Two modes of Cue2-mediated mRNA cleavage with distinct substrate recognition initiate no-go decay

Ribosome collisions are recognized by E3 ubiquitin ligase Hel2/ZNF598, leading to RQC (ribosome-associated quality control) and to endonucleolytic cleavage and degradation of the mRNA termed NGD (no-go decay). NGD in yeast requires the Cue2 endonuclease and occurs in two modes, either coupled to RQC (NGD^RQC+) or RQC uncoupled (NGD^RQC−). This is mediated by an unknown mechanism of substrate recognition by Cue2. Here, we show that the ubiquitin binding activity of Cue2 is required for NGD^RQC− but not for NGD^RQC+, and that it involves the first two N-terminal Cue domains. In contrast, Trp122 of Cue2 is crucial for NGD^RQC+. Moreover, Mbf1 is required for quality controls by preventing +1 ribosome frameshifting induced by a rare codon staller. We propose that in Cue2-dependent cleavage upstream of the collided ribosomes (NGD^RQC−), polyubiquitination of eS7 is recognized by two N-terminal Cue domains of Cue2. In contrast, for the cleavage within collided ribosomes (NGD^RQC+), the UBA domain, Trp122 and the interaction between Mbf1 and uS3 are critical.     

C9ORF72 Mutations in Neurodegenerative Diseases

Recent works have demonstrated an expansion of the GGGGCC hexanucleotide repeat in the first intron of chromosome 9 open reading frame 72 (C9ORF72), encoding an unknown C9ORF72 protein, which was responsible for an unprecedented large proportion of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) cases of European ancestry. C9ORF72 is expressed in most tissues including the brain. Emerging evidence has demonstrated that C9ORF72 mutations could reduce the level of C9ORF72 variant 1, which may influence protein expression and the formation of nuclear RNA foci. The spectrum of mutations is broad and provides new insight into neurological diseases. Clinical manifestations of diseases related with C9ORF72 mutations can vary from FTD, ALS, primary lateral sclerosis (PLS), progressive muscular atrophy (PMA), Huntington disease-like syndrome (HDL syndrome), to Alzheimer's disease. In this article, we will review the brief characterizations of the C9ORF72 gene, the expansion mutations, the related disorders, and their features, followed by a discussion of the deficiency knowledge of C9ORF72 mutations. Based on the possible pathological mechanisms of C9ORF72 mutations in ALS and FTD, we can find new targets for the treatment of C9ORF72 mutation-related diseases. Future studies into the mechanisms, taking into consideration the discovery of those disorders, will significantly accelerate new discoveries in this field, including targeting identification of new therapy.     

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.     

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.     

Neuroimmune dysfunction in frontotemporal dementia: Insights from progranulin and C9orf72 deficiency

Neuroimmune dysfunction is a cardinal feature of neurodegenerative diseases. But how immune dysregulation in the brain and peripheral organs contribute to neurodegeneration remains unclear. Here, we discuss the recent advances highlighting neuroimmune dysfunction as a key disease-driving factor in frontotemporal dementia (FTD). We provide an overview of the clinical observations supporting a high prevalence of autoimmune diseases in FTD patients with mutations in GRN or C9orf72. We then focus on a myriad of evidence from human genetic studies, mouse models, in vitro assays, and multi-omics platform, which indicate that haploinsufficiency in GRN and C9orf72 promotes neuroimmune dysfunction and contributes to neurodegeneration and premature death. These compelling data provide key insights to disease mechanisms, biomarker discovery, and therapeutic interventions for FTD (120 words).     

Lost & found: C9ORF72 and the autophagy pathway in ALS/FTD

C9ORF72 expression is reduced in a substantial number of patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), which may contribute to disease pathogenesis. However, its normal molecular function remains unknown. In this issue of The EMBO Journal, Sellier et al ( 2016 ) identified a novel protein complex consisting of C9ORF72, WDR41, and SMCR8 that acts as a GDP‐GTP exchange factor (GEF) for RAB8a and RAB39b and is regulated by TBK1, whose partial loss of function also causes ALS and FTD. They further reveal a potential modulatory role for this novel complex in macroautophagy (autophagy), especially in the context of ataxin‐2 toxicity.     

Protein products of nonstop mRNA disrupt nucleolar homeostasis

Stalled mRNA translation results in the production of incompletely synthesized proteins that are targeted for degradation by ribosome-associated quality control (RQC). Here we investigated the fate of defective proteins translated from stall-inducing, nonstop mRNA that escape ubiquitylation by the RQC protein LTN1. We found that nonstop protein products accumulated in nucleoli and this localization was driven by polylysine tracts produced by translation of the poly(A) tails of nonstop mRNA. Nucleolar sequestration increased the solubility of invading proteins but disrupted nucleoli, altering their dynamics, morphology, and resistance to stress in cell culture and intact flies. Our work elucidates how stalled translation may affect distal cellular processes and may inform studies on the pathology of diseases caused by failures in RQC and characterized by nucleolar stress.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12192-021-01200-w.     

Downregulation of MicroRNA-9 in iPSC-Derived Neurons of FTD/ALS Patients with TDP-43 Mutations

Transactive response DNA-binding protein 43 (TDP-43) is a major pathological protein in frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). There are many disease-associated mutations in TDP-43, and several cellular and animal models with ectopic overexpression of mutant TDP-43 have been established. Here we sought to study altered molecular events in FTD and ALS by using induced pluripotent stem cell (iPSC) derived patient neurons. We generated multiple iPSC lines from an FTD/ALS patient with the TARDBP A90V mutation and from an unaffected family member who lacked the mutation. After extensive characterization, two to three iPSC lines from each subject were selected, differentiated into postmitotic neurons, and screened for relevant cell-autonomous phenotypes. Patient-derived neurons were more sensitive than control neurons to 100 nM straurosporine but not to other inducers of cellular stress. Three disease-relevant cellular phenotypes were revealed under staurosporine-induced stress. First, TDP-43 was localized in the cytoplasm of a higher percentage of patient neurons than control neurons. Second, the total TDP-43 level was lower in patient neurons with the A90V mutation. Third, the levels of microRNA-9 (miR-9) and its precursor pri-miR-9-2 decreased in patient neurons but not in control neurons. The latter is likely because of reduced TDP-43, as shRNA-mediated TDP-43 knockdown in rodent primary neurons also decreased the pri-miR-9-2 level. The reduction in miR-9 expression was confirmed in human neurons derived from iPSC lines containing the more pathogenic TARDBP M337V mutation, suggesting miR-9 downregulation might be a common pathogenic event in FTD/ALS. These results show that iPSC models of FTD/ALS are useful for revealing stress-dependent cellular defects of human patient neurons containing rare TDP-43 mutations in their native genetic contexts.     

1,3-Di(quinolin-2-yl)guanidine binds to GGCCCC hexanucleotide repeat DNA in C9ORF72

Aberrant expansion of GGGGCC (G4C2) hexanucleotide repeat (HNR) in the first intron of C9ORF72 has been found in frontotemporal dementia and amyotrophic lateral sclerosis (FTD/ALD). The non-canonical DNA structures of the expanded repeats are causative to repeat instability leading to contraction and expansion. We demonstrate that 1,3-di(quinolin-2-yl)guanidine (DQG) binds to GGCCCC/GGCCCC (G2C4/G2C4) motif in double stranded DNA and also antisense G2C4 HNR DNA in C9ORF72. Large increase in the melting temperature of dsDNA containing the G2C4/G2C4 motif was confirmed by the binding of DQG. The marked CD spectral changes indicated structural transition of d(G2C4)₉ from i-motifs and/or hairpins to DQG-stabilized d(G2C4)₉ structures.     

Dysregulated molecular pathways in amyotrophic lateral sclerosis–frontotemporal dementia spectrum disorder

Frontotemporal dementia (FTD), the second most common form of dementia in people under 65 years of age, is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD overlaps extensively with the motor neuron disease amyotrophic lateral sclerosis (ALS), especially at the genetic level. Both FTD and ALS can be caused by many mutations in the same set of genes; the most prevalent of these mutations is a GGGGCC repeat expansion in the first intron of C9ORF72. As shown by recent intensive studies, some key cellular pathways are dysregulated in the ALS‐FTD spectrum disorder, including autophagy, nucleocytoplasmic transport, DNA damage repair, pre‐mRNA splicing, stress granule dynamics, and others. These exciting advances reveal the complexity of the pathogenic mechanisms of FTD and ALS and suggest promising molecular targets for future therapeutic interventions in these devastating disorders.     

Deletion of C9ORF72 Results in Motor Neuron Degeneration and Stress Sensitivity in C. elegans

An expansion of the hexanucleotide GGGGCC repeat in the first intron of C9ORF72 gene was recently linked to amyotrophic lateral sclerosis. It is not known if the mutation results in a gain of function, a loss of function or if, perhaps both mechanisms are linked to pathogenesis. We generated a genetic model of ALS to explore the biological consequences of a null mutation of the Caenorhabditis elegans C9ORF72 orthologue, F18A1.6, also called alfa-1. alfa-1 mutants displayed age-dependent motility defects leading to paralysis and the specific degeneration of GABAergic motor neurons. alfa-1 mutants showed differential susceptibility to environmental stress where osmotic stress provoked neurodegeneration. Finally, we observed that the motor defects caused by loss of alfa-1 were additive with the toxicity caused by mutant TDP-43 proteins, but not by the mutant FUS proteins. These data suggest that a loss of alfa-1/C9ORF72 expression may contribute to motor neuron degeneration in a pathway associated with other known ALS genes.     

Long non-coding RNA ZNF667-AS1 retards the development of esophageal squamous cell carcinoma via modulation of microRNA-1290-mediated PRUNE2

Abnormal long non-coding RNAs (lncRNAs) have been detected in esophageal squamous cell carcinoma (ESCC). Here, we focused on lncRNA ZNF667-AS1 and its downstream mechanism in ESCC progression. Differentially expressed lncRNAs in ESCC were predicted by bioinformatics analysis. ZNF667-AS1, microRNA-1290 (miR-1290), and prune homolog 2 with BCH domain (PRUNE2) expression was determined with their relationship in cell biological processes analyzed also by means of gain- and loss-of-function assays. Xenograft mouse models were performed to re-produce the in vitro findings. We found a decline in ZNF667-AS1 expression in ESCC tissues and cell lines. ZNF667-AS1 overexpression indicated a favorable prognosis of ESCC sufferers. ZNF667-AS1 overexpression suppressed ESCC cell malignant potentials. ZNF667-AS1 reduced miR-1290 to result in upregulation of the miR-1290 target gene PRUNE2. The inhibiting property of ZNF667-AS1 on the malignant characteristics of ESCC cells was achieved by disrupting the miR-1290-mediated downregulation of PRUNE2. ZNF667-AS1 suppressed the tumorigenesis of ESCC in vivo. Collectively, our study demonstrates that ZNF667-AS1 can function as a tumor suppressor in ESCC by upregulating PRUNE2 and downregulating miR-1290.     

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.