scholarly journals C9orf72 arginine-rich dipeptide repeat proteins disrupt importin β-mediated nuclear import

2019 ◽  
Author(s):  
Lindsey R. Hayes ◽  
Lauren Duan ◽  
Kelly Bowen ◽  
Petr Kalab ◽  
Jeffrey D. Rothstein
Keyword(s):  
Nuclear Import ◽  
Genetic Modifier ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dipeptide Repeat Proteins ◽  
Hexanucleotide Repeat ◽  
Repeat Proteins ◽  
Hexanucleotide Repeat Expansion ◽  
Dipeptide Repeat

AbstractDisruption of nucleocytoplasmic transport (NCT), including mislocalization of the importin β cargo, TDP-43, is a hallmark of amyotrophic lateral sclerosis (ALS), including ALS caused by a hexanucleotide repeat expansion in C9orf72. However, the mechanism(s) remain unclear. Importin β and its cargo adaptors have been shown to co-precipitate with the C9orf72-arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to NCT protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with nuclear transport receptors in the vicinity of the nuclear envelope.

scholarly journals C9orf72 arginine-rich dipeptide repeat proteins disrupt karyopherin-mediated nuclear import

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Lindsey R Hayes ◽  
Lauren Duan ◽  
Kelly Bowen ◽  
Petr Kalab ◽  
Jeffrey D Rothstein
Keyword(s):  
Nuclear Import ◽  
Genetic Modifier ◽  
Nucleocytoplasmic Transport ◽  
Nuclear Pore ◽  
Dipeptide Repeat Proteins ◽  
Hexanucleotide Repeat ◽  
Repeat Proteins ◽  
Importin Α ◽  
Dipeptide Repeat

Disruption of nucleocytoplasmic transport is increasingly implicated in the pathogenesis of neurodegenerative diseases, including ALS caused by a C9orf72 hexanucleotide repeat expansion. However, the mechanism(s) remain unclear. Karyopherins, including importin β and its cargo adaptors, have been shown to co-precipitate with the C9orf72 arginine-containing dipeptide repeat proteins (R-DPRs), poly-glycine arginine (GR) and poly-proline arginine (PR), and are protective in genetic modifier screens. Here, we show that R-DPRs interact with importin β, disrupt its cargo loading, and inhibit nuclear import of importin β, importin α/β, and transportin cargoes in permeabilized mouse neurons and HeLa cells, in a manner that can be rescued by RNA. Although R-DPRs induce widespread protein aggregation in this in vitro system, transport disruption is not due to nucleocytoplasmic transport protein sequestration, nor blockade of the phenylalanine-glycine (FG)-rich nuclear pore complex. Our results support a model in which R-DPRs interfere with cargo loading on karyopherins.

scholarly journals C9-ALS/FTD-linked proline–arginine dipeptide repeat protein associates with paraspeckle components and increases paraspeckle formation

2019 ◽  
Vol 10 (10) ◽  
Author(s):  
Hiroaki Suzuki ◽  
Yoshio Shibagaki ◽  
Seisuke Hattori ◽  
Masaaki Matsuoka
Keyword(s):  
Dna Binding Protein ◽  
Repeat Expansion ◽  
Hexanucleotide Repeat ◽  
Repeat Proteins ◽  
Non Coding Rna ◽  
Heterogeneous Nuclear Ribonucleoproteins ◽  
Hexanucleotide Repeat Expansion ◽  
Lateral Sclerosis ◽  
Dipeptide Repeat

Abstract A GGGGCC hexanucleotide repeat expansion in the C9ORF72 gene has been identified as the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. The repeat expansion undergoes unconventional translation to produce five dipeptide repeat proteins (DPRs). Although DPRs are thought to be neurotoxic, the molecular mechanism underlying the DPR-caused neurotoxicity has not been fully elucidated. The current study shows that poly-proline-arginine (poly-PR), the most toxic DPR in vitro, binds to and up-regulates nuclear paraspeckle assembly transcript 1 (NEAT1) that plays an essential role as a scaffold non-coding RNA during the paraspeckle formation. The CRISPR-assisted up-regulation of endogenous NEAT1 causes neurotoxicity. We also show that the poly-PR modulates the function of several paraspeckle-localizing heterogeneous nuclear ribonucleoproteins. Furthermore, dysregulated expression of TAR DNA-binding protein 43 (TDP-43) up-regulates NEAT1 expression and induces neurotoxicity. These results suggest that the increase in the paraspeckle formation may be involved in the poly-PR- and TDP-43-mediated neurotoxicity.

scholarly journals TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Kathleen M Cunningham ◽  
Kirstin Maulding ◽  
Kai Ruan ◽  
Mumine Senturk ◽  
Jonathan C Grima ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Amyotrophic Lateral Sclerosis ◽  
Nuclear Import ◽  
Transcriptional Regulator ◽  
Nucleocytoplasmic Transport ◽  
Repeat Expansion ◽  
Disease Pathogenesis ◽  
Hexanucleotide Repeat ◽  
Hexanucleotide Repeat Expansion ◽  
Lateral Sclerosis

Disrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which disrupted NCT causes neurodegeneration remain unclear. In a Drosophila screen, we identified ref(2)P/p62, a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. Our data suggest that the C9orf72-HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD.

scholarly journals Nuclear Import Receptors Directly Bind to Arginine-Rich Dipeptide Repeat Proteins and Suppress Their Pathological Interactions

Cell Reports ◽  
2020 ◽  
Vol 33 (12) ◽  
pp. 108538
Author(s):  
Saskia Hutten ◽  
Sinem Usluer ◽  
Benjamin Bourgeois ◽  
Francesca Simonetti ◽  
Hana M. Odeh ◽  
...  
Keyword(s):  
Nuclear Import ◽  
Dipeptide Repeat Proteins ◽  
Repeat Proteins ◽  
Import Receptors ◽  
Dipeptide Repeat

[P1-203]: C9ORF72 DIPEPTIDE REPEAT PROTEINS DISRUPT NUCLEOCYTOPLASMIC TRANSPORT

2017 ◽  
Vol 13 (7S_Part_6) ◽  
pp. P320-P320
Author(s):  
Sarah Ryan ◽  
Sara Rollinson ◽  
Janis Bennion Callister ◽  
Eleanor Hobbs ◽  
Stuart Pickering-Brown
Keyword(s):  
Nucleocytoplasmic Transport ◽  
Dipeptide Repeat Proteins ◽  
Repeat Proteins ◽  
Dipeptide Repeat

scholarly journals TFEB/Mitf links impaired nuclear import to autophagolysosomal dysfunction in C9-ALS

2020 ◽  
Author(s):  
Kathleen M. Cunningham ◽  
Ke Zhang ◽  
Kai Ruan ◽  
Kirstin Maulding ◽  
Mumine Senturk ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
Amyotrophic Lateral Sclerosis ◽  
Nuclear Import ◽  
Transcriptional Regulator ◽  
Nucleocytoplasmic Transport ◽  
Repeat Expansion ◽  
Disease Pathogenesis ◽  
Hexanucleotide Repeat ◽  
Hexanucleotide Repeat Expansion ◽  
Lateral Sclerosis

AbstractDisrupted nucleocytoplasmic transport (NCT) has been implicated in neurodegenerative disease pathogenesis; however, the mechanisms by which impaired NCT causes neurodegeneration remain unclear. In a Drosophila screen, we identified Ref(2)p/p62, a key regulator of autophagy, as a potent suppressor of neurodegeneration caused by the GGGGCC hexanucleotide repeat expansion (G4C2 HRE) in C9orf72 that causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). We found that p62 is increased and forms ubiquitinated aggregates due to decreased autophagic cargo degradation. Immunofluorescence and electron microscopy of Drosophila tissues demonstrate an accumulation of lysosome-like organelles that precedes neurodegeneration. These phenotypes are partially caused by cytoplasmic mislocalization of Mitf/TFEB, a key transcriptional regulator of autophagolysosomal function. Additionally, TFEB is mislocalized and downregulated in human cells expressing GGGGCC repeats and in C9-ALS patient motor cortex. Our data suggest that the C9orf72-HRE impairs Mitf/TFEB nuclear import, thereby disrupting autophagy and exacerbating proteostasis defects in C9-ALS/FTD.

scholarly journals The Nup358-RanGAP Complex Is Required for Efficient Importin α/β-dependent Nuclear Import

2008 ◽  
Vol 19 (5) ◽  
pp. 2300-2310 ◽  
Author(s):  
Saskia Hutten ◽  
Annette Flotho ◽  
Frauke Melchior ◽  
Ralph H. Kehlenbach
Keyword(s):  
Nuclear Import ◽  
Protein Transport ◽  
Nucleocytoplasmic Transport ◽  
Limiting Factor ◽  
Dual Function ◽  
Nuclear Pore ◽  
Transport Reaction ◽  
Importin Α

In vertebrate cells, the nucleoporin Nup358/RanBP2 is a major component of the filaments that emanate from the nuclear pore complex into the cytoplasm. Nup358 forms a complex with SUMOylated RanGAP1, the GTPase activating protein for Ran. RanGAP1 plays a pivotal role in the establishment of a RanGTP gradient across the nuclear envelope and, hence, in the majority of nucleocytoplasmic transport pathways. Here, we investigate the roles of the Nup358-RanGAP1 complex and of soluble RanGAP1 in nuclear protein transport, combining in vivo and in vitro approaches. Depletion of Nup358 by RNA interference led to a clear reduction of importin α/β-dependent nuclear import of various reporter proteins. In vitro, transport could be partially restored by the addition of importin β, RanBP1, and/or RanGAP1 to the transport reaction. In intact Nup358-depleted cells, overexpression of importin β strongly stimulated nuclear import, demonstrating that the transport receptor is the most rate-limiting factor at reduced Nup358-concentrations. As an alternative approach, we used antibody-inhibition experiments. Antibodies against RanGAP1 inhibited the enzymatic activity of soluble and nuclear pore–associated RanGAP1, as well as nuclear import and export. Although export could be fully restored by soluble RanGAP, import was only partially rescued. Together, these data suggest a dual function of the Nup358-RanGAP1 complex as a coordinator of importin β recycling and reformation of novel import complexes.

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