The MuSK agonist antibody protects the neuromuscular junction and extends the lifespan in C9orf72-ALS mice.

The disassembly of the neuromuscular junction (NMJ) is an early event in amyotrophic lateral sclerosis (ALS), ultimately leading to motor dysfunction and lethal respiratory paralysis. The hexanucleotide GGGGCC repeat expansion in the C9orf72 gene is the most common genetic mutation, and the dipeptide repeat (DPR) proteins have been shown to cause neurodegeneration. While no drugs can treat ALS patients efficiently, new treatment strategies are urgently needed. Here, we report that a MuSK agonist antibody alleviates poly-PR-induced NMJ deficits in C9orf72-ALS mice. The HB9-PRF/F mice, which express poly-PR proteins in motor neurons, exhibited impaired motor behavior and NMJ deficits. Mechanistically, poly-PR proteins interacted with Agrin to disrupt the interaction between Agrin and Lrp4, leading to attenuated activation of MuSK. Treatment with a MuSK agonist antibody rescued NMJ deficits, and extended the lifespan of C9orf72-ALS mice. Moreover, impaired NMJ transmission was observed in C9orf72-ALS patients. These findings identify the mechanism by which poly-PR proteins attenuate MuSK activation and NMJ transmission, highlighting the potential of promoting MuSK activation with an agonist antibody as a therapeutic strategy to protect NMJ function and prolong the lifespan of ALS patients.

Neuromuscular organoids model spinal neuromuscular pathologies in C9orf72 amyotrophic lateral sclerosis.

Hexanucleotide repeat expansions in the C9orf72 gene are the most common cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Due to the lack of trunk neuromuscular organoids (NMOs) from ALS patients' induced pluripotent stem cells (iPSCs), an organoid system was missing to model the trunk spinal neuromuscular neurodegeneration. With the C9orf72 ALS patient-derived iPSCs and isogenic controls, we used an NMO system containing trunk spinal cord neural and peripheral muscular tissues to show that the ALS NMOs could model peripheral defects in ALS, including contraction weakness, neural denervation, and loss of Schwann cells. The neurons and astrocytes in ALS NMOs manifested the RNA foci and dipeptide repeat proteins. Acute treatment with the unfolded protein response inhibitor GSK2606414 increased the glutamatergic muscular contraction 2-fold and reduced the dipeptide repeat protein aggregation and autophagy. This study provides an organoid system for spinal neuromuscular pathologies in ALS and its application for drug testing.

Glycine-Alanine Dipeptide Repeat Protein from C9-ALS Interacts with Sulfide Quinone Oxidoreductase (SQOR) to Induce the Activity of the NLRP3 Inflammasome in HMC3 Microglia: Irisflorentin Reverses This Interaction.

Amyotrophic lateral sclerosis (ALS) is a fatal rare disease of progressive degeneration of motor neurons. The most common genetic mutation in ALS is the hexanucleotide repeat expansion (HRE) located in the first intron of the C9orf72 gene (C9-ALS). HRE can produce dipeptide repeat proteins (DPRs) such as poly glycine-alanine (GA) in a repeat-associated non-ATG (RAN) translation. GA-DPR has been shown to be toxic to motor neurons in various biological models. However, its effects on microglia involved in C9-ALS have not been reported. Here, we show that GA-DPR (GA50) activates the NLR family pyrin domain containing 3 (NLRP3) inflammasome in a human HMC3 microglia model. MCC950 (specific inhibitor of the NLRP3) treatment can abrogate this activity. Next, using yeast two-hybrid screening, we identified sulfide quinone oxidoreductase (SQOR) as a GA50 interacting protein. SQOR knockdown in HMC3 cells can significantly induce the activity of the NLRP3 inflammasome by upregulating the level of intracellular reactive oxygen species and the cytoplasmic escape of mitochondrial DNA. Furthermore, we obtained irisflorentin as an effective blocker of the interaction between SQOR and GA50, thus inhibiting NLRP3 inflammasome activity in GA50-expressing HMC3 cells. These results imply the association of GA-DPR, SQOR, and NLRP3 inflammasomes in microglia and establish a treatment strategy for C9-ALS with irisflorentin.

Poly(GR) interacts with key stress granule factors promoting its assembly into cytoplasmic inclusions.

C9orf72 repeat expansions are the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). Poly(GR) proteins are toxic to neurons by forming cytoplasmic inclusions that sequester RNA-binding proteins including stress granule (SG) proteins. However, little is known of the factors governing poly(GR) inclusion formation. Here, we show that poly(GR) infiltrates a finely tuned network of protein-RNA interactions underpinning SG formation. It interacts with G3BP1, the key driver of SG assembly and a protein we found is critical for poly(GR) inclusion formation. Moreover, we discovered that N6-methyladenosine (m6A)-modified mRNAs and m6A-binding YTHDF proteins not only co-localize with poly(GR) inclusions in brains of c9FTD/ALS mouse models and patients with c9FTD, they promote poly(GR) inclusion formation via the incorporation of RNA into the inclusions. Our findings thus suggest that interrupting interactions between poly(GR) and G3BP1 or YTHDF1 proteins or decreasing poly(GR) altogether represent promising therapeutic strategies to combat c9FTD/ALS pathogenesis.

Glucose Hypometabolism Prompts RAN Translation and Exacerbates C9orf72-related ALS/FTD Phenotypes.

The most prevalent genetic cause of both amyotrophic lateral sclerosis and frontotemporal dementia is a (GGGGCC)n nucleotide repeat expansion (NRE) occurring in the first intron of the C9orf72 gene (C9). Brain glucose hypometabolism is consistently observed in C9-NRE carriers, even at pre-symptomatic stages, although its potential role in disease pathogenesis is unknown. Here, we identified alterations in glucose metabolic pathways and ATP levels in the brain of asymptomatic C9-BAC mice. We found that, through activation of the GCN2 kinase, glucose hypometabolism drives the production of dipeptide repeat proteins (DPRs), impairs the survival of C9 patient-derived neurons, and triggers motor dysfunction in C9-BAC mice. We also found that one of the arginine-rich DPRs (PR) can directly contribute to glucose metabolism and metabolic stress. These findings provide a mechanistic link between energy imbalances and C9-ALS/FTD pathogenesis and support a feedforward loop model that opens several opportunities for therapeutic intervention.

How villains are made: The translation of dipeptide repeat proteins in C9ORF72-ALS/FTD.

A hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic alteration associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). These neurodegenerative diseases share genetic, clinical and pathological features. The mutation in C9ORF72 appears to drive pathogenesis through a combination of loss of C9ORF72 normal function and gain of toxic effects due to the repeat expansion, which result in aggregation prone expanded RNAs and dipeptide repeat (DPR) proteins. Studies in cellular and animal models indicate that the DPR proteins are the more toxic species. Thus, a large body of research has focused on identifying the cellular pathways most directly impacted by these toxic proteins, with the goal of characterizing disease pathogenesis and nominating potential targets for therapeutic development. The preventative block of the production of the toxic proteins before they can cause harm is a second strategy of intense focus. Despite the considerable amount of effort dedicated to this prophylactic approach, it is still unclear how the DPR proteins are synthesized from RNAs harboring repeat expansions. In this review, we summarize our current knowledge of the specific protein translation mechanisms shown to account for the synthesis of DPR proteins. We will then discuss how enhanced understanding of the composition of these toxic effectors could help in refining disease mechanisms, and paving the way to identify and design effective prophylactic therapies for C9ORF72 ALS-FTD.

Comprehensive evaluation of human-derived anti-poly-GA antibodies in cellular and animal models of C9orf72 disease.

Hexanucleotide G4C2 repeat expansions in the C9orf72 gene are the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Dipeptide repeat proteins (DPRs) generated by translation of repeat-containing RNAs show toxic effects in vivo as well as in vitro and are key targets for therapeutic intervention. We generated human antibodies that bind DPRs with high affinity and specificity. Anti-GA antibodies engaged extra- and intra-cellular poly-GA and reduced aggregate formation in a poly-GA overexpressing human cell line. However, antibody treatment in human neuronal cultures synthesizing exogenous poly-GA resulted in the formation of large extracellular immune complexes and did not affect accumulation of intracellular poly-GA aggregates. Treatment with antibodies was also shown to directly alter the morphological and biochemical properties of poly-GA and to shift poly-GA/antibody complexes to more rapidly sedimenting ones. These alterations were not observed with poly-GP and have important implications for accurate measurement of poly-GA levels including the need to evaluate all centrifugation fractions and disrupt the interaction between treatment antibodies and poly-GA by denaturation. Targeting poly-GA and poly-GP in two mouse models expressing G4C2 repeats by systemic antibody delivery for up to 16 mo was well-tolerated and led to measurable brain penetration of antibodies. Long-term treatment with anti-GA antibodies produced improvement in an open-field movement test in aged C9orf72450 mice. However, chronic administration of anti-GA antibodies in AAV-(G4C2)149 mice was associated with increased levels of poly-GA detected by immunoassay and did not significantly reduce poly-GA aggregates or alleviate disease progression in this model.

Dipeptide Repeat Pathology in C9orf72-ALS Is Associated with Redox, Mitochondrial and NRF2 Pathway Imbalance.

The hexanucleotide expansion of the C9orf72 gene is found in 40% of familial amyotrophic lateral sclerosis (ALS) patients. This genetic alteration has been connected with impaired management of reactive oxygen species. In this study, we conducted targeted transcriptional profiling in leukocytes from C9orf72 patients and control subjects by examining the mRNA levels of 84 redox-related genes. The expression of ten redox genes was altered in samples from C9orf72 ALS patients compared to healthy controls. Considering that Nuclear factor erythroid 2-Related Factor 2 (NRF2) modulates the expression of a wide range of redox genes, we further investigated its status on an in vitro model of dipeptide repeat (DPR) toxicity. This model mimics the gain of function, toxic mechanisms attributed to C9orf72 pathology. We found that exposure to DPRs increased superoxide levels and reduced mitochondrial potential as well as cell survival. Importantly, cells overexpressing DPRs exhibited reduced protein levels of NRF2 and its target genes upon inhibition of the proteasome or its canonical repressor, the E3 ligase adapter KEAP1. However, NRF2 activation was sufficient to recover cell viability and redox homeostasis. This study identifies NRF2 as a putative target in precision medicine for the therapy of ALS patients harboring C9orf72 expansion repeats.

Repeat-Associated Non-AUG Translation of AGAGGG Repeats that Cause X-Linked Dystonia-Parkinsonism.

BACKGROUND: X-linked dystonia-parkinsonism (XDP) is a neurodegenerative disorder caused by the intronic insertion of a SINE-VNTR-Alu (SVA) retrotransposon carrying an (AGAGGG)n repeat expansion in the TAF1 gene. The molecular mechanisms by which this mutation causes neurodegeneration remain elusive. OBJECTIVES: We investigated whether (AGAGGG)n repeats undergo repeat-associated non-AUG (RAN) translation, a pathogenic mechanism common among repeat expansion diseases. METHODS: XDP-specific RAN translation reporter plasmids were generated, transfected in HEK293 cells, and putative dipeptide repeat proteins (DPRs) were detected by Western blotting. Immunocytochemistry was performed in COS-7 cells to determine the subcellular localization of one DPR. RESULTS: We detected putative DPRs from two reading frames, supporting the translation of poly-(Glu-Gly) and poly-(Arg-Glu) species. XDP RAN translation initiates within the (AGAGGG)n sequence and poly-(Glu-Gly) DPRs formed nuclear inclusions in transfected cells. CONCLUSIONS: In summary, our work provides the first in-vitro proof of principle that the XDP-linked (AGAGGG)n repeat expansions can undergo RAN translation. © 2022 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

Preclinical evaluation of WVE-004, aninvestigational stereopure oligonucleotide forthe treatment of C9orf72-associated ALS or FTD.

A large hexanucleotide (G4C2) repeat expansion in the first intronic region of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Several mechanisms have been proposed to explain how the repeat expansion drives disease, and we hypothesize that a variant-selective approach, in which transcripts affected by the repeat expansion are preferentially decreased, has the potential to address most of them. We report a stereopure antisense oligonucleotide, WVE-004, that executes this variant-selective mechanism of action. WVE-004 dose-dependently and selectively reduces repeat-containing transcripts in patient-derived motor neurons carrying a C9orf72-repeat expansion, as well as in the spinal cord and cortex of C9 BAC transgenic mice. In mice, selective transcript knockdown was accompanied by substantial decreases in dipeptide-repeat proteins, which are pathological biomarkers associated with the repeat expansion, and by preservation of healthy C9orf72 protein expression. These in vivo effects were durable, persisting for at least 6 months. These data support the advancement of WVE-004 as an investigational stereopure antisense oligonucleotide targeting C9orf72 for the treatment of C9orf72-associated ALS or FTD.

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.

Modeling C9orf72-Related Frontotemporal Dementia and Amyotrophic Lateral Sclerosis in Drosophila.

An intronic hexanucleotide (GGGGCC) expansion in the C9orf72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). In the decade following its discovery, much progress has been made in enhancing our understanding of how it precipitates disease. Both loss of function caused by reduced C9orf72 transcript levels, and gain of function mechanisms, triggered by the production of repetitive sense and antisense RNA and dipeptide repeat proteins, are thought to contribute to the toxicity. Drosophila models, with their unrivaled genetic tractability and short lifespan, have played a key role in developing our understanding of C9orf72-related FTD/ALS. There is no C9orf72 homolog in fly, and although this precludes investigations into loss of function toxicity, it is useful for elucidating mechanisms underpinning gain of function toxicity. To date there are a range of Drosophila C9orf72 models, encompassing different aspects of gain of function toxicity. In addition to pure repeat transgenes, which produce both repeat RNA and dipeptide repeat proteins (DPRs), RNA only models and DPR models have been generated to unpick the individual contributions of RNA and each dipeptide repeat protein to C9orf72 toxicity. In this review, we discuss how Drosophila models have shaped our understanding of C9orf72 gain of function toxicity, and address opportunities to utilize these models for further research.

Epigenetic Small Molecules Rescue Nucleocytoplasmic Transport and DNA Damage Phenotypes in C9ORF72 ALS/FTD.

Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease with available treatments only marginally slowing progression or improving survival. A hexanucleotide repeat expansion mutation in the C9ORF72 gene is the most commonly known genetic cause of both sporadic and familial cases of ALS and frontotemporal dementia (FTD). The C9ORF72 expansion mutation produces five dipeptide repeat proteins (DPRs), and while the mechanistic determinants of DPR-mediated neurotoxicity remain incompletely understood, evidence suggests that disruption of nucleocytoplasmic transport and increased DNA damage contributes to pathology. Therefore, characterizing these disturbances and determining the relative contribution of different DPRs is needed to facilitate the development of novel therapeutics for C9ALS/FTD. To this end, we generated a series of nucleocytoplasmic transport "biosensors", composed of the green fluorescent protein (GFP), fused to different classes of nuclear localization signals (NLSs) and nuclear export signals (NESs). Using these biosensors in conjunction with automated microscopy, we investigated the role of the three most neurotoxic DPRs (PR, GR, and GA) on seven nuclear import and two export pathways. In addition to other DPRs, we found that PR had pronounced inhibitory effects on the classical nuclear export pathway and several nuclear import pathways. To identify compounds capable of counteracting the effects of PR on nucleocytoplasmic transport, we developed a nucleocytoplasmic transport assay and screened several commercially available compound libraries, totaling 2714 compounds. In addition to restoring nucleocytoplasmic transport efficiencies, hits from the screen also counteract the cytotoxic effects of PR. Selected hits were subsequently tested for their ability to rescue another C9ALS/FTD phenotype-persistent DNA double strand breakage. Overall, we found that DPRs disrupt multiple nucleocytoplasmic transport pathways and we identified small molecules that counteract these effects-resulting in increased viability of PR-expressing cells and decreased DNA damage markers in patient-derived motor neurons. Several HDAC inhibitors were validated as hits, supporting previous studies that show that HDAC inhibitors confer therapeutic effects in neurodegenerative models.

The RNA helicase DHX36-G4R1 modulates C9orf72 GGGGCC hexanucleotide repeat-associated translation.

GGGGCC (G4C2) hexanucleotide repeat expansions in the endosomal trafficking gene C9orf72 are the most common genetic cause of ALS and frontotemporal dementia. Repeat-associated non-AUG (RAN) translation of this expansion through near-cognate initiation codon usage and internal ribosomal entry generates toxic proteins that accumulate in patients' brains and contribute to disease pathogenesis. The helicase protein DEAH-box helicase 36 (DHX36-G4R1) plays active roles in RNA and DNA G-quadruplex (G4) resolution in cells. As G4C2 repeats are known to form G4 structures in vitro, we sought to determine the impact of manipulating DHX36 expression on repeat transcription and RAN translation. Using a series of luciferase reporter assays both in cells and in vitro, we found that DHX36 depletion suppresses RAN translation in a repeat length-dependent manner, whereas overexpression of DHX36 enhances RAN translation from G4C2 reporter RNAs. Moreover, upregulation of RAN translation that is typically triggered by integrated stress response activation is prevented by loss of DHX36. These results suggest that DHX36 is active in regulating G4C2 repeat translation, providing potential implications for therapeutic development in nucleotide repeat expansion disorders.

Emerging Perspectives on Dipeptide Repeat Proteins in C9ORF72 ALS/FTD.

The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a hexanucleotide expansion in the chromosome 9 open reading frame 72 gene (C9ORF72). This hexanucleotide expansion consists of GGGGCC (G4C2) repeats that have been implicated to lead to three main modes of disease pathology: loss of function of the C9ORF72 protein, the generation of RNA foci, and the production of dipeptide repeat proteins (DPRs) through repeat-associated non-AUG (RAN) translation. Five different DPRs are currently known to be formed: glycine-alanine (GA) and glycine-arginine (GR) from the sense strand, proline-alanine (PA), and proline-arginine (PR) from the antisense strand, and glycine-proline (GP) from both strands. The exact contribution of each DPR to disease pathology is currently under intense scrutiny and is still poorly understood. However, recent advances in both neuropathological and cellular studies have provided us with clues enabling us to better understand the effect of individual DPRs on disease pathogenesis. In this review, we compile the current knowledge of specific DPR involvement on disease development and highlight recent advances, such as the impact of arginine-rich DPRs on nucleolar protein quality control, the correlation of poly-GR with neurodegeneration, and the possible involvement of chimeric DPR species. Further, we discuss recent findings regarding the mechanisms of RAN translation, its modulators, and other promising therapeutic options.

SFPQ regulates the accumulation of RNA foci and dipeptide repeat proteins from the expanded repeat mutation in C9orf72.

The expanded GGGGCC repeat mutation in the C9orf72 gene is the most common genetic cause of the neurodegenerative diseases amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The expansion is transcribed to sense and antisense RNA, which form RNA foci and bind cellular proteins. This mechanism of action is considered cytotoxic. Translation of the expanded RNA transcripts also leads to the accumulation of toxic dipeptide repeat proteins (DPRs). The RNA-binding protein splicing factor proline and glutamine rich (SFPQ), which is being increasingly associated with ALS and FTD pathology, binds to sense RNA foci. Here, we show that SFPQ plays an important role in the C9orf72 mutation. Overexpression of SFPQ resulted in higher numbers of both sense and antisense RNA foci and DPRs in transfected human embryonic kidney (HEK) cells. Conversely, reduced SPFQ levels resulted in lower numbers of RNA foci and DPRs in both transfected HEK cells and C9orf72 mutation-positive patient-derived fibroblasts and lymphoblasts. Therefore, we have revealed a role of SFPQ in regulating the C9orf72 mutation that has implications for understanding and developing novel therapeutic targets for ALS and FTD.This article has an associated First Person interview with the first author of the paper.

Fluid Biomarkers of Frontotemporal Lobar Degeneration.

A timely diagnosis of frontotemporal degeneration (FTD) is frequently challenging due to the heterogeneous symptomatology and poor phenotype-pathological correlation. Fluid biomarkers that reflect FTD pathophysiology could be instrumental in both clinical practice and pharmaceutical trials. In recent years, significant progress has been made in developing biomarkers of neurodegenerative diseases: amyloid-ß and tau in cerebrospinal fluid (CSF) can be used to exclude Alzheimer's disease, while neurofilament light chain (NfL) is emerging as a promising, albeit nonspecific, marker of neurodegeneration in both CSF and blood. Gene-specific biomarkers such as PGRN in GRN mutation carriers and dipeptide repeat proteins in C9orf72 mutation carriers are potential target engagement markers in genetic FTD trials. Novel techniques capable of measuring very low concentrations of brain-derived proteins in peripheral fluids are facilitating studies of blood biomarkers as a minimally invasive alternative to CSF. A major remaining challenge is the identification of a biomarker that can be used to predict the neuropathological substrate in sporadic FTD patients.

HDAC6 Interacts With Poly (GA) and Modulates its Accumulation in c9FTD/ALS.

The aberrant translation of a repeat expansion in chromosome 9 open reading frame 72 (C9orf72), the most common cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS), results in the accumulation of toxic dipeptide repeat (DPR) proteins in the central nervous system We have found that, among the sense DPR proteins, HDAC6 specifically interacts with the poly (GA) and co-localizes with inclusions in both patient tissue and a mouse model of this disease (c9FTD/ALS). Overexpression of HDAC6 increased poly (GA) levels in cultured cells independently of HDAC6 deacetylase activity, suggesting that HDAC6 can modulate poly (GA) pathology through a mechanism that depends upon their physical interaction. Moreover, decreasing HDAC6 expression by stereotaxic injection of antisense oligonucleotides significantly reduced the number of poly (GA) inclusions in c9FTD/ALS mice. These findings suggest that pharmacologically reducing HDAC6 levels could be of therapeutic value in c9FTD/ALS.

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

Nuclear import receptors, also called importins, mediate nuclear import of proteins and chaperone aggregation-prone cargoes (e.g., neurodegeneration-linked RNA-binding proteins [RBPs]) in the cytoplasm. Importins were identified as modulators of cellular toxicity elicited by arginine-rich dipeptide repeat proteins (DPRs), an aberrant protein species found in C9orf72-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Mechanistically, the link between importins and arginine-rich DPRs remains unclear. Here, we show that arginine-rich DPRs (poly-GR and poly-PR) bind directly to multiple importins and, in excess, promote their insolubility and condensation. In cells, poly-GR impairs Impα/ß-mediated nuclear import, including import of TDP-43, an RBP that aggregates in C9orf72-ALS/FTD patients. Arginine-rich DPRs promote phase separation and insolubility of TDP-43 in vitro and in cells, and this pathological interaction is suppressed by elevating importin concentrations. Our findings suggest that importins can decrease toxicity of arginine-rich DPRs by suppressing their pathological interactions.

Soluble and insoluble dipeptide repeat protein measurements in C9orf72-frontotemporal dementia brains show regional differential solubility and correlation of poly-GR with clinical severity.

A C9orf72 repeat expansion is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis. One of the suggested pathomechanisms is toxicity from dipeptide repeat proteins (DPRs), which are generated via unconventional translation of sense and antisense repeat transcripts with poly-GA, poly-GP and poly-GR being the most abundant dipeptide proteins. Animal and cellular studies highlight a neurotoxic role of poly-GR and poly-PR and to a lesser degree of poly-GA. Human post-mortem studies in contrast have been much less clear on a potential role of DPR toxicity but have largely focused on immunohistochemical methods to detect aggregated DPR inclusions. This study uses protein fractionation and sensitive immunoassays to quantify not only insoluble but also soluble poly-GA, poly-GP and poly-GR concentrations in brain homogenates of FTD patients with C9orf72 mutation across four brain regions. We show that soluble DPRs are less abundant in clinically affected areas (i.e. frontal and temporal cortices). In contrast, the cerebellum not only shows the largest DPR load but also the highest relative DPR solubility. Finally, poly-GR levels and poly-GP solubility correlate with clinical severity. These findings provide the first cross-comparison of soluble and insoluble forms of all sense DPRs and shed light on the distribution and role of soluble DPRs in the etiopathogenesis of human C9orf72-FTD.