C9/ALS Human Embryonic Stem Cells and C9/ALS Induced Pluripotent Stem Cells
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ABSTRACT: We established two HESC lines with a C9 mutation (SZ-ALS1, SZ-ALS3) from embryos, which were obtained through preimplantation genetic diagnosis (PGD) and donated for cell line derivation by a family in which the mother was an expansion carrier (originally diagnosed as a carrier of an expansion with >40 repeats in blood by a repeat primed PCR). In addition, we generated halo-identical and unrelated C9/ALS iPSCs from a skin biopsies of the C9 carrier mother (patient H, 30 years-old), and from an unrelated C9/ALS patient, 2 years following disease-onset (patient M, 65 years-old).
Project description:Dysregulation of RNA processing contributes to neurodegenerative diseases, especially amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Expansion of an intronic (GGGGCC)n repeat within the C9ORF72 gene is the most common cause of both FTD and ALS (C9-FTD/ALS), characterized with aberrant repeat RNA foci in the nucleus and noncanonical translation-produced dipeptide repeat (DPR) protein inclusions in the cytoplasm. Here we elucidate that the (GGGGCC)n repeat RNA co-localizes with nuclear speckles and alters their phase separation properties and granule dynamics. Moreover, the essential nuclear speckle scaffold protein SRRM2 is sequestered into the poly-GR cytoplasmic inclusions in C9-FTD/ALS mouse model and patient postmortem tissues, exacerbating the nuclear speckle dysfunction. Impaired nuclear speckle integrity induces global exon-skipping and intron retention in human iPSC-derived neurons. Similar alternative splicing changes can be found in patient postmortem tissues. This work identified novel molecular mechanism of global RNA splicing defects by impaired nuclear speckle function in C9-FTD/ALS and revealed novel potential biomarkers or therapeutic targets.
Project description:Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are strongly influenced by inherited genetic variation, but environmental and epigenetic factors also play key roles in the course of these diseases. A hexanucleotide repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of ALS and FTD. To determine the cellular alterations associated with the C9 repeat expansion, we performed single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem samples of motor and frontal cortices from C9-ALS and C9-FTD donors. We found pervasive alterations of gene expression across multiple cortical cell types in C9-ALS, with the largest number of affected genes in astrocytes and excitatory neurons. Astrocytes increased expression of markers of activation and pathways associated with structural remodeling. Excitatory neurons in upper and deep layers increased expression of genes related to proteostasis, metabolism, and protein expression, and decreased expression of genes related to neuronal function. Epigenetic analyses revealed concordant changes in chromatin accessibility, histone modifications, and gene expression in specific cell types. C9-FTD patients had a distinct pattern of changes, including loss of neurons in frontal cortex and altered expression of thousands of genes in astrocytes and oligodendrocyte-lineage cells. Overall, these findings demonstrate a context-dependent molecular disruption in C9-ALS and C9-FTD, resulting in distinct effects across cell types, brain regions, and disease phenotypes.
Project description:Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are strongly influenced by inherited genetic variation, but environmental and epigenetic factors also play key roles in the course of these diseases. A hexanucleotide repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of ALS and FTD. To determine the cellular alterations associated with the C9 repeat expansion, we performed single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem samples of motor and frontal cortices from C9-ALS and C9-FTD donors. We found pervasive alterations of gene expression across multiple cortical cell types in C9-ALS, with the largest number of affected genes in astrocytes and excitatory neurons. Astrocytes increased expression of markers of activation and pathways associated with structural remodeling. Excitatory neurons in upper and deep layers increased expression of genes related to proteostasis, metabolism, and protein expression, and decreased expression of genes related to neuronal function. Epigenetic analyses revealed concordant changes in chromatin accessibility, histone modifications, and gene expression in specific cell types. C9-FTD patients had a distinct pattern of changes, including loss of neurons in frontal cortex and altered expression of thousands of genes in astrocytes and oligodendrocyte-lineage cells. Overall, these findings demonstrate a context-dependent molecular disruption in C9-ALS and C9-FTD, resulting in distinct effects across cell types, brain regions, and disease phenotypes.
Project description:Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are strongly influenced by inherited genetic variation, but environmental and epigenetic factors also play key roles in the course of these diseases. A hexanucleotide repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of ALS and FTD. To determine the cellular alterations associated with the C9 repeat expansion, we performed single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem samples of motor and frontal cortices from C9-ALS and C9-FTD donors. We found pervasive alterations of gene expression across multiple cortical cell types in C9-ALS, with the largest number of affected genes in astrocytes and excitatory neurons. Astrocytes increased expression of markers of activation and pathways associated with structural remodeling. Excitatory neurons in upper and deep layers increased expression of genes related to proteostasis, metabolism, and protein expression, and decreased expression of genes related to neuronal function. Epigenetic analyses revealed concordant changes in chromatin accessibility, histone modifications, and gene expression in specific cell types. C9-FTD patients had a distinct pattern of changes, including loss of neurons in frontal cortex and altered expression of thousands of genes in astrocytes and oligodendrocyte-lineage cells. Overall, these findings demonstrate a context-dependent molecular disruption in C9-ALS and C9-FTD, resulting in distinct effects across cell types, brain regions, and disease phenotypes.
Project description:Neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), are strongly influenced by inherited genetic variation, but environmental and epigenetic factors also play key roles in the course of these diseases. A hexanucleotide repeat expansion in the C9orf72 (C9) gene is the most common genetic cause of ALS and FTD. To determine the cellular alterations associated with the C9 repeat expansion, we performed single nucleus transcriptomics (snRNA-seq) and epigenomics (snATAC-seq) in postmortem samples of motor and frontal cortices from C9-ALS and C9-FTD donors. We found pervasive alterations of gene expression across multiple cortical cell types in C9-ALS, with the largest number of affected genes in astrocytes and excitatory neurons. Astrocytes increased expression of markers of activation and pathways associated with structural remodeling. Excitatory neurons in upper and deep layers increased expression of genes related to proteostasis, metabolism, and protein expression, and decreased expression of genes related to neuronal function. Epigenetic analyses revealed concordant changes in chromatin accessibility, histone modifications, and gene expression in specific cell types. C9-FTD patients had a distinct pattern of changes, including loss of neurons in frontal cortex and altered expression of thousands of genes in astrocytes and oligodendrocyte-lineage cells. Overall, these findings demonstrate a context-dependent molecular disruption in C9-ALS and C9-FTD, resulting in distinct effects across cell types, brain regions, and disease phenotypes.
Project description:Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) represent two ends of a disease spectrum with shared clinical, genetic and pathological features. These include near ubiquitous pathological inclusions of the RNA binding protein (RBP) TDP-43, and often the presence of a GGGGCC expansion in the C9ORF72 (C9) gene. Here we show unexpectedly that the signature of hnRNP H sequestration and altered splicing of target transcripts we identified in C9ALS patients (Conlon et al. 2016) also occurs in fully half of 50 post-mortem sporadic, non-C9 ALS/FTD post-mortem brains. Furthermore, and equally surprisingly, these “like-C9” brains also contained correspondingly high amounts of insoluble TDP-43, as well as several other disease-related RBPs, and this correlates with widespread global splicing defects. Finally, we show that the like-C9 sporadic patients, like actual C9ALS patients, were much more likely to have developed FTD. We propose that these unexpected links between C9 and sporadic ALS/FTD define a common mechanism in this disease spectrum.
Project description:Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that affects motor neurons (MNs). It was shown that human astrocytes with mutations in genes associated with ALS, like C9orf72 (C9) or SOD1, reduce survival of MNs. Astrocyte toxicity may be related to their dysfunction or the release of neurotoxic factors. We used human induced pluripotent stem cell-derived astrocytes from ALS patients carrying C9orf72 mutations and non-affected donors. We utilized these cells to investigate astrocytic induced neuronal toxicity, changes in astrocyte transcription profile as well as changes in secretome profiles. We report that C9-mutated astrocytes are toxic to MNs via soluble factors. The toxic effects of astrocytes are positively correlated with the length of astrocyte propagation in culture, consistent with the age-related nature of ALS. We show that C9-mutated astrocytes downregulate the secretion of several antioxidant proteins. In line with these findings, we show increased astrocytic oxidative stress and senescence. Importantly, media conditioned by C9-astrocytes increased oxidative stress in wild type MNs. Our results suggest that dysfunction of C9-astrocytes leads to oxidative stress of themselves and MNs, which probably contributes to neurodegeneration. Our findings suggest that therapeutic strategies in familial ALS must not only target MNs but also focus on astrocytes to abrogate nervous system injury.
Project description:A hexanucleotide repeat expansion (HRE) in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Human imaging and experimental studies hint at early changes in the brain in C9-ALS/FTD, which remain poorly understood. To define these changes, we used cerebral organoid models derived from C9-ALS/FTD patients and controls to create a single cell RNA sequencing dataset at day 90. Together, these dataset will help to shed light on initial pathologies crucial for understanding disease onset and the design of therapeutic strategies.
Project description:The GGGGCC nucleotide repeat expansion (NRE) mutation in the C9orf72 (C9) gene is the most common cause of ALS and FTD. Neuronal activity plays an essential role in shaping biological processes within both healthy and neurodegenerative disease scenarios. Here, we show that at baseline conditions, C9-NRE iPSC-cortical neurons display aberrations in several pathways, including synaptic signaling and transcriptional machinery, potentially priming diseased neurons for an altered response to neuronal stimulation. Indeed, exposure to two pathophysiologically relevant stimulation modes, prolonged membrane depolarization, or a blockade of K+ channels, followed by RNA sequencing, induces a temporally divergent activity-dependent transcriptome of C9-NRE cortical neurons compared to healthy controls. These findings reveal the impact of neuronal activity on the ALS/FTD-associated transcriptome and may reveal pathways necessary for conferring neuronal resilience or degeneration.
Project description:Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative condition characterized by loss of motor neurons in the brain and spinal cord. Expansions of a hexanucleotide repeat (GGGGCC) in the noncoding region of the C9ORF72 gene are the most common cause of the familial form of ALS (C9-ALS), as well as frontotemporal lobar degeneration and other neurological diseases. How the repeat expansion causes disease remains unclear, with both loss of function (haploinsufficiency) and gain of function (either toxic RNA or protein products) proposed. We report a cellular model of C9-ALS with motor neurons differentiated from induced pluripotent stem cells (iPSCs) derived from ALS patients carrying the C9ORF72 repeat expansion. No significant loss of C9ORF72 expression was observed, and knockdown of the transcript was not toxic to cultured human motor neurons. Transcription of the repeat was increased, leading to accumulation of GGGGCC repeat–containing RNA foci selectively in C9-ALS iPSC-derived motor neurons. Repeat-containing RNA foci colocalized with hnRNPA1 and Pur-?, suggesting that they may be able to alter RNA metabolism. C9-ALS motor neurons showed altered expression of genes involved in membrane excitability including DPP6, and demonstrated a diminished capacity to fire continuous spikes upon depolarization compared to control motor neurons. Antisense oligonucleotides targeting the C9ORF72 transcript suppressed RNA foci formation and reversed gene expression alterations in C9-ALS motor neurons. These data show that patient-derived motor neurons can be used to delineate pathogenic events in ALS. Transcriptome profiling from iPSC derived motor neurons compared to controls