scholarly journals Seeded propagation of alpha-synuclein aggregation in mouse brain using protein misfolding cyclic amplification

2019 ◽  
Author(s):  
Simon Nicot ◽  
Jérémy Verchère ◽  
Maxime Belondrade ◽  
Charly Mayran ◽  
Dominique Bétemps ◽  
...  
Keyword(s):  
Mouse Brain ◽  
Protein Misfolding ◽  
Wild Type Mouse ◽  
Molecular Study ◽  
Alpha Synuclein ◽  
Proteinase K ◽  
Wild Type ◽  
Protein Misfolding Cyclic Amplification ◽  
Alpha Synuclein Aggregation

Alpha-synuclein (α-syn) protein aggregation is associated with several neurodegenerative disorders collectively referred to as synucleinopathies, including Parkinson disease. We used protein misfolding cyclic amplification (PMCA) to study α-syn aggregation in brain homogenates of wild-type or transgenic mice expressing normal (D line) or A53T mutant (M83 line) human α-syn. We found that sonication-incubation cycles of M83 mouse brain gradually produce large quantities of SDS-resistant α-syn aggregates, involving both human and mouse proteins. These PMCA products, containing partially proteinase K resistant α-syn species, are competent to accelerate the onset of neurological symptoms after intracerebral inoculation to young M83 mice and to seed aggregate formation of α-syn following PMCA, including in D and wild-type mouse brain substrates. Our data indicate that similar to prions, PMCA can reproduce some characteristics of α-syn aggregation and seeded-propagation in vitro in a complex milieu. This opens new opportunities for the molecular study of synucleinopathies.

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.

The Compound ATH434 Prevents Alpha-Synuclein Toxicity in a Murine Model of Multiple System Atrophy

2021 ◽  
pp. 1-11
Author(s):  
David I. Finkelstein ◽  
Jay J. Shukla ◽  
Robert A. Cherny ◽  
Jessica L. Billings ◽  
Eiman Saleh ◽  
...  
Keyword(s):  
Multiple System Atrophy ◽  
Pharmacokinetic Profile ◽  
Alpha Synuclein ◽  
Redox Activity ◽  
Substantia Nigra Pars Compacta ◽  
Drug Candidate ◽  
Older Volunteers ◽  
Control Food ◽  
Alpha Synuclein Aggregation

Background: An elevation in iron levels, together with an accumulation of α-synuclein within the oligodendrocytes, are features of the rare atypical parkinsonian disorder, Multiple System Atrophy (MSA). We have previously tested the novel compound ATH434 (formally called PBT434) in preclinical models of Parkinson’s disease and shown that it is brain-penetrant, reduces iron accumulation and iron mediated redox activity, provides neuroprotection, inhibits alpha synuclein aggregation and lowers the tissue levels of alpha synuclein. The compound was also well-tolerated in a first-in-human oral dosing study in healthy and older volunteers with a favorable, dose-dependent pharmacokinetic profile. Objective: To evaluate the efficacy of ATH434 in a mouse MSA model. Methods: The PLP-α-syn transgenic mouse overexpresses α-synuclein, demonstrates oligodendroglial pathology, and manifests motor and non-motor aspects of MSA. Animals were provided ATH434 (3, 10, or 30 mg/kg/day spiked into their food) or control food for 4 months starting at 12 months of age and were culled at 16 months. Western blot was used to assess oligomeric and urea soluble α-synuclein levels in brain homogenates, whilst stereology was used to quantitate the number of nigral neurons and glial cell inclusions (GCIs) present in the substantia nigra pars compacta. Results: ATH434 reduced oligomeric and urea soluble α-synuclein aggregation, reduced the number of GCIs, and preserved SNpc neurons. In vitro experiments suggest that ATH434 prevents the formation of toxic oligomeric species of synuclein. Conclusion: ATH434 is a promising small molecule drug candidate that has potential to move forward to trial for treating MSA.

Impaired mechanical stability, migration and contractile capacity in vimentin-deficient fibroblasts

1998 ◽  
Vol 111 (13) ◽  
pp. 1897-1907 ◽  
Author(s):  
B. Eckes ◽  
D. Dogic ◽  
E. Colucci-Guyon ◽  
N. Wang ◽  
A. Maniotis ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Mechanical Stability ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Cellular Functions ◽  
Primary Fibroblasts ◽  
In Vitro Systems ◽  
Filament Network ◽  
Collagen Lattices

Loss of a vimentin network due to gene disruption created viable mice that did not differ overtly from wild-type littermates. Here, primary fibroblasts derived from vimentin-deficient (-/-) and wild-type (+/+) mouse embryos were cultured, and biological functions were studied in in vitro systems resembling stress situations. Stiffness of -/- fibroblasts was reduced by 40% in comparison to wild-type cells. Vimentin-deficient cells also displayed reduced mechanical stability, motility and directional migration towards different chemo-attractive stimuli. Reorganization of collagen fibrils and contraction of collagen lattices were severely impaired. The spatial organization of focal contact proteins, as well as actin microfilament organization was disturbed. Thus, absence of a vimentin filament network does not impair basic cellular functions needed for growth in culture, but cells are mechanically less stable, and we propose that therefore they are impaired in all functions depending upon mechanical stability.

Abstract 470: Recording Cell Migration Dynamics in Mouse Tissues With Cells Secreting Fluorescence Protein-tagged Collagen

2020 ◽  
Vol 127 (Suppl_1) ◽  
Author(s):  
Zhongming Chen
Keyword(s):  
Cell Migration ◽  
Cell Lines ◽  
Wild Type Mouse ◽  
Cardiac Progenitor Cells ◽  
Wild Type ◽  
Mouse Tissues ◽  
Migration Dynamics ◽  
Fluorescence Protein

Background: Cell migration is an important step involved in heart regeneration and many cardiovascular diseases. However, cell migration dynamics in vivo is poorly understood due to the challenges from mammal hearts, which are opaque and fast beating, and thus individual cardiac cells cannot be imaged or tracked. Aims: In this study, cell migration dynamics in the heart is recorded with a novel strategy, in which fluorescence protein-tagged collagen is secreted from cells and deposited into extracellular matrix, forming visible trails when cells are moving in tissues. As a proof-of-concept, transplanted migration dynamics of cardiac progenitor cells in mouse hearts were investaged. Methods: Stable cell lines expressing mCherry-tagged type I collagen were generated from isolated cardiac progenitor cells, ABCG2 + CD45 - CD31 - cells (side populations), or c-kit + CD45 - CD31 - cells (c-kit + CPCs). The cell migration dynamics were monitored and measured based on the cell trails after cell transplantation into mouse tissues. Results: The stable cell lines form red cell trails both in vitro and in vivo (Fig. 1A & 1B, Green: GFP; Red: mCherry-collagen I, Blue: DAPI, bar: 50 microns). In culture dishes, the cells form visible cell trails of fluorescence protein. The cell moving directions are random, with a speed of 288 +/- 79 microns/day (side populations, n=3) or 143 +/-37 microns/day (c-kit + CPCs, n=3). After transplantation into wild-type mouse hearts, the cells form highly tortuous trails along the gaps between the heart muscle fibers. Angle between a cell trail and a muscle fiber is 16+/-16 degree (n=3). Side populations migrate twice as fast as c-kit+ CPCs in the heart (16.0 +/-8.7 microns/day vs. 8.1+/-0.0 microns/day, n=3, respectively), 18 time slower than the respective speeds in vitro . Additionally, side populations migrate significantly faster in the heart than in the skeletal muscles (26.4+/-5.8 microns/day, n=3). The side populations move significantly faster in immunodeficient mouse hearts (36.7+/-13.3 microns/day, n=3, typically used for studying cell therapies) than in wild-type mouse hearts. Conclusion: For the first time, cell migration dynamics in living hearts is monitored and examined with genetically modified cell lines. This study may greatly advance the fields of cardiovascular biology.

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 Prion replication without host adaptation during interspecies transmissions

2017 ◽  
Vol 114 (5) ◽  
pp. 1141-1146 ◽  
Author(s):  
Jifeng Bian ◽  
Vadim Khaychuk ◽  
Rachel C. Angers ◽  
Natalia Fernández-Borges ◽  
Enric Vidal ◽  
...  
Keyword(s):  
Disease Transmission ◽  
Protein Misfolding ◽  
Guanidine Hydrochloride ◽  
Host Adaptation ◽  
Wild Type ◽  
Epidemiological Features ◽  
Significant Barrier ◽  
Prion Transmission ◽  
Selection Of

Adaptation of prions to new species is thought to reflect the capacity of the host-encoded cellular form of the prion protein (PrPC) to selectively propagate optimized prion conformations from larger ensembles generated in the species of origin. Here we describe an alternate replicative process, termed nonadaptive prion amplification (NAPA), in which dominant conformers bypass this requirement during particular interspecies transmissions. To model susceptibility of horses to prions, we produced transgenic (Tg) mice expressing cognate PrPC. Although disease transmission to only a subset of infected TgEq indicated a significant barrier to EqPrPCconversion, the resulting horse prions unexpectedly failed to cause disease upon further passage to TgEq. TgD expressing deer PrPCwas similarly refractory to deer prions from diseased TgD infected with mink prions. In both cases, the resulting prions transmitted to mice expressing PrPCfrom the species of prion origin, demonstrating that transmission barrier eradication of the originating prions was ephemeral and adaptation superficial in TgEq and TgD. Horse prions produced in vitro by protein misfolding cyclic amplification of mouse prions using horse PrPCalso failed to infect TgEq but retained tropism for wild-type mice. Concordant patterns of neuropathology and prion deposition in susceptible mice infected with NAPA prions and the corresponding prion of origin confirmed preservation of strain properties. The comparable responses of both prion types to guanidine hydrochloride denaturation indicated this occurs because NAPA precludes selection of novel prion conformations. Our findings provide insights into mechanisms regulating interspecies prion transmission and a framework to reconcile puzzling epidemiological features of certain prion disorders.

A Novel ENU-Induced Intronic Mutation of Ank1 Causing Quantitative Defect of Ankyrin-1 Models Hemolytic Hereditary Spherocytosis in Mouse

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 989-989
Author(s):  
Xin Du ◽  
Hua Huang ◽  
Lara Krieg
Keyword(s):  
Cell Biology ◽  
Hereditary Spherocytosis ◽  
Conflicts Of Interest ◽  
Membrane Surface ◽  
Regulatory Region ◽  
Wild Type Mouse ◽  
Wild Type ◽  
Intronic Mutation ◽  
Vertical Linkages

Abstract Abstract 989 The ability of red blood cells (RBCs) to maintain the surface area and deformability is vital for their survival. The maintenance of membrane surface is dependent upon the strong cohesion between the lipid bilayer and the skeletal network, achieved by vertical linkages between transmembrane proteins and spectrin tetramers. Mutations causing functional deficiencies in these proteins have been identified in various hemolytic anemias. Here we reported a mild hereditary spherocytosis (HS) and hemolytic anemia phenotype in mouse, named hema6, induced by N-ethyl-N-nitrosourea (ENU) mutagenesis. Hema6 phenotype is transmitted as a semidominant trait as heterozygous mice are less severely affected than homozygotes. The causal mutation was traced to a single nucleotide transition in the deep intronic region of intron 13 of gene Ank1, encoding the anchorage protein ankyrin-1 in RBC. In vitro minigene assay revealed two abnormally spliced transcripts in addition to wild-type mRNA. The wild-type Ank1 transcript was detected in the homozygous mutant mouse with 30% reduction in expression level compared to that in wild type mouse. The aberrant transcripts presumably encoded a 509 amino acids protein, which lacks beta-spectrin binding domain and C-terminal regulatory region. The truncated protein was not detected by western blotting using currently available antibodies against full-length ankyrin-1 in homozygous hema6 erythrocyte ghosts, whereas the wild-type Ankyrin-1 are present with reduced quantity. Employing biochemical and cell biology assays, we characterized the mechanism by which Ank1hema6 mutation causes hemolytic hereditary spherocytosis in mouse. Hema6 strain provides a novel tool to study ankyrin-1 and its pathogenesis role in HS. Disclosures: No relevant conflicts of interest to declare.

Dynamics of GPIIb/IIIa-mediated platelet-platelet interactions in platelet adhesion/thrombus formation on collagen in vitro as revealed by videomicroscopy

Blood ◽  
2003 ◽  
Vol 101 (3) ◽  
pp. 929-936 ◽  
Author(s):  
Dipti Patel ◽  
Heikki Väänänen ◽  
Markéta Jiroušková ◽  
Thomas Hoffmann ◽  
Carol Bodian ◽  
...  
Keyword(s):  
Platelet Adhesion ◽  
Thrombus Formation ◽  
Wild Type Mouse ◽  
Poisson Model ◽  
Human Platelets ◽  
Wild Type ◽  
Initial Adhesion ◽  
Platelet Deposition ◽  
Coated Surfaces

Abstract The conventional description of platelet interactions with collagen-coated surfaces in vitro, based on serial static measurements, is that platelets first adhere and spread to form a monolayer and then recruit additional layers of platelets. To obtain dynamic information, we studied gravity-driven platelet deposition in vitro on purified type 1 collagen by video phase-contrast microscopy at 22°C. With untreated human and wild-type mouse platelets, soon after the initial adhesion of a small number of “vanguard” platelets, “follower” platelets attached to the spread-out vanguard platelets. Follower platelets then adhered to and spread onto nearby collagen or over the vanguard platelets. Thus, thrombi formed as a concerted process rather than as sequential processes. Treatment of human platelets with monoclonal antibody (mAb) 7E3 (anti–GPIIb/IIIa (αIIbβ3) + αVβ3) or tirofiban (anti–GPIIb/IIIa) did not prevent platelet adhesion but nearly eliminated the deposition of follower platelets onto vanguard platelets and platelet thrombi. Similar results were obtained with Glanzmann thrombasthenia platelets. Wild-type mouse platelets in the presence of mAb 1B5 (anti–GPIIb/IIIa) and platelets from β3-null mice behaved like human platelets in the presence of 7E3 or tirofiban. Deposition patterns of untreated human and wild-type mouse platelets were consistent with random distributions under a Poisson model, but those obtained with 7E3- and tirofiban-treated human platelets, 1B5-treated mouse platelets, or β3-null platelets demonstrated a more uniform deposition than predicted. Thus, in this model system, absence or blockade of GPIIb/IIIa receptors interferes with thrombus formation and alters the pattern of platelet deposition.

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