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

Table 3.

Publications retrieved concerning findings in the cerebellum

Study	Methodology	Main findings
Al-Sarraj et al. [25]	Post-mortem tissue from MND/ALS, FTLD and ALS-FTLD cases with, and FTLD cases without C9 expansion mutation Neuropathological assessment, IHC and IF studies	• p62 positive, p-TDP-43 negative inclusions were reported in cerebellar Purkinje cells and molecular layer of C9-positive cases
Ash et al. [10]	Human C9orf72 tissue Meso scale discovery assay, IHC, IF; generation of novel antibodies to poly-GA, GP and -GR	• C9orf72 RAN translation products (now called DPRs) were detected in high molecular weight aggregates specific to C9orf72 expansion and not other neurodegenerative or CAG expansion disorders • DPRs were abundant in the cerebellum of C9orf72 cases
Belzil et al. [103]	C9orf72 patient tissue, skin biopsies and blood samples with derivation of fibroblasts qRT-PCR, ddPCR and chromatin immunoprecipitation	• Both the frontal cortices and cerebellum had reductions in C9orf72 mRNA in pathogenic expansions • C9orf72 with expansions increases the rate of binding to trimethylated lysine residues within histones H3 (H3K9/K27/K79) and H4 (H4K20) which is detectable in the blood of patients
Chew et al. [94]	Transgenic mouse model expressing (G₄C₂)₆₆ throughout the CNS by means of AAV-mediated somatic brain transgenesis Behavioural testing; RNA FISH detection of RNA foci, immunoassays for DPRs	• RNA foci were abundant in Purkinje cells of (G₄C₂)₆₆ mice at 6 months, with 40–54% of Purkinje cells foci-positive, and to a lesser degree, in granular and molecular layers of cerebellum • Poly-GA, poly-GP and poly-GR inclusions were detected in the cerebellum, accompanied by a loss (11% decrease) of Purkinje cells in (G₄C₂)₆₆ mice • A decrease in overall brain weight, indicative of repeat expansion-mediated atrophy, and behavioural/ motor skill abnormalities were also reported
Cooper-Knock et al. [109]	Samples from ALS/FTD cases with and without C9 expansion mutation, healthy controls and asymptomatic C9 carriers Identification of nuclear and cytoplasmic RNA foci using FISH, investigation of RNA foci binding partners using mass spectrometry, pulldown assays and IHC	• RNA foci were found to be abundant in the cerebellar granule cells of C9-ALS/FTD patients and absent in cases without C9 mutation and healthy controls (although the levels varied based on clinical presentation) • A number of putative binding partners of RNA foci were identified, including hnRNP, which was shown to co-localise with RNA foci in cerebellar granule cells
Cooper-Knock et al. [112]	C9orf72 patient tissue IHC, FISH, RNA-binding assays	• Antisense RNA foci are predominantly present in the cerebellar Purkinje cells and motor neurons. In motor neurons, antisense foci (and not sense) correlated with TDP-43 pathology • Sense RNA foci localised predominantly to the granular cells of the cerebellum • DPRs were present with the greatest frequency in granular cells of the cerebellum and then the motor neurons of the spinal cord • Antisense RNA foci co-localised with SRSF2, hnRNP A1, hnRNP A/F, ALYREF and hnRNP K
Corcia et al. [60]	C9orf72 mutation carrier presenting with pure cerebellar syndrome Case study report	• Female with family history of ALS and other neurodegenerative disorders presents with symptoms of pure cerebellar syndrome — locomotor disturbance, cerebellar vermis atrophy and no cognitive dysfunction, and is subsequently found to be a carrier of C9orf72 mutation • Demonstrating a link between C9 mutation and cerebellar pathology
Davidson et al. [89]	Post-mortem tissue from FTLD-tau, FTLD-TDP type A, B and C, and C9orf72 expansion, MAPT or GRN mutation carriers, and healthy controls IHC staining and pathological assessment	• Investigated the patter of hnRNP A1, A2/B1 and A3 staining across brain regions in various subtypes of FTLD cases, including carriers of C9orf72 mutation • No difference in the intensity and staining pattern of hnRNP A1 or hnRNP A2/B1 was observed in C9orf72-positive cases compared to other FTLD cases, with no hnRNP A1 or A2/B1 immunoreactive inclusions reported • hnRNP A3-positive inclusions were seen in cerebellum of some C9orf72 cases
Davidson et al. [105]	C9orf72 patient tissue and other disorders (Huntington’s/FTD-TDP and Alzheimer’s) IHC, antibody comparison	• Confirming previous studies, C9-L antibodies labelling diffuse cytoplasmic staining and speckles in the Purkinje neurons of the cerebellum • Additionally, C9-S labelled the nuclear membrane only • ProteinTech’s monoclonal and polyclonal antibody as well as GeneTex achieved similar staining to that of C9-L • Nuclear membrane staining of C9-S was shifted to the plasma membrane in the spinal cord of sufferers • Commercial antibodies were unable to recapitulate C9-S staining
DeJesus-Hernandez et al. [37]	Cerebellum and frontal cortex tissue of C9 expansion mutation carriers Detection and visualisation of RNA foci by RNA FISH and IF staining followed by computer-assisted quantification and co-localisation	• RNA foci were abundant in the cerebellum of C9 carriers, with 23% and 1% of granule cells containing sense and antisense RNA foci, respectively • RNA foci were most abundantly present in cerebellar Purkinje cells, with approximately 70% of all cells containing RNA foci • Increased percentage of Purkinje cells containing antisense RNA foci was associated with delayed age at disease onset
Fogel et al. [59]	Adult-onset sporadic ataxia cases Ataxia patients were screened for common genetic spinocerebellar ataxias and tested for C9orf72 expansion mutation	• The majority of patients were diagnosed as having pure cerebellar ataxia • Out of the 209 ataxia patients tested for C9 mutation, only one positive case was identified • The C9-positive ataxia patient has shown no motor neuron deterioration or cognitive dysfunction
Gendron et al. [27]	Post-mortem tissue from ALS, FTLD, MND and neurodegenerative disease with and without C9 mutation; HeLa cells and HEK293T cells transfected with either (C₄G₂)₆₆ or (C₄G₂)₂ expression vectors Generation of novel antibodies to visualise C₄G₂ RAN proteins – poly-PR, -GP and -PA, FISH to detect RNA foci formed from antisense transcripts in cultured cells, FISH detection of RNA foci and IF staining of human post-mortem tissue	• Ectopic expression of (C₄G₂)₆₆ leads to formation of RNA foci and synthesis of RAN proteins in cultured cells • (C₄G₂)_n RNA foci were detected in the cerebellum of C9-ALS/FTD patients, predominantly in the Purkinje cell layer, in both astrocytes and neurons • Poly-PA, poly-GP and poly-PR inclusions synthesised from antisense repeat were present in the brain of human C9-ALS/ FTD cases • The cerebellar granule cells of C9-ALS/FTD patients were shown to be particularly abundant in poly-GP, whereas poly-PA and poly-PR pathology was markedly less pronounced
Gendron et al. [29]	Post-mortem tissue from C9orf72 mutation carriers with a diagnosis of ALS, FTLD and FTLD-MND Quantitative immunoassays; IHC; analysis of patients’ clinical data	• Poly-GP levels were highest in the cerebellum of C9orf72 cases • Cerebellar poly-GP load was markedly lower in patients with ALS as compared to those with FTLD or FTLD-MND • There was an association between cerebellar poly-GP levels and cognitive scores in C9-ALS cases
Goldman et al. [58]	Family with multiple system atrophy (MSA) and ALS, positive for C9 expansion mutation Case study report	• Carriers of C9orf72 expansion mutation can present with both MSA and ALS, highlighting the possibility of large phenotypic variability associated with the C9 mutation • Draws a link between C9 mutation, ALS and cerebellar ataxia
Lee et al. [36]	(G₄C₂)_n transfected human non-neuronal cell lines and rat primary cortical neurons; zebrafish embryos injected with EGFP constructs containing 8x, 38 × and 72 × G₄C₂ repeats; human cerebellum tissue from ALS and FTD cases with confirmed C9 expansion mutation Detection of RNA foci by FISH, immunoprecipitation, FACS and ICC analysis for expression of apoptosis markers, detection and co-localisation of DPRs	• RNA containing 38 × and 72 × G₄C₂ repeats caused cellular toxicity in a length-dependent manner in transfect cell lines and in vivo zebrafish model • G₄C₂ foci induced apoptotic cell death, resulting in loss of foci-positive cells and increased expression of Annexin V and Caspase-3 apoptotic markers • RNA foci were found to be abundant in the cerebellum of ALS/FTD cases with C9 expansion mutation • Intranuclear neuronal RNA foci were larger (~ 500 nm) in the cerebellum tissue of C9-ALS/FTD than cortex (~ 200 nm), with over 70% of cerebellar foci co-localised with hnRNP-A3 • Co-localisation of poly-GA with poly-GR and -PR was reported to occur infrequently • Poly-GA was capable of sequestering poly-GP and -PA
Mackenzie et al. [30]	Cohort of 35 cases with a broad spectrum of clinical phenotypes, positive for C9orf72 mutation Characterised novel monoclonal antibodies against poly-GA; immunoblotting	• The pattern of DPR expression was similar in all cases, regardless of the diagnosis • Highest abundance of DPRs was found in the cerebellum, neocortex and hippocampus • There was no correlation between DPR pathology burden and the severity of neurodegeneration
Mahoney et al. [80]	Cases with C9orf72 mutation and syndromic diagnosis within FTLD spectrum Clinical, histopathological and neuroimaging analysis of C9orf72 expansion mutation	• Large clinical heterogeneity was observed among C9orf72 patients • Anxiety and memory impairments were commonly reported • Extensive thinning of frontal, parietal, occipital lobes and cerebellar atrophy was observed • Abundant expression of p62 inclusions was seen in the hippocampus and cerebellum
Mann et al. [31]	Post-mortem tissue from C9orf72 mutation-positive cases with confirmed diagnosis of FTLD or MND and p62-positive inclusions IHC; southern blotting	• DPRs were shown to be major components of p62-positive inclusions in the cerebellum and hippocampus of C9-FTLD and MND cases • There was some evidence of antisense translated DPRs; however, the poly-AP staining was variable
Mizielinska et al. [108]	Post-mortem tissue from C9-FTLD cases, neurodegenerative disease and healthy controls FISH and protein immuno-staining to detect, quantify and determine the subcellular localisation of sense and antisense RNA foci	• The presence of both sense and antisense RNA foci was reported in the brains of C9-FTLD cases including the cerebellum • RNA foci occurred more frequently in neurons than glia • RNA foci were seen in TDP-43 and p62-positive neurons (which were particularly abundant in the cerebellum), but the frequency was not greater than would be expected to occur by chance
Mori et al. [85]	Post-mortem tissue from C9orf72-positive ALS/FTD cases, patients with other neurodegenerative diseases and healthy controls Generation of novel antibodies against antisense-translated DPRs and putative carboxy-terminal tails of poly-GP, -GR and -GA reading frames; IHC analysis; ELISA and immunoblotting	• Demonstrated that G₄C₂ repeat is bidirectionally translated into co-aggregating DPRs in patients carrying C9orf72 expansion mutation • Non-ATG translation extends past the G₄C₂ repeat region in C9orf72 patients, as demonstrated using novel antibodies raised specifically against the putative carboxy-terminal tail of DPRs in poly-GA, -GP and -GR reading frames • The pattern of poly-GR inclusion pathology was shown to follow a rostro-causal gradient, with neuronal cytoplasmic inclusions (NCIs) abundantly found in the molecular and granular layer of the cerebellum, but rarely in Purkinje cells
Quaegebeur et al. [91]	Brain homogenates of FTD patients with C9orf72 expansion mutation Meso scale discovery (MSD) immunoassay	• The highest abundance of DPRs was detected in the cerebellum of C9-FTD cases • Relative DPR solubility was highest in the cerebellum • Levels of poly-GR and relative solubility of poly-GP were correlated with clinical parameters
Renton et al. [2]	ALS-FTD cases with a positive linkage to the chromosome 9p21 region Next-generation sequencing of chromosome 9p21 region; FISH analysis; expression arrays; RT-PCR	• Identified GGGGCCC hexanucleotide repeat expansion in C9orf72 gene as the cause of 9p21-linked ALS-FTD disease • Highest expression levels of C9orf72 RNA were detected in the cerebellum
Schludi et al. [88]	Transgenic mice expressing (poly-GA)₁₄₉ conjugated with cyan fluorescent protein (CFP) under the control of Thy1 promotor — Thy1 (GA)₁₄₉-CFP mice; post-mortem C9orf72-ALS/FTD patient samples; primary hippocampal neurons from rats transduced with lentiviral vector containing (GA)₁₇₅-GFP cDNA IHC and IF staining, immunoassays; qRT-PCR; behavioural and clinical assessment of mouse motor function	• Poly-GA aggregates were abundant in the spinal cord and brainstem of (GA)₁₄₉-CFP mice at 4–6 months of age. Poly-GA inclusions were also detected in the cerebellar nuclei but not the molecular or granular layer of the cerebellum of those animals • The majority of poly-GA inclusions were p62-positive and frequently co-localised with Rad23b • There was no evidence of Unc119 sequestration and no nucleolar pathology • M1f2 (chaperone-associated protein) was shown to co-aggregate with a large proportion of poly-GA inclusions in the spinal cord of (GA)₁₄₉-CFP mice, which was not the case in human C9orf72 patients • Motor behaviour deficits were observed in (GA)₁₄₉-CFP mice including balance and gait and decreased locomotor activity • Upregulation of neuroinflammatory markers was detected in (GA)₁₄₉-CFP mice at 6 months • No neuronal loss and no signs of motor neuron degeneration were observed
Tan et al. [9]	ALS and FTLD cases mostly without the C9orf72 expansion mutation, and healthy controls Cognitive, neuropsychiatric and functional assessment of patients; neuroimaging data	• Atrophy of cerebellar grey matter was observed across the spectrum of ALS-FTD • Correlation between neuropsychiatric function and atrophy of the crus and superior lobule of cerebellum was found • Motor symptoms were associated with atrophy of the inferior lobules
Tan et al. [70]	Post-mortem cerebellum tissue from ATXN2- and C9-ALS cases, sporadic ALS disease and sporadic muscular atrophy Histopathological analysis of cerebellar Purkinje and granule cell integrity	• Significant loss of Purkinje cells was observed in ATXN2-ALS • Despite a markedly higher abundance of TDP-43, p62- positive and poly-GA inclusions in the cerebellum of C9 mutation carriers compared to ATXN2-ALS cases, neuronal integrity appeared intact with no loss of Purkinje nor granule cells
Troakes et al. [20]	C9orf72 patient tissue Case study, IHC, Western blot	• Star-shaped p62 inclusions in the cortex, basal ganglia and hippocampus • Few TDP-43 inclusions in the brain, pathology more abundant in the spinal cord such as other types of ALS • Cerebellum granular cells had p62-positive TDP-43 negative inclusions
van Blitterswijk et al. [[92]	Post-mortem cerebellar and/ or frontal cortex tissue from C9orf72 expansion mutation carriers qRT-PCR and digital molecular barcoding techniques to assess total C9 transcript and variant (1–3) levels; immunoassay of DPRs	• A decrease in the abundance of total C9orf72 transcript and variants 1 and 2 were detected in C9 carriers compared to controls • The strongest effect was seen in variant 2 — with qRT-PCR and digital barcoding showing 43% and 31% reduction in cerebellum, respectively • Intron-containing transcripts were associated with poly-GP and poly-GA levels in cerebellum of C9 carriers
Waite et al. [93]	Subjects with confirmed diagnosis of ALS, FTLD or ALS-FTD Southern blot detection of C9orf72 expansion; qPCR analysis of C9 transcript levels; generation of polyclonal antibody against C9orf72; immunoblotting analysis of C9 protein level	• Southern blotting analysis of C9orf72 repeat expansion size revealed cerebellar tissue to have reduced modal expansion size compared to other brain regions • Expression of C9 transcript was significantly reduced in the cerebellum • Significant reduction in 48-kDa isoform of C9 protein was reported in the frontal cortex, but not in the cerebellum
Xiao et al. [104]	Post-mortem tissue from ALS cases with confirmed C9orf72 mutation, non-C9-ALS cases and healthy control Development of novel antibodies against C9-L and C9-S isoforms of C9orf72; investigation of the properties, abundance and subcellular localisation of C9 isoforms using IHC, Western blotting and immunoprecipitation	• No significant changes in the abundance of C9-L or C9-S were reported in the cerebellum of C9-ALS cases vs non-C9-ALS • Distinct subcellular localisation of the two isoforms was reported • C9-L isoform exhibited diffuse labelling in the cytoplasm of cerebellar Purkinje cells, with a striking labelling of numerous speckles observed in the neuronal perikarya and dendritic processes of both C9-carriers and non-carriers • C9-S antibody gave a very specific labelling of the nuclear membrane • C9 isoforms interacted with β1 and Ran‐GTPase components of the nuclear pore complex and thus were suggested to play a role in the disruption of the nucleocytoplasmic transport • No evidence of cerebellar neurodegeneration nor loss of Purkinje cells
Zhang et al. [65]	Transgenic mouse model expressing GFP-(GR)₁₀₀ in the brain; transfected HEK293T cells expressing GFP-(GR)₁₀₀ constructs; post-mortem cerebellar and cortical tissue from C9-ALS/FTD patients Mouse behavioural test; IHC and IF staining; Western blot; FISH; RNA-Seq and gene ontology; qPCR; RT-PCR; in vivo SUnSET assay	• GFP-(GR)₁₀₀ mice accumulated diffuse, cytoplasmic poly-GRs which were associated with age-dependent neurodegeneration, brain atrophy, memory and locomotor deficits • Loss of cerebellar Purkinje cells was observed in GFP-(GR)₁₀₀ mice • Poly-GR was found to co-localise with ribosomal subunits of eIF3η in GFP-(GR)₁₀₀ mice and post-mortem tissue from C9-ALS/FTD patients • Poly-GR induced the formation of stress granules in transfected HEK293T cells
Zhang et al. [69]	Mouse model expressing poly-PR mediated through AAV1 viral injection of GFP-PR₅₀; post-mortem frontal cortical tissue from ALS and FTD cases with C9 mutation; human iPSC-derived neurons Mouse behavioural testing, histopathological analysis, RNA, protein and IHC and IF analysis, gene ontology	• GFP-PR₅₀ mice showed motor dysfunction and cognitive deficits, accompanied by reduced brain weight, age-dependent loss of poly-PR expressing Purkinje cells and cortical neurons at 3 months of age, suggestive of poly-PR-induced cell-autonomous neuron death • Increased astrogliosis and microgliosis in the cortex and cerebellum of GFP-PR₅₀ animals were also reported • Poly-PR localised to heterochromatin, causing abnormal histone H3 methylation in mice and C9-ALS/FTD tissue

Study

Methodology

Main findings

Al-Sarraj et al. [25]

Post-mortem tissue from MND/ALS, FTLD and ALS-FTLD cases with, and FTLD cases without C9 expansion mutation

Neuropathological assessment, IHC and IF studies

• p62 positive, p-TDP-43 negative inclusions were reported in cerebellar Purkinje cells and molecular layer of C9-positive cases

Ash et al. [10]

Human C9orf72 tissue

Meso scale discovery assay, IHC, IF; generation of novel antibodies to poly-GA, GP and -GR

• C9orf72 RAN translation products (now called DPRs) were detected in high molecular weight aggregates specific to C9orf72 expansion and not other neurodegenerative or CAG expansion disorders

• DPRs were abundant in the cerebellum of C9orf72 cases

Belzil et al. [103]

C9orf72 patient tissue, skin biopsies and blood samples with derivation of fibroblasts

qRT-PCR, ddPCR and chromatin immunoprecipitation

• Both the frontal cortices and cerebellum had reductions in C9orf72 mRNA in pathogenic expansions

• C9orf72 with expansions increases the rate of binding to trimethylated lysine residues within histones H3 (H3K9/K27/K79) and H4 (H4K20) which is detectable in the blood of patients

Chew et al. [94]

Transgenic mouse model expressing (G₄C₂)₆₆ throughout the CNS by means of AAV-mediated somatic brain transgenesis

Behavioural testing; RNA FISH detection of RNA foci, immunoassays for DPRs

• RNA foci were abundant in Purkinje cells of (G₄C₂)₆₆ mice at 6 months, with 40–54% of Purkinje cells foci-positive, and to a lesser degree, in granular and molecular layers of cerebellum

• Poly-GA, poly-GP and poly-GR inclusions were detected in the cerebellum, accompanied by a loss (11% decrease) of Purkinje cells in (G₄C₂)₆₆ mice

• A decrease in overall brain weight, indicative of repeat expansion-mediated atrophy, and behavioural/ motor skill abnormalities were also reported

Cooper-Knock et al. [109]

Samples from ALS/FTD cases with and without C9 expansion mutation, healthy controls and asymptomatic C9 carriers

Identification of nuclear and cytoplasmic RNA foci using FISH, investigation of RNA foci binding partners using mass spectrometry, pulldown assays and IHC

• RNA foci were found to be abundant in the cerebellar granule cells of C9-ALS/FTD patients and absent in cases without C9 mutation and healthy controls (although the levels varied based on clinical presentation)

• A number of putative binding partners of RNA foci were identified, including hnRNP, which was shown to co-localise with RNA foci in cerebellar granule cells

Cooper-Knock et al. [112]

C9orf72 patient tissue

IHC, FISH, RNA-binding assays

• Antisense RNA foci are predominantly present in the cerebellar Purkinje cells and motor neurons. In motor neurons, antisense foci (and not sense) correlated with TDP-43 pathology

• Sense RNA foci localised predominantly to the granular cells of the cerebellum

• DPRs were present with the greatest frequency in granular cells of the cerebellum and then the motor neurons of the spinal cord

• Antisense RNA foci co-localised with SRSF2, hnRNP A1, hnRNP A/F, ALYREF and hnRNP K

Corcia et al. [60]

C9orf72 mutation carrier presenting with pure cerebellar syndrome

Case study report

• Female with family history of ALS and other neurodegenerative disorders presents with symptoms of pure cerebellar syndrome — locomotor disturbance, cerebellar vermis atrophy and no cognitive dysfunction, and is subsequently found to be a carrier of C9orf72 mutation

• Demonstrating a link between C9 mutation and cerebellar pathology

Davidson et al. [89]

Post-mortem tissue from FTLD-tau, FTLD-TDP type A, B and C, and C9orf72 expansion, MAPT or GRN mutation carriers, and healthy controls

IHC staining and pathological assessment

• Investigated the patter of hnRNP A1, A2/B1 and A3 staining across brain regions in various subtypes of FTLD cases, including carriers of C9orf72 mutation

• No difference in the intensity and staining pattern of hnRNP A1 or hnRNP A2/B1 was observed in C9orf72-positive cases compared to other FTLD cases, with no hnRNP A1 or A2/B1 immunoreactive inclusions reported

• hnRNP A3-positive inclusions were seen in cerebellum of some C9orf72 cases

Davidson et al. [105]

C9orf72 patient tissue and other disorders (Huntington’s/FTD-TDP and Alzheimer’s)

IHC, antibody comparison

• Confirming previous studies, C9-L antibodies labelling diffuse cytoplasmic staining and speckles in the Purkinje neurons of the cerebellum

• Additionally, C9-S labelled the nuclear membrane only

• ProteinTech’s monoclonal and polyclonal antibody as well as GeneTex achieved similar staining to that of C9-L

• Nuclear membrane staining of C9-S was shifted to the plasma membrane in the spinal cord of sufferers

• Commercial antibodies were unable to recapitulate C9-S staining

DeJesus-Hernandez et al. [37]

Cerebellum and frontal cortex tissue of C9 expansion mutation carriers

Detection and visualisation of RNA foci by RNA FISH and IF staining followed by computer-assisted quantification and co-localisation

• RNA foci were abundant in the cerebellum of C9 carriers, with 23% and 1% of granule cells containing sense and antisense RNA foci, respectively

• RNA foci were most abundantly present in cerebellar Purkinje cells, with approximately 70% of all cells containing RNA foci

• Increased percentage of Purkinje cells containing antisense RNA foci was associated with delayed age at disease onset

Fogel et al. [59]

Adult-onset sporadic ataxia cases

Ataxia patients were screened for common genetic spinocerebellar ataxias and tested for C9orf72 expansion mutation

• The majority of patients were diagnosed as having pure cerebellar ataxia

• Out of the 209 ataxia patients tested for C9 mutation, only one positive case was identified

• The C9-positive ataxia patient has shown no motor neuron deterioration or cognitive dysfunction

Gendron et al. [27]

Post-mortem tissue from ALS, FTLD, MND and neurodegenerative disease with and without C9 mutation; HeLa cells and HEK293T cells transfected with either (C₄G₂)₆₆ or (C₄G₂)₂ expression vectors

Generation of novel antibodies to visualise C₄G₂ RAN proteins – poly-PR, -GP and -PA, FISH to detect RNA foci formed from antisense transcripts in cultured cells, FISH detection of RNA foci and IF staining of human post-mortem tissue

• Ectopic expression of (C₄G₂)₆₆ leads to formation of RNA foci and synthesis of RAN proteins in cultured cells

• (C₄G₂)_n RNA foci were detected in the cerebellum of C9-ALS/FTD patients, predominantly in the Purkinje cell layer, in both astrocytes and neurons

• Poly-PA, poly-GP and poly-PR inclusions synthesised from antisense repeat were present in the brain of human C9-ALS/ FTD cases

• The cerebellar granule cells of C9-ALS/FTD patients were shown to be particularly abundant in poly-GP, whereas poly-PA and poly-PR pathology was markedly less pronounced

Gendron et al. [29]

Post-mortem tissue from C9orf72 mutation carriers with a diagnosis of ALS, FTLD and FTLD-MND

Quantitative immunoassays; IHC; analysis of patients’ clinical data

• Poly-GP levels were highest in the cerebellum of C9orf72 cases

• Cerebellar poly-GP load was markedly lower in patients with ALS as compared to those with FTLD or FTLD-MND

• There was an association between cerebellar poly-GP levels and cognitive scores in C9-ALS cases

Goldman et al. [58]

Family with multiple system atrophy (MSA) and ALS, positive for C9 expansion mutation

Case study report

• Carriers of C9orf72 expansion mutation can present with both MSA and ALS, highlighting the possibility of large phenotypic variability associated with the C9 mutation

• Draws a link between C9 mutation, ALS and cerebellar ataxia

Lee et al. [36]

(G₄C₂)_n transfected human non-neuronal cell lines and rat primary cortical neurons; zebrafish embryos injected with EGFP constructs containing 8x, 38 × and 72 × G₄C₂ repeats; human cerebellum tissue from ALS and FTD cases with confirmed C9 expansion mutation

Detection of RNA foci by FISH, immunoprecipitation, FACS and ICC analysis for expression of apoptosis markers, detection and co-localisation of DPRs

• RNA containing 38 × and 72 × G₄C₂ repeats caused cellular toxicity in a length-dependent manner in transfect cell lines and in vivo zebrafish model

• G₄C₂ foci induced apoptotic cell death, resulting in loss of foci-positive cells and increased expression of Annexin V and Caspase-3 apoptotic markers

• RNA foci were found to be abundant in the cerebellum of ALS/FTD cases with C9 expansion mutation

• Intranuclear neuronal RNA foci were larger (~ 500 nm) in the cerebellum tissue of C9-ALS/FTD than cortex (~ 200 nm), with over 70% of cerebellar foci co-localised with hnRNP-A3

• Co-localisation of poly-GA with poly-GR and -PR was reported to occur infrequently

• Poly-GA was capable of sequestering poly-GP and -PA

Mackenzie et al. [30]

Cohort of 35 cases with a broad spectrum of clinical phenotypes, positive for C9orf72 mutation

Characterised novel monoclonal antibodies against poly-GA; immunoblotting

• The pattern of DPR expression was similar in all cases, regardless of the diagnosis

• Highest abundance of DPRs was found in the cerebellum, neocortex and hippocampus

• There was no correlation between DPR pathology burden and the severity of neurodegeneration

Mahoney et al. [80]

Cases with C9orf72 mutation and syndromic diagnosis within FTLD spectrum

Clinical, histopathological and neuroimaging analysis of C9orf72 expansion mutation

• Large clinical heterogeneity was observed among C9orf72 patients

• Anxiety and memory impairments were commonly reported

• Extensive thinning of frontal, parietal, occipital lobes and cerebellar atrophy was observed

• Abundant expression of p62 inclusions was seen in the hippocampus and cerebellum

Mann et al. [31]

Post-mortem tissue from C9orf72 mutation-positive cases with confirmed diagnosis of FTLD or MND and p62-positive inclusions

IHC; southern blotting

• DPRs were shown to be major components of p62-positive inclusions in the cerebellum and hippocampus of C9-FTLD and MND cases

• There was some evidence of antisense translated DPRs; however, the poly-AP staining was variable

Mizielinska et al. [108]

Post-mortem tissue from C9-FTLD cases, neurodegenerative disease and healthy controls

FISH and protein immuno-staining to detect, quantify and determine the subcellular localisation of sense and antisense RNA foci

• The presence of both sense and antisense RNA foci was reported in the brains of C9-FTLD cases including the cerebellum

• RNA foci occurred more frequently in neurons than glia

• RNA foci were seen in TDP-43 and p62-positive neurons (which were particularly abundant in the cerebellum), but the frequency was not greater than would be expected to occur by chance

Mori et al. [85]

Post-mortem tissue from C9orf72-positive ALS/FTD cases, patients with other neurodegenerative diseases and healthy controls

Generation of novel antibodies against antisense-translated DPRs and putative carboxy-terminal tails of poly-GP, -GR and -GA reading frames; IHC analysis; ELISA and immunoblotting

• Demonstrated that G₄C₂ repeat is bidirectionally translated into co-aggregating DPRs in patients carrying C9orf72 expansion mutation

• Non-ATG translation extends past the G₄C₂ repeat region in C9orf72 patients, as demonstrated using novel antibodies raised specifically against the putative carboxy-terminal tail of DPRs in poly-GA, -GP and -GR reading frames

• The pattern of poly-GR inclusion pathology was shown to follow a rostro-causal gradient, with neuronal cytoplasmic inclusions (NCIs) abundantly found in the molecular and granular layer of the cerebellum, but rarely in Purkinje cells

Quaegebeur et al. [91]

Brain homogenates of FTD patients with C9orf72 expansion mutation

Meso scale discovery (MSD) immunoassay

• The highest abundance of DPRs was detected in the cerebellum of C9-FTD cases

• Relative DPR solubility was highest in the cerebellum

• Levels of poly-GR and relative solubility of poly-GP were correlated with clinical parameters

Renton et al. [2]

ALS-FTD cases with a positive linkage to the chromosome 9p21 region

Next-generation sequencing of chromosome 9p21 region; FISH analysis; expression arrays; RT-PCR

• Identified GGGGCCC hexanucleotide repeat expansion in C9orf72 gene as the cause of 9p21-linked ALS-FTD disease

• Highest expression levels of C9orf72 RNA were detected in the cerebellum

Schludi et al. [88]

Transgenic mice expressing (poly-GA)₁₄₉ conjugated with cyan fluorescent protein (CFP) under the control of Thy1 promotor — Thy1 (GA)₁₄₉-CFP mice; post-mortem C9orf72-ALS/FTD patient samples; primary hippocampal neurons from rats transduced with lentiviral vector containing (GA)₁₇₅-GFP cDNA

IHC and IF staining, immunoassays; qRT-PCR; behavioural and clinical assessment of mouse motor function

• Poly-GA aggregates were abundant in the spinal cord and brainstem of (GA)₁₄₉-CFP mice at 4–6 months of age. Poly-GA inclusions were also detected in the cerebellar nuclei but not the molecular or granular layer of the cerebellum of those animals

• The majority of poly-GA inclusions were p62-positive and frequently co-localised with Rad23b

• There was no evidence of Unc119 sequestration and no nucleolar pathology

• M1f2 (chaperone-associated protein) was shown to co-aggregate with a large proportion of poly-GA inclusions in the spinal cord of (GA)₁₄₉-CFP mice, which was not the case in human C9orf72 patients

• Motor behaviour deficits were observed in (GA)₁₄₉-CFP mice including balance and gait and decreased locomotor activity

• Upregulation of neuroinflammatory markers was detected in (GA)₁₄₉-CFP mice at 6 months

• No neuronal loss and no signs of motor neuron degeneration were observed

Tan et al. [9]

ALS and FTLD cases mostly without the C9orf72 expansion mutation, and healthy controls

Cognitive, neuropsychiatric and functional assessment of patients; neuroimaging data

• Atrophy of cerebellar grey matter was observed across the spectrum of ALS-FTD

• Correlation between neuropsychiatric function and atrophy of the crus and superior lobule of cerebellum was found

• Motor symptoms were associated with atrophy of the inferior lobules

Tan et al. [70]

Post-mortem cerebellum tissue from ATXN2- and C9-ALS cases, sporadic ALS disease and sporadic muscular atrophy

Histopathological analysis of cerebellar Purkinje and granule cell integrity

• Significant loss of Purkinje cells was observed in ATXN2-ALS

• Despite a markedly higher abundance of TDP-43, p62- positive and poly-GA inclusions in the cerebellum of C9 mutation carriers compared to ATXN2-ALS cases, neuronal integrity appeared intact with no loss of Purkinje nor granule cells

Troakes et al. [20]

C9orf72 patient tissue

Case study, IHC, Western blot

• Star-shaped p62 inclusions in the cortex, basal ganglia and hippocampus

• Few TDP-43 inclusions in the brain, pathology more abundant in the spinal cord such as other types of ALS

• Cerebellum granular cells had p62-positive TDP-43 negative inclusions

van Blitterswijk et al. [[92]

Post-mortem cerebellar and/ or frontal cortex tissue from C9orf72 expansion mutation carriers

qRT-PCR and digital molecular barcoding techniques to assess total C9 transcript and variant (1–3) levels; immunoassay of DPRs

• A decrease in the abundance of total C9orf72 transcript and variants 1 and 2 were detected in C9 carriers compared to controls

• The strongest effect was seen in variant 2 — with qRT-PCR and digital barcoding showing 43% and 31% reduction in cerebellum, respectively

• Intron-containing transcripts were associated with poly-GP and poly-GA levels in cerebellum of C9 carriers

Waite et al. [93]

Subjects with confirmed diagnosis of ALS, FTLD or ALS-FTD

Southern blot detection of C9orf72 expansion; qPCR analysis of C9 transcript levels; generation of polyclonal antibody against C9orf72; immunoblotting analysis of C9 protein level

• Southern blotting analysis of C9orf72 repeat expansion size revealed cerebellar tissue to have reduced modal expansion size compared to other brain regions

• Expression of C9 transcript was significantly reduced in the cerebellum

• Significant reduction in 48-kDa isoform of C9 protein was reported in the frontal cortex, but not in the cerebellum

Xiao et al. [104]

Post-mortem tissue from ALS cases with confirmed C9orf72 mutation, non-C9-ALS cases and healthy control

Development of novel antibodies against C9-L and C9-S isoforms of C9orf72; investigation of the properties, abundance and subcellular localisation of C9 isoforms using IHC, Western blotting and immunoprecipitation

• No significant changes in the abundance of C9-L or C9-S were reported in the cerebellum of C9-ALS cases vs non-C9-ALS

• Distinct subcellular localisation of the two isoforms was reported

• C9-L isoform exhibited diffuse labelling in the cytoplasm of cerebellar Purkinje cells, with a striking labelling of numerous speckles observed in the neuronal perikarya and dendritic processes of both C9-carriers and non-carriers

• C9-S antibody gave a very specific labelling of the nuclear membrane

• C9 isoforms interacted with β1 and Ran‐GTPase components of the nuclear pore complex and thus were suggested to play a role in the disruption of the nucleocytoplasmic transport

• No evidence of cerebellar neurodegeneration nor loss of Purkinje cells

Zhang et al. [65]

Transgenic mouse model expressing GFP-(GR)₁₀₀ in the brain; transfected HEK293T cells expressing GFP-(GR)₁₀₀ constructs; post-mortem cerebellar and cortical tissue from C9-ALS/FTD patients

Mouse behavioural test; IHC and IF staining; Western blot; FISH; RNA-Seq and gene ontology; qPCR; RT-PCR; in vivo SUnSET assay

• GFP-(GR)₁₀₀ mice accumulated diffuse, cytoplasmic poly-GRs which were associated with age-dependent neurodegeneration, brain atrophy, memory and locomotor deficits

• Loss of cerebellar Purkinje cells was observed in GFP-(GR)₁₀₀ mice

• Poly-GR was found to co-localise with ribosomal subunits of eIF3η in GFP-(GR)₁₀₀ mice and post-mortem tissue from C9-ALS/FTD patients

• Poly-GR induced the formation of stress granules in transfected HEK293T cells

Zhang et al. [69]

Mouse model expressing poly-PR mediated through AAV1 viral injection of GFP-PR₅₀; post-mortem frontal cortical tissue from ALS and FTD cases with C9 mutation; human iPSC-derived neurons

Mouse behavioural testing, histopathological analysis, RNA, protein and IHC and IF analysis, gene ontology

• GFP-PR₅₀ mice showed motor dysfunction and cognitive deficits, accompanied by reduced brain weight, age-dependent loss of poly-PR expressing Purkinje cells and cortical neurons at 3 months of age, suggestive of poly-PR-induced cell-autonomous neuron death

• Increased astrogliosis and microgliosis in the cortex and cerebellum of GFP-PR₅₀ animals were also reported

• Poly-PR localised to heterochromatin, causing abnormal histone H3 methylation in mice and C9-ALS/FTD tissue

Abbreviations: AAV, adenovirus; ALS, amyotrophic lateral sclerosis; ALYREF, Aly/REF export factor; ATXN2, Ataxin-2; ddPCR, droplet digital PCR; DPR, dipeptide protein; EGFP, enhanced green fluorescent protein; ELISA, enzyme-linked immunosorbent assay; FACS, fluorescence-activated cell sorting; FISH, fluorescent in-situ hybridization; FTD, frontotemporal dementia; FTLD, frontotemporal lobar dementia; GRN, progranulin; HEK293T, human embryonic kidney 293 cells; hnRNP, heterogeneous nuclear ribonucleoproteins; ICC, immunocytochemistry; IF, immunofluorescence; IHC, immunohistochemistry; iPSCs, induced pluripotent stem cells; MAPT, microtubule-associated protein Tau; MND, motor neuron disease; p62, ubiquitin binding protein p62; p-TDP-43, phosphorylated TAR-DNA binding protein 43; qRT-PCR, quantitative real time PCR; Rad23b, UV excision repair protein homolog B; RAN, repeat-associated non-AUG; RNA, ribonucleic acid; RNA-Seq, RNA sequencing; RT-PCR, real time PCR; SRSF2, serine and arginine rich splicing factor 2; SUnSET, surface sensing of translation; TDP-43, TAR-DNA binding protein 43; Unc119, uncoordinated 119