Analysis of the composition of the external late endosomal transcriptome and its alteration in ALS-like FUS P525L Motor Neurons

The endo-lysosomal system is a critical cellular network involved in intracellular trafficking, recycling and catabolic activities. Central to this system, late endosomes act as hubs, mediating the delivery of cargo to lysosomes for degradation or recycling to other cellular compartments. While traditionally studied for roles in protein degradation, recent evidence suggests that late endosomes also transport mRNA molecules, particularly in neurons, cells in which efficient intracellular transport is essential. This process represents a specialized mechanism for RNA localization and protein synthesis, but its regulation remains poorly understood. Disruptions in vesicular trafficking are linked to neurodegenerative diseases like Amyotrophic lateral sclerosis (ALS), where its failure contribute to neuronal dysfunction. ALS-related mutations of the RNA-binding protein FUS have been implicated in the disruption of axonal transport and aberrant RNA-protein granule formation. This project investigates the mechanisms through which RNAs are recruited onto late endosomes and how the FUS P525L mutation affects RNA recruitment to those organelles, employing an APEX2-seq proximity labeling strategy in motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs). By fusing the APEX2 peroxidase to the late endosomal marker RAB7A, we selectively labeled and isolated RNAs localized to the surface of late endosomes, enabling us to map their external transcriptome. This study provides insights into how RNAs are recruited and transported on late endosomes, elucidating mechanisms of long-distance RNA transport in neurons, and further expands the understanding of how FUS mutations contribute to the pathogenesis of ALS. In particular, we defined for the first time late endosomal transcriptome in MNs and identified length, presence of putative “zip-code” sequences, and involvement in axon and synapse functionality as common features of enriched transcripts. Our data indicate that FUS mutation strongly impairs the correct recruitment of several RNAs onto those vesicles providing crucial information and novel explanations about its impact on RNA transport and ALS pathology. Understanding these molecular interactions between late endosomes, RNA, and RNA-binding proteins could reveal novel therapeutic targets for neurodegenerative diseases, offering potential strategies to restore proper RNA transport and mitigate cellular dysfunctions associated with ALS and related conditions.