically, ensuring more uniform delivery to cells. Secondly, chemical reprogramming is less invasive and can be fine-tuned with dosage adjustments, reducing the risk of unintended consequences like uncontrolled cell proliferation. Thirdly, it circumvents some of the safety concerns associated with viral vectors and genetic modifications, potentially offering a safer and more accessible approach to rejuvenation therapies.

Furthermore, the flexibility of chemical compounds allows for the combination of multiple molecules to target various epigenetic pathways simultaneously, enhancing the efficacy of the reprogramming process. Research continues to optimize the specific combinations and concentrations of these molecules to achieve the desired effects without compromising cellular function or identity.

For example, one study found that partial chemical reprogramming in mouse fibroblasts led to widespread changes, including upregulated mitochondrial function and reduced aging-related metabolites, effectively reducing biological age and improving cellular functions.⁴⁸ Another study identified six chemical cocktails that, within a week, restored a youthful transcript profile in human cells without altering the genome, demonstrating the potential of chemical reprogramming for cellular rejuvenation.⁴⁹

Overall, chemical reprogramming presents a promising alternative to genetic reprogramming that is potentially cheaper, safer, and more efficient. It simplifies the delivery process, reduces the risk of adverse effects, and offers greater flexibility in targeting multiple epigenetic pathways. If partial reprogramming becomes a widespread intervention for aging, it is likely to be through these chemical methods.

Obstacles / Criticisms of Partial Reprogramming

Looking at this data and how we can rejuvenate damaged tissues and extend the lifespan of some mice might have gotten you excited. But we are nowhere near partial reprogramming being used as an actual therapy. There are scores of boxes we have to tick off first. Some of these boxes are:

• Safety & Cancer: This is the single biggest concern for partial reprogramming being used as an aging intervention. The reprogramming process is meant to generate pluripotent stem cells, which aren’t supposed to exist in the adult body. If a single pluripotent stem cell were present in an adult body, it would likely begin to differentiate according to the environment it is in. However, the differentiation process is incredibly complex and relies on a plethora of signaling events that coordinate it during embryonic development. In the adult body, no such environment exists. Pluripotent stem cells share several characteristics with cancer cells, such as the ability to proliferate indefinitely and bypass normal cellular controls. Without the proper signals for differentiation, these stem cells can lose their pluripotency and start to behave more like cancer cells.⁵⁰ In fact one of the best tests one can perform to see if a stem cell is indeed pluripotent is injecting some of the cells they’ve generated into mice and see if they’ve formed a tumor with cells from different embryonic linages^.51  Instead of differentiating correctly, the stem cell might enter a state of uncontrolled proliferation. This uncontrolled proliferation can lead to the formation of tumors, as the cell divides rapidly without the usual checks and balances that regulate cell growth and development. The lack of proper differentiation cues can result in the cell becoming stuck in a proliferative state, where it continuously multiplies and accumulates mutations, further increasing the risk of cancer. 

• Efficiency: Cellular reprogramming is quite inefficient, with fewer and fewer cells making it through each stage of the process. In fact, only about 0.1% of the starting cells manage to become fully reprogrammed stem cells.⁵² For partial reprogramming, more cells reach the desired stage because it happens earlier in the process. However, it’s still not perfect—only a portion of the cells get reprogrammed and rejuvenated, while many remain aged. To make this more effective, we need better protocols that can reprogram a larger and more consistent percentage of cells, ensuring a more uniform rejuvenation across the entire cell population.

• Delivery: Efficiently delivering reprogramming factors to cells is a major challenge. Current methods often fail to target all cells uniformly, resulting in only a fraction of the population receiving the necessary factors. This uneven delivery means that not all cells are reprogrammed or rejuvenated as intended. To improve outcomes, we need more reliable delivery techniques that ensure all cells in a population are uniformly reprogrammed. Chemical reprogramming is one of the possible solutions to these problems. It’s a simpler, cheaper, and more controllable method that could help achieve a more consistent rejuvenation process across all cells, addressing many of the delivery issues we face with traditional methods.

• Long-term Stability: Making sure reprogrammed cells stay rejuvenated over the long term is essential. Even if we manage to reprogram cells successfully, there’s a chance they could revert to their aged state or develop other issues over time. For these therapies to be truly effective, we need to ensure that the rejuvenated cells remain stable and functional for the long haul. This means figuring out how to lock in the benefits of reprogramming and prevent any backsliding or unexpected problems, ensuring the cells stay healthy and youthful for extended periods.

• Reprogramming Heterogeneity: Several of the reasons above lead to an overall heterogeneity observed in reprogramming. Cells reprogram at different speeds and reach various stages of reprogramming at different times.⁵³ This variability can result in a mixed population of cells, some fully reprogrammed, some partially reprogrammed, and others not reprogrammed at all. This heterogeneity poses a challenge for achieving consistent and predictable outcomes. To improve the effectiveness of reprogramming therapies, we need to develop protocols that minimize this variability, ensuring a more uniform reprogramming process and more reliable results across the entire cell population.

Future of Partial Reprogramming

Partial reprogramming is a borderline science fiction-like therapy proposed for treating aging. While it’s a promising concept, we’re still a long way from seeing it used in clinics. For nearly two decades, research has focused on cellular reprogramming, not for rejuvenation. Instead, the goal was to generate stem cells. Compared to this, research into partial reprogramming has been relatively limited in both time and attention. It’s only recently that we’ve discovered this protocol, initially designed to create stem cells, can also rejuvenate some aspects of aging.

One major challenge we need to tackle to use partial reprogramming as an aging therapy is separating the rejuvenation effects from the loss of cellular identity. It’s possible that losing cellular identity is necessary for the rejuvenation we see with reprogramming. Figuring out how to decouple these effects may come from various sources. One potential solution involves using different transcription factors. The original discovery of the four reprogramming factors focused on generating stem cells, but other factors can also induce this process. In some studies, scientists have successfully replaced some reprogramming factors with others. For partial reprogramming, a common practice is to exclude the transcription factor c-Myc, which is a known oncogene.⁵⁴

Researchers have also discovered that the transcription factor SRSF1 can rejuvenate cells by reprogramming their transcriptome to a younger state, improving cellular function, wound healing, and longevity.⁵⁵ NewLimit, an aging research company founded in 2021, aims to find more optimal combinations of reprogramming factors for rejuvenation.⁵⁶ According to their latest progress report, they’ve tested over 100 reprogramming factor sets and already have their first iteration of sets nominated for further testing.⁵⁷

Besides genetic approaches, chemical reprogramming is another promising alternative. By using small molecules to modulate cellular pathways, chemical reprogramming can simplify the process and potentially reduce the risks associated with genetic modifications. This method could complement existing strategies, offering a more controllable and cost-effective approach to cellular rejuvenation.

The future of partial reprogramming lies in overcoming these challenges and exploring diverse methods. From optimizing transcription factor combinations to integrating chemical reprogramming techniques, we’re getting closer to unlocking the full potential of partial reprogramming as a viable therapy for aging.

Heterochronic Parabiosis

The list of aging interventions doesn’t end here. Although it may not be strictly epigenetic, they still have a significant impact on the epigenome. One such intervention, arguably more extreme than partial reprogramming, is heterochronic parabiosis (HPB). This method, which predates most modern therapies, involves surgically joining the circulatory systems of a young mouse and an old mouse. The result? Rejuvenation in the old mouse and accelerated aging in the young one. Recent studies have taken HPB to new lengths. Extended HPB over three months, followed by a two-month separation period, showed that old mice exhibited improved physiological parameters, reduced epigenetic age in blood and liver tissues, and increased lifespan.⁵⁸ Remarkably, these effects persisted even after detachment. Additionally, exposure to young blood has been shown to reverse brain aging at the molecular, structural, and cognitive levels. It improved synaptic plasticity and cognitive functions in aged mice.⁵⁹ In fact, the beneficial effects of young blood have already been embraced by some, such as tech millionaire Bryan Johnson. He claims to have regular blood transfusions from his teenage son to decrease his biological age.⁶⁰, ⁶¹ There have also been attempts to create companies that sell “young” blood at high prices.⁶², ⁶³ While young blood transfusions might not be a feasible aging intervention for the masses, identifying and isolating the beneficial, “young” molecules from young blood could lead to the development of drugs that offer similar benefits.

The Future of Epigenetic Aging Interventions

There is indisputable evidence that the epigenome plays a central role in the aging process and its potential rejuvenation. As research advances, precision epigenome editing emerges as a promising frontier. Using technologies like CRISPR scientists can target and modify specific epigenetic marks without altering the underlying DNA sequence.⁶⁴, ⁶⁵, ⁶⁶This method offers the potential to make precise changes to the epigenome, potentially reversing age-related alterations and restoring youthful function.

But before we can fully harness the power of precision epigenome editing for rejuvenation, several crucial steps must be taken. First, we need to identify the key epigenetic changes associated with aging and determine whether they are universal across different cell types and individuals. Understanding these changes will allow us to target the most impactful modifications for reversing the aging process. Additionally, defining the ideal “young” epigenetic state is essential. This will serve as a benchmark for successful interventions, guiding the development of therapies that can restore this youthful state.

In the future, precision epigenome editing could be used to fine-tune the epigenetic landscape, reversing harmful changes and promoting healthy aging. Combined with other interventions like partial reprogramming and chemical reprogramming, this approach holds the potential to significantly extend healthspan and lifespan.

Conclusion

The field of aging research is rapidly advancing as we gain a better understanding of our biology and learn to manipulate it to our advantage. From lifestyle modifications to cutting-edge pharmacological and genetic interventions, numerous strategies are emerging to combat the aging process. While existing interventions show promise, the journey towards effective and safe age-reversal therapies is still in its early stages. Future research must focus on refining these interventions, ensuring their safety, and understanding the long-term implications of altering the epigenome. Even though these interventions seem promising, a comprehensive approach that integrates multiple strategies targeting various aspects of aging will likely be necessary to achieve meaningful results. As we continue to explore these possibilities, the ultimate goal remains not only to extend lifespan but also to enhance the quality of life, allowing individuals to enjoy healthier years for longer. 

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