scholarly journals C9orf72 poly(GR) aggregation induces TDP-43 proteinopathy

2020 ◽  
Vol 12 (559) ◽  
pp. eabb3774 ◽  
Author(s):  
Casey N. Cook ◽  
Yanwei Wu ◽  
Hana M. Odeh ◽  
Tania F. Gendron ◽  
Karen Jansen-West ◽  
...  
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Independent Manner ◽  
Neurofilament Light ◽  
Inclusion Formation ◽  
Repeat Expansions ◽  
Transport Factors ◽  
Insight Into ◽  
Lateral Sclerosis

TAR DNA-binding protein 43 (TDP-43) inclusions are a pathological hallmark of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), including cases caused by G4C2 repeat expansions in the C9orf72 gene (c9FTD/ALS). Providing mechanistic insight into the link between C9orf72 mutations and TDP-43 pathology, we demonstrated that a glycine-arginine repeat protein [poly(GR)] translated from expanded G4C2 repeats was sufficient to promote aggregation of endogenous TDP-43. In particular, toxic poly(GR) proteins mediated sequestration of full-length TDP-43 in an RNA-independent manner to induce cytoplasmic TDP-43 inclusion formation. Moreover, in GFP-(GR)200 mice, poly(GR) caused the mislocalization of nucleocytoplasmic transport factors and nuclear pore complex proteins. These mislocalization events resulted in the aberrant accumulation of endogenous TDP-43 in the cytoplasm where it co-aggregated with poly(GR). Last, we demonstrated that treating G4C2 repeat–expressing mice with repeat-targeting antisense oligonucleotides lowered poly(GR) burden, which was accompanied by reduced TDP-43 pathology and neurodegeneration, including lowering of plasma neurofilament light (NFL) concentration. These results contribute to clarification of the mechanism by which poly(GR) drives TDP-43 proteinopathy, confirm that G4C2-targeted therapeutics reduce TDP-43 pathology in vivo, and demonstrate that alterations in plasma NFL provide insight into the therapeutic efficacy of disease-modifying treatments.

scholarly journals Loss of C9orf72 perturbs the Ran-GTPase gradient and generates compositionally diverse cytoplasmic Importin β-1 granules in motor and cortical neurons in vivo

2021 ◽  
Author(s):  
Philip McGoldrick ◽  
Agnes Lau ◽  
Zhipeng You ◽  
Thomas M Durcan ◽  
Janice Robertson
Keyword(s):  
Cortical Neurons ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Ran Gtpase ◽  
Repeat Expansions ◽  
Neuronal Subtype ◽  
Link Loss ◽  
Lateral Sclerosis

Repeat expansions in C9orf72 cause Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) eliciting toxic effects through generation of RNA foci, dipeptide repeat proteins and/or loss of C9orf72 protein. Defects in nucleocytoplasmic transport (NCT) have been implicated as a pathogenic mechanism underlying repeat expansion toxicity. Here, we show that loss of C9orf72 causes neuronal specific phenotypes, disrupting the Ran-GTPase gradient both in vitro and in vivo. We describe compositionally different types of cytoplasmic Importin β-1 granules that exhibit neuronal subtype-specific properties in vivo. We show that the abundance of Importin β-1 granules is increased in the context of C9orf72 deficiency, disrupting interactions with nuclear pore complex proteins. These granules appear to bud from the nuclear envelope and are co-immunoreactive for G3BP1 and K63-ubiquitin. These findings link loss of C9orf72 protein to gain-of-function mechanisms and defects in NCT.

scholarly journals Modulation of actin polymerization affects nucleocytoplasmic transport in multiple forms of Amyotrophic Lateral Sclerosis

2018 ◽  
Author(s):  
Anthony Giampetruzzi ◽  
Eric W. Danielson ◽  
Maryangel Jeon ◽  
Valentia Gumina ◽  
Sivakumar Boopathy ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Neurodegenerative Diseases ◽  
Actin Polymerization ◽  
Molecular Mechanisms ◽  
Nucleocytoplasmic Transport ◽  
Unknown Etiology ◽  
Nuclear Pore ◽  
Repeat Expansions ◽  
Lateral Sclerosis ◽  
Actin Homeostasis

ABSTRACTAmyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease of unknown etiology. Although defects in nucleocytoplasmic transport (NCT) may be central to the pathogenesis of ALS and other neurodegenerative diseases, the molecular mechanisms modulating the nuclear pore function are still largely unknown. Here we show that genetic and pharmacological modulation of actin polymerization disrupts nuclear pore integrity, nuclear import, and downstream pathways such as mRNA post-transcriptional regulation. Importantly, we demonstrate that modulation of actin homeostasis can rescue nuclear pore instability and dysfunction caused by mutant PFN1 as well as by C9ORF72 repeat expansions, the most common mutations in ALS patients. Collectively, our data link NCT defects to ALS-associated pathology and propose the regulation of actin homeostasis as a novel therapeutic strategy for ALS and other neurodegenerative diseases.

scholarly journals Probing the nucleoporin FG repeat network defines structural and functional features of the nuclear pore complex

2011 ◽  
Vol 195 (2) ◽  
pp. 183-192 ◽  
Author(s):  
Philipp Stelter ◽  
Ruth Kunze ◽  
Jessica Fischer ◽  
Ed Hurt
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Binding Motif ◽  
Dynein Light Chain ◽  
Transport Channel ◽  
Functional Features ◽  
And Function ◽  
Insight Into

Unraveling the organization of the FG repeat meshwork that forms the active transport channel of the nuclear pore complex (NPC) is key to understanding the mechanism of nucleocytoplasmic transport. In this paper, we develop a tool to probe the FG repeat network in living cells by modifying FG nucleoporins (Nups) with a binding motif (engineered dynein light chain–interacting domain) that can drag several copies of an interfering protein, Dyn2, into the FG network to plug the pore and stop nucleocytoplasmic transport. Our method allows us to specifically probe FG Nups in vivo, which provides insight into the organization and function of the NPC transport channel.

scholarly journals Dissecting in vivo steady-state dynamics of karyopherin-dependent nuclear transport

2016 ◽  
Vol 27 (1) ◽  
pp. 167-176 ◽  
Author(s):  
Ogheneochukome Lolodi ◽  
Hiroya Yamazaki ◽  
Shotaro Otsuka ◽  
Masahiro Kumeta ◽  
Shige H. Yoshimura
Keyword(s):  
Steady State ◽  
Molecular Transport ◽  
Nucleocytoplasmic Transport ◽  
Live Cells ◽  
Release Mechanism ◽  
Nuclear Pore ◽  
Catch And Release ◽  
Import And Export ◽  
Nucleocytoplasmic Distribution

Karyopherin-dependent molecular transport through the nuclear pore complex is maintained by constant recycling pathways of karyopherins coupled with the Ran-dependent cargo catch-and-release mechanism. Although many studies have revealed the bidirectional dynamics of karyopherins, the entire kinetics of the steady-state dynamics of karyopherin and cargo is still not fully understood. In this study, we used fluorescence recovery after photobleaching and fluorescence loss in photobleaching on live cells to provide convincing in vivo proof that karyopherin-mediated nucleocytoplasmic transport of cargoes is bidirectional. Continuous photobleaching of the cytoplasm of live cells expressing NLS cargoes led to progressive decrease of nuclear fluorescence signals. In addition, experimentally obtained kinetic parameters of karyopherin complexes were used to establish a kinetic model to explain the entire cargo import and export transport cycles facilitated by importin β. The results strongly indicate that constant shuttling of karyopherins, either free or bound to cargo, ensures proper balancing of nucleocytoplasmic distribution of cargoes and establishes effective regulation of cargo dynamics by RanGTP.

scholarly journals Simple biophysics underpins collective conformations of the intrinsically disordered proteins of the Nuclear Pore Complex

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Andrei Vovk ◽  
Chad Gu ◽  
Michael G Opferman ◽  
Larisa E Kapinos ◽  
Roderick YH Lim ◽  
...  
Keyword(s):  
Spatial Organization ◽  
Intrinsically Disordered Proteins ◽  
Nucleocytoplasmic Transport ◽  
Transport Proteins ◽  
Disordered Proteins ◽  
Biophysical Model ◽  
Nuclear Pore ◽  
Intrinsically Disordered

Nuclear Pore Complexes (NPCs) are key cellular transporter that control nucleocytoplasmic transport in eukaryotic cells, but its transport mechanism is still not understood. The centerpiece of NPC transport is the assembly of intrinsically disordered polypeptides, known as FG nucleoporins, lining its passageway. Their conformations and collective dynamics during transport are difficult to assess in vivo. In vitro investigations provide partially conflicting results, lending support to different models of transport, which invoke various conformational transitions of the FG nucleoporins induced by the cargo-carrying transport proteins. We show that the spatial organization of FG nucleoporin assemblies with the transport proteins can be understood within a first principles biophysical model with a minimal number of key physical variables, such as the average protein interaction strengths and spatial densities. These results address some of the outstanding controversies and suggest how molecularly divergent NPCs in different species can perform essentially the same function.

scholarly journals Sigma-1 receptor chaperones rescue nucleocytoplasmic transport deficit seen in cellular and Drosophila ALS/FTD models

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Pin-Tse Lee ◽  
Jean-Charles Liévens ◽  
Shao-Ming Wang ◽  
Jian-Ying Chuang ◽  
Bilal Khalil ◽  
...  
Keyword(s):  
Amyotrophic Lateral Sclerosis ◽  
Molecular Chaperone ◽  
Nucleocytoplasmic Transport ◽  
Repeat Expansion ◽  
Nuclear Pore ◽  
Sigma 1 Receptor ◽  
Sigma 1 ◽  
Cytoplasmic Accumulation ◽  
Nuclear Pore Assembly ◽  
Lateral Sclerosis

ABSTRACT In a subgroup of patients with amyotrophic lateral sclerosis (ALS)/Frontotemporal dementia (FTD), the (G4C2)-RNA repeat expansion from C9orf72 chromosome binds to the Ran-activating protein (RanGAP) at the nuclear pore, resulting in nucleocytoplasmic transport deficit and accumulation of Ran in the cytosol. Here, we found that the sigma-1 receptor (Sig-1R), a molecular chaperone, reverses the pathological effects of (G4C2)-RNA repeats in cell lines and in Drosophila. The Sig-1R colocalizes with RanGAP and nuclear pore proteins (Nups) and stabilizes the latter. Interestingly, Sig-1Rs directly bind (G4C2)-RNA repeats. Overexpression of Sig-1Rs rescues, whereas the Sig-1R knockout exacerbates, the (G4C2)-RNA repeats-induced aberrant cytoplasmic accumulation of Ran. In Drosophila, Sig-1R (but not the Sig-1R-E102Q mutant) overexpression reverses eye necrosis, climbing deficit, and firing discharge caused by (G4C2)-RNA repeats. These results on a molecular chaperone at the nuclear pore suggest that Sig-1Rs may benefit patients with C9orf72 ALS/FTD by chaperoning the nuclear pore assembly and sponging away deleterious (G4C2)-RNA repeats.

scholarly journals Nuclear transporters in a multinucleated organism: functional and localization analyses in Aspergillus nidulans

2011 ◽  
Vol 22 (20) ◽  
pp. 3874-3886 ◽  
Author(s):  
Ane Markina-Iñarrairaegui ◽  
Oier Etxebeste ◽  
Erika Herrero-García ◽  
Lidia Araújo-Bazán ◽  
Javier Fernández-Martínez ◽  
...  
Keyword(s):  
Nuclear Envelope ◽  
Aspergillus Nidulans ◽  
Molecular Mechanisms ◽  
Model Organism ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Transport Pathways ◽  
Bidirectional Transport ◽  
Distribution Studies ◽  
Insight Into

Nuclear transporters mediate bidirectional macromolecule traffic through the nuclear pore complex (NPC), thus participating in vital processes of eukaryotic cells. A systematic functional analysis in Aspergillus nidulans permitted the identification of 4 essential nuclear transport pathways of a hypothetical number of 14. The absence of phenotypes for most deletants indicates redundant roles for these nuclear receptors. Subcellular distribution studies of these carriers show three main distributions: nuclear, nucleocytoplasmic, and in association with the nuclear envelope. These locations are not specific to predicted roles as exportins or importins but indicate that bidirectional transport may occur coordinately in all nuclei of a syncytium. Coinciding with mitotic NPC rearrangements, transporters dynamically modified their localizations, suggesting supplementary roles to nucleocytoplasmic transport specifically during mitosis. Loss of transportin-SR and Mex/TAP from the nuclear envelope indicates absence of RNA transport during the partially open mitosis of Aspergillus, whereas nucleolar accumulation of Kap121 and Kap123 homologues suggests a role in nucleolar disassembly. This work provides new insight into the roles of nuclear transporters and opens an avenue for future studies of the molecular mechanisms of transport among nuclei within a common cytoplasm, using A. nidulans as a model organism.

scholarly journals Mapping the native organization of the yeast nuclear pore complex using nuclear radial intensity measurements

2019 ◽  
Vol 116 (29) ◽  
pp. 14606-14613 ◽  
Author(s):  
Pascal Vallotton ◽  
Sasikumar Rajoo ◽  
Matthias Wojtynek ◽  
Evgeny Onischenko ◽  
Annemarie Kralt ◽  
...  
Keyword(s):  
Nuclear Pore Complex ◽  
Nucleocytoplasmic Transport ◽  
Live Cells ◽  
Nuclear Pore ◽  
Intrinsically Disordered ◽  
Intensity Measurements ◽  
Composition And Structure ◽  
Associated Proteins ◽  
Multiple Copies

Selective transport across the nuclear envelope (NE) is mediated by the nuclear pore complex (NPC), a massive ∼100-MDa assembly composed of multiple copies of ∼30 nuclear pore proteins (Nups). Recent advances have shed light on the composition and structure of NPCs, but approaches that could map their organization in live cells are still lacking. Here, we introduce an in vivo method to perform nuclear radial intensity measurements (NuRIM) using fluorescence microscopy to determine the average position of NE-localized proteins along the nucleocytoplasmic transport axis. We apply NuRIM to study the organization of the NPC and the mobile transport machinery in budding yeast. This reveals a unique snapshot of the intact yeast NPC and identifies distinct steady-state localizations for various NE-associated proteins and nuclear transport factors. We find that the NPC architecture is robust against compositional changes and could also confirm that in contrast to Chlamydomonas reinhardtii, the scaffold Y complex is arranged symmetrically in the yeast NPC. Furthermore, NuRIM was applied to probe the orientation of intrinsically disordered FG-repeat segments, providing insight into their roles in selective NPC permeability and structure.

The molecular mechanism of translocation through the nuclear pore complex is highly conserved

2002 ◽  
Vol 115 (14) ◽  
pp. 2997-3005
Author(s):  
Carl Feldherr ◽  
Debra Akin ◽  
Trevor Littlewood ◽  
Murray Stewart
Keyword(s):  
Nuclear Import ◽  
Amoeba Proteus ◽  
Nuclear Pore ◽  
Hnrnp A1 ◽  
Transport Mechanisms ◽  
Evolutionary Changes ◽  
Transport Receptors ◽  
Importin Α ◽  
Transport Factors

In this report we investigated the activity of vertebrate nuclear transport factors in a primitive organism, Amoeba proteus, to better understand evolutionary changes in the transport mechanisms of organisms expected to have different requirements for nucleocytoplasmic exchange. It was initially determined that FxFG-containing nucleoporins and Ran, both of which are essential for nuclear import in vertebrates, as well as yeast, are also present and functional in amoebae. This suggests that there are fundamental similarities in the transport process; however, there are also significant differences. Transport substrates containing either the hnRNP A1 M9 shuttling signal (a GST/GFP/M9 fusion protein) or the classical bipartite NLS (colloidal gold coated with BSA-bipartite NLS conjugates), both of which are effectively transported in vertebrate cells, are excluded from the nucleus when microinjected into amoebae. However, when these substrates are injected along with transportin or importin α/β, respectively, the vertebrate receptors for these signals, they readily accumulate in the nucleoplasm. These results indicate that although the molecular recognition of substrates is not well conserved between vertebrates and amoebae, vertebrate transport receptors are functional in A. proteus, showing that the translocation machinery is highly conserved. Since selected nuclear import pathways can be investigated in the absence of competing endogenous transport, A. proteus might provide a useful in vivo system for investigating specific molecular interactions involved in trafficking.

scholarly journals Interactions and structure of the nuclear pore complex revealed by cryo-electron microscopy.

1989 ◽  
Vol 109 (3) ◽  
pp. 955-970 ◽  
Author(s):  
C W Akey
Keyword(s):  
Electron Microscopy ◽  
Mirror Symmetry ◽  
Nuclear Lamina ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Effective Diameter ◽  
Nuclear Pore Complexes ◽  
Cryo Electron Microscopy ◽  
Symmetrical Configuration

Nuclear pore complexes (NPCs) play a central role in mediating nucleocytoplasmic transport and exchange processes in eukaryotic cells. The arrangement and interactions of NPCs within amphibian nuclear envelopes have been studied using cryo-electron microscopy of unfixed and frozen hydrated specimens. The nuclear lamina in Necturus forms an orthogonal network with crossover distances which vary between 1,600 and 4,000 A and which may be related to the basic filament repeat of lamins. Furthermore, the NPCs are attached randomly within the confines of the lamin network, presumably by their nucleoplasmic rings. Image analysis of edge-on and en face projections of detergent-extracted NPCs has been combined with data on the coaxial thin rings to provide a quantitative evaluation of the triple ring model of NPC architecture proposed previously (Unwin, P. N. T., and R. Milligan. 1982. J. Cell Biol. 93:63-75). Additional details of the complex have been visualized including an intimate association of the inner spoke domains as an inner spoke ring, extensive domains within the spokes and coaxial thin rings, and interestingly, a central channel-like feature. Membrane-associated NPCs and detergent-extracted NPCs both possess peripherally located radial arms resulting in an effective diameter of approximately 1,450-1,500 A. In projection, the radial arms possess approximate mirror symmetry suggesting that they originate from both sides of the assembly. Moreover, membrane-associated NPCs are asymmetric at most radii and right-handed as viewed from the cytoplasm; detergent-extracted NPCs appear to be symmetric and have approximately 822 symmetry. Taken together, the data suggests that the framework of membrane-associated NPCs is perturbed from a symmetrical configuration, either during isolation of nuclei or by interactions with the lamina and the nuclear envelope in vivo. However, detergent extraction of nuclei appears to result in a more symmetrical alignment of components in apposing halves of the assembly.

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