The Experts below are selected from a list of 20177076 Experts worldwide ranked by ideXlab platform
Stephen Moore - One of the best experts on this subject based on the ideXlab platform.
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adar2 mislocalization and widespread rna editing aberrations in C9orf72 mediated als ftd
Acta Neuropathologica, 2019Co-Authors: Stephen Moore, Eric Alsop, Ileana Lorenzini, Alexander Starr, Benjamin E Rabichow, Emily Mendez, Jennifer Levy, Camelia Burciu, Rebecca ReimanAbstract:The hexanucleotide repeat expansion GGGGCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent findings suggest that dysfunction of nuclear-cytoplasmic trafficking could affect the transport of RNA binding proteins in C9orf72 ALS/FTD. Here, we provide evidence that the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is mislocalized in C9orf72 repeat expansion mediated ALS/FTD. ADAR2 is responsible for adenosine (A) to inosine (I) editing of double-stranded RNA, and its function has been shown to be essential for survival. Here we show the mislocalization of ADAR2 in human induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) from C9orf72 patients, in mice expressing (G4C2)149, and in C9orf72 ALS/FTD patient postmortem tissue. As a consequence of this mislocalization we observe alterations in RNA editing in our model systems and across multiple brain regions. Analysis of editing at 408,580 known RNA editing sites indicates that there are vast RNA A to I editing aberrations in C9orf72-mediated ALS/FTD. These RNA editing aberrations are found in many cellular pathways, such as the ALS pathway and the crucial EIF2 signaling pathway. Our findings suggest that the mislocalization of ADAR2 in C9orf72 mediated ALS/FTD is responsible for the alteration of RNA processing events that may impact vast cellular functions, including the integrated stress response (ISR) and protein translation.
Rosa Rademakers - One of the best experts on this subject based on the ideXlab platform.
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C9orf72 promoter hypermethylation is reduced while hydroxymethylation is acquired during reprogramming of als patient cells
Experimental Neurology, 2016Co-Authors: Rustam Esanov, Rosa Rademakers, Leonard Petrucelli, Veronique V Belzil, Marka Van Blitterswijk, Dennis W. Dickson, Kinsley C Belle, Kevin B Boylan, Derek M Dykxhoorn, Joanne WuuAbstract:Among several genetic mutations known to cause amyotrophic lateral sclerosis (ALS), a hexanucleotide repeat expansion in the C9orf72 gene is the most common. In approximately 30% of C9orf72-ALS cases, 5-methylcytosine (5mC) levels within the C9orf72 promoter are increased, resulting in a modestly attenuated phenotype. The developmental timing of C9orf72 promoter hypermethylation and the reason why it occurs in only a subset of patients remain unknown. In order to model the acquisition of C9orf72 hypermethylation and examine the potential role of 5-hydroxymethylcytosine (5hmC), we generated induced pluripotent stem cells (iPSCs) from an ALS patient with C9orf72 promoter hypermethylation. Our data show that 5mC levels are reduced by reprogramming and then re-acquired upon neuronal specification, while 5hmC levels increase following reprogramming and are highest in iPSCs and motor neurons. We confirmed the presence of 5hmC within the C9orf72 promoter in post-mortem brain tissues of hypermethylated patients. These findings show that iPSCs are a valuable model system for examining epigenetic perturbations caused by the C9orf72 mutation and reveal a potential role for cytosine demethylation.
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Expanded C9orf72 Hexanucleotide Repeat in Depressive Pseudodementia
JAMA Neurology, 2014Co-Authors: Kevin F. Bieniek, Rosa Rademakers, Matthew C. Baker, Leonard Petrucelli, Marka Van Blitterswijk, Dennis W. DicksonAbstract:IMPORTANCE Expanded hexanucleotide repeats in C9orf72 are a common genetic cause of frontotemporal dementia and amyotrophic lateral sclerosis. Repeat expansions have also been detected infrequently in other disorders, including Alzheimer disease, dementia with Lewy bodies, and parkinsonian disorders. OBSERVATIONS A consecutive series of 31 cases from the brain bank for neurodegenerative disorders at Mayo Clinic was screened to assess the incidence of the expanded C9orf72 repeat in cases of depressive pseudodementia. The presence of the hexanucleotide repeat was established using immunohistochemistry with a highly disease-specific antibody (C9RANT), and was further validated in carriers using repeat-primed polymerase chain reaction and Southern blotting. Two individuals harbored the C9orf72 repeat expansion. Both patients were men with refractory depression. One patient experienced drug-induced parkinsonism and sudden-onset dementia, while the other patient had a more insidious disease course suspected to be Alzheimer disease. CONCLUSIONS AND RELEVANCE This report increases the range of clinicopathologic presentations of C9orf72 expanded hexanucleotide repeat to include psychiatric disorders such as depressive pseudodementia.
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reduced C9orf72 gene expression in c9ftd als is caused by histone trimethylation an epigenetic event detectable in blood
Acta Neuropathologica, 2013Co-Authors: Tania F. Gendron, Matthew C. Baker, Veronique V Belzil, Peter Bauer, Mercedes Prudencio, Caroline Stetler, Irene K Yan, Luc Pregent, Lillian M Daughrity, Rosa RademakersAbstract:Individuals carrying (GGGGCC) expanded repeats in the C9orf72 gene represent a significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Elucidating how these expanded repeats cause “c9FTD/ALS” has since become an important goal of the field. Toward this end, we sought to investigate whether epigenetic changes are responsible for the decrease in C9orf72 expression levels observed in c9FTD/ALS patients. We obtained brain tissue from ten c9FTD/ALS individuals, nine FTD/ALS cases without a C9orf72 repeat expansion, and nine disease control participants, and generated fibroblastoid cell lines from seven C9orf72 expanded repeat carriers and seven participants carrying normal alleles. Chromatin immunoprecipitation using antibodies for histone H3 and H4 trimethylated at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) revealed that these trimethylated residues bind strongly to C9orf72 expanded repeats in brain tissue, but not to non-pathogenic repeats. Our finding that C9orf72 mRNA levels are reduced in the frontal cortices and cerebella of c9FTD/ALS patients is consistent with trimethylation of these histone residues, an event known to repress gene expression. Moreover, treating repeat carrier-derived fibroblasts with 5-aza-2-deoxycytidine, a DNA and histone demethylating agent, not only decreased C9orf72 binding to trimethylated histone residues, but also increased C9orf72 mRNA expression. Our results provide compelling evidence that trimethylation of lysine residues within histones H3 and H4 is a novel mechanism involved in reducing C9orf72 mRNA expression in expanded repeat carriers. Of importance, we show that mutant C9orf72 binding to trimethylated H3K9 and H3K27 is detectable in blood of c9FTD/ALS patients. Confirming these exciting results using blood from a larger cohort of patients may establish this novel epigenetic event as a biomarker for c9FTD/ALS.
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frontotemporal lobar degeneration with tdp 43 proteinopathy and chromosome 9p repeat expansion in C9orf72 clinicopathologic correlation
Neuropathology, 2013Co-Authors: Eileen H Bigio, Matt Baker, Saman S Ahmadian, Manjari Mishra, Rakhee Ganti, Rosa Rademakers, Bing Bing Weitner, Alfred W. Rademaker, Sandra Weintraub, M.-marsel MesulamAbstract:Mutations in C9orf72 resulting in expanded hexanucleotide repeats were recently reported to be the underlying genetic abnormality in chromosome 9p-linked frontotemporal lobar degeneration with TAR DNA-binding protein of 43 kD (TDP-43) proteinopathy (FTLD-TDP), amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration with motor neuron disease (FTLD-MND). Several subsequent publications described the neuropathology as being similar to that seen in cases of FTLD-TDP and ALS without C9orf72 mutations, except that cases with mutations have p62 and ubiquitin positive, TDP-43 negative inclusions in cerebellum, hippocampus, neocortex, and basal ganglia. The identity of this protein is as yet unknown, and its significance is unclear. With the goal of potentially uncovering the significance of these TDP-43 negative inclusions, we compared the clinical, pathologic, and genetic characteristics in 5 cases of FTLD-TDP and FTLD-MND with C9orf72 mutations to 20 cases without mutations. We confirmed the apparent specificity of p62 positive, TDP-43 negative inclusions in cerebellum, hippocampus, cortex, and basal ganglia to FTLD with C9orf72 mutations. p62 positive, TDP-43 negative inclusions in hippocampus correlated with hippocampal atrophy, but no additional correlations were uncovered. However, although ambiguity of TDP sub-typing has previously been reported in cases with C9orf72 mutations, this is the first report to show that although most FTLD cases with C9orf72 mutations were TDP type B, some of the pathologic characteristics in these cases were more similar to TDP types A and C than to TDP type B FTLD cases without mutations. These features include greater cortical and hippocampal atrophy, greater ventricular dilatation, more neuronal loss and gliosis in temporal lobe and striatum, and TDP-43 positive fine neuritic profiles in the hippocampus in FTLD cases with C9orf72 mutations compared to FTLD-TDP type B cases without mutations, implying that the C9orf72 mutation modifies the pathologic phenotype of FTLD-TDP type B.
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frontotemporal lobar degeneration with tdp 43 proteinopathy and chromosome 9p repeat expansion in C9orf72 clinicopathologic correlation
Neuropathology, 2013Co-Authors: Eileen H Bigio, Matt Baker, Saman S Ahmadian, Rosa Rademakers, Bing Bing Weitner, Alfred W. Rademaker, Sandra Weintraub, Qinwen Mao, Kyung Hwa Lee, Manjari MishraAbstract:Mutations in C9orf72 resulting in expanded hexanucleotide repeats were recently reported to be the underlying genetic abnormality in chromosome 9p-linked frontotemporal lobar degeneration with TAR DNA-binding protein of 43 kD (TDP-43) proteinopathy (FTLD-TDP), amyotrophic lateral sclerosis (ALS), and frontotemporal lobar degeneration with motor neuron disease (FTLD-MND). Several subsequent publications described the neuropathology as being similar to that of FTLD-TDP and ALS without C9orf72 mutations, except that cases with mutations have p62 and ubiquitin positive, TDP-43 negative inclusions in cerebellum, hippocampus, neocortex, and basal ganglia. The identity of this protein is as yet unknown, and its significance is unclear. With the goal of potentially uncovering the significance of these inclusions, we compared the clinical, pathologic and genetic characteristics in cases with C9orf72 mutations to those without. We confirmed the apparent specificity of p62 positive, TDP-43 negative inclusions to cases with C9orf72 mutations. In hippocampus, these inclusions correlated with hippocampal atrophy. No additional correlations were uncovered. However, this is the first report to show that although most cases with C9orf72 mutations were TDP type B, some of the pathologic characteristics in these cases were more similar to TDP types A and C than to type B cases. These include greater cortical and hippocampal atrophy, greater ventricular dilatation, more neuronal loss and gliosis in temporal lobe and striatum, and TDP-43 positive fine neuritic profiles in the hippocampus, implying that the C9orf72 mutation modifies the pathologic phenotype of FTLD-TDP type B.
Nidaa A Ababneh - One of the best experts on this subject based on the ideXlab platform.
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impairment of mitochondrial calcium buffering links mutations in C9orf72 and tardbp in ips derived motor neurons from patients with als ftd
Stem cell reports, 2020Co-Authors: Ruxandra Dafinca, Jakub Scaber, Nidaa A Ababneh, Jane Vowles, Paola Barbagallo, Lucy Farrimond, Ana Candalija, Chaitra Sathyaprakash, Sally A CowleyAbstract:TDP-43 dysfunction is common to 97% of amyotrophic lateral sclerosis (ALS) cases, including those with mutations in C9orf72. To investigate how C9orf72 mutations drive cellular pathology in ALS and to identify convergent mechanisms between C9orf72 and TARDBP mutations, we analyzed motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) from patients with ALS. C9orf72 iPSC-MNs have higher Ca2+ release after depolarization, delayed recovery to baseline after glutamate stimulation, and lower levels of calbindin compared with CRISPR/Cas9 genome-edited controls. TARDBP iPS-derived MNs show high glutamate-induced Ca2+ release. We identify here, by RNA sequencing, that both C9orf72 and TARDBP iPSC-MNs have upregulation of Ca2+-permeable AMPA and NMDA subunits and impairment of mitochondrial Ca2+ buffering due to an imbalance of MICU1 and MICU2 on the mitochondrial Ca2+ uniporter, indicating that impaired mitochondrial Ca2+ uptake contributes to glutamate excitotoxicity and is a shared feature of MNs with C9orf72 or TARDBP mutations.
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correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by crispr cas9 genome editing using homology directed repair
bioRxiv, 2019Co-Authors: Nidaa A Ababneh, Ruxandra Dafinca, Jakub Scaber, Andrew G L Douglas, Sally A Cowley, Rowan Flynn, Martin R Turner, David Sims, Kevin TalbotAbstract:The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 and non-homologous end-joining (NHEJ) by deleting the repeat region with the risk of creating indels and genomic instability. In this study we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/FTD patient using CRISPR/Cas9 genome editing and homology directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines, and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared to its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the KEGG ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9 mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.
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C9orf72 hexanucleotide expansions are associated with altered endoplasmic reticulum calcium homeostasis and stress granule formation in induced pluripotent stem cell derived neurons from patients with amyotrophic lateral sclerosis and frontotemporal
Stem Cells, 2016Co-Authors: Ruxandra Dafinca, Jakub Scaber, Nidaa A Ababneh, Tatjana Lalic, Gregory Weir, Helen C Christian, Jane Vowles, Andrew G L Douglas, Alexandra Fletcherjones, Cathy BrowneAbstract:An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons, decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis, reduced levels of the antiapoptotic protein Bcl-2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063-2078.
Tania F. Gendron - One of the best experts on this subject based on the ideXlab platform.
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reduced C9orf72 function exacerbates gain of toxicity from als ftd causing repeat expansion in C9orf72
Nature Neuroscience, 2020Co-Authors: Qiang Zhu, Jie Jiang, Tania F. Gendron, Lu-lin Jiang, Sandra Diaz Garcia, Somasish Ghosh Dastidar, Melissa Mcalonisdownes, Amy TaylorAbstract:Hexanucleotide expansions in C9orf72, which encodes a predicted guanine exchange factor, are the most frequent genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Although repeat expansion has been established to generate toxic products, mRNAs encoding the C9orf72 protein are also reduced in affected individuals. In this study, we tested how C9orf72 protein levels affected repeat-mediated toxicity. In somatic transgenic mice expressing 66 GGGGCC repeats, inactivation of one or both endogenous C9orf72 alleles provoked or accelerated, respectively, early death. In mice expressing a C9orf72 transgene with 450 repeats that did not encode the C9orf72 protein, inactivation of one or both endogenous C9orf72 alleles exacerbated cognitive deficits, hippocampal neuron loss, glial activation and accumulation of dipeptide-repeat proteins from translation of repeat-containing RNAs. Reduced C9orf72 was shown to suppress repeat-mediated elevation in autophagy. These efforts support a disease mechanism in ALS/FTD resulting from reduced C9orf72, which can lead to autophagy deficits, synergizing with repeat-dependent gain of toxicity.
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gain of toxicity from als ftd linked repeat expansions in C9orf72 is alleviated by antisense oligonucleotides targeting ggggcc containing rnas
Neuron, 2016Co-Authors: Qiang Zhu, Jie Jiang, Tania F. Gendron, Melissa Mcalonisdownes, Shahram Saberi, Amanda Seelman, Jennifer E Stauffer, Paymaan Jafarnejad, Kevin DrennerAbstract:Hexanucleotide expansions in C9orf72 are the most frequent genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Disease mechanisms were evaluated in mice expressing C9orf72 RNAs with up to 450 GGGGCC repeats or with one or both C9orf72 alleles inactivated. Chronic 50% reduction of C9orf72 did not provoke disease, while its absence produced splenomegaly, enlarged lymph nodes, and mild social interaction deficits, but not motor dysfunction. Hexanucleotide expansions caused age-, repeat-length-, and expression-level-dependent accumulation of RNA foci and dipeptide-repeat proteins synthesized by AUG-independent translation, accompanied by loss of hippocampal neurons, increased anxiety, and impaired cognitive function. Single-dose injection of antisense oligonucleotides (ASOs) that target repeat-containing RNAs but preserve levels of mRNAs encoding C9orf72 produced sustained reductions in RNA foci and dipeptide-repeat proteins, and ameliorated behavioral deficits. These efforts identify gain of toxicity as a central disease mechanism caused by repeat-expanded C9orf72 and establish the feasibility of ASO-mediated therapy.
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human C9orf72 hexanucleotide expansion reproduces rna foci and dipeptide repeat proteins but not neurodegeneration in bac transgenic mice
Neuron, 2015Co-Authors: Tania F. Gendron, Helene Tran, Owen Morgan Peters, Gabriela Toro Cabrera, Jeanne E Mckeon, Jake Metterville, Alexandra Weiss, Nicholas Wightman, Johnny Salameh, Juhyun KimAbstract:A non-coding hexanucleotide repeat expansion in the C9orf72 gene is the most common mutation associated with familial amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). To investigate the pathological role of C9orf72 in these diseases, we generated a line of mice carrying a bacterial artificial chromosome containing exons 1 to 6 of the human C9orf72 gene with approximately 500 repeats of the GGGGCC motif. The mice showed no overt behavioral phenotype but recapitulated distinctive histopathological features of C9orf72 ALS/FTD, including sense and antisense intranuclear RNA foci and poly(glycine-proline) dipeptide repeat proteins. Finally, using an artificial microRNA that targets human C9orf72 in cultures of primary cortical neurons from the C9BAC mice, we have attenuated expression of the C9BAC transgene and the poly(GP) dipeptides. The C9orf72 BAC transgenic mice will be a valuable tool in the study of ALS/FTD pathobiology and therapy.
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C9orf72 bac transgenic mice display typical pathologic features of als ftd
Neuron, 2015Co-Authors: Jacqueline G Orourke, Andrew Austin, Elaine Liu, Laurent P Bogdanik, A K M G Muhammad, Jonah Zarrow, Tania F. Gendron, Kevin Kim, Janet Cady, Sharday GrantAbstract:Noncoding expansions of a hexanucleotide repeat (GGGGCC) in the C9orf72 gene are the most common cause of familial amyotrophic lateral sclerosis and frontotemporal dementia. Here we report transgenic mice carrying a bacterial artificial chromosome (BAC) containing the full human C9orf72 gene with either a normal allele (15 repeats) or disease-associated expansion (∼100-1,000 repeats; C9-BACexp). C9-BACexp mice displayed pathologic features seen in C9orf72 expansion patients, including widespread RNA foci and repeat-associated non-ATG (RAN) translated dipeptides, which were suppressed by antisense oligonucleotides targeting human C9orf72. Nucleolin distribution was altered, supporting that either C9orf72 transcripts or RAN dipeptides promote nucleolar dysfunction. Despite early and widespread production of RNA foci and RAN dipeptides in C9-BACexp mice, behavioral abnormalities and neurodegeneration were not observed even at advanced ages, supporting the hypothesis that RNA foci and RAN dipeptides occur presymptomatically and are not sufficient to drive neurodegeneration in mice at levels seen in patients.
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reduced C9orf72 gene expression in c9ftd als is caused by histone trimethylation an epigenetic event detectable in blood
Acta Neuropathologica, 2013Co-Authors: Tania F. Gendron, Matthew C. Baker, Veronique V Belzil, Peter Bauer, Mercedes Prudencio, Caroline Stetler, Irene K Yan, Luc Pregent, Lillian M Daughrity, Rosa RademakersAbstract:Individuals carrying (GGGGCC) expanded repeats in the C9orf72 gene represent a significant portion of patients suffering from amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Elucidating how these expanded repeats cause “c9FTD/ALS” has since become an important goal of the field. Toward this end, we sought to investigate whether epigenetic changes are responsible for the decrease in C9orf72 expression levels observed in c9FTD/ALS patients. We obtained brain tissue from ten c9FTD/ALS individuals, nine FTD/ALS cases without a C9orf72 repeat expansion, and nine disease control participants, and generated fibroblastoid cell lines from seven C9orf72 expanded repeat carriers and seven participants carrying normal alleles. Chromatin immunoprecipitation using antibodies for histone H3 and H4 trimethylated at lysines 9 (H3K9), 27 (H3K27), 79 (H3K79), and 20 (H4K20) revealed that these trimethylated residues bind strongly to C9orf72 expanded repeats in brain tissue, but not to non-pathogenic repeats. Our finding that C9orf72 mRNA levels are reduced in the frontal cortices and cerebella of c9FTD/ALS patients is consistent with trimethylation of these histone residues, an event known to repress gene expression. Moreover, treating repeat carrier-derived fibroblasts with 5-aza-2-deoxycytidine, a DNA and histone demethylating agent, not only decreased C9orf72 binding to trimethylated histone residues, but also increased C9orf72 mRNA expression. Our results provide compelling evidence that trimethylation of lysine residues within histones H3 and H4 is a novel mechanism involved in reducing C9orf72 mRNA expression in expanded repeat carriers. Of importance, we show that mutant C9orf72 binding to trimethylated H3K9 and H3K27 is detectable in blood of c9FTD/ALS patients. Confirming these exciting results using blood from a larger cohort of patients may establish this novel epigenetic event as a biomarker for c9FTD/ALS.
Ruxandra Dafinca - One of the best experts on this subject based on the ideXlab platform.
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impairment of mitochondrial calcium buffering links mutations in C9orf72 and tardbp in ips derived motor neurons from patients with als ftd
Stem cell reports, 2020Co-Authors: Ruxandra Dafinca, Jakub Scaber, Nidaa A Ababneh, Jane Vowles, Paola Barbagallo, Lucy Farrimond, Ana Candalija, Chaitra Sathyaprakash, Sally A CowleyAbstract:TDP-43 dysfunction is common to 97% of amyotrophic lateral sclerosis (ALS) cases, including those with mutations in C9orf72. To investigate how C9orf72 mutations drive cellular pathology in ALS and to identify convergent mechanisms between C9orf72 and TARDBP mutations, we analyzed motor neurons (MNs) derived from induced pluripotent stem cells (iPSCs) from patients with ALS. C9orf72 iPSC-MNs have higher Ca2+ release after depolarization, delayed recovery to baseline after glutamate stimulation, and lower levels of calbindin compared with CRISPR/Cas9 genome-edited controls. TARDBP iPS-derived MNs show high glutamate-induced Ca2+ release. We identify here, by RNA sequencing, that both C9orf72 and TARDBP iPSC-MNs have upregulation of Ca2+-permeable AMPA and NMDA subunits and impairment of mitochondrial Ca2+ buffering due to an imbalance of MICU1 and MICU2 on the mitochondrial Ca2+ uniporter, indicating that impaired mitochondrial Ca2+ uptake contributes to glutamate excitotoxicity and is a shared feature of MNs with C9orf72 or TARDBP mutations.
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correction of amyotrophic lateral sclerosis related phenotypes in induced pluripotent stem cell derived motor neurons carrying a hexanucleotide expansion mutation in C9orf72 by crispr cas9 genome editing using homology directed repair
bioRxiv, 2019Co-Authors: Nidaa A Ababneh, Ruxandra Dafinca, Jakub Scaber, Andrew G L Douglas, Sally A Cowley, Rowan Flynn, Martin R Turner, David Sims, Kevin TalbotAbstract:The G4C2 hexanucleotide repeat expansion (HRE) in C9orf72 is the commonest cause of familial amyotrophic lateral sclerosis (ALS). A number of different methods have been used to generate isogenic control lines using CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 and non-homologous end-joining (NHEJ) by deleting the repeat region with the risk of creating indels and genomic instability. In this study we demonstrate complete correction of an induced pluripotent stem cell (iPSC) line derived from a C9orf72-HRE positive ALS/FTD patient using CRISPR/Cas9 genome editing and homology directed repair (HDR), resulting in replacement of the excised region with a donor template carrying the wild-type repeat size to maintain the genetic architecture of the locus. The isogenic correction of the C9orf72 HRE restored normal expression and methylation at the C9orf72 locus, reduced intron retention in the edited lines, and abolished pathological phenotypes associated with the C9orf72 HRE expansion in iPSC derived motor neurons (iPSMNs). RNA sequencing of the mutant line identified 2220 differentially expressed genes compared to its isogenic control. Enrichment analysis demonstrated an over-representation of ALS relevant pathways, including calcium ion dependent exocytosis, synaptic transport and the KEGG ALS pathway, as well as new targets of potential relevance to ALS pathophysiology. Complete correction of the C9orf72 HRE in iPSMNs by CRISPR/Cas9 mediated HDR provides an ideal model to study the earliest effects of the hexanucleotide expansion on cellular homeostasis and the key pathways implicated in ALS pathophysiology.
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C9orf72 hexanucleotide expansions are associated with altered endoplasmic reticulum calcium homeostasis and stress granule formation in induced pluripotent stem cell derived neurons from patients with amyotrophic lateral sclerosis and frontotemporal
Stem Cells, 2016Co-Authors: Ruxandra Dafinca, Jakub Scaber, Nidaa A Ababneh, Tatjana Lalic, Gregory Weir, Helen C Christian, Jane Vowles, Andrew G L Douglas, Alexandra Fletcherjones, Cathy BrowneAbstract:An expanded hexanucleotide repeat in a noncoding region of the C9orf72 gene is a major cause of amyotrophic lateral sclerosis (ALS), accounting for up to 40% of familial cases and 7% of sporadic ALS in European populations. We have generated induced pluripotent stem cells (iPSCs) from fibroblasts of patients carrying C9orf72 hexanucleotide expansions, differentiated these to functional motor and cortical neurons, and performed an extensive phenotypic characterization. In C9orf72 iPSC-derived motor neurons, decreased cell survival is correlated with dysfunction in Ca(2+) homeostasis, reduced levels of the antiapoptotic protein Bcl-2, increased endoplasmic reticulum (ER) stress, and reduced mitochondrial membrane potential. Furthermore, C9orf72 motor neurons, and also cortical neurons, show evidence of abnormal protein aggregation and stress granule formation. This study is an extensive characterization of iPSC-derived motor neurons as cellular models of ALS carrying C9orf72 hexanucleotide repeats, which describes a novel pathogenic link between C9orf72 mutations, dysregulation of calcium signaling, and altered proteostasis and provides a potential pharmacological target for the treatment of ALS and the related neurodegenerative disease frontotemporal dementia. Stem Cells 2016;34:2063-2078.