Crystal structure of a tetrameric RNA G-quadruplex formed by hexanucleotide repeat expansions of C9orf72 in ALS/FTD.

The abnormal GGGGCC hexanucleotide repeat expansions (HREs) in C9orf72 cause the fatal neurodegenerative diseases including amyotrophic lateral sclerosis and frontotemporal dementia. The transcribed RNA HREs, short for r(G4C2)n, can form toxic RNA foci which sequestrate RNA binding proteins and impair RNA processing, ultimately leading to neurodegeneration. Here, we determined the crystal structure of r(G4C2)2, which folds into a parallel tetrameric G-quadruplex composed of two four-layer dimeric G-quadruplex via 5'-to-5' stacking in coordination with a K+ ion. Notably, the two C bases locate at 3'- end stack on the outer G-tetrad with the assistance of two additional K+ ions. The high-resolution structure reported here lays a foundation in understanding the mechanism of neurological toxicity of RNA HREs. Furthermore, the atomic details provide a structural basis for the development of potential therapeutic agents against the fatal neurodegenerative diseases ALS/FTD.

Formation of RNA G-wires by G4C2 repeats associated with ALS and FTD.

In the neurodegenerative disorders amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), expansion of the G4C2 hexanucleotide repeat in the gene C9orf72 is a most common known cause of the disease. Here we use atomic force microscopy (AFM) and gel electrophoresis to visualize the formation of higher-order structures by RNA G4C2 repeats in physiologically relevant conditions. For the RNA sequence r[G4C2G4], we observed G-wires with left-handed undulating features of 4.4-nm periodicity and a uniform height which is consistently higher than that of a duplex B-DNA. These higher-order structures were not degraded fully when treated with a mixture of RNase A and RNase T1. Similarly, higher-order structures were observed for sequences containing three or four G4C2 repeats, pointing towards their potential formation in longer sequence contexts. Our observations suggest that RNA G-quadruplex blocks and G-wires can accumulate in cells containing G4C2 repeat transcripts.

Optical control of protein delivery and partitioning in the nucleolus.

The nucleolus is a subnuclear membraneless compartment intimately involved in ribosomal RNA synthesis, ribosome biogenesis and stress response. Multiple optogenetic devices have been developed to manipulate nuclear protein import and export, but molecular tools tailored for remote control over selective targeting or partitioning of cargo proteins into subnuclear compartments capable of phase separation are still limited. Here, we report a set of single-component photoinducible nucleolus-targeting tools, designated pNUTs, to enable rapid and reversible nucleoplasm-to-nucleolus shuttling, with the half-lives ranging from milliseconds to minutes. pNUTs allow both global protein infiltration into nucleoli and local delivery of cargoes into the outermost layer of the nucleolus, the granular component. When coupled with the amyotrophic lateral sclerosis (ALS)-associated C9ORF72 proline/arginine-rich dipeptide repeats, pNUTs allow us to photomanipulate poly-proline-arginine nucleolar localization, perturb nucleolar protein nucleophosmin 1 and suppress nascent protein synthesis. pNUTs thus expand the optogenetic toolbox by permitting light-controllable interrogation of nucleolar functions and precise induction of ALS-associated toxicity in cellular models.

Order controls disordered droplets: structure-function relationships in C9ORF72-derived poly(PR).

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) have been thought as two distinct neurodegenerative diseases. However, recent genetic screening and careful investigations found the genetic and pathological overlap among these disorders. Hexanucleotide expansions in intron 1 of C9ORF72 are a leading cause of familial ALS and familial FTD. These expansions facilitate the repeat-associated non-ATG-initiated translation (RAN translation), producing five dipeptide repeat proteins (DRPs), including Arg-rich poly(PR: Pro-Arg) and poly(GR: Gly-Arg) peptides. Arg is a positively charged, highly polar amino acid that facilitates interactions with anionic molecules such as nucleic acids and acidic amino acids via electrostatic forces and aromatic amino acids via cation-π interaction, suggesting that Arg-rich DRPs underlie the pathophysiology of ALS via Arg-mediated molecular interactions. Arg-rich DRPs have also been reported to induce neurodegeneration in cellular and animal models via multiple mechanisms; however, it remains unclear why the Arg-rich DRPs exhibit such diverse toxic properties, because not all Arg-rich peptides are toxic. In this mini-review, we discuss the current understanding of the pathophysiology of Arg-rich C9ORF72 DRPs and introduce recent findings on the role of Arg distribution as a determinant of the toxicity and its contribution to the pathogenesis of ALS.

Synthetic hydrogel mimics of the nuclear pore complex for the study of nucleocytoplasmic transport defects in C9orf72 ALS/FTD.

Dipeptide repeats (DPRs) associated with C9orf72 repeat expansions perturb nucleocytoplasmic transport and are implicated in the pathogenesis of amyotrophic lateral sclerosis. We present a synthetic hydrogel platform that can be used to analyze aspects of the molecular interaction of dipeptide repeats and the phenylalanine-glycine (FG) phase of the nuclear pore complex (NPC). Hydrogel scaffolds composed of acrylamide and copolymerized with FG monomers are first formed to recapitulate key FG interactions found in the NPC. With labeled probes, we find evidence that toxic arginine-rich DPRs (poly-GR and poly-PR), but not the non-toxic poly-GP, target NPC hydrogel mimics and block selective entry of a key nuclear transport receptor, importin beta (Impß). The ease with which these synthetic hydrogel mimics can be adjusted/altered makes them an invaluable tool to dissect complex molecular interactions that underlie cellular transport processes and their perturbation in disease.

C9orf72-derived arginine-rich poly-dipeptides impede phase modifiers.

Nuclear import receptors (NIRs) not only transport RNA-binding proteins (RBPs) but also modify phase transitions of RBPs by recognizing nuclear localization signals (NLSs). Toxic arginine-rich poly-dipeptides from C9orf72 interact with NIRs and cause nucleocytoplasmic transport deficit. However, the molecular basis for the toxicity of arginine-rich poly-dipeptides toward NIRs function as phase modifiers of RBPs remains unidentified. Here we show that arginine-rich poly-dipeptides impede the ability of NIRs to modify phase transitions of RBPs. Isothermal titration calorimetry and size-exclusion chromatography revealed that proline:arginine (PR) poly-dipeptides tightly bind karyopherin-ß2 (Kapß2) at 1:1 ratio. The nuclear magnetic resonances of Kapß2 perturbed by PR poly-dipeptides partially overlapped with those perturbed by the designed NLS peptide, suggesting that PR poly-dipeptides target the NLS binding site of Kapß2. The findings offer mechanistic insights into how phase transitions of RBPs are disabled in C9orf72-related neurodegeneration.

Molecular interactions between C9ORF72 and SMCR8: A local energetic frustration perspective.

The hexanucleotide repeat expansion in C9orf72 represents a major cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). C9orf72, together with SMCR8 and WDR41, can form a stable complex that regulates autophagy and membrane trafficking. Very recently, the cryo-EM structure of C9orf72-SMCR8-WDR41 helps in understanding the structure-function relationship of C9orf72. This protein complex is indispensable to several cellular processes and is strongly linked to familial ALS and FTD. Understanding the molecular basis of the C9orf72-SMCR8 protein-protein interaction is thus important to comprehend their function. To establish a basis for understanding the relationships between sequence, structure, and function of the C9orf72, this study reports a local frustration analysis on the C9orf72-SMCR8 complex structure. An analysis of local frustration profiles indicated that (1) the structural domains in C9orf72 are minimally-frustrated and relatively conserved, (2) high frustration patches on the protein-protein interface (3) increased frustration in the C-terminal helices involved in the dimerization of C9orf72 structures.

Crystal structure of parallel G-quadruplex formed by the two-repeat ALS- and FTD-related GGGGCC sequence.

The hexanucleotide repeat expansion, GGGGCC (G4C2), within the first intron of the C9orf72 gene is known to be the most common genetic cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The G4C2 repeat expansions, either DNA or RNA, are able to form G-quadruplexes which induce toxicity leading to ALS/FTD. Herein, we report a novel crystal structure of d(G4C2)2 that self-associates to form an eight-layer parallel tetrameric G-quadruplex. Two d(G4C2)2 associate together as a parallel dimeric G-quadruplex which folds into a tetramer via 5'-to-5' arrangements. Each dimer consists of four G-tetrads connected by two CC propeller loops. Especially, the 3'-end cytosines protrude out and form C·C+•C·C+/ C·C•C·C+ quadruple base pair or C•C·C+ triple base pair stacking on the dimeric block. Our work sheds light on the G-quadruplexes adopted by d(G4C2) and yields the invaluable structural details for the development of small molecules to tackle neurodegenerative diseases, ALS and FTD.

Variant-selective stereopure oligonucleotides protect against pathologies associated with C9orf72-repeat expansion in preclinical models.

A large G4C2-repeat expansion in C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Neuronal degeneration associated with this expansion arises from a loss of C9orf72 protein, the accumulation of RNA foci, the expression of dipeptide repeat (DPR) proteins, or all these factors. We report the discovery of a new targeting sequence that is common to all C9orf72 transcripts but enables preferential knockdown of repeat-containing transcripts in multiple cellular models and C9BAC transgenic mice. We optimize stereopure oligonucleotides that act through this site, and we demonstrate that their preferential activity depends on both backbone stereochemistry and asymmetric wing design. In mice, stereopure oligonucleotides produce durable depletion of pathogenic signatures without disrupting protein expression. These oligonucleotides selectively protect motor neurons harboring C9orf72-expansion mutation from glutamate-induced toxicity. We hypothesize that targeting C9orf72 with stereopure oligonucleotides may be a viable therapeutic approach for the treatment of C9orf72-associated neurodegenerative disorders.

Structure of the C9orf72 ARF GAP complex that is haploinsufficient in ALS and FTD.

Mutation of C9orf72 is the most prevalent defect associated with amyotrophic lateral sclerosis and frontotemporal degeneration1. Together with hexanucleotide-repeat expansion2,3, haploinsufficiency of C9orf72 contributes to neuronal dysfunction4-6. Here we determine the structure of the C9orf72-SMCR8-WDR41 complex by cryo-electron microscopy. C9orf72 and SMCR8 both contain longin and DENN (differentially expressed in normal and neoplastic cells) domains7, and WDR41 is a ß-propeller protein that binds to SMCR8 such that the whole structure resembles an eye slip hook. Contacts between WDR41 and the DENN domain of SMCR8 drive the lysosomal localization of the complex in conditions of amino acid starvation. The structure suggested that C9orf72-SMCR8 is a GTPase-activating protein (GAP), and we found that C9orf72-SMCR8-WDR41 acts as a GAP for the ARF family of small GTPases. These data shed light on the function of C9orf72 in normal physiology, and in amyotrophic lateral sclerosis and frontotemporal degeneration.

The C9orf72-SMCR8-WDR41 complex is a GAP for small GTPases.

Massive expansions of the hexanucleotide in C9orf72 are the primary genetic origins of familial amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). Current studies have found that this repeat sequence participates in the disease process by producing neurotoxic substances and reducing the level of C9orf72 protein; however, the progress in the functional study of C9orf72 is slow. Recently, a stable complex, consisting of C9orf72, SMCR8, and WDR41, has been implicated in regulating membrane trafficking and macroautophagy. We reported the cryo-electron microscopy (cryo-EM) structure of the C9orf72-SMCR8-WDR41 complex (CSW complex), unveiling that the CSW complex is a dimer of heterotrimers. Intriguingly, in the heterotrimer of the C9orf72-SMCR8-WDR41, C9orf72 interacts with SMCR8 in a manner similar to the FLCN-FNIP2 complex. Nevertheless, WDR41 is connected to the DENN domain of SMCR8 through its N-terminal ß-strand and C-terminal helix but does not directly interact with C9orf72. Notably, the C9orf72-SMCR8 complex was demonstrated to act as a GAP for RAB8A and RAB11A in vitro.

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

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 ß 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 ß, disrupt its cargo loading, and inhibit nuclear import of importin ß, importin α/ß, 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.

A synthetic Pur-based peptide binds and alters G-quadruplex secondary structure present in the expanded RNA repeat of C9orf72 ALS/FTD.

Increased Pur-alpha (Pura) protein levels in animal models alleviate certain cellular symptoms of the disease spectrum amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD). Pura is a member of the Pur family of evolutionarily conserved guanine-rich polynucleotide binding proteins containing a repeated signature PUR domain of 60-80 amino acids. Here we have employed a synthetic peptide, TZIP, similar to a Pur domain, but with sequence alterations based on a consensus of evolutionarily conserved Pur family binding domains and having an added transporter sequence. A major familial form of ALS/FTD, C9orf72 (C9), is due to a hexanucleotide repeat expansion (HRE) of (GGGGCC), a Pur binding element. We show by circular dichroism that RNA oligonucleotides containing this purine-rich sequence consist largely of parallel G-quadruplexes. TZIP peptide binds this repeat sequence in both DNA and RNA. It binds the RNA element, including the G-quadruplexes, with a high degree of specificity versus a random oligonucleotide. In addition, TZIP binds both linear and G-quadruplex repeat RNA to form higher order G-quadruplex secondary structures. This change in conformational form by Pur-based peptide represents a new mechanism for regulating G quadruplex secondary structure within the C9 repeat. TZIP modulation of C9 RNA structural configuration may alter interaction of the complex with other proteins. This Pur-based mechanism provides new targets for therapy, and it may help to explain Pura alleviation of certain cellular pathological aspects of ALS/FTD.

Structural Diversity of Sense and Antisense RNA Hexanucleotide Repeats Associated with ALS and FTLD.

The hexanucleotide expansion GGGGCC located in C9orf72 gene represents the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar dementia (FTLD). Since the discovery one of the non-exclusive mechanisms of expanded hexanucleotide G4C2 repeats involved in ALS and FTLD is RNA toxicity, which involves accumulation of pathological sense and antisense RNA transcripts. Formed RNA foci sequester RNA-binding proteins, causing their mislocalization and, thus, diminishing their biological function. Therefore, structures adopted by pathological RNA transcripts could have a key role in pathogenesis of ALS and FTLD. Utilizing NMR spectroscopy and complementary methods, we examined structures adopted by both guanine-rich sense and cytosine-rich antisense RNA oligonucleotides with four hexanucleotide repeats. While both oligonucleotides tend to form dimers and hairpins, the equilibrium of these structures differs with antisense oligonucleotide being more sensitive to changes in pH and sense oligonucleotide to temperature. In the presence of K+ ions, guanine-rich sense RNA oligonucleotide also adopts secondary structures called G-quadruplexes. Here, we also observed, for the first time, that antisense RNA oligonucleotide forms i-motifs under specific conditions. Moreover, simultaneous presence of sense and antisense RNA oligonucleotides promotes formation of heterodimer. Studied structural diversity of sense and antisense RNA transcripts not only further depicts the complex nature of neurodegenerative diseases but also reveals potential targets for drug design in treatment of ALS and FTLD.

Three-Dimensional Structure of RNA Monomeric G-Quadruplex Containing ALS and FTD Related G4C2 Repeat and Its Binding with TMPyP4 Probed by Homology Modeling based on Experimental Constraints and Molecular Dynamics Simulations.

The G-quadruplex-forming hexanucleotide repeat expansion (HRE), d(G4C2)n, within the human C9orf72 gene is the root cause for familial amyotrophic lateral sclerosis-frontotemporal dementia (ALS-FTD). A recent study has shown that TMPyP4 has good potential to work as a RNA G-quadruplex binder in treating ALS and FTD. Although the high-resolution structure of the monomeric DNA antiparallel G-quadruplex form of the monomeric hexanucleotide repeat was recently solved, the RNA parallel G-quadruplex structure and its complex with TMPyP4 are not available yet. In this study, we first constructed the homology model for the parallel monomeric RNA G-quadruplex of r(G4C2)3G4 based on experimental constraints and the parallel monomeric G-quadruplex DNA crystal structure. Although the G-tetra core of the homology model was stable observed in 15 µs molecular dynamics (MD) simulations, we observed that the loops adopt additional conformations besides the initial crystal conformation, where TMPyP4 binding was found to reduce the loop fluctuation of the RNA monomeric G-quadruplex. Next, we probed the elusive binding behavior of TMPyP4 to the RNA monomeric G-quadruplex. Encouragingly, the binding modes observed are similar to the modes observed in two experimental complexes of a parallel DNA G-quadruplex with TMPyP4. We also constructed a Markov state model to provide insights into the binding pathways. Together, the findings from our study may assist future development of G-quadruplex-specific ligands in the treatment of neurodegenerative diseases like ALS and FTD.

C9orf72 Proteins Regulate Autophagy and Undergo Autophagosomal or Proteasomal Degradation in a Cell Type-Dependent Manner.

Dysfunctional autophagy or ubiquitin-proteasome system (UPS) are suggested to underlie abnormal protein aggregation in neurodegenerative diseases. Frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS)-associated C9orf72 is implicated in autophagy, but whether it activates or inhibits autophagy is partially controversial. Here, we utilized knockdown or overexpression of C9orf72 in mouse N2a neuroblastoma cells or cultured neurons to elucidate the potential role of C9orf72 proteins in autophagy and UPS. Induction of autophagy in C9orf72 knockdown N2a cells led to decreased LC3BI to LC3BII conversion, p62 degradation, and formation of LC3-containing autophagosomes, suggesting compromised autophagy. Proteasomal activity was slightly decreased. No changes in autophagy nor proteasomal activity in C9orf72-overexpressing N2a cells were observed. However, in these cells, autophagy induction by serum starvation or rapamycin led to significantly decreased C9orf72 levels. The decreased levels of C9orf72 in serum-starved N2a cells were restored by the proteasomal inhibitor lactacystin, but not by the autophagy inhibitor bafilomycin A1 (BafA1) treatment. These data suggest that C9orf72 undergoes proteasomal degradation in N2a cells during autophagy. Lactacystin significantly elevated C9orf72 levels in N2a cells and neurons, further suggesting UPS-mediated regulation. In rapamycin and BafA1-treated neurons, C9orf72 levels were significantly increased. Altogether, these findings corroborate the previously suggested regulatory role for C9orf72 in autophagy and suggest cell type-dependent regulation of C9orf72 levels via UPS and/or autophagy.

C9orf72 and triplet repeat disorder RNAs: G-quadruplex formation, binding to PRC2 and implications for disease mechanisms.

Some neurological disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), fragile X syndrome, Huntington's disease, myotonic dystrophy, and various ataxias, can be caused by expansions of short nucleic acid sequence repeats in specific genes. A possible disease mechanism involves the transcribed repeat RNA binding an RNA-binding protein (RBP), resulting in its sequestration and thus dysfunction. Polycomb repressive complex 2 (PRC2), the histone methyltransferase that deposits the H3K27me3 mark of epigenetically silenced chromatin, binds G-rich RNAs and has especially high affinity for G-quadruplex (G-Q) structures. Here, we find that PRC2 target genes are derepressed and the RNA binding subunit EZH2 largely insoluble in postmortem brain samples from ALS/FTD patients with C9ORF72 (C9) repeat expansions, leading to the hypothesis that the (G4C2)n repeat RNA might be sequestering PRC2. Contrary to this expectation, we found that C9 repeat RNAs (n = 6 or 10) bind weakly to purified PRC2, and studies with the G-Q specific BG4 antibody and circular dichroism studies both indicated that these C9 RNAs have little propensity to form G-Qs in vitro. Several GC-rich triplet-repeat expansion RNAs also have low affinity for PRC2 and do not appreciably form G-Qs in vitro. The results are consistent with these sequences forming hairpin structures that outcompete G-Q folding when the repeat length is sufficiently large. We suggest that binding of PRC2 to these GC-rich RNAs is fundamentally weak but may be modulated in vivo by protein factors that affect secondary structure, such as helicases and other RBPs.

A Chemical Screen Identifies Compounds Limiting the Toxicity of C9ORF72 Dipeptide Repeats.

The expansion of GGGGCC repeats within the first intron of C9ORF72 constitutes the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Through repeat-associated non-ATG translation, these expansions are translated into dipeptide repeats (DPRs), some of which accumulate at nucleoli and lead to cell death. We here performed a chemical screen to identify compounds reducing the toxicity of ALS-related poly(PR) peptides. Our screening identified sodium phenylbutyrate, currently in clinical trials, and BET Bromodomain inhibitors as modifiers of poly(PR) toxicity in cell lines and developing zebrafish embryos. Mechanistically, we show that BET Bromodomain inhibitors rescue the nucleolar stress induced by poly(PR) or actinomycin D, alleviating the effects of the DPR in nucleolus-related functions such as mRNA splicing or translation. Our work suggests that BET Bromodomain inhibitors might have beneficial effects in diseases linked to nucleolar stress such as ALS/FTD.

Characterization of membrane penetration and cytotoxicity of C9orf72-encoding arginine-rich dipeptides.

Cell-penetrating peptides (CPPs) including arginine-rich peptides are attracting a lot of attention due to their potential as a novel intracellular drug delivery tool without substantial toxicity. On the other hand, disease-associated arginine-rich CPPs, such as poly-PR and poly-GR translated from C9orf72 gene, also efficiently enter neuronal cells and then kill them. Although both non-harmful CPPs and harmful poly-PR/GR penetrate the plasma membrane using same arginine residues, little is known about the factors which determine the toxicity of the pathogenic CPPs. Here, we show that poly-PR and poly-GR, but not other Arg-rich CPPs, specifically distributed to nucleolus via interaction with RNA. Importantly, C9orf72-dipeptides, but not other Arg-rich CPPs, caused inhibition of protein translation and cell death. Raising extracellular pH enhanced the cell penetration of poly-PR. The repeat number of (PR) affected the secondary structure and determined the intracellular delivery rate and neurotoxicity, and enforced intracellular delivery of non-penetrating short poly-PR peptide caused cell death, suggesting that modulation of extracellular environment to inhibit the uptake of Arg-rich dipeptides might be a drug target against poly-PR/GR-mediated neurotoxicity.

C9orf72 Dipeptide Repeats Cause Selective Neurodegeneration and Cell-Autonomous Excitotoxicity in Drosophila Glutamatergic Neurons.

The arginine-rich dipeptide repeats (DPRs) are highly toxic products from the C9orf72 repeat expansion mutations, which are the most common causes of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, the effects of DPRs in the synaptic regulation and excitotoxicity remain elusive, and how they contribute to the development of FTD is primarily unknown. By expressing DPRs with different toxicity strength in various neuronal populations in a Drosophila model, we unexpectedly found that Glycine-Arginine/Proline-Arginine (GR/PR) with 36 repeats could lead to neurodegenerative phenotypes only when they were expressed in glutamatergic neurons, including motor neurons. We detected increased extracellular glutamate and intracellular calcium levels in GR/PR-expressing larval ventral nerve cord and/or adult brain, accompanied by significant increase of synaptic boutons and active zones in larval neuromuscular junctions. Inhibiting the vesicular glutamate transporter expression or blocking the NMDA receptor in presynaptic glutamatergic motor neurons could effectively rescue the motor deficits and shortened life span caused by poly GR/PR, thus indicating a cell-autonomous excitotoxicity mechanism. Therefore, our results have revealed a novel mode of synaptic regulation by arginine-rich C9 DPRs expressed at more physiologically relevant toxicity levels and provided a mechanism that could contribute to the development of C9-related ALS and FTD.SIGNIFICANCE STATEMENT C9orf72 dipeptide repeats (DPRs) are key toxic species causing ALS/FTD, but their roles in synaptic regulation and excitotoxicity are unclear. Using C9orf72 DPRs with various toxicity strength, we have found that the arginine-rich DPRs cause selective degeneration in Drosophila glutamatergic neurons and revealed an NMDA receptor-dependent cell-autonomous excitotoxicity mechanism. Therefore, this study has advanced our understanding of C9orf72 DPR functions in synaptic regulation and excitotoxicity and provided a new mechanism that could contribute to the development of C9-related ALS and FTD.