Thesis etd-05022017-130118 |
Link copiato negli appunti
Thesis type
Tesi di laurea magistrale
Author
OLLA, IVANA
URN
etd-05022017-130118
Thesis title
Screening of potentially therapeutic compounds against Tau pathology by exploiting the CST reporter system
Department
BIOLOGIA
Course of study
BIOLOGIA APPLICATA ALLA BIOMEDICINA
Supervisors
relatore Dott.ssa Di Primio, Cristina
relatore Prof. Cattaneo, Antonino
relatore Prof.ssa Ori, Michela
controrelatore Prof.ssa Raffa, Vittoria
relatore Prof. Cattaneo, Antonino
relatore Prof.ssa Ori, Michela
controrelatore Prof.ssa Raffa, Vittoria
Keywords
- hyperphosphorylation
- Tau
Graduation session start date
29/05/2017
Availability
Full
Summary
Tau is a neuronal microtubule-associated protein (MAP), since its key biological role is promoting of assembly and stability of microtubules (MTs)1 in neurons. This ability depends on Tau degree of phosphorylation, which is the result of the action of both protein kinases and protein phosphatases on it. However, hyperphosphorylated Tau detaches from MTs, undergoes missorting and tends to constitute intracellular inclusions known as paired helical filaments (PHFs), which are successively converted into neurofibrillary tangles (NFTs). Such abnormalities are characteristics of a family of neurodegenerative disorders known as Tauopathies, which include Alzheimer’s Disease (AD) 2. Despite Tau protein constitutes a promising therapeutic target for the treatment of such pathologies3, to date, no drugs are available to cure or prevent them. Here, I developed a new setup to screen potential therapeutic compounds against Tau pathology exploiting the CST biosensor, a chimerical protein built on the Tau full-length, where a CFP and a YFP are fused with the N-terminal and the C-terminal of the protein, respectively. Depending on Tau conformation, the fluorophores of the sensor can generate a FRET signal, providing information on the Tau status in different experimental condition. Neuroblastoma cells transfected with the CST and differentiated with retinoic acid, correctly express the sensor, which co-localizes with the MTs network, constituting a neuronal CST reporter system. When associated to MTs, the CST generates a FRET signal, suggesting that when Tau binds to MTs assumes a loop-like conformation. In order to develop Tau missorting assay setup and to investigate both the homotypic and the heterotypic seeding effects on Tau, differentiated SH-SY5Y cells expressing the CST have been treated with Tau oligomers or Aβ-oligomers (AβOs), respectively. The CST sensor is released into the cytoplasm, mislocalizes in the somatodendritic compartment of the cells and tends to constitute little aggregates, reproducing what happens to Tau in pathological contexts, when cells are treated with Tau seeds. However, no effect on the CST sorting can be detected after the treatment with AβOs. Since Tau results hyperphosphorylated in pathological condition, selected kinases inhibitors have been tested exploiting the developed setup. First, DJNKI-1, a selective JNK inhibitor, has been analyzed. Such inhibitor, administrated after the induction of an homotypic seeding via Tau seeds, restores the CST sorting and its attachment to MTs. Moreover, the treatment with DJNKI-1 reduces the phosphorylation level of the CST at AD-related sites. Therefore, DJNKI-1 shows a potential therapeutic ability. On the other hand, following the treatment with AβOs, DJNKI-1 do not alter the CST attachment to MTs and its sorting along processes, which results increased. Successively, PD0325901, a selective MEK/ERK inhibitor never tested in relation to AD, has been analyzed. Following the treatment with Tau seeds, PD0325901 restores the CST binding to MTs and its physiological sorting. Following the treatment with AβOs, PD0325901 enlarges the biosensor distribution along processes. Therefore, PD0325901 may have a therapeutic effect as well. In conclusion, these setups constitute a new tool to analyze the effects of selected compounds on Tau in living cells.
File
Nome file | Dimensione |
---|---|
Tesi_definitiva.pdf | 3.11 Mb |
Contatta l’autore |