RESUMO
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease. Clinical heterogeneity and complex genetics pose challenges to understanding disease mechanisms and producing effective cures. To model clinical heterogeneity, we generated human induced pluripotent stem cells (iPSCs) from two sporadic ALS patients (sporadic ALS and sporadic ALS with frontotemporal dementia), two familial ALS patients (familial SOD1 mutation positive and familial C9orf72 repeat expansion positive), and four age- and sex-matched healthy controls. These iPSCs can be used to generate 2D and 3D in vitro models of ALS to investigate mechanisms of disease and screen for therapeutics.
Assuntos
Esclerose Lateral Amiotrófica , Proteína C9orf72 , Demência Frontotemporal , Células-Tronco Pluripotentes Induzidas , Superóxido Dismutase-1 , Humanos , Esclerose Lateral Amiotrófica/genética , Esclerose Lateral Amiotrófica/patologia , Esclerose Lateral Amiotrófica/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Demência Frontotemporal/genética , Demência Frontotemporal/patologia , Demência Frontotemporal/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Superóxido Dismutase-1/genética , Superóxido Dismutase-1/metabolismo , Feminino , Masculino , Linhagem Celular , Pessoa de Meia-IdadeRESUMO
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease in which the TDP-43 protein is believed to play a central role in disease pathophysiology. Using the CRISPR-Cas9 system, we introduced the heterozygous c.1144G > A (p.A382T) missense mutation in exon 6 of the TARDBP gene into an iPSC line derived from a healthy individual. These edited iPSCs displayed normal cellular morphology, expressed major pluripotency markers, were capable of tri-lineage differentiation, and possessed a normal karyotype.
Assuntos
Esclerose Lateral Amiotrófica , Células-Tronco Pluripotentes Induzidas , Doenças Neurodegenerativas , Humanos , Esclerose Lateral Amiotrófica/genética , Sistemas CRISPR-Cas/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Pluripotentes Induzidas/citologia , Mutação , Mutação de Sentido Incorreto , Doenças Neurodegenerativas/genéticaRESUMO
Amyotrophic lateral sclerosis is characterized by the degeneration of upper and lower motor neurons, yet an increasing number of studies in both mouse models and patients with amyotrophic lateral sclerosis suggest that altered metabolic homeostasis is also a feature of disease. Pre-clinical and clinical studies have shown that modulation of energy balance can be beneficial in amyotrophic lateral sclerosis. However, the capacity to target specific metabolic pathways or mechanisms requires detailed understanding of metabolic dysregulation in amyotrophic lateral sclerosis. Here, using the superoxide dismutase 1, glycine to alanine substitution at amino acid 93 (SOD1G93A) mouse model of amyotrophic lateral sclerosis, we demonstrate that an increase in whole-body metabolism occurs at a time when glycolytic muscle exhibits an increased dependence on fatty acid oxidation. Using myotubes derived from muscle of amyotrophic lateral sclerosis patients, we also show that increased dependence on fatty acid oxidation is associated with increased whole-body energy expenditure. In the present study, increased fatty acid oxidation was associated with slower disease progression. However, within the patient cohort, there was considerable heterogeneity in whole-body metabolism and fuel oxidation profiles. Thus, future studies that decipher specific metabolic changes at an individual patient level are essential for the development of treatments that aim to target metabolic pathways in amyotrophic lateral sclerosis.
RESUMO
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the deterioration of motor neurons. However, this complex disease extends beyond the boundaries of the central nervous system, with metabolic alterations being observed at the systemic and cellular level. While the number of studies that assess the role and impact of metabolic perturbations in ALS is rapidly increasing, the use of metabolism biomarkers in ALS remains largely underinvestigated. In this review, we discuss current and potential metabolism biomarkers in the context of ALS. Of those for which data does exist, there is limited insight provided by individual markers, with specificity for disease, and lack of reproducibility and efficacy in informing prognosis being the largest drawbacks. However, given the array of metabolic markers available, the potential exists for a panel of metabolism biomarkers, which may complement other current biomarkers (including neurophysiology, imaging, as well as CSF, blood and urine markers) to overturn these limitations and give rise to new diagnostic and prognostic indicators.
RESUMO
Lipids are a fundamental class of organic molecules implicated in a wide range of biological processes related to their structural diversity, and based on this can be broadly classified into five categories; fatty acids, triacylglycerols (TAGs), phospholipids, sterol lipids and sphingolipids. Different lipid classes play major roles in neuronal cell populations; they can be used as energy substrates, act as building blocks for cellular structural machinery, serve as bioactive molecules, or a combination of each. In amyotrophic lateral sclerosis (ALS), dysfunctions in lipid metabolism and function have been identified as potential drivers of pathogenesis. In particular, aberrant lipid metabolism is proposed to underlie denervation of neuromuscular junctions, mitochondrial dysfunction, excitotoxicity, impaired neuronal transport, cytoskeletal defects, inflammation and reduced neurotransmitter release. Here we review current knowledge of the roles of lipid metabolism and function in the CNS and discuss how modulating these pathways may offer novel therapeutic options for treating ALS.
RESUMO
Precise and accurate gene correction is crucial for enabling iPSC-based therapies, and Cas9-Nickase based approaches are increasingly considered for in vivo correction of diseases such as beta-thalassemia. Here, we generate footprint-free induced pluripotent stem cells from a patient with a beta-thalassemia mutation (IVSII-1 G > A) and employ a double Cas9nickase-mediated correction strategy combined with a piggyBac transposon-modified donor vector for gene correction. Our approach further aimed to minimize the formation of adjacent single-strand breaks at the targeted allele through the destruction of the binding site for one guide and the use of a synonymous protospacer adjacent motif blocking mutation (canonical PAM sequence 5'-NGG-3' is changed to 5'-NCG-3', where N indicates any nucleobase) for the other guide. We show that this strategy indeed not only permits bi-allelic seamless repair of the beta-globin gene splice site mutation and negligible off-target mutagenesis or re-editing of the targeted allele but also results in unexpected on-target mutagenesis with some guide RNAs (gRNAs) in several targeted clones. This study thus not only validates a framework for seamless gene correction with enhanced specificity and accuracy but also highlights potential safety concerns associated with Cas9-nickase based gene correction.