Multiple pathways of toxicity induced by C9orf72 dipeptide repeat aggregates and G4C2 RNA in a cellular model

The most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G4C2 repeat expansion in the C9orf72 gene. This expansion gives rise to translation of aggregating dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G4C2 RNA. Here, we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G4C2 RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G4C2 RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G4C2 RNA predominate in the cellular model used, DPRs exert additive effects that may contribute to pathology.

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Multiple pathways of toxicity induced by C9orf72 dipeptide repeat aggregates and G4C2 RNA in a cellular model

10.1101/2020.09.14.297036 ◽

2020 ◽

Author(s):

Frédéric Frottin ◽

Manuela Pérez-Berlanga ◽

F. Ulrich Hartl ◽

Mark S. Hipp

Keyword(s):

Protein Transport ◽

Protein Quality ◽

Protein Biosynthesis ◽

Abundant Species ◽

Cellular Model ◽

Protein Biogenesis ◽

Pathways Of Toxicity ◽

Lateral Sclerosis ◽

Dipeptide Repeat ◽

Pronounced Inhibition

AbstractThe most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia is a G4C2 repeat expansion in the C9orf72 gene. This expansion gives rise to translation of aggregating dipeptide repeat (DPR) proteins, including poly-GA as the most abundant species. However, gain of toxic function effects have been attributed to either the DPRs or the pathological G4C2 RNA. Here we analyzed in a cellular model the relative toxicity of DPRs and RNA. Cytoplasmic poly-GA aggregates, generated in the absence of G4C2 RNA, interfered with nucleocytoplasmic protein transport, but had little effect on cell viability. In contrast, nuclear poly-GA was more toxic, impairing nucleolar protein quality control and protein biosynthesis. Production of the G4C2 RNA strongly reduced viability independent of DPR translation and caused pronounced inhibition of nuclear mRNA export and protein biogenesis. Thus, while the toxic effects of G4C2 RNA predominate, DPRs exert additive effects that may contribute to pathology.

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Proline/arginine dipeptide repeat polymers derail protein folding in amyotrophic lateral sclerosis

Nature Communications ◽

10.1038/s41467-021-23691-y ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Maria Babu ◽

Filippo Favretto ◽

Alain Ibáñez de Opakua ◽

Marija Rankovic ◽

Stefan Becker ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Protein Folding ◽

Neurodegenerative Diseases ◽

Catalyst Activity ◽

Coding Region ◽

Prolyl Isomerase ◽

X Ray Crystallography ◽

Hexanucleotide Repeat ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractAmyotrophic lateral sclerosis and frontotemporal dementia are two neurodegenerative diseases with overlapping clinical features and the pathological hallmark of cytoplasmic deposits of misfolded proteins. The most frequent cause of familial forms of these diseases is a hexanucleotide repeat expansion in the non-coding region of the C9ORF72 gene that is translated into dipeptide repeat polymers. Here we show that proline/arginine repeat polymers derail protein folding by sequestering molecular chaperones. We demonstrate that proline/arginine repeat polymers inhibit the folding catalyst activity of PPIA, an abundant molecular chaperone and prolyl isomerase in the brain that is altered in amyotrophic lateral sclerosis. NMR spectroscopy reveals that proline/arginine repeat polymers bind to the active site of PPIA. X-ray crystallography determines the atomic structure of a proline/arginine repeat polymer in complex with the prolyl isomerase and defines the molecular basis for the specificity of disease-associated proline/arginine polymer interactions. The combined data establish a toxic mechanism that is specific for proline/arginine dipeptide repeat polymers and leads to derailed protein homeostasis in C9orf72-associated neurodegenerative diseases.

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Neurodegeneration induced by complex I inhibition in a cellular model of familial amyotrophic lateral sclerosis

Brain Research Bulletin ◽

10.1016/j.brainresbull.2006.02.013 ◽

2006 ◽

Vol 69 (4) ◽

pp. 465-474 ◽

Cited By ~ 19

Author(s):

Milena Rizzardini ◽

Monica Lupi ◽

Alessandra Mangolini ◽

Elisabetta Babetto ◽

Paolo Ubezio ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Complex I ◽

Familial Amyotrophic Lateral Sclerosis ◽

Cellular Model ◽

Lateral Sclerosis

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Ribosome inhibition by C9ORF72-ALS/FTD-associated poly-PR and poly-GR proteins revealed by cryo-EM

10.1101/2020.08.30.274597 ◽

2020 ◽

Author(s):

Anna B. Loveland ◽

Egor Svidritskiy ◽

Denis Susorov ◽

Soojin Lee ◽

Alexander Park ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Specific Binding ◽

Structural Basis ◽

Transferase Activity ◽

Peptidyl Transferase ◽

Peptidyl Transferase Center ◽

Ribosomal Tunnel ◽

Cellular Translation ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractToxic dipeptide repeat (DPR) proteins are produced from expanded G4C2 hexanucleotide repeats in the C9ORF72 gene, which cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Two DPR proteins, poly-PR and poly-GR, repress cellular translation but the molecular mechanism remains unknown. Here we show that poly-PR and poly-GR of ≥ 20 repeats inhibit the ribosome’s peptidyl-transferase activity at nanomolar concentrations, comparable to specific translation inhibitors. High-resolution cryo-EM structures reveal that poly-PR and poly-GR block the polypeptide tunnel of the ribosome, extending into the peptidyl-transferase center. Consistent with these findings, the macrolide erythromycin, which binds in the tunnel, competes with the DPR proteins and restores peptidyl-transferase activity. Our results demonstrate that strong and specific binding of poly-PR and poly-GR in the ribosomal tunnel blocks translation, revealing the structural basis of their toxicity in C9ORF72-ALS/FTD.

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The nuclear ubiquitin ligase adaptor SPOP is a conserved regulator of C9orf72 dipeptide toxicity

10.1101/2021.03.09.434618 ◽

2021 ◽

Author(s):

Carley Snoznik ◽

Valentina Medvedeva ◽

Jelena Mojsilovic-Petrovic ◽

Paige Rudich ◽

James Oosten ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Nuclear Localization ◽

Ubiquitin Ligase ◽

E3 Ubiquitin Ligase ◽

Repeat Expansion ◽

Dipeptide Repeat Proteins ◽

C Elegans ◽

Repeat Proteins ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractA hexanucleotide repeat expansion in the C9orf72 gene is the most common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Unconventional translation of the C9orf72 repeat produces dipeptide repeat proteins (DPRs). Previously, we showed that the DPRs (PR)50 and (GR)50 are highly toxic when expressed in C. elegans and this toxicity depends on nuclear localization of the DPR. In an unbiased genome-wide RNAi screen for suppressors of (PR)50 toxicity, we identified 12 genes that consistently suppressed either the developmental arrest and/or paralysis phenotype evoked by (PR)50 expression. All of these genes have vertebrate homologs and 7/12 contain predicted nuclear localization signals. One of these genes was spop-1, the C. elegans homolog of SPOP, a nuclear localized E3 ubiquitin ligase adaptor only found in metazoans. SPOP is also required for (GR)50 toxicity and functions in a genetic pathway that includes cul-3, which is the canonical E3 ligase partner for SPOP. Genetic or pharmacological inhibition of SPOP in mammalian primary spinal cord motor neurons suppressed DPR toxicity without affecting DPR expression levels. Finally, we find that genetic inhibition of bet-1, the C. elegans homolog of the known SPOP ubiquitination targets BRD2/3/4, suppresses the protective effect of SPOP mutations. Together, these data suggest a model in which SPOP promotes the DPR-dependent ubiquitination and degradation of BRD proteins. We speculate the pharmacological manipulation of this pathway, which is currently underway for multiple cancer subtypes, could also represent a novel entry point for therapeutic intervention to treat C9 FTD/ALS.Significance statementThe G4C2 repeat expansion in the C9orf72 gene is a major cause of Fronto-Temporal Dementia (FTD) and Amyotrophic Lateral Sclerosis (ALS). Unusual translation of the repeat sequence produces two highly toxic dipeptide repeat proteins, PRX and GRX, which accumulate in the brain tissue of individuals with these diseases. Here, we show that PR and GR toxicity in both C. elegans and mammalian neurons depends on the E3 ubiquitin ligase adaptor SPOP. SPOP acts through the bromodomain protein BET-1 to mediate dipeptide toxicity. SPOP inhibitors, which are currently being developed to treat SPOP-dependent renal cancer, also protect neurons against DPR toxicity. Our findings identify a highly conserved and ‘druggable’ pathway that may represent a new strategy for treating these currently incurable diseases.

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Aggregation and the Intrinsic Structural Disorder of Dipeptide Repeat Peptides of C9orf72-Related Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Characterized by NMR

The Journal of Physical Chemistry B ◽

10.1021/acs.jpcb.1c08149 ◽

2021 ◽

Author(s):

Bankala Krishnarjuna ◽

Magdalena I. Ivanova ◽

Ayyalusamy Ramamoorthy

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Frontotemporal Dementia ◽

Structural Disorder ◽

Lateral Sclerosis ◽

Dipeptide Repeat

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Phase separation and toxicity of C9orf72 poly(PR) depends on alternate distribution of arginine

The Journal of Cell Biology ◽

10.1083/jcb.202103160 ◽

2021 ◽

Vol 220 (11) ◽

Author(s):

Chen Chen ◽

Yoshiaki Yamanaka ◽

Koji Ueda ◽

Peiying Li ◽

Tamami Miyagi ◽

...

Keyword(s):

Phase Separation ◽

Charge Distribution ◽

Protein Interactions ◽

Protein Protein Interactions ◽

C9orf72 Gene ◽

And Diffusion ◽

Lateral Sclerosis ◽

Dipeptide Repeat ◽

Liquid Liquid Phase Separation

Arg (R)-rich dipeptide repeat proteins (DPRs; poly(PR): Pro-Arg and poly(GR): Gly-Arg), encoded by a hexanucleotide expansion in the C9ORF72 gene, induce neurodegeneration in amyotrophic lateral sclerosis (ALS). Although R-rich DPRs undergo liquid–liquid phase separation (LLPS), which affects multiple biological processes, mechanisms underlying LLPS of DPRs remain elusive. Here, using in silico, in vitro, and in cellulo methods, we determined that the distribution of charged Arg residues regulates the complex coacervation with anionic peptides and nucleic acids. Proteomic analyses revealed that alternate Arg distribution in poly(PR) facilitates entrapment of proteins with acidic motifs via LLPS. Transcription, translation, and diffusion of nucleolar nucleophosmin (NPM1) were impaired by poly(PR) with an alternate charge distribution but not by poly(PR) variants with a consecutive charge distribution. We propose that the pathogenicity of R-rich DPRs is mediated by disturbance of proteins through entrapment in the phase-separated droplets via sequence-controlled multivalent protein–protein interactions.

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Lack of TNF-alpha receptor type 2 protects motor neurons in a cellular model of amyotrophic lateral sclerosis and in mutant SOD1 mice but does not affect disease progression

Journal of Neurochemistry ◽

10.1111/jnc.13154 ◽

2015 ◽

Vol 135 (1) ◽

pp. 109-124 ◽

Cited By ~ 16

Author(s):

Massimo Tortarolo ◽

Antonio Vallarola ◽

Dario Lidonnici ◽

Elisa Battaglia ◽

Francesco Gensano ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Disease Progression ◽

Motor Neurons ◽

Receptor Type ◽

Tnf Alpha ◽

Cellular Model ◽

Mutant Sod1 ◽

Lateral Sclerosis

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Function of theBorrelia burgdorferiFtsH Homolog Is Essential for Viability bothIn VitroandIn Vivoand Independent of HflK/C

mBio ◽

10.1128/mbio.00404-16 ◽

2016 ◽

Vol 7 (2) ◽

Cited By ~ 5

Author(s):

Chen-Yi Chu ◽

Philip E. Stewart ◽

Aaron Bestor ◽

Bryan Hansen ◽

Tao Lin ◽

...

Keyword(s):

Lyme Disease ◽

Membrane Proteins ◽

Borrelia Burgdorferi ◽

Cellular Response ◽

Protein Transport ◽

Protein Quality ◽

Laboratory Culture ◽

Biologically Active ◽

Infectious Cycle

ABSTRACTIn many bacteria, the FtsH protease and its modulators, HflK and HflC, form a large protein complex that contributes to both membrane protein quality control and regulation of the cellular response to environmental stress. Both activities are crucial to the Lyme disease pathogenBorrelia burgdorferi, which depends on membrane functions, such as motility, protein transport, and cell signaling, to respond to rapid changes in its environment. Using an inducible system, we demonstrate that FtsH production is essential for both mouse and tick infectivity and forin vitrogrowth ofB. burgdorferi. FtsH depletion inB. burgdorfericells resulted in membrane deformation and cell death. Overproduction of the protease did not have any detectable adverse effects onB. burgdorferigrowthin vitro, suggesting that excess FtsH does not proteolytically overwhelm its substrates. In contrast, we did not observe any phenotype for cells lacking the protease modulators HflK and HflC (ΔHflK/C), although we examined morphology, growth rate, growth under stress conditions, and the complete mouse-tick infectious cycle. Our results demonstrate that FtsH provides an essential function in the life cycle of the obligate pathogenB. burgdorferibut that HflK and HflC do not detectably affect FtsH function.IMPORTANCELyme disease is caused byBorrelia burgdorferi, which is maintained in nature in an infectious cycle alternating between small mammals andIxodesticks.B. burgdorferiproduces specific membrane proteins to successfully infect and persist in these diverse organisms. We hypothesized thatB. burgdorferihas a specific mechanism to ensure that membrane proteins are properly folded and biologically active when needed and removed if improperly folded or dysfunctional. Our experiments demonstrate that FtsH, a protease that fulfills this role in other microorganisms, is essential toB. burgdorferiviability. Cells depleted of FtsH do not survive in laboratory culture medium and cannot colonize mice or ticks, revealing an absolute requirement for this protease. However, the loss of two potential modulators of FtsH activity, HflK and HflC, does not detectably affectB. burgdorferiphysiology. Our results provide the groundwork for the identification of FtsH substrates that are critical for the bacterium’s viability.

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