Exploring Biomarker Correlations in the Aging Process

  

    
Biomarkers 
    
Associated Hallmark of Aging
  
  

    
Telomere Length 
    
Telomere Attrition
  
  

    
AMP-activated protein    kinase (AMPK)
    
Autophagy 
  
  

    
Sirtuin 6 (SIRT6)
    
Mitochondrial Dysfunction & Genomic Instability
  
  

    
Secreted Protein Acidic and Rich    in Cysteine (SPARC)
    
Cell Senescence
  
  

    
4-Hydroxy-2-nonenal (4-HNE) 
    
Inflammation and Cell Senescence
  
  

    
SRY-Box Transcription Factor 2 (SOX2) 
    
Stem cell exhaustion
  
  

    
C-reactive protein (CRP)
    
Inflammation 
  
  

    
Taurine 
    
Increased Cellular Senescence,    Telomerase Deficiency, Mitochondrial Dysfunction, DNA Damage, and Inflammation
  
  

    
Interleukin-6 
    
immune-senescence 
  
  

    
Mammalian/mechanistic target of rapamycin (mTOR)
    
Cell senescence/ Mitochondrial dysfunction/Autophagy 
  
  

    
DNA (cytosine-5)-methyltransferase 1 (DNMT-1) 
    
Epigenetics/Methylation 
  
  

    
Aconitase-2 
    
Mitochondrial dysfunction, Autophagy 
  
  

    
Interleukin 8
    
Cell senescence, Inflammation
  
  

    
Phosphatidylinositol 3-kinase (PI3K)
    
genome instability, telomere erosion, epigenetic    alterations, and mitochondrial dysfunction.
  

Table 1: Genomic and Proteomic Biomarkers and their associated hallmarks of aging.

Genomic Biomarkers

Based on extensive large-scale studies conducted over several decades, longevity appears to be only moderately heritable. The genetic influences on longevity are likely non-additive, while the environmental factors contributing to it are non-shared [14, 15]. Despite this, the genetic component of lifespan variation across the general population has been estimated to account for approximately 25% [16].

One key area where genetic factors intersect with the biology of aging is the structure and function of telomeres. Telomeres are repetitive DNA sequences (TTAGGG) that protect chromosomes during cell division and are essential for DNA replication. However, mammalian cells experience telomere shortening with each division, limiting their capacity to divide. As a result, cells lose their proliferative potential and enter a state of irreversible cell cycle arrest, known as replicative senescence.

Telomere length (TL) is a critical determinant of cellular replicative capacity, particularly in tissues like the skin, which are highly susceptible to accelerated telomere shortening due to external DNA-damaging agents such as solar radiation, pollution, and reactive oxygen species (ROS) [17]. In multicellular organisms, TLs are highly heterogeneous across tissues and cell types, reflecting differences in tissue-specific proliferation rates. Nevertheless, telomere shortening occurs universally in all proliferating tissues with age [18]. Telomereinduced senescence has been identified as a potential key driver of aging [19].

A large community-based study in Scotland found TL to be significantly associated with age and eight measures of physical and cognitive functioning, as well as overall health status—factors closely tied to normal aging [20]. TL serves as a marker of cellular senescence and chronic disease-related oxidative stress. Accelerated telomere shortening and reduced telomerase activity have been linked to age-related skeletal pathologies like osteoporosis and osteoarthritis, caused by abnormal subchondral bone remodelling [21]. Similarly, shorter TLs elevate the risk of pathologies involving restricted cellular proliferation and tissue degeneration, such as atherosclerosis-related cardiovascular disorders [22].

Both genetic and environmental factors influence TL and the rate of age-related telomere shortening [22]. Developmental experiences, such as unfavorable intrauterine conditions [23], TL at birth [24], and early life adversity (e.g., low socioeconomic status, neglect, or abuse) [25], have profound long-term effects on telomere dynamics. Additionally, adult-life exposures, including infections [26], psychoemotional stress [27, 28], nutrition [29], physical activity [30], smoking [31], and alcohol consumption [32], significantly impact TL and overall cellular aging. While telomeres provide critical insights into cellular senescence and aging, another emerging and equally significant area of research focuses on epigenetic modifications, particularly DNA methylation, which offers a dynamic and reversible layer of regulation influencing the aging process. Recent studies have identified a measure of DNA methylation age, also referred to as the epigenetic clock, as a viable biological age predictor.

Among these, the Horvath (2013) and Hannum (2013) epigenetic clocks are currently considered the most robust and reliable predictors of chronological age. Both show high correlations with age (r = 0.96 for Horvath and r = 0.91 for Hannum) and minimal mean deviations from calendar age (3.6 years and 4.9 years, respectively) in their respective validation cohorts [33, 34]. The Horvath clock was developed using a large sample size of approximately 8,000 individuals spanning the entire adult lifespan and multiple ethnic populations. It is a multitissue predictor that utilizes methylation levels of 353 CpG sites from the Illumina 27k array. In contrast, the Hannum clock, based on 656 individuals, focuses on 71 CpG sites from the Illumina 450k array and is most accurate with whole blood samples [35].

What sets these clocks apart is their ability to predict allcause mortality, independent of traditional risk factors. A metaanalysis across 13 cohorts, comprising a total of 13,089 individuals, demonstrated that the epigenetic clock could predict all-cause mortality independent of factors such as age, BMI, education, smoking, physical activity, alcohol use, and comorbidities [36].

Genomic biomarkers such as telomere length and epigenetic biomarkers not only reveal the cumulative effects of genetic and environmental factors but also underline the dynamic interplay between molecular integrity and aging processes. Furthermore, accumulation of genomic instability from DNA damage caused by endogenous factors (e.g., replication errors, reactive oxygen species) and exogenous stressors (e.g., radiation, toxins), also characterize aging. This genomic instability, which is one of hallmarks of aging, leads to mutations, chromosomal abnormalities, and impaired cellular function, driving aging and age-related diseases.

In addition to genomic factors, the aging process is intricately influenced by changes in the proteome—the complete set of proteins expressed in a cell or organism. Proteins serve as the functional molecules of life, mediating critical biological processes and cellular communication. However, aging disrupts proteostasis, another hallmark of aging, the delicate balance between protein synthesis, folding, and degradation, leading to the accumulation of damaged or misfolded proteins. In the next section, we will explore key proteomic biomarkers and their roles in unravelling the hallmarks of aging, focusing on their significance in maintaining cellular function and systemic health.

Proteomic Biomarkers

Proteomics offers a comprehensive and quantitative view of the entire protein expression landscape within an organism, reflecting the dynamic state of cellular processes and their responses to environmental or biological perturbations [37]. As humans progress through different stages of life—from neonates to adulthood—an array of proteins is differentially expressed, showcasing the profound impact of age on the proteomic profile [38]. Proteomic biomarkers provide real-time insights into the mechanisms underlying the hallmarks of aging and the disruptions associated with age-related processes. Beyond proteostasis, specific proteomic biomarkers can illuminate diverse hallmarks of aging, guiding targeted approaches to address these biological changes. By analyzing protein expression patterns, modifications, and interactions, researchers can identify unique signatures of aging as well as early indicators of age-associated diseases, advancing our understanding and management of the aging process.

A twin-based cohort study identified four replicating proteins that demonstrated significant independent associations with age, highlighting the potential of proteomic biomarkers to serve as robust indicators of the aging process [39]. These findings underscore the value of proteomic studies in identifying proteins that not only reflect biological age but also provide insights into the molecular mechanisms underlying age-related changes. Such proteins could serve as critical tools in monitoring aging trajectories and developing interventions to promote healthy aging.

Building on the foundational understanding of proteomics, we now delve deeper into specific protein biomarkers associated with individual hallmarks of aging. By examining key protein biomarkers associated with various hallmarks of aging, we can gain targeted insights into the molecular disruptions driving age-related changes and identify potential pathways for intervention and therapeutic strategies.

AMP-activated Protein Kinase

One such critical protein biomarker that bridges metabolic regulation and aging is AMP-activated protein kinase (AMPK). This regulator of cellular energy homeostasis plays a pivotal role in modulating various hallmarks of aging, making it a key focus in aging research and therapeutic development. AMPK plays a crucial role in cellular energy regulation by responding to energy stress through phosphorylation, which restores ATP levels by inhibiting energy-consuming pathways and activating ATP-generating catabolic pathways. AMPK also influences longevity and inter-tissue communication, as intestine-specific AMPK upregulation activates autophagy both locally and in distant tissues like the brain, reduces proteotoxicity, and extends lifespan [40]. Autophagy, regulated by AMPK, is vital for degrading and recycling cellular components, maintaining homeostasis, and facilitating inter-tissue communication via the release of cytosolic molecules [41]. Tools such as Western blotting to measure AMPK Thr

(172) phosphorylation and mRNA expression analysis of AMPK subunits are pivotal for studying its pathway [42].

Overall, AMPK-mediated pathways not only maintain cellular energy homeostasis but also modulate aging and tissue health in a non-cell-autonomous manner, underscoring its significance in health and disease. While AMPK plays a central role in maintaining cellular energy homeostasis and autophagy, another key regulator, SIRT6, contributes to preserving mitochondrial function and genomic stability—two critical hallmarks of aging.

SIRT6

SIRT6 is a multifunctional protein that has garnered significant attention for its role in aging and longevity. It is known to possess several enzymatic activities that contribute to its ability to protect cells, tissues, and organs from the effects of aging. One of the key mechanisms by which SIRT6 exerts its anti-aging effects is by promoting DNA repair. Studies have shown that SIRT6 is involved in the repair of DNA damage [43, 44, 45], which is a crucial aspect of maintaining cellular function and preventing the onset of age-related diseases. In addition to DNA repair, SIRT6 plays a critical role in maintaining the normal structure of chromosomes [46, 47, 48], which is essential for the proper functioning of cells throughout an individual's lifespan. Furthermore, SIRT6 is involved in regulating energy metabolism [49, 50], which is a vital process for maintaining cellular homeostasis and function. By influencing metabolic pathways, SIRT6 helps to ensure that cells can efficiently manage their energy requirements, thereby supporting overall health and longevity. SIRT6 also plays a role in regulating the senescence-associated secretory phenotype (SASP), which is the process by which senescent cells secrete pro-inflammatory cytokines, growth factors, and proteases that can contribute to tissue dysfunction and aging [51, 52]. By modulating SASP, SIRT6 helps to mitigate the detrimental effects of cellular senescence on the aging process. In addition to its roles in DNA repair, chromosome stability, metabolism, and SASP regulation, SIRT6 has been shown to inhibit immunosenescence, a process in which the immune system becomes less effective as we age [53]. Immunosenescence is associated with a decline in immune function, which can make the body more susceptible to infections, cancers, and other age-related diseases. By inhibiting immunosenescence, SIRT6 helps to maintain a robust immune system throughout the aging process. Moreover, SIRT6 has the ability to influence the differentiation and function of immune cells. It regulates post-translational modifications (PTMs) that affect various aspects of immune cell biology, including their differentiation, maturation, and function. SIRT6 also plays a role in immunometabolism, the process by which immune cells regulate their metabolic activity to support immune responses [54]. This makes SIRT6 an essential player in the immune system’s ability to adapt to different challenges throughout life. Despite these well-documented functions, further studies are required to fully understand the role of SIRT6 in regulating inflammation, particularly its impact on different immune cells in various diseases or at different stages of aging. Research is also needed to elucidate how SIRT6 influences the differentiation, maturation, and function of immune cells under different conditions. This will provide a deeper understanding of how SIRT6 may be leveraged to enhance immune function and improve health outcomes across the lifespan [54].

In addition to mitochondrial dysfunction and genomic instability, aging is also characterized by the damaging effects of oxidative stress and lipid peroxidation. 4-Hydroxy-2-nonenal (4-HNE), a byproduct of these processes, stands out as a significant biomarker linking oxidative damage, inflammation, and cellular aging.

4-Hydroxy-2-nonenal (4-HNE)

Building on the role of SIRT6 in preserving mitochondrial function and genomic stability, 4-Hydroxy-2-nonenal (4-HNE) emerges as a significant marker linking lipid peroxidation, oxidative stress, inflammation, and cellular aging. A major a, β-unsaturated aldehyde produced during lipid peroxidation, 4-HNE acts as a potent messenger in various signalling pathways [55]. Its binding to proteins (4-HNE-protein adducts) serves as a critical marker of lipid peroxidation, with levels increasing in brain tissues and fluids during aging. This accumulation is associated with hallmark aging disorders, particularly neurodegenerative diseases. Elevated 4-HNE inhibits telomerase and proteasomes, promoting telomere shortening and protein accumulation—key contributors to neurodegenerative and degenerative conditions.

A study by Maciejczyk et al. analyzing salivary and plasma biomarkers across 180 healthy individuals in six age groups (6–13, 14–19, 20–39, 40–59, 60–79, and 80–100 years) found that 4-HNE levels rise with age, influencing the expression of senescence-related pathways [56]. As a byproduct of lipid peroxidation caused by dyshomeostasis in reactive oxygen species (ROS), excessive 4-HNE exacerbates aging-related damage [55,57]. Elevated 4-HNE levels heighten the risk of inflammaging (chronic inflammation associated with aging) and several age-related disorders, including Alzheimer’s and Parkinson’s diseases, dry eye, macular degeneration, hearing loss, and cancer. These conditions arise due to the acceleration of oxidative stress-induced damage [57]. Factors contributing to abnormal 4-HNE levels include increased cellular oxidative stress, which disrupts the balance seen in healthy aging individuals. To mitigate these effects, interventions such as glutathione supplementation, an antioxidantrich diet, and regular physical activity are recommended to restore 4-HNE levels to a healthy range.

Building on the role of 4-HNE in linking oxidative stress, inflammation, and cellular aging, Taurine emerges as another key biomarker, addressing not only systemic inflammation but also cellular senescence, mitochondrial dysfunction, telomerase deficiency, and DNA damage.

Taurine

Taurine (2-aminoethanesulfonic acid) is a versatile biomarker that plays a pivotal role in multiple biological processes and aging mechanisms, such as cellular senescence, telomerase deficiency, mitochondrial dysfunction, DNA damage, and inflammation (Figure 2). Although taurine is the most abundant free amino acid in animal tissues, it is not incorporated into proteins. Its best-known biochemical role is as a precursor of the bile acid taurocholic acid [58]. In mammalian cells, taurine is synthesized from cysteine through the enzyme cysteine sulfinic acid decarboxylase (CSAD) [59].

    

    

    Figure 2: Singh et.al, illustrated effect of taurine and taurine derived
biomolecules (in red) on various hallmarks of aging.