DTA

Digital Theses Archive

 

Tesi etd-03252023-103254

Type of thesis
Dottorato
Author
MIGNUCCI, VALERIO
URN
etd-03252023-103254
Title
Characterization of the effects of hypergravity on tau aggregation in a neuronal cell model in vitro.
Scientific disciplinary sector
BIO/11
Course
Istituto di Scienze della Vita - PHD IN MEDICINA TRASLAZIONALE
Committee
relatore Prof.ssa ANGELONI, DEBORA
Keywords
  • HT22
  • Hypergravity
  • Neurodegeneration
  • TAU
Exam session start date
06/12/2023;
Availability
parziale
Abstract
Tau is a microtubule-associated protein (MAP) highly expressed in neurons where it binds and stabilizes microtubules, necessary for proper cellular functions. When tau is deregulated, mainly at the post-translational level, it tends to aggregate into neurofibrillary tangles (NFTs), a notable lesion in several neurodegenerative disorders. The impact of mechanical forces, including the force of gravity, on neuronal cells is poorly investigated although some modifications, mainly at the level of cytoskeleton, are well documented. Moreover, although the impact of gravity on protein aggregation in solution was observed, no studies were conducted to evaluate the effects of hypergravity stimulation in an in vitro model of neurodegeneration. Therefore, the aim of this project is to evaluate how gravitational loading affects our in vitro model, tested with different hypergravity protocols. The HT22 cell line was used as neuronal model. Gravitational loading was obtained thanks to the Large Diameter Centrifuge (LDC) located at ESA-ESTEC facility, and bench centrifuges in the home lab. Hypergravity protocols consisted of gravitational loading of 50g, 20g and 10g for three hours. Neurodegeneration was obtained in vitro with transfection in HT22 cell of the plasmid encoding the conformational-sensitive tau (CST) sensor, carrying an aggregation-prone mutation (P301S), and the transfection of NFTs seeds. HT22 cells tolerated the hypergravity protocols without detrimental effects in cell viability. The analyses of physical aspects of cells and nuclei revealed no significative change in terms of size and shape upon this stimulation. At the level of the cytoskeleton, cells treated with hypergravity showed mild differences in the intensity of fibrillary actin. Hypergravity treatments did not determine a change in the proportion of the various found in HT22 transfected with CST and NFTs phenotypes (no aggregates, small number of aggregates, high number of aggregates). Interestingly, we found a significative reduction in the dimension of CST aggregates in HT22 treated simultaneously with gravitational loading the drug PD-901, with a role in the inhibition of ERK. These effects were dose-dependent. The observation about the impact of combining treatments with the kinase inhibitor PD-901 and hypergravity in the reduction of tau aggregates in HT22 cells lays the foundation for other experiments to evaluate if this phenotype implies a functional positive outcome.
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