. 2021 Sep 7;21(4):681–714. doi: 10.1007/s12311-021-01320-0

Table 4.

Publications retrieved concerning C9orf72-related pathology

Study	Methodology	Main findings
Atanasio et al. [97]	C9orf72 haploinsufficiency model (C9orf72^−/−) generated by replacing mouse C9orf72 coding sequence and introns with lacZ reporter Behavioural and clinical examination of motor function; H&E and IHC analysis; RNA-seq	• C9orf72^−/− mice showed mild motor dysfunction including progressive muscle weakness observed at 40 weeks of age, tremor, rigidity and reduced locomotor behaviour • Enlargement of cervical lymph nodes and splenomegaly was observed, as well as inflammatory infiltrates in multiple organs • Serum levels of cytokines were elevated • Enrichment of immune-related transcripts, indicative of systemic immune response, was detected in C9orf72^−/− mice • C9orf72^−/− mice exhibited autoimmunity and age-related proliferative glomerulonephropathy
Darling et al. [78]	NSC34 cells transfected with DPR-containing plasmids; primary mouse neurons IF; Western blot; isothermal titration calorimetry (ITC); circular dichroism (CD) spectroscopy; fluorescence spectroscopy; dynamic light scattering (DLS); nanoparticle tracking analysis; transmission electron microscopy (TEM)	• Co-expression of DPRs results in altered cellular outcomes as compared to expression of single DPRs, suggesting that complex interaction occurring between individual DPRs can change their intrinsic properties and toxicity • Dual expression of poly-PR and -GA resulted in altered subcellular localization, morphology and amelioration of -PR-induced cytotoxicity
DeJesus-Hernandez et al. [1]	Patients with a C9orf72 expansion mutation; post-mortem patient tissue PCR and qPCR, g/cDNA sequencing, western blotting, IHC and FISH	• First report of the C9orf72 expansion as a cause of ALS/FTD • The most common genetic cause of FTD (11.7%) and ALS (22.5%) • Expansion in C9orf72 led to nuclear foci formation in patients in the frontal cortex and spinal cord
Farg et al. [102]	Neuronal cell lines — murine neuro2a and human SH-SY5Y transfected with C9orf72 siRNA; primary cortical neurons of C57B1/6 mice; post-mortem spinal cord tissue from C9orf72 patients IF and immunoblotting to investigate subcellular localisation of C9orf72 protein; ICC, IHC and immunoprecipitation to detect co-localisation of C9 protein with Rabs; siRNA transfection of SH-SY5Y cells; transfection of neuro2a cell lines with C9orf72-GFP and LC3; mass spectroscopy to identify C9-interacting proteins	• Investigated cellular function and subcellular expression of C9orf72 protein • Evidence for C9orf72 involvement in intracellular trafficking and protein degradation • Demonstrated co-localisation of C9orf72 with Rab proteins — involved in autophagy and endosomal trafficking — in cell lines, mouse primary neurons and spinal cord of C9orf72 patients • siRNA-induced depletion of C9 protein in transfected cells inhibited endocytosis • Ubiquilin-2, hnRNPA1 and hnRNPA2/B1 were shown to interact with endogenous C9orf72 in vitro • Inhibition of the proteasome promoted co-localization of C9orf72 and ubiqilin-2 in neuro2a cells treated with lactacystin • Inhibition of the proteasome in neuro2a cells transfected with C9orf72-GFP constructs induced the formation of stress granules and C9-aggregates
Koppers et al. [98]	Conditional C9orf72 knockout mouse model — C9orf72^fl/fl mice crossed with Nestin-Cre mice to selectively ablate expression of C9orf72 from neurons and glial cells IHC analysis of motor neurons and neuromuscular junction integrity, gliosis and TDP-43 inclusions; FISH, Western blotting; motor function testing	• Conditional knockout of C9orf72 gene resulted in significant loss of body weight in mice but was not sufficient to reduce survival, induce neurodegeneration or affect motor function • No inflammatory responses or other pathological hallmarks of C9-ALS/FTD were detected in those mice
Lopez-Gonzalez et al. [84]	Transgenic Drosophila models including Vg-Gal4-GR_80, UAS-(G₄C₂)₅₈, GMR-Gal5, UAS-(G₄C₂)₅₈/TM6B and Tb lines; CRISPR-Cas9-edited iPSCs lines generated from C9orf72 patients Genetic modifier screen; Drosophila eye phenotyping; ELISA; immunoblotting; generation of lentiviral particles expression Ku80 shRNA and sdRNA	• A genetic modifier screen using transgenic Drosophila as a model identified 19 genes whose partial loss of function suppressed poly-GR toxicity, one of which was Ku80, a key DNA repair protein • The levels of Ku80 expression were markedly elevated in poly-GR expressing flies and C9orf72 iPSC-derived neurons • This was associated with increased levels of P53, phosphorylated ATM and apoptotic markers in C9orf72 patient neurons • Partial loss of function of Ku80 suppressed poly-GR-induced neuronal cell death in poly-GR expressing flies • CRISPR-Cas9-mediated deletion of G₄C₂ expansion repeats prevented elevation of Ku80 expression and downstream apoptotic markers • Small RNAs-mediated knockdown and CRISPR-Cas9-mediated ablation of Ku80 resulted in suppression of the apoptotic pathway
Mehta et al. [110]	C9orf72 patient tissue BaseScope™ ISH	• BaseScope is a highly sensitive form of in situ hybridisation that improves signal and detection of RNA foci • Sense foci are associated with TDP-43 aggregation in spinal cord motor neurons but not spinal cord glia or indeed the motor cortical neurons • No correlation between TDP-43 and foci in areas outside of motor control was seen
Mizielinska et al. [64]	Drosophila generated to express DPRs under UAS promotor with 36 or 103 repeats; neuronal transfection with poly-GR₁₀₀ and -PR₁₀₀ Northern blotting, FISH, Immunoblotting, egg-adult viability, eye phenotyping, lifespan assay	• Gene expression of DPRs was switched on post-development in flies and caused fly death within 30 days. Reducing protein output of DPRs attenuated the lifespan reduction • Protein only poly-GR and -PR were compared to poly-GA and -PA with the former causing lethality and the latter having no effect on the fly • Neuron viability was reduced upon transfection with poly-GR₁₀₀ and poly-PR₁₀₀ • Poly-GA inclusion was a poor predictor of neurodegeneration
Mori et al. [85]	C9orf72 patient tissue Filter trap assay; immunoblotting; RT-PCR; qPCR; IHC; IF	• Discovered that most of the TDP-43-negative inclusions characteristic of C9-ALS/FTD contain predominantly poly-GA proteins and to a lesser extent poly-GP and poly-GA
Mori et al. [59]	HEK293 cells transfected with plasmids containing 7, 17 or 23 repeats of G₄C₂; in vitro transcription of RNA probes; post-mortem tissue from C9orf72 cases LC–MS identification and quantification of proteins; Western blotting; IF and IHC	• 20 RNA-binding proteins were identified as capable of binding to G₄C₂ repeats in vitro • Out of those, a few were selected to be further validated in post-mortem brain tissue of C9orf72 cases including hnRNP A3 which was found to form neuronal cytoplasmic and intranuclear inclusions in the hippocampus • hnRNP A3-positive inclusions were of the p62-positive/TDP-43-negative type
Rudich et al. [72]	C. elegans model expressing 50 repeats of poly-GA, -PA, -GR or -PR DPRs Detection of DPRs by fluorescence microscopy; FRAP studies; paralysis and thrashing assays; brood size assays; neurodegeneration assays	• Expression of arginine-rich, but not alanine-rich DPRs induced toxicity in neuronal and non-neuronal contexts • This poly-PR and -GR-induced toxicity was dependent on the nuclear localization of the DPRs • The toxicity of -PR and -GR was found to be age dependent • Uncoupling of physiological aging from chronological aging ameliorated -PR but not -GR toxicity
Schludi et al. [32]	Transfected rat primary neuronal cultures expressing GA₁₇₅, GR_149, GP₈₀ or PR₁₇₅; post-mortem brain and spinal cord tissue from C9orf72 mutation patients IHC; RNA FISH; quantitative analysis of inclusion pathology	• Overexpression of poly-GA induced formation of p62-positive neuronal cytoplasmic inclusions in rat primary neurons • Overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions • In C9-ALS patient tissue, neuronal inclusions of poly-GR, -GP and -GA co-localised with Unc119 • The authors noticed a correlation between the abundance of poly-GA and Unc119 inclusions and the diagnosis of FTLD vs MND
Sudria-Lopez et al. [100]	C9orf72 knockout mouse model with full ablation of C9orf72 in all tissues Histopathological analysis	• C9orf72 knockout mice exhibited decreased body weight, enlarged lymph nodes and splenomegaly • Multiple organs in those mice contained macrophage and lymphocyte infiltrates • Neoplastic events were also reported • No evidence of motor neuron degeneration, gliosis or TDP-43 inclusions
Wen et al. [73]	Rat primary cortical and motor neuron cultures transfected with PR₅₀ cDNA and C9orf72 shRNA constructs; transgenic Drosophila model expressing poly-PR₅₀; iPSC-derived neurons transfected with PR₅₀; spinal cord tissue from C9-ALS/FTD patients IHC; quantification of nucleoli and P-bodies	• Due to its intrinsic properties including aggregation in the nucleolus, formation of stress granules and reduction in the number of processing bodies, poly-PR was found to be the most toxic DPR • Nuclear aggregates of poly-PR were found in an iPSC-derived motor neuron from C9-ALS/FTD patients and post-mortem spinal cord tissue of patients
Yamakawa et al. [75]	Synthetic cDNA encoding 100 repeats of poly-GA, poly-GP, poly-GR, poly-PR and poly-PA, without G₄C₂ repeats, was used to study the effects of those DPRs on transfected cultured neuronal cell lines (HeLa and HEK293) in vitro and mouse cortical neurons IHC, IF and immunoblot detection and characterisation of DPRs in cells, in utero electroporation, IHC of brain slices	• Out of the five DPRs, poly-GA was found to have the highest tendency to form aggregates and inclusions, in a poly-GA repeat length-dependent manner, in cultured neuronal lines • Poly-GA inclusions were p62 and ubiquitin positive but negative for TDP-43 • Poly-GR and poly-PR formed ubiquitin- p62-negative cytoplasmic inclusions co-localised with TDP-43 • Overexpression of poly-GA, -GP and -GR caused dysregulation of the cellular ubiquitin–proteasome system, which is crucial to protein homeostasis
Zhang et al. [43]	Drosophila expressing 30 G₄C₂ repeats; S2 cells expressing Drosophila RanGAP protein; C9-ALS patient-derived iPSNs; post-mortem brain tissue from C9orf72 patients Western blot; electrophysiological recording; RNA FISH; IF, IHC and Phalloidin staining; FRAP analysis; electrophoretic mobility shift assays	• Candidate-based genetic screen, in Drosophila expressing (G₄C₂)₃₀ repeats, identified RanGAP (orthologue of human RanGAP1 — regulator of nucleocytoplasmic transport) as a potential suppressor of C9-mediated neurodegeneration • RanGAP was found to interact with G₄C₂ RNA and mislocalise in (G₄C₂)₃₀ Drosophila, iPSNs and brains of C9-ALS cases • G₄C₂ repeat expansion induced impairment of nuclear import in the fly model and C9-ALS patient-derived iPSNs • Small molecules and antisense oligonucleotides targeting G₄C₂ repeat expansion G-quadruplex rescued deficits in nuclear import
Zhang et al. [33]	Transgenic mouse model expressing 50 repeats of poly-GA by means of AAV1 viral injection; primary neuronal cultures; HEK293T cells transfected with GFP-(GA)₅₀; post-mortem tissue from C9-ALS cases IHC; IF; immunoelectron microscopy; quantification of neuropathology; silver staining; RT-qPCR; co-immunoprecipitation; immunoblotting; mouse behavioural testing	• Poly-GA toxicity was accompanied by behavioural abnormalities and neurodegeneration in mice expressing (GA)₅₀ • Aggregation of poly-GA was required for the manifestation of phenotypes resembling C9-ALS pathology in these mice • Poly-GA was found to sequester HR23 proteins which are involved in proteasomal degradation in (GA)₅₀ mice • HR23A and HR23B co-localised with poly-GA inclusions in post-mortem tissue of C9-ALS cases • Aggregation of poly-GA and poly-GA-induced toxicity were attenuated in neuronal cultures when HR23B levels were restored
Zu et al. [12]	HEK293T cells transfected with antisense (G-₂C₄)_40/50; C9orf72 patient tissue IF, IHC, FISH, Western blot, protein dot blot, cell toxicity and viability assays, RT-PCR	• Antisense transcripts of C9orf72 are increased in C9orf72 patients and accumulate into antisense foci • Sense and antisense foci were detectable in the blood acting as a potential biomarker • DPRs can also present as antisense giving rise to poly-PR, -PA and -GP. Poly-GP is a repeat as it encoded in the sense direction as well. These accumulate in the frontal and motor cortices as well as the spinal cord and hippocampus

Study

Methodology

Main findings

Atanasio et al. [97]

C9orf72 haploinsufficiency model (C9orf72^−/−) generated by replacing mouse C9orf72 coding sequence and introns with lacZ reporter

Behavioural and clinical examination of motor function; H&E and IHC analysis; RNA-seq

• C9orf72^−/− mice showed mild motor dysfunction including progressive muscle weakness observed at 40 weeks of age, tremor, rigidity and reduced locomotor behaviour

• Enlargement of cervical lymph nodes and splenomegaly was observed, as well as inflammatory infiltrates in multiple organs

• Serum levels of cytokines were elevated

• Enrichment of immune-related transcripts, indicative of systemic immune response, was detected in C9orf72^−/− mice

• C9orf72^−/− mice exhibited autoimmunity and age-related proliferative glomerulonephropathy

Darling et al. [78]

NSC34 cells transfected with DPR-containing plasmids; primary mouse neurons

IF; Western blot; isothermal titration calorimetry (ITC); circular dichroism (CD) spectroscopy; fluorescence spectroscopy; dynamic light scattering (DLS); nanoparticle tracking analysis; transmission electron microscopy (TEM)

• Co-expression of DPRs results in altered cellular outcomes as compared to expression of single DPRs, suggesting that complex interaction occurring between individual DPRs can change their intrinsic properties and toxicity

• Dual expression of poly-PR and -GA resulted in altered subcellular localization, morphology and amelioration of -PR-induced cytotoxicity

DeJesus-Hernandez et al. [1]

Patients with a C9orf72 expansion mutation; post-mortem patient tissue

PCR and qPCR, g/cDNA sequencing, western blotting, IHC and FISH

• First report of the C9orf72 expansion as a cause of ALS/FTD

• The most common genetic cause of FTD (11.7%) and ALS (22.5%)

• Expansion in C9orf72 led to nuclear foci formation in patients in the frontal cortex and spinal cord

Farg et al. [102]

Neuronal cell lines — murine neuro2a and human SH-SY5Y transfected with C9orf72 siRNA; primary cortical neurons of C57B1/6 mice; post-mortem spinal cord tissue from C9orf72 patients

IF and immunoblotting to investigate subcellular localisation of C9orf72 protein; ICC, IHC and immunoprecipitation to detect co-localisation of C9 protein with Rabs; siRNA transfection of SH-SY5Y cells; transfection of neuro2a cell lines with C9orf72-GFP and LC3; mass spectroscopy to identify C9-interacting proteins

• Investigated cellular function and subcellular expression of C9orf72 protein

• Evidence for C9orf72 involvement in intracellular trafficking and protein degradation

• Demonstrated co-localisation of C9orf72 with Rab proteins — involved in autophagy and endosomal trafficking — in cell lines, mouse primary neurons and spinal cord of C9orf72 patients

• siRNA-induced depletion of C9 protein in transfected cells inhibited endocytosis

• Ubiquilin-2, hnRNPA1 and hnRNPA2/B1 were shown to interact with endogenous C9orf72 in vitro

• Inhibition of the proteasome promoted co-localization of C9orf72 and ubiqilin-2 in neuro2a cells treated with lactacystin

• Inhibition of the proteasome in neuro2a cells transfected with C9orf72-GFP constructs induced the formation of stress granules and C9-aggregates

Koppers et al. [98]

Conditional C9orf72 knockout mouse model — C9orf72^fl/fl mice crossed with Nestin-Cre mice to selectively ablate expression of C9orf72 from neurons and glial cells

IHC analysis of motor neurons and neuromuscular junction integrity, gliosis and TDP-43 inclusions; FISH, Western blotting; motor function testing

• Conditional knockout of C9orf72 gene resulted in significant loss of body weight in mice but was not sufficient to reduce survival, induce neurodegeneration or affect motor function

• No inflammatory responses or other pathological hallmarks of C9-ALS/FTD were detected in those mice

Lopez-Gonzalez et al. [84]

Transgenic Drosophila models including Vg-Gal4-GR_80, UAS-(G₄C₂)₅₈, GMR-Gal5, UAS-(G₄C₂)₅₈/TM6B and Tb lines; CRISPR-Cas9-edited iPSCs lines generated from C9orf72 patients

Genetic modifier screen; Drosophila eye phenotyping; ELISA; immunoblotting; generation of lentiviral particles expression Ku80 shRNA and sdRNA

• A genetic modifier screen using transgenic Drosophila as a model identified 19 genes whose partial loss of function suppressed poly-GR toxicity, one of which was Ku80, a key DNA repair protein

• The levels of Ku80 expression were markedly elevated in poly-GR expressing flies and C9orf72 iPSC-derived neurons

• This was associated with increased levels of P53, phosphorylated ATM and apoptotic markers in C9orf72 patient neurons

• Partial loss of function of Ku80 suppressed poly-GR-induced neuronal cell death in poly-GR expressing flies

• CRISPR-Cas9-mediated deletion of G₄C₂ expansion repeats prevented elevation of Ku80 expression and downstream apoptotic markers

• Small RNAs-mediated knockdown and CRISPR-Cas9-mediated ablation of Ku80 resulted in suppression of the apoptotic pathway

Mehta et al. [110]

C9orf72 patient tissue

BaseScope™ ISH

• BaseScope is a highly sensitive form of in situ hybridisation that improves signal and detection of RNA foci

• Sense foci are associated with TDP-43 aggregation in spinal cord motor neurons but not spinal cord glia or indeed the motor cortical neurons

• No correlation between TDP-43 and foci in areas outside of motor control was seen

Mizielinska et al. [64]

Drosophila generated to express DPRs under UAS promotor with 36 or 103 repeats; neuronal transfection with poly-GR₁₀₀ and -PR₁₀₀

Northern blotting, FISH, Immunoblotting, egg-adult viability, eye phenotyping, lifespan assay

• Gene expression of DPRs was switched on post-development in flies and caused fly death within 30 days. Reducing protein output of DPRs attenuated the lifespan reduction

• Protein only poly-GR and -PR were compared to poly-GA and -PA with the former causing lethality and the latter having no effect on the fly

• Neuron viability was reduced upon transfection with poly-GR₁₀₀ and poly-PR₁₀₀

• Poly-GA inclusion was a poor predictor of neurodegeneration

Mori et al. [85]

C9orf72 patient tissue

Filter trap assay; immunoblotting; RT-PCR; qPCR; IHC; IF

• Discovered that most of the TDP-43-negative inclusions characteristic of C9-ALS/FTD contain predominantly poly-GA proteins and to a lesser extent poly-GP and poly-GA

Mori et al. [59]

HEK293 cells transfected with plasmids containing 7, 17 or 23 repeats of G₄C₂; in vitro transcription of RNA probes; post-mortem tissue from C9orf72 cases

LC–MS identification and quantification of proteins; Western blotting; IF and IHC

• 20 RNA-binding proteins were identified as capable of binding to G₄C₂ repeats in vitro

• Out of those, a few were selected to be further validated in post-mortem brain tissue of C9orf72 cases including hnRNP A3 which was found to form neuronal cytoplasmic and intranuclear inclusions in the hippocampus

• hnRNP A3-positive inclusions were of the p62-positive/TDP-43-negative type

Rudich et al. [72]

C. elegans model expressing 50 repeats of poly-GA, -PA, -GR or -PR DPRs

Detection of DPRs by fluorescence microscopy; FRAP studies; paralysis and thrashing assays; brood size assays; neurodegeneration assays

• Expression of arginine-rich, but not alanine-rich DPRs induced toxicity in neuronal and non-neuronal contexts

• This poly-PR and -GR-induced toxicity was dependent on the nuclear localization of the DPRs

• The toxicity of -PR and -GR was found to be age dependent

• Uncoupling of physiological aging from chronological aging ameliorated -PR but not -GR toxicity

Schludi et al. [32]

Transfected rat primary neuronal cultures expressing GA₁₇₅, GR_149, GP₈₀ or PR₁₇₅; post-mortem brain and spinal cord tissue from C9orf72 mutation patients

IHC; RNA FISH; quantitative analysis of inclusion pathology

• Overexpression of poly-GA induced formation of p62-positive neuronal cytoplasmic inclusions in rat primary neurons

• Overexpressed poly-GR and poly-PR formed nucleolar p62-negative inclusions

• In C9-ALS patient tissue, neuronal inclusions of poly-GR, -GP and -GA co-localised with Unc119

• The authors noticed a correlation between the abundance of poly-GA and Unc119 inclusions and the diagnosis of FTLD vs MND

Sudria-Lopez et al. [100]

C9orf72 knockout mouse model with full ablation of C9orf72 in all tissues

Histopathological analysis

• C9orf72 knockout mice exhibited decreased body weight, enlarged lymph nodes and splenomegaly

• Multiple organs in those mice contained macrophage and lymphocyte infiltrates

• Neoplastic events were also reported

• No evidence of motor neuron degeneration, gliosis or TDP-43 inclusions

Wen et al. [73]

Rat primary cortical and motor neuron cultures transfected with PR₅₀ cDNA and C9orf72 shRNA constructs; transgenic Drosophila model expressing poly-PR₅₀; iPSC-derived neurons transfected with PR₅₀; spinal cord tissue from C9-ALS/FTD patients

IHC; quantification of nucleoli and P-bodies

• Due to its intrinsic properties including aggregation in the nucleolus, formation of stress granules and reduction in the number of processing bodies, poly-PR was found to be the most toxic DPR

• Nuclear aggregates of poly-PR were found in an iPSC-derived motor neuron from C9-ALS/FTD patients and post-mortem spinal cord tissue of patients

Yamakawa et al. [75]

Synthetic cDNA encoding 100 repeats of poly-GA, poly-GP, poly-GR, poly-PR and poly-PA, without G₄C₂ repeats, was used to study the effects of those DPRs on transfected cultured neuronal cell lines (HeLa and HEK293) in vitro and mouse cortical neurons

IHC, IF and immunoblot detection and characterisation of DPRs in cells, in utero electroporation, IHC of brain slices

• Out of the five DPRs, poly-GA was found to have the highest tendency to form aggregates and inclusions, in a poly-GA repeat length-dependent manner, in cultured neuronal lines

• Poly-GA inclusions were p62 and ubiquitin positive but negative for TDP-43

• Poly-GR and poly-PR formed ubiquitin- p62-negative cytoplasmic inclusions co-localised with TDP-43

• Overexpression of poly-GA, -GP and -GR caused dysregulation of the cellular ubiquitin–proteasome system, which is crucial to protein homeostasis

Zhang et al. [43]

Drosophila expressing 30 G₄C₂ repeats; S2 cells expressing Drosophila RanGAP protein; C9-ALS patient-derived iPSNs; post-mortem brain tissue from C9orf72 patients

Western blot; electrophysiological recording; RNA FISH; IF, IHC and Phalloidin staining; FRAP analysis; electrophoretic mobility shift assays

• Candidate-based genetic screen, in Drosophila expressing (G₄C₂)₃₀ repeats, identified RanGAP (orthologue of human RanGAP1 — regulator of nucleocytoplasmic transport) as a potential suppressor of C9-mediated neurodegeneration

• RanGAP was found to interact with G₄C₂ RNA and mislocalise in (G₄C₂)₃₀ Drosophila, iPSNs and brains of C9-ALS cases

• G₄C₂ repeat expansion induced impairment of nuclear import in the fly model and C9-ALS patient-derived iPSNs

• Small molecules and antisense oligonucleotides targeting G₄C₂ repeat expansion G-quadruplex rescued deficits in nuclear import

Zhang et al. [33]

Transgenic mouse model expressing 50 repeats of poly-GA by means of AAV1 viral injection; primary neuronal cultures; HEK293T cells transfected with GFP-(GA)₅₀;

post-mortem tissue from C9-ALS cases

IHC; IF; immunoelectron microscopy; quantification of neuropathology; silver staining; RT-qPCR; co-immunoprecipitation; immunoblotting; mouse behavioural testing

• Poly-GA toxicity was accompanied by behavioural abnormalities and neurodegeneration in mice expressing (GA)₅₀

• Aggregation of poly-GA was required for the manifestation of phenotypes resembling C9-ALS pathology in these mice

• Poly-GA was found to sequester HR23 proteins which are involved in proteasomal degradation in (GA)₅₀ mice

• HR23A and HR23B co-localised with poly-GA inclusions in post-mortem tissue of C9-ALS cases

• Aggregation of poly-GA and poly-GA-induced toxicity were attenuated in neuronal cultures when HR23B levels were restored

Zu et al. [12]

HEK293T cells transfected with antisense (G-₂C₄)_40/50; C9orf72 patient tissue

IF, IHC, FISH, Western blot, protein dot blot, cell toxicity and viability assays, RT-PCR

• Antisense transcripts of C9orf72 are increased in C9orf72 patients and accumulate into antisense foci

• Sense and antisense foci were detectable in the blood acting as a potential biomarker

• DPRs can also present as antisense giving rise to poly-PR, -PA and -GP. Poly-GP is a repeat as it encoded in the sense direction as well. These accumulate in the frontal and motor cortices as well as the spinal cord and hippocampus

Abbreviations: ALS, amyotrophic lateral sclerosis; ATM, ataxia telangiectasia mutated; Cas9, CRISPR-associated 9; cDNA, circular DNA; CRISPR, clustered regularly interspaced short palindromic repeats; DPR, dipeptide repeat; ELISA, enzyme-linked immunosorbent assays; FISH, fluorescent in-situ hybridisation; FRAP, fluorescence recovery after photobleaching; FTD, frontotemporal dementia; Gal4, galactose 4; GFP, green fluorescent protein; GMR, glass multiple reporter; H&E, haematoxylin and eosin; HEK293, human embryonic kidney cells; hnRNP, heterogenous nuclear ribonucleoproteins; HR23, UV excision repair protein RAD23 homolog B; ICC, immunocytochemistry; IF, immunofluorescence; IHC, immunohistochemistry; iPSC, induced pluripotent cells; ISH, in situ hybridisation; Ku80, Lupus Ku autoantigen protein p80; lacZ, lactose operon Z; LC3, microtubule-associated proteins 1A/1B light chain 3B; LC–MS, liquid chromatography mass spectrometry; MND, motor neuron disease; NSC-34, motor neuron-like hybrid line; p53, tumour protein 53; p62, ubiquitin-binding protein; PCR, polymerase chain reaction; qPCR, quantitative polymerase chain reaction; Rab, ras-associated binding protein; RNA, ribonucleic acid; RNP, ribonucleoprotein; RT-PCR, real-time PCR; sdRNA, small self-deliverable interference RNA; shRNA, short hairpin RNA; siRNA, small interfering RNA; TDP-43, tar-DNA binding protein 43; TM6B, third chromosome balancer; UAS, upstream activation sequence; Unc119, uncoordinated 119