Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive degeneration of motor neurons (MNs) in the corticospinal, bulbar, and spinal regions, ultimately resulting in paralysis and death within 3–5 years. The etiology of ALS is multifactorial, with over 25 known mutant genes contributing to approximately 70% of familial cases and 15% of sporadic cases (Goutman et al., 2023). Mutations in FUS are particularly associated with early-onset, aggressive forms of ALS. To date, more than 50 different FUS mutations have been identified in ALS patients, collectively accounting for approximately 4% of familial cases and 2% of sporadic ALS cases (Korobeynikov et al., 2022).

Fused in sarcoma (FUS) is a protein expressed ubiquitously, primarily located in the nucleus, with multiple functions in DNA damage repair and various aspects of RNA metabolism, including transcription, mRNA transport, pre-mRNA splicing, stability, and translation of microRNA and other non-coding RNAs (Dormann and Haass, 2013). It shuttles between the nucleus and cytoplasm, operating at a low cytoplasmic level under physiological conditions. However, in FUS-ALS patients (Kapeli et al., 2017), mutant FUS proteins mislocalize to the cytoplasm of MNs. The functional consequences of these ALS-associated mutations remain unclear. Evidence suggests that while loss-of-function alone is insufficient to cause MN death, toxic gain-of-function, including phenomena such as liquid–liquid phase separation (LLPS), stress granule (SG) formation, and involvement in the lysosomal autophagic system, contributes significantly (Scekic-Zahirovic et al., 2016). FUS’s nuclear import is facilitated by the C-terminal nuclear localization signal (NLS). Most ALS-causing mutations in FUS occur in this region, each displaying distinct molecular phenotypes. For example, arginine mutations impair RNA binding, while glycine mutations lead to rapid loss of fluidity (Niaki et al., 2020). Additionally, FUS-NLS mutations alter LLPS and SG associations, making FUS less sensitive to the chaperone activity of the nuclear import receptor transportin (TNPO1) (Hofweber et al., 2018). Previous reports suggest that the severity of the effect of FUS mutations on NLS function may correlate with the onset time and severity of ALS disease, as seen in variants such as p.R503fs in a 17-year-old juvenile amyotrophic lateral sclerosis (JALS) patient (Chen et al., 2020) and p.R495QfsX527 in a 19-year-old JALS patient (Belzil et al., 2012). Thus, understanding the structural and physicochemical properties of mutant FUS could guide laboratory research directions.

Our previous study identified a novel heterozygous FUS mutation, c.1509dupA:p.R503fs, in a 17-year-old female patient diagnosed with sporadic JALS who succumbed to the disease 15 months after onset (Chen et al., 2020). The early age of onset and rapid progression prompted further investigation. We observed increased FUS expression and suspicious accumulation in iPSC-derived MNs from the patient (Chen et al., 2020). However, the mechanism underlying JALS caused by the FUS^R503fs mutation warrants exploration. This mutation leads to the abnormal translation and early termination of the FUS protein in the NLS terminal, likely playing a pivotal role in disease development and progression. In this study, we explored the mutant FUS^R503fs extensively using bioinformatics analysis. We conducted electrophysiological tests on iPSC-derived MNs and examined their response to ropinirole treatment. Transcriptomic analysis revealed the downregulation of genes related to neural function maintenance, such as the autophagolysosomal-associated ATP6V1C2 gene. Ropinirole treatment partially alleviated the electrophysiological decline in FUS^R503fs MNs. Our findings suggest that the electrophysiological and spontaneous calcium activities of FUS^R503fs MNs are diminished, with ropinirole treatment showing slight improvement in some electrophysiological properties. Moreover, the decrease in spontaneous calcium activity signals may be independent of the glutamatergic receptor system, likely caused by other unknown mechanisms. The transcriptomic analysis also revealed the downregulation of genes related to neural function maintenance, along with the upregulation of genes associated with stress and viral infection. This study sheds light on the structural and physicochemical properties of this novel mutation and attempts to explore the pathophysiological characteristics of FUS-JALS in cell models, providing a basis and direction for further research into the pathological mechanisms.

The normal full-length FUS protein consists of 256 amino acid residues, including an LC domain, three RGG domains (RGG1, RGG2, and RGG3), an RRM domain, a zinc finger (ZnF) domain, and a NLS. The RGG1-RRM domain can bind RNA, while the RGG2-ZnF-RGG3 domain has a strong affinity for RNA and single-stranded DNA (ssDNA) (Schwartz et al., 2015). The majority of ALS-associated FUS mutations are missense mutations affecting the NLS. Additionally, some frameshift mutations resulting in complete NLS deletions have been reported. Our study identified a novel mutation, c.1509dupA:pR503fs, located in the NLS, leading to a truncated FUS protein of 503 amino acids with an aberrant sequence of RWLWPWQDGFQG. The addition of a novel “tail” of amino acids at the FUS C-terminus due to frameshift would alter the properties of truncated FUS proteins. Evidence suggests that mutations such as c.1432_1478del, c.1506dupA, and c.1509_1510delAG result in a frameshift peptide WLWPWQDGFQG, which may increase nuclear retention of mutant FUS (An et al., 2020). The c.1509dupA mutation results in truncated FUS proteins with a “tail” RWLWPWQDGFQG containing the peptide WLWPWQDGFQG, possibly explaining the accumulation of FUS in the nucleus and cytoplasm of differentiated MNs in our previous study (Chen et al., 2020). Although the presence of this peptide can partially rescue cytoplasmic FUS deposition, the accumulation of mutant FUS in the nucleus can lead to toxic gain-of-function effects. FUS nuclear import is mediated by its NLS sequence binding to the nuclear transporter TNPO1 (Transportin-1 and TNPO1) (Springhower et al., 2020), while nuclear export is independent of Exportin 1 (Exportin 1 and XPO1) (Fahrenkrog and Harel, 2018). This disruption severely affects the normal shuttling of FUS between the cytoplasm and nucleus, impacting its physiological function and causing cytotoxicity. The severity and rapid progression of ALS associated with this mutation may result from a combination of loss-of-function and gain-of-toxic function.

Changes in physicochemical properties and modification sites likely contribute to the pathophysiological effects of FUS mutations. Phosphorylation of FUS can occur in specific domains to prevent oligomerization, which affects its ability to regulate transcription. Arginine residues in the LC domain are also necessary for FUS maturation in cellular SGs (Bogaert et al., 2018). FUS functions not only through chemical interactions but also through its unique physical structure. The ability of FUS to bind G-quadruplexes depends on the β-spiral structure of the RGG domain (Yagi et al., 2018). Specifically, modified tyrosine in the RGG domain of FUS binds to G-quadruplex RNA, recognizing the 2′-OH of the ribose in the loops of G-quadruplexes (Takahama and Oyoshi, 2013). Our physicochemical and structural analyses (Figure 1) revealed significant differences in FUS^R503fs, particularly in the tertiary structure, hydrophilicity/hydrophobicity, and O-glycosylation sites. Previous studies have shown that EOGT mutations in ALS reduce O-GlcNAcylation, leading to the hyperphosphorylation of neurofilaments (Moll et al., 2019). Additionally, de-glycosylation is associated with a block in endoplasmic reticulum-to-Golgi traffic during autophagy progress (Fahie and Zachara, 2016). The structural abnormalities, reduced phosphorylation sites, and decreased hydrophilicity observed in the NLS domain of FUS^R503fs could be related to nuclear import disorders and dysregulation of transcriptional regulation. The significant reductions in O-glycosylation sites and hydrophilic changes may contribute to the aggregation of aberrant proteins and FUS.

Our MEA and calcium activity imaging experiments on MNs showed a significant decrease in the spontaneous electrical activity and spontaneous calcium activity signal of FUS^R503fs mutant neurons (Figure 3). The decrease in MN spontaneous electrical activity (DIV 3–4 weeks) occurred earlier than that of iPSCs derived from FUS^{R521L,R521C,R495QfsX527} genetic mutations, which typically occurs between DIV 7 and 10 weeks (Gunes et al., 2020). Considering that ALS patients usually develop symptoms in adolescence or adulthood and that iPSC-induced neurons can survive and grow in vitro for more than 70 days, we have performed MEA since DIV 24 and calcium activity imaging since DIV 30 to observe more obvious pathological manifestations. In our experiment, FUS^R503fs MNs (>DIV 24) exhibited hypoexcitability, while it has been reported that iPSC-derived MN genome-edited from a healthy cell line (FUS^H517D/H517D) showed hyperexcitability on day 7 by MEA and increased calcium activity on day 14 by calcium activity imaging (Kondo et al., 2023). The discrepancy in results may be due to the different lengths of culture time in vitro, suggesting that the excitability and activity of neurons in the early and late stages of the disease may differ. This hypothesis needs to be confirmed by large-scale studies with time gradients.

Transcriptomic analysis (Figure 4) of MNs revealed upregulated genes associated with viral infection and a downregulation of genes related to the maintenance of neural function, such as the ATP6V1C2 gene. ATP6V1C2 (ATPase H⁺ transporting V1 subunit C2) belongs to the vacuolar ATPase (v-ATPase) family and is significantly reduced in FUS^R503fs neurons mutated with FUS^R503fs (Log2fc < −2, p < 0.0001). Simultaneously, the expression of autophagosome/lysosome network components, ATP-binding cassette subfamily A8(ABCA8) and ATP-binding cassette subfamily C6(ABCC6) also declined (Log2fc < −1, p < 0.01). Maintaining a lysosomal acidic environment and autophagy flow is an energy-intensive process, relying on ATP breakdown facilitated by ABCA8 and ABCC6 (França et al., 2020). A study in Nature Cell Biology on the autophagy phenotype related to a common ALS-associated mutation in UBQLN2 highlights the role of abnormal v-ATPase-MTOR-mediated autophagy in ALS pathogenesis (Yang and Klionsky, 2020). Ubiquilins are crucial for v-ATPase-mediated lysosomal acidification (Senturk et al., 2019). A genome-wide CRISPR screen identified v-ATPase as a potential drug target to reduce levels of the ALS protein ataxin-2 (Kim et al., 2022), suggesting that FUS^R503fs mutations likely affect the autophagic lysosomal system.

We evaluated the therapeutic impact of ropinirole on induced MNs using MEA analysis. The results indicated that the decrease in burst frequency observed in mutated neurons could be partially reversed (Figure 5). Because only one concentration and one treatment duration were tested, these results might not fully represent the drug’s efficacy. However, they suggest that this cell line could serve as a tool for drug screening and hint at the potential therapeutic value of ropinirole in FUS-ALS. Ropinirole, a non-ergot dopamine receptor agonist, has been reported to activate autophagy, possibly through a Beclin-1-dependent pathway (Okano et al., 2020). It would be interesting to explore if ropinirole-induced activation of D2R/D3R receptors could induce autophagy, leading to the degradation of abnormal RNA–protein complexes in ALS MNs.

The data presented in this study has been deposited in the SRA on NCBI repository, accession number PRJNA1097276.

The studies involving humans were approved by Ethics Committee of Shanghai Sixth People’s Hospital. The studies were conducted in accordance with the local legislation and institutional requirements. The participants provided their written informed consent to participate in this study.

LC: Data curation, Formal analysis, Funding acquisition, Writing – original draft, Writing – review & editing. GC: Methodology, Software, Writing – review & editing. MZ: Methodology, Writing – review & editing. XZ: Supervision, Writing – review & editing.