| Popis: |
Background. The main pathological features of Parkinson's disease (PD) are the death of dopaminergic neurons and the diffuse accumulation of alpha-synuclein (aS) aggregates in neurons. The NEDD4 E3 ubiquitin ligase has been shown to promote aS degradation by the endosomal/lysosomal route. Interestingly, NEDD4 is protective against human aS toxicity in evolutionary distant models. Furthermore, a small molecule able to activate NEDD4 functions, was neuroprotective in evolutionary distant models of aS toxicity. While activation of E3s cannot be easily obtained pharmacologically, their flexibility and the lack of consensus motifs for ubiquitin (Ub) conjugation allow the development of engineered Ub-ligases able to target proteins of interest. The aim of our study was to exploit chimeric Ub-ligases, named ubiquibodies, specifically targeting aS to prove the protective role of aS degradation pathway towards the development of innovative strategies and rescue the normal physiology of neurons. Methods. To this end, we have developed lentiviral vectors encoding well characterized human scFvs fused in frame to the NEDD4 catalytic domain, in order to obtain enzymes that specifically ubiquitinate aS either in its monomeric and/or oligomeric form. We also generated two lentiviral vectors expressing wild type aS and a mutant form (A53T aS), known to play a role in PD pathogenesis, either alone or fused in frame with the reporter protein EGFP. Furthermore, we adopted different in vitro models including human and murine dopaminergic cell lines and three different lines of human induced pluripotent stem cells (hiPSCs) derived from a healthy donor and from Parkinson's patients, transduced with lentiviral particles expressing ubiquibodies, with the aim of analyzing their ability to rescue of hiPSCs-derived dopaminergic neurons physiological features. Results And Conclusions. We are able to demonstrated that: i) the recombinant proteins are expressed in human embryonic 293T cells, as well as in the human and mouse dopaminergic neuroblastoma, SH-SY5Y and MN9D in cell line respectively; ii) recombinant lentiviral particles transduce not only 293T, MN9D and SH-SY5Y cells, but also in human hiPSCs, neural stem cells (NSCs) and NSC-derived dopaminergic neurons; iii) ubiquibodies interacts in a specific manner with overexpressed aS also in dopaminergic cell lines; iv) a specific degradation of aS takes place in 293T cells transduced with one of the developed the chimeric Ub-ligases (Nac32HECTWT) that was selected based on the achieved results on its characteristics. Finally, experiment conducted in hiPSCs-derived NSCs indicate that aS overexpression and mutation have a negative effect on NSCs differentiation into dopaminergic neurons with an increase in cell mortality. The phenotype can be rescued by Nac32HECTWT expression. The results achieved so far represent a starting point strongly supporting the validity of the strategy we intend to adopt in order to obtain a specific degradation of aS. |
