2^nd EURO GEROSCIENCE Conference Oral Communications

Oral Communications

OC1 — Exploiting Metabolic Reprogramming in Cellular Senescence as a Therapeutic Target

José Américo Nabuco L F De Freitas¹, Khaled Tighanimine², Yvan Nemazanyy², Yara Bou Saada¹, Delphine Benarroch-Popivker³, Aaron Mendez-Bermudez³, Stefano Fumagalli², Bertrand Friguet¹, Eric Gilson³, Mario Pende², Oliver Bischof⁴ (1. Sorbonne Université, Cnrs, Inserm, Institut De Biologie Paris-Seine, Biological Adaptation And Ageing, B2a-Ibps — Paris (France), 2. Institut Necker-Enfants Malades, Université Paris Descartes, Sorbonne Paris Cité — Paris (France), 3. School Of Medicine, Cnrs, Inserm, Ircan, Université Côte D’azur — Nice (France), 4. Cnrs Délégation Ile De France — Villejuif (France))

Backgrounds: Cellular senescence (CS) is a response to diverse forms of nonlethal cellular stress. The phenotypic transformations occurring in CS include a stable cell cycle arrest, an inflammatory response, and a complex metabolic shift. Among the most prominent intrinsic stimuli are genotoxic and oncogenic perturbations, many of which can initiate or promote aging and age-related diseases. Thus, the accumulation of senescent cells in tissues emerges as a critical driver of aging. Consistently, senescent cells can be found in the affected tissues of patients with age-related diseases such as osteoarthritis, pulmonary fibrosis, atherosclerosis, and Alzheimer’s disease. Conversely, recent data show that senescent cell elimination in healthy animals prolongs lifespan. Despite the important contribution of senescence to tumor suppression and aging in animals and humans, we have only begun to define the underlying mechanisms, and many unresolved questions remain. For example, 1) What are the senescence clocks that reflect biological rather than chronological cell age 2) Is the senescence clock/program common across, or specific to, different stresses, cell types, and species? 3) How do senescent cells evolve, and 5) Are there senescence biomarkers that are more specific/robust to senescent cells other than currently used “soft” biomarkers, such as senescence-associated beta-galactosidase? Objectives: Our understanding of the metabolic reprogramming that occurs during cellular senescence is still highly fragmentary. Senescent cells show a plethora of metabolic changes, including increased glucose consumption and lactate production (Warburg effect) along with a reduction in dNTPs as well as reduced NAD+/NADH and AMP/ATP ratios. Compounds that elevate NAD+, including nicotinamide mononucleotide (NMN), have been proposed as possible therapeutics for preventing several age-related pathologies. Given the complexities of metabolism in general, and the role of metabolism in regulating both the causes and consequences of the senescence response, the field is ripe for applications of holistic approaches. In addition, there is a growing body of evidence that metabolic changes also influence epigenetic modifications. For example, α-ketoglutarate (α-KG), and cofactors such as acetyl-coenzyme A (acetyl-CoA), S-adenosylmethionine (SAM), ATP, UDP, flavin adenine dinucleotide (FAD), and nicotinamide adenine dinucleotide (NAD+), influence the activity of various epigenetic modifying enzymes, thereby coupling chromatin-dependent gene regulation with the metabolic state of the cell. Results and Methods: Here, we provide comprehensive and dynamic profiling of the senescence metabolome using mass spectrometry across different cell types and during the time-course of different senescence induction conditions and perform transcriptomic and metabolomic data integration revealing uniquely enriched pathways in senescent cells. We identify a communal set of metabolites that distinguish senescent cells from proliferating and quiescent cells, including metabolites that act as substrates or cofactors for chromatin and DNA-modifying enzymes, providing a potential cross-talk between metabolism and epigenetic regulation of gene expression. Furthermore, modulating the levels of some of these metabolites using drugs profoundly impact the senescence phenotype including a reprogramming of the senescence transcriptome. Conclusion: Together, our analysis identifies senescence metabolome biomarkers, potential metabolic liabilities of senescent cells and thus, opens therapeutic avenues to exploit metabolic senescence targeting for health benefit.

OC02 — Telomerase Preserves Lung Function in Old Mice by Preventing Senescence of Endothelial Cells

Vincent Geli¹, Marielle Breau¹, Lipskaia Larissa², Churikov Dmitri¹, Cayroux Christelle¹, Fouillade Charles³, London-Vallejo Arturo³, Adnot Serge⁴ (1. Crcm — Marseille (France), 2. Hôpital Mondor — Creteil (France), 3. Institut Curie — Orsay (France), 4. Hôpital Mondor (France))

Accumulation of senescent cells has been causally linked to the development of age-related pathologies. Variations in telomere state are critical for cell senescence, stem cell biology and the development of several age-related diseases. What would happen if cells would escape cell senescence through the conditional expression telomerase «only when necessary» and what would be the consequences of such expression? To answer these questions, we generated a mouse model (called p21+/mTert) that expresses the telomerase reverse transcriptase (Tert) under the control of the p21Cdkn1apromoter, a major actor of cellular senescence in response to telomere dysfunction and DNA damage. The goal was to create a unique fine-tuned regulatory loop allowing the conditional expression of telomerase that would specifically avoid replicative senescence in cells with dysfunctional telomeres. We report that this conditional expression of Tert reduces the accumulation of senescent endothelial cells (EC) with age. This is correlated with a reduction in age-related alveolar space enlargement and lung fibrosis. The protection against emphysema depends on TERT catalytic activity and is associated with an increased proliferation of pulmonary EC and capillary density. Single-cell RNA sequencing of lung cells revealed that TERT expression is associated with the enrichment of ECs expressing genes involved in vessel regeneration. These findings reveal unique insights by which active telomerase prevents emphysema and preserve lung functions in old mice.

OC03 — A Single Short Reprogramming Early in Life Improves Fitness and Increases Lifespan in Old Age

Quentin Alle¹, Enora Le Borgne², Paul Bensadoun¹, Camille Lemey², Nelly Bechir², Melissa Gabanou², Fanny Estermann¹, Christelle Bertrand-Gaday³, Laurence Pessemesse³, Karine Toupet⁴, Jerome Vialaret⁵, Christophe Hirtz⁶, Daniele Noel⁴, Christian Jorgensen⁴, Francois Casas³, Ollivier Milhavet¹, Jean-Marc Lemaitre¹ (1. INSERM/IRMB — Montpellier (France), 2. UM/IRMB — Montpellier (France), 3. Metamus/INRAE — Montpellier (France), 4. Ecell France/IRMB — Montpellier (France), 5. Ppc/CHU — Montpellier (France), 6. Ppc/UM — Fort Worth (United States))

Although, the iPSCs reprogramming process, using the 4 Yamanaka factors, OCT4, SOX2, KLF4, C-MYC (OSKM) (1), has been described to favour senescence, establishing senescence as a barrier to reprogramming, we were able to derive iPSCs efficiently from senescent cells and from centenarian donor cells, using an optimized reprogramming strategy based on 6 factors (OCT4, SOX2, KLF4, C-MYC, NANOG, LIN28) (2). This strategy allowed to overcome the senescent state and both gene expression patterns, telomere length and metabolism were rejuvenated after reprogramming into iPSCs and re-differentiation into fibroblasts. We further investigated for a different reprogramming regimen using OSKM factors to avoid senescence promotion. Consistent with previous results, we show that inducing transiently OSKM factors in fibroblasts decreases DNA damage and senescence and activate autophagy, in vitro (3). To reproduce this activity in vivo, we derived a mouse transgenic mice model, allowing both a controlled expression of OSKM by doxycycline and recapitulating the human phenotype of the accelerated aging syndrome of Hutchinson-Gilford Progeria (HGPS). We firstly established a specific induction protocol that significantly extend the life expectancy of this accelerating aging mice confirming our hypothesis and previous results (4). Then, we investigated for tissue integrity improvement. Unexpectedly, we observed that a single transient reprogramming induction for a short period of time, in the early life was able to improve body composition and functional capacities of mice over the entire lifespan. In addition, treated mice have improved tissue structures such as bone, cartilage, lung, spleen, kidney and skin, leading to an increased lifespan of 15%, in old age. Altogether, our results indicate that a single short reprogramming, applied early in life might initiate and propagate an epigenetically related rejuvenated cell physiology, to promote a healthy lifespan (5). This new reprogramming strategy is a pertinent approach to explore potential rejuvenation mechanisms and its propagation to prevent age-related pathologies and promote healthy aging. 1. Takahashi et a., Cell 2006; 2. Lapasset et al., Genes and development 2011; 3. Sarkar et al., Nature Communications 2020; 4. Ocampo et al., Cell 2016; 5. Alle et al., Biorvx 2021.

OC04 — Novel Approaches for Single-Cell and Cost-Effective Epigenetic Age Profiling

Alexandre Trapp¹, Csaba Kerepesi¹, Vadim Gladyshev¹ (1. Harvard Medical School — Boston (United States))

Backgrounds: How can we track biological age? This is perhaps one of the most fundamental questions in our field. In order to study aging, when it begins, and how it progresses, we desperately need methods that enable robust assessment of biological age in tissues of mammalian organisms. Since their inception in the last decade, epigenetic clocks have materialized into some of the most accurate trackers of biological aging. Clocks have been developed for humans, mice, and now many other mammalian species. These machine-learning based models have proved useful not only for tracking aging in adult populations, but also to evaluate and validate putative longevity and rejuvenation interventions. In effect, biological age, as measured by various clock models, has emerged as one of the most pivotal endpoint readouts for aging studies, reinforcing their increasing importance in the longevity field. However, current clock methods are inherently limited by two factors: 1) their reliance on bulk samples, masking the heterogeneity among cells and 2) their extremely high cost and relatively low throughput. Objectives: To address these fundamental limitations, we report the development of scAge, a novel platform that enables both single-cell epigenetic age profiling, as well as extremely cost-effective bulk age profiling. With this framework, we allow a new layer of resolution for biological age profiling, while simultaneously permitting democratization of age assessment with low-cost approaches. Methods: We developed scAge, a unique statistical platform that leverages single-cell or bulk shallow methylation sequencing to enable accurate epigenetic age estimations. Previous epigenetic clocks have all relied on assembling dense methylation matrices that are easily amenable to conventional elastic-net regularization approaches. However, methylation matrices from single cells (or shallow bulk data) produce a very distinct modality: primarily sparse, binary methylation values. Importantly, these approaches lead to discordant read coverage across different cells/samples, meaning that different CpGs are covered in each profile. These limitations make conventional approaches inapplicable to single-cell or shallow bulk data. To address these challenges, our platform employs a multi-step process that is particularly well-suited to this type of sparse, binary data. Inherently, we leverage age-associated changes in CpG methylation in highly-covered bulk data, which serve as reference linear models that detail how methylation is impacted by age. Next, we intersect single-cell/shallow sample profiles with this reference, picking exclusively sites that are covered in both the reference and the sample of interest. These sites are then filtered and ranked based on their association with age in the bulk data. For each selected site, we compute the probability of observing the methylation status (1, methylated or 0, unmethylated) for a wide range of ages. We harness this algorithm over many age-associated CpGs, enabling us to construct a comprehensive age likelihood distribution for each sample. Using maximum likelihood estimation, we can then directly compute the epigenetic age of a cell or shallow sample. Results: We validated our scAge approach on several single-cell datasets, as well as downsampled RRBS mouse datasets. In single young and old hepatocytes, scAge achieves remarkable predictive power (r = 0.95). We also examined muscle stem cells, which were previously shown by pseudo-bulk approaches to display minimal epigenetic aging; our new high-resolution predictions corroborate these earlier findings, suggesting attenuated epigenetic aging in murine muscle stem cells. We also used our method to uncover a stratified rejuvenation event occurring during mouse embryogenesis. Previous work from our group has revealed that early embryogenesis is characterized by epigenetic rejuvenation and damage clearance, but the specificity of this process across different embryonic cell lineages remained enigmatic. With scAge, we show that a rejuvenation event indeed occurs during embryogenesis, but that rejuvenation is stratified based on the cell lineage. Notably, supportive extra-embryonic cells do not appear to undergo rejuvenation, seemingly because they do not become part of the final embryo. Additionally, our method is amenable to assessment of epigenetic age from extremely shallow sequencing of bulk samples. We show that our tool can robustly track (r > 0.9) aging in shallow bulk samples (with only 10,000 reads/sample, representing a 1000-fold reduction in per-sample sequencing costs). We validated our platform over several datasets, and additionally observed that our method reports with high confidence the attenuated aging effect of caloric restriction and the rejuvenation effect of iPSC reprogramming. Conclusion: Overall, we present here a new platform that will enable population-scale assessment of epigenetic age, both at the highest resolution (individual cells), and in a very cost-effective manner. With this method, it will be possible to evaluate the cell and lineage-specific effects of several new and existing longevity interventions, while simultaneously enabling democratization of biological age assessment.

OC05 — Necroptosis Inhibition Counteracts Axonal Degeneration, Cognitive Decline and Key Hallmarks of Aging, Promoting Brain Rejuvenation

Macarena Arrázola^1,2, Matías Lira³, Gabriel Quiroz², Somya Iqbal⁴, Rachel A. Kline⁴, Waldo Cerpa³, Thomas M. Wishart^4,5, Felipe Court^1,2,6 (1. Center For Integrative Biology, Universidad Mayor — Santiago (Chile), 2. Geroscience Center for Brain Health and Metabolism (GERO) — Santiago (Chile), 3. Departamento de Biología Celular y Molecular, Facultad de Ciencias Biológicas, Pontificia Universidad Católica de Chile, — Santiago (Chile), 4. The Roslin Institute, University of Edinburgh — Edinburgh (United Kingdom), 5. Centro de Excelencia en Biomedicina de Magallanes (CEBIMA), Universidad de Magallanes, — Punta Arenas (Chile), 6. Buck Institute for Research on Aging, — Novato, Ca (United States))

The current rise in human life expectancy is not precisely accompanied by an equivalent increase in ‘healthspan’. The impact of age on brain function is unquestionable, being the main risk factor for the development of neurodegenerative diseases and cognitive disabilities. As a fundamental structure for human cognition, the hippocampus is particularly vulnerable to the deleterious effects of aging. Microstructural changes at the synaptic level are correlated with learning and memory impairment during aging. White matter abnormalities and axonal degeneration (AxD) have been identified in aged brains of diverse species, particularly in the hippocampus and strictly correlated with impaired memory performance in humans. Due to the importance of axonal integrity on hippocampal function and progression of cognitive decline in the elderly, it is imperative to determine the mechanism of AxD during aging. We were pioneers in identifying necroptosis as the mechanism involved in mechanical and chemical-induced AxD. Necroptosis is an alternative form of programmed cell death triggered by the tumor necrosis factor of cytokines under caspase-8 inhibitory conditions and characterized by a necrotic-like and pro-inflammatory response. Receptor-interacting kinase 1 (RIPK1) recruits and phosphorylates RIPK3 which in turn phosphorylates the mixed lineage kinase domain-like protein (MLKL). MLKL oligomerizes and translocate to the plasma membrane, disrupting membrane integrity followed by the release of cellular components, an exacerbated inflammatory response, and cell death. Age-associated increase in low-grade sterile inflammation is one of the ‘seven pillars of aging’ contributing with the development and progression of age-associated diseases. Considering the inflammatory response triggered upon necroptosis activation, the role of this pathway in the progression of normal aging has been largely overlooked. Recent studies have depicted the importance of necroptosis in the aging of the mouse male reproductive system and the epididymal white adipose tissue. In the context of brain aging, several age-related neurodegenerative conditions with prominent AxD and neuroinflammation as common features have shown increased necroptosis activation in the brain, associated with functional impairment. However, the involvement of necroptosis in the progression of normal brain aging and its cognitive consequences remain unexplored. We evaluated the role of necroptosis in the progression of AxD in the hippocampus and its impact in the brain function during aging. Necroptosis increased in hippocampal subfields with evident AxD in aged mice. Loss of Mlkl was sufficient to delay age-related AxD in the hippocampus, a youthful phenotype also displayed at the synaptic and functional level. Restored synaptic transmission and facilitation were accompanied by improved learning and memory in aged mice deficient for Mlkl. Short-term inhibition of RIPK3 in aged mice demonstrated to be extraordinarily effective on reverting AxD and hippocampus-dependent functional impairment. Finally, an unbiased quantitative proteomic analysis demonstrated that necroptosis inhibition improved the aged hippocampal proteomic profile, restoring the levels of key protein pathways associated with aging hallmarks, including a subset of biofunctions associated with brain rejuvenation. Our study demonstrates that necroptosis contributes to the age-associated deterioration of axons and their function, affecting hippocampal neuronal connectivity and cognitive function of aged mice. Finally, our pharmacological approach demonstrated that necroptosis inhibition promotes brain rejuvenation in aged mice, proposing necroptosis as an interesting target for the future development of geroprotectors to treat age-related brain disabilities.

0C07 — Short Senolytic or Senostatic Interventions Rescue Progression of Radiation-Induced Frailty and Premature Ageing in Mice

Edward Fielder¹, Satomi Miwa¹, Thomas Von Zglinicki¹ (1. Newcastle University — Newcastle Upon Tyne (United Kingdom))

Backgrounds: Cancer survivors suffer from progressive frailty, multimorbidity and premature morbidity. We hypothesize that therapy-induced senescence and senescence progression via bystander effects is a significant cause of this premature ageing phenotype. Aims: 1. Is a short adjuvant anti-senescence intervention with either a senolytic or the senostatic metformin sufficient to block long-term progression of radiation-induced frailty and disability in a pre-clinical setting? 2. Is a short senolytic intervention at a later date, after establishment of premature frailty, still effective? 3. How does metformin work as a senostatic at therapeutically achievable concentrations? Methods: Male mice were sub-lethally irradiated at 5 months of age and treated (or not) with either a senolytic drug (Navitoclax or dasatinib + quercetin) for 10 days or with the senostatic metformin for 10 weeks. Follow up was for one year. Progression of frailty, neuromuscular coordination, short-term spatial memory tumour incidence and liver damage were assessed as described (Fielder et al. Mech Ageing Dev 180 (2019) 63–69). Effects of metformin over a wide concentration range (0.1 – 5mM) on cellular stress signalling pathways, mitochondrial respiration and complex I activity, ROS production, release of SASP factors, mitophagy and NOX4 activity were measured in senescent human fibroblasts in vitro. Results: Treatments with either senolytic commencing within a month after irradiation effectively reduced frailty progression (p<0.05) and improved muscle (p<0.01) and liver (p<0.05) function as well as short-term memory (p<0.05) until advanced age with no need for repeated interventions. Senolytic interventions that started late, after radiation-induced premature frailty was manifest, still had beneficial effects on frailty (p<0.05) and short-term memory (p<0.05). Metformin was similarly effective as senolytics. At therapeutically achievable concentrations metformin acted as a senostatic neither via inhibition of mitochondrial complex I, nor via improvement of mitophagy or mitochondrial function, but by reducing non-mitochondrial ROS production via NOX4 inhibition in senescent cells. Conclusions: Our study suggests that the progression of adverse long-term health and quality-of-life effects of radiation exposure, as experienced by cancer survivors, might be rescued by short-term adjuvant anti-senescence interventions.

OC08 — Selective Targeting of Senescent Cell Mitochondria by Mitochondrial Uncouplers Enhances Specificity and Sensitivity of Senolytics

Satomi Miwa¹, Edward Fielder¹, Abbas Ishaq¹, Thomas Von Zglinicki¹ (1. Newcastle University — Newcastle Upon Tyne (United Kingdom))

Backgrounds: Mitochondrial dysfunction, characterised by decreased respirator capacity, low membrane potentials and increased reactive oxygen species production, is one of the most robust and common features of senescent cells including replicative, damage and oncogene induced senescence. Thus, the mitochondria in senescent cells are particularly sensitive to mild uncoupling by a low dose of mitochondrial uncoupler agent, which in young normal cells does not show significant effects. Drugs that selectively kill senescent cells (senolytics) have emerged as novel therapeutic modalities for a wide range of age-associated diseases in pre-clinical studies, and classic senolytics are now in about a dozen of clinical trials. The main limitations for translation are frequent dose-dependent side effects, limited specificity, and importantly the lack of rigorous evaluations of the administration protocols. Objectives: We reasoned that low membrane potentials in senescent cell mitochondria are particularly sensitive and might provide specific functional targets for senescent cells. Many known senolytic agents such as Bcl-2 family inhibitors including Navitoclax induce mitochondria-dependent apoptosis, through mitochondrial membrane depolarisation and cytochrome c release, to kill senescent cells. We tested the hypothesis that mild uncoupling of mitochondria by a low dose of an uncoupler agent combined with Bcl-2 family inhibitor senolytic may enhance specificity and sensitivity of senolytic action. We tested the hypothesis both in vitro and in vivo in a pre-clinical mouse model of radiation-induced premature ageing. Here we tested if a significantly lower dose of senolytic, when combined with a low dose of mitochondrial uncoupler agent, was sufficient to bring about the same beneficial effects as a standard dose of senolytic. Methods: Using an in vitro system, young and senescent human dermal fibroblasts were treated with various combinations of different concentrations of Bcl-2 family inhibitor senolytics (Navitoclax, A1331852, EGCG) and common mitochondrial uncouplers (FCCP, CCCP and BAM15). Cells were treated with a given treatment for 3 days and the numbers for each population of cells were recorded by microscopy, and the changes in their cell numbers are recorded. In vivo, a mouse model of premature ageing caused by radiotherapy was used. A sub-lethal dose of radiotherapy with 3 doses of 3 Gray X-ray irradiation over one week was conducted. A combination of a mitochondrial uncoupler with a proven safety record in mice (BAM15) and a low dose of Navitoclax (0.5mg/kg/day) was tested to determine if it would be effective in alleviating premature ageing phenotype in the mice, compared with a substantially higher dose of Navitoclax (5mg/kg/day). Note, a typical dose used in literature is even higher, 50mg/kg/day. Uncoupler alone, and a low dose of Navitoclax alone, were also tested as controls. Frailty index scores, neuromuscular function (hanging wire test and rotarod) and cognitive function (short-term memory by Y-maze test), were assessed using protocols described previously (Mech Age Dev, 2019, 180, 63–69). Results: Typically, in vitro, 5 – 10µM of Navitoclax was required to induce a significant amount of cell death selectively in senescent cells. However, we found that inclusion of small doses of uncouplers (e.g. FCCP 1.5µM) dramatically lowered the effective dose of Navitoclax, such that Navitoclax doses of around 10nM (i.e. 2–3 orders of magnitude lower) cause significant cell death by apoptosis in senescent but not normal, young cells. Uncouplers alone do not significantly affect cell death in either senescent or young cells. Different combinations of other tested senolytics and ucnouplers essentially showed the same results. In the in vivo mouse model, we followed the progression of frailty scores of the mice with different treatment groups over 7 months after radiotherapy. Single treatments with either low dose of Navitoclax or BAM15 did not reduce frailty progression nor did they change any parameters we tested compared with vehicle controls. In contrast, both high Navitoclax and the combination of low Navitoclax and BAM15 slowed frailty progression significantly and equally well over vehicle controls. Furthermore, short-term memory and neuromuscular function were also similarly improved. Conclusion: We identified the capability of a low dose of a mitochondrial uncoupler to depolarise mitochondrial membrane potnetials in senescent cells to a critically low level, while having little effect on mitochondria in normal cells, as a tool to specifically and sensitively target senescent cells for destruction. When combined with a senolytic agent, the mitochondrial uncoupler can aid the senolytic agent’s selective apoptosis of senescent cells. It enables the effective dose of a potentially toxic senolytic agent to be significantly reduced without adversely affecting efficacy, which decreases the risk of unwanted side effects from the senolytic agent during treatment of senescence associated diseases or disorders. We demonstrated that dysfunctional mitochondria in senescent cells, due to their distinct differences from the mitochondria in young normal cells, are particularly convenient and powerful functional target for killing senescent cells with a high specificity and sensitivity, which offers novel approaches for further developments of senescent targeted therapies.

OC09 — Biophytis BIO101 in Sarcopenia: Results of the Phase 2 Sara-Int Study

Cendrine Tourette¹, Waly Dioh¹, Carole Margalef¹, Anait Azbekyan², René Lafont^1,3, Pierre Dilda¹, Jean Mariani^1,4, Suzanna Del Signore⁵, Sam Agus², Rob Van Maanen¹, Stanislas Veillet¹ (1. Biophytis Sa — Paris (France), 2. Biophytis Inc — Cambridge (United States), 3. Sorbonne Université, CNRS — Institut de Biologie Paris Seine (BIOSIPE) — Paris (France), 4. Sorbonne Université, CNRS — IBPS (Biological Adaptation & Aging) (France), 5. Bluecompanion Ldt — London (United Kingdom))

Backgrounds: Sarcopenia is a geriatric condition characterized by a progressive loss of muscle mass and function, having high personal, social and economic burdens when untreated. Sarcopenia increases risk of falls and fractures; impairs ability to perform activities of daily living; is associated with cardiac and respiratory disease and cognitive impairment; leads to mobility disorders; and contributes to lowered quality of life, loss of independence or need for long-term care placement, and death. It is recognized as one of the five pillars of frailty. As of today, to our knowledge, only exercise and nutrition interventions seem somewhat effective interventions. Objectives: SARA-INT study is a Phase 2 study to develop a viable option to treat community-dwelling seniors suffering from age-related sarcopenia, including sarcopenic obesity. Methods: SARA-INT is a randomized double-blind three-arm study (BIO101 175 mg bid / BIO101 350 mg bid / placebo) with planned treatment duration of 6 Months; due to COVID-related measures, 49 patients continued up to 9 months of treatment. Main eligibility criteria for sarcopenia were meeting FNIH criteria and Short Physical Performance Battery (SPPB) score ≤ 8/12 in men and women aged ≥ 65 years. Primary analysis was the gait speed from the 400-meter walking test (400MWT) at month 6/9 in the FAS with secondary analyses at other timepoints, secondary endpoints were other physical activity assessments, muscle strength, muscle mass and Patient Reported Outcomes (PROs). Results: 233 participants were randomized in the study, 232 and 156 participants were included in the Full Analysis Set (FAS) and Per-Protocol (PP) populations, respectively. Due to COVID-19 pandemic, end-of-treatment assessments are missing for approximately half of the participants, impacting the treatment effect detection. In the primary analysis (at month 6/9 in the FAS population) of the primary parameter, the improvement in 400MWT compared to placebo was not statistically significant (0.0363 (0.03098) m/s and 0.0385 (0.02985) m/s in the BIO101 175 mg and 350 mg groups, p=0.2437 and p=0.2000, respectively). BIO101 350 mg bid treatment after 6 months showed a clinically relevant improvement in the 400MWT of 0.07 m/s in the FAS population (not significant) and of 0.09 m/s in the PP population (nominally statistically significant, p=0.008); this is close to the Minimal Clinically Important Difference (MCID) in sarcopenia (0.1 m/s). BIO101 350mg bid treatment effect on the 400MWT is confirmed in pre-defined sub-populations at higher risk of mobility disability such as slow walkers, obese and those with chair stand sub-score ≤2 from SPPB; trends were observed with other independent endpoints. BIO101 showed no difference in adverse events or safety laboratory parameters versus placebo (), and no severe adverse event associated with BIO101 treatment. Conclusions: After 6 to 9 months of treatment, BIO101 at 350 mg bid showed promising results with a clinically relevant improvement in the 400MWT gait speed, the primary endpoint of the study, confirmed in sub-populations at higher risk of mobility disability. BIO101 showed a very good safety profile at the doses of 175 and 350 mg bid. Biophytis is preparing to start a phase 3 program with BIO101 at 350 mg bid in 2022, targeting a similar patient population. Conflicts of interests: CT, WD, CM, RL, PD, RvM and SV are employees of Biophytis SA, AZ, and SA are employees of Biophytis Inc., JM is president of the Scientific Advisory Board of Biophytis, SDS is employee of BlueCompanion Ltd

0C10 — The Effect of Methionine Restriction on Endurance Performance in Mice

Charlotte G. Mann¹, Thomas Agius², Alban Longchamp², Michael R. Mac Arthur¹, Katrien De Bock¹, Sarah J. Mitchell¹ (1. Eidgenösschiche Technische Hochschule — Zürich (Switzerland), 2. Ecole Polytechnique Fédérale De Lausanne — Lausanne (Switzerland))

Backgrounds: Endurance exercise is linked to maintenance of metabolic fitness by lowering body weight, increasing insulin sensitivity and glucose tolerance, leading to improved health span. Endurance exercise leads to increased mitochondrial quality and is one of the only post-developmental, nonpathogenic stimuli to cause muscle neovascularization. Interestingly, there is overlap in the phenotypes of endurance exercise and dietary methionine restriction (MR). During MR, intake of the amino acids methionine and cysteine is limited to 0.1% and 0% respectively. The physiological effects of MR include reduced bodyweight, improved insulin sensitivity and glucose tolerance, leading to an overall improvement in metabolic homeostasis. Our lab has previously reported that MR increases skeletal muscle angiogenesis through activation of GCN2/ATF4 to improve recovery following hindlimb ischemia. However, the effect of MR on exercise endurance in rodents has not been shown. We hypothesized that one week of MR would be sufficient to increase endurance exercise performance in sedentary mice through the induction of metabolic changes in the muscle. Objectives: The aim of this study was to investigate whether MR can act as endurance exercise mimetic and improve endurance exercise capacity in sedentary rodents. Methods: Sedentary, 13 weeks old male C57BL6/J mice, were randomly assigned to either a semi-purified control diet (CON) or a methionine restriction (MR) diet (0.1% methionine, 0% cysteine). After 7 days, mice were subject to a treadmill maximal performance test. Time and distance until exhaustion were recorded. Energy expenditure (EE), respiratory exchange ratio (RER) and spontaneous activity were assessed using the Sable System Indirect Calorimetry System. A vO2max treadmill test was performed using an enclosed treadmill connected to an indirect calorimeter to test substrate utilization during maximal running performance. To understand the molecular mechanisms underlying the endurance exercise phenotype, bulk RNA sequencing and lipidomic analysis of soleus (SOL) and extensor digitorum longus (EDL) muscle groups was performed in CON and MR mice after 7 days of dietary intervention. Results: 7 days of MR feeding lead to a significant increase in endurance exercise capacity compared to CON (956.5 m vs. 634.8 m, p=0.0093). Indirect calorimetry measurements showed significantly higher EE in MR mice in the light cycle. RER was significantly lower in MR mice at 7 days, compared to CON mice. There was no significant difference between overall movement in MR vs CON mice as measured by infrared beam break or spontaneous wheel running after 7 days of dietary intervention. Pathway over-representation analysis of bulk RNA sequencing data showed significant upregulation of pathways associated with metabolic profile switching and fatty acid catabolism in EDL compared to SOL, 7 days on MR. In the EDL, MR induced a switching of glycolytic fast twitch fibres to more oxidative slow twitch fibres. Concurrently, glycolytic muscle fibres showed a gene expression profile indicative of increased fatty acid import and TCA cycle. These findings were confirmed at the mRNA and protein level by RT-qPCR and western blotting. Independent of muscle type, MR led to an increase in mitochondrial protein expression shown by pathway analysis of RNA sequencing data, EMI imaging and western blotting. Conclusion: 7 days of a MR diet in male mice is sufficient to significantly increase exercise performance. This phenotype is likely driven by increased OXPHOS and b-oxidation leading to prolonged usage of fatty acids as main energy source. The lengthened dependency on fatty acids rather than stored glycogen and glycolysis allows for longer endurance performance leading to a “pseudo-trained” state of the muscle in sedentary rodents upon 7 days of MR feeding. Taken together, these data suggest an overlap between the molecular phenotypes of MR and endurance exercise, and that MR can act as an exercise mimetic.

0C11 — Early-Adulthood Spike in Protein Translation Drives Proteostatic Dysfunction and Age-Related Decline Via Juvenile Hormone/Germline Stem Cell Signaling

Harper Kim¹, Danitra Parker¹, Madison Hardiman¹, Erin Munkácsy², Aric Rogers³, Yidong Bai⁴, Steven Austad¹, James Mobley¹, Andrew Pickering¹ (1. University Of Alabama At Birmingham — Birmingham (United States) — Birmingham (United States), 2. Barshop Institute For Longevity And Aging Studies-San Antonio (united States) — San Antonio (United States), 3. Mdi Biological Laboratory — Bar Harbor (United States), 4. Barshop Institute For Longevity And Aging Studies — San Antonio (United States))

Backgrounds: Increasing evidence suggests that high protein translation (PT) may contribute to age-related functional decline. Interestingly, PT is high only during young adulthood in multiple animal species, including humans. PT sharply declines thereafter, staying at low basal levels throughout mid-old ages. However, elevated PT during early adulthood has not been a major focus of aging studies due to the implicit assumption that molecular events that drive aging should occur at old ages. In addition, the age-dependent decline in PT has been regarded as a passive byproduct of aging although this has not been directly tested. Objectives: To investigate how dynamic changes in PT over time regulate the onset of aging. Methods: We used multiple (pharmacologic and genetic) approaches to transiently modify PT in different life stages of Drosophila (early adulthood, late adulthood, and the entire adult life) and investigated how this impacts their lifespan and healthspan. We used the mass spectrometry (LC/ESI-MS/MS)-driven proteomics to identify several specific proteins synthesized in early adulthood that trigger age-related proteostatic dysfunction and drive aging. Results: We propose the early-adulthood spike in PT as a novel driver of aging. In Drosophila, we show that blocking the PT spike during the critical window of early-adulthood improves life-/health-span and prevents age-related accumulations of insoluble protein aggregates. Inducing the early-life PT spike in long-lived dwarf flies abolishes their longevity/proteostasis advantage. In addition, the early-life PT rise reliably predicts future lifespan across different fly strains. Further, fly models of Alzheimer’s disease and Parkinson’s disease showed a much greater surge in PT during early adulthood. Proteomics (LC/ESI-MS/MS)-guided mechanistic investigations revealed that during the early-life PT rise, juvenile hormone triggers proteostatic dysfunction and drives aging via aggregation-prone large lipid transfer proteins (LLTPs). We show that the early-life PT spike also stimulates germline stem cell proliferation to increase fertility, which transcriptionally silences stress resistance genes essential for proteostasis maintenance and drives aging. Our findings indicate that PT is thus rapidly suppressed after early adulthood/fertility peak in order to alleviate proteostatic burden, slow down aging, and optimize life-/health-span. Conclusion: The transient PT spike during the critical window of early adulthood exerts long-lasting negative impacts on aging trajectories and proteostasis in later life. The early spike in PT may represent an antagonistic pleiotropic effect, enhancing reproductive fitness early in life at the expense of proteostasis in later life. Our findings suggest that age-dependent decline in PT may not simply be a passive byproduct of aging as previously thought but rather an adaptive response to promote healthy aging. Our work provides a new theoretical framework for understanding how lifetime PT dynamics regulate the onset of aging.

0C12 — Differences in Blood-Based Inflammatory Profile According to Longitudinal Intrinsic Capacity Trajectories in Community-Dwelling Older Adults

Wan-Hsuan Lu¹, Emmanuel Gonzalez-Bautista¹, Philipe De Souto Barreto¹ (1. Toulouse University Hospital (chu Toulouse) — Toulouse (France))

Backgrounds: An important element for the Geroscience hypothesis is the relationship between the biology of disease and aging. However, the biology of functional capacity is increasingly recognized as a relevant feature to foster healthy aging even in non-diseased individuals. Evidence about the profile of aging biomarkers according to functional status in older people, particularly with a longitudinal scope, is still lacking. One option to approach the functional trajectories of older people is by using repeated measures of intrinsic capacity (IC). IC has been defined by the World Health Organization as the aggregate of physical and cognitive functions of an individual during the aging process. IC comprehends several domains, including cognition, locomotion, psychological and vitality. Different instruments have been used to measure individual performance in these domains, but there is no consensus about a standard manner to measure the global IC. Chronic inflammation has been recognized as one of the key drivers of aging and functional decline. Both classical inflammatory markers — such as C-reactive protein (CRP), interleukin 6 (IL6), tumor necrosis factor receptor type 1 (TNFR1) — and novel biomarkers related to inflammation — such as monocyte chemoattractant protein 1 (MCP1) and growth differentiation factor 15 (GDF15) — have been associated with functional outcomes in basic and epidemiological research. However, previous findings have focused on only one functional domain (i.e., cognition, physical performance), and very few of them have presented longitudinal trajectories. Objectives: The objective of our study is a) to identify the longitudinal IC trajectories in older adults and b) to compare the inflammatory biological profile of individuals in extreme IC change with those in a stable IC status over time. Methods: This is a secondary analysis of the Multidomain Alzheimer Preventive Trial (MAPT). We included 1,380 participants aged 70 and over with four annual IC assessments. The time point of collecting plasma inflammation-related biomarkers, including CRP, IL6, TNFR1, MCP1, GDF15 was defined as the baseline. The IC was calculated as the summary performance of the following domains: cognition (cognitive composite scores), locomotion (Short Physical Performance Battery), psychological (Geriatric Depression Scale 15 items) and vitality (handgrip strength). The IC score was scaled from 0 to 10, with a higher score indicating better performance. We first used linear mixed-effect models to estimate each participant’s IC trajectory over four years. Based on this model, we obtained an intercept (baseline IC value) and a slope (degree of decline/improvement) for each participant. Then we categorized participants according to their IC trajectory slopes (flat, decrease or increase) and examined the differences of plasma inflammatory biomarkers among those categories using multilinear regressions. Results: The overall MAPT IC trajectory tended to be flat-to-very slight decline over four years (−0.3 points per year on average). We grouped participants according to their slopes: 61% (n=842) with flat-to-slight IC decline (slope within −0.6 to 0 points per year), 19% (n=262) with improved IC trajectory (slope >0), and 20% (n=276) showed accelerated IC decline with a slope three times steeper than the average (−0.9 IC points per year on average). Regarding plasma biomarkers, we observed significant associations of CRP, IL6, TNFR1 and GDF15 with baseline IC after adjusting for demographic confounders. We found significant differences in GDF15 between participants with improved IC trajectory and those with flat-to-slight decline trajectory, but no difference in plasma biomarkers between those with accelerated IC decline and those with flat-to-slight decline trajectory. In other words, lower levels of GDF15 were significantly associated with higher baseline IC and an improvement of IC over time. Conclusion: We observed that IC tended to be stable or decreased slowly for the majority of our community-dwelling cohort. About 1/5 subjects improved their IC after the four-year follow-up. Cross-sectional associations between inflammatory biomarkers and IC might be explained by an acute event triggering both inflammation and the loss of function. Lower baseline GDF-15 levels were associated with having a trajectory of improved IC, which might serve as an indicator of the better physiological reserve.

OC13 — Epigenetic Clocks Reveal Reversible Changes in Biological Age in Response to Stress

Jesse Poganik¹, Vadim Gladyshev¹ (1. Brigham And Women’s Hospital, Harvard Medical School — Boston (United States))

Background: In contrast to chronological age, biological age is dynamic and influenced by numerous intrinsic and extrinsic factors. However, the extent to which biological age undergoes reversible changes over relatively short time periods throughout life remains unexplored. Objectives: We sought to understand how biological age fluctuates in response to stressful stimuli and to identify situations in which biological age is transiently increased. Methods: We leveraged DNA methylation (DNAm) clocks to explore the concept of reversible changes in biological age. Using a combination of mouse models, human clinical samples, and analysis of publicly available human methylation data, we applied several DNAm biomarkers to longitudinally assess biological age changes in response to stressful stimuli and following recovery. Results: We found that reversible biological age changes can be modeled experimentally using heterochronic parabiosis in mice. This procedure induced a biological age increase that resolved following surgical detachment. We further identified several situations including emergency trauma surgery, pregnancy, and COVID-19 disease, in which a transient, reversible increase in biological age is induced. Conclusion: Our data indicate that biological age undergoes rapid increases in response to stressful stimuli that are reversed following recovery from stress. These data highlight a new layer of biological age dynamics and suggest that increased biological age caused by stress may be an actionable target for future anti-aging interventions.

OC14 — Cryptic Nature-Phenomenal Animalesque Human Torpor: Forever-Young Inner Fountain-Of-Youth?

Sébastien Murat¹ (1. Charles Sturt University — Bathurst (Australia))

Background: Dormancy, termed torpor in birds in mammals, i.e., profound metabolic downregulation below the approximately 36°C-normothermic diurnal circadian rhythm basal metabolic rate (BMR), confers very different animals (e.g., hummingbirds, dormice, bats, primates) quintessential broad-spectrum shielding against an unmatched variety of time-dose dependent environmental threat agents, including acute & chronic infectious & non-infectious diseases as well as lifestyle diseases, e.g., MDR-bacteria, RNA-viruses, inflammation, neurodegeneration, obesity. This low, slow & cool, fat-burning, caloric restricted (fasted) & autophagic physiological state affords more time for body system repairs since physiological times are basically the simple inverse of mass-specific metabolic rate (Lindsted, & Calder III, 1981); cold-shock repair proteins (e.g., RBM3) are upregulated & the apparent effective physiological insult exposure duration is relatively reduced. This capability allows torpid critters phenomenal feats, e.g., scarless wound-healing (Iaizzo et al., 2012), more prolonged healthspan (Blanco, & Zehr, 2015), stalled aging (Pinho et al., 2021) & limits of longevity dictated by purely size-related allometric BMR law far exceeded (Al-Attar, & Storey, 2020). Indeed, there is a clear association between the MR-reducing fall in core body temperature & lifespan (Keil, Cummings, & de Magalhaes, 2015). Moreover, the blood serum of torpid mammals imparts at least some of the qualities to both autologous & heterologous biologics, e.g., human stem-cells in torpid serum experience slowed aging, including inflammaging (Berg von Linde et al., 2021). Evidently little appreciated, a latent human diurnal (nocturnal) animalesque human torpor (HT) capacity of certain physiological outliers, e.g., Australian Central Desert Aboriginals is known (Hicks, & Matters, 1933; Scholander et al., 1958; Hammel et al., 1959; LeBlanc, 1975), which may account for anecdotal reports of prolonged, seemingly non-aging good-health &, suspected, extreme lifespan (Giles, 1873; Blyton, 2009). Objectives: It is possible that HT blood serum might likewise express some of these animalesque capabilities. Presented here, a case-study glimpse-insight of the HT immuno-metabolic latent potential as it relates to inflammation by various immuno-challenges. Methods: A (trained) adult torpor-capable human volunteer was in/ex-vivo challenged with various inflammation-& fever- or fever-like-inducing analogs, e.g., known-potent pro-inflammatory & pyrexic bacterial, Escherichia coli, lipopolysaccharide (LPS) endotoxin, whole-body superficial & deep-core electromagnetic resonant radio-frequency diathermy (heating). SubBMR, anapyrexia & anti/pro-inflammation were repeat-measured (pooled triplicates, mean ± s.d.) via indirect whole-body O2-respirometry (Metalyzer 3B, CORTEX Biophysik, Germany, ± 5 %), ocular inner canthum (brain temperature tunnel) thermal imaging thermometry (Compact Pro, SEEK, USA, ±0.1°C), ingestible pill thermometry (e-Celsius, BodyCap, France, ± 0.2 °C), cerebral magnetic resonance spectroscopy thermometry (1.5 Tesla, Achieva, Philips, The Netherlands, 0.4 °C), & ELISA cytokine assay (IL-8, IL-6, TNF-α, MILLIPLEX, Merck-Millipore, Germany, CV < 7 %). Results: HT expressed profoundly & ultra-fast, minutes-fast, 35 % of BMR in some 15 minutes. This was associated with a minor reduction in core body temperature & which were highly selective, prioritized, towards more vital organs before stabilizing: 0.9±0.4°C (gastrointestinal), −2.0 to −3.5±0.5°C (cerebral), & 3.0±0.1°C (inner canthum). SubBMR rates could be realized even at elevated, 26°C-ambient, tropical temmperatures, termed, hot torpor. Compared to normal (control) human blood serum, ex-vivo LPS-challenged (10 ng/ml) blood mono/lymphocytes remained viable & showed little-to-no pro-inflammatory IL-6 & TNF-a pro-inflammatory cytokine response, whereas anti-inflammatory IL-8 was upregulated. Likewise, human heterologous macrophages cultures in 10% torpor serum at 37°C remained viable & showed little-to-inflammation. Follow-up intravenous LPS administration (2 ng/kg) in torpor likewise proved anti-inflammatory & asymptomatic. Conclusions: In this HT case-study, torpor manifested with a minor temperature-dependent contribution; Q10 effects might have contributed no more than 20–25% to the overall subBMR suppression since MR falls by no more than 6–8% per °C. This is in line with observations made in large bradymetabolic mammals, e.g., hibernating bears (Tøien et al., 2011) & diving seals (Scholander, Irving, & Grinnell, 1942). In regards the inflammatory response, it expressed no differently to what has already been observed in various other torpid mammals, including, primates (Canale, & Henry, 2006). Unlike other animals, however, HT can be triggered consciously, on-demand. Though N-of-1-limited, this study tentatively suggests that the outlier HT phenotype can suppress inflammation. Moreover, as in other torpid mammals, HT may generate blood-borne biologics that may confer broad biomedical shielding, including possibly against inflammaging. Furthermore, this serum may contain biologics that can preserve & extend the no-cold-chain shelf-life tolerance of various biologics, possibly including, e.g., cell, tissue or organ cultures. The enigmatic biologic(s) responsible for this remarkable & largely temperature-independent immuno-metabolic modulating capability remain to be identified. Given the considerable supporting animal evidence, the concept of HT as an (inner) fountain-of-youth & torpor-in-a-bottle have the potential to perhaps offer a more effective & holistic anti-aging strategy than the more invasive state-of-the-art pollution dilution solution (Mehdipour et al., 2020).

OC15 — Sexual Identity of Enterocytes Regulates Rapamycin-Mediated Intestinal Homeostasis and Lifespan Extension

Mary Yu-Xuan Lu¹, Jennifer C. Regan^2,3, Enric Ureña², Ralf Meilenbrock¹, James H. Catterson², Disna Kißler¹, Linda Partridge^1, (1. Max Planck Institute For Biology Of Ageing — Cologne (Germany), 2. Institute Of Healthy Ageing, Department Of Genetics, Evolution And Environment, University College London — London (United Kingdom), 3. University of Edinburgh — Edinburgh (United Kingdom))

Background: Pharmacological intervention of ageing by rapamycin and other compounds presents a promising route for the extension of lifespan and delay of ageing-related pathologies, including intestinal cancers. Many of these geroprotective compounds exhibit sexual dimorphic responses. In mice, rapamycin shows remarked stronger lifespan extension in females than in males and the sexually dimorphic effects on ageing pathologies, specifically cancer incidence and type. However, the physiological bases for these dimorphic responses to geroprotective compounds are not well-understood. Objectives: Understanding how sex influences both development of age-related disease and responses to treatment will be key to move forward with the development of geroprotective therapeutics. Methods: In our study, we used Drosophila melanogaster, which shows a strong lifespan extension in females treated with rapamycin, as a tractable system for understanding tissue-specific contributions to ageing dimorphisms, and dimorphic responses to anti-ageing therapeutics. We performed physiological, genetic and molecular experiments to systematically assess different responses between females and males to rapamycin treatment. In addition, we performed qPCR and 16S sequencing to measure if the load and the composition of microbiome are sexually different and in response to rapamycin treatment. Results: We show that rapamycin treatment in Drosophila extends lifespan in females but not in males, regardless of their genetic background. Both sexes show a significant reduction in p-S6K levels in response to rapamycin, and there is no significant interaction between sex and treatment, which suggests the dimorphic response of lifespan to rapamycin is therefore likely not due to sex differences in suppression of mTORC1 signalling by the drug. Female-specific, age-related gut pathology and impaired intestinal barrier function are both markedly slowed by rapamycin treatment. Interestingly, the size and composition of the microbiome is sex- and age-dependent but does not change significantly upon treatment of rapamycin. These suggest that the sexually dimorphic effects of rapamycin treatment on gut pathology and barrier function are not achieved through remodelling of the microbiome. We further show that this sex dimorphic age-related structural and functional decline in the gut in response to rapamycin is mediated by intestinal autophagy. Upon rapamycin treatment, female intestinal enterocytes increase autophagy, via the H3/ H4 histone-Bchs axis, while male enterocytes show high basal levels of autophagy that do not increase further upon rapamycin treatment. Suppressing autophagy by knockdown of Atg5 in enterocytes reduces barrier function and decreases lifespan in males only. These data reveal the dimorphic regulation of autophagy in enterocytes and its impact on gut pathology and lifespan. Strikingly, sexual identity of enterocytes alone, determined by the expression of transformerFemale, dictates sexually dimorphic H3/H4-Bchs expression, basal rates of autophagy, fecundity, intestinal homeostasis and extension of lifespan in response to rapamycin. Feminised males show a lifespan extension upon treatment with rapamycin that is not observed in control males. In contrast, masculinized females do not extend lifespan in response to rapamycin. Altogether, these results suggest that the intrinsic sexual identity of enterocytes determines the effect of rapamycin on intestinal homeostasis and lifespan, regardless of organismal sex. Conclusion: Understanding sex differential responses to geroprotective interventions gives an understanding of the mechanistic underpinnings of sex differences in the intrinsic rate of ageing in specific tissues, including sex-specific tradeoffs. Our study shows sex dimorphic of basal autophagy but not microbiome in the gut plays a key role in intestinal homeostasis and lifespan in response to rapamycin treatment. Our work, therefore, highlights that tissue sex determines the regulation of metabolic processes by mTOR and the efficacy of mTOR-targeted, anti-ageing drug treatments, which will allow for the development of sex-optimised treatments.

OC16 — Exploring the Interplay of Sex, Immunity and Ageing Using Drosophila Melanogaster

Mary-Kate Corbally¹, Nahian Majlish², Stanislava Chtarbanova-Rudloff², David Duneau³, Jennifer C. Regan¹ (1. University Of Edinburgh — Edinburgh (United Kingdom), 2. University Of Alabama — Tuscaloosa (United States), 3. Instituto Gulbenkian de Ciencia — Oeiras (Portugal))

Background: The sexes age differently: they have different lifespans and show unequal risk to develop ageing pathologies. One key example is the age-related dysregulation of immune function, immunosenescence, which impacts not only responses to infection (highlighted by the current pandemic), but also the risk to develop inflammation-driven diseases such as neurodegeneration. Sexually dimorphic immunity and aging are observed across the animal kingdom, and may represent different investments by the sexes into immune strategy and homeostasis. Objectives: Using the genetic model organism, Drosophila melanogaster, that shows sex differences in both immune responses and ageing, we aimed to leverage dimorphisms in immunosenescence to better understand underlying mechanisms. In particular, we ask how the sexes differ in age-related decline in immune strategies (resistance and tolerance) and how inflammation over ageing (‘inflammageing’) impacts tissue-specific pathology such as neurodegeneration. Methods: Using multiple isogenic lines from the fully-sequenced Drosophila Genetic Resource Panel (DGRP), that represent natural variation in phenotypic traits such as ageing and immunity, we have tested the effect of sex on immunosenescenc e. Through high-powered, controlled infections with a natural bacterial pathogen, Providencia rettgeri, in young and old individuals, we can assay immunocompetence over ageing. Correlating lethal pathogen load with hazard ratio in each sex and line allows us to estimate whether decreased resistance or tolerance mechanisms underpin age-related changes in survival. To correlate immune phenotypes with tissue-specific pathology, brains of each line were sampled at the same timepoints to assess sterile, inflammation-driven neurodegeneration. Results: Immunosenescence is observed across the panel, with genotype driving differences in the magnitude of increased susceptibility to infection. Importantly, mechanisms are sex-specific, with age affecting tolerance and resistance strategies differently in males and females. We have focussed on individual lines that lose resistance to infection in one sex only, to explore physiological, tissue-specific, mechanisms of immunosenescence and pathology. Conclusion: Immune decline is both cause and consequence of ageing. The sexes age differently across taxa — we can use a simple, genetic model system to better understand dimorphisms in immune ageing and their underlying mechanisms. Males and females both increase their susceptibility to infection over age but do so in different ways, by loss of efficiency of different immune strategies, impacting pathology and ultimately, survival.

OC17 — Explainable Machine Learning Pipeline for Personalized Estimation of Physiological

Paul Monsarrat¹, David Bernard², Doumard Doumard², Julien Aligon³, Sylvain Cussac-Blanc⁴, Cyrille Delpierre⁵, Luc Penicaud², Isabelle Ader², Philippe Kemoun², Louis Casteilla² (1. Restore, Aniti — Toulouse (France), 2. Restore — Toulouse (France), 3. Irit — Toulouse (France), 4. Irit, Aniti — Toulouse (France), 5. Cerpop — Toulouse (France))

Background: Aging is a multifactorial process affecting differently each individual over time. The challenge to achieve healthy aging for each individual is to monitor their physiological and not chronological age as early as possible to adapt healthcare management. Several strategies have been proposed but the advent of Machine Learning (ML) allows a better consideration of a very large number of biological parameters and their interactions, even if there are major issues around explainability, limiting their uses and applications in the medical field. Objective: Using simple intrinsic biological variables, we propose a comprehensive ML-based analytical framework to define an explainable, Personalized Physiological Age (PPA). Methods: A new consistent largest possible dataset has been built from the complete National Health and Nutrition Examination Survey (NHANES 1999–2018) database (60,402 individuals from 12 to 80 years-old with 48 simple laboratory variables and 0.6% of missing data). A specific algorithm has been optimized and trained to defined PPA with simple laboratory variables as the predicted age that estimates the relative aging deviation of one individual from the general population. The explainability profile has been generated using the SHapley Additive exPlanations (SHAP) model to quantify what is the relative contribution of each variable to predict the individuals older or younger than their respective chronological ages. Deviation of PA from chronological age is therefore the sum of the SHAP contributions of all the laboratory variables. Results: PPA deviation was validated as predictor of lifespan but also, risk factor for chronic diseases and decline of functions as health span monitoring. Furthermore, partial dependence plots of SHAP values also identify precise range of values and thresholds for each variable and age group delineating accelerating or reduced aging. Clustering of SHAP values identifies specific PPA profiles. Finally, using recursive feature selection, we could restrict the list of relevant variables without significant loss of model performance to 26 simple biological variables out of 49 initial variables only suitable to define a personalized and explainable monitoring of physiological age to achieve healthy aging. Conclusion: Altogether these data demonstrate that Personalized Physiological Age could provide a very new, robust quantitative and explainable ML-based tool to define a new metric to efficiently monitor personalized health status. Disclosure: The authors have no conflict of interest

OC18 — Precise Machine Learning Suggests that Brain Cells Facing a Neurodegenerative Insult are the Subject of Molecular Decompensation and Aging

Lucile Mégret¹, Barbara Gris², Satish Sasidharan Nair¹, Jasmin Cevost¹, Mary Wertz³, Jeff Aaronson⁴ , Jim Rosinski⁴, Thomas F. Vogt⁴, Hilary Wilkinson³, Myriam Heiman³, Christian Néri¹ (1. Sorbonne Université, Centre National de la Recherche Scientifique UMR 8256, INSERM ERL U1164, Brain-C Lab — Paris (France), 2. Sorbonne Université, Centre National de la Recherche Scientifique, Laboratoire Jacques-Louis Lyons (LJLL), Paris (France), 3. MIT, Broad Institute Cambridge, MA (USA); 4. CHDIFoundation — Princeton, NJ (USA))

Loss of cellular homeostasis has been implicated in the etiology of several neurodegenerative diseases (ND). However, the molecular mechanisms that underlie this loss remain poorly understood on a systems-level in each case. More largely, acceleration of neuronal aging is suspected to be involved in ND pathogenesis, but the molecular features (e.g. size and strength) of this acceleration remain poorly understood on a systems level and across brain cell types. To investigate these questions, we used a novel computational approach (Geomic) based on the application of shape deformation concepts to the analysis of complex omics data. We used Geomic to analyze and integrate dimensional RNA-seq and in vivo neuron survival data obtained in the striatum of Huntington’s disease (HD) model mice. We mapped the temporal dynamics of homeostatic and pathogenic responses in four striatal cell types of HD model mice. We found that most pathogenic responses are mitigated and most homeostatic responses are decreased over time, suggesting that neuronal death in HD is primarily driven by the loss of homeostatic responses. Moreover, we found that different cell types may lose similar homeostatic processes, e.g. endosome biogenesis and mitochondrial quality-control in Drd1-expressing-neurons and astrocytes. The relevance of these data to HD was validated by human stem cell, GWAS and postmortem brain data. These findings provide a new paradigm and a database of future targets to probe for therapeutic discovery in HD and other NDs, based on remodeling stress-response and re-instating neuronal resilience. These findings also provide systems-level evidence for molecular aging to be a highly dynamic phenomenon that may significantly affect several cell types in the neurodegenerative brain, particularly in the early phases of ND processes. Reference: https://elifesciences.org/articles/64984; Database: http://www.broca.inserm.fr/geomic/index.php

OC19 — Development of Transformative Oral Drugs to Regenerate Aging Muscles

Stan Watowich¹ (1. Ridgeline Therapeutics — Houston (United States))

Background: Every adult over 40 years of age faces the relentless loss of skeletal muscle strength caused primarily by the inability of aging muscles to fully regenerate. In the elderly, decreased muscle strength leads to reduced mobility and physical activity, placing older adults at high risk for sarcopenia, frailty, falls, fractures, chronic diseases, assisted living, and cognitive disorders. Muscle stem cells (muSC) are responsible for the muscle regeneration and repair that maintains muscle strength and function. Unfortunately, muSC become increasingly senescent during aging and cannot sufficiently regenerate and repair skeletal muscle to maintain muscle strength. Current recommendations to reduce age-linked muscle weakness include resistance exercise and protein-rich diets; these approaches provide modest benefits at best. Given the critical unmet need to prevent muscle weakness during aging, we are developing transformative drugs to reactivate aged muSC and reverse muscle degeneration, weakness, and aging in older adults. Objectives: Our oral drugs utilize a novel mechanism-of-action to reactivate aged muSC. We discovered that the enzyme nicotinamide N-methyltransferase (NNMT) is upregulated in skeletal muscle during aging and disrupts muSC energetics and function. In numerous paradigm-shifting preliminary studies with aged animals, we are testing the extent that NNMT inhibitors reactivate senescent muSC and regenerate skeletal muscles. Methods: Using complementary muscle regeneration and resistance-exercise models that are highly predictive of human muscle function, we have tested NNMT inhibitor drugs in dose-ranging efficacy studies with aged animals. Results: We have rigorously demonstrated that our NNMT inhibitor drugs reactivate senescent and dysfunctional muSC, significantly improving muscle regeneration, growth, and peak strength output in aged mice. In paradigm-shifting preliminary studies, we demonstrated that NNMT inhibitors could reactivate aged muSC, resulting in significantly increased muscle strength, size, and function. Using a muscle regeneration (i.e., BaCl2-induced muscle injury) model, we observed that 1–3-week treatment of aged (24-mo-old) mice with NNMT inhibitor drugs could produce 2-fold increases in muSC activity and fusion index, ∼80% increase in myofiber size, and an approximately 2-fold increase in peak muscle strength. In parallel studies with highly aged (27-mo-old) mice, we observed a greater than 50% reduction in intramyocellular lipid (IMCL) levels in aged skeletal muscle tissues and increased muscle mitochondrial respiratory capacity (assessed via the Oroboros high respirometry system), suggesting important secondary effects of NNMT inhibitor treatment in improving aged muscle quality. Additionally, 2-week treatment with NNMT inhibitors reduced the effective muscle age of old animals by more ∼10-weeks relative to control animals. In a murine sarcopenia model, 6-wk drug treatment of aged (22-mo-old) mice dramatically increased muscle strength by ∼36% and muscle mass by ∼15%, relative to age-matched controls. In contrast, extensive resistance exercise training in this model increased muscle strength of the aged animals by less than 20%. Further, combining drug treatment with resistance exercise training produced a supraadditive effect, increasing muscle strength by ∼60% and aerobic running metrics by >3-fold compared to controls. Conclusion: The enzyme NNMT is upregulated in aged skeletal muscle. NNMT upregulation disrupts cellular energetics and metabolic functions. In aged animal models, our NNMT inhibitor drugs can double muscle strength, size, and muscle stem cell regenerative capacity, while reducing epigenetic age.

OC20 — Longitudinal Analysis of Aging Trajectories in Big Biomed Data Reveals Progressive Loss of Resilience and Predicts Human Lifespan Limit

Peter Fedichev¹ (1. Gero.ai — Singapore (Singapore))

We investigated the dynamic properties of the organism state fluctuations along individual aging trajectories in a large longitudinal database of CBC measurements from a consumer diagnostics laboratory. To simplify the analysis, we used a log-linear mortality estimate from the CBC variables as a single quantitative measure of the aging process, henceforth referred to as dynamic organism state indicator (DOSI). We observed, that the age-dependent population DOSI distribution broadening could be explained by a progressive loss of physiological resilience measured by the DOSI auto-correlation time. Extrapolation of this trend suggested that DOSI recovery time and variance would simultaneously diverge at a critical point of 120 – 150 years of age corresponding to a complete loss of resilience. The observation was immediately confirmed by the independent analysis of correlation properties of intraday physical activity levels fluctuations collected by wearable devices. Moreover, the fraction of individuals suffering from the loss of resilience increased exponentially and doubled every 8 years, as expected from Gompertz’s mortality law. We conclude that the criticality resulting in the end of life is an intrinsic biological property of an organism that is independent of stress factors and signifies a fundamental or absolute limit of human lifespan. On the practical side, the revealed in the study allow for a novel and unsupervised way of training biological age models from big longitudinal biomed data in the absence of follow-up information.

OC21 — Counteracting Age-Related VEGF Signaling Insufficiency Promotes Healthy Aging and Extends Life Span

Myriam Grunewald¹ (1. The Hebrew University — Jerusalem (Israel))

Background: All body cells rely on blood vessels (BVs) for the provision of oxygen and other blood-borne substances and, in certain settings, also for the provision of endothelial derived paracrine factors. Like other organ systems, the vascular system undergoes aging, which leads to progressive functional deterioration. Given the centrality of BVs to organ homeostasis, it has been hypothesized that vascular aging is an upstream, founding factor in organismal aging, but experimental support for this proposition is limited. Vascular aging impacts both large and small vessels, with the latter marked by capillary rarefaction, i.e., age-related failure to maintain adequate microvascular density (MVD). A key homeostatic mechanism preventing MVD reduction relies on the angiogenic activity of the vascular endothelial growth factor (VEGF), which by virtue of its hypoxic inducibility, constantly acts to replenish lost vessels and match vascular supply to the tissue needs. The reason(s) that VEGF fails to do so during aging is unknown. Objectives: Compromised vascular function is expected to perturb organ homeostasis in ways conducive for the development of age-related frailties and diseases. Accordingly, counteracting critical facets of vascular aging might be a useful approach for their alleviation. The presumption that insufficient vascular supply in aging is underlined by VEGF signaling insufficiency, primarily (but not exclusively) because of its indispensable role in preventing capillary loss, led us to investigate whether securing a young-like level of VEGF signaling might rectify capillary loss and its sequelae. On the premise that deteriorated vascular function is an upstream driver of multiorgan malfunctioning, it is envisioned that its rectification might confer comprehensive geroprotection. Methods and results: Although VEGF production is not significantly reduced during mouse aging, longitudinal monitoring revealed that VEGF signaling was greatly reduced in multiple key organs. This was associated with increased production of soluble VEGFR1 (sVEGFR1) generated through an age-related shift in alternative splicing of VEGFR1 mRNA and its activity as a VEGF trap. A modest increase in circulatory VEGF using a transgenic VEGF gain-of- function system or adeno-associated virus (AAV)-assisted VEGF transduction maintained a more youthful level of VEGF signaling and provided protection from age-related capillary loss, compromised perfusion, and reduced tissue oxygenation. Aging hallmarks such as mitochondrial dysfunction, compromised metabolic flexibility, endothelial cell senescence, and inflammaging were alleviated in VEGF-treated mice. Conversely, VEGF loss of function by conditional induction of transgenic sFlt1 in endothelial cells accelerated the development of these adverse age-related phenotypes. VEGF-treated mice lived longer and had an extended health span, as reflected by reduced abdominal fat accumulation, reduced liver steatosis, reduced muscle loss (sarcopenia) associated with better preservation of muscle generating force, reduced bone loss (osteoporosis), reduced kyphosis, and reduced burden of spontaneous tumors. Conclusion: The study provides compelling evidence for the proposition that vascular aging is a hierarchically high driver of overall organismal aging. It places VEGF signaling insufficiency at center stage to multi-organ aging and suggests that its undoing might confer comprehensive geroprotection.

OC22 — Nutrition as a Tool to Counteract Inflammaging: Results and Challenges from the Nu-Age Project

Aurelia Santoro¹, Claudio Franceschi¹ (1. University Of Bologna — Bologna (Italy))

Background: Inflammaging, the age-related chronic, low-grade inflammatory state, is one of the main drivers of the aging process. The basic hypothesis of Geroscience is that the mechanisms leading to ageing and age-related diseases largely overlap, and seven common mechanisms/pathways called ‘pillars’ (adaptation to stress, epigenetics, inflammation, macromolecular damage, metabolism, proteostasis, stem cells and regeneration) have been identified. Interestingly, these pillars are highly and tightly interconnected and converge on inflammation, as an impairment of any one pillar fuels inflammation, which subsequently can feed back to all the other pillars. The most common triggers of inflammaging include chronic infections, physical inactivity, (visceral) obesity, intestinal dysbiosis, unbalanced diet, psychological stress, disturbed sleep and circadian rhythm and exposure to environmental stressors. There is an urgent need to find personalized strategies to counteract inflammaging and thus prolong health span. Among the non-pharmacological approaches, dietary and physical activity interventions are the most encouraged. Objectives: The NU-AGE dietary intervention (ClinicalTrials.gov, NCT01754012) investigated whether a newly designed, personally tailored diet, designed to meet the nutritional needs of European people over 65 years of age can counteract or slow down inflammaging and modify a series of health-related parameters. Methods: The EU-funded project NU-AGE (2011–2016) is a parallel randomized, single-blind, one-year trial with two arms (controls and intervention) investigating the effect of a one-year elderly-tailored Mediterranean Diet (MedDiet) in 1296 healthy elderly (65–79 years old) recruited in 5 European countries (Italy, the Netherland, Poland, France, UK). At baseline and one-year follow-up all the subjects have had a comprehensive panel of measurements and information collected: phenotypic traits, including demographics, medical and health conditions, medication, anthropometry, education, cognitive and physical status, blood pressure, among others. Blood, urine, and faecal samples were collected and stored. Moreover, an in-depth set of omics (genetics, metabolomics, lipidomics, metagenomics, epigenetics) before and after the NU-AGE dietary intervention was performed. In total more than 4500 variables were collected for each subject and stored in the NU-AGE database. Results: Since 2014 up today, the NU-AGE project consortium published 25 papers describing the project and the main outcomes of the trial and further analyses are still ongoing. In summary, results showed that the compliance to NU-AGE diet and the consumption of typical Mediterranean diet foods (such as whole grain cereals, vegetables, legumes, fish and extra virgin olive oil) increased significantly in diet group, revealing that it is still possible to shift the diet of older old to healthy habits. The NU-AGE diet has also proven to be an effective strategy to slow cognitive decline and osteoporosis, to improve the inflammatory profile, blood pressure, to keep the efficiency of proteasome activity and to improve properties of the intestinal microbiome related to inflammation and cognitive state. It also «rejuvenated» the biological age, measured by DNA methylation, in a subgroup of female participants. Moreover, anthropometric measures (weight, BMI, waist and hip circumference) significantly decreased and body composition improved when adherence to NU-AGE diet increases. The NU-AGE study design (different countries with different dietary traditions and habits) and the large number of collected data allowed to evaluate the impact of relevant variables usually poorly investigated (age, sex, ethnicity/genetics, as well as individual characteristics) on the effect of MedDiet. However, consistent country- and sex-differences emerged either at baseline and after one year with MedDiet in term of: i) adherence to diet; ii) body composition; iii) blood pressure; iv) cognitive status; v) blood levels of nutrients (amino acids, fatty acids, vitamins and minerals); vi) markers of frailty vii) seasonality and viii) iron and selenium status. Conclusion: The NU-AGE dietary intervention emerged as an effective strategy to reduce the age-related increase of pro-inflammatory parameters and improve the health status of old population in Europe. However, such large number of NU-AGE results let emerge a number of critical questions that will represent a new challenge for researchers in the aging field. The most important take home message is that, despite its overall beneficial impact on health parameters, the effect of MedDiet appears to have an elevated granularity, as it is influenced and modulated by a number of intertwined variables such as geography/ethnicity, sex/gender and related trait (genetics, metagenomics, epigenetics, among others), thus paving the way to and the need of a personalized approach. Moreover, variables such as education, socio-economic status and physical activity need a more accurate investigation. Lastly, given the high number of variables collected in NU-AGE, a follow-up after several years from the baseline characterization would be highly informative to investigate the long-term effect of the NU-AGE diet on the health status of elderly people.

OC23 — LOMECEL-B as a Geroscience Therapeutic Candidate: Clinical Trial Results

Joshua Hare¹, Kevin Ramdas¹, Dan Gincel¹, Lisa Mcclain-Moss¹, Jorge Ruiz², Anthony Oliva¹ (1. Longeveron Inc. — Miami (United States), 2. Bruce W. Carter Miami Vamc — Miami (United States))

Background: Lomecel-B is a proprietary medicinal signaling cell (MSC) formulation under clinical evaluation for multiple aging-related indications. Like other MSC, Lomecel-B has geroscience therapeutic promise, and there are accumulating clinical trials testing their potential. MSCs possess pro-vascular properties, anti-inflammatory properties without leading to toxic immunosuppression, and pro-regenerative properties. Since aging-related indications, such as frailty and Alzheimer’s disease (AD), are associated with endothelial dysfunction and inflammation, we have hypothesized that MSCs hold potential to target these and other pathological features of these aging-related indications. Objectives: We have completed a Phase 2b trial for frailty in aging adults (n=149), and phase 1 trial for mild AD (n=33), to investigate safety and therapeutic potential of Lomecel-B for these aging-related indications; ancillary biomarker and mechanism of action studies were also built into trial design. The primary endpoint of the frailty trial was change in physical function assessed via the 6-minute walk test (6MWT), and the primary objective of the AD trial was safety. Both also examined objectives and endpoints in various other domains, including biomarkers, cognitive, physical function, and quality-of-life. Methods: These trials were double-blind, randomized, and placebo-controlled. Subjects received a single intravenous infusion of Lomecel-B at doses ranging from 2 × 107 cells to 2 × 108, and follow-up was conducted for 1 year. Patient and caregiver (for the AD trial) assessments were performed. Blood-based biomarkers were analyzed, as well as magnetic resonance imaging (MRI) biomarkers for AD. The frailty trial was designed to evaluate the dose-response relationship of Lomecel-B, and the AD trial was a safety trial designed to obtain provisional efficacy information to inform a next-phase trial. Results: The frailty trial revealed a significant dose-response relationship of Lomecel-B to increases in 6MWT. These effects were sustained, despite just the single dose, in which the greatest response was found with the highest dose group (2.0 × 108 cells, “200M”), and no significant differences from placebo were seen with the lowest dose (2.5 × 107 cells, “25M”). At 9 months post-treatment, the 6MWT increased by 48.7±75.5 m from baseline in the 200M Lomecel-B arm (p=0.0115; 95% CI 10.9 – 84.9). This was significantly different from placebo by 63.4 m (p=0.0077; 95% CI 17.1 – 109.6 m), the latter of which decreased by 19.1±97.5 m (p=0.2728; 95% CI −43.3 − 12.3 m). These changes in 6MWT also showed a significant dose-response curve to Lomecel-B. There were detectable changes in serum-based biomarkers in a dose-dependent manner. Among these was soluble TIE-2 (sTIE-2), in which the 200M Lomecel-B arm decreased by − 403.6±003.6 pg/mL from baseline by 9 months, versus an increase in placebo by 356.0±1018.9 pg/mL, and a significant difference between these of −936.9 pg/mL (p=0.0095; 95% CI −1640.3 − 233.4). Vasoactive endothelial growth factor-D (VEGF-D) also increased, nearly doubling at 9 months post-treatment in the 200M Lomecel-B arm (p=0.013). Increases in sTIE-2 is indicative of vascular dysfunction and inflammation, whereas increases in VEGF-D are pro-vascular. Similarly in the AD trial, significant and sustained changes versus placebo were seen in multiple biomarkers indicative of vascular and inflammatory improvement, including VEGF, IL-4, IL-6, IL-10, IL-12, and sIL-2Rα. There were also significant improvements in multiple clinical assessments, including a difference of 5.0±1.5 points in the low-dose Lomecel-B arm (2 × 107 cells) over placebo at 1 year post-infusion (p=0.0317; 2-sided 95% CI −11.09 − −0.77) in the Mini-Mental State Exam (MMSE). Conclusion: Our results support the potential of Lomecel-B to improve clinical outcomes in aging-related frailty and AD as example indications of aging-related physical and cognitive disorders. The changes in 6MWT exceed previously calculated minimal clinically important differences (MCID) of 19 – 40 m, and the change in MMSE exceeds the MCID of 1.4. Changes in pro-vascular and anti-inflammatory biomarkers may prove useful to monitor therapeutic efficacy in future trials. Together, these results support the geroscience therapeutic potential of Lomecel-B, and have potential applicability to a broad range of aging-related indications. Next-phase trials are warranted to investigate these further.

OC24 — Exercise Rejuvenates the Skeletal Muscle Methylome and Transcriptome in Humansy

Sarah Voisin¹, Macsue Jacques¹, Shanie Landen¹, Nicholas Harvey², Larisa Haupt³, Lyn Griffiths³, Kevin Ashton², Vernon Coffey², Jamie-Lee Thompson², Thomas Doering⁴, Malene Lindholm⁵, Ola Hansson⁶, Olof Asplund⁶, Sara Blocquiaux⁷, Martine Thomis⁷ , Adam Sharples⁸, Steve Horvath⁹, Nir Eynon¹⁰ (1. Victoria University — Melbourne (Australia), 2. Bond University — Gold Coast (Australia), 3. Queensland University Of Technology — Brisbane (Australia), 4. Central Queensland University — Rockhampton (Australia), 5. Stanford University — Stanford (United States), 6. Lund University — Lund (Sweden), 7. Ku Leuven — Leuven (Belgium), 8. School Of Sport Sciences — Oslo (Norway), 9. Ucla — Los Angeles (United States), 10. Victoria University — Footscray (Australia))

Backgrounds: While human lifespan has increased by ∼3.5 years/decade since 1900, healthspan has not increased as much. Maximising healthspan should therefore be a global priority from an economic and healthcare perspective. Exercise training is one of the most affordable and effective ways to promote healthy aging, but we have an incomplete understanding of the fundamental mechanisms by which physical activity delays the age-related decline in skeletal muscle function. Methods: We performed a large-scale data mining of online databases that we combined with our own original data from the Gene SMART study to characterise the effect of cardiorespiratory fitness, exercise training and inactivity on human skeletal muscle aging across three OMIC layers. We first compiled a list of age-related changes in DNA methylation, mRNA and protein expression from a total of 1,617 human muscle samples across 57 datasets. Using a cross-sectional association between cardiorespiratory fitness and age-related OMIC profiles from 783 samples across 6 datasets, we then investigated whether fitter individuals displayed ‘younger’ profiles at age-related CpGs, mRNAs and proteins, compared with less fit individuals. Finally, we investigated more directly whether exercise training and its “reverse”, i.e. muscle disuse, caused shifts towards ‘younger’ or ‘older’ OMIC profiles in human skeletal muscle, using high-resolution longitudinal data collected from more than 1,832 human samples across 83 datasets. Results: First, we investigated whether fitter individuals displayed ‘younger’ OMIC profiles in skeletal muscle, compared with less fit individuals. Six percent (2,311/40,113) of all CpGs that change with age in human skeletal muscle were also associated with VO2max (FDR < 0.005), 96% of which showed contrasting effects for age and VO2max. At those CpGs, an increase in 1 mL/min/kg of VO2max was associated, on average, with DNA methylation changes equivalent to a reduction of ∼3 years of age. Two percent (13/740) of all mRNAs that change with age in human skeletal muscle were associated with VO2max (FDR < 0.005), and they all showed contrasting effects for age and VO2max. At those 13 mRNAs, an increase in 1 mL/min/kg of VO2max was associated, on average, with mRNA expression fold changes equivalent to a reduction of ∼2 years of age. Proteomics analyses are ongoing. As cross-sectional analyses can be confounded by unmeasured factors (e.g. lifelong dietary patterns), we tested more directly whether an increase in physical activity could shift OMIC profiles towards a younger state. We also tested whether age-related OMIC profiles were altered following a decrease in physical activity, caused by muscle disuse. We did not observe any significant change in DNA methylation levels at age-related CpGs following exercise training (FDR < 0.005), but we noted an inflation of low p-values, suggesting a true effect of exercise training on age-related CpGs, which was not captured due to low statistical power. The levels of 95 age-related mRNAs changed following exercise training (FDR < 0.005), and they all changed in the direction opposite to that of ageing. Furthermore, the levels of 42 age-related mRNAs changed following muscle disuse (FDR < 0.005), and all but two changed in the same direction as that of ageing. Proteomics analyses are ongoing. Finally, we integrated results across the three OMIC layers, and across the effects of age, aerobic fitness, exercise training and muscle disuse. We identified CACNB1 as a central gene whose DNA methylation levels decrease with age but increase with VO2max, while its mRNA levels increase with age but decrease with VO2max and following exercise training. Functional validation assays are ongoing to confirm the implication of this gene in ageing and muscle function, and to investigate the causal relationship between DNA methylation and mRNA expression at this gene. Conclusion: We showed that higher cardiorespiratory fitness was associated with younger epigenetic and transcriptomic profiles in human skeletal muscle, and that execise training rejuvenates while muscle disuse ages the muscle methylome and transcriptome. This work provides a comprehensive and integrative view of the effect of physical activity on the age-related changes in fundamental processes controlling gene expression in human muscle.

OC25 — Lipidclock: A Lipid-Based Predictor of Biological Age

Maximilian Unfried¹, Li Fang Ng², Amaury Cazenave-Gassiot¹, Krishna Chaithanya Batchu², Brian K. Kennedy¹, Markus R. Wenk¹, Nicholas Tolwinski², Jan Gruber² (1. National University Of Singapore — Singapore (Singapore), 2. Yale-Nus College — Singapore (Singapore))

Background: Complexity is a defining feature of many biological systems. Biological aging is a prime example of a process that exhibits complex behaviour across multiple scales of biological organisation. The advent of -omics techniques such as lipidomics has enabled us to directly capture biological complexity by recording molecular diversity across time. However, this approach yields datasets that encode much of the original biological complexity, posing new challenges in data reduction and analysis. On an organismal level, ageing is defined by an exponential increase in morbidity and mortality, yet, these systemic changes are the consequence of slow, cumulative and detrimental changes at different levels of biological organization. Objectives: The slow temporal dynamics of biological changes related to ageing can be exploited to extract key insights into its underlying biology. Here we explore this idea by using data on changes in lipid composition across the lifespan of an organism to construct and test “lipid-clocks” for model organisms. Methods: The data used to develop the lipid clock were derived from cohorts of aging C. elegans, sampled at different timepoints. To validate the lipid clock, we compared animals of four different strains that are characterized by different lifespans. We used standard wild-type (WT) Bristol N2 as controls undergoing normal aging. We utilized mev-1 as short-lived strain. Mev-1 animals carry a defect in complex II of the electron transfer chain, produce excessive amounts of reactive oxygen species, and suffer from elevated oxidative damage, as well as defective energy metabolism have shorter lifespans. We selected two strains with extended lifespan and healthspans; age-1 and eat-2. Lipidomics analysis was performed using an Agilent 1260-Ultra Performance Liquid chromatography system coupled to Triple Quad Mass spectrometer with dynamic multiple reaction monitoring for lipid quantification. The total dataset comprised data on 168 lipid species, for 54 samples covering three mutant strains and wild type at ages between 3 and 20 days. At the core of the LipidClock is a coordinate transformation by Principal Component Analysis (PCA) followed by supervised learning via Elastic net regression. PCA is a method widely used for linear dimensionality reduction. It is a prominent Eigenvalue method that is used to factorize a matrix into its principal components (PCs). PCA rotates the coordinate system of feature space (lipid species) to align the main axes with directions in feature space along which the covariance between samples is maximal. We trained an elastic net regression model using the 54 linearly independent PC coordinates for each sample as input (intendent variables) and chronological age as a dependent variable. For training we only used data from WT cohorts and young (day 3) mutants. Model parameters were optimized using 10-fold cross validation by Grid search. Following constructing of our lipid clock, we determined aging rates for mutant strains relative to WT based on the biological age determined by the lipid clock and used these estimates of relative aging rates to scale the mortality rate doubling time for each mutant strain. We then calculated estimated survival curves using a simple Monte Carlo approach based on the scaled strain-dependent mortality dynamic. Results: By construction, coordinates along all the 54 PCs are able to reconstruct the complete dataset, reproducing 100% of the variance. However, PC1 alone captured 49% of the overall variance and, combined 10 PCs explained 92% of the variance. Following transformation of all samples into principal component space, we therefore determined the linear correlation of each PC coordinate with age in both WT and mutant cohorts. We considered correlation strong when the absolute r-value was larger or equal to r=0.7. We found that, among the first 5 PCs, three showed strong correlations with chronological age in WT cohorts. Interestingly, conservation of such patterns was variable in mutant strains, suggesting that, as expected, mutations affecting lifespan impact aging mechanisms captured by the main PCs. PC2 was unequivocally correlated to aging across all strains while PCs 1 and 3 also showed tendencies to correlate with age but were less conserved across strains. Consistent with other recent developments in aging clocks, we find that dimensionality reduction by PCA prior to regression functions well in constructing aging clocks. The mean absolute error on the repeated 10- Fold cross validation was 1.45 days, meaning that within this dataset, cohort age was predicted with an error of less than 2 days. We found that predicted lifespan curves under these conditions were in good agreement with observed survival data for age-1 and mev-1, but the simulated data somewhat overestimated eat-2 survival. Conclusion: In this study we successfully constructed and tested a lipid clock using C. elegans. Our findings suggest that lipid clocks may be a promising addition to the emerging field of -omics-derived aging clocks.

ROC01 — TIME-SEQ Enables Inexpensive and Scalable Epigenetic Age Predictions for Large Studies in Mice and Humans

Patrick Griffin¹, Alice Kane¹, Alexandre Trapp², Jien Li¹, Maeve Mcnamara¹, Margarita Meer³, Michael Macarthur⁴, Sarah Mitchell⁴, Amber Mueller¹, Colleen Carmody¹, Daniel Vera¹, Csaba Kerepesi², Nicole Noren Hooten⁵, James Mitchell⁴, Michele Evans⁵, Vadim Gladyshev², David Sinclair¹ (1. Blavatnik Institute, Dept. Of Genetics, Paul F. Glenn Center For Biology Of Aging Research At Harvard Medical School — Boston (United States), 2. Brigham And Women’s Hospital, Division Of Genetics, Department Of Medicine, Harvard Medical School — Boston (United States), 3. Yale University School Of Medicine, Department Of Pathology — New Haven (United States), 4. Department Of Health Sciences And Technology, Eth Zurich — Zürich (Switzerland), 5. Laboratory Of Epidemiology And Population Science, National Institute On Aging, National Institutes Of Health — Bethesda (United States))

Background: Epigenetic clocks based on DNA methylation are the most robust and widely used category of aging biomarker. They have been built for numerous species and can reflect decelerated aging from longevity interventions. However, conventional approaches to measure clocks are expensive and not easily applied to large cohorts. This has limited the use of epigenetic clocks to relatively small or costly studies. Objectives: Develop a flexible and robust method for inexpensive and high-throughput epigenetic age prediction. Use the method to assess longevity interventions and discover correlates with epigenetic age in large cohorts. Methods: We designed a highly scalable and inexpensive epigenetic age prediction method called Tagmentation-based Indexing for Methylation Sequencing (TIME-Seq). TIME-Seq is a targeted methylation sequencing approach that rapidly indexes samples via barcoded Tn5 adaptor insertion that is compatible with bisulfite conversion and Illumina sequencing. TIME-Seq also relies upon targeted enrichment of epigenetic clock loci via biotinylated-RNA bait hybridization. We applied TIME-Seq to predict age in thousands of human and mouse samples, including late-life dietary interventions and longitudinally tracked mice. Using TIME-Seq libraries, we also attempted to predict age from extremely shallow sequencing (e.g., 10,000 reads) using scAge, a probabilistic age prediction algorithm first applied to single cells. Results: TIME-Seq is capable of highly accurate age prediction in mice and humans, streamlines large-scale experiments, and reduces the cost of epigenetic age analysis more than two orders of magnitude. Age prediction from shallow sequencing using scAge was surprisingly accurate, approaching the predictive power of traditional elastic-net based clocks. The longevity benefit from late-life caloric restriction was reflected in epigenetic age predictions from our TIME-Seq mouse blood clock, and we did not see correlation between epigenetic age and blood cell composition or frailty index, an alternative age-related biomarker. Conclusion: TIME-Seq promises to enable larger-scale and more routine studies using epigenetic clocks. It is a flexible method that can be applied to build low-cost clocks in numerous species. Currently, we are using TIME-Seq clocks to track epigenetic age in large cohorts of mice with high temporal resolution during longevity interventions. We are also investigating correlates of predicted age in genetically diverse mice and mice tracked until death.

ROC02 — The NAD+-MITOPHAGY Axis in Ageing and Alzheimer’s Disease and Artificial Intelligence-Based Drug Development

Evandro Fei Fang¹ (1. University Of Oslo And Akershus University Hospital — Oslo (Norway))

Background: Ageing is arguably the highest risk factor for numerous human diseases, including Alzheimer’s disease (AD). Understanding the molecular mechanisms of human aging holds the promise of developing interventional and therapeutic strategies for many diseases simultaneously, promoting healthy longevity. Accumulation of damaged mitochondria is a hallmark of aging and age-related neurodegeneration, including AD. The molecular mechanisms of impaired mitochondrial homeostasis and their relationship to AD are elusive. Mitophagy is the cellular self-clearing process of damaged and superfluous mitochondria, and therefore plays a fundamental role in maintaining neuronal function and survival (1). Objectives: We hypothesize that age-susceptible defective mitophagy causes accumulation of damaged mitochondria, which in combination with the two AD-defining pathologies, Aβ plaques and tau tangles, further exacerbates AD progression. Methods: We used a cross-species strategy, covering the use of AD iPSC, C. elegans, and mouse models. Behavioural studies, imaging systems, and genetic manipulation approaches were used to quantify mitophagy, memory, and for mechanistic exploration. Results: Restoration of mitophagy through upregulation of cellular NAD+, a primary molecule in human health and life, and genetic approaches, forestalls pathology and cognitive decline in C. elegans and three mouse models of AD and improves mitochondrial function in the AD iPSC neurons (2, 3). We are now involved in more than 5 clinical trials on the use of NAD+ precursors to treat AD, and premature ageing diseases, among others (4). Additionally, we are using artificial intelligence (AI) to propel drug screening and drug design targeting AD and ageing pathways (5). Conclusions: Impaired mitophagy is a risk factor of AD, while turning up mitophagy to physiological level of young age may serve as a novel therapeutic strategy for AD. Acknowledgements: This project was supported by Helse Sør-Øst (#2017056, #2020001, #2021021), the Research Council of Norway (#262175 and #277813), the National Natural Science Foundation of China (#81971327), Akershus University Hospital (#269901, #261973), the Civitan Norges Forskningsfond for Alzheimers sykdom (#281931), the Czech Republic-Norway KAPPA programme (with Martin Vyhnálek, #TO01000215), and the Rosa sløyfe/Norwegian Cancer Society & Norwegian Breast Cancer Society (#207819) to EFF. Declaration of Interests: E.F.F. has CRADA arrangement with ChromaDex, and is consultant to Aladdin Healthcare Technologies, Vancouver Dementia Prevention Centre, Intellectual Labs, and MindRank AI Ltd. References: 1. Aman, Y. et al. Autophagy in healthy ageing and disease. Nat Aging 1, 634–650 (2021). 2. Fang, E.F. et al. Mitophagy inhibits amyloid-beta and tau pathology and reverses cognitive deficits in models of Alzheimer’s disease. Nat Neurosci 22, 401–412 (2019). 3. Lautrup, S., Sinclair, D.A., Mattson, M.P. & Fang, E.F. NAD(+) in Brain Aging and Neurodegenerative Disorders. Cell Metab 30, 630–655 (2019). 4. Reiten, O.K., Wilvang, M.A., Mitchell, S.J., Hu, Z. & Fang, E.F. Preclinical and clinical evidence of NAD(+) precursors in health, disease, and ageing. Mech Ageing Dev, 111567 (2021). 5. Xie, C. et al. Amelioration of Alzheimer’s disease pathology by mitophagy inducers identified via machine learning and a cross-species workflow. Nat Biomed Eng (In press).

ROC03 — Metabolic Implications on Neuroinflammation in the Pathological Aging Context. Dietary Restriction, Glial Autophagy and Metformin in Alzheimer’s Disease

Juan Beauquis¹, Carlos Pomilio¹, Angeles Vinuesa¹, Amal Gregosa¹, Nicolas Gonzalez-Perez¹, Flavia E Saravia¹ (1. Conicet & Uba — Buenos Aires (Argentina))

Overnutrition and modern diets containing high proportions of saturated fat are among the major factors contributing to a low-grade state of inflammation, hyperglycemia and dyslipidemia. In the last decades, the global rise of type 2 diabetes (T2D) and obesity prevalence has elicited a great interest in understanding how changes in metabolic function lead to an increased risk for premature brain aging and the development of neurodegenerative disorders such as Alzheimer’s disease (AD). Cognitive impairment and decreased neurogenic capacity could be a consequence of metabolic disturbances. In these scenarios, the interplay between inflammation and insulin resistance could represent a potential therapeutic target to prevent or ameliorate neurodegeneration and cognitive ability. Dietary restriction (DR) has been proposed as a potential therapeutic strategy for age-associated diseases. We analyzed memory performance and hippocampal alterations in an animal model of familial AD, the PDAPP-J20 transgenic mouse (Tg) and evaluated the effects of a periodic DR protocol (3 cycles of 40% DR for 5 days and ad libitum (AL) diet for 9 days). We observed cognitive impairment, impaired adult neurogenesis and progressive amyloid beta (AB) deposition in the hippocampus of AL-fed Tg mice. Periodic DR was associated to cognitive improvement, increased hippocampal neurogenesis, and reduced hippocampal amyloid load. Through immunofluorescence for LC3 specific marker for autophagosomes and GFAP (astrocytes), we found that autophagy is modulated in Tg mice with a high proportion of LC3 localized in astrocytes. We also found that astrocytes contained AB co-localizing with LC3. From these results we hypothesized that the reduction in amyloid plaques associated to DR would be partly mediated by astroglial autophagy. This data was in line with in vitro results from cultured astrocytes exposed to a nutrient restriction (NR; 2% FBS) or not (10% FBS), where we found evidence of glial autophagy implication promoted by NR, strongly suggesting a link between metabolic pathways and neuroinflammation in AD. In this line, some therapeutic strategies employed on T2D subjects could be beneficial on AD patients. We evaluated the effect of the antidiabetic drug metformin -considered a DR mimetic- on patients enrolled in ADNI, an observational and longitudinal study including patients from all around the world. We employed data from patients diagnosed with mild cognitive impairment (MCI) due to AD and we performed a principal component analysis focusing on biomarkers associated to AD measured in cerebrospinal fluid. We concluded that MCI metformin-treated patients were globally characterized as subjects with a better CSF biomarkers profile than the mean population of MCI patients (p<0.05). On the other hand, control subjects and T2D patients were paired by age, gender, ApoE allele and years of education, defining three groups: MCI, MCI+T2D and MCI+T2D+metformin. We evaluated the effect of T2D and metformin treatment employing the PACC score, and composites defined from standardized ADNI variables to evaluate the memory and learning function. We found that MCI+T2D patients have a worse cognitive performance than MCI patients (p<0.01), but this deleterious effect was not observed in MCI+T2D+metformin patients. These cognitive variations were associated with changes in cortical thickness and hippocampal volume obtained from Magnetic Resonance Images (p<0.001). To summary, our preliminary study shows a beneficial effect of metformin treatment on cognitive performance, CSF biomarkers profile and neuroanatomical measures in MCI due to AD patients. The molecular targets of metformin are still not completely characterized, although one candidate is the AMP-activated protein kinase (AMPK). AMPK- a highly conserved regulator of energy homeostasis is a key nutrient sensor that plays an important role in metabolism and the regulation of whole body energy balance and is also crucial in DR effects. Further studies are needed to provide more evidence about different strategies targeting this pathway with a promising gerotherapeutic role.

ROC04 — Targeting Cellular Senescence with Novel Senotherapeutics by Design to Extend Healthspan

Lei Zhang¹, Brian Hughes¹, Wandi Xu¹, Sara J. McGowan¹, Luise Angelini¹, Ryan O’Kelly¹, Matthew Yousefzadeh¹, Theodore M. Kamenecka², Laura J. Niedernhofer¹, Paul D. Robbins¹ (1. University Of Minnesota — Minneapolis (United States), 2. The Scripps Research Institute — Florida (United States))

Background: Aging is associated with declines in health and physiological functions, leading to an increase in the number of many chronic diseases. One of the critical pillars or underlying mechanisms of aging is cellular senescence. Senescence is a phenomenon in which cells lose the ability to proliferate under different cellular stress conditions, resulting in a state of stable cell cycle arrest. With age, senescent cells accumulate in many tissues and produce different deleterious signals to neighboring cells, including pro-inflammatory cytokines, chemokines and proteases, termed senescence-associated secretory phenotypes (SASPs). The SASPs released by senescent cells can lead to tissue dysfunction and even turn healthy cells senescent, further driving aging and promoting many age-related pathologies. More importantly, removing senescent cells either genetically or pharmacologically using a class of drugs termed senolytics has been demonstrated as an effective strategy to extend healthspan and treat age-related diseases. Objectives: Despite their promising potential, only a handful of senolytics have been reported, including a natural flavonoid fisetin discovered by our group. Fisetin has been shown to reduce senescence, suppress age-related pathology, and extend healthspan in aged mice. However, the moderate potency and poor bioavailability of fisetin may limit its effectiveness in clinical applications. Therefore, there is a clear need to identify better and safer fisetin analogs (FAs) that can be translated into clinical applications for the treatment of age-related diseases. This project aims to develop novel FAs with more potent senolytic activity, safer toxicity profiles, and improved physicochemical properties. In addition, the optimized FAs will be tested for reducing senescence and extending healthspan with different in vitro, ex vivo, and in vivo models of senescence and aging. Methods: The biological properties of a compound are determined by its chemical structure. The moderate potency and poor pharmacokinetic profiles of fisetin can attribute to its polyphenol structures. Medicinal chemistry is a powerful approach to optimize molecular properties through chemical structures. Therefore, we take advantage of medicinal chemistry and drug design to optimize the senolytic activity of fisetin using detailed structure-activity relationship (SAR) studies. In addition, an efficient cell-based drug screening platform equipped with high-content fluorescent image analysis that I have optimized will be utilized to evaluate the senolytic activities. After identifying improved fisetin analogs (FAs) in cell culture, their senotherapeutic potential for reducing cellular senescence and extending healthspan will be investigated using human tissue explants ex vivo as well as accelerated aging and naturally aged mice in vivo. Results: With several rounds of SAR studies including drug design, chemical synthesis and biological evaluation, we have identified several improved analogs of fisetin. The best FAs showed not only improved senolytic activity than fisetin (2.9 and 27.5 folds more potent respectively) but also better physicochemical properties including enhanced lipophilicity and lower topological polar surface area (tPSA) values, indicating better permeability of the blood-brain barrier (BBB). As a starting point to confirm the in vivo therapeutic potential of the two promising fisetin analogs, we used > 2-year-old C57Bl/6 mice. The mice were acutely treated with vehicle, fisetin, the best fisetin analogs SR29384 and SR31133 at 20 mg/kg of body weight for five days. Molecular analysis of different tissues found that fisetin had only a marginal effect on senescence in these tissues, whereas the two fisetin analogs, especially SR29384, significantly decreased the expression of multiple senescence and SASP factors in different tissues of mice including kidney, brain, and lung. To evaluate the effect on healthspan, SR29384 was selected for chronic treatment in the Ercc1-/Δ mouse model of accelerated aging for 5 weeks. Results showed that SR29384 can suppress the composite score of aging symptoms that reflect the overall health condition of the Ercc1-/Δ mice. Moreover, gene expression analysis demonstrated that SR29384 can reduce senescence and SASP factors significantly or with a treading to significance in multiple tissues including kidney, liver, lung, and brain. Conclusion: In summary, with a multidisciplinary approach, we have successfully developed several optimized analogs of fisetin with improved potency and enhanced physicochemical properties. The senotherapeutic potential of these FAs has been confirmed in both naturally aged and accelerated aging mice in vivo. These novel FAs with improved senolytic activity and reduced toxicity in nonsenescent cells have clinical potential to slow aging and reduce the severity of age-related diseases driven by senescence.

ROC05 — Resveratrol: A Potential Nutritional Polyphenol for Age-Related Macular Degeneration Treatment

Agustina Alaimo¹, Guadalupe Garcia Liñares², Ana Paula Dominguez Rubio¹, Oscar Edgardo Pérez¹ (1. Biological Department, Faculty Of Exact And Natural Sciences, Univeristy Of Buenos Aires. Iquibicen-Conicet — Buenos Aires (Argentina), 2. Organic Department, Faculty Of Exact And Natural Sciences, Univeristy Of Buenos Aires. Umymfor-Conicet — Buenos Aires (Argentina))

Age-related macular degeneration (AMD) is a late-onset retinal disease and the leading cause of central vision loss in the elderly. Degeneration of retinal pigment epithelial cells (RPE) is a crucial contributing factor responsible for the onset and progression of AMD. The toxic fluorophore N-retinyl-N-retinylidene ethanolamine (A2E), a major lipofuscin component, accumulates in RPE cells with age. Phytochemicals with antioxidant properties may have a potential role in both the prevention and treatment of this age-related ocular disease. Particularly, there is an increased interest in the therapeutic effects of Resveratrol (RSV), a naturally occurring polyphenol (3,4′,5-Trihydroxystilbene). However, the underlying mechanism of the RSV antioxidative effect in ocular diseases has not been well explored. We hypothesized that this bioactive compound may have beneficial effects for AMD. To this end, to investigate the potential profits of RSV against A2E-provoked oxidative damage, we used human RPE cell line (ARPE-19). RSV (25 µM) attenuates the cytotoxicity and the typical morphological characteristics of apoptosis observed in 25 µM A2E-laden cells. RSV pretreatment strengthened cell monolayer integrity through the preservation of the transepithelial electrical resistance and reduced the fluorescein isothiocyanate (FITC)-dextran diffusion rate as well as cytoskeleton architecture. In addition, RSV exhorts protective effects against A2E-induced modifications in the intracellular redox balance. Finally, RSV also prevented A2E-induced mitochondrial dynamics alteration. These findings reinforce the idea that RSV represents an attractive bioactive for therapeutic intervention against ocular diseases associated with oxidative stress such as AMD.