scholarly journals A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia

PLoS Genetics ◽  
2021 ◽  
Vol 17 (11) ◽  
pp. e1009882
Author(s):  
Jorge Azpurua ◽  
Enas Gad El-Karim ◽  
Marvel Tranquille ◽  
Josh Dubnau
Keyword(s):  
Motor Neurons ◽  
Nuclear Import ◽  
Nuclear Export ◽  
Rna Binding ◽  
Negative Effects ◽  
Number Of Genes ◽  
Age Dependent ◽  
Neurological Phenotype ◽  
Lateral Sclerosis ◽  
Downstream Mediator

Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), ~40% of frontotemporal dementia (FTD) and in many cases of Alzheimer’s disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, we expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, we conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, our screen also yielded genes involved in chromatin regulation and nuclear/import export- pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. Our identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator.

scholarly journals A behavioral screen for mediators of age-dependent TDP-43 neurodegeneration identifies SF2/SRSF1 among a group of potent suppressors in both neurons and glia.

2021 ◽  
Author(s):  
Jorge Azpurua ◽  
Enas Gad El-Karim ◽  
Marvel Tranquille ◽  
josh dubnau
Keyword(s):  
Motor Neurons ◽  
Nuclear Export ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Negative Effects ◽  
Age Dependent ◽  
Neurological Phenotype ◽  
C9orf72 Gene ◽  
Lateral Sclerosis

Cytoplasmic aggregation of Tar-DNA/RNA binding protein 43 (TDP-43) occurs in 97 percent of amyotrophic lateral sclerosis (ALS), about 40 percent of frontotemporal dementia (FTD) and in many cases of Alzheimers disease (AD). Cytoplasmic TDP-43 inclusions are seen in both sporadic and familial forms of these disorders, including those cases that are caused by repeat expansion mutations in the C9orf72 gene. To identify downstream mediators of TDP-43 toxicity, we expressed human TDP-43 in a subset of Drosophila motor neurons. Such expression causes age-dependent deficits in negative geotaxis behavior. Using this behavioral readout of locomotion, we conducted an shRNA suppressor screen and identified 32 transcripts whose knockdown was sufficient to ameliorate the neurological phenotype. The majority of these suppressors also substantially suppressed the negative effects on lifespan seen with glial TDP-43 expression. In addition to identification of a number of genes whose roles in neurodegeneration were not previously known, our screen also yielded genes involved in chromatin regulation and nuclear import-export pathways that were previously identified in the context of cell based or neurodevelopmental suppressor screens. A notable example is SF2, a conserved orthologue of mammalian SRSF1, an RNA binding protein with roles in splicing and nuclear export. Our identification SF2/SRSF1 as a potent suppressor of both neuronal and glial TDP-43 toxicity also provides a convergence with C9orf72 expansion repeat mediated neurodegeneration, where this gene also acts as a downstream mediator.

scholarly journals Mutant VAPB: Culprit or Innocent Bystander of Amyotrophic Lateral Sclerosis?

Contact ◽  
2021 ◽  
Vol 4 ◽  
pp. 251525642110225
Author(s):  
Nica Borgese ◽  
Francesca Navone ◽  
Nobuyuki Nukina ◽  
Tomoyuki Yamanaka
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Dominant Negative ◽  
Negative Effects ◽  
Membrane Contact Sites ◽  
Familial Form ◽  
Main Driver ◽  
Mechanistic Basis ◽  
Lateral Sclerosis

Nearly twenty years ago a mutation in the VAPB gene, resulting in a proline to serine substitution (p.P56S), was identified as the cause of a rare, slowly progressing, familial form of the motor neuron degenerative disease Amyotrophic Lateral Sclerosis (ALS). Since then, progress in unravelling the mechanistic basis of this mutation has proceeded in parallel with research on the VAP proteins and on their role in establishing membrane contact sites between the ER and other organelles. Analysis of the literature on cellular and animal models reviewed here supports the conclusion that P56S-VAPB, which is aggregation-prone, non-functional and unstable, is expressed at levels that are insufficient to support toxic gain-of-function or dominant negative effects within motor neurons. Instead, insufficient levels of the product of the single wild-type allele appear to be required for pathological effects, and may be the main driver of the disease. In light of the multiple interactions of the VAP proteins, we address the consequences of specific VAPB depletion and highlight various affected processes that could contribute to motor neuron degeneration. In the future, distinction of specific roles of each of the two VAP paralogues should help to further elucidate the basis of p.P56S familial ALS, as well as of other more common forms of the disease.

scholarly journals RBM45 Modulates the Antioxidant Response in Amyotrophic Lateral Sclerosis through Interactions with KEAP1

2015 ◽  
Vol 35 (14) ◽  
pp. 2385-2399 ◽  
Author(s):  
Nadine Bakkar ◽  
Arianna Kousari ◽  
Tina Kovalik ◽  
Yang Li ◽  
Robert Bowser
Keyword(s):  
Oxidative Stress ◽  
Spinal Cord ◽  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Antioxidant Response ◽  
Cytoplasmic Inclusions ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by the selective loss of motor neurons. Various factors contribute to the disease, including RNA binding protein dysregulation and oxidative stress, but their exact role in pathogenic mechanisms remains unclear. We have recently linked another RNA binding protein, RBM45, to ALS via increased levels of protein in the cerebrospinal fluid of ALS patients and its localization to cytoplasmic inclusions in ALS motor neurons. Here we show RBM45 nuclear exit in ALS spinal cord motor neurons compared to controls, a phenotype recapitulatedin vitroin motor neurons treated with oxidative stressors. We find that RBM45 binds and stabilizes KEAP1, the inhibitor of the antioxidant response transcription factor NRF2. ALS lumbar spinal cord lysates similarly show increased cytoplasmic binding of KEAP1 and RBM45. Binding of RBM45 to KEAP1 impedes the protective antioxidant response, thus contributing to oxidative stress-induced cellular toxicity. Our findings thus describe a novel link between a mislocalized RNA binding protein implicated in ALS (RBM45) and dysregulation of the neuroprotective antioxidant response seen in the disease.

scholarly journals Image-based deep learning reveals the responses of human motor neurons to stress and ALS

2021 ◽  
Author(s):  
Colombine Verzat ◽  
Jasmine Harley ◽  
Rickie Patani ◽  
Raphaëlle Luisier
Keyword(s):  
Deep Learning ◽  
Motor Neurons ◽  
Rna Binding ◽  
Relevant Information ◽  
Morphological Attributes ◽  
Fluorescent Markers ◽  
Human Motor ◽  
Microscopy Data ◽  
Key Aspects ◽  
Lateral Sclerosis

SUMMARYAlthough morphological attributes of cells and their substructures are recognized readouts of physiological or pathophysiological states, these have been relatively understudied in amyotrophic lateral sclerosis (ALS) research. In this study we integrate multichannel fluorescence high-content microscopy data with deep-learning imaging methods to reveal - directly from unsegmented images - novel neurite-associated morphological perturbations associated with (ALS-causing) VCP-mutant human motor neurons (MNs). Surprisingly, we reveal that previously unrecognized disease-relevant information is withheld in broadly used and often considered ‘generic’ biological markers of nuclei (DAPI) and neurons (βIII-tubulin). Additionally, we identify changes within the information content of ALS-related RNA binding protein (RBP) immunofluorescence imaging that is captured in VCP-mutant MN cultures. Furthermore, by analyzing MN cultures exposed to different extrinsic stressors, we show that heat stress recapitulates key aspects of ALS. Our study therefore reveals disease-relevant information contained in a range of both generic and more specific fluorescent markers, and establishes the use of image-based deep learning methods for rapid, automated and unbiased testing of biological hypotheses.

scholarly journals RNA Is a Double-Edged Sword in ALS Pathogenesis

2021 ◽  
Vol 15 ◽  
Author(s):  
Benjamin L. Zaepfel ◽  
Jeffrey D. Rothstein
Keyword(s):  
Motor Neurons ◽  
Cortical Neurons ◽  
Binding Proteins ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Rna Metabolism ◽  
Small Subset ◽  
Toxic Rna ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease that affects upper and lower motor neurons. Familial ALS accounts for a small subset of cases (<10–15%) and is caused by dominant mutations in one of more than 10 known genes. Multiple genes have been causally or pathologically linked to both ALS and frontotemporal dementia (FTD). Many of these genes encode RNA-binding proteins, so the role of dysregulated RNA metabolism in neurodegeneration is being actively investigated. In addition to defects in RNA metabolism, recent studies provide emerging evidence into how RNA itself can contribute to the degeneration of both motor and cortical neurons. In this review, we discuss the roles of altered RNA metabolism and RNA-mediated toxicity in the context of TARDBP, FUS, and C9ORF72 mutations. Specifically, we focus on recent studies that describe toxic RNA as the potential initiator of disease, disease-associated defects in specific RNA metabolism pathways, as well as how RNA-based approaches can be used as potential therapies. Altogether, we highlight the importance of RNA-based investigations into the molecular progression of ALS, as well as the need for RNA-dependent structural studies of disease-linked RNA-binding proteins to identify clear therapeutic targets.

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 Widespread FUS mislocalization is a molecular hallmark of ALS

2019 ◽  
Author(s):  
Giulia E. Tyzack ◽  
Raphaelle Luisier ◽  
Doaa M. Taha ◽  
Jacob Neeves ◽  
Miha Modic ◽  
...  
Keyword(s):  
Motor Neurons ◽  
Nuclear Export ◽  
Rna Binding ◽  
Rna Binding Proteins ◽  
Intron Retention ◽  
Direct Interaction ◽  
Spinal Cord Tissue ◽  
Cytoplasmic Inclusions ◽  
Sporadic Als ◽  
Fus Protein

AbstractAmyotrophic lateral sclerosis (ALS)-causing mutations clearly implicate ubiquitously expressed and predominantly nuclear RNA binding proteins (RBPs), which form pathological cytoplasmic inclusions in this context. However, the possibility that wild-type RBPs mislocalize without necessarily becoming constituents of ALS cytoplasmic inclusions themselves remains unexplored. We hypothesized that nuclear-to-cytoplasmic mislocalization of the RBP Fused in Sarcoma (FUS), in an unaggregated state, may occur more widely in ALS that previously recognized. To address this hypothesis, we analysed motor neurons (MNs) from an human ALS induced-pluripotent stem cells (iPSC) 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.

scholarly journals Nuclear depletion of RNA binding protein ELAVL3 (HuC) in sporadic and familial amyotrophic lateral sclerosis

2021 ◽  
Author(s):  
Sandra Diaz-Garcia ◽  
Vivian I. Ko ◽  
Sonia Vazquez-Sanchez ◽  
Ruth Chia ◽  
Olubankole Aladesuyi Arogundade ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Motor Neurons ◽  
Rna Binding ◽  
Binding Protein ◽  
Rna Binding Protein ◽  
Loss Of Function ◽  
Cryptic Exon ◽  
Protein Levels ◽  
Sporadic Als ◽  
Lateral Sclerosis

Amyotrophic lateral sclerosis is a progressive fatal neurodegenerative disease caused by loss of motor neurons and characterized neuropathologically in almost all cases by nuclear depletion and cytoplasmic aggregation of TDP-43, a nuclear RNA binding protein (RBP). We identified ELAVL3 as one of the most downregulated genes in our transcriptome profiles of laser captured microdissection of motor neurons from sporadic ALS nervous systems and the top dysregulated RBPs. Neuropathological characterizations showed ELAVL3 nuclear depletion in a great percentage of remnant motor neurons, sometimes accompanied by cytoplasmic accumulations. These abnormalities were common in sporadic cases with and without intermediate expansions in ATXN2 and familial cases carrying mutations in C9orf72 and SOD1. Depletion of ELAVL3 occurred at both the RNA and protein levels and a short protein isoform was identified but it is not related to a TDP-43-dependent cryptic exon in intron 3. Strikingly, ELAVL3 abnormalities were more frequent than TDP-43 abnormalities and occurred in motor neurons still with normal nuclear TDP-43 present, but all neurons with abnormal TDP-43 also had abnormal ELAVL3. In a neuron-like cell culture model using SH-SY5Y cells, ELAVL3 mislocalization occurred weeks before TDP-43 abnormalities were seen. We interrogated genetic databases but did not identify association of ELAVL3 genetic structure associated with ALS. Taken together, these findings suggest that ELAVL3 is an important RBP in ALS pathogenesis acquired early and the neuropathological data suggest it is involved by loss of function rather than cytoplasmic toxicity.

Nucleo-cytoplasmic distribution of human hnRNP proteins: a search for the targeting domains in hnRNP A1

1995 ◽  
Vol 108 (2) ◽  
pp. 545-555 ◽  
Author(s):  
F. Weighardt ◽  
G. Biamonti ◽  
S. Riva
Keyword(s):  
Nuclear Import ◽  
Nuclear Export ◽  
Rna Binding ◽  
Subcellular Localisation ◽  
Hnrnp A1 ◽  
Reporter Protein ◽  
Binding Domains ◽  
Rich Domain ◽  
Hnrnp Proteins ◽  
Active Transcription

hnRNP A1 (34 kDa) is an RNA binding protein consisting of two tandemly arranged RNA binding domains C-terminally linked to a glycine-rich auxiliary domain (2 × RBD-Gly). A1 belongs to the set of polypeptides that bind nascent hnRNA in the nucleus to form the so called hnRNP complexes. These complexes seem to be involved both in pre-mRNA processing and in the nuclear export of mRNA. In fact A1, along with other hnRNP proteins, is exported from the nucleus probably bound to mRNA and is immediately re-imported. A1 nuclear re-import, which requires active transcription, is not mediated by a canonical nuclear localisation signal (NLS). To identify the determinants of A1 subcellular localisation we developed an expression vector for studying the localisation, in transiently transfected cells, of the different structural motifs of A1 fused to a small reporter protein (chloramphenicol acetyltransferase, CAT; 26 kDa). We demonstrate that a 30 amino acid sequence in the glycine-rich domain (YNDFGNYNNQSSNFGPMKGGNFGGRSSGPY), which bears no resemblance to canonical NLS, is necessary and sufficient to target the protein to the nucleus. Our data suggest that this targeting sequence might act by mediating the interaction of A1 with a NLS-containing nuclear import complex. On the other hand, the nuclear export of A1 requires at least one RNA binding domain in accord with the hypothesis that A1 exits from the nucleus bound to mRNA. We propose a mechanism for the nucleo-cytoplasmic shuttling of A1 that envisages a specific role for the different structural domains and can explain the dependence of nuclear import from active transcription.

scholarly journals Identification and Functional Characterization of a Novel Nuclear Localization Signal Present in the Yeast Nab2 Poly(A)+ RNA Binding Protein

1998 ◽  
Vol 18 (3) ◽  
pp. 1449-1458 ◽  
Author(s):  
Ray Truant ◽  
Robert A. Fridell ◽  
R. Edward Benson ◽  
Hal Bogerd ◽  
Bryan R. Cullen
Keyword(s):  
Nuclear Localization ◽  
Nuclear Import ◽  
Nuclear Localization Signal ◽  
Nuclear Export ◽  
Mammalian Cells ◽  
Rna Binding ◽  
Yeast Cells ◽  
Vitro Assay ◽  
Localization Signal

ABSTRACT The nuclear import of proteins bearing a basic nuclear localization signal (NLS) is dependent on karyopherin α/importin α, which acts as the NLS receptor, and karyopherin β1/importin β, which binds karyopherin α and mediates the nuclear import of the resultant ternary complex. Recently, a second nuclear import pathway that allows the rapid reentry into the nucleus of proteins that participate in the nuclear export of mature mRNAs has been identified. In mammalian cells, a single NLS specific for this alternate pathway, the M9 NLS of heterogeneous nuclear ribonucleoprotein A1 (hnRNPA1), has been described. The M9 NLS binds a transport factor related to karyopherin β1, termed karyopherin β2 or transportin, and does not require a karyopherin α-like adapter protein. A yeast homolog of karyopherin β2, termed Kap104p, has also been described and proposed to play a role in the nuclear import of a yeast hnRNP-like protein termed Nab2p. Here, we define a Nab2p sequence that binds to Kap104p and that functions as an NLS in both human and yeast cells despite lacking any evident similarity to basic or M9 NLSs. Using an in vitro nuclear import assay, we demonstrate that Kap104p can direct the import into isolated human cell nuclei of a substrate containing a wild-type, but not a defective mutant, Nab2p NLS. In contrast, other NLSs, including the M9 NLS, could not function as substrates for Kap104p. Surprisingly, this in vitro assay also revealed that human karyopherin β1, but not the Kap104p homolog karyopherin β2, could direct the efficient nuclear import of a Nab2p NLS substrate in vitro in the absence of karyopherin α. These data therefore identify a novel NLS sequence, active in both yeast and mammalian cells, that is functionally distinct from both basic and M9 NLS sequences.

Sign in / Sign up

Export Citation Format

Share Document