scholarly journals Misfolded α-synuclein causes hyperactive respiration without functional deficit in live neuroblastoma cells

2019 ◽  
Author(s):  
C.L Ugalde ◽  
S.J Annesley ◽  
S Gordon ◽  
K Mroczek ◽  
M.A Perugini ◽  
...  
Keyword(s):  
Mitochondrial Respiration ◽  
Protein Misfolding ◽  
Neuroblastoma Cells ◽  
Strong Support ◽  
Inner Mitochondrial Membrane ◽  
Biologically Relevant ◽  
Functional Deficit ◽  
Protein Misfolding Cyclic Amplification ◽  
Potential Binding ◽  
Summary Statement

AbstractThe misfolding and aggregation of the largely disordered protein, α-synuclein, is a central pathogenic event that occurs in the synucleinopathies; a group of neurodegenerative disorders that includes Parkinson’s disease. While there is a clear link between protein misfolding and neuronal vulnerability, the precise pathogenic mechanisms employed by disease-associated α-synuclein are unresolved. Here, we studied the pathogenicity of misfolded α-synuclein produced using the Protein Misfolding Cyclic Amplification (PMCA) assay. To do this, previous published methods were adapted to allow PMCA-induced protein fibrillization to occur under non-toxic conditions. Insight into potential intracellular targets of misfolded α-synuclein was obtained using an unbiased lipid screen of 15 biologically relevant lipids that identified cardiolipin (CA) as a potential binding partner for PMCA-generated misfolded α-synuclein. To investigate if such an interaction can impact the properties of α-synuclein misfolding, protein fibrillization was carried out in the presence of the lipid. We show CA both accelerates the rate of α-synuclein fibrillization and produces species that harbour enhanced resistance to proteolysis. Because CA is virtually exclusively expressed in the inner mitochondrial membrane, we then assessed the ability of these misfolded species to alter mitochondrial respiration in live non-transgenic SH-SY5Y neuroblastoma cells. Extensive analysis revealed misfolded α-synuclein causes hyperactive mitochondrial respiration without causing any functional deficit. These data give strong support for the mitochondrion as a target for misfolded α-synuclein and reveals persistent, hyperactive respiration as a potential up-stream pathogenic event associated with the synucleinopathies.Summary statementMisfolded α-synuclein that was produced using the Protein Misfolding Cyclic Amplification (PMCA) assay was found to associate with cardiolipin and cause hyperactive respiration in neuronal cells.

scholarly journals Sensitive protein misfolding cyclic amplification of sporadic Creutzfeldt–Jakob disease prions is strongly seed and substrate dependent

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Maxime Bélondrade ◽  
Simon Nicot ◽  
Charly Mayran ◽  
Lilian Bruyere-Ostells ◽  
Florian Almela ◽  
...  
Keyword(s):  
Prion Protein ◽  
Protein Misfolding ◽  
Protein Misfolding Cyclic Amplification ◽  
Bank Voles ◽  
Substrate Dependence ◽  
Brain Extracts ◽  
Jakob Disease ◽  
Human Prion Protein

AbstractUnlike variant Creutzfeldt–Jakob disease prions, sporadic Creutzfeldt–Jakob disease prions have been shown to be difficult to amplify in vitro by protein misfolding cyclic amplification (PMCA). We assessed PMCA of pathological prion protein (PrPTSE) from 14 human sCJD brain samples in 3 substrates: 2 from transgenic mice expressing human prion protein (PrP) with either methionine (M) or valine (V) at position 129, and 1 from bank voles. Brain extracts representing the 5 major clinicopathological sCJD subtypes (MM1/MV1, MM2, MV2, VV1, and VV2) all triggered seeded PrPTSE amplification during serial PMCA with strong seed- and substrate-dependence. Remarkably, bank vole PrP substrate allowed the propagation of all sCJD subtypes with preservation of the initial molecular PrPTSE type. In contrast, PMCA in human PrP substrates was accompanied by a PrPTSE molecular shift during heterologous (M/V129) PMCA reactions, with increased permissiveness of V129 PrP substrate to in vitro sCJD prion amplification compared to M129 PrP substrate. Combining PMCA amplification sensitivities with PrPTSE electrophoretic profiles obtained in the different substrates confirmed the classification of 4 distinct major sCJD prion strains (M1, M2, V1, and V2). Finally, the level of sensitivity required to detect VV2 sCJD prions in cerebrospinal fluid was achieved.

scholarly journals Protein misfolding cyclic amplification of infectious prions

2012 ◽  
Vol 7 (7) ◽  
pp. 1397-1409 ◽  
Author(s):  
Rodrigo Morales ◽  
Claudia Duran-Aniotz ◽  
Rodrigo Diaz-Espinoza ◽  
Manuel V Camacho ◽  
Claudio Soto
Keyword(s):  
Protein Misfolding ◽  
Protein Misfolding Cyclic Amplification

Optimal Geometric Control Applied to the Protein Misfolding Cyclic Amplification Process

2014 ◽  
Vol 135 (1) ◽  
pp. 145-173 ◽  
Author(s):  
Monique Chyba ◽  
Jean-Michel Coron ◽  
Pierre Gabriel ◽  
Alain Jacquemard ◽  
Geoff Patterson ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Geometric Control ◽  
Protein Misfolding Cyclic Amplification ◽  
Amplification Process

The detection of seeds alpha-synuclein using protein misfolding cyclic amplification(PMCA)

2017 ◽  
Vol 381 ◽  
pp. 972-973
Author(s):  
S. Yoshinaga ◽  
T. Yamanaka ◽  
Y. Furukawa ◽  
N. Nukina
Keyword(s):  
Protein Misfolding ◽  
Alpha Synuclein ◽  
Protein Misfolding Cyclic Amplification

scholarly journals Characterization of Anchorless Human PrP With Q227X Stop Mutation Linked to Gerstmann-Sträussler-Scheinker Syndrome In Vivo and In Vitro

2020 ◽  
Vol 58 (1) ◽  
pp. 21-33
Author(s):  
Pingping Shen ◽  
Johnny Dang ◽  
Zerui Wang ◽  
Weiguanliu Zhang ◽  
Jue Yuan ◽  
...  
Keyword(s):  
Protein Misfolding ◽  
Neuroblastoma Cells ◽  
Cellular Prion Protein ◽  
Wild Type ◽  
Gpi Anchor ◽  
Two Dimensional Gel Electrophoresis ◽  
Molecular Weights ◽  
Prion Propagation

AbstractAlteration in cellular prion protein (PrPC) localization on the cell surface through mediation of the glycosylphosphatidylinositol (GPI) anchor has been reported to dramatically affect the formation and infectivity of its pathological isoform (PrPSc). A patient with Gerstmann-Sträussler-Scheinker (GSS) syndrome was previously found to have a nonsense heterozygous PrP-Q227X mutation resulting in an anchorless PrP. However, the allelic origin of this anchorless PrPSc and cellular trafficking of PrPQ227X remain to be determined. Here, we show that PrPSc in the brain of this GSS patient is mainly composed of the mutant but not wild-type PrP (PrPWt), suggesting pathological PrPQ227X is incapable of recruiting PrPWt in vivo. This mutant anchorless protein, however, is able to recruit PrPWt from humanized transgenic mouse brain but not from autopsied human brain homogenates to produce a protease-resistant PrPSc-like form in vitro by protein misfolding cyclic amplification (PMCA). To further investigate the characteristics of this mutation, constructs expressing human PrPQ227X or PrPWt were transfected into neuroblastoma cells (M17). Fractionation of the M17 cells demonstrated that most PrPWt is recovered in the cell lysate fraction, while most of the mutant PrPQ227X is recovered in the medium fraction, consistent with the results obtained by immunofluorescence microscopy. Two-dimensional gel-electrophoresis and Western blotting showed that cellular PrPQ227X spots clustered at molecular weights of 22–25 kDa with an isoelectric point (pI) of 3.5–5.5, whereas protein spots from the medium are at 18–26 kDa with a pI of 7–10. Our findings suggest that the role of GPI anchor in prion propagation between the anchorless mutant PrP and wild-type PrP relies on the cellular distribution of the protein.

scholarly journals Two distinct prions in fatal familial insomnia and its sporadic form

2019 ◽  
Vol 1 (1) ◽  
Author(s):  
Atsuko Takeuchi ◽  
Shirou Mohri ◽  
Hideaki Kai ◽  
Akira Tamaoka ◽  
Atsushi Kobayashi ◽  
...  
Keyword(s):  
Prion Protein ◽  
Protein Misfolding ◽  
Protein Gene ◽  
Clinical Findings ◽  
Fatal Familial Insomnia ◽  
Pathological Feature ◽  
Prion Protein Gene ◽  
Successful Transmission ◽  
Protein Misfolding Cyclic Amplification ◽  
Transmission Studies

Abstract Fatal familial insomnia is a genetic prion disease, which is associated with the aspartic acid to asparagine substitution at codon 178 of the prion protein gene. Although the hallmark pathological feature is thalamic and olivary degeneration, there is a patient with an atypical fatal familial insomnia without the hallmark feature. The cause of the pathological variability is unclear. We analysed a Japanese fatal familial insomnia kindred and compared one atypical clinicopathological fatal familial insomnia phenotype case and typical fatal familial insomnia phenotype cases with transmission studies using multiple lines of knock-in mice and with protein misfolding cyclic amplification. We also analysed the transmissibility and the amplification properties of sporadic fatal insomnia. Transmission studies revealed that the typical fatal familial insomnia with thalamic and olivary degeneration showed successful transmission only using knock-in mice expressing human–mouse chimeric prion protein gene. The atypical fatal familial insomnia with spongiform changes showed successful transmission only using knock-in mice expressing bank vole prion protein gene. Two sporadic fatal insomnia cases with thalamic and olivary degeneration showed the same transmissibility as the typical fatal familial insomnia phenotype. Interestingly, one sporadic fatal insomnia case with thalamic/olivary degeneration and spongiform changes showed transmissibility of both the typical and atypical fatal familial insomnia phenotypes. Protein misfolding cyclic amplification could amplify both typical fatal familial insomnia cases and sporadic fatal insomnia cases but not the atypical fatal familial insomnia phenotype or other sporadic Creutzfeldt–Jakob disease subtypes. In addition to clinical findings and neuropathological features, the transmission properties and the amplification properties were different between the typical and atypical fatal familial insomnia phenotypes. It is suggested that two distinct prions were associated with the diversity in the fatal familial insomnia phenotype, and these two prions could also be detected in sporadic fatal insomnia.

scholarly journals Detection of prion protein in the cerebrospinal fluid of elk (Cervus canadensis nelsoni) with chronic wasting disease using protein misfolding cyclic amplification

2012 ◽  
Vol 24 (4) ◽  
pp. 746-749 ◽  
Author(s):  
Tracy A. Nichols ◽  
Terry R. Spraker ◽  
Tom Gidlewski ◽  
Jenny G. Powers ◽  
Glenn C. Telling ◽  
...  
Keyword(s):  
Cerebrospinal Fluid ◽  
Prion Protein ◽  
Protein Misfolding ◽  
Chronic Wasting Disease ◽  
Wasting Disease ◽  
Protein Misfolding Cyclic Amplification ◽  
Cervus Canadensis
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