Biology subjects

Chincisan, A.

Publications and source records attributed to Chincisan, A..

5 recordsLinked to original sources

bioRxiv2022

An Arrayed Genome-Wide Perturbation Screen Identifies the Ribonucleoprotein hnRNP K As Rate-Limiting for Prion Propagation

A defining characteristic of mammalian prions is their capacity for self-sustained propagation. Theoretical considerations and experimental evidence suggest that prion propagation is modulated by cell-autonomous and non-autonomous modifiers. Using a novel quantitative phospholipase protection assay (QUIPPER) for high-throughput prion measurements, we performed an arrayed genome-wide RNA interference (RNAi) screen aimed at detecting modifiers of prion propagation. We exposed prion-infected cells in high-density microplates to 35364 ternary pools of 52746 siRNAs targeting 17582 genes representing the mouse protein-coding transcriptome. We identified 1191 modulators of prion propagation. While 1151 of these modified the expression of both the pathological prion protein, PrPSc, and its cellular counterpart PrPC, 40 genes affected selectively PrPSc. Of the latter, 20 genes augmented prion production when suppressed. A prominent limiter of prion propagation was the heterogeneous nuclear ribonucleoprotein Hnrnpk. Psammaplysene A (PSA), which binds Hnrnpk, reduced prion levels in cultured cells and protected them from cytotoxicity. PSA also reduced prion levels in infected cerebellar organotypic slices and alleviated locomotor deficits in prion-infected Drosophila melanogaster expressing ovine PrPC. Hence, genome-wide QUIPPER-based perturbations can discover actionable cellular pathways involved in prion propagation. Finally, the unexpected identification of a prioncontrolling ribonucleoprotein suggests a role for RNA in the generation of infectious prions.

neuroscience↗

bioRxiv2021

The landscape of PrPC biosynthesis revealed by an arrayed genome-wide interference screen

The cellular prion protein PrPC is necessary for prion replication, and its reduction greatly increases life expectancy in animal models of prion infection. Hence the factors controlling the levels of PrPC may represent therapeutic targets against human prion diseases. Here we performed an arrayed whole-transcriptome RNA interference screen to identify modulators of PrPC expression. We cultured human U251-MG glioblastoma cells in the presence of 64752 unique siRNAs targeting 21584 annotated human genes, and measured PrPC using a one-pot fluorescence-resonance energy transfer immunoassay in 51128 individual microplate wells. This screen yielded 743 candidate regulators of PrPC. When downregulated, 563 of these candidates reduced and 180 enhanced PrPC expression. Recursive candidate attrition through multiple secondary screens yielded 54 novel regulators of PrPC, 9 of which were confirmed by CRISPR interference as robust regulators of PrPC biosynthesis and degradation. The phenotypes of 6 of the 9 candidates were inverted in response to transcriptional activation using CRISPRa. The RNA-binding post-transcriptional repressor Pumilio-1 was identified as a potent limiter of PrPC expression through the degradation of PRNP mRNA. Because of its hypothesis-free design, this comprehensive genetic-perturbation screen delivers an unbiased landscape of the genes regulating PrPC levels in cells, most of which were unanticipated, and some of which may be amenable to pharmacological targeting in the context of antiprion therapies.

neuroscience↗

bioRxiv2020

Protective anti-prion antibodies in human immunoglobulin repertoires

Prion immunotherapy may hold great potential, but antibodies against certain PrP epitopes can be neurotoxic. Here we identified >6000 PrP-binding antibodies in a synthetic human Fab phage display library, 49 of which we characterized in detail. Antibodies directed against the flexible tail of PrP conferred neuroprotection against infectious prions. We then mined published repertoires of circulating B cells from healthy humans and found antibodies similar to the protective phage-derived antibodies. When expressed recombinantly, these antibodies exhibited anti-PrP reactivity. Furthermore, we surveyed 48718 samples from 37894 hospital patients for the presence of anti-PrP IgGs, and found 21 high-titer individuals. The clinical files of these individuals did not reveal any enrichment of specific pathologies, suggesting that anti-PrP autoimmunity is innocuous. The existence of protective anti-prion antibodies in unbiased human immunological repertoires, combined with the reported lack of such antibodies in carriers of disease-associated PRNP mutations, suggests a link to the low incidence of spontaneous prion diseases in human populations.

neuroscience↗

bioRxiv2020

Genome-wide transcriptomics identifies an early preclinical signature of prion infection

The clinical course of prion diseases is accurately predictable despite long latency periods, suggesting that prion pathogenesis is driven by precisely timed molecular events. We constructed a searchable genome-wide atlas of mRNA abundance, splicing and editing alterations during the course of disease in prion-inoculated mice. Prion infection induced transient changes in mRNA abundance and processing already at eight weeks post inoculation, well ahead of any neuropathological and clinical signs. In contrast, microglia-enriched genes displayed an increase simultaneous with the appearance of clinical symptoms, whereas neuronal-enriched transcripts remained unchanged until the very terminal stage of disease. This suggests that glial pathophysiology, rather than neuronal demise, represents the final driver of disease. The administration of young plasma attenuated the occurrence of early mRNA abundance alterations and delayed symptoms in the terminal phase of the disease. The early onset of prion-induced molecular changes might thus point to novel biomarkers and potential interventional targets.

neuroscience↗

bioRxiv2019

An integrated genomic approach to dissect the genetic landscape regulating the cell-to-cell transfer of a-synuclein

Neuropathological and experimental evidence suggests that the cell-to-cell transfer of a-synuclein has an important role in the pathogenesis of Parkinsons disease (PD). However, the mechanism underlying this phenomenon is not fully understood. We undertook an siRNA, genome-wide high throughput screen to identify genes regulating the cell-to-cell transfer of a-synuclein. We transiently transfected HEK cells stably overexpressing a-synuclein with a construct encoding GFP-2a-aSynuclein-RFP. The cells expressing a-synuclein-RFP through transfection were double positive for GFP and RFP fluorescence, whereas the cells receiving it through transfer were positive only for RFP fluorescence. The amount of a-synuclein transfer was quantified by high content microscopy. A series of unbiased screens confirmed the involvement of 38 genes in the regulation of a-synuclein-RFP transfer. One of those hits was ITGA8, a candidate gene recently identified through a large PD genome wide association study (GWAS). Weighted gene co-expression network analysis (WGCNA) and weighted protein-protein network interaction analysis (WPPNIA) showed that the hits clustered in networks that included known PD Mendelian and GWAS risk genes more frequently than expected than random chance. Given the genetic overlap between a-synuclein transfer and PD, those findings provide supporting evidence for the importance of the cell-to-cell transfer of a-synuclein in the pathogenesis of PD, and expand our understanding of the mechanism of a-synuclein spread.

neuroscience↗