C9orf72 ablation in mice does not cause motor neuron degeneration or motor deficits.
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ABSTRACT:
SUBMITTER: Koppers M
PROVIDER: S-EPMC4744979 | biostudies-other | 2015 Sep
REPOSITORIES: biostudies-other
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Project description:Mutations in C9orf72 are the most common genetic cause of amyotrophic lateral sclerosis (ALS). Accumulating evidence implicates astrocytes as important non-cell autonomous contributors to ALS pathogenesis, although the potential deleterious effects of astrocytes on the function of motor neurons remains to be determined in a completely humanized model of C9orf72-mediated ALS. Here, we use a human iPSC-based model to study the cell autonomous and non-autonomous consequences of mutant C9orf72 expression by astrocytes. We show that mutant astrocytes both recapitulate key aspects of C9orf72-related ALS pathology and, upon co-culture, cause motor neurons to undergo a progressive loss of action potential output due to decreases in the magnitude of voltage-activated Na+ and K+ currents. Importantly, CRISPR/Cas-9 mediated excision of the C9orf72 repeat expansion reverses these phenotypes, confirming that the C9orf72 mutation is responsible for both cell-autonomous astrocyte pathology and non-cell autonomous motor neuron pathophysiology.
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Project description: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.
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Project description:Axonal dysfunction is a common phenotype in neurodegenerative disorders, including in amyotrophic lateral sclerosis (ALS), where the key pathological cell-type, the motor neuron (MN), has an axon extending up to a metre long. The maintenance of axonal function is a highly energy-demanding process, raising the question of whether MN cellular energetics is perturbed in ALS, and whether its recovery promotes axonal rescue. To address this, we undertook cellular and molecular interrogation of multiple patient-derived induced pluripotent stem cell lines and patient autopsy samples harbouring the most common ALS causing mutation, C9orf72. Using paired mutant and isogenic expansion-corrected controls, we show that C9orf72 MNs have shorter axons, impaired fast axonal transport of mitochondrial cargo, and altered mitochondrial bioenergetic function. RNAseq revealed reduced gene expression of mitochondrially encoded electron transport chain transcripts, with neuropathological analysis of C9orf72-ALS post-mortem tissue importantly confirming selective dysregulation of the mitochondrially encoded transcripts in ventral horn spinal MNs, but not in corresponding dorsal horn sensory neurons, with findings reflected at the protein level. Mitochondrial DNA copy number was unaltered, both in vitro and in human post-mortem tissue. Genetic manipulation of mitochondrial biogenesis in C9orf72 MNs corrected the bioenergetic deficit and also rescued the axonal length and transport phenotypes. Collectively, our data show that loss of mitochondrial function is a key mediator of axonal dysfunction in C9orf72-ALS, and that boosting MN bioenergetics is sufficient to restore axonal homeostasis, opening new potential therapeutic strategies for ALS that target mitochondrial function.
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Project description:BackgroundThe functional deterioration and loss of motor neurons are tightly associated with degenerative motor neuron diseases and aging-related muscle wasting. Motor neuron diseases or aging-related muscle wasting in turn contribute to increased risk of adverse health outcomes in the elderly. Cdon (cell adhesion molecule-downregulated oncogene) belongs to the immunoglobulin superfamily of cell adhesion molecule and plays essential roles in multiple signalling pathways, including sonic hedgehog (Shh), netrin, and cadherin-mediated signalling. Cdon as a Shh coreceptor plays a critical role in motor neuron specification during embryonic development. However, its role in adult motor neuron function is unknown.MethodsHb9-Cre recombinase-driven motor neuron-specific Cdon deficient mice (mnKO) and a compound mutant mice (mnKO::SOD1G93A ) were generated to investigate the role of Cdon in motor neuron degeneration. Motor neuron regeneration was examined by using a sciatic nerve crush injury model. To investigate the phenotype, physical activity, compound muscle action potential, immunostaining, and transmission electron microscopy were carried out. In the mechanism study, RNA sequencing and RNA/protein analyses were employed.ResultsMice lacking Cdon in motor neurons exhibited middle age onset lethality and aging-related decline in motor function. In the sciatic nerve crush injury model, mnKO mice exhibited an impairment in motor function recovery evident by prolonged compound muscle action potential duration (4.63 ± 0.35 vs. 3.93 ± 0.22 s for f/f, P < 0.01) and physical activity. Consistently, neuromuscular junctions of mnKO muscles were incompletely occupied (49.79 ± 5.74 vs. 79.39 ± 3.77% fully occupied neuromuscular junctions for f/f, P < 0.0001), suggesting an impaired reinnervation. The transmission electron microscopy analysis revealed that mnKO sciatic nerves had smaller axon diameter (0.88 ± 0.13 vs. 1.43 ± 0.48 μm for f/f, P < 0.0001) and myelination defects. RNA sequencing of mnKO lumbar spinal cords showed alteration in genes related to neurogenesis, inflammation and cell death. Among the altered genes, ErbB4 and FgfR expressions were significantly altered in mnKO as well as in Cdon-depleted NSC34 motor neuron cells. Consistently, Cdon-depleted NSC34 cells exhibited elevated levels of cleaved Caspase3 and γH2AX proteins, as well as Bax transcription. Cdon-depleted NSC34 cells also exhibited impaired activation of Akt in response to neuregulin-1 (NRG1) treatment.ConclusionsOur current data demonstrate the functional importance of Cdon in motor neuron function and nerve repair. Cdon ablation causes alterations in neurotrophin signalling that leads to motor neuron degeneration.
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Project description:Translation of the expanded (ggggcc)n repeat in C9orf72 patients with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) causes abundant poly-GA inclusions. To elucidate their role in pathogenesis, we generated transgenic mice expressing codon-modified (GA)149 conjugated with cyan fluorescent protein (CFP). Transgenic mice progressively developed poly-GA inclusions predominantly in motoneurons and interneurons of the spinal cord and brain stem and in deep cerebellar nuclei. Poly-GA co-aggregated with p62, Rad23b and the newly identified Mlf2, in both mouse and patient samples. Consistent with the expression pattern, 4-month-old transgenic mice showed abnormal gait and progressive balance impairment, but showed normal hippocampus-dependent learning and memory. Apart from microglia activation we detected phosphorylated TDP-43 but no neuronal loss. Thus, poly-GA triggers behavioral deficits through inflammation and protein sequestration that likely contribute to the prodromal symptoms and disease progression of C9orf72 patients.
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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
2013-11-07 | E-GEOD-52202 | biostudies-arrayexpress
Project description:ObjectiveDuchenne muscular dystrophy (DMD) is a devastating muscle disease caused by a mutation in DMD encoding dystrophin. Oxidative stress accounts for dystrophic muscle pathologies in DMD. We examined the effects of molecular hydrogen in mdx mice, a model animal for DMD.MethodsThe pregnant mother started to take supersaturated hydrogen water (>5 ppm) ad libitum from E15.5 up to weaning of the offspring. The mdx mice took supersaturated hydrogen water from weaning until age 10 or 24 weeks when they were sacrificed.ResultsHydrogen water prevented abnormal body mass gain that is commonly observed in mdx mice. Hydrogen improved the spontaneous running distance that was estimated by a counter-equipped running-wheel, and extended the duration on the rota-rod. Plasma creatine kinase activities were decreased by hydrogen at ages 10 and 24 weeks. Hydrogen also decreased the number of central nuclei of muscle fibers at ages 10 and 24 weeks, and immunostaining for nitrotyrosine in gastrocnemius muscle at age 24 weeks. Additionally, hydrogen tended to increase protein expressions of antioxidant glutathione peroxidase 1, as well as anti-apoptotic Bcl-2, in skeletal muscle at age 10 weeks.DiscussionAlthough molecular mechanisms of the diverse effects of hydrogen remain to be elucidated, hydrogen potentially improves muscular dystrophy in DMD patients.
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