Selective regulation of the prion protein by endolysosomal pathways identified via genome-wide CRISPR screening

Introduction:
Prion diseases are fatal neurodegenerative disorders driven by the misfolding of the cellular prion protein (PrPC) into pathogenic PrPSc. As PrPC expression is rate-limiting for disease progression, understanding its regulation is essential for developing therapeutic strategies.

Objectives:
To systematically identify and characterize genetic regulators of PrPC expression and elucidate the underlying mechanisms, particularly those involving protein degradation and trafficking.

Methods:
We conducted an arrayed genome-wide CRISPR activation screen in U251-MG cells using a custom library targeting 19,839 human genes. PrPC levels were quantified via TR-FRET, and 50 top hits were validated using orthogonal assays including Western blotting, flow cytometry, and proteomics. Multi-omics and AI-based imaging analyses were performed to dissect regulatory mechanisms.

Results:
The screen identified 531 PrPC modulators (80 upregulators, 451 downregulators), with >90% validation among 50 prioritized genes. Proteomic profiling showed these regulators specifically modulate PrPC without broadly affecting other GPI-anchored proteins, indicating high target specificity. PrPC downregulators acted through three major mechanisms: (1) increased trafficking of PrPC to lysosomes via upregulation of the HOPS-CORVET complexes and downregulation of the retromer complex; (2) enhanced perinuclear lysosome localization with increased lysosomal acidification and degradative activity; and (3) LRP1-mediated, PrPC-specific endocytosis. Co-regulation with APP-processing proteins further suggests mechanistic convergence with amyloid beta pathways.

Conclusion:
This study uncovers a lysosome-centered and PrPC-specific regulatory network. The identified genes represent therapeutic targets with strong mechanistic rationale and specificity, enabling selective PrPC modulation without perturbing broader GPI-anchored protein homeostasis. These findings offer a framework for targeted interventions for prion and related neurodegenerative diseases.