Biology subjects

Bouris, V.

Publications and source records attributed to Bouris, V..

5 recordsLinked to original sources

bioRxiv2026

PODXL regulates the neuronal entry of α-synuclein aggregates

Cell-to-cell transmission of -synuclein (-Syn) aggregates is a key event in Parkinson's disease (PD) pathogenesis, yet the neuronal surface factors that govern uptake of pathogenic -Syn fibrils remain undefined. We developed a flow cytometry-based CRISPR activation screen for modifiers of -Syn preformed fibril (PFF) uptake by human iPSC-derived cortical neurons. By interrogating 68 genes encoding cell-surface proteins prioritised by PD genetic associations, we identified podocalyxin-like protein 1 (PODXL) as a strong suppressor of -Syn fibril uptake. High-content imaging and PODXL cDNA overexpression confirmed reduced PFF uptake. Stable PODXL overexpression also reduced PFF-seeded pSynSer129 accumulation and uptake of structurally distinct recombinant and disease-derived -Syn fibrils. Conversely, shRNA-mediated PODXL knockdown increased -Syn PFF uptake, and analysis of PODXL domain-deletion and N-glycosylation mutants suggested that the ectodomain and N-glycosylation contribute to the PODXL-mediated reduction in uptake. Transcriptomic profiling further identified membrane metalloendopeptidase (MME) as a PODXL-responsive candidate mediator. MME activation and treatment with conditioned medium from MME-activated neurons reduced PFF uptake. Thiorphan, an MME inhibitor, partially attenuated the PODXL-associated reduction in uptake. Together, our findings identify PODXL as a neuronal brake on -Syn uptake and implicate its glycan-dependent surface properties together with MME-associated extracellular activity.

neuroscience↗

bioRxiv2026

Genome-wide arrayed CRISPR activation screen for prion protein modulators

The cellular prion protein (PrPC) is an essential substrate for prion propagation, and its abundance strongly influences susceptibility to prion disease. To systematically identify genetic regulators of PrPC abundance, we performed an arrayed genome-wide CRISPR activation (CRISPRa) screen targeting 19,839 human protein-coding genes in human glioblastoma cells. Using a quantitative time-resolved fluorescence resonance energy transfer (TR-FRET) immunoassay, we measured PrPC levels across 22,442 individual genetic perturbations. This screen identified 531 genes whose activation significantly modulated PrPC abundance. A curated subset of 50 candidates was subjected to validation using independent TR-FRET assays and Western blotting, confirming 45 (90%) of tested hits. All raw and processed screening data, metadata, and analysis code are publicly available. This dataset provides a comprehensive resource for studying genetic regulation of PrPC homeostasis and enables reuse for investigations of membrane protein trafficking, proteostasis, and prion biology.

neuroscience↗

bioRxiv2025

Large-scale bidirectional arrayed genetic screens identify OXR1 and EMC4 as modifiers of α-synuclein aggregation

In Parkinsons disease and other synucleinopathies, -synuclein (-Syn) misfolds and forms Ser129-phosphorylated aggregates (pSyn129). The factors controlling this process are largely unknown. Here, we used arrayed CRISPR-mediated gene activation and ablation to discover new pSyn129 modulators. Using quadruple-guide RNAs (qgRNAs) and Cas9, or an inactive Cas9 version fused to a synthetic transactivator, we ablated 2304 and activated 2428 human genes related to mitochondrial, trafficking and motility function in HEK293 cells. After exposure of cells to -Syn fibrils, pSyn129 signals were recorded by high-throughput fluorescence microscopy and aggregates were identified by image analysis. We found that pSyn129 was increased by activating the mitochondrial protein OXR1, which decreased ATP levels and altered the mitochondrial membrane potential. Instead, pSyn129 was reduced by ablation of the endoplasmic reticulum (ER)-associated protein EMC4, which enhanced ER-driven autophagic flux and lysosomal clearance. OXR1 activation preferentially modulated cellular reactions to fibrils derived from multiple system atrophy (MSA) patients, whereas EMC4 ablation broadly reduced pSyn129 across diverse -Syn polymorphs. These findings were confirmed in human iPSC-derived cortical and dopaminergic neurons, where OXR1 preferentially promoted somatic aggregation and EMC4 reduced both somatic and neuritic aggregates. These results uncover previously unrecognized roles for OXR1 and EMC4 in -Syn aggregation, thereby broadening our mechanistic understanding of synucleinopathies.

neuroscience↗

bioRxiv2024

TFAP2C and HNRNPK control mTOR cell metabolism and prion propagation

Heterogeneous Nuclear Ribonucleoprotein K (hnRNP K) is a limiting factor for prion propagation. However, little is known about the function of hnRNP K except that it is essential to cell survival. Here, we performed a synthetic-viability CRISPR ablation screen to identify epistatic interactors of HNRNPK. We found that deletion of Transcription Factor AP-2{gamma} (TFAP2C) suppressed the death of hnRNP K-depleted LN-229 and U-251 MG cells, whereas its overexpression hypersensitized cells to hnRNP K loss. HNRNPK ablation decreased cellular ATP, downregulated genes related to lipid and glucose metabolism, and enhanced autophagy. Co-occurrent deletion of TFAP2C reversed these effects, restoring transcriptional balance and alleviating energy deficiency. We linked HNRNPK and TFAP2C interaction to mTOR signaling, observing that HNRNPK ablation inhibited mTORC1 activity through downregulation of mTOR and Rptor, while TFAP2C overexpression enhanced mTORC1 downstream functions. In prion-infected cells, TFAP2C activation reduced prion levels and countered the increased prion propagation caused by HNRNPK suppression. Short-term pharmacological inhibition of mTOR also elevated prion levels and partially mimicked the effects of HNRNPK silencing. Our study identifies TFAP2C as a genetic interactor of HNRNPK, implicates their roles in mTOR metabolic regulation, and establishes a causative link between these activities and prion propagation.

cell biology↗

bioRxiv2019

Nutrient transporter translocation to the plasma membrane via a Golgi-independent unconventional route

Nutrient transporters are believed to traffic from their site of synthesis, the ER, to the plasma membrane, through the Golgi, using the conventional vesicular trafficking pathway. However, here we report that the UapA purine transporter in Aspergillus nidulans, follows a Golgi-independent, unconventional new route, which does not involve key Rab GTPases, AP adaptors, microtubules or endosomes, but is dependent on functional COPII, clathrin heavy chain (ClaH) and actin. The role of ClaH in transporter secretion is shown to be unrelated to that performing, together with Rab11/AP-1, at the Golgi, and is seemingly due to an effect in actin network functioning. Our findings are discussed within the frame of a model that rationalizes why the trafficking mechanism uncovered herein might hold true for transporters in higher organisms. One Sentence SummaryFungal transporters use a non-polar, COPII- and actin-dependent, route for subcellular traffic to the cell membrane.

cell biology↗