The pathogenesis of a disease involves a stochastic refolding of the etiologic protein into a misfolded infectious state known as prion. Recently, there has been renewed interest in the possibility that proteins causing neurodegeneration are all prions. The β-sheet rich pathological α-synuclein (α-syn) can cross from the neurons of transplanted patients into the grafted cells, and induce a change in the structure of α-syn in Parkinson’s disease (PD) is an example. The convergence of studies showing the presence of prions in the pathogenesis of common neurodegenerative maladies has since been remarkable. Studies on synthetic prions showed that recombinant (rec) prion protein (PrP) is refolded into infectious conformations in vitro. This synthetic prion protein stimulates the conversion of cellular PrP into nascent pathological PrP and induces the accumulation of the isoform that causes neurodegeneration in vivo. Using defined biophysical and biochemical conditions in vitro, we developed methods for the pathological conversion of recPrP into PrPSc, and we established whether synthetic pathological agents of rec human α-syn amyloids can be infectious, as Legname et al. showed for the first time in production of mammalian synthetic prions. The pathological conversion process of both PrP and α-syn required only purified recombinant proteins and common chemicals. We generated putative infectious materials that possess different conformational structures. Moreover, we designed a novel build-in screening methodology for amyloid preparations to achieve putative infectious materials using amyloid-infected-cell culture assay. At fifth cell passage after single infection, prion amyloid fibrils from different preparations induced endogenous PrPC to convert into PrPSc in both non-infected mouse hypothalamic GT1 and mouse neuroblastoma N2a cell lines. Moreover, these variant synthetic proteinaceous infectious agents can replicate and be detected by protein misfolding cyclic amplification (PMCA). Through this methodology that was used to obtain synthetic mammalian prions, we also tested whether recombinant human α-syn amyloids can infect neuronal cell lines in vitro, and wild-type mice in vivo. A single exposure to amyloid fibrils of human α-syn was sufficient to induce aggregation of endogenous α-syn in human neuroblastoma SH-SY5Y cells, mouse hypothalamic GT1 cells and mouse brains. Interestingly, we found pathological phosphorylated α-syn in amyloid-infected cells and in neurons and neurites of mice. These results suggest that recombinant human α-syn amyloids can promote endogenous α-syn aggregation and pathological post-translational modification. Upon subsequent passages, mice inoculated with either human α-syn amyloid or diseased mouse brain homogenates showed marked neurological symptoms resembling those of PD, as well as neuropathological α-syn inclusions in neurons.
|Autori interni:||Le, Tran Thanh Nhat|
|Autori:||Nhat, Le Tran Thanh|
|Titolo:||From Misfolded Recombinant Proteins in vitro to Pathological Agents in vivo|
|Data di pubblicazione:||27-mar-2014|
|Appare nelle tipologie:||8.1 PhD thesis|
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