Early lethality and neuronal proteinopathy in mice expressing cytoplasm-targeted FUS that lacks the RNA recognition motif.
ABSTRACT: Mutations to the RNA binding protein, fused in sarcoma (FUS) occur in ?5% of familial ALS and FUS-positive cytoplasmic inclusions are commonly observed in these patients. Altered RNA metabolism is increasingly implicated in ALS, yet it is not understood how the specificity with which FUS interacts with RNA in the cytoplasm can affect its aggregation in vivo. To further understand this, we expressed, in mice, a form of FUS (FUS ?RRMcyt) that lacked the RNA recognition motif (RRM), thought to impart specificity to FUS-RNA interactions, and carried an ALS-associated point mutation, R522G, retaining the protein in the cytoplasm. Here we report the phenotype and results of histological assessment of the brain of transgenic mice expressing this isoform of FUS. Results demonstrated that neuronal expression of FUS ?RRMcyt caused early lethality often preceded by severe tremor. Large FUS-positive cytoplasmic inclusions were found in many brain neurons; however, neither neuronal loss nor neuroinflammatory response was observed. In conclusion, the extensive FUS proteinopathy and severe phenotype of these mice suggests that affecting the interactions of FUS with RNA in vivo may augment its aggregation in the neuronal cytoplasm and the severity of disease processes.
Project description:Juvenile amyotrophic lateral sclerosis (ALS) with basophilic inclusions is a form of ALS characterized by protein deposits in motor neurons that are morphologically and tinctorially distinct from those of classic sporadic ALS. The nosologic position of this type of ALS in the molecular pathologic and genetic classification of ALS is unknown.We identified neuropathologically 4 patients with juvenile ALS with basophilic inclusions and tested the hypothesis that specific RNA binding protein pathology may define this type of ALS. Immunohistochemical findings prompted us to sequence the fused in sarcoma (FUS) gene.Motor symptoms began between ages 17 and 22. Disease progression was rapid without dementia. No family history was identified. Basophilic inclusions were strongly positive for FUS protein but negative for TAR DNA binding protein 43 (TDP-43). Granular and compact FUS deposits were identified in glia and neuronal cytoplasm and nuclei. Ultrastructure of aggregates was in keeping with origin from fragmented rough endoplasmic reticulum. Sequencing of all 15 exons of the FUS gene in 3 patients revealed a novel deletion mutation (c.1554_1557delACAG) in 1 individual and the c.1574C>T (P525L) mutation in 2 others.Juvenile ALS with basophilic inclusions is a FUS proteinopathy and should be classified as ALS-FUS. The FUS c.1574C>T (P525L) and c.1554_1557delACAG mutations are associated with this distinct phenotype. The molecular genetic relationship with frontotemporal lobar degeneration with FUS pathology remains to be clarified.
Project description:TDP-43 and FUS are RNA-binding proteins that form cytoplasmic inclusions in some forms of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Moreover, mutations in TDP-43 and FUS are linked to ALS and FTLD. However, it is unknown whether TDP-43 and FUS aggregate and cause toxicity by similar mechanisms. Here, we exploit a yeast model and purified FUS to elucidate mechanisms of FUS aggregation and toxicity. Like TDP-43, FUS must aggregate in the cytoplasm and bind RNA to confer toxicity in yeast. These cytoplasmic FUS aggregates partition to stress granule compartments just as they do in ALS patients. Importantly, in isolation, FUS spontaneously forms pore-like oligomers and filamentous structures reminiscent of FUS inclusions in ALS patients. FUS aggregation and toxicity requires a prion-like domain, but unlike TDP-43, additional determinants within a RGG domain are critical for FUS aggregation and toxicity. In further distinction to TDP-43, ALS-linked FUS mutations do not promote aggregation. Finally, genome-wide screens uncovered stress granule assembly and RNA metabolism genes that modify FUS toxicity but not TDP-43 toxicity. Our findings suggest that TDP-43 and FUS, though similar RNA-binding proteins, aggregate and confer disease phenotypes via distinct mechanisms. These differences will likely have important therapeutic implications.
Project description:Mutations in the gene encoding the RNA-binding protein fused in sarcoma (FUS) can cause familial and sporadic amyotrophic lateral sclerosis (ALS) and rarely frontotemproal dementia (FTD). FUS accumulates in neuronal cytoplasmic inclusions (NCIs) in ALS patients with FUS mutations. FUS is also a major pathologic marker for a group of less common forms of frontotemporal lobar degeneration (FTLD), which includes atypical FTLD with ubiquitinated inclusions (aFTLD-U), neuronal intermediate filament inclusion disease (NIFID) and basophilic inclusion body disease (BIBD). These diseases are now called FUS proteinopathies, because they share this disease marker. It is unknown how FUS mutations cause disease and the role of FUS in FTD-FUS cases, which do not have FUS mutations. In this paper we report the development of somatic brain transgenic (SBT) mice using recombinant adeno-associated virus (rAAV) to investigate how FUS mutations lead to neurodegeneration.We compared SBT mice expressing wild-type human FUS (FUSWT), and two ALS-linked mutations: FUSR521C and FUS?14, which lacks the nuclear localization signal. Both FUS mutants accumulated in the cytoplasm relative to FUSWT. The degree of this shift correlated with the severity of the FUS mutation as reflected by disease onset in humans. Mice expressing the most aggressive mutation, FUS?14, recapitulated many aspects of FUS proteinopathies, including insoluble FUS, basophilic and eosiniphilic NCIs, and other pathologic markers, including ubiquitin, p62/SQSTM1, ?-internexin, and the poly-adenylate(A)-binding protein 1 (PABP-1). However, TDP-43 did not localize to inclusions.Our data supports the hypothesis that ALS or FTD-linked FUS mutations cause neurodegeneration by increasing cyotplasmic FUS. Accumulation of FUS in the cytoplasm may retain RNA targets and recruit additional RNA-binding proteins, such as PABP-1, into stress-granule like aggregates that coalesce into permanent inclusions that could negatively affect RNA metabolism. Identification of mutations in other genes that cause ALS/FTD, such as C9ORF72, sentaxin, and angiogenin, lends support to the idea that defective RNA metabolism is a critical pathogenic pathway. The SBT FUS mice described here will provide a valuable platform for dissecting the pathogenic mechanism of FUS mutations, define the relationship between FTD and ALS-FUS, and help identify therapeutic targets that are desperately needed for these devastating neurodegenerative disorders.
Project description:Fused in sarcoma (FUS)-immunoreactive neuronal and glial inclusions define a novel molecular pathology called FUS proteinopathy. FUS has been shown to be a component of inclusions of familial amyotrophic lateral sclerosis with FUS mutation and three frontotemporal lobar degeneration entities, including neuronal intermediate filament inclusion disease (NIFID). The pathogenic role of FUS is unknown. In addition to FUS, many neuronal cytoplasmic inclusions (NCI) of NIFID contain aggregates of ?-internexin and neurofilament proteins. Herein, we have shown that: (1) FUS becomes relatively insoluble in NIFID and there are no apparent posttranslational modifications, (2) there are no pathogenic abnormalities in the FUS gene in NIFID, and (3) immunoelectron microscopy demonstrates the fine structural localization of FUS in NIFID which has not previously been described. FUS localized to euchromatin, and strongly with paraspeckles, in nuclei, consistent with its RNA/DNA-binding functions. NCI of varying morphologies were observed. Most frequent were the "loosely aggregated cytoplasmic inclusions," 81% of which had moderate or high levels of FUS immunoreactivity. Much rarer "compact cytoplasmic inclusions" and "tangled twine ball inclusions" were FUS-immunoreactive at their granular peripheries, or heavily FUS-positive throughout, respectively. Thus, FUS may aggregate in the cytoplasm and then admix with neuronal intermediate filament accumulations.
Project description:Mutations in the RNA-binding protein FUS/TLS (FUS) have been linked to the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Although predominantly nuclear, this heterogenous nuclear ribonuclear protein (hnRNP) has multiple functions in RNA processing including intracellular trafficking. In ALS, mutant or wild-type (WT) FUS can form neuronal cytoplasmic inclusions. Asymmetric arginine methylation of FUS by the class 1 arginine methyltransferase, protein arginine methyltransferase 1 (PRMT1), regulates nucleocytoplasmic shuttling of FUS. In motor neurons of primary spinal cord cultures, redistribution of endogenous mouse and that of ectopically expressed WT or mutant human FUS to the cytoplasm led to nuclear depletion of PRMT1, abrogating methylation of its nuclear substrates. Specifically, hypomethylation of arginine 3 of histone 4 resulted in decreased acetylation of lysine 9/14 of histone 3 and transcriptional repression. Distribution of neuronal PRMT1 coincident with FUS also was detected in vivo in the spinal cord of FUS(R495X) transgenic mice. However, nuclear PRMT1 was not stable postmortem obviating meaningful evaluation of ALS autopsy cases. This study provides evidence for loss of PRMT1 function as a consequence of cytoplasmic accumulation of FUS in the pathogenesis of ALS, including changes in the histone code regulating gene transcription.
Project description:Mutations in fused in sarcoma (FUS) have been reported to cause a subset of familial amyotrophic lateral sclerosis (ALS) cases. Wild-type FUS is mostly localized in the nuclei of neurons, but the ALS mutants are partly mislocalized in the cytoplasm and can form inclusions. We demonstrate that the C-terminal 32 amino acid residues of FUS constitute an effective nuclear localization sequence (NLS) as it targeted beta-galactosidase (LacZ, 116 kDa) to the nucleus. Deletion of or the ALS mutations within the NLS caused cytoplasmic mislocalization of FUS. Moreover, we identified the poly-A binding protein (PABP1), a stress granule marker, as an interacting partner of FUS. Large PABP1-positive cytoplasmic foci (i.e. stress granules) colocalized with the mutant FUS inclusions but were absent in wild-type FUS-expressing cells. Processing bodies, which are functionally related to stress granules, were adjacent to but not colocalized with the mutant FUS inclusions. Our results suggest that the ALS mutations in FUS NLS can impair FUS nuclear localization, induce cytoplasmic inclusions and stress granules, and potentially perturb RNA metabolism.
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: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:Amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD) are relentlessly progressive neurodegenerative disorders with overlapping clinical, genetic and pathological features. Cytoplasmic inclusions of fused in sarcoma (FUS) are the hallmark of several forms of FTLD and ALS patients with mutations in the FUS gene. FUS is a multifunctional, predominantly nuclear, DNA and RNA binding protein. Here, we report that transgenic mice overexpressing wild-type human FUS develop an aggressive phenotype with an early onset tremor followed by progressive hind limb paralysis and death by 12 weeks in homozygous animals. Large motor neurons were lost from the spinal cord accompanied by neurophysiological evidence of denervation and focal muscle atrophy. Surviving motor neurons in the spinal cord had greatly increased cytoplasmic expression of FUS, with globular and skein-like FUS-positive and ubiquitin-negative inclusions associated with astroglial and microglial reactivity. Cytoplasmic FUS inclusions were also detected in the brain of transgenic mice without apparent neuronal loss and little astroglial or microglial activation. Hemizygous FUS overexpressing mice showed no evidence of a motor phenotype or pathology. These findings recapitulate several pathological features seen in human ALS and FTLD patients, and suggest that overexpression of wild-type FUS in vulnerable neurons may be one of the root causes of disease. Furthermore, these mice will provide a new model to study disease mechanism, and test therapies.
Project description:Mutations causing amyotrophic lateral sclerosis (ALS) clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins, which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RNA binding proteins mislocalize without necessarily becoming constituents of cytoplasmic inclusions themselves remains relatively unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RNA binding protein fused in sarcoma (FUS), in an unaggregated state, may occur more widely in ALS than previously recognized. To address this hypothesis, we analysed motor neurons from a human ALS induced-pluripotent stem cell model caused by the VCP mutation. Additionally, we examined mouse transgenic models and post-mortem tissue from human sporadic ALS cases. We report nuclear-to-cytoplasmic mislocalization of FUS in both VCP-mutation related ALS and, crucially, in sporadic ALS spinal cord tissue from multiple cases. Furthermore, we provide evidence that FUS protein binds to an aberrantly retained intron within the SFPQ transcript, which is exported from the nucleus into the cytoplasm. Collectively, these data support a model for ALS pathogenesis whereby aberrant intron retention in SFPQ transcripts contributes to FUS mislocalization through their direct interaction and nuclear export. In summary, we report widespread mislocalization of the FUS protein in ALS and propose a putative underlying mechanism for this process.