scholarly journals FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

2021 ◽  
Vol 7 (30) ◽  
pp. eabf8660
Author(s):  
Nicol Birsa ◽  
Agnieszka M. Ule ◽  
Maria Giovanna Garone ◽  
Brian Tsang ◽  
Francesca Mattedi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Motor Neurons ◽  
Rna Binding ◽  
Fragile X ◽  
Protein Translation ◽  
Fused In Sarcoma ◽  
Mental Retardation Protein ◽  
Lateral Sclerosis

FUsed in Sarcoma (FUS) is a multifunctional RNA binding protein (RBP). FUS mutations lead to its cytoplasmic mislocalization and cause the neurodegenerative disease amyotrophic lateral sclerosis (ALS). Here, we use mouse and human models with endogenous ALS-associated mutations to study the early consequences of increased cytoplasmic FUS. We show that in axons, mutant FUS condensates sequester and promote the phase separation of fragile X mental retardation protein (FMRP), another RBP associated with neurodegeneration. This leads to repression of translation in mouse and human FUS-ALS motor neurons and is corroborated in vitro, where FUS and FMRP copartition and repress translation. Last, we show that translation of FMRP-bound RNAs is reduced in vivo in FUS-ALS motor neurons. Our results unravel new pathomechanisms of FUS-ALS and identify a novel paradigm by which mutations in one RBP favor the formation of condensates sequestering other RBPs, affecting crucial biological functions, such as protein translation.

scholarly journals FUS-ALS mutants alter FMRP phase separation equilibrium and impair protein translation

2020 ◽  
Author(s):  
Nicol Birsa ◽  
Agnieszka M Ule ◽  
Maria Giovanna Garone ◽  
Brian Tsang ◽  
Francesca Mattedi ◽  
...  
Keyword(s):  
Phase Separation ◽  
Motor Neurons ◽  
Rna Binding ◽  
Protein Translation ◽  
Translation Regulation ◽  
Condensed State ◽  
In Vivo Model ◽  
Rna Targets

Mutations in the RNA binding protein (RBP) FUS cause amyotrophic lateral sclerosis (ALS) and result in its nuclear depletion and cytoplasmic mislocalisation, with cytoplasmic gain of function thought to be crucial in pathogenesis. Here, we show that expression of mutant FUS at physiological levels drives translation inhibition in both mouse and human motor neurons. Rather than acting directly on the translation machinery, we find that mutant FUS forms cytoplasmic condensates that promote the phase separation of FMRP, another RBP associated with neurodegeneration and robustly involved in translation regulation. FUS and FMRP co-partition and repress translation in vitro. In our in vivo model, FMRP RNA targets are depleted from ribosomes. Our results identify a novel paradigm by which FUS mutations favour the condensed state of other RBPs, impacting on crucial biological functions, such as protein translation.

scholarly journals Astrocytic expression of ALS-causative mutant FUS leads to TNFa-dependent neurodegeneration in vivo

2021 ◽  
Author(s):  
Brigid K Jensen ◽  
Kevin J McAvoy ◽  
Nicolette M Heinsinger ◽  
Angelo C Lepore ◽  
Hristelina Ilieva ◽  
...  
Keyword(s):  
Motor Neuron ◽  
Motor Neurons ◽  
Neutralizing Antibodies ◽  
Rna Binding ◽  
Neuronal Damage ◽  
Specific Expression ◽  
Fused In Sarcoma ◽  
Knockout Animals

Genetic mutations that cause Amyotrophic Lateral Sclerosis (ALS), a progressively lethal motor neuron disease, are commonly found in ubiquitously expressed genes. In addition to direct defects within motor neurons, growing evidence suggests that dysfunction of non-neuronal cells is also an important driver of disease. Previously, we demonstrated that mutations in DNA/RNA binding protein Fused in Sarcoma (FUS) induce neurotoxic phenotypes in astrocytes in vitro, via activation of the NF-κB pathway and release of pro-inflammatory cytokine TNFα. Here, we developed an intraspinal cord injection model to test whether astrocyte-specific expression of ALS-causative FUSR521G variant (mtFUS) causes neuronal damage in vivo. We show that mtFUS expression causes TNFα upregulation, motor function deficits, and spinal motor neuron loss. We further demonstrate a lack of phenotype in TNFα knockout animals expressing mtFUS, and prevention of neurodegeneration in mtFUS-transduced animals through administration of TNFα neutralizing antibodies. Together, these studies strengthen evidence that astrocytes contribute to disease in ALS, establish that FUS-ALS astrocytes induce pathogenic changes to motor neurons in vivo, and provide insights identifying FUS-ALS specific potential therapeutic targets.

scholarly journals Identification of a Novel Interaction of FUS and Syntaphilin May Explain Synaptic and Mitochondrial Abnormalities Caused by ALS Mutations

2021 ◽  
Author(s):  
Caroline Vance ◽  
Shaakir Salam ◽  
Sara Tacconelli ◽  
Bradley Smith ◽  
Jacqueline Mitchell ◽  
...  
Keyword(s):  
Physiological Role ◽  
Protein Translation ◽  
Vital Role ◽  
Primary Neurons ◽  
Fused In Sarcoma ◽  
Mitochondrial Defects ◽  
Synaptic Changes ◽  
Lateral Sclerosis

Abstract Aberrantly expressed fused in sarcoma (FUS) is a hallmark of FUS-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Wildtype FUS localises to synapses and interacts with mitochondrial proteins while mutations have been shown to cause to pathological changes affecting mitochondria, synapses and the neuromuscular junction (NMJ). This indicates a crucial physiological role for FUS in regulating synaptic and mitochondrial function that is currently poorly understood. In this paper we provide evidence that mislocalised cytoplasmic FUS causes mitochondrial and synaptic changes and that FUS plays a vital role in maintaining neuronal health in vitro and in vivo. Overexpressing mutant FUS altered synaptic numbers and neuronal complexity in both primary neurons and zebrafish models. The degree to which FUS was mislocalised led to differences in the synaptic changes which was mirrored by changes in mitochondrial numbers and transport. Furthermore, we showed that FUS interacts with the mitochondrial tethering protein Syntaphilin (SNPH), and that mutations in FUS affect this relationship. Finally, we demonstrated mutant FUS led to changes in global protein translation. This interaction between FUS and SNPH could explain the synaptic and mitochondrial defects observed leading to global protein translation defects. Importantly, our results support the ‘gain-of-function’ hypothesis for disease pathogenesis in FUS-related ALS.

scholarly journals Identification of a novel interaction of FUS and syntaphilin may explain synaptic and mitochondrial abnormalities caused by ALS mutations

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Shaakir Salam ◽  
Sara Tacconelli ◽  
Bradley N. Smith ◽  
Jacqueline C. Mitchell ◽  
Elizabeth Glennon ◽  
...  
Keyword(s):  
Physiological Role ◽  
Protein Translation ◽  
Vital Role ◽  
Primary Neurons ◽  
Fused In Sarcoma ◽  
Mitochondrial Defects ◽  
Synaptic Changes ◽  
Lateral Sclerosis

AbstractAberrantly expressed fused in sarcoma (FUS) is a hallmark of FUS-related amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Wildtype FUS localises to synapses and interacts with mitochondrial proteins while mutations have been shown to cause to pathological changes affecting mitochondria, synapses and the neuromuscular junction (NMJ). This indicates a crucial physiological role for FUS in regulating synaptic and mitochondrial function that is currently poorly understood. In this paper we provide evidence that mislocalised cytoplasmic FUS causes mitochondrial and synaptic changes and that FUS plays a vital role in maintaining neuronal health in vitro and in vivo. Overexpressing mutant FUS altered synaptic numbers and neuronal complexity in both primary neurons and zebrafish models. The degree to which FUS was mislocalised led to differences in the synaptic changes which was mirrored by changes in mitochondrial numbers and transport. Furthermore, we showed that FUS co-localises with the mitochondrial tethering protein Syntaphilin (SNPH), and that mutations in FUS affect this relationship. Finally, we demonstrated mutant FUS led to changes in global protein translation. This localisation between FUS and SNPH could explain the synaptic and mitochondrial defects observed leading to global protein translation defects. Importantly, our results support the ‘gain-of-function’ hypothesis for disease pathogenesis in FUS-related ALS.

scholarly journals Glial Cells—The Strategic Targets in Amyotrophic Lateral Sclerosis Treatment

2020 ◽  
Vol 9 (1) ◽  
pp. 261 ◽  
Author(s):  
Tereza Filipi ◽  
Zuzana Hermanova ◽  
Jana Tureckova ◽  
Ondrej Vanatko ◽  
Miroslava Anderova
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Current Knowledge ◽  
Gene Mutations ◽  
Cell Types ◽  
In Vivo Models ◽  
Cell Therapies ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurological disease, which is characterized by the degeneration of motor neurons in the motor cortex and the spinal cord and subsequently by muscle atrophy. To date, numerous gene mutations have been linked to both sporadic and familial ALS, but the effort of many experimental groups to develop a suitable therapy has not, as of yet, proven successful. The original focus was on the degenerating motor neurons, when researchers tried to understand the pathological mechanisms that cause their slow death. However, it was soon discovered that ALS is a complicated and diverse pathology, where not only neurons, but also other cell types, play a crucial role via the so-called non-cell autonomous effect, which strongly deteriorates neuronal conditions. Subsequently, variable glia-based in vitro and in vivo models of ALS were established and used for brand-new experimental and clinical approaches. Such a shift towards glia soon bore its fruit in the form of several clinical studies, which more or less successfully tried to ward the unfavourable prognosis of ALS progression off. In this review, we aimed to summarize current knowledge regarding the involvement of each glial cell type in the progression of ALS, currently available treatments, and to provide an overview of diverse clinical trials covering pharmacological approaches, gene, and cell therapies.

scholarly journals Kinase pathway inhibition restores PSD95 induction in neurons lacking fragile X mental retardation protein

2019 ◽  
pp. 201812056
Author(s):  
Ying Yang ◽  
Yang Geng ◽  
Dongyun Jiang ◽  
Lin Ning ◽  
Hyung Joon Kim ◽  
...  
Keyword(s):  
Protein Synthesis ◽  
Mental Retardation ◽  
Rna Binding ◽  
Fragile X ◽  
Alternative Pathways ◽  
Fragile X Mental Retardation ◽  
Mrna Targets ◽  
Mental Retardation Protein ◽  
Pathway Inhibition

Fragile X syndrome (FXS) is the leading monogenic cause of autism and intellectual disability. FXS is caused by loss of expression of fragile X mental retardation protein (FMRP), an RNA-binding protein that regulates translation of numerous mRNA targets, some of which are present at synapses. While protein synthesis deficits have long been postulated as an etiology of FXS, how FMRP loss affects distributions of newly synthesized proteins is unknown. Here we investigated the role of FMRP in regulating expression of new copies of the synaptic protein PSD95 in an in vitro model of synaptic plasticity. We find that local BDNF application promotes persistent accumulation of new PSD95 at stimulated synapses and dendrites of cultured neurons, and that this accumulation is absent in FMRP-deficient mouse neurons. New PSD95 accumulation at sites of BDNF stimulation does not require known mechanisms regulating FMRP–mRNA interactions but instead requires the PI3K-mTORC1-S6K1 pathway. Surprisingly, in FMRP-deficient neurons, BDNF induction of new PSD95 accumulation can be restored by mTORC1-S6K1 blockade, suggesting that constitutively high mTORC1-S6K1 activity occludes PSD95 regulation by BDNF and that alternative pathways exist to mediate induction when mTORC1-S6K1 is inhibited. This study provides direct evidence for deficits in local protein synthesis and accumulation of newly synthesized protein in response to local stimulation in FXS, and supports mTORC1-S6K1 pathway inhibition as a potential therapeutic approach for FXS.

scholarly journals Neuronal over-expression of Oxr1 is protective against ALS-associated mutant TDP-43 mislocalisation in motor neurons and neuromuscular defects in vivo

2019 ◽  
Vol 28 (21) ◽  
pp. 3584-3599 ◽  
Author(s):  
Matthew G Williamson ◽  
Mattéa J Finelli ◽  
James N Sleigh ◽  
Amy Reddington ◽  
David Gordon ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Neurodegenerative Disorder ◽  
Late Onset ◽  
Gene Encoding ◽  
Over Expression ◽  
Neuromuscular Abnormalities ◽  
And Function ◽  
Lateral Sclerosis

Abstract A common pathological hallmark of amyotrophic lateral sclerosis (ALS) and the related neurodegenerative disorder frontotemporal dementia, is the cellular mislocalization of transactive response DNA-binding protein 43 kDa (TDP-43). Additionally, multiple mutations in the TARDBP gene (encoding TDP-43) are associated with familial forms of ALS. While the exact role for TDP-43 in the onset and progression of ALS remains unclear, the identification of factors that can prevent aberrant TDP-43 localization and function could be clinically beneficial. Previously, we discovered that the oxidation resistance 1 (Oxr1) protein could alleviate cellular mislocalization phenotypes associated with TDP-43 mutations, and that over-expression of Oxr1 was able to delay neuromuscular abnormalities in the hSOD1G93A ALS mouse model. Here, to determine whether Oxr1 can protect against TDP-43-associated phenotypes in vitro and in vivo, we used the same genetic approach in a newly described transgenic mouse expressing the human TDP-43 locus harbouring an ALS disease mutation (TDP-43M337V). We show in primary motor neurons from TDP-43M337V mice that genetically-driven Oxr1 over-expression significantly alleviates cytoplasmic mislocalization of mutant TDP-43. We also further quantified newly-identified, late-onset neuromuscular phenotypes of this mutant line, and demonstrate that neuronal Oxr1 over-expression causes a significant reduction in muscle denervation and neuromuscular junction degeneration in homozygous mutants in parallel with improved motor function and a reduction in neuroinflammation. Together these data support the application of Oxr1 as a viable and safe modifier of TDP-43-associated ALS phenotypes.

scholarly journals Pathological Roles of Wild-Type Cu, Zn-Superoxide Dismutase in Amyotrophic Lateral Sclerosis

2012 ◽  
Vol 2012 ◽  
pp. 1-6 ◽  
Author(s):  
Yoshiaki Furukawa
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Superoxide Dismutase ◽  
Motor Neurons ◽  
Wild Type ◽  
Sporadic Als ◽  
Familial Form ◽  
Recent Developments ◽  
Lateral Sclerosis

Dominant mutations in a Cu, Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (ALS). While it remains controversial how SOD1 mutations lead to onset and progression of the disease, manyin vitroandin vivostudies have supported a gain-of-toxicity mechanism where pathogenic mutations contribute to destabilizing a native structure of SOD1 and thus facilitate misfolding and aggregation. Indeed, abnormal accumulation of SOD1-positive inclusions in spinal motor neurons is a pathological hallmark in SOD1-related familial ALS. Furthermore, similarities in clinical phenotypes and neuropathology of ALS cases with and without mutations insod1gene have implied a disease mechanism involving SOD1 common to all ALS cases. Although pathogenic roles of wild-type SOD1 in sporadic ALS remain controversial, recent developments of novel SOD1 antibodies have made it possible to characterize wild-type SOD1 under pathological conditions of ALS. Here, I have briefly reviewed recent progress on biochemical and immunohistochemical characterization of wild-type SOD1 in sporadic ALS cases and discussed possible involvement of wild-type SOD1 in a pathomechanism of ALS.

scholarly journals Temporal-specific roles of fragile X mental retardation protein in the development of the hindbrain auditory circuit

Development ◽  
2020 ◽  
Vol 147 (21) ◽  
pp. dev188797
Author(s):  
Xiaoyu Wang ◽  
Ayelet Kohl ◽  
Xiaoyan Yu ◽  
Diego A. R. Zorio ◽  
Avihu Klar ◽  
...  
Keyword(s):  
Mental Retardation ◽  
Rna Binding ◽  
Fragile X ◽  
Neuronal Cell ◽  
High Specificity ◽  
Fragile X Mental Retardation ◽  
Midline Crossing ◽  
Mental Retardation Protein ◽  
Axonal Targeting

ABSTRACTFragile X mental retardation protein (FMRP) is an RNA-binding protein abundant in the nervous system. Functional loss of FMRP leads to sensory dysfunction and severe intellectual disabilities. In the auditory system, FMRP deficiency alters neuronal function and synaptic connectivity and results in perturbed processing of sound information. Nevertheless, roles of FMRP in embryonic development of the auditory hindbrain have not been identified. Here, we developed high-specificity approaches to genetically track and manipulate throughout development of the Atoh1+ neuronal cell type, which is highly conserved in vertebrates, in the cochlear nucleus of chicken embryos. We identified distinct FMRP-containing granules in the growing axons of Atoh1+ neurons and post-migrating NM cells. FMRP downregulation induced by CRISPR/Cas9 and shRNA techniques resulted in perturbed axonal pathfinding, delay in midline crossing, excess branching of neurites, and axonal targeting errors during the period of circuit development. Together, these results provide the first in vivo identification of FMRP localization and actions in developing axons of auditory neurons, and demonstrate the importance of investigating early embryonic alterations toward understanding the pathogenesis of neurodevelopmental disorders.

scholarly journals Conformational-Dependent and Independent RNA Binding to the Fragile X Mental Retardation Protein

2011 ◽  
Vol 2011 ◽  
pp. 1-14 ◽  
Author(s):  
Xin Yan ◽  
Robert B. Denman
Keyword(s):  
Mental Retardation ◽  
Rna Binding ◽  
Fragile X ◽  
In Vitro Transcription ◽  
Rna Transcripts ◽  
Fragile X Mental Retardation ◽  
Mental Retardation Protein ◽  
Arginine Residues ◽  
Rnase Digestion

The interaction between the fragile X mental retardation protein (FMRP) and BC1 RNA has been the subject of controversy. We probed the parameters of RNA binding to FMRP in several ways. Nondenaturing agarose gel analysis showed that BC1 RNA transcripts produced by in vitro transcription contain a population of conformers, which can be modulated by preannealing. Accordingly, FMRP differentially binds to the annealed and unannealed conformer populations. Using partial RNase digestion, we demonstrate that annealed BC1 RNA contains a unique conformer that FMRP likely binds. We further demonstrate that this interaction is 100-fold weaker than that the binding of eEF-1A mRNA and FMRP, and that preannealing is not a general requirement for FMRP's interaction with RNA. In addition, binding does not require the N-terminal 204 amino acids of FMRP, methylated arginine residues and can be recapitulated by both fragile X paralogs. Altogether, our data continue to support a model in which BC1 RNA functions independently of FMRP.

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