Biology subjects

Andreoletti, O.

Publications and source records attributed to Andreoletti, O..

4 recordsLinked to original sources

bioRxiv2025

Proteomic and Kinetic Characterization of Prion Seeding in Distinct Human CJD Strains Unveils Early Diagnostic Biomarkers

To enhance understanding of early diagnosis, differential disease progression rates, and the molecular profiles of human prion strains, we analyzed prion seeding activity over time in Creutzfeldt-Jakob disease (CJD)-infected mice using the real-time quaking-induced conversion (RT-QuIC) assay. Our previous work highlighted pre- clinical alterations in endocytic machinery and cytoskeleton-associated responses in CJD-affected brain regions. In this study, infectious prion strains derived from human CJD-MM1 and VV2 brain tissues were inoculated into tg340, tg361 (expressing approximately four times the human PrP-M129 and PrP-V129, respectively), and tg650 (expressing approximately six times the human PrP-M129) mice. A total of 188 brain samples (cortex) were analyzed from confirmed CJD-infected mice and control mice at both pre-clinical and clinical stages of the disease. Notably, we observed region- specific and PrP strain-specific differences in seeding activity at the pre-clinical stage of disease in CJD-MM1 and VV2 infected mice. The lag phase between the positive response ranged from 7.5 to 24.5 hours across all regions and disease stages. In the cortex, CJD-MM1-infected tg340 mice exhibited a prolonged lag phase ([~]24.5 hours), while CJD-VV2-infected tg341 mice showed minimal seeding response and relative fluorescence unit (RFU) signal rates. In contrast, the cerebellum of VV2 clinical stage mice exhibited a shorter lag phase, and VV2 preclinical stage mice showed significantly lower RFU signal rates. Proteomic profiling via SWATH-MS identified 500 and 682 differentially expressed proteins in the MM1 and VV2 models, respectively. Key proteins such as Gnl1, Stxbp1, Pllp, Gps1, Nefh, Ahsa1, Rala, Cacybp, Pdk1, Unc13a, Rab21, Rraga, Ppp1r9a, Eif4b, Atp2b2, Vps51, H2afx in CJD-MM1 and Gm10358, Tnc, Calb2, Ppm1h, Dnaja1, Gm45808, Hpcal1, Prkca, Dock3, Syn2, Agap2, Tmem126a, Fdps, Ndufa4 in CJD- VV2 showed significant alterations at the early pre-clinical stages, correlating with detectable prion replication. These molecular shifts highlight potential early-stage diagnostic biomarkers. Functional analyses revealed that both MM1 and VV2 subtypes engage early compensatory responses; however, MM1 primarily involves metabolic reprogramming and enhanced vesicle clearance, while VV2 is characterized by pronounced disturbances in calcium signaling and structural integrity at early stage of the disease. These findings emphasize the utility of RT-QuIC and proteomics in characterizing prion seeding and progression, providing valuable insights into the molecular mechanisms underlying prion diseases and potential early diagnostic markers.

neuroscience↗

bioRxiv2023

Cleavage site-directed antibodies reveal the prion protein in humans is shed by ADAM10 at Y226 and associates with misfolded protein deposits in neurodegenerative diseases

Proteolytic cell surface release ( shedding) of the prion protein (PrP), a broadly expressed GPI-anchored glycoprotein, by the metalloprotease ADAM10 impacts on neurodegenerative and other diseases in animal and in vitro models. Recent studies employing the latter also suggest shed PrP (sPrP) to be a ligand in intercellular communication and critically involved in PrP-associated physiological tasks. Although expectedly an evolutionary conserved event, and while soluble forms of PrP are present in human tissues and body fluids, neither proteolytic PrP shedding and its cleavage site nor involvement of ADAM10 or the biological relevance of this process have been demonstrated for the human body thus far. In this study, cleavage site prediction and generation (plus detailed characterization) of sPrP-specific antibodies enabled us to identify PrP cleaved at tyrosin 226 as the physiological and strictly ADAM10-dependent shed form in humans. Using cell lines, neural stem cells and brain organoids, we show that shedding of human PrP can be stimulated by PrP-binding ligands without targeting the protease, which may open novel therapeutic perspectives. Site-specific antibodies directed against human sPrP also detect the shed form in brains of cattle, sheep and deer, hence in all most relevant species naturally affected by fatal and transmissible prion diseases. In human and animal prion diseases, but also in patients with Alzheimers disease, sPrP relocalizes from a physiological diffuse tissue pattern to intimately associate with extracellular aggregates of misfolded proteins characteristic for the respective pathological condition. Findings and research tools presented here will accelerate novel insight into the roles of PrP shedding (as a process) and sPrP (as a released factor) in neurodegeneration and beyond.

neuroscience↗

bioRxiv2023

Pathogenicity, strain properties and interspecies transmission capacity of pure recombinant prion protein assemblies

The pathogenicity of fibrillar assemblies derived from bacterially expressed recombinant prion protein (rPrP) has been key to the demonstration that prions are infectious proteins responsible for human and animal transmissible spongiform encephalopathies. Yet, their use in identifying which structural PrP features are important for prion biology, including strain properties and capacity to transmit between species, has been hampered by their limited transmissibility de novo. We report the generation of prions with distinct biological characteristics from rPrP assemblies differing only in their primary structure (hamster, mouse and human amino acid sequence). These rPrP assemblies were transmissible to transgenic mice expressing hamster PrP, causing a clinical disease at full attack rate, brain deposition of pathological prion protein PrPSc and spongiform degeneration. Their adaptation process on serial sub-passaging seemed to depend, as for genuine prions, on the presence of a species/transmission barrier, due notably to PrP sequence mismatch. Remarkably, one of the strains obtained is an unprecedented shortened prion, lacking the 90-140 amino-acid region which is believed to be key to infectivity and structural stability of disease-associated PrP assemblies. Finally, we provide evidence that rPrP prionogenicity lies in the structural organization and/or heterogeneity of the rPrP assemblies. These preparations of rPrP offer unprecedented opportunities for meaningful studies correlating the dynamicity and structures of PrPSc assemblies to prion pathobiology. Author summaryPrions are infectious proteins, causing rapidly progressive neurodegenerative diseases in animals and humans. They are formed from the assisted-refolding and aggregation of the host-encoded prion protein (PrP). During the propagation of the disease, pathological PrP forces normal PrP to adopt its own conformation by a self-templating process. In infected host, different pathological structures of PrP or strains are found, causing diseases with specific biological phenotypes. Prions can also transmit between species. This capacity is limited by a species barrier, which critically depends on the infecting strain and PrP primary structure. How strain biological information is encoded in PrP structural fold remains unknown. We describe here the generation of different bona fide prion strains with markedly distinct adaptation capacities by transmission of refolded assemblies derived from bacterially-derived recombinant PrP (rPrP) of different species. We provide evidence that pathogenicity lies in the structural organization and/or heterogeneity of rPrP assemblies. Pathological PrP from one of the generated strains exhibited unique molecular features, including absence of domains that are thought to be key to prion infectivity, according to most recent ultrastructural studies. Our findings provide new insights for generating prion infectious material and resolving mechanisms of infectivity acquisition during PrP conversion process.

microbiology↗

bioRxiv2020

Prion infection, transmission and cytopathology modelled in a low-biohazard human cell line

Transmission of prion infectivity to susceptible murine cell lines has simplified prion titration assays and has greatly reduced the need for animal experimentation. However, murine cell models suffer from technical and biological constraints. Human cell lines might be more useful, but they are much more biohazardous and are often poorly infectible. Here we describe the human clonal cell line hovS, which lacks the human PRNP gene and expresses instead the ovine PRNP VRQ allele. HovS cells were highly susceptible to the PG127 strain of sheep-derived murine prions, reaching up to 90% infected cells in any given culture, and were maintained in a continuous infected state for at least 14 passages. Infected hovS cells produced proteinase K-resistant prion protein (PrPSc), pelletable PrP aggregates and bona fide infectious prions capable of infecting further generations of naive hovS cells and mice expressing the VRQ allelic variant of ovine PrPC. Infection in hovS led to prominent cytopathic vacuolation akin to the spongiform changes observed in individuals suffering from prion diseases. In addition to expanding the toolbox for prion research to human experimental genetics, the hovS cell line provides a human-derived system that does not require human prions. Hence, the manipulation of scrapie-infected hovS cells may present fewer biosafety hazards than that of genuine human prions.

neuroscience↗