Telomere shortening was the first demonstrated cause of cell aging, also known as cellular

senescence. The causative engagement of telomeres was next extended to long but damaged telomeres. It is now well established that short and/or damaged telomeres accumulates with aging in proliferating and non-proliferating tissues in mammals and, in its exacerbated form, this is associated, often causatively, with several human age-related diseases and aging itself.

Animal models carrying different kinds of telomere dysfunction often recapitulate human pathologies caused by telomeric damage.

Our recent development of tools able to reduce the consequences of telomere dysfunction, by selective inhibition of DNA damage response (DDR) activation at telomeres, allowed us to determine the contribution of telomere dysfunction in vivo in a number of experimental settings.

In addition to telomeres, other “clocks” measuring aging have been proposed, with those based on DNA methylation changes being most widely used. Whether telomere biology and epigenetic aging pathways interact is unclear.

We will discuss these subjects and our progresses on these matters.

Background: Loss of proteostasis and mitochondrial dysfunction are two key cell-intrinsic drivers of aging whose interconnection is elusive. We uncovered an unprecedented role for the eumetazoan autophagic receptor and hub adapter protein SQSTM1/p62, previously described to promote longevity in mice, in regulating mitochondrial structure and function.

Methods: Combining proteomics, biochemistry, molecular biology, electron microscopy (EM) and functional assays, we defined the changes in endogenous p62 interactome under various proteostasis stressors, including proteasome, autophagy and deubiquitinases (DUB) inhibitors, as well as the expression of an aggregation-prone synuclein mutant. Our approach revealed a new cellular mechanism linking protein and mitochondrial homeostasis. We then challenged the preclinical and clinical relevance of this mechanisms in aging pathophysiology using suitable genetically modified mouse models and through geriatric assessment of elderly human individuals carrying p62 mutations.

Results: Proteomic analysis upon p62 immunoprecipitation revealed the unanticipated interaction with the mitochondrial intermembrane bridging (MIB) super-complex, comprising the outer membrane SAM (Sorting and Assembly Machinery) and the inner membrane MICOS (Mitochondrial contact site and Cristae Organizing System) complexes. Pharmacologic or mutant-driven protein aggregation diverted p62 from mitochondria, leading to loss of the MICOS core protein mitofilin, MIB disassembly, and mitochondrial dysfunction. Targeted mutagenesis of p62 proved its ubiquitin-binding activity (UBA) domain necessary for proteotoxicity-induced mitochondrial dissociation and defined the minimal MIB-binding moiety within its ZZ-type zinc finger domain. Attesting to an essential and direct positive effect on mitochondrial function, genetic p62 ablation disrupted mitochondrial cristae and suppressed oxidative phosphorylation, effects rescued by its exogenous re-expression. In vivo, EM showed profound defects of mitochondrial cristae in the brain cortex of aged p62 knock-out mice, as compared to age-matched controls, while old p62 P394L (UBA-impaired) homozygous knock-in mice had preserved mitochondrial cristae and ATP yield. Attesting to relevance to human aging, elderly subjects carrying SQSTM1 UBA-inactivating mutations appeared protected from declining skeletal muscle and heart function and from disability as compared to elderly controls with intact SQSTM1.

Conclusions: Our findings reveal a previously unappreciated intracellular mechanism whereby protein homeostasis regulates mitochondrial architecture and function via the adapter protein SQSTM1/p62 of clinical relevance in aging pathophysiology.

High Mobility Group Box 1 (HMGB1) is a nuclear protein released by cells upon stress or damage to mediate sterile inflammation and regeneration according to its redox state. Notably, altered levels of HMGB1 inside and outside the cells are tightly linked to hallmarks of aging, and HMGB1 has been recently included in the panel of biomarkers that may contribute to the development of frailty. Hence, our working hypothesis is that cells tend to release HMGB1 upon aging due to constant and increasing cell stress and damage, leading to alteration of cell intrinsic homeostasis as well as inappropriate inflammatory signalling and damage of cells/tissues. Total-body HMGB1 knockout (tKO) mice and their respective controls (Ctr) were employed to investigate the role of HMGB1 in aging by monitoring body parameters, motor performance, and cognitive function up to 20 months of age. Interestingly, tKO mice did not show significant changes in body weight during aging, in contrast to Ctr mice, which exhibited progressive weight gain starting from 10 months. This difference was associated with a lower fat mass percentage in tKO mice compared to controls. Additionally, the elevated triglyceride levels observed in both young and aged tKO mice indicate a key role for HMGB1 in the regulation of lipid metabolism. Functionally, tKO mice displayed reduced muscle strength and increased muscular fatigue compared to controls, pointing to an early decline in muscle function. However, this difference became less pronounced with aging, as the absence of HMGB1 did not exacerbate the age-related decline in muscle strength. Moreover, proteomic analysis of tibialis anterior muscles from 20 months-old tKO and control mice identified differential expression of key signaling pathways involved in muscle maintenance, reinforcing the specific role of HMGB1 in sustaining muscle homeostasis and function. To confirm the muscle-specific role of HMGB1, we utilized a skeletal muscle-specific HMGB1 knockout (mKO) mouse model. Interestingly, mKO mice exhibited similar muscle defects to those observed in 10-month-old tKO mice, and no significant differences between mKO and Ctr at 20 months of age. Regarding body parameters, the absence of HMGB1 specifically in muscle did not appear to affect fat mass percentage during aging, but rather had an impact on lean mass, which was significantly higher in 20-month-old mKO mice compared to controls. Given the interplay between lipid metabolism and muscle function—through effects on energy availability, mitochondrial efficiency, and metabolic homeostasis—these alterations may contribute to muscle fatigue and loss of homeostasis. Further studies are necessary to elucidate the mechanistic link between HMGB1, lipid metabolism, and muscle physiology during aging.

Only 10% of the human genome is well characterized in terms of function. Among the 20,000 potential protein encoding genes, only 2,000 have been well studied by scientists. About 5,000 genes are completely unknown and the remaining 13,000 genes are poorly described/characterized by researchers. We looked at the neglected genome and screened the 5,000 unknown genes for potential candidates that may play a major role in aging. We identified a novel gene whose expression was under the control of one of the most important longevity pathways. The number of this unknown human gene was C16ORF70 (Chromosome 16 Open Reading Frame 70) and we renamed it with the acronym MYTHO (MacroAutophagy and Youth Optimizer).MYTHO has some unique features including: 1) The amino acid sequence is highly conserved among species ranging from worms to humans, 2) its expression is higher in extremely long-lived subjects both humans and mice, 3) when Mytho was inhibited by a genetic approach in different species such as worms, fish, mice, and mammalian cells in vitro all the hallmarks of premature aging including the alteration of mitochondrial function, proteostasis and cell replication appeared; 4) consequently, inhibition of Mytho not only induced premature aging but also it reduced the life-span premature mortality in worms, fish and rodents. On the other hand, activation of Mytho induces a significant increase in health span

Physiological brain aging is characterized by cognitive decline correlated to progressive synaptic degradation. Zebrafish, due to an aging progression similar to the one in humans, is an ideal model to combine behavioral with neurobiological studies to explore the interplay among aging, behaviour, synaptic integrity and environmental stress conditions.

We combined behavioral analysis based on deep learning with confocal microscopy synaptic reconstruction in zebrafish, to correlate behavioral and synaptic changes in response to social isolation, a recognized environmental stressor, at different ages. We reported aging-related modifications in zebrafish social preference and in the adaptation to social deprivation. In juvenile control fish, which are social and prone to explore novel context, social isolation induced conspecifics avoidance and increased freezing behavior, a hallmark of anxiety. Differently, aged fish showed reduced social preference in control and social isolation did not alter their behavior or increase anxiety responses.

In juvenile isolated animals, respect to their controls, the analysis of zebrafish amygdala homologue (a brain region specifically involved in stress responses) revealed an increased density in dendritic spines, which is indicative for synaptic plasticity. Such an effect was not detected in aged individuals, suggesting that the reduced neurobehavioral responsiveness to environmental changes is due to altered synaptic plasticity in the aged brain. With our integrative studies on zebrafish we may gain insights in synaptic integrity, cognitive function and anxiety in late life.