Search

The GGGGCC hexanucleotide repeat expansion (HRE) of the C9orf72 gene is the most frequent cause of amyotrophic lateral sclerosis (ALS), a devastative neurodegenerative disease characterized by motor neuron degeneration. C9orf72 HRE is associated with lowered levels of C9orf72 expression and its translation results in the production of dipeptide-repeats (DPRs). To recapitulate C9orf72 -related ALS disease in vivo , we developed a zebrafish model where we expressed glycine-proline (GP) DPR in a c9orf72 knockdown context. We report that C9orf72 gain- and loss-of-function properties act synergistically to induce motor neuron degeneration and paralysis with poly(GP) accumulating preferentially within motor neurons along with Sqstm1/p62 aggregation indicating macroautophagy/autophagy deficits. Poly(GP) levels were shown to accumulate upon c9orf72 downregulation and were comparable to levels assessed in autopsy samples of patients carrying C9orf72 HRE. Chemical boosting of autophagy using rapamycin or apilimod, is able to rescue motor deficits. Proteomics analysis of zebrafish-purified motor neurons unravels mitochondria dysfunction confirmed through a comparative analysis of previously published C9orf72 iPSC-derived motor neurons. Consistently, 3D-reconstructions of motor neuron demonstrate that poly(GP) aggregates colocalize to mitochondria, thus inducing their elongation and swelling and the failure of their processing by mitophagy, with mitophagy activation through urolithin A preventing locomotor deficits. Finally, we report apoptotic-related increased amounts of cleaved Casp3 (caspase 3, apoptosis-related cysteine peptidase) and rescue of motor neuron degeneration by constitutive inhibition of Casp9 or treatment with decylubiquinone. Here we provide evidence of key pathogenic steps in C9ALS-FTD that can be targeted through pharmacological avenues, thus raising new therapeutic perspectives for ALS patients.

Amyotrophic lateral sclerosis (ALS) is a debilitating motor neuron disease and lacks effective disease-modifying treatments. This study utilizes a comprehensive multiomic approach to investigate the early and sex-specific molecular mechanisms underlying ALS. By analyzing the prefrontal cortex of 51 patients with sporadic ALS and 50 control subjects, alongside four transgenic mouse models (C9orf72-, SOD1-, TDP-43-, and FUS-ALS), we have uncovered significant molecular alterations associated with the disease. Here, we show that males exhibit more pronounced changes in molecular pathways compared to females. Our integrated analysis of transcriptomes, (phospho)proteomes, and miRNAomes also identified distinct ALS subclusters in humans, characterized by variations in immune response, extracellular matrix composition, mitochondrial function, and RNA processing. The molecular signatures of human subclusters were reflected in specific mouse models. Our study highlighted the mitogen-activated protein kinase (MAPK) pathway as an early disease mechanism. We further demonstrate that trametinib, a MAPK inhibitor, has potential therapeutic benefits in vitro and in vivo, particularly in females, suggesting a direction for developing targeted ALS treatments., (© 2024. The Author(s).)

An intronic (G 4 C 2 ) n expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal dementia primarily through gain-of-function mechanisms: the accumulation of sense and antisense repeat RNA foci and dipeptide repeat (DPR) proteins (poly-GA/GP/GR/PA/PR) translated from repeat RNA. To therapeutically block this pathway, we screen a library of 1,430 approved drugs and known bioactive compounds in patient-derived induced pluripotent stem cell-derived neurons (iPSC-Neurons) for inhibitors of DPR expression. The clinically used guanosine/cytidine analogs decitabine, entecavir, and nelarabine reduce poly-GA/GP expression, with decitabine being the most potent. Hit compounds nearly abolish sense and antisense RNA foci and reduce expression of the repeat-containing nascent C9orf72 RNA transcript and its mature mRNA with minimal effects on global gene expression, suggesting that they specifically act on repeat transcription. Importantly, decitabine treatment reduces (G 4 C 2 ) n foci and DPRs in C9orf72 BAC transgenic mice. Our findings suggest that nucleoside analogs are a promising compound class for therapeutic development in C9orf72 repeat-expansion-associated disorders., Competing Interests: Declaration of interests T.K. and J.M. are employees of Orion Pharma. The other authors declare no competing interests., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

Expansion of a (G 4 C 2 ) n repeat in C9orf72 causes amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), but the link of the five repeat-encoded dipeptide repeat (DPR) proteins to neuroinflammation, TDP-43 pathology, and neurodegeneration is unclear. Poly-PR is most toxic in vitro, but poly-GA is far more abundant in patients. To directly compare these in vivo, we created congenic poly-GA and poly-PR mice. 40% of poly-PR mice were affected with ataxia and seizures, requiring euthanasia by 6 weeks of age. The remaining poly-PR mice were asymptomatic at 14 months of age, likely due to an 80% reduction of the transgene mRNA in this subgroup. In contrast, all poly-GA mice showed selective neuron loss, inflammation, as well as muscle denervation and wasting requiring euthanasia before 7 weeks of age. In-depth analysis of peripheral organs and blood samples suggests that peripheral organ failure does not drive these phenotypes. Although transgene mRNA levels were similar between poly-GA and affected poly-PR mice, poly-GA aggregated far more abundantly than poly-PR in the CNS and was also found in skeletal muscle. In addition, TDP-43 and other disease-linked RNA-binding proteins co-aggregated in rare nuclear inclusions in the hippocampus and frontal cortex only in poly-GA mice. Transcriptome analysis revealed activation of an interferon-responsive pro-inflammatory microglial signature in end-stage poly-GA but not poly-PR mice. This signature was also found in all ALS patients and enriched in C9orf72 cases. In summary, our rigorous comparison of poly-GA and poly-PR toxicity in vivo indicates that poly-GA, but not poly-PR at the same mRNA expression level, promotes interferon responses in C9orf72 disease and contributes to TDP-43 abnormalities and neuron loss selectively in disease-relevant regions.

The C9orf72 repeat expansion causes amyotrophic lateral sclerosis and frontotemporal dementia, but the poor correlation between C9orf72-specific pathology and TDP-43 pathology linked to neurodegeneration hinders targeted therapeutic development. Here, we addressed the role of the aggregating dipeptide repeat proteins resulting from unconventional translation of the repeat in all reading frames. Poly-GA promoted cytoplasmic mislocalization and aggregation of TDP-43 non-cell-autonomously, and anti-GA antibodies ameliorated TDP-43 mislocalization in both donor and receiver cells. Cell-to-cell transmission of poly-GA inhibited proteasome function in neighboring cells. Importantly, proteasome inhibition led to the accumulation of TDP-43 ubiquitinated within the nuclear localization signal (NLS) at lysine 95. Mutagenesis of this ubiquitination site completely blocked poly-GA-dependent mislocalization of TDP-43. Boosting proteasome function with rolipram reduced both poly-GA and TDP-43 aggregation. Our data from cell lines, primary neurons, transgenic mice, and patient tissue suggest that poly-GA promotes TDP-43 aggregation by inhibiting the proteasome cell-autonomously and non-cell-autonomously, which can be prevented by inhibiting poly-GA transmission with antibodies or boosting proteasome activity with rolipram., (© 2020 The Authors. Published under the terms of the CC BY NC ND 4.0 license.)

The C9orf72 repeat expansion is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD). Non-canonical translation of the expanded repeat results in abundant poly-GA inclusion pathology throughout the CNS. (GA) 149 -CFP expression in mice triggers motor deficits and neuroinflammation. Since poly-GA is transmitted between cells, we investigated the therapeutic potential of anti-GA antibodies by vaccinating (GA) 149 -CFP mice. To overcome poor immunogenicity, we compared the antibody response of multivalent ovalbumin-(GA) 10 conjugates and pre-aggregated carrier-free (GA) 15 . Only ovalbumin-(GA) 10 immunization induced a strong anti-GA response. The resulting antisera detected poly-GA aggregates in cell culture and patient tissue. Ovalbumin-(GA) 10 immunization largely rescued the motor function in (GA) 149 -CFP transgenic mice and reduced poly-GA inclusions. Transcriptome analysis showed less neuroinflammation in ovalbumin-(GA) 10 -immunized poly-GA mice, which was corroborated by semiquantitative and morphological analysis of microglia/macrophages. Moreover, cytoplasmic TDP-43 mislocalization and levels of the neurofilament light chain in the CSF were reduced, suggesting neuroaxonal damage is reduced. Our data suggest that immunotherapy may be a viable primary prevention strategy for ALS/FTD in C9orf72 mutation carriers., (© 2019 The Authors. Published under the terms of the CC BY 4.0 license.)

Repeat expansion in C9orf72 causes amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Expanded sense and antisense repeat RNA transcripts in C9orf72 are translated into five dipeptide-repeat proteins (DPRs) in an AUG-independent manner. We previously identified the heterogeneous ribonucleoprotein (hnRNP) A3 as an interactor of the sense repeat RNA that reduces its translation into DPRs. Furthermore, we found that hnRNPA3 is depleted from the nucleus and partially mislocalized to cytoplasmic poly-GA inclusions in C9orf72 patients, suggesting that poly-GA sequesters hnRNPA3 within the cytoplasm. We now demonstrate that hnRNPA3 also binds to the antisense repeat RNA. Both DPR production and deposition from sense and antisense RNA repeats are increased upon hnRNPA3 reduction. All DPRs induced DNA double strand breaks (DSB), which was further enhanced upon reduction of hnRNPA3. Poly-glycine-arginine and poly-proline-arginine increased foci formed by phosphorylated Ataxia Telangiectasia Mutated (pATM), a major sensor of DSBs, whereas poly-glycine-alanine (poly-GA) evoked a reduction of pATM foci. In dentate gyri of C9orf72 patients, lower nuclear hnRNPA3 levels were associated with increased DNA damage. Moreover, enhanced poly-GA deposition correlated with reduced pATM foci. Since cytoplasmic pATM deposits partially colocalized with poly-GA deposits, these results suggest that poly-GA, the most frequent DPR observed in C9orf72 patients, differentially causes DNA damage and that poly-GA selectively sequesters pATM in the cytoplasm inhibiting its recruitment to sites of DNA damage. Thus, mislocalization of nuclear hnRNPA3 caused by poly-GA leads to increased poly-GA production, which partially depletes pATM, and consequently enhances DSB.

Cell-to-cell transmission of protein aggregates is an emerging theme in neurodegenerative disease. Here, we analyze the dipeptide repeat (DPR) proteins that form neuronal inclusions in patients with hexanucleotide repeat expansion C9orf72 , the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). Sense and antisense transcripts of the (G4C2) n repeat are translated by repeat-associated non-ATG (RAN) translation in all reading frames into five aggregating DPR proteins. We show that the hydrophobic DPR proteins poly-GA, poly-GP, and poly-PA are transmitted between cells using co-culture assays and cell extracts. Moreover, uptake or expression of poly-GA induces nuclear RNA foci in (G4C2) 80 -expressing cells and patient fibroblasts, suggesting an unexpected positive feedback loop. Exposure to recombinant poly-GA and cerebellar extracts of C9orf72 patients increases repeat RNA levels and seeds aggregation of all DPR proteins in receiver cells expressing (G4C2) 80 Treatment with anti-GA antibodies inhibits intracellular poly-GA aggregation and blocks the seeding activity of C9orf72 brain extracts. Poly-GA-directed immunotherapy may thus reduce DPR aggregation and disease progression in C9orf72 ALS/FTD., (© 2017 The Authors. Published under the terms of the CC BY 4.0 license.)