Toxic PR Poly-Dipeptides Encoded by the C9orf72 Repeat Expansion Target LC Domain Polymers.

Two complementary approaches were used in search of the intracellular targets of the toxic PR poly-dipeptide encoded by the repeat sequences expanded in the C9orf72 form of amyotrophic lateral sclerosis. The top categories of PRn-bound proteins include constituents of non-membrane invested cellular organelles and intermediate filaments. PRn targets are enriched for the inclusion of low complexity (LC) sequences. Evidence is presented indicating that LC sequences represent the direct target of PRn binding and that interaction between the PRn poly-dipeptide and LC domains is polymer-dependent. These studies indicate that PRn-mediated toxicity may result from broad impediments to the dynamics of cell structure and information flow from gene to message to protein.

C9orf72 Dipeptide Repeats Impair the Assembly, Dynamics, and Function of Membrane-Less Organelles.

Expansion of a hexanucleotide repeat GGGGCC (G4C2) in C9ORF72 is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Transcripts carrying (G4C2) expansions undergo unconventional, non-ATG-dependent translation, generating toxic dipeptide repeat (DPR) proteins thought to contribute to disease. Here, we identify the interactome of all DPRs and find that arginine-containing DPRs, polyGly-Arg (GR) and polyPro-Arg (PR), interact with RNA-binding proteins and proteins with low complexity sequence domains (LCDs) that often mediate the assembly of membrane-less organelles. Indeed, most GR/PR interactors are components of membrane-less organelles such as nucleoli, the nuclear pore complex and stress granules. Genetic analysis in Drosophila demonstrated the functional relevance of these interactions to DPR toxicity. Furthermore, we show that GR and PR altered phase separation of LCD-containing proteins, insinuating into their liquid assemblies and changing their material properties, resulting in perturbed dynamics and/or functions of multiple membrane-less organelles.

Spatiotemporal Proteomic Analysis of Stress Granule Disassembly Using APEX Reveals Regulation by SUMOylation and Links to ALS Pathogenesis.

Stress granules (SGs) are cytoplasmic assemblies of proteins and non-translating mRNAs. Whereas much has been learned about SG formation, a major gap remains in understanding the compositional changes SGs undergo during normal disassembly and under disease conditions. Here, we address this gap by proteomic dissection of the SG temporal disassembly sequence using multi-bait APEX proximity proteomics. We discover 109 novel SG proteins and characterize distinct SG substructures. We reveal dozens of disassembly-engaged proteins (DEPs), some of which play functional roles in SG disassembly, including small ubiquitin-like modifier (SUMO) conjugating enzymes. We further demonstrate that SUMOylation regulates SG disassembly and SG formation. Parallel proteomics with amyotrophic lateral sclerosis (ALS)-associated C9ORF72 dipeptides uncovered attenuated DEP recruitment during SG disassembly and impaired SUMOylation. Accordingly, SUMO activity ameliorated C9ORF72-ALS-related neurodegeneration in Drosophila. By dissecting the SG spatiotemporal proteomic landscape, we provide an in-depth resource for future work on SG function and reveal basic and disease-relevant mechanisms of SG disassembly.

C9orf72 Poly(PR) Dipeptide Repeats Disturb Biomolecular Phase Separation and Disrupt Nucleolar Function.

Repeat expansion in the C9orf72 gene is the most common cause of the neurodegenerative disorder amyotrophic lateral sclerosis (C9-ALS) and is linked to the unconventional translation of five dipeptide-repeat polypeptides (DPRs). The two enriched in arginine, poly(GR) and poly(PR), infiltrate liquid-like nucleoli, co-localize with the nucleolar protein nucleophosmin (NPM1), and alter the phase separation behavior of NPM1 in vitro. Here, we show that poly(PR) DPRs bind tightly to a long acidic tract within the intrinsically disordered region of NPM1, altering its phase separation with nucleolar partners to the extreme of forming large, soluble complexes that cause droplet dissolution in vitro. In cells, poly(PR) DPRs disperse NPM1 from nucleoli and entrap rRNA in static condensates in a DPR-length-dependent manner. We propose that R-rich DPR toxicity involves disrupting the role of phase separation by NPM1 in organizing ribosomal proteins and RNAs within the nucleolus.

Massively parallel identification of sequence motifs triggering ribosome-associated mRNA quality control.

Decay of mRNAs can be triggered by ribosome slowdown at stretches of rare codons or positively charged amino acids. However, the full diversity of sequences that trigger co-translational mRNA decay is poorly understood. To comprehensively identify sequence motifs that trigger mRNA decay, we use a massively parallel reporter assay to measure the effect of all possible combinations of codon pairs on mRNA levels in S. cerevisiae. In addition to known mRNA-destabilizing sequences, we identify several dipeptide repeats whose translation reduces mRNA levels. These include combinations of positively charged and bulky residues, as well as proline-glycine and proline-aspartate dipeptide repeats. Genetic deletion of the ribosome collision sensor Hel2 rescues the mRNA effects of these motifs, suggesting that they trigger ribosome slowdown and activate the ribosome-associated quality control (RQC) pathway. Deep mutational scanning of an mRNA-destabilizing dipeptide repeat reveals a complex interplay between the charge, bulkiness, and location of amino acid residues in conferring mRNA instability. Finally, we show that the mRNA effects of codon pairs are predictive of the effects of endogenous sequences. Our work highlights the complexity of sequence motifs driving co-translational mRNA decay in eukaryotes, and presents a high throughput approach to dissect their requirements at the codon level.

Discovery of cyanophycin dipeptide hydrolase enzymes suggests widespread utility of the natural biopolymer cyanophycin.

Cyanophycin is a bacterial polymer mainly used for nitrogen storage. It is composed of a peptide backbone of L-aspartate residues with L-arginines attached to their side chains through isopeptide bonds. Cyanophycin is degraded in two steps: Cyanophycinase cleaves the polymer into ß-Asp-Arg dipeptides, which are hydrolyzed into free Asp and Arg by enzymes possessing isoaspartyl dipeptide hydrolase activity. Two unrelated enzymes with this activity, isoaspartyl dipeptidase (IadA) and isoaspartyl aminopeptidase (IaaA) have been shown to degrade ß-Asp-Arg dipeptides, but bacteria which encode cyanophycin-metabolizing genes can lack iaaA and iadA genes. In this study, we investigate a previously uncharacterized enzyme whose gene can cluster with cyanophycin-metabolizing genes. This enzyme, which we name cyanophycin dipeptide hydrolase (CphZ), is specific for dipeptides derived from cyanophycin degradation. Accordingly, a co-complex structure of CphZ and ß-Asp-Arg shows that CphZ, unlike IadA or IaaA, recognizes all portions of its ß-Asp-Arg substrate. Bioinformatic analyses showed that CphZ is found in very many proteobacteria and is homologous to an uncharacterized protein encoded in the "arginine/ornithine transport" (aot) operon of many pseudomonas species, including Pseudomonas aeruginosa. In vitro assays show that AotO is indeed a CphZ, and in cellulo growth experiments show that this enzyme and the aot operon allow P. aeruginosa to take up and use ß-Asp-Arg as a sole carbon and nitrogen source. Together the results establish the novel, highly specific enzyme subfamily of CphZs, suggesting that cyanophycin is potentially used by a much wider range of bacteria than previously appreciated.

Mammalian telomeric RNA (TERRA) can be translated to produce valine-arginine and glycine-leucine dipeptide repeat proteins.

Mammalian telomeres consist of (TTAGGG)n repeats. Transcription of the C-rich strand generates a G-rich RNA, termed TERRA, containing G-quadruplex structures. Recent discoveries in several human nucleotide expansion diseases revealed that RNA transcripts containing long runs of 3 or 6 nt repeats which can form strong secondary structures can be translated in multiple frames to generate homopeptide or dipeptide repeat proteins, and multiple studies have shown them to be toxic in cells. We noted that the translation of TERRA would generate two dipeptide repeat proteins: highly charged repeating valine-arginine (VR)n and hydrophobic repeating glycine-leucine (GL)n. Here, we synthesized these two dipeptide proteins and raised polyclonal antibodies to VR. The VR dipeptide repeat protein binds nucleic acids and localizes strongly to replication forks in DNA. Both VR and GL form long 8-nm filaments with amyloid properties. Using labeled antibodies to VR and laser scanning confocal microscopy, threefold to fourfold more VR was observed in the nuclei of cell lines containing elevated TERRA as contrasted to a primary fibroblast line. Induction of telomere dysfunction via knockdown of TRF2 led to higher amounts of VR, and alteration of TERRA levels using a locked nucleic acid (LNA) GapmeR led to large nuclear VR aggregates. These observations suggest that telomeres, in particular in cells undergoing telomere dysfunction, may express two dipeptide repeat proteins with potentially strong biological properties.

eIF5 stimulates the CUG initiation of RAN translation of poly-GA dipeptide repeat protein (DPR) in C9orf72 FTLD/ALS.

Tandem GGGGCC repeat expansion in C9orf72 is a genetic cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Transcribed repeats are translated into dipeptide repeat proteins via repeat-associated non-AUG (RAN) translation. However, the regulatory mechanism of RAN translation remains unclear. Here, we reveal a GTPase-activating protein, eukaryotic initiation factor 5 (eIF5), which allosterically facilitates the conversion of eIF2-bound GTP into GDP upon start codon recognition, as a novel modifier of C9orf72 RAN translation. Compared to global translation, eIF5, but not its inactive mutants, preferentially stimulates poly-GA RAN translation. RAN translation is increased during integrated stress response, but the stimulatory effect of eIF5 on poly-GA RAN translation was additive to the increase of RAN translation during integrated stress response, with no further increase in phosphorylated eIF2α. Moreover, an alteration of the CUG near cognate codon to CCG or AUG in the poly-GA reading frame abolished the stimulatory effects, indicating that eIF5 primarily acts through the CUG-dependent initiation. Lastly, in a Drosophila model of C9orf72 FTLD/ALS that expresses GGGGCC repeats in the eye, knockdown of endogenous eIF5 by two independent RNAi strains significantly reduced poly-GA expressions, confirming in vivo effect of eIF5 on poly-GA RAN translation. Together, eIF5 stimulates the CUG initiation of poly-GA RAN translation in cellular and Drosophila disease models of C9orf72 FTLD/ALS.

Therapeutic reduction of GGGGCC repeat RNA levels by hnRNPA3 suppresses neurodegeneration in Drosophila models of C9orf72-linked ALS/FTD.

The abnormal expansion of GGGGCC hexanucleotide repeats within the C9orf72 gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The accumulation of GGGGCC repeat-containing RNAs as RNA foci, and the deposition of dipeptide repeat proteins (DPR) produced from these repeat RNAs by unconventional translation are major pathological hallmarks of C9orf72-linked ALS/FTD (C9-ALS/FTD), and are both thought to play a crucial role in the pathogenesis of these diseases. Because GGGGCC repeat RNA is likely to be the most upstream therapeutic target in the pathogenic cascade of C9-ALS/FTD, lowering the cellular level of GGGGCC repeat RNA is expected to mitigate repeat RNA toxicity, and will therefore be a disease-modifying therapeutic strategy for the treatment of C9-ALS/FTD. In this study, we demonstrated using a Drosophila model of C9-ALS/FTD that elevated expression of a subset of human RNA-binding proteins that bind to GGGGCC repeat RNA, including hnRNPA3, IGF2BP1, hnRNPA2B1, hnRNPR and SF3B3, reduces the level of GGGGCC repeat RNA, resulting in the suppression of neurodegeneration. We further showed that hnRNPA3-mediated reduction of GGGGCC repeat RNA suppresses disease pathology, such as RNA foci and DPR accumulation. These results demonstrate that hnRNPA3 and other RNA-binding proteins negatively regulate the level of GGGGCC repeat RNA, and mitigate repeat RNA toxicity in vivo, indicating the therapeutic potential of the repeat RNA-lowering approach mediated by endogenous RNA-binding proteins for the treatment of C9-ALS/FTD.

Disrupted phase behavior of FUS underlies poly-PR-induced DNA damage in amyotrophic lateral sclerosis.

GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the first intron of the chromosome 9 open reading frame 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Among the five dipeptide repeat proteins translated from G4C2 HRE, arginine-rich poly-PR (proline:arginine) is extremely toxic. However, the molecular mechanism responsible for poly-PR-induced cell toxicity remains incompletely understood. Here, we found that poly-PR overexpression triggers severe DNA damage in cultured cells, primary cortical neurons, and the motor cortex of a poly-PR transgenic mouse model. Interestingly, we identified a linkage between poly-PR and RNA-binding protein fused in sarcoma (FUS), another ALS-related gene product associated with DNA repair. Poly-PR interacts with FUS both in vitro and in vivo, phase separates with FUS in a poly-PR concentration-dependent manner, and impairs the fluidity of FUS droplets in vitro and in cells. Moreover, poly-PR impedes the recruitment of FUS and its downstream protein XRCC1 to DNA damage foci after microirradiation. Importantly, overexpression of FUS significantly decreased the level of DNA damage and dramatically reduced poly-PR-induced cell death. Our data suggest the severe DNA damage caused by poly-PR and highlight the interconnection between poly-PR and FUS, enlightening the potential therapeutic role of FUS in alleviating poly-PR-induced cell toxicity.

Genome mining for unknown-unknown natural products.

Genome mining of biosynthetic pathways with no identifiable core enzymes can lead to discovery of the so-called unknown (biosynthetic route)-unknown (molecular structure) natural products. Here we focused on a conserved fungal biosynthetic pathway that lacks a canonical core enzyme and used heterologous expression to identify the associated natural product, a highly modified cyclo-arginine-tyrosine dipeptide. Biochemical characterization of the pathway led to identification of a new arginine-containing cyclodipeptide synthase (RCDPS), which was previously annotated as a hypothetical protein and has no sequence homology to non-ribosomal peptide synthetase or bacterial cyclodipeptide synthase. RCDPS homologs are widely encoded in fungal genomes; other members of this family can synthesize diverse cyclo-arginine-Xaa dipeptides, and characterization of a cyclo-arginine-tryptophan RCDPS showed that the enzyme is aminoacyl-tRNA dependent. Further characterization of the biosynthetic pathway led to discovery of new compounds whose structures would not have been predicted without knowledge of RCDPS function.

Poly-dipeptides produced from C9orf72 hexanucleotide repeats cause selective motor neuron hyperexcitability in ALS.

SignificanceThe GGGGCC hexanucleotide repeat expansion in the chromosome 9 open reading frame 72 (C9orf72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS). Despite myriad studies on the toxic effects of poly-dipeptides produced from the C9orf72 repeats, the mechanisms underlying the selective hyperexcitability of motor cortex that characterizes the early stages of C9orf72 ALS patients remain elusive. Here, we show that the proline-arginine poly-dipeptides cause hyperexcitability in cortical motor neurons by increasing persistent sodium currents conducted by the Nav1.2/ß4 sodium channel complex, which is highly expressed in the motor cortex. These findings provide the basis for understanding how the C9orf72 mutation causes motor neuron hyperactivation that can lead to the motor neuron death in C9orf72 ALS.

c-Jun N-Terminal Kinase Promotes Stress Granule Assembly and Neurodegeneration in C9orf72-Mediated ALS and FTD.

Stress granules are the RNA/protein condensates assembled in the cells under stress. They play a critical role in the pathogenesis of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, how stress granule assembly is regulated and related to ALS/FTD pathomechanism is incompletely understood. Mutation in the C9orf72 gene is the most common cause of familial ALS and FTD. C9orf72 mutation causes the formation of toxic dipeptide repeats. Here we show that the two most toxic dipeptide repeats [i.e., poly(GR) and poly(PR)] activate c-Jun N-terminal kinase (JNK) via the ER-stress response protein IRE1 using fly and cellular models. Further, we show that activated JNK promotes stress granule assembly in cells by promoting the transcription of one of the key stress granule proteins (i.e., G3BP1) by inducing histone 3 phosphorylation. Consistent with these findings, JNK or IRE1 inhibition reduced stress granule formation, histone 3 phosphorylation, G3BP1 mRNA and protein levels, and neurotoxicity in cells overexpressing poly(GR) and poly(PR) or neurons derived from male and female C9ALS/FTD patient-induced pluripotent stem cells. Our findings connect ER stress, JNK activation, and stress granule assembly in a unified pathway contributing to C9ALS/FTD neurodegeneration.SIGNIFICANCE STATEMENT c-Jun N-terminal kinase (JNK) is a part of the mitogen-activated protein kinase pathway, which is the central node for the integration of multiple stress signals. Cells are under constant stress in neurodegenerative diseases, and how these cells respond to stress signals is a critical factor in determining their survival or death. Previous studies have shown JNK as a major contributor to cellular apoptosis. Here, we show the role of JNK in stress granule assembly. We identify that toxic dipeptide repeats produced in ALS/FTD conditions activate JNK. The activated JNK in the nucleus can induce histone modifications which increase G3BP1 expression, thus promoting stress granule assembly and neurodegeneration.

Artificial microRNA suppresses C9ORF72 variants and decreases toxic dipeptide repeat proteins in vivo.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons, causing progressive muscle weakness and respiratory failure. The presence of an expanded hexanucleotide repeat in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent mutation causing familial ALS and frontotemporal dementia (FTD). To determine if suppressing expression of C9ORF72 gene products can reduce toxicity, we designed a set of artificial microRNAs (amiRNA) targeting the human C9ORF72 gene. Here we report that an AAV9-mediated amiRNA significantly suppresses expression of the C9ORF72 mRNA, protein, and toxic dipeptide repeat proteins generated by the expanded repeat in the brain and spinal cord of C9ORF72 transgenic mice.

Reduced autophagy upon C9ORF72 loss synergizes with dipeptide repeat protein toxicity in G4C2 repeat expansion disorders.

Expansion of G4C2 repeats within the C9ORF72 gene is the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Such repeats lead to decreased expression of the autophagy regulator C9ORF72 protein. Furthermore, sense and antisense repeats are translated into toxic dipeptide repeat (DPR) proteins. It is unclear how these repeats are translated, and in which way their translation and the reduced expression of C9ORF72 modulate repeat toxicity. Here, we found that sense and antisense repeats are translated upon initiation at canonical AUG or near-cognate start codons, resulting in polyGA-, polyPG-, and to a lesser degree polyGR-DPR proteins. However, accumulation of these proteins is prevented by autophagy. Importantly, reduced C9ORF72 levels lead to suboptimal autophagy, thereby impairing clearance of DPR proteins and causing their toxic accumulation, ultimately resulting in neuronal cell death. Of clinical importance, pharmacological compounds activating autophagy can prevent neuronal cell death caused by DPR proteins accumulation. These results suggest the existence of a double-hit pathogenic mechanism in ALS/FTD, whereby reduced expression of C9ORF72 synergizes with DPR protein accumulation and toxicity.

Impaired ribosome-associated quality control of C9orf72 arginine-rich dipeptide-repeat proteins.

Protein quality control pathways have evolved to ensure the fidelity of protein synthesis and efficiently clear potentially toxic protein species. Defects in ribosome-associated quality control and its associated factors have been implicated in the accumulation of aberrant proteins and neurodegeneration. C9orf72 repeat-associated non-AUG translation has been suggested to involve inefficient translation elongation, lead to ribosomal pausing and activation of ribosome-associated quality control pathways. However, the role of the ribosome-associated quality control complex in the processing of proteins generated through this non-canonical translation is not well understood. Here we use reporter constructs containing the C9orf72-associated hexanucleotide repeat, ribosome-associated quality control complex deficient cell models and stain for ribosome-associated quality control markers in C9orf72-expansion carrier human tissue to understand its role in dipeptide-repeat protein pathology. Our studies show that canonical ribosome-associated quality control substrates products are efficiently cleared by the ribosome-associated quality control complex in mammalian cells. Furthermore, using stalling reporter constructs, we show that repeats associated with the C9orf72-expansion induce ribosomal stalling when arginine (R)-rich dipeptide-repeat proteins are synthesized in a length-dependent manner. However, despite triggering this pathway, these arginine-rich dipeptide-repeat proteins are not efficiently processed by the core components of the ribosome-associated quality control complex (listerin, nuclear-export mediator factor and valosin containing protein) partly due to lack of lysine residues, which precludes ubiquitination. Deficient processing by this complex may be implicated in C9orf72-expansion associated disease as dipeptide-repeat protein inclusions were observed to be predominantly devoid of ubiquitin and co-localize with nuclear-export mediator factor in mutation carriers' frontal cortex and cerebellum tissue. These findings suggest that impaired processing of these arginine-rich dipeptide-repeat proteins derived from repeat-associated non-AUG translation by the ribosome-associated quality control complex may contribute to protein homeostasis dysregulation observed in C9orf72-expansion amyotrophic lateral sclerosis and frontotemporal degeneration neuropathogenesis.

C9orf72 poly-PR helps p53 escape from the ubiquitin-proteasome system and promotes its stability.

C9orf72-derived dipeptide repeats (DPRs) proteins have been regarded as the pathogenic cause of neurodegeneration in amyotrophic lateral sclerosis and frontotemporal dementia (C9-ALS/FTD). As the most toxic DPRs in C9-ALS/FTD, poly-proline-arginine (poly-PR) is associated with the stability and accumulation of p53, which consequently induces neurodegeneration. However, the exact molecular mechanism via which C9orf72 poly-PR stabilizes p53 remains unclear. In this study, we showed that C9orf72 poly-PR induces not only neuronal damage but also p53 accumulation and p53 downstream gene activation in primary neurons. C9orf72 (PR)50 also slows down p53 protein turnover without affecting the p53 transcription level and thus promotes its stability in N2a cells. Interestingly, the ubiquitin-proteasome system but not the autophagy function was impaired in (PR)50 transfected N2a cells, resulting in defective p53 degradation. Moreover, we found that (PR)50 induces mdm2 mistranslocation from the nucleus to the cytoplasm and competitively binds to p53, reducing mdm2-p53 interactions in the nucleus in two different (PR)50 transfected cells. Our data strongly indicate that (PR)50 reduces mdm2-p53 interactions and causes p53 to escape from the ubiquitin-proteasome system, promoting its stability and accumulation. Inhibiting or at least downregulating (PR)50 binding with p53 may be therapeutically exploited for the treatment of C9-ALS/FTD.

C9orf72 poly(PR) aggregation in nucleus induces ALS/FTD-related neurodegeneration in cynomolgus monkeys.

Poly(PR) is a dipeptide repeat protein comprising proline and arginine residues. It is one of the translational product of expanded G4C2 repeats in the C9orf72 gene, and its accumulation is contributing to the neuropathogenesis of C9orf72-associated amyotrophic lateral sclerosis and/or frontotemporal dementia (C9-ALS/FTD). In this study, we demonstrate that poly(PR) protein alone is sufficient to induce neurodegeneration related to ALS/FTD in cynomolgus monkeys. By delivering poly(PR) via AAV, we observed that the PR proteins were located within the nucleus of infected cells. The expression of (PR)50 protein, consisting of 50 PR repeats, led to increased loss of cortical neurons, cytoplasmic lipofuscin, and gliosis in the brain, as well as demyelination and loss of ChAT positive neurons in the spinal cord of monkeys. While, these pathologies were not observed in monkeys expressing (PR)5, a protein comprising only 5 PR repeats. Furthermore, the (PR)50-expressing monkeys exhibited progressive motor deficits, cognitive impairment, muscle atrophy, and abnormal electromyography (EMG) potentials, which closely resemble clinical symptoms seen in C9-ALS/FTD patients. By longitudinally tracking these monkeys, we found that changes in cystatin C and chitinase-1 (CHIT1) levels in the cerebrospinal fluid (CSF) corresponded to the phenotypic progression of (PR)50-induced disease. Proteomic analysis revealed that the major clusters of dysregulated proteins were nuclear-localized, and downregulation of the MECP2 protein was implicated in the toxic process of poly(PR). This research indicates that poly(PR) expression alone induces neurodegeneration and core phenotypes associated with C9-ALS/FTD in monkeys, which may provide insights into the mechanisms of disease pathogenesis.

C9orf72-associated arginine-rich dipeptide repeats induce RNA-dependent nuclear accumulation of Staufen in neurons.

RNA-binding proteins (RBPs) play essential roles in diverse cellular processes through post-transcriptional regulation of RNAs. The subcellular localization of RBPs is thus under tight control, the breakdown of which is associated with aberrant cytoplasmic accumulation of nuclear RBPs such as TDP-43 and FUS, well-known pathological markers for amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). Here, we report in Drosophila model for ALS/FTD that nuclear accumulation of a cytoplasmic RBP Staufen may be a new pathological feature. We found that in Drosophila C4da neurons expressing PR36, one of the arginine-rich dipeptide repeat proteins (DPRs), Staufen accumulated in the nucleus in Importin- and RNA-dependent manner. Notably, expressing Staufen with exogenous NLS-but not with mutated endogenous NLS-potentiated PR-induced dendritic defect, suggesting that nuclear-accumulated Staufen can enhance PR toxicity. PR36 expression increased Fibrillarin staining in the nucleolus, which was enhanced by heterozygous mutation of stau (stau+/-), a gene that codes Staufen. Furthermore, knockdown of fib, which codes Fibrillarin, exacerbated retinal degeneration mediated by PR toxicity, suggesting that increased amount of Fibrillarin by stau+/- is protective. stau+/- also reduced the amount of PR-induced nuclear-accumulated Staufen and mitigated retinal degeneration and rescued viability of flies expressing PR36. Taken together, our data show that nuclear accumulation of Staufen in neurons may be an important pathological feature contributing to the pathogenesis of ALS/FTD.

Interactome screening of C9orf72 dipeptide repeats reveals VCP sequestration and functional impairment by polyGA.

Repeat expansions in the C9orf72 gene are a common cause of amyotrophic lateral sclerosis and frontotemporal lobar degeneration, two devastating neurodegenerative disorders. One of the proposed mechanisms of GGGGCC repeat expansion is their translation into non-canonical dipeptide repeats, which can then accumulate as aggregates and contribute to these pathologies. There are five different dipeptide repeat proteins (polyGA, polyGR, polyPR, polyPA and polyGP), some of which are known to be neurotoxic. In the present study, we used BioID2 proximity labelling to identify the interactomes of all five dipeptide repeat proteins consisting of 125 repeats each. We identified 113 interacting partners for polyGR, 90 for polyGA, 106 for polyPR, 25 for polyPA and 27 for polyGP. Gene Ontology enrichment analysis of the proteomic data revealed that these target interaction partners are involved in a variety of functions, including protein translation, signal transduction pathways, protein catabolic processes, amide metabolic processes and RNA-binding. Using autopsy brain tissue from patients with C9orf72 expansion complemented with cell culture analysis, we evaluated the interactions between polyGA and valosin containing protein (VCP). Functional analysis of this interaction revealed sequestration of VCP with polyGA aggregates, altering levels of soluble valosin-containing protein. VCP also functions in autophagy processes, and consistent with this, we observed altered autophagy in cells expressing polyGA. We also observed altered co-localization of polyGA aggregates and p62 in cells depleted of VCP. All together, these data suggest that sequestration of VCP with polyGA aggregates contributes to the loss of VCP function, and consequently to alterations in autophagy processes in C9orf72 expansion disorders.