Aging Hyperspace

Successful control of the aging rate requires the development of a satisfying, comprehensive model of the process, explaining the facts of reality. The following facts require explanation:

1. Aging is reversible and reaches minimal value in the early embryonic period. At that point, all vestiges of parental identity are gone, parental epigenetic patterns are dismantled, parental molecular defects are diluted, and the embryo is at an early gastrula stage. Aging, as measured by epigenetic clocks, begins to progress from this point. 
2. The aging of a cloned nucleus extracted from somatic cells is reversed by the ovular environment; it treats the donated diploid somatic nucleus in the same manner as a naturally fertilized ovum, reprogramming a sperm nucleus. The cloned – or even multiply re-cloned – progeny lives as much as normal sexually produced progeny – despite mutations accumulating in somatic cells tenfold more frequently than in the germline.
3. The genes most differentially expressed between the old and young individuals of the same species are those involved in organism development and organ function. 
4. The cells of longer-living organisms are more difficult to reprogram. Older cells are easier to reprogram than younger cells. The rigidity of the epigenetic landscape can be controlled by overexpression or inhibition of morphogens such as p53, p16, ARF, and other tumor suppressors. Murine models overexpressing morphogens can live longer, up to 10%. The cells of older individuals begin to lose differentiation identity after certain ages.
5. The longer-living species also develop at a slower rate, with a longer period of gestation and childhood. 
6. The protein, mRNA, hormone, and metabolite levels continue to change over the lifespan, some continuously increasing and some continuously decreasing. The ratios of these levels form age clocks comparable to or better than the epigenetic clocks.
7. The genes associated with reaching centenarian ages are scattered in the genome without a preference for a particular chromosome and are also randomly spaced along the chromosome length. 
8. The contact of tumor cells with differentiating embryonic tissues related to the tumor identity reverses the malignant phenotype and normalizes the tumor cells. They can even become a part of the developing embryo, which grows normally. Tumors are typically age-accelerated (epigenetically older) than normal tissues; thus, the contact between the differentiating embryonic environment and the aged, de-differentiated cell ends in both differentiation and rejuvenation of the latter.
9. The transplantation of old muscles into young recipients results in the old transplanted muscles gaining the strength of the young transplant. The transplantation of the young muscles in the old recipients leads to the opposite results. Many forms of contact between young and old tissues lead to a partial equalization of biological ages between the contacting sides. 
10. Social insects form colonies where all members are genetically identical. Nevertheless, the breeding females live much longer than the twin sisters, who become workers. In certain species of termites, the lifespans of workers are a few weeks, while the lifespans of queens and kings are 20–30 years. These dramatic differences in lifespan and cellular composition are entirely attributed to non-genetic, endocrine factors, defining which route of gene expression is optimal for colony survival and setting the aging rate of all members. 

The facts above are explicable by a generalized, eclectic aging theory postulating the participation of multiple mechanisms in the total outcome. The outcome is the departure of the system’s state from the optimum, called “the prime of life.” When this departure becomes too significant, the organism self-destructs. The departure can be graphically presented as a trajectory in a parametric, multidimensional space.

Figure 1: The structure of the aging process. Black arrows indicate the dimensions of a hyperspace characterizing the aging process. The blue cylinder in the center indicates the state of the organism at the gastrula stage, when aging experiences a complete natural reversal (“embryonic reset” or “cloning reset”). Green cylinders near the origin indicate the hypothetical induced rejuvenated states (“induced youth”) arising due to the action of improved combinatorial cocktails or other methods like OSKM reprogramming (below). Waving blue arrows lead to the alternative aging states located at the same distance from the embryonic reset origin but including different combinations of principal components.

The realistic aging hyperspace is non-orthogonal; different dimensions of the total process are coupled, and changes in one direction can project in other directions. The realistic aging hyperspace is a hyper-parallelogram, not a hypercube where all vertices are orthogonal.

Embryonic reset.

The scheme in Figure 1 defines the embryonic reset as a central event of life. Indeed, this is the point where a previous generation that experienced the entire spectrum of aging processes presents its gametes or even somatic cells, which also aged and accumulated some damage, and produces a brand new next generation, with all damage erased as if it never occurred. Why is a baby younger than her mother? After all, she was born of the cells that were part of her aging parents and that inherited at least a fraction of this aging. If these fractions were allowed to accumulate through the millions of generations, life would simply disappear. Perhaps the species where this mechanism of aging defense becomes corroded become extinct. The embryonic reset operates by reversing all pathways to aging, as shown in Figure 1. Reversing aging in an entire organism is perhaps impossible, but at the level of one rapidly dividing cell colony, it is possible. This division dilutes the damage, like in bacterial colonies, which are technically immortal. In a thought experiment, if one bacterium had a “personality” and was placed in a rich medium, it would have experienced uninterrupted “self-awareness” because the mother cell and its daughter are the same. The same applies to yeast, fungi, stem cells, and cancer cells.

The apparatus of life’s immortality was identified in 2006 by Dr. Shinya Yamanaka. The 2012 Nobel Prize in Physiology or Medicine was awarded jointly to Sir John B. Gurdon and Shinya Yamanaka “for the discovery that mature cells can be reprogrammed to become pluripotent.” Ironically, this effort took off to replace embryonic stem cells, banned for harvesting and use in the USA by the decree of President George W. Bush.

The article (Takahashi et al.) is cited > 32000 times by 2024, and it describes the introduction of 24 candidate transcription factors in murine embryonic fibroblasts (MEF), first separately with no results, and next all together, producing stem-cell-like transformants, which were termed iPSC (induced pluripotent stem cells). The initial cocktail of factors was simplified, and, in the end, only 4 factors (OCT4, SOX2, KLF4, and c-MYC, known as OSKM) were found to be sufficient for converting a differentiated somatic cell into a stem cell. The conversion is real; the induced stem cells form normally developing embryos (Boland et al.). The article by Boland et al. describes the most stringent test of whether a stem cell line has sufficient developmental potential to generate all tissues required for the survival of an organism (termed full pluripotency). The test is tetraploid embryonic complementation (TEC). The test involves the electrofusion of two-cell embryos to generate tetraploid (4n) one-cell embryos that can be cultured in vitro to the blastocyst stage. Diploid (2n) pluripotent stem cells (e.g., ESCs or iPSCs) are then injected into the blastocoel cavity of the tetraploid blastocyst and transferred to a female recipient. The tetraploid component of the complemented embryo contributes to the placenta and yolk sac, whereas the iPSC diploid cells constitute the embryo proper, resulting in a fetus derived entirely from the stem cells of interest. Boland et al. report that iPSC lines give rise to viable pups with efficiencies of 5–13%, which is comparable to the natural embryonic stem cells.

The properties of rejuvenated cells, returned to the ground zero of development, depend on the choice of reprogramming factors, and later the OSKM set was reduced to OSK or SKM sets, also sufficient for the purpose. Most importantly, the dangerous procedure of transfection by a viral vector that expresses the reprogramming transcription factors was replaced by small chemicals, which exert a similar effect on the adult cells and return them to the ground zero of development (Yang et al.). When added to 8-cell very early embryos, the chemically reprogrammed cells became seamless components and developed normally to later stages (Hu et al.). “Somatic embryogenesis”—natural reprogramming of adult cells in the embryonic cells exists in plants and in animals (Asghar et al.). In animals, the jellyfish Turritopsis dohrnii spontaneously returns into embryonic form directly from the whole-body adult stage without producing a facilitating single-cell form (fertilized ovum), as we do (Hasegawa et al.)

It is apparent that Nature found its way to create a literally immortal Tree of Life, but this is possible at the price of destroying any personality, which requires connectivity, complexity, and stable structure. Such requirements make total renewal impossible. Nevertheless, combining awakened reprogramming machinery with normal adult differentiation seems to be the path to capturing the most of both worlds.

Such ideas materialized and are exemplified by the work of Ocampo et al., while other reports use similar methodology, applying either a combination of transfected protein factors or chemical reprogramming cocktails in a pulsed manner, avoiding the induction of complete dedifferentiation, which is rapidly lethal in an adult organism (Ocampo et al.). The group used a bacterially derived promoter activating in the presence of antibiotic doxycycline and initiating the expression of embryonic factors OSKM (above). Once doxycycline is fed to the mice for 2 days, the adult tissues experience rejuvenation and reprogramming. The cycles repeat after a rest of 4-5 days. A progeroid murine model lived 30% longer in this regime, and a genetically heterogeneous, more normal mouse lived 10% longer.

The same level of modest success achieved by such a promising method is recorded in more recent works, and a natural question is: why?

Chromatin entropy accumulation and control of lifespan.

One principal component of aging is the state of chromatin entropy, typically measured by the epigenetic clock. The components defining active and open euchromatin, as well as the components defining closed heterochromatin, are located in the respective bands along the length of the chromosome. Random breaks, radicals, or radiation damage demand immediate attention to prevent the incorrect reconnection of broken chromosome ends. Such reconnection will lead to cancerous growth or apoptosis and is urgently prevented by the recombination machinery typically associated with heterochromatin. Mobilization of DNA repair components denudes the chromatin bands normally closed for reading. Such components may not completely return to the previous binding sites and instead silence the genes meant to be opened by the differentiated state of the cell. The more DNA damage takes place, the more subunit migration occurs between euchromatin and heterochromatin, eroding the epigenetic pattern. Yang et al. developed a system called “ICE” (inducible changes to the epigenome), producing DNA breaks outside of coding sequences. Induction of DNA damage accelerated the progression of the epigenetic clock and the aging of the model organisms. Disappearing differences in the expression signatures of the aging somatic cells of vital organs demonstrate that aging is a process of gradual cell differentiation loss, sometimes leading to amnesia of the original differentiation state (Izgi et al.). The report by Izgi et al. describes developmental trajectories of gene expression that reverse in their direction during aging, a phenomenon previously linked to cellular identity loss. The analysis of the cerebral cortex, lung, liver, and muscle transcriptomes of 16 mice, covering development and aging intervals, revealed widespread but tissue-specific aging-associated expression reversals. Cumulatively, these reversals create a unique phenomenon: mammalian tissue transcriptomes diverge from each other during postnatal development, but during aging, they tend to converge towards similar expression levels. This process was most prevalent among tissue-specific genes and associated with loss of tissue identity, which was confirmed using data from independent mouse and human datasets. Similar observations are in Donertas et al., Huner et al., and Traxler et al. The increase in the prevalence of oncological disease with age is consistent with increasing de-differentiation of dividing tissues, which leads to the spontaneous acquisition of cancerous phenotypes (Wu et al.).

The organisms that maintain the rigidity of epigenetic patterns live longer than those with a higher plasticity of differentiation (Matheu et al.). In this well-known article, Matheu et al. show that genetically manipulated mice with increased, but otherwise normally regulated, levels of Arf and p53 (morphogens, tumor suppressors) present strong cancer resistance and have decreased levels of aging-associated damage. These observations extend the protective role of Arf/p53 to aging, revealing a previously unknown anti-aging mechanism and providing a rationale for the co-evolution of cancer resistance and longevity.

In my view, these results show that in the absence of a cancer threat, the p53 pathway remains constrained to its other principal role, which is to be a “guardian of the genome” and maintain the fidelity of differentiation. Delayed loss of this fidelity extends the lifespan (Figure 2 of Matheu et al., 10%). Other interesting findings in this article are that the mice overexpressing “the guardians of the genome” were more resistant to oxidant poisons such as herbicide paraquat and lived twice as long under its lethal dose (Figure 4). The engineered mice express more antioxidants and can protect genomes more effectively against free radicals.

With multiple mechanisms of protection, one would expect greater lifespan effects than reported, but alas. This is important for understanding aging. Remarkably, murine embryonic fibroblasts spontaneously immortalize when propagated (and human cells never do). Figure 1 of Matheu et al. shows that this process was delayed by the expression of the p53/ARF pathway, making the murine epigenome more “rigid.” The epigenome in humans is much more rigid than in mice, and we live ~ 40 fold longer. Another model rodent organism, the mole rat, lives > 30 years and is very resistant to cancer. Almost as expected, its cells are epigenetically very rigid and are hard to reprogram (Miura et al.). By contrast, the cells of progeroid animals (with mutations accelerating aging) are more plastic and easy to reprogram than those of normal animals. The same applies to the naturally old cells (easy to reprogram) vs. the young cells (more difficult to reprogram). In another report, García‐Cao et al. present p53-overexpressing mice under normal control who are also cancer-resistant and better surviving the injection with fibrosarcoma cells. The lifespan of these mice was not extended, but they were more resistant to DNA damage.

Why did the cancer-resistant mice of Garcia-Cao et al. live the same and not longer than the wild type? Perhaps in this p53 construct, some accelerated aging effect is superimposed on the reduced cancer risk, producing a neutral result. Remarkably, the super-p53 mice were resistant to viral infections (Munoz-Fontela et al.), explaining why the “guardians of the genome” can shorten lifespan if these systems sense DNA damage due to radiation, viral infection, or chromosomal translocations inherent to cancer. The role of tumor suppressors is indeed longevity, but not of individuals. They guard the collective genome of species, following a very ancient function of antiviral defense. Our very distant ancestors probably split from the viral world > 4 billion years ago and have since had to repel viral invasions. The cellular clones that could not resist were wiped out. The survivors developed defenses, including p53, p16, p21, ARF, and more. They later repurposed these functions for the elimination of cancer cells by leading them into apoptosis and for aborting the division of damaged cells by leading them into senescence. The ability to blockade the expression of undesired genes was later repurposed one more time by incorporating tumor suppressors into the morphogenic functions when multicellular organisms appeared and began to develop from simple to complex during embryogenesis.

The criteria for longevity will be incomplete without discussion of telomerase. Chromosomes in dividing cells require flanking sequences to attach the replication machinery. The attachment regions are “blind spots,” and they cannot be reproduced during chromosome duplication that precedes division. The chromosome progressively shortens on the flanks, and by reaching 50–70 divisions, the shortening begins to damage the informative material. This level of shortening is perceived as a threat due to the cancer risk, and the cell rapidly loses its ability to propagate due to senescence. When telomerase is expressed, the telomeres are constantly built up, and a cell can divide indefinitely without triggering an alarm because its functional chromosomal regions are intact. Tomas-Loba et al. used the cancer-resistant mice of Matheu et al., overexpressing p53, p16, and p19ARF as a basis and introduced an inducible TERT subunit of telomerase for expression in the adult differentiated cells. They overexpressed only TERT, a protein component of telomerase, without expressing TERC, the ribonucleic component annealed to the chromosome flanks. This partial overexpression did not prevent telomere elongation in these mice, despite the absence of the TERC component needed for a complete enzyme structure. Nevertheless, the endogenous TERC gene was present, and when it was ablated, the TERT effects also ceased. The TERT-overexpressing cancer-resistant mice demonstrated outstanding longevity and health. The addition of TERT increased the lifespan of p53-expressing mice by 9%, but for p53/p16/p19ARF mice, the increase was 26%. The effect was >40% comparing a p53 control with a p53, p16/p19ARF/TERT transgene. Both median and maximal lifespans were impacted. Considering that the p53 transgene is also longer-lived (by 10%) compared to the basal wild type, the overall effect of the p53/p16/p19ARF/TERT transgene approaches 50% lifespan extension. The cancers started later and proceeded at a lower frequency in this model; the neurodegeneration was also postponed; the integrity of all biological barriers was improved; and inflammation was reduced. IGF1 is typically associated with aging in mice, but it was increased in these mutants. The general biological implications are that the best longevity results are achieved when:

1. Cancer and the loss of epigenetic rigidity are controlled by a combination of morphogens.
2. A potential oncogene (TERT) plays a stimulating role in the cancer-resistant context.
3. One of the OSKM members, the oncogene MYC, is the main regulator of glucose metabolism in the central nervous system, on the side of activation.
4. Once cancer-preventing interventions are mastered, activations of multiple oncogenes can be synergistic, signaling a return to the rejuvenating embryonic reset in the adult tissues but not causing the dedifferentiation or cancer growth that would follow in the absence of anti-cancer protection. As chemical activators of OSKM and other oncogenes are known, equally potent activators of tumor suppressors are needed. Strengthening these two opposing forces produces the best life-extension result.

Systemic control of lifespan.

Another informational component of life is systemic cell-to-cell and tissue-to-tissue communications. The extracellular matrix is linked to genomic stability through focal kinase, integrin, and catenin pathways. Hypothetical twin cells will diverge upon contacting the old and the young extracellular matrix. This dimension is relatively controversial as compared to the chromatin entropy factor. Many publications describe the impressive heterochronic parabiosis in mice (Ma et al.), but in humans, the results do not seem to favor these systemic effects. For example, Jimenez-Romero et al. conclude that patient and graft survival rates are not affected by donor age; well-selected older donor livers can be safely used if they show good function and preharvesting conditions. Edgren et al. conclude that donor age and sex are not associated with patient survival and need not be considered in blood allocation. Even in mice, the effects on lifespan are consistent with an alternative common-sense hypothesis that an invasive long-term procedure may adversely impact the young without benefiting the old (Yankova et al.). Stronger and more consistent evidence remains for the effects of embryonic environments on the normalization of cancer cell transplants (Proietti et al.; Bizzarri et al.; Li et al.; Hendrix et al.).

Hochedlinger et al. describe nuclear transplantation to test whether oocytes can reestablish the developmental pluripotency of malignant cancer cells through reprogramming activity. The nuclei of leukemia, lymphoma, and breast cancer cells could support normal preimplantation development to the blastocyst stage but failed to produce embryonic stem (ES) cells. However, a blastocyst cloned from a RAS-inducible melanoma nucleus gave rise to normal embryonic stem cells with the potential to differentiate in vivo into melanocytes, lymphocytes, and fibroblasts. Chimeras produced from these ES cells developed cancer with higher penetrance, shorter latency, and an expanded tumor spectrum when compared with the donor mouse model. These results demonstrate that cancer cells can be reversed back to normal by non-genomic information carriers, but the accumulated mutations due to malignant clone progression cannot be neglected or reversed. Nevertheless, the normalized converted cells (former cancer!) were forming most of normal tissues in a new organism. This normalization was maintained by the contact with oocyte environment, and later maintained by a healthy cell-to-cell interaction context.

Equally consistent evidence exists regarding the lifespans of the genetically identical but hormonally distinct casts of social insects (Tasaki et al.). Reproducing queens (and, in termites, also kings) can live for several decades, whereas sterile workers often have a lifespan of only a few weeks, despite identical genomes (Elsner et al.). The group of Elsner et al. studied aging in the wild in a highly social insect, the termite Macrotermes bellicosus, which has one of the most pronounced longevity differences between reproductives and workers. According to Elsner et al., reproductive systems seem to be protected by a process that normally silences transposable elements (ancient viral invasions) in the germline of animals. This suggests that natural selection used a mechanism from the germline to protect whole animals. According to Koubová et al., long-lived termite kings and queens activate telomerase in somatic organs. Telomerase is another stem cell/germline protein typically associated with immortalization. Similar literature points to multiple mechanisms cooperating to ensure lifespan differentials ~ 200–300 fold in the genetically identical progeny of the same mother queen (Tasaki et al.). Interestingly, this partially immortalized phenotype is suppressed in working termites by cuticular hydrocarbon hormones and proteinaceous signaling (Hanus et al.). Do humans have similar suppressing signaling? What would happen when it is removed?

The evidence for cancer reversals and insect cast conversions demonstrates that life is equipped with switches that turn on partial immortalization in adult tissues in response to external signals arising in the process of cell-to-cell communication. These signals can be decoded and used for rejuvenation. In humans, the biological information encoded in the circulating molecules maintains the young, well-differentiated state, which begins to corrupt when the secretory profile erodes with time. The identity of our tissues is supported by soluble morphogenic signaling in the same sense as the initial tissue formation in an embryo is supported by morphogenic gradients; it is just less intense and more fragile in adults.

Control of lifespan by nervous system.

The brain regulates homeostasis through its direct signaling of hormones, micro-RNAs, and neuropeptides (respiratory center, innervation of organs, growth control, and puberty onset). The review by Mattson et al. and Wolkow et al. suggests several mechanisms of neuroendocrine lifespan control. Causative data pointing to some routes of brain regulation of lifespan are presented by Tokizane et al., who identified a key neuronal subpopulation in the dorsomedial hypothalamus (DMH), marked by Ppp1r17 expression (DMH^Ppp1r17 neurons), that regulates aging and longevity in mice (~ 10% effect, Figure 6). The review by Satoh et al. suggests that molecules and signaling pathways, including sirtuin, the mechanistic target of rapamycin (mTOR), and nuclear factor-κB (NF-κB), specific areas of the hypothalamus (the dorsomedial/lateral hypothalamus, mediobasal hypothalamus, and arcuate nucleus), neural stem cells, and temperature-sensitive neurons within the hypothalamus, play a role in the regulation of mammalian longevity.

Neurons express the rejuvenating machinery typically reserved for expression in self-renewing lineages such as stem cells and germline. Eitan et al. report the presence of active telomerase in the cytoplasm and nucleus of cerebellar Purkinje neurons in adult and old mice. TERT protein levels are reduced with age, whereas in the nucleus, TERT activity is increased. These findings suggest that telomerase plays a role in the aging of nondividing cells. The combination of OSKM is known as “Yamanaka factors,”  converting differentiated cells into pluripotent stem cells. Neurons express 3 out of 4 Yamanaka factors. Poitras et al. describe c-Myc, N-Myc (Yamanaka’s “M”), Max, and Mad expressed in adult sensory neurons and in partnering stem cells. In vitro knockdown (KD) of either Mad1 or Max, competitive inhibitors of Myc, unleashed heightened neurite outgrowth in both naive, uninjured, and preconditioned adult neurons. Cheng et al. describe Yamanaka’s “K”—KLF4—as expressed in the adult brain and playing a role in Alzheimer’s disease as a regulator. KLF4 is directly expressed in adult neurons, not just in neurogenic stem cells (Imbernon et al.). Another member of the OSKM group, SOX2, can also be expressed in selected adult neuron populations (Mercurio et al.).

With so many embryonic genes expressed in neurons, it is not surprising that they can outlive the source organism when transplanted into another host (Magrassi et al.). Strong correlations at genetic levels exist between intellect and longevity (Deary et al., Savage et al.) and between intellect and the presence of chronic disease or mortality (Deary et al., 2010). Finally, the nervous system interacts with stem cell niches in multiple organs (Katayama et. al., Davis et al., Davis et al., 2018, Oben et al.). Apparently, the sympathetic autonomous nervous system inhibits stem cell niches, including cancer stem cells, while the parasympathetic nervous system activates them (Fielding et al., Wang et al.). The expression of similar self-renewal machinery in neurons, stem cells, germ cells, and cancer cells creates a unique communication hub that can control mammalian lifespan through withholding the rejuvenating exosome signaling by the stem cell niches (Shen et al., Chen et al., Lei et al., Kulkarni et al.) and/or allowing cancer development (Ondicova et al., Kuol et al., Erin et al., Zahalka et al.). These two factors alone can serve as a lifespan control mechanism. Unsurprisingly, brain age is closely correlated with mortality rates (Cole et al.). Neuroimaging and epigenetic measures of aging can provide complementary data regarding health outcomes (Cole et al.), suggesting independent coordinates of the aging hyperspace.

Figure 2: The scheme of lifespan regulation by the nervous system through the control of stem cell niches and cancer stem cells by nerve signaling. The brain timer communicates with the autonomous nervous system, which in turn communicates directly with the stem cells. Stem cells represent the depository of corrected biological information retained since the embryonic reset and have the ability to normalize or rejuvenate somatic tissue through the production of exosomes, lipid vesicles loaded with micro-RNAs, which in turn silence senescence-promoting genes. Stem cells operate in a similar manner as the ovular environment, reprogramming older tissues into the state of relative youth, just with less vigor. Withholding of this corrective signaling sharply accelerates the senescence of somatic tissues. This theory explains why a better-developed nervous system (in more intelligent biological species and in more intelligent individuals within the same species) facilitates longevity and health. Humans live ~ 5-fold longer than mammals of comparable weight, longer than other primates. Smarter, more purposeful, and more educated individuals within the human population enjoy better health and a longer lifespan. The genes involved in the development of the central nervous system are involved both in longevity and in intellect and need to be considered targets for future pharmacological manipulation of these characteristics. Our big brains evolved in the horrific conditions of our fight for survival, and civilization sharply removed the selective pressure that maintained intelligence throughout the Paleolithic period. H.G. Wells, in his amazing “Time Machine,” described the Eloi, the Morlocks, and the scariest of all, the Rabbit Man, as dystopian models that we should avoid through scientific pursuit of countermeasures.

Runaway dysregulation.

According to the GenAge database (https://genomics.senescence.info/genes/index.html), there are 307 genes linked to human aging and 2,205 genes associated with aging in model organisms. The list of 4,243 genes is reported by Gene Cards (https://www.genecards.org/Search/Keyword?queryString=longevity) in response to the search term “longevity.” The Gene Cards search for [longevity AND lifespan AND mortality AND centenarian] identifies “only” 466 results. By all accounts, aging does not have a single master switch or a singularly important pathway. Instead, it is a polygenic process with the implicated gene candidates scattered across the genome and located on different chromosomes (https://genomics.senescence.info/longevity/; Longevity Map). A question arises about how this plurality of small effects interact to result in the final lifespan. Lifespans vary in a prodigious range, from days in yeast and fungi to weeks in insects, decades in animals, and centuries in plants. There are ideas of specialized timers, binding lifespan to circadian rhythms, movements of heavenly bodies, and other physical factors, or simply proposing a literal biological clock, specially designed to terminate lifespan (Olovnikov, 2007; Olovnikov, 2022). But Occam’s razor would eliminate extra assumptions. Biological timing (including the clock in our brain) arises directly from most general regulation schemes of biological processes (Figure 3).

Figure 3: General scheme of biological regulation. Any biological pathway includes a substrate, a product, an activator, and a repressor. The activators and repressors can be individual molecules or pathways.

We will attempt to obtain a time-dependent solution for the repressor-activator relationships by writing a general form for the system in Figure 3 with the understanding that the constants are positive and the signs point to the direction of the processes.

dS/dt = -k₄[A]; S(t) = -k₄ ∫ [A] dt                                                                                                        (1)

The rate of substrate S disappearance is proportional to the level of activator.

dP/dt = -k₃[R]; P(t) = -k₃ ∫ [R] dt                                                                                                       (2)

The rate of product P buildup decreases with the increase of repressor.

dA/dt = k₅ [A] – k₈ [R] + k₂[S]                                                                                                            (3)

The rate of activation A buildup is proportional to the pre-existing activator level (autocrine loop), decreased by the repressor R level, and stimulated (or repressed) by the substrate S level.

dR/dt = k₆ [R] – k₇ [A] + k₁[P]                                                                                                          (4)

The rate of repressor R buildup is proportional to the pre-existing repressor level (autocrine loop), decreased by the activator A level, and stimulated (or decreased) by product P level. Combining the equations (1)–(4) and assuming non-interaction between R and A (k₇ = k₈ = 0), we obtain:

dA/dt – k₅ [A] = k₂[S]                                                                                                                         (5)

dR/dt – k₆ [R] = k₁[P]                                                                                                                           (6)

Combining (5) and (6) with (1) and (2) produces, after elementary transformations:

d²A/dt² – k₅ d[A]/dt + k₂k₄ [A] = 0                                                                                                      (7)

d²R/dt² – k₆ d[R]/dt + k₁k₃ [R] = 0                                                                                                      (8)

The equations (7) and (8) are homogenous second order, where activator and repressor levels can be found explicitly as a function of the constants k₁-k₆, boundary conditions, and time. The auxiliary equations for (7) and (8) are:

l₁² – k₅l₁ + k₂k₄ = 0                                                                                                                          (9)

l₂² – k₅l₂ + k₁k₃ = 0                                                                                                                            (10)

A(t) = A(0) exp (l₁t)                                                                                                                           (11)

R(t) = R(0) exp (l₂t)                                                                                                                           (12)

D₁ = k₅² – 4 k₂k₄                                                                                                                                (13)

D₂ = k₆² – 4 k₁k₃                                                                                                                                (14)

Depending on the sign of the discriminants D₁ and D₂, the solutions (11) and (12) become either exponential or harmonic (through the Euler transformation) or mixed, if we consider interactions between A and R. Harmonic profiles of regulators are observed in stem and germ cells, intended for the preservation of biological information (Stringari et al., Suzuki et al., Rodriguez-Brenes et al.), and can be considered a marker of self-renewal, including malignant transformation. Somatically differentiated cells follow the path of optimal adaptation and development, which requires an exponential solution. Other authors also noted the role of the runaway positive feedback dynamic in the development of aging and chronic disease (Belikov, Kandhaya-Pillai et al., Littlejohn et al.), but a non-cyclic or open profile is required for development in general and begins in the early embryonic period.

In the article by Mayburd (2009), the author measured the variability of expression of disease-associated genes analyzed in health and found that it was higher than the variability of random genes for all chronic pathologies. Anti-cancer FDA-approved targets were displaying much higher variability as a class compared to random genes. The same is true for the magnitude of gene expression. The genes known to participate in multiple chronic disorders demonstrated the highest variability. Disease-related gene categories, which displayed on average more intricate regulation of biological function vs. random reference, were enriched in adaptive and transient functions as well as positive feedback relationships.

The literature supports the conclusion that the developing and adaptive function of the somatic tissues requires them to respond to the stimuli in an escalating manner, rapidly reaching the desired level of response. A positive feedback mechanism is ideal for an exponential buildup of reactions. However, the price of this flexibility is the runaway wiring of differentiated cells. As time progresses, the state of differentiated tissues never remains constant, and the critical ratios correlating with the vitality of organisms change exponentially, recapturing the Gompertz law.

The timer of life thus does not need a special anatomical localization; it is inherent in the wiring of the constants k₁–k₆. When the values of the constants are high and the autocrine processes are uncompensated, the critical ratios of R(t) and A(t) are achieved quickly, defining a shorter lifespan. By contrast, when D₁ and D₂ are negative, critical levels oscillate near optimal values. The value of response constants is proportional to metabolic rate; therefore, organisms with a faster metabolic rate generally achieve shorter lifespans than those with a slower metabolic rate. But the factor of balancing and defining the final exponent l is also important, and it explains why some organisms with a higher metabolic rate may live longer than those with slower rates (Munshi-South et al.). As predicted by the theory above, the longer-living but rapidly metabolizing organisms must dampen the runaway processes; inflammation is among the autocrine processes that require special attention (Banerjee et al., Kacprzyk et al.).

Smaller organisms typically have a higher surface-to-volume ratio, with hostile stimuli (temperature, evaporation, loss of nutrients, exposure to poisons or radiation) proportional to surface, while the buffer of the system is proportional to volume. To provide identical protection, the response rates must be scaled in proportion to the surface-to-volume ratio, or M^-1/3, where M is the mass of the organism. These response rates define the constants k₁–k₆. The analysis predicts the scaling of lifespan with M^1/3 across multiple species. The measured scaling exponent for the relationship between lifespan and body mass is between 0.15 and 0.3 (Speakman), close to the predicted 0.33.

Equations (7) and (8) predict essentially quantum states of life, when a quantum state is defined as a discontinuous bistable transition. Indeed,

l₁ = (k₅ ± (k₅² – 4 k₂k₄)^1/2)/2                                                                                                              (15)

l₂ = (k₆ ± (k₆² – 4 k₁k₃)^1/2)/2                                                                                                              (16)

With two parametric roots, each profile (11)–(12) has two exponents, defining the rate of departure from the initial youthful state for the activator and repressor. The predicted multi-stability in living organisms is well known (Piedrafita et al., Chavez et al., Sobie et al., Laurent et al., and more). Lifespan is terminated when the organism reaches a critical quotient Q in the number of essential pathways, defining vitality.

Q = ∏ R(t)/A(t)                                                                                                                                (17)

Where Q is the biomarker quotient measuring the position of the organism on the lifespan trajectory, R(t) and A(t) are the critical ratios of the most relevant biomarkers. It is clear from (11), (12), and (17) that the solution based on a runaway exponential regulatory process will recapture the Gompertz Law, with Q ~ H(t), where H(t) is the annual probability of mortality. With many phenomena predicted by the theory above, it also predicts the pattern of the response of the aging process to a combinational treatment. Aging consists of multiple semi-independent contours, expanding exponentially (Figure 4).

Figure 4: Comparison of the relative size of the controlled and uncontrolled aging contours as a function of time. The gray-colored contours are pharmacologically controlled; the blue-colored contours develop freely. Occupation of the entire area by a contour is incompatible with life. A reduction in the number of uncontrolled contours will reduce earlier-age mortality by keeping more surfaces unoccupied. However, the retention of even one uncompensated contour would still mean an exponential increase in risk, just shifted in the future. Only the blockade of all mapped pathways would produce a qualitative change.

Interestingly, the rate of annual mortality acceleration dH(t)/dt due to exposure to a late-life uncompensated contour is higher than the rate due to exposure at younger ages. Indeed,

H(t) ~ ∏(R(0)/A(0)) x exp ∑(l₂-l₁)t; where ∑(l₂-l₁)t > 0                                                                               (18)

dH(t)/dt ~∑ (l₂-l₁)∏ (R(0)/A(0)) x exp ∑(l₂-l₁)t                                                                                          (19)

The equation (19) shows that the derivative dH(t)/dt is greater at a later period t. Mortality suppressed over a longer period will reassert itself at a later age, according to (19). This is really observed; see “Human Data.” Only multimodal suppression of all aging contours, based on a correct and comprehensive understanding of the global aging structure, can introduce a different kinetic of decline.

Molecular defects accumulation.

Life follows the Second Law of Thermodynamics, which prescribes the accumulation of entropy (disorder) in isolated systems. However, life can be a completely open system, like a rapidly growing embryo or a bacterial colony in the exponential stage of growth. In completely open systems, entropy may decrease in time by washing away with the material flows. Differentiated cells are in a gray zone between the open and closed thermodynamic systems. They are capable of renewal (autophagy, exosome export of debris, molecular repair systems) and can grow de-novo from stem cell niche precursors; in that aspect, they resemble open systems. On the other hand, they are bound by complex structures and are busy performing vital organ functions.

A successful radical anti-aging approach would try to stop the accumulation of entropy in a mature functioning organism and also reverse the flow of dysregulation by altering the regulatory constants k₁–k₆, considered above. The success of this procedure will not create an immortal complex organism since the factor of random fluctuations in its parameters will early or later materialize on a cosmic scale. But freezing the level of entropy and reversing the exponential dysregulation would dramatically decrease the annual probability of mortality H(T), for example, at the level of a 30 or 40-year-old.

The challenge to this idea stems from the complexity of biological molecules, each of which is vulnerable to degradation by multiple routes. There is only one path by which a protein molecule can fold correctly, but multiple paths exist by which it can form irreversible aggregates via hydrophobic patches, dialdehyde links, or disulfide bridges, disabling its functional activity. What does nature envisage to protect the building blocks of life against degradation?

The most obvious is turnover. Once a molecule becomes non-functional, it is labeled for recycling and will be either enzymatically cleaved or excreted. Unfortunately, depositions of defective biomolecules accumulate over time, and the reversibility of these processes is an interesting question.

Lipofuscin is a yellowish-brown pigment composed of highly oxidized proteins, lipids, and metals. The rate of lipofuscin accumulation correlates negatively with longevity (Brunk et al., Mannel et al., Nakano et al., Porta et al., Strehler et al.). Accumulation of this pigment is not only a reliable marker of aging but is also a part of pathology such as retinal atrophy when the deposits interfere with the vitality of retinal cells. The retinal pigment epithelium (RPE) is a single layer of cells that acts as a barrier between the retina and the choroid. It’s essential for the survival and function of the retina’s photoreceptor cells. Accumulation of lipofuscin in RPE is a frequent symptom of macular degeneration.

Remarkably, Fang et al. report that remofuscin (soraprazan) reverses lipofuscin accumulation in aged primary human RPE cells. The removal of lipofuscin after a single intravitreal injection of Remofuscin results in a rescue from retinal degeneration in a mouse model of Stargardt disease (the pigment builds up in the macula). Fang et al. show that lipofuscin dissolution may involve the reactive oxygen species generated via the presence of remofuscin. Similar results with the same agent are reported in monkey RPE by Julien et al. The results of a successful human trial (RCT) of remofuscin are reported in Peters et al. A mechanism of myocardial cell self-purification by lipofuscin excretion is reported in Wang et al. Lipofuscin may or may not accumulate in the aging tissues, depending on the health and wellness status. The immunosuppressor drug rapamycin is an effective lifespan modifier in rodents, stimulating autophagy, the process of digesting defective molecules by the cell. In the presence of rapamycin, the accumulation of lipofuscin in the rat hearts was delayed (Li et al.).

Another group of molecular defects previously deemed irreversible are AGEs (advanced glycation age products), reviewed in Twarda-Clapa et al. These defects arise by non-enzymatic random binding of the sugar (glucose) moieties in the aldehyde tautomer form to the N-termini and lysine or arginine protein side chains (Millard reaction). The modified proteins lose function and promote further epigenetic aging (Perrone et al.). Remarkably, multiple pharmaceuticals were discovered to arrest and reverse the AGE product accumulation (Abbas et al., Khan et al.). The examples are pentoxifylline (70% AGE prevention at 1 micromole, 40% at 0.01 micromoles) and pioglitazone (40% AGE inhibition at 0.01 micromole). The dicarbonyl-reactive compounds 2-aminoguanidine (2-AG), semicarbazide, and o-phenyl-enediamine (OPD) were able to inhibit the process of protein glycation at relatively low concentrations (1–2 mM).

Another group of molecular defects are protein aggregates. Normally, proteins are naturally folded as micelles, exposing polar side chains and burying the hydrophobic (non-polar) side chains in the interior of the protein structure. A misfolded protein would form hydrophobic patches that reduce water-exposed surfaces by aggregating. These dead protein aggregates will sporadically form due to thermal fluctuations of the protein structure, producing “breathing” conformational motions. Accumulation of misfolded proteins is the main cause of neurodegenerative disease and a direct factor terminating a lifespan (Ross et al.). Furthermore, the protein aggregates can convert correctly folded twins into poorly digestible conformers, or prions, which are infectious while not even alive (Jucker et al.). The disruption of correct folding may be a result of genetic mutations and, as such, may occur early in life. More typically, it occurs late in life as an event termed “proteostasis collapse” (Hipp et al.), which occurs due to increasing entropy and dysregulation of the cellular and systemic environment. The sources of defects are oxidation and cross-linking of sidechains, thermal fluctuations, complexation with heavy metals, mRNA isoforms or defects, resulting in truncated proteins, incompatibility of the defective proteins with their organelle addresses in the cell, making them “homeless” and available for aggregation, inherently unstable proteins with flexible domains, etc.

Nature counters this problem by labeling and trafficking the defective proteins to special disposal sites—proteosomes (Tanaka et al.). In addition, Nature destroys the protein debris in autophagosomes, membrane vesicles filled with proteolytic enzymes and merging with lysosomes and the endoplasmic reticulum (Xie et al.). As in any complex, coordinated system, there is a probability of malfunction and non-detection. Eventually, a non-dividing, long-living post-mitotic cell like a neuron or cardiomyocyte will accumulate a critical mass of non-detected defective proteins incompatible with life. The mitochondrial-lysosomal axis theory of aging posits that the buildup of lipofuscin within lysosomes inhibits autophagy, resulting in reduced turnover of old mitochondria. The latter produce increased amounts of reactive oxygen species, prompting lipofuscin formation (a feedback loop). Moreover, defective and enlarged mitochondria avoid autophagy and constitute a growing population of badly functioning organelles. The progress of these changes seems to result in enhanced oxidative stress, decreased ATP production, and the collapse of the cellular catabolic machinery, which eventually is incompatible with survival (Terman et al.). The collapse of energy production results in an explosion of misfolded proteins, especially in neurons, where the high aspect ratio already hinders genomic information transfer and normal protein synthesis. All transcripts (m-RNA) must travel—and avoid degradation—across a length of ~1 m into the dendrites and along the axon. Most proteins are synthesized in the cell soma, and only a minority are produced locally in the dendrites and synapses. This majority needs to be trafficked across 1 m at ~ 38 C in the brain, outside of stabilizing participation in supramolecular complexes (before reaching the destination). The energy required for maintaining the structural stability of the central nervous system and the integrity of its very proteins is immense (the brain weighs 2% of the body but consumes 20% of metabolic energy; see Atwell et al.). There is perhaps a structural limitation on how individually smart we can be.

Mitochondria represent another source of molecular damage: free radicals and reactive oxygen species (ROS), resulting in accelerated DNA breaks and the need for frequent DNA repair, which in turn induces chromatin entropy. As a progeny of symbiotic bacteria, mitochondria encode some of their future proteins using the internal mitochondrial DNA, inherited exclusively through the maternal cytosol. Mitochondria divide by cell fission in a manner tightly coordinated with the host cell (Taanman). Mitochondria participate in the regulation of the cell cycle and control the suicide program of a cell—apoptosis. Mitochondria must import most of the required proteins through the outer membrane, and any error in the imported proteins results in suboptimal, “smoky” organelles producing genotoxic ROS.

The state of youth in the non-dividing vital cells requires perfect quality control of the mitochondrial population (which can self-renew by division but depends on the state of the cell for the average quality of the newly formed organelles). Another criterion is the reversal of protein aggregation by intensification of autophagy and proteosome disposal of defective molecules, as well as the export of the remaining defects outside of the critical compartments into less important compartments. Of these processes, the average concentration of protein defects in the cell and the intensity of mitochondrial autophagy are the most causative.

Kapetanou et al. experimented with Wharton’s jelly- and adipose-derived human adult mesenchymal stem cells (hMSCs), and the study found a significant age-related decline in proteasome content and peptidase activities. Kapetanou et al. found that the loss of proliferative capacity and stemness of hMSCs can be counteracted through proteasome activation. A key OSKM factor, Oct4, binds at the promoter region of β2 and β5 proteasome subunits and thus possibly regulates their expression. The relationship is thus mutual: in the presence of embryonic reset machinery (Oct4), protein quality control is improved, while either a greater count of proteosomes or a defect-free environment fosters stemness and self-renewal. Djordjevic et al. describe a treatment of a transgenic murine model of Alzheimer’s disease with 2.18 alpha-glycyrrhetinic acid (18α-GA, Sigma) and omega-3 fatty acids. 18α-GA was given intraperitoneally at a concentration of 10 μg/g of body weight in an 8% DMSO solution. Shortly after proteasome activation, the significantly reduced amyloid beta load correlated with improved motor functions, reduced anxiety, and frailty levels. Chocron et al. report a transgenic mouse with neuronal-specific proteasome overexpression that, when crossed with an Alzheimer’s disease mouse model, showed reduced mortality and cognitive deficits. A set of proteasome-activating peptidomimetics stably penetrated the blood-brain barrier and enhanced 20S/26S proteasome activity. These agonists protected against cell death, cognitive decline, and mortality in cell culture, fly, and mouse AD models. Munkácsy et al. also report the extension of Drosophila melanogaster fruit fly lifespan (Figure 4, 20%) by overexpression of the proteasome β5 subunit.

Autophagy activation was addressed in the report by Pyo et al. In this report, whole-body overexpression of Atg5, a protein essential for autophagosome formation, extends the median lifespan of mice by 17.2%. Overexpression of Atg5 in mice indeed enhanced autophagy, and Atg5 transgenic mice demonstrated anti-aging markers such as leanness, increased insulin sensitivity, and improved motor function. Furthermore, mouse embryonic fibroblasts cultured from Atg5 transgenic mice are more tolerant to oxidative damage and cell death induced by oxidative stress, and this tolerance was reversible by treatment with an autophagy inhibitor.

Autophagy inhibition is illustrated by extensive literature, and less is reported for autophagy activation. The term “chemical modulation” appears to capture both directions (Fan et al., He et al., Kim et al., Whitmarsh-Everiss et al.). Autophagy and proteosome inhibition are well-known anti-cancer approaches, relying on the accumulation of defective protein molecules in anarchic cancer cells and eventually inhibiting them. Will proteosomes and autophagy stimulate cancer via induced stemness? This is an important question, stressing the need for rapid development of anti-cancer prophylactic cocktails. The absence of such tools restricts the development of the longevity field.

Summary

Life exists in two modalities: the immortalized germline and the mortal somatic line. The immortality of life is maintained by a complete rejuvenation event called the embryonic reset, which reverts all characteristics of aging to zero. Germline and its continuation in adult tissues—stem cell niches—operate in oscillatory regimes of regulators, minimizing aging rates. Somatic cells are responsible for the physical preservation of germline; they are compelled by evolution to take an exponential path of development that at some point results in dysregulation by autocrine runaway regimes.

Evidence in the literature suggests that every dimension of the aging process is reversible, and aging continues forward because there is no procedure so far that would act simultaneously against all components of the aging process. If only some components are allowed to progress, the total result does not differ much from the non-intervention control (Figure 3). Even one significant contour, if allowed to progress unchecked, would, at some point, create a degree of destabilization that is incompatible with the continuation of life. The rate of destabilization increases with time. The lone overlooked contour at a later point may be as destabilizing as multiple contours at an earlier point.

The rejuvenation program is turned on by the activated ovular environment and reaches its peak performance by 2-3 weeks of age. In a milder form, the program persists after birth in the stem cells, possibly in macrophages and neurons. It is gradually extinguished by competing senescence processes, but the result may perhaps become different if the rejuvenation program is artificially reinforced and senescence processes are artificially delayed. The direction of daily balance may temporarily or permanently change, from continuous overall senescence to continuous overall stability or rejuvenation. This is the gray zone, the domain of hot research. In the next section, we will consider what can be done to alter this balance.

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