Large C9orf72 Hexanucleotide Repeat Expansions Are Seen in Multiple Neurodegenerative Syndromes and Are More Frequent Than Expected in the UK Population

Hexanucleotide repeat expansions in C9orf72 are a major cause of frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Understanding the disease mechanisms and a method for clinical diagnostic genotyping have been hindered because of the difficulty in estimating the expansion size. We found 96 repeat-primed PCR expansions: 85/2,974 in six neurodegenerative diseases cohorts (FTLD, ALS, Alzheimer disease, sporadic Creutzfeldt-Jakob disease, Huntington disease-like syndrome, and other nonspecific neurodegenerative disease syndromes) and 11/7,579 (0.15%) in UK 1958 birth cohort (58BC) controls. With the use of a modified Southern blot method, the estimated expansion range (smear maxima) in cases was 800–4,400. Similarly, large expansions were detected in the population controls. Differences in expansion size and morphology were detected between DNA samples from tissue and cell lines. Of those in whom repeat-primed PCR detected expansions, 68/69 were confirmed by blotting, which was specific for greater than 275 repeats. We found that morphology in the expansion smear varied among different individuals and among different brain regions in the same individual. Expansion size correlated with age at clinical onset but did not differ between diagnostic groups. Evidence of instability of repeat size in control families, as well as neighboring SNP and microsatellite analyses, support multiple expansion events on the same haplotype background. Our method of estimating the size of large expansions has potential clinical utility. C9orf72-related disease might mimic several neurodegenerative disorders and, with potentially 90,000 carriers in the United Kingdom, is more common than previously realized.     

Can ALS-Associated C9orf72 Repeat Expansions Be Diagnosed on a Blood DNA Test Alone?

Gene mutations that preferentially affect the CNS have been implicated in a number of neurological disorders. This leads to the possibility that a disease-causing mutation present only in CNS tissues could be missed if it were tested in a blood DNA sample only. The commonest mutation in amyotrophic lateral sclerosis (ALS) is an expansion of the hexanucleotide repeats of C9orf72. To find out if CNS-specific mutations of this gene could cause some cases of ALS we looked for differences in the size of C9orf72 repeats between DNA from the CNS and from white blood cells (WBCs) of 38 sporadic ALS patients, using a repeat-primed PCR screening test. We also looked for differences in C9orf72 repeats in WBC DNA from 6 ALS-discordant and 1 ALS-concordant monozygotic twins. Abnormally expanded C9orf72 repeats were found in 13% of the ALS CNS samples, as well as in their paired WBC DNA. The 87% of ALS CNS samples with normal-sized C9orf72 repeats had the same number of repeats in paired WBC samples. All ALS-discordant twins had the same normal numbers of WBC C9orf72 repeats. Although previous work suggests some tissue mosaicism in C9orf72 repeat size is probably present, this study indicates that this is not likely to be of sufficient magnitude to result in a normal C9orf72 repeat length in blood but an abnormally expanded repeat length in the CNS. This suggests that a blood DNA test alone will usually be sufficient to make a diagnosis of C9orf72 repeat-related ALS.     

Hypermethylation of the CpG Island Near the G4C2 Repeat in ALS with a C9orf72 Expansion

The G₄C₂ repeat expansion in C9orf72 is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). We tested the hypothesis that the repeat expansion causes aberrant CpG methylation near the G₄C₂ repeat, which could be responsible for the downregulation of gene expression. We investigated the CpG methylation profile by two methods using genomic DNA from the blood of individuals with ALS (37 expansion carriers and 64 noncarriers), normal controls (n = 76), and family members of 7 ALS probands with the expansion. We report that hypermethylation of the CpG island 5′ of the G₄C₂ repeat is associated with the presence of the expansion (p < 0.0001). A higher degree of methylation was significantly correlated with a shorter disease duration (p < 0.01), associated with familial ALS (p = 0.009) and segregated with the expansion in 7 investigated families. Notably, we did not detect methylation for either normal or intermediate alleles (up to 43 repeats), bringing to question the current cutoff of 30 repeats for pathological alleles. Our study raises several important questions for the future investigation of large data sets, such as whether the degree of methylation corresponds to clinical presentation (ALS versus FTLD).     

Reduced hnRNPA3 increases C9orf72 repeat RNA levels and dipeptide‐repeat protein deposition

Intronic hexanucleotide (G₄C₂) repeat expansions in C9orf72 are genetically associated with frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). The repeat RNA accumulates within RNA foci but is also translated into disease characterizing dipeptide repeat proteins (DPR). Repeat‐dependent toxicity may affect nuclear import. hnRNPA3 is a heterogeneous nuclear ribonucleoprotein, which specifically binds to the G₄C₂ repeat RNA. We now report that a reduction of nuclear hnRNPA3 leads to an increase of the repeat RNA as well as DPR production and deposition in primary neurons and a novel tissue culture model that reproduces features of the C9orf72 pathology. In fibroblasts derived from patients carrying extended C9orf72 repeats, nuclear RNA foci accumulated upon reduction of hnRNPA3. Neurons in the hippocampus of C9orf72 patients are frequently devoid of hnRNPA3. Reduced nuclear hnRNPA3 in the hippocampus of patients with extended C9orf72 repeats correlates with increased DPR deposition. Thus, reduced hnRNPA3 expression in C9orf72 cases leads to increased levels of the repeat RNA as well as enhanced production and deposition of DPR proteins and RNA foci.     

C9ORF72 Repeat Expansion in Australian and Spanish Frontotemporal Dementia Patients

A hexanucleotide repeat expansion in C9ORF72 has been established as a common cause of frontotemporal dementia (FTD). However, the minimum repeat number necessary for disease pathogenesis is not known. The aims of our study were to determine the frequency of the C9ORF72 repeat expansion in two FTD patient collections (one Australian and one Spanish, combined n = 190), to examine C9ORF72 expansion allele length in a subset of FTD patients, and to examine C9ORF72 allele length in ‘non-expansion’ patients (those with <30 repeats). The C9ORF72 repeat expansion was detected in 5–17% of patients (21–41% of familial FTD patients). For one family, the expansion was present in the proband but absent in the mother, who was diagnosed with dementia at age 68. No association was found between C9ORF72 non-expanded allele length and age of onset and in the Spanish sample mean allele length was shorter in cases than in controls. Southern blotting analysis revealed that one of the nine ‘expansion-positive’ patients examined, who had neuropathologically confirmed frontotemporal lobar degeneration with TDP-43 pathology, harboured an ‘intermediate’ allele with a mean size of only ∼65 repeats. Our study indicates that the C9ORF72 repeat expansion accounts for a significant proportion of Australian and Spanish FTD cases. However, C9ORF72 allele length does not influence the age at onset of ‘non-expansion’ FTD patients in the series examined. Expansion of the C9ORF72 allele to as little as ∼65 repeats may be sufficient to cause disease.     

Analysis of the GGGGCC Repeat Expansions of the C9orf72 Gene in SCA3/MJD Patients from China

Neurodegenerative disorders are a heterogeneous group of chronic progressive diseases and have pathological mechanisms in common. A certain causative gene identified for a particular disease may be found to play roles in more than one neurodegenerative disorder. We analyzed the GGGGCC repeat expansions of C9orf72 gene in patients with SCA3/MJD from mainland China to determine whether the C9orf72 gene plays a role in the pathogenesis of SCA3/MJD. In our study, there were no pathogenic repeats (>30 repeats) detected in either the patients or controls. SCA3/MJD patients with intermediate/intermediate or short/intermediate genotype (short: <7 repeats; intermediate: 7-30 repeats) of the GGGGCC repeats had an earlier onset compared with those with short/short genotype. The presence of the intermediate allele of the GGGGCC repeats in the patients decreased the age at onset by nearly 3 years. Our study firstly demonstrate that the development of SCA3/MJD may involve some physiological functions of the C9orf72 gene and provide new evidence to the hypothesis that a specific mutation identified in one of the neurodegenerative disorders may be a modulator in this class of diseases.     

The Glycine-Alanine Dipeptide Repeat from C9orf72 Hexanucleotide Expansions Forms Toxic Amyloids Possessing Cell-to-Cell Transmission Properties

Hexanucleotide expansions, GGGGCC, in the non-coding regions of the C9orf72 gene were found in major frontotemporal lobar dementia and amyotrophic lateral sclerosis patients (C9FTD/ALS). In addition to possible RNA toxicity, several dipeptide repeats (DPRs) are translated through repeat-associated non-ATG-initiated translation. The DPRs, including poly(GA), poly(GR), poly(GP), poly(PR), and poly(PA), were found in the brains and spinal cords of C9FTD/ALS patients. Among the DPRs, poly(GA) is highly susceptible to form cytoplasmic inclusions, which is a characteristic of C9FTD/ALS. To elucidate DPR aggregation, we used synthetic (GA)₁₅ DPR as a model system to examine the aggregation and structural properties in vitro. We found that (GA)₁₅ with 15 repeats fibrillates rapidly and ultimately forms flat, ribbon-type fibrils evidenced by transmission electron microscopy and atomic force microscopy. The fibrils are capable of amyloid dye binding and contain a characteristic cross-β sheet structure, as revealed by x-ray scattering. Furthermore, using neuroblastoma cells, we demonstrated the neurotoxicity and cell-to-cell transmission property of (GA)₁₅ DPR. Overall, our results show the structural and toxicity properties of GA DPR to facilitate future DPR-related therapeutic development.     

Disruption of ER‐mitochondria tethering and signalling in C9orf72‐associated amyotrophic lateral sclerosis and frontotemporal dementia

Hexanucleotide repeat expansions in C9orf72 are the most common cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). The mechanisms by which the expansions cause disease are not properly understood but a favoured route involves its translation into dipeptide repeat (DPR) polypeptides, some of which are neurotoxic. However, the precise targets for mutant C9orf72 and DPR toxicity are not fully clear, and damage to several neuronal functions has been described. Many of these functions are regulated by signalling between the endoplasmic reticulum (ER) and mitochondria. ER‐mitochondria signalling requires close physical contacts between the two organelles that are mediated by the VAPB‐PTPIP51 ‘tethering’ proteins. Here, we show that ER‐mitochondria signalling and the VAPB‐PTPIP51 tethers are disrupted in neurons derived from induced pluripotent stem (iPS) cells from patients carrying ALS/FTD pathogenic C9orf72 expansions and in affected neurons in mutant C9orf72 transgenic mice. In these mice, disruption of the VAPB‐PTPIP51 tethers occurs prior to disease onset suggesting that it contributes to the pathogenic process. We also show that neurotoxic DPRs disrupt the VAPB‐PTPIP51 interaction and ER‐mitochondria contacts and that this may involve activation of glycogen synthase kinases‐3β (GSK3β), a known negative regulator of VAPB‐PTPIP51 binding. Finally, we show that these DPRs disrupt delivery of Ca²⁺ from ER stores to mitochondria, which is a primary function of the VAPB‐PTPIP51 tethers. This delivery regulates a number of key neuronal functions that are damaged in ALS/FTD including bioenergetics, autophagy and synaptic function. Our findings reveal a new molecular target for mutant C9orf72‐mediated toxicity.     

Selective inhibition of MBNL1�CCCUG interaction by small molecules toward potential therapeutic agents for myotonic dystrophy type 2 (DM2)

Myotonic dystrophy type 2 (DM2) is an incurable neuromuscular disease caused by expanded CCUG repeats that may exhibit toxicity by sequestering the splicing regulator MBNL1. A series of triaminotriazine- and triaminopyrimidine-based small molecules (ligands 1–3) were designed, synthesized and tested as inhibitors of the MBNL1–CCUG interaction. Despite the structural similarities of the triaminotriazine and triaminopyrimidine units, the triaminopyrimidine-based ligands bind with low micromolar affinity to CCUG repeats (K_d ∼ 0.1–3.6 µM) whereas the triaminotriazine ligands do not bind CCUG repeats. Importantly, these simple and small triaminopyrimidine ligands exhibit both strong inhibition (K_i ∼ 2 µM) of the MBNL1–CCUG interaction and high selectivity for CCUG repeats over other RNA targets. These experiments suggest these compounds are potential lead agents for the treatment of DM2.     

Structure of the human C9orf72-SMCR8 complex reveals a multivalent protein interaction architecture

A major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) spectrum disorder is the hexanucleotide G₄C₂ repeat expansion in the first intron of the C9orf72 gene. Many underlying mechanisms lead to manifestation of disease that include toxic gain-of-function by repeat G₄C₂ RNAs, dipeptide repeat proteins, and a reduction of the C9orf72 gene product. The C9orf72 protein interacts with SMCR8 and WDR41 to form a trimeric complex and regulates multiple cellular pathways including autophagy. Here, we report the structure of the C9orf72-SMCR8 complex at 3.8 Å resolution using single-particle cryo-electron microscopy (cryo-EM). The structure reveals 2 distinct dimerization interfaces between C9orf72 and SMCR8 that involves an extensive network of interactions. Homology between C9orf72-SMCR8 and Folliculin-Folliculin Interacting Protein 2 (FLCN-FNIP2), a GTPase activating protein (GAP) complex, enabled identification of a key residue within the active site of SMCR8. Further structural analysis suggested that a coiled-coil region within the uDenn domain of SMCR8 could act as an interaction platform for other coiled-coil proteins, and its deletion reduced the interaction of the C9orf72-SMCR8 complex with FIP200 upon starvation. In summary, this study contributes toward our understanding of the biological function of the C9orf72-SMCR8 complex.

Structural and biochemical characterisation of the C9orf72-SMCR8 complex sheds light on its overall architecture and highlights its role as a multi-functional scaffold for coordinating autophagy.     

The C9orf72-SMCR8-WDR41 complex is a GAP for small GTPases

ABSTRACT

Massive expansions of the hexanucleotide in C9orf72 are the primary genetic origins of familial amyotrophic lateral sclerosis (ALS) and frontal temporal dementia (FTD). Current studies have found that this repeat sequence participates in the disease process by producing neurotoxic substances and reducing the level of C9orf72 protein; however, the progress in the functional study of C9orf72 is slow. Recently, a stable complex, consisting of C9orf72, SMCR8, and WDR41, has been implicated in regulating membrane trafficking and macroautophagy. We reported the cryo-electron microscopy (cryo-EM) structure of the C9orf72-SMCR8-WDR41 complex (CSW complex), unveiling that the CSW complex is a dimer of heterotrimers. Intriguingly, in the heterotrimer of the C9orf72-SMCR8-WDR41, C9orf72 interacts with SMCR8 in a manner similar to the FLCN-FNIP2 complex. Nevertheless, WDR41 is connected to the DENN domain of SMCR8 through its N-terminal β-strand and C-terminal helix but does not directly interact with C9orf72. Notably, the C9orf72-SMCR8 complex was demonstrated to act as a GAP for RAB8A and RAB11A in vitro.     

Current knowledge and recent insights into the genetic basis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is the most frequent motor neuron disease, affecting the upper and/or lower motor neurons. However, extramotor symptoms can also occur; cognitive deficits are present in more than 40% of patients and 5–8% of ALS patients develop frontotemporal dementia. There is no effective treatment for ALS and median survival is 2–3 years after onset.Amyotrophic lateral sclerosis is a genetically heterogeneous disorder with monogenic forms as well as complex genetic etiology. Currently, complex genetic risk factors are of minor interest for routine diagnostic testing or counseling of patients and their families. By contrast, a monogenic cause can be identified in 70% of familial and 10% of sporadic ALS cases. The most frequent genetic cause is a noncoding hexanucleotide repeat expansion in the C9orf72 gene. In recent years, high-throughput sequencing technologies have helped to identify additional monogenic and complex risk factors of ALS.Genetic counseling should be offered to all ALS patients and their first- and possibly second-degree relatives, and should include information about the possibilities and limitations of genetic testing. Routine diagnostic testing should at least encompass the most frequently mutated disease genes (C9orf72, SOD1, TDP-43, FUS). Targeted sequencing approaches including further disease genes may be applied. Caution is warranted as the C9orf72 repeat expansion cannot be detected by routine sequencing technologies and testing by polymerase chain reaction (PCR) is failure-prone.Predictive testing is possible in families in which a genetic cause has been identified, but the limitations of genetic testing (i.?e., the problems of incomplete penetrance, variable expressivity and possible oligogenic inheritance) have to be explained to the families.     

Distinct Muscle Biopsy Findings in Genetically Defined Adult-Onset Motor Neuron Disorders

The objective of this study was to characterize and compare muscle histopathological findings in 3 different genetic motor neuron disorders. We retrospectively re-assessed muscle biopsy findings in 23 patients with autosomal dominant lower motor neuron disease caused by p.G66V mutation in CHCHD10 (SMAJ), 10 X-linked spinal and bulbar muscular atrophy (SBMA) and 11 autosomal dominant c9orf72-mutated amyotrophic lateral sclerosis (c9ALS) patients. Distinct large fiber type grouping consisting of non-atrophic type IIA muscle fibers were 100% specific for the late-onset spinal muscular atrophies (SMAJ and SBMA) and were never observed in c9ALS. Common, but less specific findings included small groups of highly atrophic rounded type IIA fibers in SMAJ/SBMA, whereas in c9ALS, small group atrophies consisting of small-caliber angular fibers involving both fiber types were more characteristic. We also show that in the 2 slowly progressive motor neuron disorders (SMAJ and SBMA) the initial neurogenic features are often confused with considerable secondary “myopathic” changes at later disease stages, such as rimmed vacuoles, myofibrillar aggregates and numerous fibers reactive for fetal myosin heavy chain (dMyHC) antibodies. Based on our findings, muscle biopsy may be valuable in the diagnostic work-up of suspected motor neuron disorders in order to avoid a false ALS diagnosis in patients without clear findings of upper motor neuron lesions.     

The most prevalent genetic cause of ALS-FTD,C9orf72 synergizes the toxicity of ATXN2 intermediate polyglutamine repeats through the autophagy pathway

The most common genetic cause for amyotrophic lateral sclerosis and frontotemporal dementia (ALS-FTD) is repeat expansion of a hexanucleotide sequence (GGGGCC) within the C9orf72 genomic sequence. To elucidate the functional role of C9orf72 in disease pathogenesis, we identified certain molecular interactors of this factor. We determined that C9orf72 exists in a complex with SMCR8 and WDR41 and that this complex acts as a GDP/GTP exchange factor for RAB8 and RAB39, 2 RAB GTPases involved in macroautophagy/autophagy. Consequently, C9orf72 depletion in neuronal cultures leads to accumulation of unresolved aggregates of SQSTM1/p62 and phosphorylated TARDBP/TDP-43. However, C9orf72 reduction does not lead to major neuronal toxicity, suggesting that a second stress may be required to induce neuronal cell death. An intermediate size of polyglutamine repeats within ATXN2 is an important genetic modifier of ALS-FTD. We found that coexpression of intermediate polyglutamine repeats (30Q) of ATXN2 combined with C9orf72 depletion increases the aggregation of ATXN2 and neuronal toxicity. These results were confirmed in zebrafish embryos where partial C9orf72 knockdown along with intermediate (but not normal) repeat expansions in ATXN2 causes locomotion deficits and abnormal axonal projections from spinal motor neurons. These results demonstrate that C9orf72 plays an important role in the autophagy pathway while genetically interacting with another major genetic risk factor, ATXN2, to contribute to ALS-FTD pathogenesis.