Biology subjects

Supattapone, S.

Publications and source records attributed to Supattapone, S..

8 recordsLinked to original sources

bioRxiv2026

Glycogen Synthase Kinase-3β Regulates Cellular Prion Protein Levels

The normal cellular prion protein (PrPC) is an essential substrate in all forms of prion diseases and a receptor for A{beta} oligomers in Alzheimers disease. However, it is not fully understood how cells regulate PrPC levels. Recently, we identified glycogen synthase kinase-3{beta} (GSK-3{beta}) as a potential regulator of PrPC levels in a whole genome knockout screen. Here, we show that both cell surface and total PrPC levels can be reduced either by siRNA-mediated Gsk3b (but not Gsk3a) knockdown or by CRISPR-mediated Gs3b knockout. Whole cell mass spectrometric analysis showed that PrPC was the 60th most significantly reduced protein (out of 7227 total proteins detected) in Gsk3b knockout cells, compared to wild-type cells. Two different GSK-3 inhibitors, laduviglusib (CHIR-99021) and AZD-1080, reduced PrPC levels in mouse CAD5 and human BE(2)-C cells, both in undifferentiated and differentiated states. PrPC levels were similarly reduced by cycloheximide treatment in both Gsk3b knockout and WT cells, indicating that GSK-3{beta} regulates PrPC levels through a post-translational mechanism. Finally, treatment with either laduviglusib or AZD-1080 reduced PrPSc levels in CAD5 cells infected with three different rodent prion strains. Overall, the results reveal that GSK-3{beta} activity controls PrPC levels in living cells, revealing a novel regulatory mechanism and promising therapeutic target.

cell biology↗

bioRxiv2026

Fluorescent non-canonical amino acid as a site-specific conformational probe of prion formation

The pathogenic conversion of the cellular prion protein (PrPC) into the {beta}-sheet-rich isoform PrPSc is the pivotal pathogenic event in prion disease, yet the molecular steps that govern this structural transition remain elusive. In this study, we introduce a new approach to monitor site-specific conformational transitions that occur during infectious prion formation. The method relies on genetically encoded substitution of a fluorescent, environmentally sensitive non-canonical amino acid, L-(7-hydroxycoumarin-4-yl)ethylglycine (7-HCAA), into recombinant PrP substate molecules, allowing real-time monitoring of structural changes in high-efficiency in vitro PrPSc conversion reactions. As proof of principle, we show that the W99 7-HCAA recPrP substate efficiently propagates two different PrPSc conformers (infectious cofactor PrPSc and non-infectious protein-only PrPSc). Bioassays in knock-in mice expressing bank vole (BV) PrP confirm that W99 7-HCAA cofactor PrPSc produced by serial propagation is infectious, causing scrapie with an incubation period and neuropathological profile like those induced by wild-type cofactor PrPSc. Marked differences in fluorescence intensity were observed between native, misfolded, and denatured states of W99 7-HCAA PrP, confirming that 7-HCAA reports on local changes in PrP conformation. Together, these findings establish 7-HCAA as a site-specific and sensitive probe of local PrP conformation. Moreover, the results suggest a new and broadly applicable strategy for studying conformational dynamics in amyloid-forming proteins.

biochemistry↗

bioRxiv2026

Generation of Infectious Prions Amenable to Site-specific Click Chemistry

Prion diseases are a group of fatal neurodegenerative diseases that proceed through the templated conversion of the normal PrPC protein to a self-propagating and infectious form, termed PrPSc. This conversion process is central to disease progression. However, due to difficulties in producing functional PrPSc molecules that can be selectively modified with chemical probes, many aspects of PrPSc biology cannot be directly studied. To overcome this limitation, we substituted p-azido-L-phenylalanine (AzF), a small click chemistry-reactive amino acid, for tryptophan residue 99 of PrPC. W99AzF PrPC substrate can efficiently and faithfully propagate either infectious or non-infectious PrPSc conformers in vitro. Critically, W99AzF PrPSc amyloid fibrils remain amenable to click chemistry by various ligands after the prion conversion process. Through the combination of site-specific substitution, the modularity of click chemistry, and the functional diversity of click labels, a multitude of modified prions can now be produced to ask targeted questions about the biochemical and biological basis of prion infectivity. O_FIG O_LINKSMALLFIG WIDTH=191 HEIGHT=200 SRC="FIGDIR/small/717205v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@562b13org.highwire.dtl.DTLVardef@12620e2org.highwire.dtl.DTLVardef@38e775org.highwire.dtl.DTLVardef@1e3c0cc_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

bioRxiv2026

Energy Flux Regulates Cell Death Induced by California Serogroup Orthobunyaviruses

The California serogroup (CSG) of orthobunyaviruses includes neuroinvasive viruses with varying pathogenicity. La Crosse virus (LACV) is a leading cause of pediatric arboviral encephalitis in the USA, while Inkoo virus (INKV) is widespread in Northern Europe but rarely causes disease. The reassortment potential of CSG viruses raises concerns about emerging virulent strains and highlights the need to develop therapies that are broadly effective against multiple CSG viruses. To identify host factors mediating viral neurotoxicity, we performed genome-wide CRISPR-Cas9 knockout screens in human neuroblastoma cells infected with LACV or INKV. Analysis revealed largely overlapping host dependency factors for both viruses. Unexpectedly, the screens identified mitochondrial energy production as a major pathway required for both LACV- and INKV-induced cell death. Reducing host cell energy production with mild hypothermia or sugar source substitution prolonged cell survival during viral infection with additive effects mediated by different mechanisms. Both manipulations also protected neuroblastoma cells from the Bunyamwera virus (BUNV), a non-CSG orthobunyavirus; and mild hypothermia protected mature human neurons from LACV. These results highlight host energy metabolism as a key modulator of CSG virus cytotoxicity and suggest novel avenues for general non-invasive therapeutic intervention against current and future strains of these and other orthobunyaviruses.

microbiology↗

bioRxiv2025

Genome-Wide Screens Identify Core Regulators of Cell Surface Prion Protein Expression

Expression of the cellular prion protein, PrPC, on the surface of neurons plays an important role in the pathogenesis of prion disease. We performed genome-wide CRISPR/Cas9 knockout screens in prion-infectible cells of neuronal origin (CAD5) to identify regulators of cell surface PrPC expression. We identified and validated 46 positive and 21 negative regulators of cell surface PrPC expression in undifferentiated CAD5 cells. Pathway analysis of the screening dataset showed that genes involved in the glycophosphatidylinositol (GPI) anchor and N-glycosylation biosynthetic pathways were overrepresented as positive regulators of cell surface PrPC. We also sought to determine whether the same or different genes regulate cell surface PrPC in CAD5 cells that have been differentiated to a more neuronal state and validated 41 positive and 13 negative regulators of CAD5 cell surface PrPC expression in the differentiated state. We identified 23 core genes as shared between the undifferentiated and differentiated cell states, including many positive regulators involved in GPI anchor biosynthesis. Intriguingly, unique regulators were also identified in the undifferentiated and differentiated cell states, suggesting that some mechanisms regulating cell surface PrPC expression in CAD5 cells are dependent on cell state. This list of core genes involved in regulating cell surface PrPC expression in a prion-susceptible, neuron-like cell type offers a valuable guide for future research and may help identify potential therapeutic targets for prion disease and other neurodegenerative diseases.

genomics↗

bioRxiv2025

Oligosaccharyltransferase (OST) complex inhibition effectively treats rodent and human prions

Prion diseases are invariably fatal neurodegenerative diseases that occur when the prion protein misfolds into a pathogenic form. There are currently no clinical treatments or cures for prion disease. Current challenges in the development of prion therapeutics include prion strain specificity, which can cause the emergence of drug-resistant prions, and lack of efficacy in treating human prions despite promising results in rodent models. Here we identify a novel therapeutic target for prion disease: the oligosaccharyltransferase (OST) complex. The OST complex is responsible for transferring the mature glycan to the acceptor polypeptide during Nglycosylation. We found that inhibiting OST effectively treats rodent prions in various dividing and non-dividing cell types. Importantly, we also demonstrate efficacy in treating human sCJD prions in non-dividing cerebral organoids. Inhibition of OST results in a 50% reduction in cell surface expression of the prion protein, PrPC. In addition, lysates of cells treated with the OST inhibitor NGI-1 were unable to amplify PrPSc seeds in Protein Misfolding Cyclic Amplification (PMCA) reactions. In summary, our results identify OST as a novel therapeutic target that regulates both the abundance of cell surface PrPC as well as its ability to convert into multiple strains of PrPSc, including human prions, in various in vitro systems. Author SummaryPrion diseases, such as Creutzfeldt-Jakob disease, are fatal brain disorders caused when a normal protein (PrPC) misfolds into a harmful form that spreads through the brain. There are no effective treatments, and drug development has been hampered by "strain" differences in prions that can lead to resistance and by therapies that work in rodents but not in humans. This study identifies a new treatment target: the oligosaccharyltransferase (OST) complex, a cellular machine that adds sugar groups to proteins. Blocking OST with a small molecule (NGI1) limited prion growth in multiple types of rodent cells, including non-dividing cells, and-- critically--also worked against human sporadic CJD prions in laboratory-grown human brain organoids. OST inhibition cut the amount of normal prion protein on cell surfaces in half, reducing the raw material available to convert into the disease-causing form. In addition, extracts from NGI-1-treated cells could no longer drive prion formation in a sensitive lab amplification test. These results suggest that targeting OST may offer a new strategy that works across different prion strains, including human ones.

cell biology↗

bioRxiv2023

Anti-prion drugs do not improve survival in knock-in models of inherited prion disease

Prion diseases uniquely manifest in three distinct forms: inherited, sporadic, and infectious. Wild-type prions are responsible for the sporadic and infectious versions, while mutant prions cause inherited variants like fatal familial insomnia (FFI) and familial Creutzfeldt-Jakob disease (fCJD). Although some drugs can prolong prion incubation times up to four-fold in rodent models of infectious prion diseases, no effective treatments for FFI and fCJD have been found. In this study, we evaluated the efficacy of various anti-prion drugs on newly-developed knock-in mouse models for FFI and fCJD. These models express bank vole prion protein (PrP) with the pathogenic D178N and E200K mutations. We applied various drug regimens known to be highly effective against wild-type prions in vivo as well as a brain-penetrant compound that inhibits mutant PrPSc propagation in vitro. None of the regimens tested (Anle138b, IND24, Anle138b + IND24, cellulose ether, and PSCMA) significantly extended disease-free survival or prevented mutant PrPSc accumulation in either knock-in mouse model, despite their ability to induce strain adaptation of mutant prions. Paradoxically, the combination of Anle138b and IND24 appeared to accelerate disease by 16% and 26% in kiBVIE200K and kiBVID178N mice, respectively, and accelerated the aggregation of mutant PrP molecules in vitro. Our results show that anti-prion drugs originally developed to treat infectious prion diseases do not necessarily work for inherited prion diseases, and that the recombinant sPMCA is not a reliable platform for identifying compounds that target mutant prions. This work underscores the need to develop therapies and validate screening assays specifically for mutant prions.

pharmacology and toxicology↗

bioRxiv2023

Convergent generation of atypical prions in knock-in mouse models of genetic prion disease

Most cases of human prion disease arise due to spontaneous misfolding of wild-type or mutant prion protein. Though recapitulating spontaneous prion conversion in animal models has proven challenging, transgenic mice expressing the misfolding-prone bank vole prion protein (BVPrP) recreate certain key aspects of sporadic and genetic prion disease. However, it remains unclear whether spontaneous prion generation can occur in the absence of protein over-expression and how disease-causing mutations affect prion strain properties. To address these issues, we generated knock-in mice expressing physiological levels of either wild-type or mutant BVPrP with isoleucine at codon 109. While mice expressing wild-type BVPrP remained free from neurological disease, a subset of knock-in mice expressing BVPrP with mutations that cause either fatal familial insomnia (D178N) or familial Creutzfeldt-Jakob disease (E200K) developed progressive neurological illness. Brains from spontaneously ill knock-in mice contained prion disease-specific neuropathological changes as well as atypical protease-resistant prion protein. Moreover, brain extracts from spontaneously ill D178N- or E200K-mutant BVPrP knock-in mice transmitted disease to mice expressing wild-type BVPrP. Surprisingly, the properties of the D178N- and E200K-mutant prions appeared identical both pre- and post-transmission, suggesting that both mutations guide the formation of a highly similar atypical prion strain. These findings imply that knock-in mice expressing mutant BVPrP spontaneously develop a bona fide prion disease and that mutations causing prion diseases may share a uniform initial mechanism of action. Therefore, these mice represent useful tools for studying the early stages of genetic prion diseases.

neuroscience↗