Role and therapeutic potential of liquid-liquid phase separation in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a late-onset neurodegenerative disease selectively affecting motor neurons, leading to progressive paralysis. Although most cases are sporadic, ∼10% are familial. Similar proteins are found in aggregates in sporadic and familial ALS, and over the last decade, research has been focused on the underlying nature of this common pathology. Notably, TDP-43 inclusions are found in almost all ALS patients, while FUS inclusions have been reported in some familial ALS patients. Both TDP-43 and FUS possess 'low-complexity domains' (LCDs) and are considered as 'intrinsically disordered proteins', which form liquid droplets in vitro due to the weak interactions caused by the LCDs. Dysfunctional 'liquid-liquid phase separation' (LLPS) emerged as a new mechanism linking ALS-related proteins to pathogenesis. Here, we review the current state of knowledge on ALS-related gene products associated with a proteinopathy and discuss their status as LLPS proteins. In addition, we highlight the therapeutic potential of targeting LLPS for treating ALS.

MiR-133a-5p inhibits androgen receptor (AR)-induced proliferation in prostate cancer cells via targeting FUsed in Sarcoma (FUS) and AR.

Androgens and androgen receptors are vital factors involved in prostate cancer progression, and androgen ablation therapies are commonly employed to treat advanced prostate cancer. Previously, FUsed in Sarcoma (FUS) was identified as an AR-interacting protein that enhances AR transcriptional activity. In the present study, we attempted to identify miRNAs that might target both FUS and AR to inhibit FUS and AR expression. Based on TCGA data and the online tools UALCAN, Kaplan Meier-plotter (KMplot), LncTar and miRWalk prediction, miR-133a-5p was selected. MiR-133a-5p expression was significantly downregulated in prostate cancer, and low miR-133a-5p expression was correlated with low survival probability. As predicted by LncTar and miRWalk, miR-133a-5p could bind to the 3'UTR of FUS and AR to inhibit their expression. MiR-133a-5p overexpression significantly suppressed the cell viability of the AR-positive prostate cancer cell lines VCaP and LNCaP, inhibited the expression of FUS, AR, as well as AR downstream targets IGF1R and EGFR. More importantly, miR-133a inhibition increased cancer cell proliferation as well as the expression of AR and AR downstream factors, while FUS knockdown exerted an opposite effect; the effect of miR-133a on cancer cell proliferation and AR could be significantly reversed by FUS knockdown. Moreover, IGF1R and EGFR knockdown reversed the effect of the miR-133a-5p inhibition. In summary, miR-133a-5p inhibits AR-positive prostate cancer cell proliferation by targeting FUS/AR, thus improving the resistance of prostate cancer to androgen ablation therapies, which requires further in vivo validation. We provided a novel miRNA regulation mechanism for proliferation regulation in AR-positive prostate cancer cells.

Silencing of FUS in the common marmoset (Callithrix jacchus) brain via stereotaxic injection of an adeno-associated virus encoding shRNA.

Fused in sarcoma (FUS) is an RNA binding protein that is involved in frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). To establish the common marmoset (Callithrix jacchus) as a model for FTLD, we generated a stereotaxic injection-based marmoset model of FUS-silencing. We designed shRNAs against the marmoset FUS gene and generated an AAV9 virus encoding the most effective shRNA against FUS (shFUS). The AAV encoding shFUS (AAV-shFUS) was introduced into the frontal cortex of young adult marmosets, whereas AAV encoding a control shRNA was injected into the contralateral side. We obtained approximately 70-80% silencing of FUS following AAV-shFUS injection. Interestingly, FUS-silencing provoked a proliferation of astrocytes and microglias. Since FTLD is characterized by various emotional deficits, it would be helpful to establish a marmoset model of FUS-silencing in various brain tissues for investigating the pathomechanism of higher cognitive and behavioral dysfunction.

The oncofusion protein FUS-ERG targets key hematopoietic regulators and modulates the all-trans retinoic acid signaling pathway in t(16;21) acute myeloid leukemia.

The ETS transcription factor ERG has been implicated as a major regulator of both normal and aberrant hematopoiesis. In acute myeloid leukemias harboring t(16;21), ERG function is deregulated due to a fusion with FUS/TLS resulting in the expression of a FUS-ERG oncofusion protein. How this oncofusion protein deregulates the normal ERG transcription program is unclear. Here, we show that FUS-ERG acts in the context of a heptad of proteins (ERG, FLI1, GATA2, LYL1, LMO2, RUNX1 and TAL1) central to proper expression of genes involved in maintaining a stem cell hematopoietic phenotype. Moreover, in t(16;21) FUS-ERG co-occupies genomic regions bound by the nuclear receptor heterodimer RXR:RARA inhibiting target gene expression and interfering with hematopoietic differentiation. All-trans retinoic acid treatment of t(16;21) cells as well as FUS-ERG knockdown alleviate the myeloid-differentiation block. Together, the results suggest that FUS-ERG acts as a transcriptional repressor of the retinoic acid signaling pathway.

A phosphomimetic mutant TDP-43 (S409/410E) induces Drosha instability and cytotoxicity in Neuro 2A cells.

Two DNA/RNA binding proteins, TDP-43 and FUS/TLSU, are involved in RNA processing, and their aberrant mutations induce inherited amyotrophic lateral sclerosis and frontotemporal lobar degeneration with ubiquitinated inclusions. Wild type TDP-43 and FUS (wtTDP-43 and wtFUS) are mainly localized in the nucleus and biochemically interact with the microRNA processing enzyme Drosha. In this study, we investigated Drosha stability in Neuro 2A cells by gain and loss of function studies of wtTDP-43 and wtFUS and cycloheximide mediated protein degradation assay. We also generated three different phosphomimetic mutants of TDP-43 (S379E, S403/404E and S409/410E) by using a site-directed mutagenesis method and examined Drosha stability to elucidate a correlation between the phosphorylated TDP-43 mutants and Drosha stability. Overexpression of wtTDP-43 and/or wtFUS increased Drosha stability in Neuro 2A cells and double knockdown of wtTDP-43 and wtFUS reduced its stability. However, knockdown of wtTDP-43 or wtFUS did not affect Drosha stability in Neuro 2A cells. Interestingly, a phosphomimetic mutant TDP-43 (S409/410E) significantly reduced Drosha stability via prevention of protein-protein interactions between wtFUS and Drosha, and induced cytotoxicity in Neuro 2A cells. Our findings suggest that TDP-43 and FUS controls Drosha stability in Neuro 2A cells and that a phosphomimetic mutant TDP-43 (S409/410E) which is associated with Drosha instability can induce neuronal toxicity.

FUS is sequestered in nuclear aggregates in ALS patient fibroblasts.

Mutations in the RNA-binding protein FUS have been shown to cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). We investigate whether mutant FUS protein in ALS patient-derived fibroblasts affects normal FUS functions in the nucleus. We investigated fibroblasts from two ALS patients possessing different FUS mutations and a normal control. Fibroblasts from these patients have their nuclear FUS protein trapped in SDS-resistant aggregates. Genome-wide analysis reveals an inappropriate accumulation of Ser-2 phosphorylation on RNA polymerase II (RNA Pol II) near the transcription start sites of 625 genes for ALS patient cells and after small interfering RNA (siRNA) knockdown of FUS in normal fibroblasts. Furthermore, both the presence of mutant FUS protein and siRNA knockdown of wild-type FUS correlate with altered distribution of RNA Pol II within fibroblast nuclei. A loss of FUS function in orchestrating Ser-2 phosphorylation of the CTD of RNA Pol II is detectable in ALS patient-derived fibroblasts expressing mutant FUS protein, even when the FUS protein remains largely nuclear. A likely explanation for this loss of function is the aggregation of FUS protein in nuclei. Thus our results suggest a specific mechanism by which mutant FUS can have biological consequences other than by the formation of cytoplasmic aggregates.

Targeting RNA binding proteins involved in neurodegeneration.

Dysfunctions at the level of RNA processing have recently been shown to play a fundamental role in the pathogenesis of many neurodegenerative diseases. Several proteins responsible for these dysfunctions (TDP-43, FUS/TLS, and hnRNP A/Bs) belong to the nuclear class of heterogeneous ribonucleoproteins (hnRNPs) that predominantly function as general regulators of both coding and noncoding RNA metabolism. The discovery of the importance of these factors in mediating neuronal death has represented a major paradigmatic shift in our understanding of neurodegenerative processes. As a result, these discoveries have also opened the way toward novel biomolecular screening approaches in our search for therapeutic options. One of the major hurdles in this search is represented by the correct identification of the most promising targets to be prioritized. These may include aberrant aggregation processes, protein-protein interactions, RNA-protein interactions, or specific cellular pathways altered by disease. In this review, we discuss these four major options together with their various advantages and drawbacks.

FUS-SMN protein interactions link the motor neuron diseases ALS and SMA.

Mutations in the RNA binding protein FUS cause amyotrophic lateral sclerosis (ALS), a fatal adult motor neuron disease. Decreased expression of SMN causes the fatal childhood motor neuron disorder spinal muscular atrophy (SMA). The SMN complex localizes in both the cytoplasm and nuclear Gems, and loss of Gems is a cellular hallmark of fibroblasts in patients with SMA. Here, we report that FUS associates with the SMN complex, mediated by U1 snRNP and by direct interactions between FUS and SMN. Functionally, we show that FUS is required for Gem formation in HeLa cells, and expression of FUS containing a severe ALS-causing mutation (R495X) also results in Gem loss. Strikingly, a reduction in Gems is observed in ALS patient fibroblasts expressing either mutant FUS or TDP-43, another ALS-causing protein that interacts with FUS. The physical and functional interactions among SMN, FUS, TDP-43, and Gems indicate that ALS and SMA share a biochemical pathway, providing strong support for the view that these motor neuron diseases are related.

Multiple functional domains of the oncoproteins Spi-1/PU.1 and TLS are involved in their opposite splicing effects in erythroleukemic cells.

The hematopoietic transcription factor Spi-1/PU.1 is an oncoprotein participating to the malignant transformation of proerythroblasts in the Friend erythroleukemia or in the erythroleukemic process developed in spi-1 transgenic mice. Overexpression of Spi-1 in proerythroblasts blocks their differentiation. We have shown that Spi-1 promotes the use of the proximal 5'-splice site during the E1A pre-mRNA splicing and interferes with the effect of TLS (Translocated in LipoSarcoma) in this splicing assay. TLS was identified from chromosomal translocations in human liposarcoma and acute myeloid leukemia. Here, we determine the function of Spi-1 domains in splicing and in the interference with TLS. In transient transfection assays in erythroid cells, we show that the DNA binding domain cooperates with the transactivation domain or the PEST region of Spi-1 to modify the function of TLS in splicing. Interestingly, the 27 C-terminal amino acids, which determine the DNA binding activity of Spi-1, are necessary for the splicing function of Spi-1 as well as for its ability to interfere with TLS. Finally, we demonstrate that in leukemic proerythroblasts overexpressing Spi-1, TLS has lost its splicing effect. Thus, we hypothesize that oncogenic pathways in proerythroblasts may involve the ability of Spi-1 to alter splicing.