Unaffected mosaic C9orf72 case

ObjectiveSuggested C9orf72 disease mechanisms for amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration include C9orf72 haploinsufficiency, G4C2/C4G2 RNA foci, and dipeptide repeat (DPR) proteins translated from the G4C2 expansion; however, the role of small expansions (e.g., 30–90 repeats) is unknown and was investigated here.MethodsWe conducted a molecular and pathology study of a family in which the father (unaffected at age 90) carried a 70-repeat allele in blood DNA that expanded to ≈1,750 repeats in his children, causing ALS.ResultsSouthern blotting revealed different degrees of mosaicism of small and large expansions in the father's tissues from the CNS. Surprisingly, in each mosaic tissue, C9orf72 mRNA levels were significantly increased compared to an ALS-affected daughter with a large expansion. Increased expression correlated with higher levels of the 70-repeat allele (the upregulation was also evident at the protein level). Remarkably, RNA foci and DPR burdens were similar or even significantly increased (in cerebellum) in the unaffected father compared to the daughter with ALS. However, the father did not display TDP-43 pathology and signs of neurodegeneration.ConclusionThe presence of RNA foci and DPR pathology was insufficient for disease manifestation and TDP-43 pathology in the mosaic C9orf72 carrier with upregulated C9orf72 expression. It is important to conduct an investigation of similar cases, which could be found among unaffected parents of sporadic C9orf72 patients (e.g., 21% among Finnish patients with ALS). Caution should be taken when consulting carriers of small expansions because disease manifestation could be dependent on the extent of the somatic instability in disease-relevant tissues.

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

Journal of Cell Science ◽

10.1242/jcs.256602 ◽

2021 ◽

Vol 134 (4) ◽

pp. jcs256602 ◽

Cited By ~ 1

Author(s):

Mirjana Malnar ◽

Boris Rogelj

Keyword(s):

Antisense Rna ◽

Rna Binding ◽

Dipeptide Repeat Proteins ◽

Rna Transcripts ◽

Repeat Proteins ◽

Rna Foci ◽

Hek Cells ◽

Lateral Sclerosis ◽

Dipeptide Repeat

ABSTRACTThe 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.

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Order controls disordered droplets: structure-function relationships in C9orf72-derived poly(PR)

AJP Cell Physiology ◽

10.1152/ajpcell.00372.2021 ◽

2021 ◽

Author(s):

Kohsuke Kanekura ◽

Yuhei Hayamizu ◽

Masahiko Kuroda

Keyword(s):

Amino Acids ◽

Aromatic Amino Acids ◽

Polar Amino Acid ◽

Repeat Proteins ◽

Intron 1 ◽

Lateral Sclerosis ◽

Ran Translation ◽

Dipeptide Repeat ◽

Positively Charged

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-pi 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.

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C9orf72-generated poly-GR and poly-PR do not directly interfere with nucleocytoplasmic transport

Scientific Reports ◽

10.1038/s41598-019-52035-6 ◽

2019 ◽

Vol 9 (1) ◽

Cited By ~ 14

Author(s):

Joni Vanneste ◽

Thomas Vercruysse ◽

Steven Boeynaems ◽

Adria Sicart ◽

Philip Van Damme ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Motor Neurons ◽

Hela Cells ◽

Nuclear Import ◽

Nucleocytoplasmic Transport ◽

Repeat Expansions ◽

Import And Export ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Abstract Repeat expansions in the C9orf72 gene cause amyotrophic lateral sclerosis and frontotemporal dementia characterized by dipeptide-repeat protein (DPR) inclusions. The toxicity associated with two of these DPRs, poly-GR and poly-PR, has been associated with nucleocytoplasmic transport. To investigate the causal role of poly-GR or poly-PR on active nucleocytoplasmic transport, we measured nuclear import and export in poly-GR or poly-PR expressing Hela cells, neuronal-like SH-SY5Y cells and iPSC-derived motor neurons. Our data strongly indicate that poly-GR and poly-PR do not directly impede active nucleocytoplasmic transport.

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

Acta Neuropathologica Communications ◽

10.1186/s40478-020-01036-y ◽

2020 ◽

Vol 8 (1) ◽

Author(s):

Annelies Quaegebeur ◽

Idoia Glaria ◽

Tammaryn Lashley ◽

Adrian M. Isaacs

Keyword(s):

Frontotemporal Dementia ◽

Potential Role ◽

Brain Regions ◽

Clinical Severity ◽

Repeat Proteins ◽

Differential Solubility ◽

Shed Light ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Abstract 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.

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C9orf72-derived poly-GA DPRs undergo endocytic uptake in iNPC-derived astrocytes and spread to motor neurons

10.1101/2021.10.11.463891 ◽

2021 ◽

Author(s):

Paolo M. Marchi ◽

Lara Marrone ◽

Laurent Brasseur ◽

Luc Bousset ◽

Christopher P. Webster ◽

...

Keyword(s):

Motor Neurons ◽

Disease Spread ◽

C9orf72 Gene ◽

Cell Propagation ◽

Β Sheet ◽

Lateral Sclerosis ◽

Dipeptide Repeat ◽

Axonal Swellings

Dipeptide repeat proteins (DPRs) are aggregation-prone polypeptides encoded by the pathogenic G4C2 repeat expansion in the C9orf72 gene, the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia (ALS/FTD). In this study, we focus on the role of poly-GA DPRs in disease spread. We demonstrate that recombinant poly-GA oligomers can directly convert into solid-like aggregates and form characteristic β-sheet fibrils in vitro. To dissect the process of cell-to-cell DPR transmission, we closely follow the fate of poly-GA DPRs in either their oligomeric or fibrillized form after administration in the cell culture medium. We observe that poly-GA DPRs are taken up via dynamin-dependent and -independent endocytosis, eventually converging at the lysosomal compartment and leading to axonal swellings in neurons. We then use a co-culture system to demonstrate astrocyte-to-motor neuron DPR propagation, showing that astrocytes may internalise and release aberrant peptides in disease pathogenesis. Overall, our results shed light on the mechanisms of poly-GA cellular uptake and cell-to-cell propagation, suggesting lysosomal impairment as a possible feature underlying the cellular pathogenicity of these DPR species.

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Transcription elongation factor AFF2/FMR2 regulates expression of expanded GGGGCC repeat-containing C9ORF72 allele in ALS/FTD

Nature Communications ◽

10.1038/s41467-019-13477-8 ◽

2019 ◽

Vol 10 (1) ◽

Cited By ~ 12

Author(s):

Yeliz Yuva-Aydemir ◽

Sandra Almeida ◽

Gopinath Krishnan ◽

Tania F. Gendron ◽

Fen-Biao Gao

Keyword(s):

Cortical Neurons ◽

Elongation Factor ◽

Premature Death ◽

Partial Loss ◽

Repeat Proteins ◽

Rna Foci ◽

Human Neurons ◽

Induced Pluripotent ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractExpanded GGGGCC (G4C2) repeats in C9ORF72 cause amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). How RNAs containing expanded G4C2 repeats are transcribed in human neurons is largely unknown. Here we describe a Drosophila model in which poly(GR) expression in adult neurons causes axonal and locomotor defects and premature death without apparent TDP-43 pathology. In an unbiased genetic screen, partial loss of Lilliputian (Lilli) activity strongly suppresses poly(GR) toxicity by specifically downregulating the transcription of GC-rich sequences in Drosophila. Knockout of AFF2/FMR2 (one of four mammalian homologues of Lilli) with CRISPR-Cas9 decreases the expression of the mutant C9ORF72 allele containing expanded G4C2 repeats and the levels of repeat RNA foci and dipeptide repeat proteins in cortical neurons derived from induced pluripotent stem cells of C9ORF72 patients, resulting in rescue of axonal degeneration and TDP-43 pathology. Thus, AFF2/FMR2 regulates the transcription and toxicity of expanded G4C2 repeats in human C9ORF72-ALS/FTD neurons.

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Hippocampal protein aggregation signatures fully distinguish pathogenic and wildtype UBQLN2 in amyotrophic lateral sclerosis

10.1101/2022.01.12.475792 ◽

2022 ◽

Author(s):

Kyrah M Thumbadoo ◽

Birger V Dieriks ◽

Helen C Murray ◽

Molly EV Swanson ◽

Ji Hun Yoo ◽

...

Keyword(s):

Amyotrophic Lateral Sclerosis ◽

Protein Aggregation ◽

Gene Mutations ◽

Repeat Proteins ◽

Familial Als ◽

Fluorescent Immunohistochemistry ◽

Self Aggregation ◽

Lateral Sclerosis ◽

Dipeptide Repeat

Mutations in the UBQLN2 gene cause X-linked dominant amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD) characterised by ubiquilin 2 aggregates in neurons of the motor cortex, hippocampus, cerebellum, and spinal cord. However, ubiquilin 2 neuropathology is also seen in sporadic and familial ALS or FTD cases not caused by UBQLN2 mutations, particularly C9ORF72-linked cases. This makes the mechanistic role of ubiquilin 2 mutations and the value of ubiquilin 2 pathology for predicting genotype unclear. Here we examine a cohort of 31 genotypically diverse ALS cases with or without FTD, including four cases with UBQLN2 mutations (resulting in p.P497H, p.P506S, and two cases with p.T487I). Using double-, triple-, and six-label fluorescent immunohistochemistry, we mapped the co-localisation of ubiquilin 2 with phosphorylated TDP-43 (pTDP-43), dipeptide repeat aggregates, and p62, in the hippocampus of controls (n=5), or ALS with or without FTD in sporadic (n=19), unknown familial (n=3), SOD1-linked (n=1), C9ORF72-linked (n=4), and UBQLN2-linked (n=4) cases. We differentiate between i) ubiquilin 2 aggregation together with, or driven by, pTDP-43 or dipeptide repeat proteins, and ii) ubiquilin 2 self-aggregation driven by UBQLN2 gene mutations. Together we describe a hippocampal protein aggregation signature that fully distinguishes mutant from wildtype ubiquilin 2 in ALS with or without FTD, whereby mutant ubiquilin 2 is more prone than wildtype to aggregate independently of driving factors. This neuropathological signature can be used to assess the pathogenicity of UBQLN2 gene mutations and to understand the mechanisms of UBQLN2-linked disease.

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The role of DNA damage response in amyotrophic lateral sclerosis

Essays in Biochemistry ◽

10.1042/ebc20200002 ◽

2020 ◽

Vol 64 (5) ◽

pp. 847-861 ◽

Cited By ~ 1

Author(s):

Yu Sun ◽

Annabel J. Curle ◽

Arshad M. Haider ◽

Gabriel Balmus

Keyword(s):

Dna Damage ◽

Amyotrophic Lateral Sclerosis ◽

Genomic Instability ◽

Dna Damage Response ◽

Disease Mechanisms ◽

Age Related ◽

Sporadic Als ◽

Damage Response ◽

Lateral Sclerosis

Abstract Amyotrophic lateral sclerosis (ALS) is a rapidly disabling and fatal neurodegenerative disease. Due to insufficient disease-modifying treatments, there is an unmet and urgent need for elucidating disease mechanisms that occur early and represent common triggers in both familial and sporadic ALS. Emerging evidence suggests that impaired DNA damage response contributes to age-related somatic accumulation of genomic instability and can trigger or accelerate ALS pathological manifestations. In this review, we summarize and discuss recent studies indicating a direct link between DNA damage response and ALS. Further mechanistic understanding of the role genomic instability is playing in ALS disease pathophysiology will be critical for discovering new therapeutic avenues.

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

Nature Communications ◽

10.1038/s41467-021-21112-8 ◽

2021 ◽

Vol 12 (1) ◽

Author(s):

Yuanjing Liu ◽

Jean-Cosme Dodart ◽

Helene Tran ◽

Shaunna Berkovitch ◽

Maurine Braun ◽

...

Keyword(s):

Motor Neurons ◽

Neuronal Degeneration ◽

Repeat Expansion ◽

Preclinical Models ◽

C9orf72 Repeat Expansion ◽

Cellular Models ◽

Rna Foci ◽

Expansion Mutation ◽

Lateral Sclerosis ◽

Dipeptide Repeat

AbstractA 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.

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