Chromosome 9 Open Reading Frame 72 Research Articles

Midpoint baseline results of the Longitudinal Early‐Onset Alzheimer’s Disease Study (LEADS)

AbstractBackgroundFewer than 5% of patients with Alzheimer’s disease are diagnosed between 40‐64 years old, making “early‐onset Alzheimer’s disease” (EOAD) an uncommon phenomenon (Zu et al., 2015). To date, only basic knowledge on EOAD is known, no uniform criteria for EOAD have been implemented, and sample sizes in research have been small. The Longitudinal Early‐Onset Alzheimer’s Disease Study (LEADS) aims to provide a comprehensive understanding of EOAD symptomology, with the current project profiling baseline clinical, cognitive, and basic genetic characteristics of the cohort nearing the data‐collection mid‐point.MethodData from 371 participants undergoing the LEADS protocol were compared based on diagnostic group classification (cognitively normal [CN], amyloid‐positive EOAD, and amyloid‐negative Early‐Onset non‐Alzheimer’s disease [EOnonAD]). Because LEADS focuses on sporadic early‐onset dementia, impaired individuals with genetic mutations in Amyloid Precursor Protein (APP), Presenilin‐1 (PSEN1) or Presenilin‐2 (PSEN2), Microtubule Associated Protein Tau (MAPT), Chromosome 9 Open Reading Frame 72 (C9ORF72), or Granulin Precursor aka Progranulin (GRN) were excluded.ResultDemographic characteristics for each group are shown in Table 1. After adjusting for age, education, and sex, EOAD performed worse than CN for all domains (ps<.001, η2s = 0.05‐0.66), and worse than EOnonAD for all domains except language (ps<.001, η2s = 0.06‐0.29). As shown in Table 2, EOAD showed worse performance on Episodic Memory (z = ‐2.38), followed by Speed Attention (z = ‐1.93) and Executive domains (z = ‐1.43) relative to CN. An amnestic presentation was common among impaired participants, with several clinical phenotypes present. The EOAD group possessed a significantly greater proportion of apolipoprotein ε4 homozygous participants than the EOnonAD group (p = .01, Cohen’s w = 0.15), and a trend of greater proportions of ε4 heterozygous participants than both CN and EOnonAD (ps = .05‐.07, Cohen’s ws = 0.11; Table 3). Finally, LEADS participants generally consented at high rates to optional trial procedures like lumbar puncture (60%) and genetic disclosure (89%).ConclusionThis work represents the best baseline characterization of sporadic EOAD in the U.S. to date. Early‐onset dementia presents with widespread cognitive impairment, characterized by several known clinical phenotypes. Apolipoprotein ε4 allele carrier status appears to remain important in EOAD. Future investigation will explore many potential associations between EOAD and cognition, neuropsychiatric symptoms, genetics, and imaging.

Artificial microRNA suppresses C9ORF72 variants and decreases toxic dipeptide repeat proteins in vivo.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease that affects motor neurons, causing progressive muscle weakness and respiratory failure. The presence of an expanded hexanucleotide repeat in chromosome 9 open reading frame 72 (C9ORF72) is the most frequent mutation causing familial ALS and frontotemporal dementia (FTD). To determine if suppressing expression of C9ORF72 gene products can reduce toxicity, we designed a set of artificial microRNAs (amiRNA) targeting the human C9ORF72 gene. Here we report that an AAV9-mediated amiRNA significantly suppresses expression of the C9ORF72 mRNA, protein, and toxic dipeptide repeat proteins generated by the expanded repeat in the brain and spinal cord of C9ORF72 transgenic mice.

Disrupted phase behavior of FUS underlies poly-PR-induced DNA damage in amyotrophic lateral sclerosis.

GGGGCC (G4C2) hexanucleotide repeat expansion (HRE) in the first intron of the chromosome 9 open reading frame 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Among the five dipeptide repeat proteins translated from G4C2 HRE, arginine-rich poly-PR (proline:arginine) is extremely toxic. However, the molecular mechanism responsible for poly-PR-induced cell toxicity remains incompletely understood. Here, we found that poly-PR overexpression triggers severe DNA damage in cultured cells, primary cortical neurons, and the motor cortex of a poly-PR transgenic mouse model. Interestingly, we identified a linkage between poly-PR and RNA-binding protein fused in sarcoma (FUS), another ALS-related gene product associated with DNA repair. Poly-PR interacts with FUS both in vitro and in vivo, phase separates with FUS in a poly-PR concentration-dependent manner, and impairs the fluidity of FUS droplets in vitro and in cells. Moreover, poly-PR impedes the recruitment of FUS and its downstream protein XRCC1 to DNA damage foci after microirradiation. Importantly, overexpression of FUS significantly decreased the level of DNA damage and dramatically reduced poly-PR-induced cell death. Our data suggest the severe DNA damage caused by poly-PR and highlight the interconnection between poly-PR and FUS, enlightening the potential therapeutic role of FUS in alleviating poly-PR-induced cell toxicity.

CRISPR interference to evaluate modifiers of C9ORF72-mediated toxicity in FTD.

Treatments for neurodegenerative disease, including Frontotemporal dementia (FTD) and Amyotrophic lateral sclerosis (ALS), remain rather limited, underscoring the need for greater mechanistic insight and disease-relevant models. Our ability to develop novel disease models of genetic risk factors, disease modifiers, and other FTD/ALS-relevant targets is impeded by the significant amount of time and capital required to develop conventional knockout and transgenic mice. To overcome these limitations, we have generated a novel CRISPRi interference (CRISPRi) knockin mouse. CRISPRi uses a catalytically dead form of Cas9, fused to a transcriptional repressor to knockdown protein expression, following the introduction of single guide RNA against the gene of interest. To validate the utility of this model we have selected the TAR DNA binding protein (TDP-43) splicing target, stathmin-2 (STMN2). STMN2 RNA is downregulated in FTD/ALS due to loss of TDP-43 activity and STMN2 loss is suggested to play a role in ALS pathogenesis. The involvement of STMN2 loss of function in FTD has yet to be determined. We find that STMN2 protein levels in familial FTD cases are significantly reduced compared to controls, supporting that STMN2 depletion may be involved in the pathogenesis of FTD. Here, we provide proof-of-concept that we can simultaneously knock down Stmn2 and express the expanded repeat in the Chromosome 9 open reading frame 72 (C9ORF72) gene, successfully replicating features of C9-associated pathology. Of interest, depletion of Stmn2 had no effect on expression or deposition of dipeptide repeat proteins (DPRs), but significantly decreased the number of phosphorylated Tdp-43 (pTdp-43) inclusions. We submit that our novel CRISPRi mouse provides a versatile and rapid method to silence gene expression in vivo and propose this model will be useful to understand gene function in isolation or in the context of other neurodegenerative disease models.

Involvement of striatal motoric subregions in familial frontotemporal dementia with parkinsonism harboring the C9orf72 repeat expansions

AbstractBackgroundThe chromosome 9 open reading frame 72 (C9ORF72) has been proposed as the causative gene of frontotemporal dementia with parkinsonism (FTDP), but its pathophysiological mechanism of parkinsonism is poorly understood.MethodTo explore the roles of striatal motor subdivisions in the pathogenesis of parkinsonism resulting from C9ORF72 repeat expansions in the FTDP, two patients with FTDP from one pedigree and seventeen healthy controls were enrolled. The participants received clinical interviews and underwent neuropsychological assessments, genetic testing, [18F]‐fluorodeoxyglucose PET/MRI, and [18F]‐dihydrotetrabenazine PET/CT. Voxel‐wise and region of interest analysis were conducted with respect to gray matter volume, metabolism, and dopamine transport function between patients and controls, focusing on the motor part of the striatum according to the Oxford‐GSK‐Imanova Striatal Connectivity Atlas.ResultPatient 1 presented with parkinsonism as the initial symptom, while patient 2 exhibited behavior disturbance as the first symptom, followed by parkinsonism within one year. Both patients had the hexanucleotide expansion detected in C9ORF72(>52 repeats). Gray matter volume atrophy, hypometabolism and dopamine dysfunction were observed in the motor areas of the striatum. Of the two patients, marked glucose hypometabolism within the striatal motor subregion was observed in patient 1, with corresponding gray matter atrophy. In addition, presynaptic dopaminergic integrity of patient 2 was deteriorated in the motor subregions which was consistent with gray matter atrophy.ConclusionThese findings provide evidence that C9orf72 repeat expansions could result in degeneration and dopaminergic dysfunction of the striatal motor subregion, which contributed to parkinsonism in FTDP.

Mutation-specific metabolic profiles in presymptomatic amyotrophic lateral sclerosis.

Growing evidence shows that ALS patients feature a disturbed energy metabolism. However, these features have rarely been investigated in the presymptomatic stage. A total of 60 presymptomatic ALS mutation carriers and 70 age- and gender-matched controls (non-mutation carriers from the same families) were recruited. All subjects underwent assessments of their metabolic profiles under fasting conditions at enrollment, including body mass index (BMI), blood pressure and serum levels of blood glucose, total cholesterol, triglycerides, high-density lipoprotein (HDL) and low-density lipoprotein. All mutations combined, no differences between presymptomatic ALS gene carriers and controls were found. From a cardiovascular point of view, presymptomatic chromosome 9 open reading frame 72 (C9ORF72) gene carriers showed lower cardiovascular risk profiles compared to healthy controls, including lower BMI (median 22.9, interquartile range [IQR] 20.6-26.1 kg/m2 vs. 24.9, IQR 22.7-30.5 kg/m2 ; p=0.007), lower systolic blood pressure (120, IQR 110-130 mmHg vs. 128, IQR 120-140 mmHg; p=0.02), lower fasting serum glucose (89.0, IQR 85.0-97.0 mg/dl vs. 96.0, IQR 89.3-102.0 mg/dl; p=0.005) and higher HDL (1.6, IQR 1.3-1.8 mmol/l vs. 1.2, IQR 1.0-1.4 mmol/l; p=0.04). However, presymptomatic superoxide dismutase 1 (SOD1) gene mutation carriers showed higher cardiovascular risk profiles compared to healthy controls, including higher BMI (28.0,IQR 26.1-31.5 kg/m2 vs. 24.9, IQR 22.7-30.5 kg/m2 ; p=0.02), higher fasting serum glucose (100.0, IQR 94.0-117.0 mg/dl vs. 96.0, IQR 89.3-102.0 mg/dl; p=0.04) and lower HDL (1.2, IQR 1.0-1.4 mmol/l vs. 1.4, IQR 1.2-1.7 mmol/l; p=0.01). These features were most prominent in patients carrying SOD1 gene mutations associated with slow disease progression. This study identified distinct metabolic profiles in presymptomatic ALS gene carriers, which might be associated with disease progression in the symptomatic phase.

Involvement of striatal motoric subregions in familial frontotemporal dementia with parkinsonism harboring the C9orf72 repeat expansions

The chromosome 9 open reading frame 72 (C9ORF72) has been proposed as the causative gene of frontotemporal dementia with parkinsonism (FTDP), but its pathophysiological mechanism of parkinsonism is poorly understood. To explore the roles of striatal motor subdivisions in the pathogenesis of parkinsonism resulting from C9ORF72 repeat expansions in the FTDP, two patients with FTDP from one pedigree and seventeen healthy controls were enrolled. The participants received clinical interviews, physical examinations, genetic testing, [18F]-fluorodeoxyglucose PET/MRI, and [18F]-dihydrotetrabenazine PET/CT. Voxel-wise and region of interest analysis were conducted with respect to gray matter volume, metabolism, and dopamine transport function between patients and controls, focusing on the motor part of the striatum according to the Oxford-GSK-Imanova Striatal Connectivity Atlas. Patient 1 presented with parkinsonism as the initial symptom, while patient 2 exhibited behavior disturbance as the first symptom, followed by parkinsonism within one year. Both patients had the hexanucleotide expansion detected in C9ORF72(>52 repeats). Gray matter volume atrophy, hypometabolism and dopamine dysfunction were observed in the motor areas of the striatum. Of the two patients, marked glucose hypometabolism within the striatal motor subregion was observed in patient 1, with corresponding gray matter atrophy. In addition, presynaptic dopaminergic integrity of patient 2 was deteriorated in the motor subregions which was consistent with gray matter atrophy. These findings imply that parkinsonism in FTDP may be associated with the degeneration and dopaminergic dysfunction of the striatal motor subregion, which might be attributed to C9orf72 repeat expansions.

Synaptic dysfunction in ALS and FTD: anatomical and molecular changes provide insights into mechanisms of disease.

Synaptic loss is a pathological feature of all neurodegenerative diseases including amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). ALS is a disease of the cortical and spinal motor neurons resulting in fatal paralysis due to denervation of muscles. FTD is a form of dementia that primarily affects brain regions controlling cognition, language and behavior. Once classified as two distinct diseases, ALS and FTD are now considered as part of a common disease spectrum based on overlapping clinical, pathological and genetic evidence. At the cellular level, aggregation of common proteins and overlapping gene susceptibilities are shared in both ALS and FTD. Despite the convergence of these two fields of research, the underlying disease mechanisms remain elusive. However, recent discovers from ALS and FTD patient studies and models of ALS/FTD strongly suggests that synaptic dysfunction is an early event in the disease process and a unifying hallmark of these diseases. This review provides a summary of the reported anatomical and cellular changes that occur in cortical and spinal motor neurons in ALS and FTD tissues and models of disease. We also highlight studies that identify changes in the proteome and transcriptome of ALS and FTD models and provide a conceptual overview of the processes that contribute to synaptic dysfunction in these diseases. Due to space limitations and the vast number of publications in the ALS and FTD fields, many articles have not been discussed in this review. As such, this review focuses on the three most common shared mutations in ALS and FTD, the hexanucleuotide repeat expansion within intron 1 of chromosome 9 open reading frame 72 (C9ORF72), transactive response DNA binding protein 43 (TARDBP or TDP-43) and fused in sarcoma (FUS), with the intention of highlighting common pathways that promote synaptic dysfunction in the ALS-FTD disease spectrum.

Correlation between C9ORF72 mutation and neurodegenerative diseases: A comprehensive review of the literature.

Chromosome 9 open reading frame 72 (C9ORF72) encodes a 54-kDa protein with unknown function that is expressed at high levels in the central nervous system. The C9ORF72 hexanucleotide amplification is one of the most recently discovered repetitive amplification diseases related to neurodegeneration. Its association with amyotrophic lateral sclerosis/frontotemporal dementia (ALS/FTD) spectrum diseases has been fully established, although a causative role for C9ORF72 in Alzheimer's disease (AD) and Parkinson's disease (PD) remains to be established. Therefore, in this article, we will review the evidence for C9ORF72 as a causative factor in neurodegenerative diseases, the underlying mechanisms, and the potential for targeting C9ORF72 as a strategy to alleviate neurodegenerative disease progression.

Co‐occurring mutations of optineurin (OPTN) and colony‐stimulating factor‐1 receptor (CSF1R) genes in a family with familial frontotemporal dementia

AbstractBackgroundWe report the clinical profiles and genetics of multiple family members from Samar, Philippines presenting with varying phenotypes of frontotemporal dementia. Literature studies report three gene mutations associated with a majority of fFTD cases, namely the microtubule associated protein tau gene (MAPT), and progranulin gene (PGRN), chromosome 9 open reading frame 72 (C9ORF72). These were all excluded in our case.MethodsClinical examination, Cranial MRI, and FDG‐PET were done. Genomic DNA was extracted from blood samples and analyzed via targeted exome sequencing.ResultA frameshift mutation in the Optineurin (OPTN Chr 10:13166090 G>GA) was identified in multiple family members. This mutation is absent in public large international databases. Clinical and genetic studies show that frontotemporal dementia and motor neuron disease, in particular amyotrophic lateral sclerosis, are part of a disease spectrum with a common pathogenesis. However, there was no clinical or diagnostic evidence of motor neuron disease in family members with Optineurin mutations. A second mutation‐ missense mutation in the Colony‐Stimulating Factor‐1R V235G genes was seen in several family members. Colony‐Stimulating Factor‐1R gene mutations are associated with leukodystrophies when occurring singly. Clinical presentation and diagnostic tests did not support any of these findings.ConclusionAt present, this is the only reported family with such combined mutations and genotype‐phenotype discordance. We are further investigating this family by conducting whole exome sequencing.

Cell-Clearing Systems Bridging Repeat Expansion Proteotoxicity and Neuromuscular Junction Alterations in ALS and SBMA.

The coordinated activities of autophagy and the ubiquitin proteasome system (UPS) are key to preventing the aggregation and toxicity of misfold-prone proteins which manifest in a number of neurodegenerative disorders. These include proteins which are encoded by genes containing nucleotide repeat expansions. In the present review we focus on the overlapping role of autophagy and the UPS in repeat expansion proteotoxicity associated with chromosome 9 open reading frame 72 (C9ORF72) and androgen receptor (AR) genes, which are implicated in two motor neuron disorders, amyotrophic lateral sclerosis (ALS) and spinal-bulbar muscular atrophy (SBMA), respectively. At baseline, both C9ORF72 and AR regulate autophagy, while their aberrantly-expanded isoforms may lead to a failure in both autophagy and the UPS, further promoting protein aggregation and toxicity within motor neurons and skeletal muscles. Besides proteotoxicity, autophagy and UPS alterations are also implicated in neuromuscular junction (NMJ) alterations, which occur early in both ALS and SBMA. In fact, autophagy and the UPS intermingle with endocytic/secretory pathways to regulate axonal homeostasis and neurotransmission by interacting with key proteins which operate at the NMJ, such as agrin, acetylcholine receptors (AChRs), and adrenergic beta2 receptors (B2-ARs). Thus, alterations of autophagy and the UPS configure as a common hallmark in both ALS and SBMA disease progression. The findings here discussed may contribute to disclosing overlapping molecular mechanisms which are associated with a failure in cell-clearing systems in ALS and SBMA.

Motor dysfunction and neurodegeneration in a C9orf72 mouse line expressing poly-PR

A GGGGCC hexanucleotide repeat expansion in intron 1 of chromosome 9 open reading frame 72 (C9ORF72) gene is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Repeat-associated non-ATG translation of dipeptide repeat proteins (DPRs) contributes to the neuropathological features of c9FTD/ALS. Among the five DPRs, arginine-rich poly-PR are reported to be the most toxic. Here, we generate a transgenic mouse line that expresses poly-PR (GFP-PR28) specifically in neurons. GFP-PR28 homozygous mice show decreased survival time, while the heterozygous mice show motor imbalance, decreased brain weight, loss of Purkinje cells and lower motor neurons, and inflammation in the cerebellum and spinal cord. Transcriptional analysis shows that in the cerebellum, GFP-PR28 heterozygous mice show differential expression of genes related to synaptic transmission. Our findings show that GFP-PR28 transgenic mice partly model neuropathological features of c9FTD/ALS, and show a role for poly-PR in neurodegeneration.

Amyotrophic Lateral Sclerosis-associated GGGGCC repeat expansion promotes Tau phosphorylation and toxicity

Microtubule-associated protein Tau (MAPT) and GGGGCC (G4C2) repeat expansion in chromosome 9 open reading frame 72 (C9ORF72) are the major known genetic causes of frontotemporal dementia (FTD) and other neurodegenerative diseases, such as Amyotrophic Lateral Sclerosis (ALS). Although expanded G4C2 repeats and Tau traditionally are associated with different clinical presentations, pathological and genetic studies have suggested a strong association between them. Here we demonstrate a strong genetic interaction between expanded G4C2 repeats and Tau. We found that co-expression of expanded G4C2 repeats and Tau could produce a synergistic deterioration of rough eyes, motor function, life span and neuromuscular junction morphological abnormalities in Drosophila. Mechanistically, compared with the normal allele containing (G4C2)3 repeats, the (G4C2)30 allele increased Tau phosphorylation levels and promoted Tau R406W aggregation. These results together suggest a potential crosstalk between expanded G4C2 repeats and Tau in modulating neurodegeneration.

18F-flortaucipir (AV-1451) tau PET in frontotemporal dementia syndromes

BackgroundThe tau positron emission tomography (PET) ligand 18F-flortaucipir binds to paired helical filaments of tau in aging and Alzheimer’s disease (AD), but its utility in detecting tau aggregates in frontotemporal dementia (FTD) is uncertain.MethodsWe performed 18F-flortaucipir imaging in patients with the FTD syndromes (n = 45): nonfluent variant primary progressive aphasia (nfvPPA) (n = 11), corticobasal syndrome (CBS) (n = 10), behavioral variant frontotemporal dementia (bvFTD) (n = 10), semantic variant primary progressive aphasia (svPPA) (n = 2) and FTD associated pathogenic genetic mutations microtubule-associated protein tau (MAPT) (n = 6), chromosome 9 open reading frame 72 (C9ORF72) (n = 5), and progranulin (GRN) (n = 1). All patients underwent MRI and β-amyloid biomarker testing via 11C-PiB or cerebrospinal fluid. 18F-flortaucipir uptake in patients was compared to 53 β-amyloid negative normal controls using voxelwise and pre-specified region of interest approaches.ResultsOn qualitative assessment, patients with nfvPPA showed elevated 18F-flortacupir binding in the left greater than right inferior frontal gyrus. Patients with CBS showed elevated binding in frontal white matter, with higher cortical gray matter uptake in a subset of β-amyloid-positive patients. Five of ten patients with sporadic bvFTD demonstrated increased frontotemporal binding. MAPT mutation carriers had elevated 18F-flortaucipir retention primarily, but not exclusively, in mutations with Alzheimer’s-like neurofibrillary tangles. However, tracer retention was also seen in patients with svPPA, and the mutations C9ORF72, GRN predicted to have TDP-43 pathology. Quantitative region-of-interest differences between patients and controls were seen only in inferior frontal gyrus in nfvPPA and left insula and bilateral temporal poles in MAPT carriers. No significant regional differences were found in CBS or sporadic bvFTD. Two patients underwent postmortem neuropathological examination. A patient with C9ORF72, TDP-43-type B pathology, and incidental co-pathology of scattered neurofibrillary tangles in the middle frontal, inferior temporal gyrus showed corresponding mild 18F-flortaucipir retention without additional uptake matching the widespread TDP-43 type B pathology. A patient with sporadic bvFTD demonstrated punctate inferior temporal and hippocampus tracer retention, corresponding to the area of severe argyrophilic grain disease pathology.Conclusions18F-flortaucipir in patients with FTD and predicted tauopathy or TDP-43 pathology demonstrated limited sensitivity and specificity. Further postmortem pathological confirmation and development of FTD tau-specific ligands are needed.

C9ORF72 dipeptide repeat poly-GA inclusions promote intracellular aggregation of phosphorylated TDP-43.

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration are neurodegenerative diseases characterized by accumulation of insoluble aggregates of phosphorylated 43kDa TAR DNA-binding protein (TDP-43) and linked with abnormal expansion of a hexanucleotide repeat in an intron of chromosome 9 open reading frame 72 (C9ORF72). However, the relationship between C9ORF72 mutations and TDP-43 aggregation remains unknown. Non-ATG-dependent translation of C9ORF72 repeats produces dipeptide repeat proteins, which form p62-positive aggregates in cerebral cortex and cerebellum of patients. Here, we show that the formation of poly-GA protein inclusions induced intracellular aggregation of endogenous and exogenous TDP-43 in cultured cells. Poly-GA aggregation preceded accumulation of phosphorylated TDP-43. These inclusions induced intracellular aggregation of phosphorylated TDP-43, but not tau or α-synuclein. Formation of phosphorylated TDP-43 aggregates depends on the number of poly-GA repeats. Detergent-insoluble fraction from cells co-expressing poly-GA and TDP-43 could function as seeds for further TDP-43 aggregation. These findings suggest a novel pathogenic mechanism that poly-GA protein aggregation directly promotes pathogenic changes of TDP-43 without the formation of nuclear RNA foci containing GGGGCC repeat expansion or loss-of-function of the C9ORF72 protein.

C9ORF72, implicated in amytrophic lateral sclerosis and frontotemporal dementia, regulates endosomal trafficking.

Intronic expansion of a hexanucleotide GGGGCC repeat in the chromosome 9 open reading frame 72 (C9ORF72) gene is the major cause of familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. However, the cellular function of the C9ORF72 protein remains unknown. Here, we demonstrate that C9ORF72 regulates endosomal trafficking. C9ORF72 colocalized with Rab proteins implicated in autophagy and endocytic transport: Rab1, Rab5, Rab7 and Rab11 in neuronal cell lines, primary cortical neurons and human spinal cord motor neurons, consistent with previous predictions that C9ORF72 bears Rab guanine exchange factor activity. Consistent with this notion, C9ORF72 was present in the extracellular space and as cytoplasmic vesicles. Depletion of C9ORF72 using siRNA inhibited transport of Shiga toxin from the plasma membrane to Golgi apparatus, internalization of TrkB receptor and altered the ratio of autophagosome marker light chain 3 (LC3) II:LC3I, indicating that C9ORF72 regulates endocytosis and autophagy. C9ORF72 also colocalized with ubiquilin-2 and LC3-positive vesicles, and co-migrated with lysosome-stained vesicles in neuronal cell lines, providing further evidence that C9ORF72 regulates autophagy. Investigation of proteins interacting with C9ORF72 using mass spectrometry identified other proteins implicated in ALS; ubiquilin-2 and heterogeneous nuclear ribonucleoproteins, hnRNPA2/B1 and hnRNPA1, and actin. Treatment of cells overexpressing C9ORF72 with proteasome inhibitors induced the formation of stress granules positive for hnRNPA1 and hnRNPA2/B1. Immunohistochemistry of C9ORF72 ALS patient motor neurons revealed increased colocalization between C9ORF72 and Rab7 and Rab11 compared with controls, suggesting possible dysregulation of trafficking in patients bearing the C9ORF72 repeat expansion. Hence, this study identifies a role for C9ORF72 in Rab-mediated cellular trafficking.

Abnormal expression of homeobox genes and transthyretin in C9ORF72 expansion carriers.

Objective:We performed a genome-wide brain expression study to reveal the underpinnings of diseases linked to a repeat expansion in chromosome 9 open reading frame 72 (C9ORF72).Methods:The genome-wide expression profile was investigated in brain tissue obtained from C9ORF72 expansion carriers (n = 32), patients without this expansion (n = 30), and controls (n = 20). Using quantitative real-time PCR, findings were confirmed in our entire pathologic cohort of expansion carriers (n = 56) as well as nonexpansion carriers (n = 31) and controls (n = 20).Results:Our findings were most profound in the cerebellum, where we identified 40 differentially expressed genes, when comparing expansion carriers to patients without this expansion, including 22 genes that have a homeobox (e.g., HOX genes) and/or are located within the HOX gene cluster (top hit: homeobox A5 [HOXA5]). In addition to the upregulation of multiple homeobox genes that play a vital role in neuronal development, we noticed an upregulation of transthyretin (TTR), an extracellular protein that is thought to be involved in neuroprotection. Pathway analysis aligned with these findings and revealed enrichment for gene ontology processes involved in (anatomic) development (e.g., organ morphogenesis). Additional analyses uncovered that HOXA5 and TTR levels are associated with C9ORF72 variant 2 levels as well as with intron-containing transcript levels, and thus, disease-related changes in those transcripts may have triggered the upregulation of HOXA5 and TTR.Conclusions:In conclusion, our identification of genes involved in developmental processes and neuroprotection sheds light on potential compensatory mechanisms influencing the occurrence, presentation, and/or progression of C9ORF72-related diseases.

In-depth clinico-pathological examination of RNA foci in a large cohort of C9ORF72 expansion carriers

A growing body of evidence suggests that a loss of chromosome 9 open reading frame 72 (C9ORF72) expression, formation of dipeptide-repeat proteins, and generation of RNA foci contribute to disease pathogenesis in amyotrophic lateral sclerosis and frontotemporal dementia. Although the levels of C9ORF72 transcripts and dipeptide-repeat proteins have already been examined thoroughly, much remains unknown about the role of RNA foci in C9ORF72-linked diseases. As such, we performed a comprehensive RNA foci study in an extensive pathological cohort of C9ORF72 expansion carriers (n = 63). We evaluated two brain regions using a newly developed computer-automated pipeline allowing recognition of cell nuclei and RNA foci (sense and antisense) supplemented by manual counting. In the frontal cortex, the percentage of cells with sense or antisense RNA foci was 26 or 12%, respectively. In the cerebellum, 23% of granule cells contained sense RNA foci and 1% antisense RNA foci. Interestingly, the highest percentage of cells with RNA foci was observed in cerebellar Purkinje cells (~70%). In general, more cells contained sense RNA foci than antisense RNA foci; however, when antisense RNA foci were present, they were usually more abundant. We also observed that an increase in the percentage of cells with antisense RNA foci was associated with a delayed age at onset in the frontal cortex (r = 0.43, p = 0.003), whereas no other associations with clinico-pathological features were seen. Importantly, our large-scale study is the first to provide conclusive evidence that RNA foci are not the determining factor of the clinico-pathological variability observed in C9ORF72 expansion carriers and it emphasizes that the distribution of RNA foci does not follow the pattern of neurodegeneration, stressing the complex interplay between different aspects of C9ORF72-related diseases.

The Role of Dipeptide Repeats in C9ORF72-Related ALS-FTD.

Expansion of a hexanucleotide (GGGGCC) repeat in the gene chromosome 9 open reading frame 72 (C9ORF72) is the most common cause of amyotrophic lateral sclerosis and frontotemporal dementia (FTD). Three non-exclusive mechanisms have been proposed to contribute to the pathology initiated by this genetic insult. First, it was suggested that decreased expression of the C9orf72 protein product may contribute to disease. Second, the recognition that C9ORF72-related disease is associated with accumulation of GGGGCC repeat-containing RNA in nuclear foci led to the suggestion that toxic gain of RNA function, perhaps related to sequestration of RNA-binding proteins, might be an important driver of disease. Third, it was subsequently appreciated that GGGGCC repeat-containing RNA undergoes unconventional translation to produce unnatural dipeptide repeat (DPR) proteins that accumulate in patient brain early in disease. DPRs translated from all six reading frames in either the sense or antisense direction of the hexanucleotide repeat result in the expression of five DPRs: glycine–alanine (GA), glycine–arginine (GR), proline–alanine (PA), proline–arginine (PR) and glycine–proline (GP; GP is generated from both the sense and antisense reading frames). However, the relative contribution of each DPR to disease pathogenesis remains unclear. Here, we review evidence for the contribution of each specific DPR to pathogenesis and examine the probable mechanisms through which these DPRs induce neurodegeneration. We also consider the association of the toxic DPRs with impaired RNA metabolism and alterations to the liquid-like state of non-membrane-bound organelles.

Genetic analysis of the SOD1 and C9ORF72 genes in Hungarian patients with amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the death of motor neurons. To date, more than 20 genes have been implicated in ALS, and of these, the 2 most frequently mutated are the superoxide dismutase 1 (SOD1) gene and the chromosome 9 open reading frame 72 (C9ORF72) gene. In this study, we aimed to investigate the contribution of these 2 Mendelian genes to the development of the disease in Hungarian ALS patients (n = 66). Direct sequencing of the SOD1 gene revealed a novel (p.Lys91ArgfsTer8) and 3 recurrent heterozygous mutations (p.Val14Met, p.Asp90Ala, and p.Leu144Phe) in 5 patients. The novel p.Lys91ArgfsTer8 mutation led to a frameshift causing the addition of 8 new amino acids, including a premature stop codon at position 99. The GGGGCC hexanucleotide repeat expansion of the C9ORF72 gene was present in 1 ALS patient. This study represents the first genetic analysis of 2 major ALS causative genes in a cohort of Hungarian ALS patients and contributes to the further understanding of the genetic and phenotypic diversity of ALS.