A Roadmap to Rejuvenation: Targeting the Hallmarks of Aging
Aging is a complex process, a river fed by several tributaries connected by countless interweaving streams. Its direction is set inexorably towards infirmity, or so it would first appear. Daunting as navigation may seem, their interrelatedness should inspire hope instead of fear.
Aging is undeniably the root of the most common and costly noncommunicable diseases in the developed world, as well as a predisposing factor to severe or fatal reactions to infectious ones. Whatever can be done to slow, halt, or reverse its course holds inestimable economic and humanitarian value (Lee, 2017).
The hallmarks of aging were assembled to broadly conceptualize what lies behind phenomena as seemingly unrelated as gray hair, wrinkles, heart disease, cognitive decline, and cancer. They serve as explanations for why everything from our joints to our eyesight steadily give out over time.
Lopez-Otin, Maria Blasco, and their colleagues originally identified nine hallmarks. Although others have since been added to different lists, and are worthy of consideration in future articles, our discussion will revolve around the original nine: genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion, and altered intercellular communication (Lopez-Otin, 2013).
Telomere attrition and stem cell exhaustion, the former being largely responsible for the latter, are the two most widely recognized hallmarks. A close second is oxidative damage (reformulated over time with an emphasis on mitochondria), due to its long history as a focal point for anti-aging research fueled by a largely misplaced preoccupation with free radical damage.
Senescent cells continue to receive sporadic media coverage thanks to fanfare for senolytics, but whether the evidence supports this enthusiasm is another question (Flores, 2010; de Grey, 1999; Kang, 2019).
What makes something a hallmark rather than a symptom?
In molecular biology, clear chains of causality are not always forthcoming, but it can be argued that some are more fundamental than others.
All of these factors are the roots of chronic disease, and some run deeper than others.
While there are several (and not always mutually exclusive reasons) for a cell to become senescent, telomere shortening is likely the most common culprit (Zhu, 2019).
Critically short telomeres activate DNA damage response pathways, inducing a senescence-associated secretory phenotype (SASP). SASP is characterized by the secretion of pro-inflammatory cytokines and chemokines that can promote tissue damage and age-related diseases (Freund, 2010).
While they are not without promise or utility, senolytics have concerning limitations and side-effects.
Their lack of specificity can be detrimental as senescent cells are also present in healthy tissues; indiscriminate destruction of senescent cells can lead to the loss of healthy cells, tissues, and organs.
Chang et al. (2016) found that treatment with dasatinib and quercetin, two common senolytics, decreased the number of senescent cells in the liver, kidney, and adipose tissue of mice. However, treatment also led to the loss of osteoblasts in the bone, without which normal bone formation and remodeling does not take place.
This same combination, while yielding improvements in cognitive function in a mouse model of Alzheimer’s, also resulted in a decrease in left ventricular ejection. Similarly, an investigation in diabetic mice, showed that, while the combination improved insulin resistance, it also produced cardiac dysfunction and metabolic inflexibility (Bussian, 2010; Song, 2018).
Moreover, why a cell is senescent should be considered: cellular senescence protects our bodies from cancer by preventing the proliferation of damaged cells, facilitating their clearance, and promoting tissue repair (Ogrodink, 2018).
“Telomeres are like the plastic tips on shoelaces that keep them from fraying; they are protective caps at the end of each strand of DNA that help prevent damage and maintain the integrity of the genome.”
- Elizabeth Blackburn, Nobel laureate and pioneer in telomere research.
What causes telomere shortening?
Telomere attrition accompanies cell division. Telomeres become slightly shorter due to incomplete DNA replication. This is the “end-replication problem.” This, however, is not the end of the story (Eppel and Blackburn, 2017).
Oxidate damage leads to the activation of multiple DNA damage response pathways. These in turn activate p53, a tumor suppressor protein.
Oxidative damage also impacts telomerase, the enzyme responsible for maintaining telomere length. This further aggravates telomere shortening and cellular aging (Ahmed, 2018).
Klotho is the queen of anti-aging proteins.
Makato Kuro-o, one of the foremost authorities on Klotho, proposed it protects against oxidative damage by promoting the production of antioxidant enzymes and inhibiting the generation of reactive oxygen species (ROS). Klotho elevates the expression of superoxide dismutase (SOD), an enzyme that converts harmful superoxide radicals into (less harmful) hydrogen peroxide (Kuro-o, 2008).
Klotho can directly scavenge ROS, which partially accounts for its effects on age-related diseases, such as cardiovascular disease, diabetes, and neurodegeneration. It is one of the reasons BioViva Science is exploring Klotho gene therapy as treatment for Alzheimer’s and other forms of dementia (Afanas’ ev, 2010).
There is already compelling human evidence for the use of Klotho and telomerase gene therapy in neurodegenerative disease (Sewell, 2019). It’s good to note that Klotho activates the production of endogenous antioxidants.
While supplements like vitamin C and E can reduce oxidative stress and in some cases improve cellular function, they do not extend median or maximum lifespan in mammalian model organisms, unlike rapamycin, metformin, caloric restriction (CR), telomerase, or follistatin.
Although it’s been proposed that CR’s effects are due mostly to diminished oxidative stress, there are compelling reasons to believe this is not the case. Any effective longevity intervention will likely have to pull on several levers.
High doses of certain antioxidants over long periods are probably deleterious. For example, vitamin E was found to increase mortality risk in older adults and increase issues in smokers. Meta-analysis, however, suggests there is simply no benefit (Abner, 2017).
There are many conditions antioxidants appear to help with, though often only marginally. Aging, the MacDaddy of them all, the true emperor of all maladies, is not one of them.
Long-lived species have evolved more efficient mechanisms for dealing with oxidative stress. Heterocephalus glaber, the naked mole rat, is a negligibly senescent mammal — meaning they remain youthful up until the end of their lives, whereas animals like ourselves experiencce a gradual (then steep) decline.
While Naked mole rats (NMRs) have higher levels of antioxidant enzymes, their DNA repair mechanisms may contribute to their remarkable longevity compared to other rodents — despite higher levels of oxidative stress. A lab mouse (Mus musculus) will live between 1.5 and 2 years, an NMR lives up to thirty (MacRae, 2015).
- NMRs have a high-fidelity DNA polymerase that replicates with extremely low error rates, reducing the likelihood of deleterious mutations.
- Their version of Ku70 better protects from double-strand DNA breaks.
- They have a unique DNA damage response pathway that involves the upregulation genes involved in DNA repair and cell cycle arrest, preventing damaged cells from proliferating.
These are all related to the first member from our list, genomic instability (Petruseva. 2017). Yet this hallmark received relatively little attention for the first few decades of serious inquiry into the causes of biological aging.
Denham Harman first proposed the oxidative stress theory of aging in the 1950s. Since then, more modern incarnations have made mitochondria their central focus, which may be a mistake (Harman, 1992).
Telomere shortening can lead to the activation of DNA damage response pathways, which in turn add to mitochondrial dysfunction by impairing mitochondrial biogenesis (creation of new mitochondria) and function: fewer mitochondria means more work falls on the backs of those left standing (Arnoult, 2018).
This does not mean that telomere shortening always takes precedence, as the reverse is also true: mitochondrial dysfunction creates oxidative stress which, in turn, damages cellular components, including telomeres. Ideally, any set of anti-aging interventions should address both, although addressing just one can affect all the others (Passos, 2007).
Mitochondrial theories of aging have enjoyed ongoing popularity because the mechanism of action for what is currently our most robust intervention is commonly chalked up to reduced ROS generation.
Caloric restriction extends healthspan and lifespan in animal models, including rodents and non-human primates.
CR suppresses ROS production while bolstering antioxidant defenses. This makes sense, as less food means less work for mitochondria, but it affects nearly all of the hallmarks. For example, it acts on the mechanistic target of rapamycin, or mTOR, a vital part of nutrient sensing ((Sinclair, 2005; Pifferi. 2019).
An old antifungal drug, in some studies rapamycin has extended the lifespans of mice more than CR. One published in Nature found rapamycin extended maximum murine lifespans by about 9–14% depending on the sex and strain of the mice, while caloric restriction extended the lifespan by about 5–6% in the same strains (Miller, 2014).
A meta-analysis showed a 15.3% average increase from CR and 9.2% from rapamycin. Despite its close association with one pathway, rapamycin achieves its ends by inducing autophagy, abating inflammation, and lubricating mitochondrial function (Tong, 2018).
Why do Great Danes die young?
Nutrient sensing is modulated by insulin/IGF-1. Central to regulating metabolism and growth, its dysregulation has been linked to age-related diseases. It is downregulated by CR (Greer, 2010; Ziv, 2011).
Larger breeds of dogs have higher serum IGF-1 concentrations compared to smaller ones. This is a general intraspecies trend, although between species the opposite is true (e.g., whales live longer than mice).
The insulin/IGF-1 pathway is activated in response to the presence of nutrients, particularly glucose, and stimulates cell growth and division. However, prolonged activation of this pathway is linked with age-related diseases, particularly type 2 diabetes, cardiovascular disease, and cancer (Abbas, 2008).
When nutrients are abundant, mTOR is activated and promotes cell growth and division, but aberrant activation is undesirable.
This isn’t to say that growth and renewal are always unwanted, just that they are one side of the coin. The disposal or recycling of cellular waste and worn-out components, or autophagy, is the other.
Autophagy, which is induced by CR, maintains cellular homeostasis by breaking things down, and its dysregulation has been linked to a slew of health issues. Yet, again, it is a balance. Like senolytics, upregulating autophagy is not a complete or one-size-fits-all solution.
Too much or too little autophagy is not good. Bisphosphonates are used to treat osteoporosis. While X-rays show healthy bones, closer inspection can tell a different story.
One possible disadvantage is they may interfere with lysosomal activity, impairing autophagy.
Contrary to earlier rodent studies, a paper in Frontiers in Endocrinology suggests bisphosphonates may impair autophagy in bone cells, leading to reduced bone formation and increased resorption. Long-term use may lead to the accumulation of dysfunctional cells, ultimately compromising bone health (Scala, 2022).
Klotho, along with its previously mentioned properties, is a regulator of mineral homeostasis: keeping calcium out of your arteries and in your bones, preventing overaccumulation of phosphate, and safeguarding blood pressure from salt intake (Citterio, 20210).
And yet, despite the impressive literature supporting its role in nearly every aspect of the aging process, Klotho depletion is not a hallmark (Kuro-o, 2012; Kuro-o, 2011).
By no means is this a suggestion to add it.
It is not fundamental enough to be one, but its versatility, coupled with its role in preventing mineral imbalances should make it a close contender, if not an officially recognized member.
Should sarcopenia, or age-related muscle loss, be a hallmark?
Published in the Proceedings of the National Academy of Sciences (PNAS), Follistatin gene therapy delivered with BioViva’s CMV vector yielded a 32.5% increase in median lifespan in mice. Mouse cytomegalovirus (MCMV) carrying exogenous TERT extended median lifespan by 41.4% (Jaijyan, 2022).
While follistatin may have other as-of-yet undiscovered longevity-enhancing properties, it is first and foremost a myostatin inhibitor. Skeletal muscle does not seem overly fundamental, yet it profoundly affects metabolic health.
In sarcopenia, the activation of mTOR is reduced, while the activation of AMPK and SIRT1 is increased, leading to a shift in favor of catabolism, the cannibalization of tissue. This feedback loop becomes a vicious cycle with very real consequences beyond frailty and falls (Tan, 2010).
Long-term inhibition of mTOR by drugs like rapamycin are associated with muscle wasting (Gyawali, 2016). Mouse models show metformin impairs mitochondrial function in skeletal muscle (Wessels, 2014). These interventions sound reasonable on paper, but have unwanted off-target effects — these are just two of them.
They lack the specificity of approaches like gene therapy.
Muscle loss predisposes us to Metabolic Syndrome, a constellation of traits including insulin insensitivity, obesity, and other forerunners to heart disease and diabetes (Collins, 2018).
Like Klotho depletion, muscular atrophy is closely associated with the hallmarks. Again, like Klotho, it is foundational enough to be a worthwhile target for anti-aging interventions.
Intercellular communication refers to the ways cells coordinate activities and respond to environmental stimuli. Thise include cell-to-cell contact, secretion of signaling molecules (e.g. hormones or cytokines), and signal transmission through extracellular matrix (Fafián-Labora, 2020).
Cellular senescence and stem cell exhaustion are exacerbated by irregularities here.
Cytokines from senescent cells disrupt communications lines, as does the changing microenvironment for stem cells. The accumulation of advanced glycation end-products (AGEs), considered a hallmark of aging by some, stiffens the extracellular matrix and impairs cell signaling (Perrone, 2020).
The extracellular matrix is a network of proteins and carbohydrates that provide structural integrity and regulate cell behavior. It influences nearby cells as the cells influence it.
We are, after all, talking about something physical.
A great deal of confusion comes from the misuse of two occasionally useful metaphors. Calling DNA software is problematic because there is no demarcation between genetic information and how it is expressed. DNA is a physical substance working in an analog system, not a string of code on a digital computer.
The irony is that those who use (or overuse) these analogies may be reluctant to think of aging as a programmed or semi-programmed process, rather than simply the result of “wear-and-tear.” The many issues of comparing a biological system with a car, besides the fact cars can’t regenerate themselves, have been dealt with elsewhere.
The epigenetic aspect of aging was in the headlines not too recently, mostly due to the ruckus around the Horvath Clock. Whatever their real value may be in predicting mortality or disease, it’s clear that epigenetic changes, changes in the way our genes are expressed, are part of the aging process (Horvath, 2018; Pal, 2016).
They come in two flavors: preprogrammed and stochastic.
Here preprogrammed refers to the changes that take us from infancy and adulthood. Although it’s hard to deny the reality of preprogrammed development forming our limbs in the womb or initiating puberty , it’s taken many people, including many scientists, a surprisingly long time to accept aging as a semi-programmed phenomenon.
The prefix semi- has to be attached because stochastic changes, to the epigenome and elsewhere, occur randomly and are influenced by diet, lifestyle, and toxins or other stressors.
Given the complexity and uniqueness of each person’s epigenome, direct interventions may be some ways away. Fortunately, its relationship with the other hallmarks is bidirectional.
From a therapeutic standpoint, doing what safely yields the most substantial benefits makes more sense than attempting to tweak processes so basic that any misstep could be disastrous.
If a solution is ever found, it will be multifaceted. Yet the evidence suggests that even today comparatively simple interventions, like telomerase, Klotho, and follistatin gene therapy can, alone or in concert, improve and extend human healthspan.
Authored by Adam Alonzi
Adam is a writer, independent researcher, documentary maker, and author of two novels. He is the director of marketing for BioViva Science. Visit adamalonzi.com for more.
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