Molecular Targets/Pathways of Interest
Various metabolic pathways are considered locus points for longevity, either in an enhancing or attenuating manner. These pathways highly influence on another, and are likely druggable targets to promote longevity
A large amount of research is conducted in nematodes (C. Elegans) with some more research conducted in mice and rats. Research in humans is somewhat nonexistent due to practical complications with monitoring a study for an entire lifespan
Telomerase is a ribonucleoprotein that mediates telomere length (telomeres being 'caps' on chromosomes), which appear to decline in length with age. Telomerase is composed of two subunits, the catalytic TERT subunit (also known as TRT or Est2) which forms the core of the enzyme integral telomerase RNA (sometimes referred to as TR, TER, TERC, or TLC1) that provides the template for TERT; the catalytic cycle involves the synthesis of a single telomere repeat at the 3′ end of the telomeric DNA primer and the regeneration of the template for the synthesis of additional repeats. There are other proteins associated with telomerase (known as accessory proteins) that are not outright required for its function.
Telomeres are investigated into longevity due to their correlation with aging, but have been demonstrated to be independent of aging in C. Elegans and nutraceutical interventions with Astragaloside IV (from astragalus membranaceus) have successfully increased telomere length in aged rats independent of longevity promotion.
Telomeres are commonly associated with aging, but may be a biomarker of aging rather than a druggable target. Increasing activity of telomerase or preserving telomere length via exogenous agents does not necessary promote longevity, although telomerase cannot be ruled out as a druggable target either
Human FOXO (ortholog of DAF-16 in C. Elegans) is a term used to refer to a group of genes known as FKHRL1 (FOXO3a), FKHR (FOXO1), and AFX (FOXO4); DAF-16 and FOXO are commonly used interchangeably, and refer to the main nuclear mechanisms that result from IIS and TOR signalling, both of those pathways (commonly seen as anti-longevity) promote nuclear exclusion of FOXO and reduce its expression of gene products.
FOXO is activated by reactive oxygen species (ROS) and appears to be a negative REDOX regulator via its gene products, and is positively modulated by AMPK (molecular target of metformin and berberine, also positively regulated by ROS)
When DAF-16/FOXO is able to signal via the genome (with its coregulator Smk-1), the gene products are associated with dauer formation and the stress response as well as life extension. Gene products that are increased following nuclear accumulation of DAF-16 (and thus are implicated in longevity promotion) include heat shock proteins (HSP-70 and HSP16-1), the SIR-2 family of genes, increased PGC-1a activity and superoxide dismutase 3 (SOD3). Other genes found to be upregulated via DAF-16 include vha-3, E3 ubiquitin ligase, zfp-1, and sarcoendoplasmic reticulum calcium ATPase (not conclusive list).
Nuclear accumulation of DAF-16 appears to induce gene products with a wide range of attributes but mostly related to stress survival and oxidant defense, with a notable increase in superoxide dismutase. There may be significant interplay between activation of DAF-16 and chaperone-mediated autophagy
PHA-4 is the C. Elegans ortholog to the human FOXA family of genes, which consists of FOXA1-3 and appears to be mandatory for the benefit of caloric restriction on longevity. PHA4 is additionally mandatory for the development of the C.Elegans pharynx that mediates feeding and influences the genome (response elements here) to induce the gene products. FOXA is also activated by hepatocyte nuclear factors (HFNs) with HFNα, β, and γ corresponding to FOXA1, FOXA2, and FOXA3 respectively.
FOXA1 appears to be regulated by steroid hormones, as FOXA1 appears to be mandatory for androgen receptor signalling as a cofactor and is highly involved in the genomic signalling of estrogen (α subset) insofar that it is mandatory for development of breast tissue.
FOXA2 is additionally activated by both glucocorticoids and cAMP, both increased during fasting. FOXA2 is also known to interact with bile acids which may explain the synergism between caloric restriction and supplemental lithocholic acid.
FOXA signalling appears to be mandatory for the benefits of caloric restriction on lifespan, although FOXA promoters are quite general and interact with a variety of hormones (including both steroid hormone classes of androgens and estrogens)
Insulin/DAF-2/IGF-1 signalling pathway
The Insulin/IGF-1 signalling pathway (IIS; C. Elegans ortholog of the receptor is Daf-2) appears to be a regulator of longevity in various research models and ultimaltely works via FOXO/DAF-16. Phosphorylation of DAF-16 by Akt (downstream of the insulin receptor and upstream of TOR) accumulates DAF-16 in the cytoplasm which prevents nuclear signalling and life promoting effects, which require nuclear accumulation (occurs when not phosphorylated). Activation of this pathway tends to reduce longevity, as mutants (with subactive levels of signalling) have prolonged vitality and life span.
Akt also activates TOR signalling (via the substrate PRAS40) and is able to phosphorylate TSC1, inactivating it and preserving mTOR activation via Rheb. These mechanisms establish Akt as a positive modulator of TOR, and the IIS pathway with the TOR pathway (next section) are intimately linked.
Signalling through the insulin receptor can activate Akt and promotes cytoplasmic accumulation and nuclear restriction of FOXO/Daf-16. Signalling via the insulin receptor is likely one of the more biologically relevant mechanisms underlying anti-longevity
Prolonged signalling via the insulin receptor towards FOXO/DAF-16 appears to upregulate components of the Akt pathway as negative feedback.
Some negative feedback potential of FOXO on the IIS may reduce the anti-longevity effects of this pathway in practical situations
Target of Rapamycin (TOR)
TOR is a protein that was identified as the target of the immunosuppresive drug Rapamycin (Sirolimus), and is called mTOR or CeTOR depending on if the protein is mammalian (mTOR) or from C. Elegans (CeTOR). TOR modulates ribosome biogenesis, autophagy and transcription and is a molecular target of leucine and the metabolite HMB.
TOR has a complex known as complex 1 (or TORC1), which is considered to be of more interest as the other complex (TORC2) is insensitive to Rapamycin; components of this complex include TOR kinase (let-363 in C. Elegans), Raptor (daf-15 in C. Elegans), and LST8 although LST8 does not appear vital to TORc1 signalling. LST8 is also involved in complex 2 and is the only subset present in both complexes, and complex 2 also includes Rictor (Rapamycin Insensitive Companion of TOR), Sin1 (stress-activated map kinase-interacting protein 1), and TOR itself.
TOR (mTOR in mammals) is a pathway intermediate in the IIS that is also a direct target of various metabolites, and is considered a druggable target due to its inhibition by rapamycin promoting longevity
TOR partially antagonizes PHA-4/FOXA signalling secondary to the PHA-4 suppressor known as ruvb-1 and as such can reduce the longevity effects of caloric restriction.
The small GTPase protein known as Rheb appears to directly stimulate TORC1 and is the intermediate by which the tumor suppressor proteins TSC1 and TSC2 inhibit TORc1, as the sequestering of Rheb by TSCs reduces activation of TORc1. Rag GTPases are also implicated in positively regulating TORC1 via the raptor subset and is inhibited by Metformin.
TOR is activated in periods of nutrient excess and by signalling molecules induced by nutrient excess, such as amino acids; activation of the TOR pathway is known to suppress the FOXA pathway, which is mandatory for caloric-restriction related benefits to longevity
Cannabinoids are endogenous fatty acid amides that signal through cannabinoid receptors, known as CB1 and CB2; these receptors are named after cannabis (Marijuana) where they were first characterized, and endogenous cannabinoids include anandamide and oleamide.
At least one study has note that abolishing the CB1 receptor has promoted longevity (C. Elegans) while either reducing N-acylethanolamine concentrations or increasing the activity of the FAAH enzyme (which metabolizes N-acylethanolamines) was able to promote lifespan although elsewhere CB1 knockout mice have shortened lifespans.
Signalling via the CB1 receptor has mixed effects on longevity
Serotonin is noted to possess potential antilongevity mechanisms, with mutants deficient in serotonin having greater nuclear accumulation of DAF-16 that is treated with exogenous serotonin or an SSRI. This is dependent on ADF chemosensory neurons and through daf-2 (insulin receptor) and appears to share signalling properties with the IIS pathway, and serotonin itself and antipsychotics have been linked to activating Akt (upstream of TOR) secondary to augmenting insulin receptor signalling.
Serotonin appears to augment signalling via IIS, TOR, and the subsequent reduction in DAF-16 nuclear accumulation and impaired FOXO signalling; serotonin may not be per se anti-longevity but merely augment the efficacy of other pathways, but still appears to be involved
A reduction in sphingolipid synthesis in yeast has been shown to increase lifespan via Sch9 (S6K human homolog) dependent mechanisms, Sch9/S6K being downstream of IIS/TOR, and possibly secondary to activation of Pkh1/2 in yeast which are known to activate Sch9.
Sphingolipids are negative regulators of longevity secondary to activating S6K, a protein downstream of TOR
The HECT E3 ubiquitin ligase WWP-1 appears to be a positive regulator of longevity, independent of the IIS pathway and able to induce longevity independent of caloric restriction. Interestingly, reducing WWP-1 or increasing levels of a metabolically inactive form of WWP-1 is able to reduce the benefits of caloric restriction; the mandatory cofactor UBC-18 is also mandatory for caloric restriction benefits.
The benefits of overexpressing WWP-1 are abolished if PHA-4 is abolished, but the reduction in life is preserved with abolishign DAF-16; WWP-1 appears to be more related to FOXA metabolism than ISS signalling.
Enzymes in the ubiquination pathway have been identified as a positive modulator of longevity in C. Elegans
SKN-1 is the C. Elegans homolog of the human Nrf gene, and Nrf2 is involved in a signalling pathway with a cytoplasmic protein known as Keap1; when Keap1 is phosphorylated, it releases Nrf2 to the nucleus to act upon the antioxidant response element (ARE) and induce production of oxidative enzymes. Nrf2 is induced by caloric restriction and is thought to underlie some hormetic benefits of caloric restriction.
Human Nrf2 (C. Elegans SKN-1) is a protein that signals for production of antioxidant enzymes and is activated by oxidative stress and fasting; a possible mechanistic link between caloric restriction and longevity
The Raf-1/MEK1/2/ERK1/2 pathway is a pathway that positively regulates autophagy as ERK1/2 phosphorylates Gα-interacting protein which accelerates the rate of GTP hydrolysis (via Gαi3 protein) which ultimately promotes autophagy; this pathway is inhibited by amino acids and a nutrient surplus, and is thought to be related to caloric restriction.
Phenomena of Interest
Cellular senesence (or sensence) is the aging of an individual cell as assessed by phenotypical and metabolic changes that slow down or cease cell functioning.
Accumulation of senescent cells appears to contribute to age-related pathology, and clearance of senescent cells has been demonstrated to revert these pathological changes.
Senesence is highly related to the tumor suppressor gene p16 and p16Ink4a appears to be a biomarker of sensecence, abolishing this protein has been noted to attenuate age-related phenotypical changes selectively in the tissue where p16Ink4a is abolished whereas the related protein p19Arf appears to preserve longevity (abolishing it augments aging). p16Ink4a may accumulate following activation of PPARγ and is induced by agonists such as rosiglitazone.
The mechanism is depedent on pmk-1 in C. Elegans (human ortholog is the p38 MAP kinase) and abolished by antioxidant compounds.
Several compounds that induce minor damage to the mitochondria (indicative by mitochondrial oxidation) appear to promote lifespan; this includes low dose arsenic, lonidamine,
Autophagy is the process of cellular structures being encapsulated and digested by autophagosomes in a manner similar to cellular recycling. Autophagosomes can be formed from the endoplasmic reticulum of a cell (called an omegasome) and the lipids that form the membrane of the autophagosome can be derived from either the mitochondria or cytoplasmic membrane. Autophagy can be microautophagic (not present in mammals), macroautophagic (major and selective autophagy of target damaged molecules), or chaperone-mediated autophagy mediated by Hsc70 and co-chaperones.
This process is mediated by autophagy-related proteins, of which over 30 have been identified, and can occur under conditions of both nutrient surplus and nutrient deficit. In nutrient deficit, autophagy is nonselective and aims to provide substrate to a cells energy state; in a nutrient excess, cargo-specific protein targets damaged organelles for removal.
Autophagy is a process of targeted cellular destruction that appears to act as quality control for a cell. In mammals it can be broken down into macroautophagy (which consists of the majority of the actions) or chaperone-mediated autophagy
Mitophagy is autophagy in application to mitochondria and is required for maintaining mitochondrial quality and function via recycling damaged mitochondria. It is conducted either via micromitophagy (invagination of the vacuolar/lysosomal boundary membrane) or macromitophagy (sequestering into autophagosomes); only the latter appears to be involved in mammals, with both active in yeast. Specifically, mitophagy is a cargo-specific autophagy rather than nonspecific.
Cells (yeast) without macromitophagy appear to have more enlarged mitochondria of round shape and shortened cristae, and under periods of caloric restriction without mitophagy an apparent accumulation of dysfunctional mitochondria and subsequent oxidation is noted. Mitophagy is proposed to maintain mitochondrial function and turnover over a long period of time, and is immediately preceded by mitochondrial fission.
Mitophagy is a cellular recycling of mitochondria that is requires for optimal long-term functioning of a cell
Mitophagy appears to be somewhat guided by NIX (NIP3-like protein X or BNIP3L) with the yeast homolog of ATG32; NIX is a protein that is located in the outer membrane of the mitochondria and can bind to LC3 and its homologue (GABARAP) acting as a membrane receptor for mitophagy.
Parkin is a protein (E3 ubiquitin ligase) that is selectively translocated to the damaged mitochondria following stressors, and Parkin overexpression appears to destroy damaged mitochondria without influencing normal ones. Parkin is recruited to damaged mitochondria by PINK1, a protein that is normally metabolized in mitochondria but accumulates in damaged mitochondria secondary to less proteolysis and PINK1 has the capacity to phosphorylate and activate Parkin which then functions as a ubiquitin in the mitochondria outer membrane; it is thought that Parkin targeting two proteins known as mitofusions (1 and 2) which stimulates mitophagy.
Parkin and PINK1 appear to be target proteins highly involved in cargo-specific mitophagy
Chaperone mediated autophagy (CMA) is a selective autophagy process for cytosolic proteins by transporting proteins across the lysosomal membrane and does not have the capacity to degrade organelles such as mitochondria; CMA, like other autophagic processes, is activated by cellular stresses and nutrient restriction but specifically nutrient deprivation activates nonselective macroautophagy and gradually switch to CMA during prolonged starvation. CMA function declines with age, can be activated by oxidative stress, and the 20-30% of lysosomes that are functional with CMA under fed conditions increases to 60-80% after three days of starvation (rat liver homogenates).
Chaperone mediated autophagy is a highly selective autophagic process mediated from the endoplasmic reticulum and maintains protein homeostasis in the cytoplasm (but not organelles) through protein unfolding responses
CMA tends to target proteins with a KFERQ-motif (such as IκBα, RNAase A, and c-Fos; consisting of about 30% of target proteins) although similar pentaamino motifs such as QREFK or VDKFQ are also recognized. Starvation, which increases CMA activity, is known to reduce the amount of cytosolic proteins with KFERQ motifs but not in skeletal muscle nor neurons (although neurons have higher baseline CMA activity).
These sequences that are expressed on cytosolic proteins are targeted by chaperones and co-chaperones, with the primary chaperone being Heat Shock Cognate protein of 70-kDa (Hsc70 or Hsp70) which also appears to be present in lysosomes and Hsc90 which is mostly associated with the luminal side; both proteins (Hsc70 and Hsc90) are chaperones that participate in protein unfolding.
Chaperones also target lysosomes via the glycoprotein receptor known as LAMP-2A (no affinity for LAMP-2B or 2C) and the rate limiting step in CMA appears to be expression of LAMP-2A; genetically ablating lamp-1A is lethal.
Chaperones target cytoplasmic proteins via an amino acid binding sequence and then target the LAMP-2A receptor on lysosomes, bringing together the lysosome and protein for degradation.
Protein degradation is known to slow with aging which leads to accumulation of altered or damaged proteins associated with reductions in chaperone mediated autophagy (among other things, but thought to play a causative role). Senescent fibroblasts appear to have reduced CMA activity that is no longer able to be upregulated by serum starvation (intrigueing, due to some molecules such as curcumin acting via caloric restriction-like mechanisms but being unable to benefit older persons) and has been noted in vivo. It appears that the rate limiting step, LAMP-2A receptor expression, is downregulated which causes reductions in CMA rate of proteolysis and is not due to transcriptional downregulation or changes in rates of synthesis and delivery of this receptor to the lysosomal compartment but rather due to dynamics and stability at the lysosomal membrane being reduced with age.
Genetically restoring LAMP-2A activity to older rats is able to remove age-related comorbidities and reduce oxidative damage in liver cells secondary to preserving CMA function. LAMP-2A has been demonstrated to be downregulated in high-fat fed mice but beyond that the mechanisms underlying the increased membrane instability of LAMP-2A (and reduction of activity with age) are largely unknown.
LAMP-2A downregulation provides a basis for how caloric restriction influences youth more than older individuals, and plays a crucial role in CMA
Currently, caloric restriction is the most reliable method of promoting longevity known and as such is usually reference in all longevity research
Mechanisms involved in Caloric Restriction
Deletion of a gene (atg32Δ) that regulates mitophagy appears to prevent caloric restriction from promoting longevity in yeast.
The essential co-regulator of DAF-16, smk-1, and FOX(A) signalling appear to be vital for caloric restriction related longevity despite DAF-16 nuclear accumulation (and FOXO signalling) per se not being mandatory.
Exercise is able to increase mRNA translation of Hsp72 as Hsp72 is increase following oxidative, heat, hypoxic/hyperoxic and physical damage to tissue; this increase has been noted in the liver, heart, leukocytes, and adrenal glands in addition to the exercised skeletal muscle and the mechanism of activation is that muscle metabolism byproducts (lactate, radicals, and glucose deprivation) and stress hormones phosphorylate a protein known as heat shock factor-1 (HSF-1) which becomes a trimer when phosphorylated and signals via the genome and produces Hsp72. Hsp72 works in manners similar to other molecular chaperones and acts to degrade proteins in the cytoplasm and maintain cellular protein homeostasis and the amount it is induced by exercise seems to correlate with the amount of metabolic damage the tissue undergoes (upregulated for 30 minutes following an hour of running and up to 24 hours after a marathon) and is upregulated more when exercising in the heat.
Increasing Hsp72 expression in skeletal muscle is thought to be protective against pathology and aging and does not appear to be increased during caloric restriction like Hsp90 is increased.
Exercise can stimulate molecular chaperones to promote cellular homeostasis of proteins in a similar manner to chaperone-mediated autophagy. The chaperones involved in the stress response to exercise appear to be different than those involved in caloric restriction
Supplements that Interact with Longevity
Melatonin is a pineal hormone best known for its role in sleep, and appears to be highly involved with longevity (can be read on the Melatonin supplement page) in both a preventative and rehabilitative manner.
Melatonin has shown efficacy in nematodes, fruit flies, and mammals (rats and mice) with feasible oral doses of 5.45mg (150lb human equivalent, based off 1mg/kg in rats) taken daily. At least according to rat studies, 10-fold the dose confers no additional protective properties and is largely equivalent.
Although the mechanism is not known, it is thought to either be direct via inhibition of telomerase or indirect secondary to the pineal gland.
Melatonin supplementation is likely to be a feasible and safe longevity promoting agent that is feasible to take daily. Repeated trials have shown longevity promoting effects in mammalian species
Both Rhodiola Rosea and Eleutherococcus senticosus have been implicated in promoting heat resistance and longevity in drosophilia, which is thought to be related to DAF-16 signalling (as this molecule mediates both heat resistance in drosophilia and longevity) and indepedent of any losses in fecundity or caloric restriction. Both adaptogen compounds listed were shown to reduce lifespan in 10-fold the oral dose.
Adaptogens appear to induce longevity secondary to DAF-16 nuclear accumulation, which is independent of caloric restriction. Currently no mammalian research
Lithocholic Acid (LCA) is a bile acid that appears to be synergistic with caloric restriction in promoting longevity in yeast related to mitophagy, as abolishing macromitophagy abolished the synergism. LCA appears to work independently of TOR and cAMP/PKA signalling (abolishing Rim15 failed to prevent the effects), and only works during certain life phases of yeast. It should be mentioned that the authors hypothesized that the targets of LCA may be inhibited by TOR and cAMP/PKA activation.
Possible explanations for the obeserved synergism include the interaction of FOXA proteins with bile acids and a reduction of lipid (free fatty acid and diacylglycerol) accumulation on the endoplastmic reticulum (as LCA was able to rescue age reducing impairments in beta-oxidation).
Lithocholic acid appears to synergistically promote longevity with caloric restriction in yeast (currently no studies in mammals) possibly related to lipid dynamics and the endoplasmic reticulum
Lithocholic acid is known to activate the Vitamin D receptor, the TGR5 bile acid receptor, the PXR receptor, and the FXRα receptor although both the VDR and PXR require concentrations above 30μM to activate. It should also be noted that lithocholic acid has demonstratable toxicity in rats when orally ingested at 0.03% of the diet.
Although promising due to synergism demonstrated with caloric restriction, is too preliminary to draw conclusions in mammalian species. Molecular targets are not fully identified yet
Caffeine (xanthine in tea and coffee) is noted in yeast to promote lifespan by a mechanism that is dependent on CREB1 phosphorylation, TORC1 inhibition, and DAF-16 nuclear accumulation.