Pur-alpha regulates cytoplasmic stress granule dynamics and ameliorates FUS toxicity.
ABSTRACT: Amyotrophic lateral sclerosis is characterized by progressive loss of motor neurons in the brain and spinal cord. Mutations in several genes, including FUS, TDP43, Matrin 3, hnRNPA2 and other RNA-binding proteins, have been linked to ALS pathology. Recently, Pur-alpha, a DNA/RNA-binding protein was found to bind to C9orf72 repeat expansions and could possibly play a role in the pathogenesis of ALS. When overexpressed, Pur-alpha mitigates toxicities associated with Fragile X tumor ataxia syndrome (FXTAS) and C9orf72 repeat expansion diseases in Drosophila and mammalian cell culture models. However, the function of Pur-alpha in regulating ALS pathogenesis has not been fully understood. We identified Pur-alpha as a novel component of cytoplasmic stress granules (SGs) in ALS patient cells carrying disease-causing mutations in FUS. When cells were challenged with stress, we observed that Pur-alpha co-localized with mutant FUS in ALS patient cells and became trapped in constitutive SGs. We also found that FUS physically interacted with Pur-alpha in mammalian neuronal cells. Interestingly, shRNA-mediated knock down of endogenous Pur-alpha significantly reduced formation of cytoplasmic stress granules in mammalian cells suggesting that Pur-alpha is essential for the formation of SGs. Furthermore, ectopic expression of Pur-alpha blocked cytoplasmic mislocalization of mutant FUS and strongly suppressed toxicity associated with mutant FUS expression in primary motor neurons. Our data emphasizes the importance of stress granules in ALS pathogenesis and identifies Pur-alpha as a novel regulator of SG dynamics.
Project description:Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disorder due to motor neuron loss. Fused in sarcoma (FUS) protein carrying ALS-associated mutations localizes to stress granules and causes their coalescence into larger aggregates. Here we show that Pur-alpha physically interacts with mutated FUS in an RNA-dependent manner. Pur-alpha colocalizes with FUS carrying mutations in stress granules of motoneuronal cells differentiated from induced pluripotent stem cells and that are derived from ALS patients. We observe that both Pur-alpha and mutated FUS upregulate phosphorylation of the translation initiation factor eukaryotic translation initiation factor 2 alpha and consistently inhibit global protein synthesis. In vivo expression of Pur-alpha in different Drosophila tissues significatively exacerbates the neurodegeneration caused by mutated FUS. Conversely, the downregulation of Pur-alpha in neurons expressing mutated FUS significatively improves fly climbing activity. All these findings suggest that Pur-alpha, through the control of mRNA translation, might be involved in the pathogenesis of ALS associated with the mutation of FUS, and that an alteration of protein synthesis may be directly implicated in the disease. Finally, in vivo RNAi-mediated ablation of Pur-alpha produced locomotion defects in Drosophila, indicating a pivotal role for this protein in the motoneuronal function.
Project description:Amyotrophic lateral sclerosis (ALS) is an uncommon neurodegenerative disease caused by degeneration of upper and lower motor neurons. Several genes, including SOD1, TDP-43, FUS, Ubiquilin 2, C9orf72 and Profilin 1, have been linked with the sporadic and familiar forms of ALS. FUS is a DNA/RNA-binding protein (RBP) that forms cytoplasmic inclusions in ALS and frontotemporal lobular degeneration (FTLD) patients' brains and spinal cords. However, it is unknown whether the RNA-binding ability of FUS is required for causing ALS pathogenesis. Here, we exploited a Drosophila model of ALS and neuronal cell lines to elucidate the role of the RNA-binding ability of FUS in regulating FUS-mediated toxicity, cytoplasmic mislocalization and incorporation into stress granules (SGs). To determine the role of the RNA-binding ability of FUS in ALS, we mutated FUS RNA-binding sites (F305L, F341L, F359L, F368L) and generated RNA-binding-incompetent FUS mutants with and without ALS-causing mutations (R518K or R521C). We found that mutating the aforementioned four phenylalanine (F) amino acids to leucines (L) (4F-L) eliminates FUS RNA binding. We observed that these RNA-binding mutations block neurodegenerative phenotypes seen in the fly brains, eyes and motor neurons compared with the expression of RNA-binding-competent FUS carrying ALS-causing mutations. Interestingly, RNA-binding-deficient FUS strongly localized to the nucleus of Drosophila motor neurons and mammalian neuronal cells, whereas FUS carrying ALS-linked mutations was distributed to the nucleus and cytoplasm. Importantly, we determined that incorporation of mutant FUS into the SG compartment is dependent on the RNA-binding ability of FUS. In summary, we demonstrate that the RNA-binding ability of FUS is essential for the neurodegenerative phenotype in vivo of mutant FUS (either through direct contact with RNA or through interactions with other RBPs).
Project description:Fused in sarcoma/translocated in liposarcoma (FUS/TLS) is one of causative genes for familial amyotrophic lateral sclerosis (ALS). In order to identify binding partners for FUS/TLS, we performed a yeast two-hybrid screening and found that protein arginine methyltransferase 1 (PRMT1) is one of binding partners primarily in the nucleus. In vitro and in vivo methylation assays showed that FUS/TLS could be methylated by PRMT1. The modulation of arginine methylation levels by a general methyltransferase inhibitor or conditional over-expression of PRMT1 altered slightly the nucleus-cytoplasmic ratio of FUS/TLS in cell fractionation assays. Although co-localized primarily in the nucleus in normal condition, FUS/TLS and PRMT1 were partially recruited to the cytoplasmic granules under oxidative stress, which were merged with stress granules (SGs) markers in SH-SY5Y cell. C-terminal truncated form of FUS/TLS (FUS-dC), which lacks C-terminal nuclear localization signal (NLS), formed cytoplasmic inclusions like ALS-linked FUS mutants and was partially co-localized with PRMT1. Furthermore, conditional over-expression of PRMT1 reduced the FUS-dC-mediated SGs formation and the detergent-insoluble aggregates in HEK293 cells. These findings indicate that PRMT1-mediated arginine methylation could be implicated in the nucleus-cytoplasmic shuttling of FUS/TLS and in the SGs formation and the detergent-insoluble inclusions of ALS-linked FUS/TLS mutants.
Project description:Mutations in proteins like FUS which cause Amyotrophic Lateral Sclerosis (ALS) result in the aberrant formation of stress granules while ALS-linked mutations in other proteins impede elimination of stress granules. Repeat expansions in C9ORF72, the major cause of ALS, reduce C9ORF72 levels but how this impacts stress granules is uncertain. Here, we demonstrate that C9ORF72 associates with the autophagy receptor p62 and controls elimination of stress granules by autophagy. This requires p62 to associate via the Tudor protein SMN with proteins, including FUS, that are symmetrically methylated on arginines. Mice lacking p62 accumulate arginine-methylated proteins and alterations in FUS-dependent splicing. Patients with C9ORF72 repeat expansions accumulate symmetric arginine dimethylated proteins which co-localize with p62. This suggests that C9ORF72 initiates a cascade of ALS-linked proteins (C9ORF72, p62, SMN, FUS) to recognize stress granules for degradation by autophagy and hallmarks of a defect in this process are observable in ALS patients.
Project description:The RNA-binding proteins TAR DNA-binding protein (TDP-43) and fused in sarcoma (FUS) play central roles in neurodegeneration associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-U). Normally localized in the nucleus, in sites affected by ALS and FTLD-U they are mislocalized to the cytoplasm and form cytoplasmic inclusions. TDP-43 and FUS are transported to the nucleus in a Ran-GTPase-dependent manner via nuclear import receptors, but they also contribute to the formation of stress granules (SGs), which are intracytoplasmic structures incorporating RNA. C-terminal truncations of TDP-43 eliminate the nuclear transport signal and cause mislocalization of the protein to the cytoplasm, where it accumulates and forms SGs. ALS-associated FUS mutations impair nuclear transport and cause mislocalization of FUS to the cytoplasm, where it also contributes to assembly of SGs. Furthermore, the ALS susceptibility factor ataxin-2, recently identified as a potent modifier of TDP-43 toxicity, is also a predicted cytoplasmic RNA-binding protein and a constituent protein of SGs, suggesting that it is a part of the common pathologic cascade formed by TDP-43 and FUS. Thus, we propose that excessive mislocalization of the RNA-binding proteins TDP-43, FUS, and ataxin-2 into the cytoplasm leads to impairment of the RNA quality control system, forming the core of the ALS/FTLD-U degenerative cascade. In this review, we discuss the molecular basis of the novel disease spectrum of ALS/FTLD-U, including the neurodegenerative mechanism of the cytoplasmic RNA-binding proteins TDP-43 and FUS and the possibility of a novel therapeutic strategy.
Project description:Stress granules (SGs) are RNA-containing cytoplasmic foci formed in response to stress exposure. Since their discovery in 1999, over 120 proteins have been described to be localized to these structures (in 154 publications). Most of these components are RNA binding proteins (RBPs) or are involved in RNA metabolism and translation. SGs have been linked to several pathologies including inflammatory diseases, cancer, viral infection, and neurodegenerative diseases such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In ALS and FTD, the majority of cases have no known etiology and exposure to external stress is frequently proposed as a contributor to either disease initiation or the rate of disease progression. Of note, both ALS and FTD are characterized by pathological inclusions, where some well-known SG markers localize with the ALS related proteins TDP-43 and FUS. We propose that TDP-43 and FUS serve as an interface between genetic susceptibility and environmental stress exposure in disease pathogenesis. Here, we will discuss the role of TDP-43 and FUS in SG dynamics and how disease-linked mutations affect this process.
Project description:Patient-derived induced pluripotent stem cells (iPSCs) provide an opportunity to study human diseases mainly in those cases for which no suitable model systems are available. Here, we have taken advantage of in vitro iPSCs derived from patients affected by amyotrophic lateral sclerosis (ALS) and carrying mutations in the RNA-binding protein FUS to study the cellular behavior of the mutant proteins in the appropriate genetic background. Moreover, the ability to differentiate iPSCs into spinal cord neural cells provides an in vitro model mimicking the physiological conditions. iPSCs were derived from FUS(R514S) and FUS(R521C) patient fibroblasts, whereas in the case of the severe FUS(P525L) mutation, in which fibroblasts were not available, a heterozygous and a homozygous iPSC line were raised by TALEN-directed mutagenesis. We show that aberrant localization and recruitment of FUS into stress granules (SGs) is a prerogative of the FUS mutant proteins and occurs only upon induction of stress in both undifferentiated iPSCs and spinal cord neural cells. Moreover, we show that the incorporation into SGs is proportional to the amount of cytoplasmic FUS, strongly correlating with the cytoplasmic delocalization phenotype of the different mutants. Therefore, the available iPSCs represent a very powerful system for understanding the correlation between FUS mutations, the molecular mechanisms of SG formation and ALS ethiopathogenesis.
Project description:Fused in sarcoma (FUS) belongs to the group of RNA-binding proteins implicated as underlying factors in amyotrophic lateral sclerosis (ALS) and certain other neurodegenerative diseases. Multiple FUS gene mutations have been linked to hereditary forms, and aggregation of FUS protein is believed to play an important role in pathogenesis of these diseases. In cultured cells, FUS variants with disease-associated amino acid substitutions or short deletions affecting nuclear localization signal (NLS) and causing cytoplasmic mislocalization can be sequestered into stress granules (SGs). We demonstrated that disruption of motifs responsible for RNA recognition and binding not only prevents SG recruitment, but also dramatically increases the protein propensity to aggregate in the cell cytoplasm with formation of juxtanuclear structures displaying typical features of aggresomes. Functional RNA-binding domains from TAR DNA-binding protein of 43 kDa (TDP-43) fused to highly aggregation-prone C-terminally truncated FUS protein restored the ability to enter SGs and prevented aggregation of the chimeric protein. Truncated FUS was also able to trap endogenous FUS molecules in the cytoplasmic aggregates. Our data indicate that RNA binding and recruitment to SGs protect cytoplasmic FUS from aggregation, and loss of this protection may trigger its pathological aggregation in vivo.
Project description:Stress granules (SGs) form during cellular stress and are implicated in neurodegenerative diseases such as amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). To yield insights into the role of SGs in pathophysiology, we performed a high-content screen to identify small molecules that alter SG properties in proliferative cells and human iPSC-derived motor neurons (iPS-MNs). One major class of active molecules contained extended planar aromatic moieties, suggesting a potential to intercalate in nucleic acids. Accordingly, we show that several hit compounds can prevent the RNA-dependent recruitment of the ALS-associated RNA-binding proteins (RBPs) TDP-43, FUS, and HNRNPA2B1 into SGs. We further demonstrate that transient SG formation contributes to persistent accumulation of TDP-43 into cytoplasmic puncta and that our hit compounds can reduce this accumulation in iPS-MNs from ALS patients. We propose that compounds with planar moieties represent a promising starting point to develop small-molecule therapeutics for treating ALS/FTD.
Project description:Amyotrophic lateral sclerosis (ALS) arises from an interplay of genetic mutations and environmental factors. ssRNA viruses are possible ALS risk factors, but testing their interaction with mutations such as in FUS, which encodes an RNA-binding protein, has been difficult due to the lack of a human disease model. Here, we use isogenic induced pluripotent stem cell (iPSC)-derived spinal neurons (SNs) to investigate the interaction between ssRNA viruses and mutant FUS. We find that rabies virus (RABV) spreads ALS phenotypes, including the formation of stress granules (SGs) with aberrant composition due to increased levels of FUS protein, as well as neurodegeneration and reduced restriction activity by FUS mutations. Consistent with this, iPSC-derived SNs harboring mutant FUS are more sensitive to human immunodeficiency virus (HIV-1) and Zika viruses (ZIKV). We demonstrate that RABV and HIV-1 exacerbate cytoplasmic mislocalization of FUS. Our results demonstrate that viral infections worsen ALS pathology in SNs with genetic risk factors, suggesting a novel role for viruses in modulating patient phenotypes.