Thesis title: IDENTIFICAZIONE DELLE ISOFORME DI PrP FISIOLOGICA E PATOLOGICA IN ARVICOLE ROSSASTRE INFETTATE CON DIVERSI CEPPI DI PRIONI
The Transmissible Spongiform Encephalopathies (TSE) are a group of rare and lethal neurodegenerative diseases, caused by the conformational conversion of the host’s physiological prion protein (PrPC) in a pathological isoform (PrPSc). TSEs exist as different strains that generate an extraordinary clinical and pathological variety in term of incubation time, involved cells and brain areas. Despite that, they are all characterised by a shared pathogenesis, which invariably lead to PrPScaccumulation and to the development of the neuropathologic “triad”.
The neurotoxicity mechanisms of prion diseases are not yet known, although all the evidences so far point to the quantitative and conformational variations of PrP. The main hypotheses suggest that a loss of function, that is the loss of the of physiological PrP functions (PrPC), an endogen proteolytic alteration of the PrPC or the acquisition of new functions from the pathological PrP (gain of function) can be the cause of the neurotoxicity encountered in the EST. To properly tackle these hypotheses, it is necessary a better understanding of the phenomena that involves the PrP during the prion disease, following both, the modifications of the PrPC isoforms and their trans-conformation in PrPSc.
Previous studies focusing on these issues used few strains or one only monoclonal antibody, obtaining limited and partially contradictory results either for the use of low-resolution methods and for the low biological and biochemical variability of the strains studied.
With the aim to deepen our knowledge on these issues, we selected seven TSE strains extremely variable for source (human and animal), incubation period, biochemical and pathological characteristics. In addition, a new separation assay has been developed based on the differential solubility between PrPC and PrPSc, following the example of the one introduced by this lab (Pirisinu et al., 2013), but optimised in order to get the best experimental conditions for the PrPC and PrPSc separation. Finally, by combining the solubility separation principle with the deglycosylation enzymatic assay and by using different mAbs that identify specific epitopes along the PrP, this project has conceived epitope-mapping techniques both in solution, through western blot, and in situ, through discriminative IHC. These techniques allowed to identify the PrPC and PrPSc isoforms in solution and localize the main PrPSc isoforms in the pathological tissue, including the specific identification of the shedded isoforms.
This methodology, even being relatively rapid, fully exploits the different solubility characteristics of the two isoforms and allowed parallel analyses of PrPC and PrPSc in many samples, where the PrPC, being in monomeric form, is in the soluble fraction (supernatant) while the PrPSc, in aggregated form, is in the insoluble fraction (pellet).
Concerning PrPC, on the basis of previous evidences of its important downregulation during prion diseases, we studied its overall expression level as well as the relative variation of its isoforms. Despite it has been observed in some strains a drastic statistical reduction of the PrPC in the CNS of the infected animals, we did not identify downregulation of PrPC as shared phenomenon for all the strains investigated, being less evident or even absent in the half of them. The results obtained on the single isoforms suggest that the PrPC decrease, when present, is mostly dependent on the full-length PrPC. At variance, for the strains in which PrPC downregulation was not identified, we found that the physiological regulation of the different PrPC isoform is unchanged with respect to the physiological conditions. These results do not support the hypotheses for which a loss of function or an alteration of the proteolytic events impacting the PrPC can represent a determinant role in the neurotoxicity induction from prions, although we can not exclude a role in the pathological context of some specific strains.
Concerning PrPSc, at first, we identified the strain-specific isoforms, which showed an extremely variable scenario among the strains, corroborating the idea that PrPSc conformational differences are at the base of their variability. This not only allowed to study the known isoforms of native PrPSc, but also to identify new ones and analyse, for the first time in quantitative terms, also the shedded isoforms of PrPSc.
We then investigated the amount of total PrPSc and of its different isoforms that accumulate in the brain of the sick animals, and how these correlate with the disease incubation period and the type of neurodegenerative pathology observed.
By analysing the amount of total PrPSc we observed important differences among strains, whit strains characterized by short incubation period (used in this study as proxy of neurotoxicity) showing lower level of PrPSc accumulation. In this sense, our results do not support the hypothesis for which PrPSc can perform a toxic action by itself.
In the contrary, the study observed interesting correlations between the incubation time of the different strains and the amount of shedded PrPSc and of fragments truncated at the N-terminus (overall defined as CTF) relative to the full-length isoform anchored on the cell surface. The strains in which these phenomena were more pronounced show a minor neurotoxicity (longer survival period) with respect to those in which these pathological events were less evident. This suggests a possible neuroprotective role of these isoforms or of the cells’ reactions phenomena involved in their production.
Then we investigated the in situ localization of these PrPSc isoforms, determining that the shedded isoforms of PrPSc accumulate in the extra-cellular space while the CTF were detected at intracellular level, probably in lysosomes where the N-terminus of PrPSc, protease sensitive, is trimmed by lysosomal enzymes. The full-length isoform of PrPSc is instead anchored at the plasma membrane.
Overall, our results lead to hypothesize that the main neurotoxic trigger can be represented by the accumulation of PrPSc on the cell membrane, also involving a decrease of PrPC in some strains. In this context, the shedding and the endocytosis of PrPSc may represent cellular reactions able to play a neuroprotective role, since they cause PrPSc dismissal from the neuron cell surface and, in the case of endocytosis, a partial PrPSc degradation.
In conclusion, this project contributes to highlight two possible therapeutics targets for prion diseases, represented by strategies aiming at increasing shedding and/or autophagy of the PrPSc in the affected cellular populations.