Fused In Sarcoma Protein Research Articles

CircFndc3b Mediates Exercise-Induced Neuroprotection by Mitigating Microglial/Macrophage Pyroptosis via the ENO1/KLF2 Axis in Stroke Mice.

Circular RNA (circRNA) plays a pivotal role in regulating neurological damage post-ischemic stroke. Previous researches demonstrated that exercise mitigates neurological dysfunction after ischemic stroke, yet the specific contributions of circRNAs to exercise-induced neuroprotection remain unclear. This study reveals that mmu_circ_0001113 (circFndc3b) is markedly downregulated in the penumbral cortex of a mouse model subjected to middle cerebral artery occlusion (MCAO). However, exercise increased circFndc3b expression in microglia/macrophages, alleviating pyroptosis, reducing infarct volume, and enhancing neurological recovery in MCAO mice. Mechanistically, circFndc3b interacted with Enolase 1 (ENO1), facilitating ENO1's binding to the 3' Untranslated Region (3'UTR) of Krüppel-like Factor 2 (Klf2) mRNA, thereby stabilizing Klf2 mRNA and increasing its protein expression, which suppressed NOD-like Receptor Family Pyrin Domain Containing 3 (NLRP3) inflammasome-mediated microglial/macrophage pyroptosis. Additionally, circFndc3b enhanced ENO1's interaction with the 3'UTR of Fused in Sarcoma (FUS) mRNA, leading to increased FUS protein levels and promoting circFndc3b cyclization. These results suggest that circFndc3b mediates exercise-induced anti-pyroptotic effects via the ENO1/Klf2 axis, and a circFndc3b/ENO1/FUS positive feedback loop may potentiate exercise's neuroprotective effects. This study unveils a novel mechanism underlying exercise-induced neuroprotection in ischemic stroke and positions circFndc3b as a promising therapeutic target for stroke management, mimicking the beneficial effects of exercise.

FUS-Mediated Inhibition of Myogenesis Elicited by Suppressing TNNT1 Production.

Myogenesis is a highly orchestrated process whereby muscle precursor cells, myoblasts, develop into muscle fibers to form skeletal muscle during embryogenesis and regenerate adult muscle. Here, we studied the RNA-binding protein FUS (fused in sarcoma), which has been implicated in muscular and neuromuscular pathologies but is poorly characterized in myogenesis. Given that FUS levels declined in human and mouse models of skeletal myogenesis, and that silencing FUS enhanced myogenesis, we hypothesized that FUS might be a repressor of myogenic differentiation. Interestingly, overexpression of FUS delayed myogenesis, accompanied by slower production of muscle differentiation markers. To identify the mechanisms through which FUS inhibits myogenesis, we uncovered RNA targets of FUS by ribonucleoprotein immunoprecipitation (RIP) followed by RNA-sequencing (RNA-seq) analysis. Stringent selection of the bound transcripts uncovered Tnnt1 mRNA, encoding troponin T1 (TNNT1), as a major effector of FUS influence on myogenesis. We found that in myoblasts, FUSretained Tnnt1 mRNA in the nucleus, preventing TNNT1 expression; however, reduction of FUS during myogenesis or by silencing FUS released Tnnt1 mRNA for export to the cytoplasm, enabling TNNT1 translation and promoting myogenesis. We propose that FUS inhibits myogenesis by suppressing TNNT1 expression through a mechanism of nuclear Tnnt1 mRNA retention.

Mutation of the ALS-/FTD-Associated RNA-Binding Protein FUS Affects Axonal Development.

Aberrant condensation and localization of the RNA-binding protein (RBP) fused in sarcoma (FUS) occur in variants of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Changes in RBP function are commonly associated with changes in axonal cytoskeletal organization and branching in neurodevelopmental disorders. Here, we asked whether branching defects also occur in vivo in a model of FUS-associated disease. We use two reported Xenopus models of ALS/FTD (of either sex), the ALS-associated mutant FUS(P525L) and a mimic of hypomethylated FUS, FUS(16R). Both mutants strongly reduced axonal complexity in vivo. We also observed an axon looping defect for FUS(P525L) in the target area, which presumably arises due to errors in stop cue signaling. To assess whether the loss of axon complexity also had a cue-independent component, we assessed axonal cytoskeletal integrity in vitro. Using a novel combination of fluorescence and atomic force microscopy, we found that mutant FUS reduced actin density in the growth cone, altering its mechanical properties. Therefore, FUS mutants may induce defects during early axonal development.

Cell-Type-Dependent Recruitment Dynamics of FUS Protein at Laser-Induced DNA Damage Sites.

Increased signs of DNA damage have been associated to aging and neurodegenerative diseases. DNA damage repair mechanisms are tightly regulated and involve different pathways depending on cell types and proliferative vs. postmitotic states. Amongst them, fused in sarcoma (FUS) was reported to be involved in different pathways of single- and double-strand break repair, including an early recruitment to DNA damage. FUS is a ubiquitously expressed protein, but if mutated, leads to a more or less selective motor neurodegeneration, causing amyotrophic lateral sclerosis (ALS). Of note, ALS-causing mutation leads to impaired DNA damage repair. We thus asked whether FUS recruitment dynamics differ across different cell types putatively contributing to such cell-type-specific vulnerability. For this, we generated engineered human induced pluripotent stem cells carrying wild-type FUS-eGFP and analyzed different derivatives from these, combining a laser micro-irradiation technique and a workflow to analyze the real-time process of FUS at DNA damage sites. All cells showed FUS recruitment to DNA damage sites except for hiPSC, with only 70% of cells recruiting FUS. In-depth analysis of the kinetics of FUS recruitment at DNA damage sites revealed differences among cellular types in response to laser-irradiation-induced DNA damage. Our work suggests a cell-type-dependent recruitment behavior of FUS during the DNA damage response and repair procedure. The presented workflow might be a valuable tool for studying the proteins recruited at the DNA damage site in a real-time course.

Cytoplasmic retention of the DNA/RNA-binding protein FUS ameliorates organ fibrosis in mice.

Uncontrolled accumulation of extracellular matrix leads to tissue fibrosis and loss of organ function. We previously demonstrated in vitro that the DNA/RNA-binding protein fused in sarcoma (FUS) promotes fibrotic responses by translocating to the nucleus, where it initiates collagen gene transcription. However, it is still not known whether FUS is profibrotic in vivo and whether preventing its nuclear translocation might inhibit development of fibrosis following injury. We now demonstrate that levels of nuclear FUS are significantly increased in mouse models of kidney and liver fibrosis. To evaluate the direct role of FUS nuclear translocation in fibrosis, we used mice that carry a mutation in the FUS nuclear localization sequence (FUSR521G) and the cell-penetrating peptide CP-FUS-NLS that we previously showed inhibits FUS nuclear translocation in vitro. We provide evidence that FUSR521G mice or CP-FUS-NLS-treated mice showed reduced nuclear FUS and fibrosis following injury. Finally, differential gene expression analysis and immunohistochemistry of tissues from individuals with focal segmental glomerulosclerosis or nonalcoholic steatohepatitis revealed significant upregulation of FUS and/or collagen genes and FUS protein nuclear localization in diseased organs. These results demonstrate that injury-induced nuclear translocation of FUS contributes to fibrosis and highlight CP-FUS-NLS as a promising therapeutic option for organ fibrosis.

Zebrafish CCNF and FUS Mediate Stress-Specific Motor Responses.

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the degeneration of motor neurons. Mutations in the cyclin F (CCNF) and fused in sarcoma (FUS) genes have been associated with ALS pathology. In this study, we aimed to investigate the functional role of CCNF and FUS in ALS by using genome editing techniques to generate zebrafish models with genetic disruptions in these genes. Sequence comparisons showed significant homology between human and zebrafish CCNF and FUS proteins. We used CRISPR/Cas9 and TALEN-mediated genome editing to generate targeted disruptions in the zebrafish ccnf and fus genes. Ccnf-deficient zebrafish exhibited abnormal motor neuron development and axonal outgrowth, whereas Fus-deficient zebrafish did not exhibit developmental abnormalities or axonopathies in primary motor neurons. However, Fus-deficient zebrafish displayed motor impairments in response to oxidative and endoplasmic reticulum stress. The Ccnf-deficient zebrafish were only sensitized to endoplasmic reticulum stress, indicating that ALS genes have overlapping as well as unique cellular functions. These zebrafish models provide valuable platforms for studying the functional consequences of CCNF and FUS mutations in ALS pathogenesis. Furthermore, these zebrafish models expand the drug screening toolkit used to evaluate possible ALS treatments.

Study on Phase Separation of Fused in Sarcoma by Fluorescence Correlation Spectroscopy.

Liquid-liquid phase separation (LLPS) of fused in sarcoma (FUS) has emerged as a fundamental principle underpinning cellular function and malfunction. However, we know little about the FUS phase transition process from individual molecules to nanoscale condensates, which plays important roles in neurodegenerative diseases. Here, we propose the fluorescence correlation spectroscopy (FCS) method to quantitatively study the phase separation process of FUS protein with the fluorescent tag-enhanced green fluorescent protein (EGFP), from individual molecules to nanoscale condensates. The characteristic diffusion time (τD) of the protein condensates can be obtained from the FCS curve, which increases with the growth of the protein hydration radius. The bigger the τD value of the protein condensates, the larger the condensates formed by the phase separation of FUS. By this method, we discovered that the critical concentration for FUS to phase separation was 20 nM. We then plotted FUS phase diagrams based on τD under different concentrations of NaCl and found that both low-salt and high-salt concentrations tended to promote FUS-EGFP phase separation. Our results showed that ATP has a good inhibitory effect on FUS phase separation, and its inhibition constant IC50 was 3.2 mM. Finally, we evaluated the inhibition efficiency of single-stranded DNA sequences (ssDNA) on FUS phase separation and demonstrated that ssDNA containing three copies of TCCCCGT had relatively strong inhibition efficiency. In summary, our work provides detailed insight into the FUS phase transition process from individual molecules to nanoscale condensates at nanomolar concentrations and can be exploited for drug screening of neurodegenerative diseases.

FUS-mediated HypEVs: Neuroprotective effects against ischemic stroke.

Few studies have investigated the properties and protein composition of small extracellular vesicles (sEVs) derived from neurons under hypoxic conditions. Presently, the extent of the involvement of these plentiful sEVs in the onset and progression of ischemic stroke remains an unresolved question. Our study systematically identified the characteristics of sEVs derived from neurons under hypoxic conditions (HypEVs) by physical characterization, sEV absorption, proteomics and transcriptomics analysis. The effects of HypEVs on neurites, cell survival, and neuron structure were assessed in vitro and in vivo by neural complexity tests, magnetic resonance imaging (MRI), Golgi staining, and Western blotting of synaptic plasticity-related proteins and apoptotic proteins. Knockdown of Fused in Sarcoma (FUS) small interfering RNA (siRNA) was used to validate FUS-mediated HypEV neuroprotection and mitochondrial mRNA release. Hypoxia promoted the secretion of sEVs, and HypEVs were more easily taken up and utilized by recipient cells. The MRI results illustrated that the cerebral infarction volume was reduced by 45% with the application of HypEVs, in comparison to the non- HypEV treatment group. Mechanistically, the FUS protein is necessary for the uptake and neuroprotection of HypEVs against ischemic stroke as well as carrying a large amount of mitochondrial mRNA in HypEVs. However, FUS knockdown attenuated the neuroprotective rescue capabilities of HypEVs. Our comprehensive dataset clearly illustrates that FUS-mediated HypEVs deliver exceptional neuroprotective effects against ischemic stroke, primarily through the maintenance of neurite integrity and the reduction of mitochondria-associated apoptosis.

“Boundary residues” between the folded RNA recognition motif and disordered RGG domains are critical for FUS–RNA binding

Fused in sarcoma (FUS) is an abundant RNA-binding protein, which drives phase separation of cellular condensates and plays multiple roles in RNA regulation. The RNA-binding ability of FUS protein is crucial to its cellular function. Here, our molecular simulation study on the FUS-RNA complex provides atomic resolution insights into the observations from biochemical studies and also illuminates our understanding of molecular driving forces that mediate the structure, stability, and interaction of the RNA recognition motif (RRM) and RGG domains of FUS with a stem-loop junction RNA. We observe clear cooperativity and division of labor among the ordered (RRM) and disordered domains (RGG1 and RGG2) of FUS that leads to an organized and tighter RNA binding. Irrespective of the length of RGG2, the RGG2-RNA interaction is confined to the stem-loop junction and the proximal stem regions. On the other hand, the RGG1 interactions are primarily with the longer RNA stem. We find that the C terminus of RRM, which make up the "boundary residues" that connect the folded RRM with the long disordered RGG2 stretch of the protein, plays a critical role in FUS-RNA binding. Our study provides high-resolution molecular insights into the FUS-RNA interactions and forms the basis for understanding the molecular origins of full-length FUS interaction with RNA.

Binding of the nuclear ribonucleoprotein family member FUS to RNA prevents R-loop RNA:DNA hybrid structures

The protein FUS (FUSed in sarcoma) is a metazoan RNA-binding protein that influences RNA production by all three nuclear polymerases. FUS also binds nascent transcripts, RNA processing factors, RNA polymerases, and transcription machinery. Here, we explored the role of FUS binding interactions for activity during transcription. Invitro run-off transcription assays revealed FUS-enhanced RNA produced by a non-eukaryote polymerase. The activity also reduced the formation of R-loops between RNA products and their DNA template. Analysis by domain mutation and deletion indicated RNA-binding was required for activity. We interpret that FUS binds and sequesters nascent transcripts to prevent R-loops from forming with nearby DNA. DRIP-seq analysis showed that a knockdown of FUS increased R-loop enrichment near expressed genes. Prevention of R-loops by FUS binding to nascent transcripts has the potential to affect transcription by any RNA polymerase, highlighting the broad impact FUS can have on RNA metabolism in cells and disease.

The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence.

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Phosphorylation sites are evolutionary checkpoints against liquid–solid transition in protein condensates

Assemblies of multivalent RNA-binding protein fused in sarcoma (FUS) can exist in the functional liquid-like state as well as less dynamic and potentially toxic amyloid- and hydrogel-like states. How could then cells form liquid-like condensates while avoiding their transformation to amyloids? Here, we show how posttranslational phosphorylation can provide a "handle" that prevents liquid-solid transition of intracellular condensates containing FUS. Using residue-specific coarse-grained simulations, for 85 different mammalian FUS sequences, we show how the number of phosphorylation sites and their spatial arrangement affect intracluster dynamics preventing conversion to amyloids. All atom simulations further confirm that phosphorylation can effectively reduce the β-sheet propensity in amyloid-prone fragments of FUS. A detailed evolutionary analysis shows that mammalian FUS PLDs are enriched in amyloid-prone stretches compared to control neutrally evolved sequences, suggesting that mammalian FUS proteins evolved to self-assemble. However, in stark contrast to proteins that do not phase-separate for their function, mammalian sequences have phosphosites in close proximity to these amyloid-prone regions. These results suggest that evolution uses amyloid-prone sequences in prion-like domains to enhance phase separation of condensate proteins while enriching phosphorylation sites in close proximity to safeguard against liquid-solid transitions.

Benchmarking Molecular Dynamics Force Fields for All-Atom Simulations of Biological Condensates.

Proteins containing intrinsically disordered regions are integral parts of the cellular signaling pathways and common components of biological condensates. Point mutations in the protein sequence, genetic at birth or acquired through aging, can alter the properties of the condensates, marking the onset of neurodegenerative diseases such as ALS and dementia. While the all-atom molecular dynamics method can, in principle, elucidate the conformational changes that arise from point mutations, the applications of this method to protein condensate systems is conditioned upon the availability of molecular force fields that can accurately describe both structured and disordered regions of such proteins. Using the special-purpose Anton 2 supercomputer, we benchmarked the efficacy of nine presently available molecular force fields in describing the structure and dynamics of a Fused in sarcoma (FUS) protein. Five-microsecond simulations of the full-length FUS protein characterized the effect of the force field on the global conformation of the protein, self-interactions among its side chains, solvent accessible surface area, and the diffusion constant. Using the results of dynamic light scattering as a benchmark for the FUS radius of gyration, we identified several force fields that produced FUS conformations within the experimental range. Next, we used these force fields to perform ten-microsecond simulations of two structured RNA binding domains of FUS bound to their respective RNA targets, finding the choice of the force field to affect stability of the RNA-FUS complex. Taken together, our data suggest that a combination of protein and RNA force fields sharing a common four-point water model provides an optimal description of proteins containing both disordered and structured regions and RNA-protein interactions. To make simulations of such systems available beyond the Anton 2 machines, we describe and validate implementation of the best performing force fields in a publicly available molecular dynamics program NAMD. Our NAMD implementation enables simulations of large (tens of millions of atoms) biological condensate systems and makes such simulations accessible to a broader scientific community.

The identification of high-performing antibodies for RNA-binding protein FUS for use in Western Blot, immunoprecipitation, and immunofluorescence

RNA-binding protein Fused-in Sarcoma (FUS) plays an essential role in various cellular processes. Mutations in the C-terminal domain region, where the nuclear localization signal (NLS) is located, causes the redistribution of FUS from the nucleus to the cytoplasm. In neurons, neurotoxic aggregates are formed as a result, contributing to neurogenerative diseases. Well-characterized anti-FUS antibodies would enable the reproducibility of FUS research, thereby benefiting the scientific community. In this study, we characterized ten FUS commercial antibodies for Western Blot, immunoprecipitation, and immunofluorescence using a standardized experimental protocol based on comparing read-outs in knockout cell lines and isogenic parental controls. We identified many high-performing antibodies and encourage readers to use this report as a guide to select the most appropriate antibody for their specific needs.

Generation of two induced pluripotent stem cell lines with heterozygous and homozygous amyotrophic lateral sclerosis-causing mutation R521G (c.1561C > G) in FUS gene

Mutations in the RNA-binding protein FUS (fused in sarcoma) are linked to amyotrophic lateral sclerosis (ALS), but the pathogenesis is not fully understood. For modeling ALS, here we generated two induced pluripotent stem cell (iPSC) lines carrying the heterozygous and homozygous R521G (c.1561C > G) mutation in the FUS gene via genetic modification of a healthy hiPSC line (WTC11, UCSFi001-A). Both lines show normal stem cell morphology and karyotype, express pluripotent markers, and can differentiate into three germ layers, providing a valuable resource in determining the pathological mechanisms underlying the FUS mutation of R521G in ALS.

DNA G-Quadruplex-Binding Protein Developed Using the RGG Domain of Translocated in Liposarcoma/Fused in Sarcoma Inhibits Transcription of bcl-2.

The G-quadruplexes (G4s) in the genome are important drug targets because they regulate gene expression and the genome structure. Several small molecules that bind the G4 have been developed, but few artificial G4 binding proteins have been reported. We previously reported a novel DNA G4 binding protein (RGGF) engineered using the Arg-Gly-Gly repeat (RGG) domain of TLS (translocated in liposarcoma), also known as FUS (fused in sarcoma) protein (TLS/FUS). Here, we show that RGGF recognizes DNA loops in the G4 and preferentially binds DNA G4 with long loops in vitro. Furthermore, RGGF binds to the DNA G4 of the bcl-2 promoter in vitro. RGGF overexpression in HeLa cells represses bcl-2 transcription. On the basis of these findings, G4 binding protein engineered from the RGG domain will be useful for investigating G4 transcriptional function in the genome.

Interactions between FUS and the C-terminal Domain of Nup62 are Sufficient for their Co-phase Separation into Amorphous Assemblies

Deficient nucleocytoplasmic transport is emerging as a pathogenic feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including in ALS caused by mutations in Fused in Sarcoma (FUS). Recently, both wild-type and ALS-linked mutant FUS were shown to directly interact with the phenylalanine-glycine (FG)-rich nucleoporin 62 (Nup62) protein, where FUS WT/ Nup62 interactions were enriched within the nucleus but ALS-linked mutant FUS/ Nup62 interactions were enriched within the cytoplasm of cells. Nup62 is a central channel Nup that has a prominent role in forming the selectivity filter within the nuclear pore complex and in regulating effective nucleocytoplasmic transport. Under conditions where FUS phase separates into liquid droplets in vitro, the addition of Nup62 caused the synergistic formation of amorphous assemblies containing both FUS and Nup62. Here, we examined the molecular determinants of this process using recombinant FUS and Nup62 proteins and biochemical approaches. We demonstrate that the structured C-terminal domain of Nup62 containing an alpha-helical coiled-coil region plays a dominant role in binding FUS and is sufficient for inducing the formation of FUS/Nup62 amorphous assemblies. In contrast, the natively unstructured, F/G repeat-rich N-terminal domain of Nup62 modestly contributed to FUS/Nup62 phase separation behavior. Expression of individual Nup62 domain constructs in human cells confirmed that the Nup62 C-terminal domain is essential for localization of the protein to the nuclear envelope. Our results raise the possibility that interactions between FUS and the C-terminal domain of Nup62 can influence the function of Nup62 under physiological and/or pathological conditions.

FUS Contributes to Nerve Injury-Induced Nociceptive Hypersensitivity by Activating NF-κB Pathway in Primary Sensory Neurons.

Dysregulation of pain-associated genes in the dorsal root ganglion (DRG) is considered to be a molecular basis of neuropathic pain genesis. Fused in sarcoma (FUS), a DNA/RNA-binding protein, is a critical regulator of gene expression. However, whether it contributes to neuropathic pain is unknown. This study showed that peripheral nerve injury caused by the fourth lumbar (L4) spinal nerve ligation (SNL) or chronic constriction injury (CCI) of the sciatic nerve produced a marked increase in the expression of FUS protein in injured DRG neurons. Blocking this increase through microinjection of the adeno-associated virus (AAV) 5-expressing Fus shRNA into the ipsilateral L4 DRG mitigated the SNL-induced nociceptive hypersensitivities in both male and female mice. This microinjection also alleviated the SNL-induced increases in the levels of phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2) and glial fibrillary acidic protein (GFAP) in the ipsilateral L4 dorsal horn. Furthermore, mimicking this increase through microinjection of AAV5 expressing full-length Fus mRNA into unilateral L3/4 DRGs produced the elevations in the levels of p-ERK1/2 and GFAP in the dorsal horn, enhanced responses to mechanical, heat and cold stimuli, and induced the spontaneous pain on the ipsilateral side of both male and female mice in the absence of SNL. Mechanistically, the increased FUS activated the NF-κB signaling pathway by promoting the translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Our results indicate that DRG FUS contributes to neuropathic pain likely through the activation of NF-κB in primary sensory neurons.SIGNIFICANCE STATEMENT In the present study, we reported that fused in sarcoma (FUS), a DNA/RNA-binding protein, is upregulated in injured dorsal root ganglion (DRG) following peripheral nerve injury. This upregulation is responsible for nerve injury-induced translocation of p65 into the nucleus and phosphorylation of p65 in the nucleus from injured DRG neurons. Because blocking this upregulation alleviates nerve injury-induced nociceptive hypersensitivity, DRG FUS participates in neuropathic pain likely through the activation of NF-κB in primary sensory neurons. FUS may be a potential target for neuropathic pain management.

Cyclic dipeptide-based small molecules modulate zinc-mediated liquid-liquid phase separation of tau.

Liquid-liquid phase separation (LLPS) is a complex physicochemical phenomenon mediated by multivalent transient weak interactions among macromolecules like polymers, proteins, and nucleic acids. It has implications in cellular physiology and disease conditions like cancer and neurodegenerative disorders. Many proteins associated with neurodegenerative disorders like RNA binding protein FUS (FUsed in Sarcoma), alpha-synuclein (α-Syn), TAR DNA binding protein 43 (TDP-43), and tau are shown to undergo LLPS. Recently, the tau protein responsible for Alzheimer's disease (AD) and other tauopathies is shown to phase separate into condensates in vitro and in vivo. The diverse noncovalent interactions among the biomolecules dictate the complex LLPS phenomenon. There are limited chemical tools to modulate protein LLPS which has therapeutic potential for neurodegenerative disorders. We have rationally designed cyclic dipeptide (CDP)-based small-molecule modulators (SMMs) by integrating multiple chemical groups that offer diverse chemical interactions to modulate tau LLPS. Among them, compound 1c effectively inhibits and dissolves Zn-mediated tau LLPS condensates. The SMM also inhibits tau condensate-to-fibril transition (tau aggregation through LLPS). This approach of designing SMMs of LLPS establishes a novel platform that has potential implication for the development of therapeutics for neurodegenerative disorders.

Investigation of a Fused in Sarcoma Splicing Mutation in a Chinese Amyotrophic Lateral Sclerosis Patient.

Genetic mutations of fused in sarcoma (FUS) causing amyotrophic lateral sclerosis (ALS) may disrupt mRNA splicing events. For example, the FUS c.1394-2delA variant was reported in two western ALS patients, but its molecular mechanism is unclear. In this study, we aim to investigate FUS splice site mutations in Chinese ALS patients. Sanger sequencing was used to identify FUS splicing mutations in Chinese ALS patients. We combined a deep learning tool (SpliceAI), RNA sequencing, and RT-PCR/RT-qPCR to analyze the effect of FUS c.1394-2delA mutation on RNA splicing and expression. AlphaFold was used to predict the protein structure of mutant FUS. In transfected cell lines, we used immunofluorescence to assess cytoplasmic mislocalization of mutant FUS protein. We identified a de novo FUS splice acceptor site mutation (c.1394-2delA, p. Gly466Valfs*14) in one Chinese sporadic ALS patient, which is linked to exon 14 skipping, and upregulated total FUS mRNA expression. The FUS splice site mutation was predicted to be translated into a truncated protein product at C-terminal. In vitro studies revealed that the FUS mutation increased cytoplasmic mislocalization in both HEK293T and SH-SY5Y cells. We identified a de novo FUS splicing mutation (c.1394-2delA, p. Gly466Valfs*14) in 1 out of 233 Chinese ALS patients. It caused abnormal RNA splicing, upregulated gene expression, truncated FUS translation, and cytosolic mislocalization. Our findings suggested that FUS splice site mutation is rare in Chinese ALS patients and extended our knowledge of molecular mechanisms of the FUS c.1394-2delA mutation.