Lysine acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the preferential death of motor neurons. Approximately 10% of ALS cases are familial and 90% are sporadic. Fused in sarcoma (FUS) is a ubiquitously expressed RNA-binding protein implicated in familial ALS and frontotemporal dementia (FTD). The physiological function and pathological mechanism of FUS are not well understood, particularly whether post-translational modifications play a role in regulating FUS function. In this study, we discovered that FUS was acetylated at lysine-315/316 (K315/K316) and lysine-510 (K510) residues in two distinct domains. Located in the nuclear localization sequence, K510 acetylation disrupted the interaction between FUS and Transportin-1, resulting in the mislocalization of FUS in the cytoplasm and formation of stress granule-like inclusions. Located in the RNA recognition motif, K315/K316 acetylation reduced RNA binding to FUS and decreased the formation of cytoplasmic inclusions. Treatment with deacetylase inhibitors also significantly reduced the inclusion formation in cells expressing ALS mutation P525L. More interestingly, familial ALS patient fibroblasts showed higher levels of FUS K510 acetylation as compared with healthy controls. Lastly, CREB-binding protein/p300 acetylated FUS, whereas both sirtuins and histone deacetylases families of lysine deacetylases contributed to FUS deacetylation. These findings demonstrate that FUS acetylation regulates the RNA binding, subcellular localization and inclusion formation of FUS, implicating a potential role of acetylation in the pathophysiological process leading to FUS-mediated ALS/FTD.

FUS regulates autophagy by mediating the transcription of genes critical to the autophagosome formation.

Fused in sarcoma (FUS) is a ubiquitously expressed RNA/DNA-binding protein that plays different roles in the cell. FUS pathology has been reported in neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mutations in FUS have also been linked to a subset of familial ALS. FUS is mainly localized in the nucleus although it shuttles between the nucleus and the cytoplasm. ALS-linked mutations cause the accumulation of the FUS protein in cytoplasm where it forms stress granule-like inclusions. The protein- and RNA-containing inclusions are reported to be positive of autophagosome markers and degraded by the autophagy pathway. However, the role of FUS in the autophagy pathway remains to be better understood. Using immunoblot and confocal imaging techniques in this study, we found that FUS knockout (KO) cells showed a decreased basal autophagy level. Rapamycin and bafilomycin A1 treatment showed that FUS KO cells were not able to initiate autophagy as efficiently as wild-type cells, suggesting that the autophagosome formation is affected in the absence of FUS. Moreover, using immunoblot and quantitative PCR techniques, we found that the mRNA and protein levels of the genes critical in the initial steps of the autophagy pathway (FIP200, ATG16L1 and ATG12) were significantly lower in FUS KO cells. Re-expressing FUS in the KO cells restored the expression of FIP200 and ATG16L1. Our findings demonstrate a novel role of FUS in the autophagy pathway, that is, regulating the transcription of genes involved in early stages of autophagy such as the initiation and elongation of autophagosomes.

Proteomic analysis of FUS interacting proteins provides insights into FUS function and its role in ALS.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease. Mutations in the Fused in Sarcoma/Translocated in Liposarcoma (FUS/TLS) gene cause a subset of familial ALS cases and are also implicated in sporadic ALS. FUS is typically localized to the nucleus. The ALS-related FUS mutations cause cytoplasmic mis-localization and the formation of stress granule-like structures. Abnormal cytoplasmic FUS localization was also found in a subset of frontotemporal dementia (FTLD) cases without FUS mutations. To better understand the function of FUS, we performed wild-type and mutant FUS pull-downs followed by proteomic identification of the interacting proteins. The FUS interacting partners we identified are involved in multiple pathways, including chromosomal organization, transcription, RNA splicing, RNA transport, localized translation, and stress response. FUS interacted with hnRNPA1 and Matrin-3, RNA binding proteins whose mutations were also reported to cause familial ALS, suggesting that hnRNPA1 and Matrin-3 may play common pathogenic roles with FUS. The FUS interactions displayed varied RNA dependence. Numerous FUS interacting partners that we identified are components of exosomes. We found that FUS itself was present in exosomes, suggesting that the secretion of FUS might contribute to the cell-to-cell spreading of FUS pathology. FUS interacting proteins were sequestered into the cytoplasmic mutant FUS inclusions that could lead to their mis-regulation or loss of function, contributing to ALS pathogenesis. Our results provide insights into the physiological functions of FUS as well as important pathways where mutant FUS can interfere with cellular processes and potentially contribute to the pathogenesis of ALS.

Clinical and experimental studies of a novel P525R FUS mutation in amyotrophic lateral sclerosis.

OBJECTIVE: To describe the clinical features of a novel fused in sarcoma (FUS) mutation in a young adult female amyotrophic lateral sclerosis (ALS) patient with rapid progression of weakness and to experimentally validate the consequences of the P525R mutation in cellular neuronal models. METHODS: We conducted sequencing of genomic DNA from the index patient and her family members. Immunocytochemistry was performed in various cellular models to determine whether the newly identified P525R mutant FUS protein accumulated in cytoplasmic inclusions. Clinical features of the index patient were compared with 19 other patients with ALS carrying the P525L mutation in the same amino acid position. RESULTS: A novel mutation c.1574C>G (p.525P>R) in the FUS gene was identified in the index patient. The clinical symptoms are similar to those in familial ALS patients with the P525L mutation at the same position. The P525R mutant FUS protein showed cytoplasmic localization and formed large stress granule-like cytoplasmic inclusions in multiple cellular models. CONCLUSIONS: The clinical features of the patient and the cytoplasmic inclusions of the P525R mutant FUS protein strengthen the notion that mutations at position 525 of the FUS protein result in a coherent phenotype characterized by juvenile or young adult onset, rapid progression, variable positive family history, and female preponderance.