RESUMEN
The peroxisomal disease adrenoleukodystrophy (X-ALD) is caused by loss of the transporter of very-long-chain fatty acids (VLCFAs), ABCD1. An excess of VLCFAs disrupts essential homeostatic functions crucial for axonal maintenance, including redox metabolism, glycolysis and mitochondrial respiration. As mitochondrial function and morphology are intertwined, we set out to investigate the role of mitochondrial dynamics in X-ALD models. Using quantitative 3D transmission electron microscopy, we revealed mitochondrial fragmentation in corticospinal axons in Abcd1- mice. In patient fibroblasts, an excess of VLCFAs triggers mitochondrial fragmentation through the redox-dependent phosphorylation of DRP1 (DRP1S616). The blockade of DRP1-driven fission by the peptide P110 effectively preserved mitochondrial morphology. Furthermore, mRNA inhibition of DRP1 not only prevented mitochondrial fragmentation but also protected axonal health in a Caenorhabditis elegans model of X-ALD, underscoring DRP1 as a potential therapeutic target. Elevated levels of circulating cell-free mtDNA in patients' CSF align this leukodystrophy with primary mitochondrial disorders. Our findings underscore the intricate interplay between peroxisomal dysfunction, mitochondrial dynamics and axonal integrity in X-ALD, shedding light on potential avenues for therapeutic intervention.
Asunto(s)
Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP , Adrenoleucodistrofia , Dinaminas , Dinámicas Mitocondriales , Adrenoleucodistrofia/metabolismo , Adrenoleucodistrofia/patología , Adrenoleucodistrofia/genética , Animales , Dinámicas Mitocondriales/fisiología , Humanos , Ratones , Dinaminas/metabolismo , Dinaminas/genética , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/genética , Caenorhabditis elegans , Mitocondrias/metabolismo , Mitocondrias/patología , Axones/patología , Axones/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Masculino , ADN Mitocondrial/genética , ADN Mitocondrial/metabolismo , Modelos Animales de Enfermedad , Tractos Piramidales/patología , Tractos Piramidales/metabolismo , Fragmentos de Péptidos , GTP FosfohidrolasasRESUMEN
Mutations in the human kinesin family member 5A (KIF5A) gene were recently identified as a genetic cause of amyotrophic lateral sclerosis (ALS). Several KIF5A ALS variants cause exon 27 skipping and are predicted to produce motor proteins with an altered C-terminal tail (referred to as ΔExon27). However, the underlying pathogenic mechanism is still unknown. Here, we confirm the expression of KIF5A mutant proteins in patient iPSC-derived motor neurons. We perform a comprehensive analysis of ΔExon27 at the single-molecule, cellular, and organism levels. Our results show that ΔExon27 is prone to form cytoplasmic aggregates and is neurotoxic. The mutation relieves motor autoinhibition and increases motor self-association, leading to drastically enhanced processivity on microtubules. Finally, ectopic expression of ΔExon27 in Drosophila melanogaster causes wing defects, motor impairment, paralysis, and premature death. Our results suggest gain-of-function as an underlying disease mechanism in KIF5A-associated ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , ADN sin Sentido/genética , Drosophila melanogaster , Mutación con Ganancia de Función , Humanos , Cinesinas/genética , Neuronas Motoras/metabolismo , Mutación , Proteína 2 Similar al Factor de Transcripción 7/metabolismoRESUMEN
Aberrant endocannabinoid signaling accompanies several neurodegenerative disorders, including multiple sclerosis. Here, we report altered endocannabinoid signaling in X-linked adrenoleukodystrophy (X-ALD), a rare neurometabolic demyelinating syndrome caused by malfunction of the peroxisomal ABCD1 transporter, resulting in the accumulation of very long-chain fatty acids (VLCFAs). We found abnormal levels of cannabinoid receptor 2 (CB2r) and related endocannabinoid enzymes in the brain and peripheral blood mononuclear cells (PBMCs) of X-ALD patients and in the spinal cord of a murine model of X-ALD. Preclinical treatment with a selective agonist of CB2r (JWH133) halted axonal degeneration and associated locomotor deficits, along with normalization of microgliosis. Moreover, the drug improved the main metabolic disturbances underlying this model, particularly in redox and lipid homeostatic pathways, including increased lipid droplets in motor neurons, through the modulation of the GSK-3ß/NRF2 axis. JWH133 inhibited Reactive Oxygen Species elicited by excess VLCFAs in primary microglial cultures of Abcd1-null mice. Furthermore, we uncovered intertwined redox and CB2r signaling in the murine spinal cords and in patient PBMC samples obtained from a phase II clinical trial with antioxidants (NCT01495260). These findings highlight CB2r signaling as a potential therapeutic target for X-ALD and perhaps other neurodegenerative disorders that present with dysregulated redox and lipid homeostasis.
Asunto(s)
Adrenoleucodistrofia , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP , Adrenoleucodistrofia/tratamiento farmacológico , Animales , Ensayos Clínicos Fase II como Asunto , Endocannabinoides/uso terapéutico , Glucógeno Sintasa Quinasa 3 beta/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/metabolismo , Receptores de Cannabinoides/metabolismo , Receptores de Cannabinoides/uso terapéuticoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurological disorder characterized by progressive muscular atrophy and respiratory failure. The G4C2 repeat expansion in the C9orf72 gene is the most prevalent genetic risk for ALS. Mutation carriers (C9ALS) display variability in phenotypes such as age-at-onset and duration, suggesting the existence of additional genetic factors. Here we introduce a three-step gene discovery strategy to identify genetic factors modifying the risk of both C9ALS and sporadic ALS (sALS) using limited samples. We first identified 135 candidate genetic modifiers of C9ALS using whole-genome sequencing (WGS) of extreme C9ALS cases diagnosed ~30 years apart. We then performed an unbiased genetic screen using a Drosophila model of the G4C2 repeat expansion with the genes identified from WGS analysis. This genetic screen identified the novel genetic interaction between G4C2 repeat-associated toxicity and 18 genetic factors, suggesting their potential association with C9ALS risk. We went on to test if 14 out of the 18 genes, those which were not known to be risk factors for ALS previously, are also associated with ALS risk in sALS cases. Gene-based-statistical analyses of targeted resequencing and WGS were performed. These analyses together reveal that rare variants in MYH15 represent a likely genetic risk factor for ALS. Furthermore, we show that MYH15 could modulate the toxicity of dipeptides produced from expanded G4C2 repeat. Our study presented here demonstrates the power of combining WGS with fly genetics to facilitate the discovery of fundamental genetic components of complex traits with a limited number of samples.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN , Drosophila/genética , Cadenas Pesadas de Miosina/genética , Adulto , Anciano , Animales , Animales Modificados Genéticamente , Proteína C9orf72/metabolismo , Proteína C9orf72/toxicidad , Dipéptidos/metabolismo , Dipéptidos/toxicidad , Modelos Animales de Enfermedad , Drosophila/citología , Drosophila/crecimiento & desarrollo , Drosophila/ultraestructura , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Humanos , Masculino , Proteínas de Microfilamentos/genética , Proteínas de Microfilamentos/metabolismo , Mutación , Cadenas Pesadas de Miosina/metabolismo , Fenotipo , Factores de Riesgo , Secuenciación Completa del GenomaRESUMEN
A GGGGCC hexanucleotide repeat expansion in the first intron of C9orf72 is the most common genetic cause of amyotrophic lateral sclerosis and frontotemporal dementia. Compelling evidence suggests that gain of toxicity from the bidirectionally transcribed repeat expanded RNAs plays a central role in disease pathogenesis. Two potential mechanisms have been proposed including RNA-mediated toxicity and/or the production of toxic dipeptide repeat proteins. In this review, we focus on the role of RNA mediated toxicity in ALS/FTD caused by the C9orf72 mutation and discuss arguments for and against this mechanism. In addition, we summarize how G4C2 repeat RNAs can elicit toxicity and potential therapeutic strategies to mitigate RNA-mediated toxicity.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Proteína C9orf72/genética , Demencia Frontotemporal/patología , ARN/toxicidad , Esclerosis Amiotrófica Lateral/genética , Animales , Expansión de las Repeticiones de ADN , HumanosRESUMEN
GGGGCC (G4C2) hexanucleotide repeat expansion in the C9ORF72 gene is the most common genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). The repeat is bidirectionally transcribed and confers gain of toxicity. However, the underlying toxic species is debated, and it is not clear whether antisense CCCCGG (C4G2) repeat expanded RNAs contribute to disease pathogenesis. Our study shows that C9ORF72 antisense C4G2 repeat expanded RNAs trigger the activation of the PKR/eIF2α-dependent integrated stress response independent of dipeptide repeat proteins that are produced through repeat-associated non-AUG-initiated translation, leading to global translation inhibition and stress granule formation. Reducing PKR levels with either siRNA or morpholinos mitigates integrated stress response and toxicity caused by the antisense C4G2 RNAs in cell lines, primary neurons, and zebrafish. Increased phosphorylation of PKR/eIF2α is also observed in the frontal cortex of C9ORF72 FTD/ALS patients. Finally, only antisense C4G2, but not sense G4C2, repeat expanded RNAs robustly activate the PKR/eIF2α pathway and induce aberrant stress granule formation. These results provide a mechanism by which antisense C4G2 repeat expanded RNAs elicit neuronal toxicity in FTD/ALS caused by C9ORF72 repeat expansions.
Asunto(s)
Esclerosis Amiotrófica Lateral , Demencia Frontotemporal , Animales , Demencia Frontotemporal/patología , Esclerosis Amiotrófica Lateral/patología , Proteína C9orf72/genética , Pez Cebra/genética , Expansión de las Repeticiones de ADN , ARN Interferente Pequeño/genéticaRESUMEN
Adrenoleukodystrophy is a neurometabolic disorder caused by a defective peroxisomal ABCD1 transporter of very long-chain fatty acids (VLCFAs). Its pathogenesis is incompletely understood. Here we characterize a nematode model of X-ALD with loss of the pmp-4 gene, the worm orthologue of ABCD1. These mutants recapitulate the hallmarks of X-ALD: i) VLCFAs accumulation and impaired mitochondrial redox homeostasis and ii) axonal damage coupled to locomotor dysfunction. Furthermore, we identify a novel role for PMP-4 in modulating lipid droplet dynamics. Importantly, we show that the mitochondria targeted antioxidant MitoQ normalizes lipid droplets size, and prevents axonal degeneration and locomotor disability, highlighting its therapeutic potential. Moreover, PMP-4 acting solely in the hypodermis rescues axonal and locomotion abnormalities, suggesting a myelin-like role for the hypodermis in providing essential peroxisomal functions for the nematode nervous system.
Asunto(s)
Adrenoleucodistrofia , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/genética , Transportadoras de Casetes de Unión a ATP/genética , Adrenoleucodistrofia/tratamiento farmacológico , Adrenoleucodistrofia/genética , Animales , Caenorhabditis elegans/genética , Ácidos Grasos , Ratones , Ratones Noqueados , Tejido SubcutáneoRESUMEN
Biotin is an essential cofactor for carboxylases that regulates the energy metabolism. Recently, high-dose pharmaceutical-grade biotin (MD1003) was shown to improve clinical parameters in a subset of patients with chronic progressive multiple sclerosis. To gain insight into the mechanisms of action, we investigated the efficacy of high-dose biotin in a genetic model of chronic axonopathy caused by oxidative damage and bioenergetic failure, the Abcd1- mouse model of adrenomyeloneuropathy. High-dose biotin restored redox homeostasis driven by NRF-2, mitochondria biogenesis and ATP levels, and reversed axonal demise and locomotor impairment. Moreover, we uncovered a concerted dysregulation of the transcriptional program for lipid synthesis and degradation in the spinal cord likely driven by aberrant SREBP-1c/mTORC1signaling. This resulted in increased triglyceride levels and lipid droplets in motor neurons. High-dose biotin normalized the hyperactivation of mTORC1, thus restoring lipid homeostasis. These results shed light into the mechanism of action of high-dose biotin of relevance for neurodegenerative and metabolic disorders.
Asunto(s)
Adrenoleucodistrofia/terapia , Biotina/farmacología , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/genética , Miembro 1 de la Subfamilia D de Transportador de Casetes de Unión al ATP/metabolismo , Adrenoleucodistrofia/genética , Adrenoleucodistrofia/metabolismo , Animales , Axones/metabolismo , Biotina/metabolismo , Línea Celular , Modelos Animales de Enfermedad , Metabolismo Energético , Homeostasis , Humanos , Lípidos , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo/fisiología , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/metabolismoRESUMEN
Parkinson's disease (PD) is associated with the degeneration of ventral midbrain dopaminergic neurons (vmDAns) and the accumulation of toxic α-synuclein. A non-cell-autonomous contribution, in particular of astrocytes, during PD pathogenesis has been suggested by observational studies, but remains to be experimentally tested. Here, we generated induced pluripotent stem cell-derived astrocytes and neurons from familial mutant LRRK2 G2019S PD patients and healthy individuals. Upon co-culture on top of PD astrocytes, control vmDAns displayed morphological signs of neurodegeneration and abnormal, astrocyte-derived α-synuclein accumulation. Conversely, control astrocytes partially prevented the appearance of disease-related phenotypes in PD vmDAns. We additionally identified dysfunctional chaperone-mediated autophagy (CMA), impaired macroautophagy, and progressive α-synuclein accumulation in PD astrocytes. Finally, chemical enhancement of CMA protected PD astrocytes and vmDAns via the clearance of α-synuclein accumulation. Our findings unveil a crucial non-cell-autonomous contribution of astrocytes during PD pathogenesis, and open the path to exploring novel therapeutic strategies aimed at blocking the pathogenic cross talk between neurons and glial cells.
Asunto(s)
Astrocitos/citología , Células Madre Pluripotentes Inducidas/citología , Enfermedad de Parkinson/fisiopatología , Astrocitos/metabolismo , Autofagia/fisiología , Diferenciación Celular/fisiología , Células Cultivadas , Técnicas de Cocultivo/métodos , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mesencéfalo/citología , Mesencéfalo/metabolismo , Neuroglía , Enfermedad de Parkinson/metabolismo , Fenotipo , alfa-Sinucleína/metabolismoRESUMEN
Sirtuin 2 (SIRT2) is a member of a family of NAD+ -dependent histone deacetylases (HDAC) that play diverse roles in cellular metabolism and especially for aging process. SIRT2 is located in the nucleus, cytoplasm, and mitochondria, is highly expressed in the central nervous system (CNS), and has been reported to regulate a variety of processes including oxidative stress, genome integrity, and myelination. However, little is known about the role of SIRT2 in the nervous system specifically during aging. Here, we show that middle-aged, 13-month-old mice lacking SIRT2 exhibit locomotor dysfunction due to axonal degeneration, which was not present in young SIRT2 mice. In addition, these Sirt2-/- mice exhibit mitochondrial depletion resulting in energy failure, and redox dyshomeostasis. Our results provide a novel link between SIRT2 and physiological aging impacting the axonal compartment of the central nervous system, while supporting a major role for SIRT2 in orchestrating its metabolic regulation. This underscores the value of SIRT2 as a therapeutic target in the most prevalent neurodegenerative diseases that undergo with axonal degeneration associated with redox and energetic dyshomeostasis.