Development and characterisation of PC12 cell lines allowing inducible expression of prion proteins carrying pathogenic mutations

• Main Author: Quaglio, Elena
• Format: Dissertation
• Language: English
• Published: Open University 2005
• DOI: 10.21954/ou.ro.000101c0

Inherited prion diseases are linked to mutations in the prion protein (PrP)p gene that are presumed to favor conversion of PrP into a neurotoxic isoform (PrpSc) Several cellular models of inherited prion diseases have been Q developed in which mutant PrP costitutively expressed acquires PrPSc-like properties but is not cytotoxic. However, the use of constitutive models does not exclude the potential toxicity of mutant PrP, as it can be speculated that only J clones resistant to PrP toxicity are selected after clonal selection. To rule out n this possibility a tetracycline-inducible (Tet-on) model was developed in this thesis, in which PrP expression is switched on after clonal selection. cDNAs encoding mouse wild-type PrP, as well as mouse PrP homologues of the human D178N (D177N/M128 and D177NA/128) and nine-octapeptide (PG14) mutations under the control of the tetracycline-responsive element, were transfected in PC12 Tet-on cells. The Tet-on system was chosen based on the lack of pleiotropic or toxic effects and because it allows a tightly regulated expression of the protein of interest. To ensure unwanted background expression of PrP in the absence of inducer, cells were co-transfected with J pTet-tTS, encoding the tet-controlled transcriptional silencer. The D177N and PG14 mutants expressed in this system displayed biochemical properties reminiscent of PrP^^, including detergent insolubility and low protease resistance. Low levels of mutant PrPs were detected on the cell surface by confocal immunofluorescence analysis compared to wild-type PrP. These mutant displayed reticular intracellular distribution, suggesting impaired delivery to the cell surface and retention in the endoplasmic reticulum (ER). Evaluation of cellular viability revealed a decrement in cell survival after 96 h of mutant PrP expression, associated with an increased number of apoptotic cells. This effect was not fully consistent and was not exacerbated by Q neuronal differentiation with NGF or by treating cells with FI2O2, tunicamycin, or by removing serum. The results presented in this thesis also demonstrate that expression of PrP does not protect PC 12 Tet-on cells from such stresses. To explore the possibility that expression of mutant PrPs triggered toxic -1 response pathways in the ER, the mRNA level of ER stress markers was measured. The results shown in this thesis indicate that, although the altered intracellular distribution of mutant PrPs suggested retention of the protein in the endoplasmic reticulum, ER stress responses were not detected.