Abstract / Summary
Abstract Background Synucleinopathies are a group of neurodegenerative disorders characterized by the misfolding, aggregation, and accumulation of α-synuclein (α-syn) into oligomeric assemblies and amyloid inclusions. Alongside neurons, glial cells can internalize pathological α-syn and play a major role in its degradation. Despite the central importance of uptake in disease progression, the molecular mechanisms regulating α-syn fibril internalization are only beginning to be defined. Given that microRNAs (miRNAs) modulate endocytic pathways and multiple aspects of α-syn biology, we performed an unbiased genome-wide microRNA mimic screen to identify miRNAs that influence α-syn fibril uptake. Methods An image-based high-throughput screen of arrayed miRNAs was conducted in the U-87 MG glioblastoma cell line. Differential fluorescent and immunolabeling distinguished intracellular from extracellular α-syn PFFs, enabling quantification of miRNA-dependent effects on α-syn uptake. The lead miRNA identified in the primary screen was subsequently validated by flow cytometry in three glioblastoma cell lines and in mature hiPSC-derived astrocytes, using fluorescently labeled α-syn PFFs, monomeric α-syn, Tau-K18 fibrils, and PD-, DLB-, and MSA-seeded α-syn fibrils. Results In our primary screen, we identified nine enhancers and eight suppressors of α-syn preformed fibril (PFF) internalization. Orthogonal validation revealed miR-517a-3p as a robust suppressor of uptake, reducing PFF internalization by ~ 73% at early time points, with significant reductions maintained after 48 and 72 h of fibril exposure. miR-517a-3p did not accelerate degradation or secretion of internalized fibrils, indicating that it primarily impairs uptake rather than downstream processing. It also decreased internalization of monomeric α-syn and Tau-K18 fibrils, as well as PD-, DLB-, and MSA-seeded α-syn fibrils, while increasing transferrin uptake, indicating a cargo-selective rather than global effect on endocytosis. The inhibitory effect was reproduced in two additional glioblastoma lines (U-251 MG, LN-229). The effect extended to mature hiPSC-derived astrocytes, where miR-517a-3p reduced α-syn PFF uptake relative to the vehicle-treated control, although not below the untransfected baseline. Conclusions Together, our findings reveal a previously unrecognized role of miRNAs in the regulation of pathogenic protein aggregate internalization, suggesting that miRNA-dependent control of endocytosis may represent a modulatory axis in the cellular handling of α-syn assemblies.