Leader: Daniela Monti (UNIFI); Others collaborator(s): Elisa Bientinesi, Sara Ristori and Gianmarco Bertoni
The project aims: 1) to study cell senescence by different stimuli and the composition of SASP in primary dermal fibroblasts from subjects of various ages. Soluble SASP factors (sSASP), small extracellular vesicles (EVs) production and their molecular cargo will be characterized and their effects investigated on proliferating fibroblasts and cancer cells; 2) to analyze the proteomic and metabolomic profiles of sSASP and EVs to identify new aging and disease biomarkers; 3) to study the effects of potential senomorphic/senolytic drugs on sSASP and EVs; 4) to investigate β-Amyloid-Induced senescence in human astrocytes of Alzheimer's disease and healthy subjects and characterize sSASP factors and EVs and their effects on neurons.
Brief description of the activities and intermediate results: We conducted studies on primary astrocyte cultures obtained from both healthy (H) and Alzheimer's disease (AD) subjects who were exposed to Aβ1-42 oligomers (characterised in collaboration with Chitis's laboratory, WP3). According to the preliminary data, the internalisation of Aβ1-42 oligomers was similar in both AD- and H-astrocytes. We also found that female astrocytes in both groups showed greater internalisation than males. Next, we evaluated whether oligomeric species could cause dysregulation of cytosolic Ca2+ homeostasis in AD- and H-astrocytes. The preliminary observations revealed increased intracellular Ca2+ levels, similar in both groups and did not show sex differences. Interestingly, the Ca2+ increase induced only in H-astrocyte apoptosis, while a comparable number of AD-astrocytes entered senescence. Female AD-astrocytes exhibited the highest number of senescent cells. Furthermore, we cultured differentiated neuroblastoma cells with conditioned media from astrocytes of Alzheimer's patients and healthy individuals treated with oligomers. Only the supernatant from Alzheimer's astrocytes reduced the viability of neuroblastoma cells. The AD senescent astrocytes partly produce SASP factors that can harm neuroblastoma cells. Therefore, the induced senescent phenotype in AD-astrocytes could contribute to neuroinflammation, leading to the progression and worsening of the disease, especially in women. These preliminary results will be presented at Age-It General meeting.
Brief description of the activities and intermediate results: In the last months, we have continued our studies on astrocytes from both healthy individuals and those with Alzheimer's disease (AD) who were exposed to Aβ oligomers. This work, in collaboration with the Chiti group (Spoke 2, WP2), has demonstrated that these oligomers elicit distinct responses in astrocytes, inducing apoptosis in healthy astrocytes and senescence in AD astrocytes, particularly in female AD subjects. We also confirmed that the expression of senescence-associated secretory phenotype (SASP) factors did not differ significantly between treated astrocytes from healthy individuals and those with AD. However, only the conditioned medium from treated AD astrocytes was found to induce apoptosis in differentiated neuroblastoma cells. We are conducting metabolomic studies with the Tenori group (Spoke 2, WP3). We are also investigating the role of the proteasome by analysing its activity following treatment with Aβ oligomers in both case series.
Brief description of the activities and intermediate results: During this period, we published a paper studying senescence induced by Doxorubicin in human WI-38 fibroblasts. Our findings showed that Doxo-induced senescent fibroblasts produced several pro-inflammatory SASP factors that increased autophagy. We investigated the senolytic effects of quercetin, a bioactive flavonoid, and found that it reduced autophagy, heightened ER stress, and partially induced apoptosis in senescent fibroblasts. Notably, we found that quercetin kills part of senescent fibroblasts, and the conditioned medium from these fibroblasts decreases the invasiveness of osteosarcoma cells and diminishes the pro-tumour effects of senescent cells. The diminished autophagic activity and heightened ER stress, a previously undescribed effect of quercetin, represent a new vulnerability of Doxo-induced senescent fibroblasts (doi: 10.1016/j.mad.2024.111957). In the coming months, we plan to investigate the new mechanism of quercetin on replicative senescence to determine whether it could be a general mechanism or specific to Doxo-induced senescence. Additionally, we will induce senescence in human primary fibroblasts from patients of various ages to examine the potential differences in the senescent phenotype and the action of senolytic substances (in collaboration with the Salvioli group, Spoke 3).
We also continued our research on astrocyte
Brief description of the activities and intermediate results: As mentioned in the previous report, we recruited a research assistant through a 14-month research grant this trimester. This will allow us to extend our activities related to senescence in human primary fibroblasts from individuals of various ages. We have continued our studies on astrocytes from healthy individuals and those with Alzheimer's disease (AD) exposed to Aβ oligomers. Our focus is on assessing the impact of these oligomers on proteasome function. We evaluated proteasome activity at 48 and 120 hours and measured the expression of key catalytic subunits (β1, β5) at 24, 48, 72, and 120 hours of treatment. Our results show that proteasome activity was significantly increased in treated astrocytes from healthy subjects (HS) at both time points compared to the control group. In contrast, astrocytes from AD patients exhibited reduced activation. The expression of the catalytic β1 subunit remained unchanged in both groups, except at 120 hours, when it was significantly upregulated in HS-astrocytes compared to AD-astrocytes. Additionally, the expression of the β5 subunit differed significantly between treated AD and HS astrocytes at all time points. Specifically, HS-astrocytes showed a significant increase in β5 expression at 48 and 120 hours compared to both control and AD astrocytes. On the other hand, AD astrocytes exhibited a significant increase in β5 expression only at 72 hours compared to both the control and HS-astrocytes. Data suggest that astrocytes from healthy subjects more effectively eliminate oligomers than those from Alzheimer's patients, likely due to enhanced proteasome activation.
Brief description of the activities and intermediate results: We have continued our studies on astrocytes from healthy donors (HS) and those with Alzheimer's disease (AD) exposed to Aβ oligomers. Our research aims to clarify the role of increased intracellular calcium in both groups following treatment; however, we demonstrated that treated astrocytes display apoptosis only in the HS group. We also assessed mitochondrial membrane potential using the JC-1 assay after 48, 72, and 120 hours of treatment. Interestingly, we observed significant mitochondrial depolarisation in astrocytes from both HS and AD after 48 and 72 hours. However, the depolarisation was noticeably more pronounced in HS astrocytes compared to AD astrocytes. No changes were observed at 120 hours. Additionally, senescence has only been previously noted in AD astrocytes at 120 hours. Nevertheless, we observed an unexpected increase in the expression of SASP in both HS and AD astrocytes following treatment. Consequently, we explored the possibility that the increased SASP factors in HS astrocytes could be linked to a chronic state of activation induced by Aβ1-42. This activation is often associated with various morphological and functional changes, including increased cell proliferation and the release of proinflammatory factors. We performed cell counts at 48, 72, and 120 hours after treatment. At 48 and 72 hours, the percentage of treated HS astrocytes significantly decreased compared to the control, consistent with previous reports indicating an increase in apoptotic cells. However, at 120 hours, the number of treated HS astrocytes exceeded that of the control, suggesting recovery and compensatory hyperproliferation. The cell count at 120 hours was significantly higher than at 48 and 72 hours. In contrast, treated AD astrocytes showed a significant reduction in cell number at both 48 and 120 hours. The presence of hyperproliferative reactive astrocytes in HS was further supported by increased expression of GFAP and Vimentin, as well as a significant positive correlation between GFAP fluorescence intensity and the number of Ki-67-positive cells, a marker of proliferation.
Within the framework of the PNRR-funded project:
- We continued our investigation into the role of astrocytes in Alzheimer’s disease (AD), focusing on their response to Aβ1-42 oligomers. Our findings confirmed that both healthy (HS) and AD astrocytes internalise Aβ1-42 through endocytosis and alternative pathways, with higher uptake observed in female AD cells. AD astrocytes exhibited impaired proteasome activity, resistance to apoptosis, and clear signs of cellular senescence—particularly in female donors—while HS astrocytes experienced a transient apoptotic phase followed by reactive gliosis and compensatory proliferation. Inflammatory profiling revealed distinct secretory phenotypes between HS and AD astrocytes, with the latter producing a pro-inflammatory, neurotoxic environment. Conditioned media from AD astrocytes reduced neuronal viability in vitro, confirming their pathogenic potential. These results, achieved thanks to PNRR support, build on previous findings and offer new insights into astrocyte-driven mechanisms in AD, supporting the development of glia-targeted therapeutic approaches. The paper is accepted with revisions and is currently being revised.
- We developed an in vitro model of astrocyte senescence using low-dose gemcitabine (1 µM) in primary cells from healthy and Alzheimer’s disease (AD) donors. Preliminary results show higher susceptibility to senescence in AD astrocytes, with increased SA-β-Gal activity and DNA damage markers. This model may help investigate astrocyte aging in AD and screen senescence-targeting therapies.
- In parallel, and as part of the broader research activities supported by the project, we published a scientific review focused on the role of nutraceuticals and functional foods as modulators of cellular senescence, with implications for neurodegenerative diseases. The review highlights the antioxidant, anti-inflammatory, and epigenetic actions of bioactive dietary compounds—such as polyphenols, vitamins, and spices—and their ability to modulate senescence-associated secretory phenotype (SASP) factors, enhance mitochondrial function, and reduce oxidative stress. While promising preclinical data exist, the review emphasises the need for more direct evidence linking nutraceuticals to reductions in senescent cell burden. This publication contributes to the dissemination of knowledge on non-pharmacological strategies for promoting healthy aging and delaying neurodegeneration, aligning with the goals of the PNRR programme.
Ongoing scientific collaborations:
- In collaboration with Prof. Salvioli (Spoke 3), I contributed to a study investigating plasma biomarkers (GDF15, FGF21, sRAGE, NfL) as indicators of frailty in older adults. In a cohort of 463 subjects (50–113 years), we found that GDF15, NfL, and FGF21 were significantly associated with frailty and survival. A machine learning model using these biomarkers predicted frailty with 82% accuracy and improved survival prediction, supporting their potential as complementary tools to traditional frailty assessments. This study has been the subject of a scientific publication in Mech. Ageing Dev. 2025
- In partnership with Marco Malavolta (INRCA), we have also submitted a review article examining the therapeutic potential of marine-derived bioactive molecules in ageing and age-related diseases. These compounds, characterised by structural uniqueness and multitarget effects, are emerging as promising agents with antioxidant, anti-inflammatory, and cytoprotective properties. The review emphasised their ability to modulate ageing hallmarks—such as cellular senescence, genomic instability, and impaired autophagy—and their potential to prevent or treat conditions including neurodegenerative, cardiovascular, metabolic, and pulmonary diseases. While many demonstrate preclinical efficacy, their clinical translation remains limited, underscoring the need for further research. (submitted to ARR)
Within the framework of the PNRR-funded project:
- The study on the role of astrocytes in Alzheimer’s disease (AD), with particular focus on their response to Aβ1–42 oligomers, was submitted to Mechanisms of Ageing and Development; the peer-review process is now complete, and the manuscript has been published. Future studies will build on these findings to further explore the molecular and functional aspects of astrocyte reactivity and senescence, with particular emphasis on the mechanisms, regulation, and pathological consequences of astrocyte hyperproliferation in neurodegeneration.
- We created an in vitro model of astrocyte senescence by treating primary cells from healthy and Alzheimer’s disease donors with low doses of gemcitabine. Gemcitabine (IC50 = 1.64 μM) reduced astrocyte viability at sub-toxic doses (0.25 to 1 μM) in both groups, with AD astrocytes more sensitive. Cell counts showed the cells stopped growing rather than dying in large numbers, though late apoptosis or necrosis increased by about 20%. Cell cycle analysis showed a lasting G1-phase arrest that persisted after the drug was removed. Gemcitabine also caused the cells to become senescent, as shown by more SA-β-gal-positive cells and higher senescence in AD astrocytes. We chose a dose of 0.5 μM for further experiments because it produced similar senescence with less toxicity. At this dose, DNA damage and markers of senescence (γH2AX, macroH2A.1) were much higher.
- The findings from these studies were disseminated through three poster presentations at the SIPmET Congress, held in Pavia from 18-20 September 2025.
Ongoing scientific collaborations:
- In collaboration with Marco Malavolta (INRCA), we authored a review on the therapeutic potential of marine-derived bioactive molecules in ageing and age-related diseases, which has been published in Ageing Research Reviews, a prestigious, high-impact journal in the field of ageing research.
Within the framework of the PNRR-funded project:
- Studies using this in vitro astrocyte model were further extended, and we demonstrated that gemcitabine induces a marked increase in oxidative stress. Specifically, intracellular reactive oxygen species (ROS) levels were significantly elevated after 48 and 72 hours of treatment, with up to a 4-fold increase in healthy donor–derived astrocytes and a 6-fold increase in astrocytes from Alzheimer’s disease patients. These findings indicate that oxidative stress is a key contributor to the gemcitabine-induced senescent response, acting in concert with its established role in enforcing cell-cycle arrest. Ongoing studies aim to uncover the molecular mechanisms underlying this phenotype. This model provides a robust platform to study astrocyte senescence in Alzheimer’s disease and to accelerate the identification of senescence-targeting therapeutic strategies.
- The findings from these studies were disseminated through three presentations at the 2ND General Meeting of Age-IT, held in Naples on 29-31 October 2025
- In collaboration with several researchers from the Age.it Project and colleagues from different universities, we published a study in Geroscience. The work shows that Italian centenarians display a significantly higher contribution of Western Hunter-Gatherer–related ancestry, suggesting that ancient pre-Neolithic genetic components may positively influence human longevity.
Publications
- Prattichizzo F, Frigé C, Pellegrini V, Scisciola L, Santoro A, Monti D, Rippo MR, Ivanchenko M, Olivieri F, Franceschi C. Organ-specific biological clocks: Ageotyping for personalized anti-aging medicine. Ageing Res Rev. 2024 Apr;96:102253. doi: 10.1016/j.arr.2024.102253. Epub 2024 Mar 4. PMID: 38447609.
- Bientinesi E, Ristori S, Lulli M, Monti D. Quercetin induces senolysis of doxorubicin-induced senescent fibroblasts by reducing autophagy, preventing their pro-tumour effect on osteosarcoma cells. Mech Ageing Dev. 2024 Aug;220:111957. doi: 10.1016/j.mad.2024.111957. Epub 2024 Jun 21. PMID:38909661.
- Ristori S, Bertoni G., Bientinesi E., Monti D. The Role of Nutraceuticals and Functional Foods in Mitigating Cellular Senescence and Its Related Aspects: A Key Strategy for Delaying or Preventing Aging and Neurodegenerative Disorders. Nutrients. 2025 May 28;17(11):1837. doi: 10.3390/nu17111837.
- Conte M, Sevini F, Conte G, Tognocchi M, Ciurca E, Trofarello L, Chiariello A, Capri M, Franceschi C, Monti D, Di Rosa M, Cherubini A, Olivieri F, Salvioli S. The combination of GDF15, FGF21, sRAGE and NfL plasma levels can identify frailty in community-dwelling people across old age. Mech Ageing Dev. 2025 Aug;226:112077. doi: 10.1016/j.mad.2025.112077. Epub 2025 Jun 4.
- Ristori S., Bertoni G., Bigi A., Cecchi C., Sollazzo M., Iommarini L., Monti D., Bientinesi E. Human astrocytes from healthy individuals and Alzheimer’s patients respond differently to Aβ1-42 oligomers, triggering distinct paths of reactivity and senescence. Mech Ageing Dev. 2025 Dec;228:112116. doi: 10.1016/j.mad.2025.112116. Epub 2025 Sep 24.
- Giuliani M.E., Giordani C., Francini E., Bigossi G., Lai S., Marcozzi S., Alimi S.S., Perini D., Piccinini A., Bertoni G., Ristori S., Bientinesi E., Barone F., Cardelli M., Indiveri C., Scalise M.F., Monti D., Malavolta M. Ageing Res Rev. 2026 Jan;113:102935. doi: 10.1016/j.arr.2025.102935. Epub 2025 Nov 5.
- Sarno S, Iannuzzi V, Sazzini M, Pirazzini C, Bacalini MG, Gentilini D, Passarino G, Mari D, Monti D, Nacmias B, Sorbi S, Pettener D, Luiselli D, Franceschi C, Garagnani P, Giuliani C. Western Hunter-Gatherer genetic ancestry contributes to human longevity in the Italian population. Geroscience. 2025 Dec 13. doi: 10.1007/s11357-025-02043-4. Epub ahead of print. PMID: 41389162.
Oral Communications
- S. Ristori, E. Bientinesi, G. Bertoni, A. Bigi, D. Monti. Differential Responses of Human Astrocytes to Amyloid β1-42 Treatment: a comparative study between astrocytes from healthy individuals and Alzheimer's patients. 2ND General Meeting Age-IT, 2025, 29-31 October, Naples
- E. Bientinesi, S. Ristori, G. Bertoni, M. Lulli, D. Monti. Quercetin induces senolysis of doxorubicin-induced senescent fibroblasts by reducing autophagy, preventing their pro-tumour effect on osteosarcoma cells. 2ND General Meeting Age-IT, 2025, 29-31 October, Naples
- G. Bertoni, S. Ristori, E. Bientinesi, D. Monti. Modelling Astrocyte Senescence in Alzheimer’s Disease Using Gemcitabine: Differential Responses in AD and Healthy Cells. 2ND General Meeting Age-IT, 2025, 29-31 October, Naples
- E. Bientinesi, S. Ristori, M. Lulli, D. Monti. Quercetin exerts a senolytic effect on Doxorubicin-induced senescent fibroblasts by reducing autophagy, thereby preventing the pro-tumour effect on osteosarcoma cells: SiPMet, 2024, 19-21 September, Udine.
- E. Bientinesi, S. Ristori, A. Bigi, C. Cecchi, F. Chiti, D. Monti. Different effects of Aβ oligomers on human astrocytes from healthy subjects and Alzheimer’s disease patients. General Meeting Age-It, 2024, 20-22 May, Venice.
Poster Presentations
- S. Ristori, E. Bientinesi, G. Bertoni, A. Bigi, D. Monti. Impact of Aβ oligomers on human astrocytes derived from healthy individuals and Alzheimer's patients. SIPMeT Translational Pathophysiology, 2025, 18-20 September, Pavia
- G. Bertoni, S. Ristori, E. Bientinesi, D. Monti. Gemcitabine Promotes Astrocyte Senescence: A Comparative Study Between Alzheimer's Disease and Healthy Cells. SIPMeT Translational Pathophysiology, 2025, 18-20 September, Pavia
- E. Bientinesi, S. Ristori, G. Bertoni, D. Monti. Aβ1-42- induced senescence in Alzheimer's Astrocytes drives neuronal death via SASP and metabolic dysregulation. SIPMeT Translational Pathophysiology, 2025, 18-20 September, Pavia
- E. Bientinesi, S. Ristori, M. Lulli, D. Monti. Quercetin triggers senolysis in doxorubicin-induced senescent fibroblasts through autophagy inhibition, limiting their pro-tumour effects on Osteosarcoma cells. Age Florence, 2024, 19 December, Florence.
- S. Ristori, E. Bientinesi, A. Bigi, D. Monti. Effects of Aβ oligomers on human primary astrocytes obtained from healthy subjects and Alzheimer’s disease patients. Age Florence, 2024, 19 December, Florence.
- E. Bientinesi, S. Ristori, M. Lulli, D. Monti. Quercetin exerts a senolytic effect on Doxorubicin-induced senescent fibroblasts by reducing autophagy, thereby preventing the pro-tumour effect on osteosarcoma cells. SiPMet, Translational Pathology, 2024, 19-21 September, Udine.
- E. Bientinesi, S. Ristori, D. Monti. Aβ-induced senescence in astrocytes from Alzheimer’s disease patients contributes to neuronal death through senescence-associated secretory phenotype (SASP) and metabolic changes. SIPMeT, Translational Pathology, 2024, 19-21 September, Udine.
- S. Ristori, E. Bientinesi, A. Bigi, M. Lulli, D. Monti. Astrocyte senescence as a pathogenetic mechanism underlying sex differences in Alzheimer’s disease onset. SIPMeT, Translational Pathology, 2024, 19-21 September, Udine.
- E. Bientinesi, S. Ristori, M. Lulli, D. Monti. Quercetin triggers senolysis of pro-tumor doxorubicin-induced senescent fibroblasts by targeting autophagy. SIPMeT Young Scientist Meeting, General Pathology: the trunk of the tree of medicine, 2023, 22-23 September, Parma.
- S. Ristori, E. Bientinesi, M. Lulli, D. Monti. Astrocyte senescence as a pathogenetic mechanism underlying sex differences in Alzheimer’s disease onset. SIPMeT Young Scientist Meeting, General Pathology: the trunk of the tree of medicine, 2023, 22-23 September, Parma.
- E. Bientinesi, S. Ristori, M. Lulli, D. Monti. Quercetin triggers senolysis of pro-tumor doxorubicin-induced senescent fibroblasts by targeting autophagy: bad news for osteosarcoma cells. Gordon Research Conference and Seminar, Evolving Concepts of Organismal Ageing, 2023, 1-7 July, Castelldefels, Barcelona.
- S. Ristori, E. Bientinesi, M. Lulli, D. Monti. Astrocyte senescence in Alzheimer’s disease as a pathogenetic mechanism underlying sex differences. Gordon Research Conference and Seminar, Evolving Concepts of Organismal Ageing, 2023, 1-7 July, Castelldefels, Barcelona.