FUS Alters circRNA Metabolism in Human Motor Neurons Carrying the ALS-Linked P525L Mutation.

Deregulation of RNA metabolism has emerged as one of the key events leading to the degeneration of motor neurons (MNs) in Amyotrophic Lateral Sclerosis (ALS) disease. Indeed, mutations on RNA-binding proteins (RBPs) or on proteins involved in aspects of RNA metabolism account for the majority of familiar forms of ALS. In particular, the impact of the ALS-linked mutations of the RBP FUS on many aspects of RNA-related processes has been vastly investigated. FUS plays a pivotal role in splicing regulation and its mutations severely alter the exon composition of transcripts coding for proteins involved in neurogenesis, axon guidance, and synaptic activity. In this study, by using in vitro-derived human MNs, we investigate the effect of the P525L FUS mutation on non-canonical splicing events that leads to the formation of circular RNAs (circRNAs). We observed altered levels of circRNAs in FUSP525L MNs and a preferential binding of the mutant protein to introns flanking downregulated circRNAs and containing inverted Alu repeats. For a subset of circRNAs, FUSP525L also impacts their nuclear/cytoplasmic partitioning, confirming its involvement in different processes of RNA metabolism. Finally, we assess the potential of cytoplasmic circRNAs to act as miRNA sponges, with possible implications in ALS pathogenesis.

Circ-Hdgfrp3 shuttles along neurites and is trapped in aggregates formed by ALS-associated mutant FUS.

CircRNAs belong to a family of RNA molecules which are conserved in evolution, have tissue-specific expression, and are abundant in neuronal cells. Here, we define several features of circ-Hdgfrp3 and describe interesting alterations occurring in motor neurons (MNs) carrying ALS-associated FUS mutations. Through a highly sensitive in situ approach we describe that circ-Hdgfrp3 traffics along neurites, while upon oxidative stress it is retained in the perinuclear region. While in wild-type stressed MNs, circ-Hdgfrp3 localizes in stress granules (SGs), in MNs carrying mutant FUS, a higher proportion of circ-Hdgfrp3 was trapped into cytoplasmic aggregates. Upon stress removal, circ-Hdgfrp3 was easily freed from SGs whereas it was less efficiently released from FUS-aggregates. We found that the human circ-Hdgfrp3 counterpart was also similarly associated to mutant FUS-aggregates in stressed neuronal cells. Overall, the alteration of circ-Hdgfrp3 trafficking adds a further layer of complexity to the role of FUS-aggregates in ALS disease.