Leader: Daniela Trisciuoglio (CNR); Other collaborator(s):
We will investigate the interplay between Ca2+ signaling and autophagy in cell homeostasis in various models of aging. By using neuronal explants or MEFs isolated from healthy mice and primary human fibroblasts, we will study the role played by ATG4, a critical autophagy gene, in the context of cellular senescence and we will investigate the intracellular machinery that links Ca2+ signaling to the regulation of ER/mitochondrial stress and autophagy, focusing on the role of Sorcin and Sigma1 Receptor. By applying a multidisciplinary approach, we aim at identifying new compounds with the potential to counteract cellular senescence. We will also generate innovative cellular and animal models to study the therapeutic potential of newly-identified molecules and to assess whether modulation of autophagy and Ca2+ might rescue aging-like phenotypes.
Brief description of the activities and of the intermediate results: ATM deficiency recapitulates most of the cellular senescence hallmarks like telomere attrition, mitochondrial dysfunction, epigenetic alteration, genomic instability, SASP and proteostasis dysregulation. We have employed a cell model system of lymphoblastoid cell lines derived from healthy donors and ATM-/- patients. In this cell context we investigated the ATM/ATG4 axis finding that ATG4C variants expression levels are controlled by ATM, and that ATG4C overexpression in a ATM -/- context rescues cell proliferation, survival to oxidative stress and mitochondrial functionality. Surprisingly, although ATG4 controls early steps of autophagic process, its overexpression only partially rescues autophagy in ATM-/- cells. We collected preliminary evidence that ATG4C could be transcriptionally regulated by oxidative stress independently on ATM. Moreover, IF analysis showed that ATG4C overexpression protect telomeres from DNA damage. Notably, IF analysis of ATG4C/D also showed the presence of nuclear spots which occasionally colocalize with telomere signals. We next measured the expression of long non-coding RNAs called TERRA (Telomere Repeats containing RNAs) transcribed at subtelomeric loci, in ATM +/+ and ATM-/- conditions, finding that TERRAs are hyper transcribed in ATM-/- cells compared to controls, suggesting that these molecules could be employed as senescence biomarkers.Aging is strongly correlated to the onset of brain diseases and pathologies. One of the most important problems in drug administration for brain diseases is the presence of the blood-brain barrier (BBB), that prevents most drugs from entering the brain. We designed and produced a ferritin-based stimuli-sensitive nanocarrier with high biocompatibility and water solubility. It can incorporate high amounts of the potent topoisomerase 1 inhibitor Genz-644282. We showed that this nanocarrier, named The-0504, can cross the BBB and specifically can deliver the payload to gliomas that express high amounts of the ferritin/transferrin receptor TfR1 (CD71). Both intranasal and intravenous administration of The-0504 reduce tumor growth and improve the survival rate of glioma-bearing mice. However, nose-to-brain administration is a simpler and less invasive route that may spare most of the healthy tissues compared to intravenous injections. For this reason, the data reported in could pave the way towards a new, safe, and direct drug delivery method for brain pathologies.
In order to investigate the role of autophagy in age-related disease, the Ataxia Telangiectasia (ATM) cell model system was employed. The deficiency of ATM in this pathology is characterised by the recapitulation of the hallmarks of cellular senescence. A cell model system comprising lymphoblastoid cell lines derived from healthy donors and ATM-deficient patients has been employed. In this cellular context, we investigated the role of the ATM/ATG4 axis. Our findings revealed that ATG4C variant expression levels are regulated by ATM, and that ATG4C overexpression in an ATM-/- context enhances cell proliferation, survival to oxidative stress and mitochondrial functionality. It was unexpected that, despite ATG4 regulating the initial stages of autophagy, its overexpression only partially restored autophagy in ATM-/- cells. The preliminary evidence suggests that ATG4C may be transcriptionally regulated by oxidative stress independently of ATM. Furthermore, immunofluorescence analysis demonstrated that ATG4C overexpression provides protection against DNA damage to telomeres. It is noteworthy that immunofluorescence analysis of ATG4C/D also demonstrated the presence of nuclear spots that occasionally colocalised with telomere signals. We proceeded to quantify the expression of long non-coding RNAs designated TERRA (Telomere Repeats Containing RNAs), which are transcribed at subtelomeric loci, in both ATM +/+ and ATM-/- conditions. Our findings revealed that TERRAs are significantly overexpressed in ATM-/- cells relative to controls, suggesting that these molecules may serve as potential biomarkers for senescence.
A further objective of this study is to examine the function of ATAT1, an enzyme involved in the acetylation of microtubules, in the cellular senescence of retinal pigment epithelial cells (RPE), which is a prominent factor in age-related macular degeneration. The integrity of the cytoskeleton is associated with a number of cellular functions, including migration, proliferation, degeneration and mitochondrial bioenergy production. It is also linked to a range of chronic disorders, such as neuronal degeneration and premature ageing. Consequently, human RPE cell lines were utilised to examine the expression of certain senescence markers in response to hydrogen peroxide (e.g the expression of two proteins, p21 and p16; the accumulation of beta galactosidase; and the levels of phosphorylated H2AX and tubulin acetylation in K40). The same approach was employed to characterise the RPE hTERT cell line that had been modified to silence ATAT1 protein expression, which is responsible for tubulin acetylation in K40. The silencing of ATAT1 has been observed to result in the modulation of several autophagic markers. It is noteworthy that ATAT1-silenced cells exhibited a markedly elevated level of reactive oxygen species (ROS), accompanied by an increase in lipid peroxides and alterations in mitochondrial dynamics. Further experiments are being conducted to elucidate the molecular mechanisms underlying senescence induced by ATAT1 depletion and the cross-talk between autophagy induction and senescence. (ref: Iuzzolino A et al. The α-tubulin acetyltransferase ATAT1: structure, cellular functions, and its emerging role in human diseases. Cell. Mol. Life Sci. 81, 193 (2024). https://doi.org/10.1007/s00018-024-05227-x)
Aging is also strongly correlated to the onset of brain diseases and pathologies. One of the most important problems in drug administration for brain diseases is the presence of the blood-brain barrier (BBB), that prevents most drugs from entering the brain. We designed and produced a ferritin-based stimuli-sensitive nanocarrier with high biocompatibility and water solubility. It can incorporate high amounts of the potent topoisomerase 1 inhibitor Genz-644282. We showed that this nanocarrier, named The-0504, can cross the BBB and specifically can deliver the payload to gliomas that express high amounts of the ferritin/transferrin receptor TfR1 (CD71). Both intranasal and intravenous administration of The-0504 reduce tumor growth and improve the survival rate of glioma-bearing mice. However, nose-to-brain administration is a simpler and less invasive route that may spare most of the healthy tissues compared to intravenous injections. For this reason, the data reported in could pave the way towards a new, safe, and direct drug delivery method for brain pathologies (ref.: Marrocco et al., Nose-to-brain selective drug delivery to glioma via ferritin-based nanovectors reduces tumor growth and improves survival rate, Cell Death & Disease 2024 Apr 13;15(4):262. doi: 10.1038/s41419-024-06653-2.)
Recently it has been reported that the ATG4s isoform predominantly expressed in the brain is ATG4D and some of its variants are associated with neurodegenerative phenotypes and are involved in oxidative stress-induced cell death. Interestingly, we found that expression of ATG4D in A-T cells rescue oxidative response defects of A-T and that ATG4D pathogenic variants are involved in cerebellar degeneration through regulation of ATM dependent oxidative stress-induced senescence.
A further objective of this study is to examine the function of ATAT1, an enzyme involved in the acetylation of microtubules, in the cellular senescence of retinal pigment epithelial cells (RPE), which is a prominent factor in age-related macular degeneration. The integrity of the cytoskeleton is associated with a number of cellular functions, including migration, proliferation, degeneration and mitochondrial bioenergy production. It is also linked to a range of chronic disorders, such as neuronal degeneration and premature ageing. Consequently, human RPE cell lines were utilised to examine the expression of certain senescence markers in response to hydrogen peroxide (e.g the expression of two proteins, p21 and p16; the accumulation of beta galactosidase; and the levels of phosphorylated H2AX and tubulin acetylation in K40). The same approach was employed to characterise the RPE hTERT cell line that had been modified to silence ATAT1 protein expression, which is responsible for tubulin acetylation in K40. Cells silenced for ATAT-1 exhibited increased susceptibility to hydrogen peroxide demonstrating that ATAT-1, through acetylation of tubulin, exerts a protective action against oxidative stress and cellular senescence. Moreover, the silencing of ATAT1 has been observed to result in the modulation of several autophagic markers. It is noteworthy that ATAT1-silenced cells exhibited a markedly elevated level of reactive oxygen species (ROS), accompanied by an increase in lipid peroxides and alterations in mitochondrial dynamics. We are now performing comprehensive proteomic analysis to characterize and define the ATAT-1-dependent acetylome, identifying the specific protein substrates and acetylation sites regulated by the alpha-tubulin acetyltransferase ATAT-1. This study have employed advanced mass spectrometry-based proteomics to compare acetylation profiles between wild-type and ATAT-1-deficient models, thereby elucidating the full spectrum of ATAT-1-mediated protein acetylation beyond its canonical role in tubulin modification.