Leader: Daniela Monti (UNIFI); Other 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.
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