Introduction: The increasing global aging population highlights the urgent need for effective strategies to prevent functional and cognitive decline. Multidomain interventions, which target multiple health areas concurrently, offer a promising approach to promote healthy aging. The IN-TEMPO (ItaliaN study with Tailored Multidomain interventions to Prevent functional and cognitive decline in community-dwelling Older adults) study, a key component of the broader Age-It project and World-Wide FINGERS network, aims to evaluate the effectiveness of personalized multidomain interventions in older adults at risk of dementia. This approach is supported by recent evidence highlighting the importance of targeting modifiable risk factors and the potential of multidomain interventions within the FINGERS framework.

Methods: IN-TEMPO is a 12-month, two-phase randomized controlled trial (RCT). Phase 1, a population-based observational study, began in September 2024. Phase 2, the efficacy study, commenced in January 2025. Participants aged ≥60 years with mild/moderate vulnerability (PC-FI 0.07-0.21) and increased dementia risk (CAIDE ≥6) are randomized to an active intervention group (personalized physical exercise, healthy diet, cognitive training, oral hygiene, sleep management, cardiovascular risk factor control, social support) or a self-guided control group via a dedicated website. The study is officially registered on ClinicalTrials.gov (NCT06248723), involving 9 clinical centers across Italy. A sample size of 1534 patients (1662 assuming a 10% dropout) is sufficient to detect a Standardized Mean Difference (SMD) of 0.127 in the mean change of the NTB z-score at 12 months with 80% power and a 5% Type I error. Adherence to interventions is monitored via electronic Case Report Forms (eCRF) on the RedCap platform, wearable devices (Garmin trackers) for physical activity, and cognitive training platforms (BrainHQ).

Preliminary Results: As of May 31, 2025, the IN-TEMPO study has made significant progress in its initial phases. A total of 1515 subjects have been screened across all participating centers, with screening activities continuing until the end of the year. Screening failure rates ranged from 46.18% to 70.37% across centers, reflecting the study's rigorous inclusion criteria. The coordinating center (IRCCS San Gerardo dei Tintori, Monza) has successfully included 128 participants into the Phase 2 intervention. Overall, 498 participants have been enrolled into Phase 2, with approximately half in the self-intervention group. Key milestones include the approval and dissemination of the first protocol amendment, successful deployment and activation of tablets and health-tracking wearable devices, and operationalization of the control group's educational website. Recruitment efforts are ongoing, with a projected continuation until mid-August for recently added centers (Catania and Palermo), aiming to meet the total recruitment target of 1665 patients. Biological samples from randomized subjects will be analyzed at UNIMIB, UPO, and UNINA laboratories.
Conclusion: IN-TEMPO is successfully progressing through its initial recruitment and intervention phases, establishing a robust framework for evaluating personalized multidomain interventions. The substantial progress in screening and initial randomization, alongside the deployment of technological tools and the establishment of support systems, lays the groundwork for a comprehensive assessment of efficacy on functional and cognitive decline in older adults. These early achievements underscore the feasibility of large-scale implementation of such complex interventions within a multicenter setting, contributing valuable insights to the Age-It project and the global FINGERS initiative. The FINGER approach has demonstrated its potential in reducing dementia risk , and IN-TEMPO's contribution to this global effort is significant

Keyworks: Healthy Ageing; Dementia Risk Reduction; Prevention; Multidomain Intervention Strategy
References:

[1] Ngandu T, Kivipelto M. Successful strategies to prevent dementia and cognitive decline. The Lancet. 2024;404(10444):2199-2214.

[2] Livingston G, Huntley J, Sommerlad A, et al. Dementia prevention, intervention, and care: 2020 report of the Lancet Commission. Lancet. 2020;396(10248):413-446.

[3] Richard E, et al. World-Wide FINGERS: A network of multidomain intervention studies to prevent cognitive decline and dementia. Alzheimer's & Dementia: The Journal of the Alzheimer's Association. 2020;16(4):741-751.

The IN-TeMPO trial (ClinicalTrials.gov ID NCT06248723) is a multicenter randomized controlled study aimed at evaluating the effectiveness of guided multidomain interventions in preventing cognitive and functional decline in older adults at increased risk of dementia. As part of the trial, both established and exploratory blood-based biomarkers linked to age-related processes—such as Alzheimer’s disease (AD), neurodegeneration, inflammation, cellular senescence, and sarcopenia—are being investigated to improve risk stratification and identify surrogate markers for intervention outcomes.

Here we focus on biomarker analyses conducted at the University of Milano-Bicocca (UNIMIB) on subjects enrolled by the Monza clinical center. Biomarkers include ApoE genotype and plasma levels of p-tau217, neurofilament light chain (NfL), and glial fibrillary acidic protein (GFAP) as indicators of AD, neurodegeneration, and inflammation, respectively. In a subset of 80 participants, additional analyses target plasma redox status, γ-H2AX expression in PBMCs, as a marker of cellular senescence, and untargeted plasma metabolomics.

As of May 2025, baseline samples from 132 participants were collected and analyzed. ApoE genotyping showed 13% carried at least one ε4 allele. Mean plasma concentrations were: p-tau217 0.12 ± 0.1 pg/mL, NfL 19.66 ± 11.08 pg/mL, and GFAP 50.34 ± 20.89 pg/mL. Plasma p-tau217 levels significantly correlated with NfL (r = 0.39, p < 0.0001) and GFAP (r = 0.33, p < 0.0005). Biomarkers will be reassessed at 12-month follow-up to evaluate intervention effects.

These findings may support the early detection of age-related decline and inform preventive strategies.

DNA methylation (DNAm), an epigenetic modification that reflects both inherited and environmental influences, plays a crucial role in regulating gene expression and is known to undergo dynamic changes with age.

The objective of this study is to test long-term predictive ability (>20 years) of existing DNAm-based epigenetic age acceleration (EAA) measures (biological age) and identifying novel blood-based DNAm age-prediction biomarkers.

EAA measures calculated from Illumina EPIC v2 950k arrays data from blood were used to evaluate biological aging through previously validated epigenetic clocks in a cohort of 161 90+ long-lived individuals (LLI) and 136 geographical matched younger controls (CG) followed up to 20 years. Survival analysis was used to evaluate the effect of EAA measures on mortality risk in these two ages groups.

Linear mixed-effects models (LMMs) were used to identify cross-sectional age-associated differentially methylated CpG positions (aDMPs). In contrast, survival analyses were used to identify DMPs longitudinal associated with mortality risk in these two age groups.

Epigenetic clocks showed significant correlation with age. EAA was significantly correlated with mortality risk regardless of the epigenetic model used.

21742 aDMPs were identified thought LMM comparing the genome-wide methylation profile between CG and LLI. Most of them were previously incorporated into several well validated epigenetic clocks.

Several methylation markers showed strong associations with long-term mortality risk. These results reinforce the role of known CpG sites, such as ELOVL2, as reliable age predictors. Additionally, newly identified markers may enhance the accuracy of current blood-based age prediction models.

BACKGROUND: Immuno-senescence is the progressive decline in immune function, which supports ageing-associated diminished capacity to mount effective immune responses, reduced vaccine efficacy, and an increased susceptibility to infections, cancer, and autoimmune diseases. A hallmark of immuno-senescence is the dysfunction of T cells, particularly characterized by impaired activation of the T cell receptor (TCR) and defective downstream signaling pathways which drive the antigen-induced immune response. Several experimental evidence demonstrate that accumulation of DNA damage and activation of the DNA-damage response (DDR) in T cells is sufficient to trigger immune-senescence and drive inflammaging in tissues (1)

However, the molecular mechanisms linking DDR activation to T cell dysfunction have not been elucidated. The regulation of receptor dynamics, i.e. the rate of its internalization and either recycling to the cell surface or sorting to the lysosome for degradation, is a key event to control cellular response to a wide range of ligands. More specifically, the extent, the kinetics and the cellular fate of T cell response to antigen stimulation are indeed regulated by the dynamics of the activated TCR. Still unpublished results from our lab indicate that the extent of DGKA expression, a well-known negative regulator of TCR signaling (2), promotes CD3z degradation, thus impairing TCR recycling. Interestingly, DGKA has been reported in fibroblasts to be induced by DNA damage.

RATIONALE: Thus, we set to investigate the hypothesis that i) DDR in T cells impairs TCR recycling to the cell surface, thus impairing TCR signaling and effector functions; ii) DGKA deletion may result in rescuing defective TCR recycling, thus re-establishing TCR signaling strength.

METHODS: We developed an in-vitro model of DDR -induced T cell senescence in primary human CD8+ T lymphocytes. DDR was induced by silencing telomeric repeat binding factor 2 (TRF2), one of the core proteins in the Shelterin complex which is responsible for the protection of the telomeric ends,. By silencing TRF2 we closely resemble physiological occurrences[VM1] inducing a persistent DNA damage response (DDR) with consequent establishment of senescence. In addition, DGKA gene has been deleted by CRISPR-CAS9 in primary CD8+ T cells.

Then cells has been stimulated with OKT3 anti-TCR antibodies, either in solution or upon tethering a single chain OKT3 tethered on the cell surface of antigen presenting cells

RESULTS: Our data show that shTRF2 T cells display a senescent phenotype (e.g impaired CD28 at cell surface and expression of senescence markers) and DNA damage establishment compared to shCTRL counterpart. Notably, upon stimulation, these cells also show impaired recycling of the TCR to the plasma membrane after receptor internalization, suggesting that DDR may interfere with TCR trafficking upon TCR engagement. Consistently with a defective TCR recycling, we observed defective activation of both PLCg and Erk ½ signaling pathways.

Finally, preliminary results indicate that DGKA deletion rescue TCR recycling at the cell surface and TCR intracellular signaling.

MAIN IMPLICATION OF THE RESEARCH: These results provide the first demonstration that impaired/aberrant TCR dynamics mediate the dysfunction of senescent T cells. This finding unveil a broad spectrum of strategies tuned to rescue T cell function. Pharmacological targeting of DGKA expression and activity may represent a promising strategy for T cells rejuvenation and delay inflammaging.
REFERENCES
1) Yousefzadeh, M. J. et al. An aged immune system drives senescence and ageing of solid organs. Nature 594, 100–105 (2021).

2).Ruffo, E. et al. Inhibition of diacylglycerol kinase α restores restimulation-induced cell death and reduces immunopathology in XLP-1. Sci Transl Med 8, 321ra7-321ra7 (2016)

In aging research, evolutionary comparative studies offer a privileged, yet underexploited approach. During the past few years, bats have emerged as innovative models as they boast the highest longevity quotient among mammals, coupled with exceptional viral tolerance, low cancer incidence, negligible macroscopic phenotypic aging, and overall poor susceptibility to age-related diseases. We have generated a biobank of bat tissues and primary and immortalized cells to identify the biological bases of bats halted aging with particular focus on their uniquely upregulated autophagic activity. Comparative unsupervised transcriptomic analysis confirmed the presence of unique adaptations within autophagy and in autophagy-regulated cellular domains, such as bioenergetics and proliferation. Following a morphological and functional characterization, multi-omics dissection of the autophagic network revealed an unprecedented remarkable activity in bat-derived primary fibroblasts compared to murine counterparts. Biochemical characterization of the expression of specific autophagosome markers and substrates under lysosomal inhibition further revealed higher autophagic flux in bat cells. We then set out to determine what supramolecular structures are degraded in bat cells by differential proteomics upon distal autophagic inhibition. Surprisingly, we failed to demonstrate accumulation of specific cellular compartments. We then hypothesized that bat cells may deploy constitutive autophagy of bulk cytoplasmic material as an allegedly uniquely evolved stress-adaptive strategy. In keeping with this, biochemical assessment of autophagic degradation of specific cytoplasmic substrates and time-lapse imaging employing a customized fluorescent cytoplasmic reporter confirmed our hypothesis. The data reveal high default autophagy as an unprecedented cellular strategy evolved to cope with high flight-associated cellular stress, ensuring prompter clearance of any damaged organelles and protein aggregates. This tonic activation of autophagy may have afforded superior stress adaptive abilities that in turn favoured the evolution of unique longevity observed within this taxonomic clade. Finally, we isolated a bat transcriptional signature of naturally-evolved healthy aging and integrated it with all the other results we generated to instruct an innovative in silico drug repositioning platform. Our data mosaiced a unique expression patterns, including molecules and pathways previously not directly involved in aging pathophysiology. The signature is being used in both data-driven and unbiased drug repositioning approaches to develop novel pharmacological strategies against human aging and age-related diseases.

Global life expectancy has significantly increased, but this has been associated with a rise in age-related diseases (ARDs), such as cancer, type 2 diabetes (T2D) and obesity. These conditions are characterized by chronic/low-grade inflammation and immune system remodeling. Cellular senescence drives both metabolic and immune aging; however, its role in shaping immune system alterations in individuals with obesity and/or T2D remains unclear. We performed an immune phenotypic characterization of obese subjects (T2D or normoglycemic) to address whether metabolic dysregulation and/or obesity could drive immune senescence in specific CD4⁺ T cell lineages. We also interrogated our previously generated DNA methylome from human PBMCs to investigate potential epigenetic alterations associated with immune senescence. Our findings suggest that obesity, particularly when combined with T2D, accelerates immune senescence in both young and elderly individuals, as testified by the increased expression of immune senescence-associated markers and molecular pathways in proinflammatory T cells of obese individuals. These findings highlight a potential mechanism by which metabolic dysfunction contributes to age-related immune decline. Furthermore, the observed CpG hypomethylation at senescence-specific gene loci raises the possibility that epigenetic modifications may contribute to transcriptional dysregulation associated with immune aging in obesity. These insights may guide future strategies to target immune senescence in metabolic diseases.