RESUMEN
Mutations in RNA/DNA-binding proteins cause amyotrophic lateral sclerosis (ALS), but the underlying disease mechanisms remain unclear. Here, we report that a set of ALS-associated proteins, namely FUS, EWSR1, TAF15, and MATR3, impact the expression of genes encoding the major histocompatibility complex II (MHC II) antigen presentation pathway. Both subunits of the MHC II heterodimer, HLA-DR, are down-regulated in ALS gene knockouts/knockdown in HeLa and human microglial cells, due to loss of the MHC II transcription factor CIITA. Importantly, hematopoietic progenitor cells (HPCs) derived from human embryonic stem cells bearing the FUSR495X mutation and HPCs derived from C9ORF72 ALS patient induced pluripotent stem cells also exhibit disrupted MHC II expression. Given that HPCs give rise to numerous immune cells, our data raise the possibility that loss of the MHC II pathway results in global failure of the immune system to protect motor neurons from damage that leads to ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral , Humanos , Esclerosis Amiotrófica Lateral/genética , Presentación de Antígeno/genética , Genes MHC Clase II , Complejo Mayor de Histocompatibilidad , Neuronas Motoras , Proteínas de Unión al ARN/genética , Proteínas Asociadas a Matriz NuclearRESUMEN
The GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Dysregulated DNA damage response and the generation of reactive oxygen species (ROS) have been postulated as major drivers of toxicity in C9ORF72 pathogenesis. Telomeres are tandem-repeated nucleotide sequences that are located at the end of chromosomes and protect them from degradation. Interestingly, it has been established that telomeres are sensitive to ROS. Here, we analyzed telomere length in neurons and neural progenitor cells from several induced pluripotent stem cell (iPSC) lines from control subjects and C9ORF72 repeat expansion carriers. We found an age-dependent decrease in telomere length in two-month-old iPSC-derived motor neurons from C9ORF72 carriers as compared to control subjects and a dysregulation in the protein levels of shelterin complex members TRF2 and POT1.
RESUMEN
Nucleocytoplasmic transport (NCT) decline occurs with aging and neurodegeneration. Here, we investigated the NCT pathway in models of amyotrophic lateral sclerosis-fused in sarcoma (ALS-FUS). Expression of ALS-FUS led to a reduction in NCT and nucleoporin (Nup) density within the nuclear membrane of human neurons. FUS and Nups were found to interact independently of RNA in cells and to alter the phase-separation properties of each other in vitro. FUS-Nup interactions were not localized to nuclear pores, but were enriched in the nucleus of control neurons versus the cytoplasm of mutant neurons. Our data indicate that the effect of ALS-linked mutations on the cytoplasmic mislocalization of FUS, rather than on the physiochemical properties of the protein itself, underlie our reported NCT defects. An aberrant interaction between mutant FUS and Nups is underscored by studies in Drosophila, whereby reduced Nup expression rescued multiple toxic FUS-induced phenotypes, including abnormal nuclear membrane morphology in neurons.
Asunto(s)
Transporte Activo de Núcleo Celular/fisiología , Neuronas/metabolismo , Proteínas de Complejo Poro Nuclear/metabolismo , Proteína FUS de Unión a ARN/metabolismo , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila , Humanos , Mutación , Proteína FUS de Unión a ARN/genéticaRESUMEN
The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC repeat expansion in the C9orf72 gene. We developed a platform to interrogate the chromatin accessibility landscape and transcriptional program within neurons during degeneration. We provide evidence that neurons expressing the dipeptide repeat protein poly(proline-arginine), translated from the C9orf72 repeat expansion, activate a highly specific transcriptional program, exemplified by a single transcription factor, p53. Ablating p53 in mice completely rescued neurons from degeneration and markedly increased survival in a C9orf72 mouse model. p53 reduction also rescued axonal degeneration caused by poly(glycine-arginine), increased survival of C9orf72 ALS/FTD-patient-induced pluripotent stem cell (iPSC)-derived motor neurons, and mitigated neurodegeneration in a C9orf72 fly model. We show that p53 activates a downstream transcriptional program, including Puma, which drives neurodegeneration. These data demonstrate a neurodegenerative mechanism dynamically regulated through transcription-factor-binding events and provide a framework to apply chromatin accessibility and transcription program profiles to neurodegeneration.
Asunto(s)
Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN/genética , Degeneración Nerviosa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Axones/metabolismo , Proteína C9orf72/genética , Muerte Celular , Células Cultivadas , Corteza Cerebral/patología , Cromatina/metabolismo , Daño del ADN , Modelos Animales de Enfermedad , Drosophila , Ratones Endogámicos C57BL , Degeneración Nerviosa/patología , Estabilidad Proteica , Transcripción Genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of motor neurons (MNs) and subsequent muscle weakness. These pathological features are associated with numerous cellular changes, including alteration in mitochondrial morphology and function. However, the molecular mechanisms associating mitochondrial structure with ALS pathology are poorly understood. In this study, we found that Dynamin-related protein 1 (Drp1) was dephosphorylated in several ALS models, including those with SOD1 and TDP-43 mutations, and the dephosphorylation was mediated by the pathological induction of protein phosphatase 1 (PP1) activity in these models. Suppression of the PP1-Drp1 cascade effectively prevented ALS-related symptoms, including mitochondrial fragmentation, mitochondrial complex I impairment, axonal degeneration, and cell death, in primary neuronal culture models, iPSC-derived human MNs, and zebrafish models in vivo. These results suggest that modulation of PP1-Drp1 activity may be a therapeutic target for multiple pathological features of ALS.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Mitocondrias/metabolismo , Proteína Fosfatasa 1/metabolismo , Animales , Muerte Celular/genética , Muerte Celular/fisiología , Modelos Animales de Enfermedad , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Mitocondriales/metabolismo , Neuronas Motoras/efectos de los fármacos , Neuronas Motoras/metabolismo , Mutación/genética , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/metabolismo , Pez CebraRESUMEN
The GGGGCC repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it is not known which dysregulated molecular pathways are primarily responsible for disease initiation or progression. We established an inducible mouse model of poly(GR) toxicity in which (GR)80 gradually accumulates in cortical excitatory neurons. Low-level poly(GR) expression induced FTD/ALS-associated synaptic dysfunction and behavioral abnormalities, as well as age-dependent neuronal cell loss, microgliosis and DNA damage, probably caused in part by early defects in mitochondrial function. Poly(GR) bound preferentially to the mitochondrial complex V component ATP5A1 and enhanced its ubiquitination and degradation, consistent with reduced ATP5A1 protein level in both (GR)80 mouse neurons and patient brains. Moreover, inducing ectopic Atp5a1 expression in poly(GR)-expressing neurons or reducing poly(GR) level in adult mice after disease onset rescued poly(GR)-induced neurotoxicity. Thus, poly(GR)-induced mitochondrial defects are a major driver of disease initiation in C9ORF72-related ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Proteína C9orf72/genética , Demencia Frontotemporal/fisiopatología , Mitocondrias/patología , ATPasas de Translocación de Protón Mitocondriales/metabolismo , Esclerosis Amiotrófica Lateral/genética , Animales , Encéfalo/metabolismo , Expansión de las Repeticiones de ADN , Modelos Animales de Enfermedad , Demencia Frontotemporal/genética , Humanos , Ratones , Ratones Transgénicos , Neuronas/metabolismoRESUMEN
GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One class of major pathogenic molecules in C9ORF72-ALS/FTD is dipeptide repeat proteins such as poly(GR), whose toxicity has been well documented in cellular and animal models. However, it is not known how poly(GR) toxicity can be alleviated, especially in patient neurons. Using Drosophila as a model system in an unbiased genetic screen, we identified a number of genetic modifiers of poly(GR) toxicity. Surprisingly, partial loss of function of Ku80, an essential DNA repair protein, suppressed poly(GR)-induced retinal degeneration in flies. Ku80 expression was greatly elevated in flies expressing poly(GR) and in C9ORF72 iPSC-derived patient neurons. As a result, the levels of phosphorylated ATM and P53 as well as other downstream proapoptotic proteins such as PUMA, Bax, and cleaved caspase-3 were all significantly increased in C9ORF72 patient neurons. The increase in the levels of Ku80 and some downstream signaling proteins was prevented by CRISPR-Cas9-mediated deletion of expanded G4C2 repeats. More importantly, partial loss of function of Ku80 in these neurons through CRISPR/Cas9-mediated ablation or small RNAs-mediated knockdown suppressed the apoptotic pathway. Thus, partial inhibition of the overactivated Ku80-dependent DNA repair pathway is a promising therapeutic approach in C9ORF72-ALS/FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral , Proteína C9orf72 , Reparación del ADN , Demencia Frontotemporal , Autoantígeno Ku , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína C9orf72/genética , Proteína C9orf72/metabolismo , Sistemas CRISPR-Cas , Modelos Animales de Enfermedad , Drosophila melanogaster , Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Autoantígeno Ku/genética , Autoantígeno Ku/metabolismo , Secuencias Repetitivas de AminoácidoRESUMEN
Hexanucleotide repeat expansion in C9ORF72 is the most frequent cause of both amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Here we demonstrate that the repeat-associated non-AUG (RAN) translation of (GGGGCC) n -containing RNAs into poly-dipeptides can initiate in vivo without a 5'-cap. The primary RNA substrate for RAN translation of C9ORF72 sense repeats is shown to be the spliced first intron, following its excision from the initial pre-mRNA and transport to the cytoplasm. Cap-independent RAN translation is shown to be upregulated by various stress stimuli through phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF2α), the core event of an integrated stress response (ISR). Compounds inhibiting phospho-eIF2α-signaling pathways are shown to suppress RAN translation. Since the poly-dipeptides can themselves induce stress, these findings support a feedforward loop with initial repeat-mediated toxicity enhancing RAN translation and subsequent production of additional poly-dipeptides through ISR, thereby promoting progressive disease.
Asunto(s)
Proteína C9orf72/genética , Factor 2 Eucariótico de Iniciación/metabolismo , Estrés Fisiológico/genética , Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/metabolismo , Expansión de las Repeticiones de ADN , Dipéptidos , Retroalimentación Fisiológica , Demencia Frontotemporal/genética , Células HeLa , Humanos , Intrones , Péptidos , Fosforilación , Biosíntesis de Proteínas , Empalme del ARN , Regulación hacia ArribaRESUMEN
Frontotemporal dementia (FTD), the second most common form of dementia in people under 65 years of age, is characterized by progressive atrophy of the frontal and/or temporal lobes. FTD overlaps extensively with the motor neuron disease amyotrophic lateral sclerosis (ALS), especially at the genetic level. Both FTD and ALS can be caused by many mutations in the same set of genes; the most prevalent of these mutations is a GGGGCC repeat expansion in the first intron of C9ORF72 As shown by recent intensive studies, some key cellular pathways are dysregulated in the ALS-FTD spectrum disorder, including autophagy, nucleocytoplasmic transport, DNA damage repair, pre-mRNA splicing, stress granule dynamics, and others. These exciting advances reveal the complexity of the pathogenic mechanisms of FTD and ALS and suggest promising molecular targets for future therapeutic interventions in these devastating disorders.
Asunto(s)
Esclerosis Amiotrófica Lateral/fisiopatología , Demencia Frontotemporal/fisiopatología , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/terapia , Animales , Fenómenos Fisiológicos Celulares , Demencia Frontotemporal/terapia , HumanosRESUMEN
Hexanucleotide repeat expansion in the C9ORF72 gene results in production of dipeptide repeat (DPR) proteins that may disrupt pre-mRNA splicing in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) patients. At present, the mechanisms underlying this mis-splicing are not understood. Here, we show that addition of proline-arginine (PR) and glycine-arginine (GR) toxic DPR peptides to nuclear extracts blocks spliceosome assembly and splicing, but not other types of RNA processing. Proteomic and biochemical analyses identified the U2 small nuclear ribonucleoprotein particle (snRNP) as a major interactor of PR and GR peptides. In addition, U2 snRNP, but not other splicing factors, mislocalizes from the nucleus to the cytoplasm both in C9ORF72 patient induced pluripotent stem cell (iPSC)-derived motor neurons and in HeLa cells treated with the toxic peptides. Bioinformatic studies support a specific role for U2-snRNP-dependent mis-splicing in C9ORF72 patient brains. Together, our data indicate that DPR-mediated dysfunction of U2 snRNP could account for as much as â¼44% of the mis-spliced cassette exons in C9ORF72 patient brains.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Proteína C9orf72/metabolismo , Dipéptidos/farmacología , Demencia Frontotemporal/genética , ARN Nuclear Pequeño/metabolismo , Esclerosis Amiotrófica Lateral/inmunología , Esclerosis Amiotrófica Lateral/metabolismo , Proteína C9orf72/genética , Expansión de las Repeticiones de ADN , Dipéptidos/metabolismo , Demencia Frontotemporal/inmunología , Demencia Frontotemporal/metabolismo , Humanos , Proteómica/métodos , Empalme del ARN , ARN Nuclear Pequeño/genética , Ribonucleoproteína Nuclear Pequeña U2/genética , Ribonucleoproteína Nuclear Pequeña U2/metabolismoRESUMEN
GGGGCC repeat expansions in C9ORF72 are the most common genetic cause of both ALS and FTD. To uncover underlying pathogenic mechanisms, we found that DNA damage was greater, in an age-dependent manner, in motor neurons differentiated from iPSCs of multiple C9ORF72 patients than control neurons. Ectopic expression of the dipeptide repeat (DPR) protein (GR)80 in iPSC-derived control neurons increased DNA damage, suggesting poly(GR) contributes to DNA damage in aged C9ORF72 neurons. Oxidative stress was also increased in C9ORF72 neurons in an age-dependent manner. Pharmacological or genetic reduction of oxidative stress partially rescued DNA damage in C9ORF72 neurons and control neurons expressing (GR)80 or (GR)80-induced cellular toxicity in flies. Moreover, interactome analysis revealed that (GR)80 preferentially bound to mitochondrial ribosomal proteins and caused mitochondrial dysfunction. Thus, poly(GR) in C9ORF72 neurons compromises mitochondrial function and causes DNA damage in part by increasing oxidative stress, revealing another pathogenic mechanism in C9ORF72-related ALS and FTD.
Asunto(s)
Esclerosis Amiotrófica Lateral/metabolismo , Daño del ADN , Demencia Frontotemporal/metabolismo , Mitocondrias/metabolismo , Neuronas Motoras/metabolismo , Estrés Oxidativo/genética , Esclerosis Amiotrófica Lateral/genética , Arginina/metabolismo , Western Blotting , Proteína C9orf72 , Línea Celular , Expansión de las Repeticiones de ADN , Dipéptidos/metabolismo , Demencia Frontotemporal/genética , Glicina/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Proteínas/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
How mutations in the microtubule-associated protein tau (MAPT) gene cause frontotemporal dementia (FTD) remains poorly understood. We generated and characterized multiple induced pluripotent stem cell (iPSC) lines from patients with MAPT IVS10+16 and tau-A152T mutations and a control subject. In cortical neurons differentiated from these and other published iPSC lines, we found that MAPT mutations do not affect neuronal differentiation but increase the 4R/3R tau ratio. Patient neurons had significantly higher levels of MMP-9 and MMP-2 and were more sensitive to stress-induced cell death. Inhibitors of MMP-9/MMP-2 protected patient neurons from stress-induced cell death and recombinant MMP-9/MMP-2 were sufficient to decrease neuronal survival. In tau-A152T neurons, inhibition of the ERK pathway decreased MMP-9 expression. Moreover, ectopic expression of 4R but not 3R tau-A152T in HEK293 cells increased MMP-9 expression and ERK phosphorylation. These findings provide insights into the molecular pathogenesis of FTD and suggest a potential therapeutic target for FTD with MAPT mutations.
Asunto(s)
Demencia Frontotemporal/genética , Demencia Frontotemporal/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Mutación , Neuronas/metabolismo , Proteínas tau/genética , Anciano , Muerte Celular/genética , Diferenciación Celular/genética , Supervivencia Celular , Reprogramación Celular , Técnicas de Reprogramación Celular , Activación Enzimática , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Demencia Frontotemporal/patología , Regulación de la Expresión Génica , Humanos , Células Madre Pluripotentes Inducidas/citología , Masculino , Metaloproteinasa 2 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/genética , Neuronas/citología , Proteínas tau/metabolismoRESUMEN
Disturbance of endoplasmic reticulum (ER) proteostasis is a common feature of amyotrophic lateral sclerosis (ALS). Protein disulfide isomerases (PDIs) areERfoldases identified as possibleALSbiomarkers, as well as neuroprotective factors. However, no functional studies have addressed their impact on the disease process. Here, we functionally characterized fourALS-linked mutations recently identified in two majorPDIgenes,PDIA1 andPDIA3/ERp57. Phenotypic screening in zebrafish revealed that the expression of thesePDIvariants induce motor defects associated with a disruption of motoneuron connectivity. Similarly, the expression of mutantPDIs impaired dendritic outgrowth in motoneuron cell culture models. Cellular and biochemical studies identified distinct molecular defects underlying the pathogenicity of thesePDImutants. Finally, targetingERp57 in the nervous system led to severe motor dysfunction in mice associated with a loss of neuromuscular synapses. This study identifiesERproteostasis imbalance as a risk factor forALS, driving initial stages of the disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Neuronas Motoras/patología , Procolágeno-Prolina Dioxigenasa/genética , Proteína Disulfuro Isomerasas/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Electromiografía , Embrión no Mamífero , Estrés del Retículo Endoplásmico/genética , Humanos , Ratones Noqueados , Mutación , Neuritas/patología , Procolágeno-Prolina Dioxigenasa/metabolismo , Proteína Disulfuro Isomerasas/metabolismo , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
The GGGGCC (G4C2) repeat expansion in a noncoding region of C9orf72 is the most common cause of sporadic and familial forms of amyotrophic lateral sclerosis and frontotemporal dementia. The basis for pathogenesis is unknown. To elucidate the consequences of G4C2 repeat expansion in a tractable genetic system, we generated transgenic fly lines expressing 8, 28 or 58 G4C2-repeat-containing transcripts that do not have a translation start site (AUG) but contain an open-reading frame for green fluorescent protein to detect repeat-associated non-AUG (RAN) translation. We show that these transgenic animals display dosage-dependent, repeat-length-dependent degeneration in neuronal tissues and RAN translation of dipeptide repeat (DPR) proteins, as observed in patients with C9orf72-related disease. This model was used in a large-scale, unbiased genetic screen, ultimately leading to the identification of 18 genetic modifiers that encode components of the nuclear pore complex (NPC), as well as the machinery that coordinates the export of nuclear RNA and the import of nuclear proteins. Consistent with these results, we found morphological abnormalities in the architecture of the nuclear envelope in cells expressing expanded G4C2 repeats in vitro and in vivo. Moreover, we identified a substantial defect in RNA export resulting in retention of RNA in the nuclei of Drosophila cells expressing expanded G4C2 repeats and also in mammalian cells, including aged induced pluripotent stem-cell-derived neurons from patients with C9orf72-related disease. These studies show that a primary consequence of G4C2 repeat expansion is the compromise of nucleocytoplasmic transport through the nuclear pore, revealing a novel mechanism of neurodegeneration.
Asunto(s)
Transporte Activo de Núcleo Celular/genética , Expansión de las Repeticiones de ADN/genética , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Sistemas de Lectura Abierta/genética , Proteínas/genética , Transporte de ARN/genética , Esclerosis Amiotrófica Lateral/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Animales Modificados Genéticamente , Proteína C9orf72 , Drosophila melanogaster/genética , Ojo/metabolismo , Femenino , Demencia Frontotemporal/genética , Demencia Frontotemporal/patología , Células HeLa , Humanos , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/metabolismo , Masculino , Músculos/citología , Músculos/metabolismo , Neuronas/citología , Neuronas/metabolismo , Poro Nuclear/genética , Poro Nuclear/metabolismo , Poro Nuclear/patología , Fenotipo , Biosíntesis de Proteínas , ARN/genética , ARN/metabolismo , Glándulas Salivales/citología , Glándulas Salivales/metabolismo , Glándulas Salivales/patologíaRESUMEN
BACKGROUND: Histamine (HA) regulates the sleep-wake cycle, synaptic plasticity and memory in adult mammals. Dopaminergic specification in the embryonic ventral midbrain (VM) coincides with increased HA brain levels. To study the effect of HA receptor stimulation on dopamine neuron generation, we administered HA to dopamine progenitors, both in vitro and in vivo. RESULTS: Cultured embryonic day 12 (E12) VM neural stem/progenitor cells expressed transcripts for HA receptors H1R, H2R and H3R. These undifferentiated progenitors increased intracellular calcium upon HA addition. In HA-treated cultures, dopamine neurons significantly decreased after activation of H1R. We performed intrauterine injections in the developing VM to investigate HA effects in vivo. HA administration to E12 rat embryos notably reduced VM Tyrosine Hydroxylase (TH) staining 2 days later, without affecting GABA neurons in the midbrain, or serotonin neurons in the mid-hindbrain boundary. qRT-PCR and Western blot analyses confirmed that several markers important for the generation and maintenance of dopaminergic lineage such as TH, Lmx1a and Lmx1b were significantly diminished. To identify the cell type susceptible to HA action, we injected embryos of different developmental stages, and found that neural progenitors (E10 and E12) were responsive, whereas differentiated dopaminergic neurons (E14 and E16) were not susceptible to HA actions. Proliferation was significantly diminished, whereas neuronal death was not increased in the VM after HA administration. We injected H1R or H2R antagonists to identify the receptor responsible for the detrimental effect of HA on dopaminergic lineage and found that activation of H1R was required. CONCLUSION: These results reveal a novel action of HA affecting dopaminergic lineage during VM development.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Histamina/farmacología , Mesencéfalo/embriología , Receptores Histamínicos H1/metabolismo , Animales , Apoptosis/efectos de los fármacos , Calcio/metabolismo , Recuento de Células , Diferenciación Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Separación Celular , Células Cultivadas , Clorfeniramina/farmacología , Cimetidina/farmacología , Neuronas Dopaminérgicas/efectos de los fármacos , Embrión de Mamíferos/citología , Femenino , Neuronas GABAérgicas/efectos de los fármacos , Neuronas GABAérgicas/metabolismo , Histamina/administración & dosificación , Espacio Intracelular/metabolismo , Células-Madre Neurales/efectos de los fármacos , Células-Madre Neurales/metabolismo , Ratas Wistar , Neuronas Serotoninérgicas/efectos de los fármacos , Neuronas Serotoninérgicas/metabolismoRESUMEN
Degeneration of motor neurons (MN) caused by disease or injury leads to paralysis and is fatal in some conditions. To date, there are no effective treatments for MN disorders; therefore, cell therapy is a promising strategy to replace lost MN. Embryonic stem (ES) cells isolated from the inner cell mass of mammalian blastocysts self-renew and are pluripotent because they differentiate into cell types of the three germinal layers. Reprogramming of adult cells to a state similar to ES cells, termed induced pluripotent stem (iPS) cells, has been recently reported. It is well established that pluripotent cell types can give rise to specialized phenotypes, including neurons. Mouse, monkey and human MN can be differentiated from ES and iPS cells using procedures generally involving embryoid bodies formation and stimulation with retinoic acid and Sonic hedgehog. Differentiated MN express characteristic molecular markers such as Islet1, HB9 and Choline acetyltransferase, exhibit electrophysiological maturity and are able to form synaptic contacts similar to neuromuscular junctions in vitro. Furthermore, transplanted MN promote functional recovery in animal models of neurodegenerative diseases and MN injury. The potential clinical applications of stem cell-derived MN was enhanced after iPS cell derivation, which makes possible the generation of patient-specific pluripotent cells for autologous cell replacement therapies and may be used for drug development and disease modeling. This review summarizes MN differentiation protocols from ES and iPS cells in regard to neuronal differentiation efficiency, expression of MN markers and functional properties in vitro, as well as their therapeutic effects after grafting.
Asunto(s)
Diferenciación Celular , Células Madre Embrionarias/fisiología , Células Madre Pluripotentes Inducidas/fisiología , Neuronas Motoras/trasplante , Enfermedades Neurodegenerativas/terapia , Animales , Técnicas de Cultivo de Célula , Humanos , Medicina Regenerativa , Enfermedades de la Médula Espinal/terapia , Factores de Transcripción/farmacología , Factores de Transcripción/fisiologíaRESUMEN
Embryonic stem (ES) cells have the capacity to differentiate into endodermal, mesodermal, and ectodermal lineages. Motor neuron (MN) differentiation of mouse ES cells involves embryoid bodies formation with addition of Sonic hedgehog and retinoic acid. In this work, using immunocytochemistry, flow cytometry, and quantitative RT-PCR, we investigated whether progesterone or 17ß-estradiol have inductive effects on ES cell-derived MN, as it has been demonstrated that these hormones modify proliferation and neural differentiation of pluripotent cells. When 100 nM progesterone was added during differentiation, we found higher proportions of MN, compared to the control condition; coincubation of progesterone with the progesterone receptor (PR) antagonist RU-486 caused a decrease in the number of MN to a percentage even lower than controls. The addition of nanomolar concentrations of 17ß-estradiol also significantly induced MN differentiation. This effect of estradiol was completely antagonized by addition of the general estrogen receptor (ER) antagonist ICI 182,780. To identify the ER subtype mediating the increase on MN differentiation, we incubated estradiol with the ER-α antagonist MPP or with the ER-ß blocker PHTPP. When we coincubated 17ß-estradiol with MPP, we found a significant decrease in the percentage of MN. In contrast, the coincubation of 17ß-estradiol with PHTPP had no effect on the induction of MN differentiation. All these effects on cell number were confirmed by significant changes in the expression of the MN markers Islet-1 and Choline acetyl transferase, assessed by real-time RT-PCR. Cell proliferation in embryoid bodies was significantly enhanced by progesterone treatment. No changes in apoptotic cell death were found in differentiating cells after progesterone or 17ß-estradiol addition. Our findings indicate that progesterone and 17ß-estradiol induce a higher proportion of MN derived from mouse ES cells through intracellular PR and ER, respectively. Furthermore, the effect of estradiol was mediated by specific activation of ER-α.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Células Madre Embrionarias/fisiología , Estradiol/farmacología , Receptor alfa de Estrógeno/metabolismo , Neuronas Motoras/citología , Progesterona/farmacología , Análisis de Varianza , Animales , Diferenciación Celular/fisiología , Colina O-Acetiltransferasa/metabolismo , Cartilla de ADN/genética , Células Madre Embrionarias/citología , Estradiol/análogos & derivados , Antagonistas de Estrógenos/farmacología , Citometría de Flujo , Fulvestrant , Inmunohistoquímica , Proteínas con Homeodominio LIM/metabolismo , Ratones , Mifepristona/farmacología , Neuronas Motoras/efectos de los fármacos , Progesterona/antagonistas & inhibidores , Pirazoles , Pirimidinas , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/metabolismoRESUMEN
Embryonic stem (ES) cells can be induced to differentiate into motor neurons (MN). Animal models resembling MN degeneration and paralysis observed in familial amyotrophic lateral sclerosis (ALS) have been previously reported. In this work, we aimed to investigate whether transplanted MN could prevent motor deterioration in transgenic rats expressing a mutant form of human superoxide dismutase 1 (hSOD1(G93A)) associated with inherited ALS. Mouse ES cells were differentiated to neurons that express green fluorescent protein (GFP) under the promoter of the MN-specific gene hb9, as well as molecular markers indicative of MN identity. Cells were grafted into the lumbar spinal cord of adult wild-type (WT) or hSOD1(G93A) rats at 10 weeks of age, when transgenic animals are presymptomatic. Grafted cells with MN phenotype can survive for at least 1 week in hSOD1(G93A) animals. To quantitatively evaluate motor performance of WT and transgenic rats, we carried out weekly rotarod tests starting when the animals were 14 weeks old. Sham and grafted WT animals showed no decline in their ability to sustain themselves on the rotating rod. In contrast, sham hSOD1(G93A) rats decreased in motor performance from week 16 onwards, reaching paralysis by week 19 of age. In grafted transgenic animals, there was a significant improvement in rotarod competence at weeks 16 and 17 when compared to sham hSOD1(G93A). However, in the following weeks, transplanted hSOD1(G93A) rats showed motor deterioration and eventually exhibited paralysis by week 19. At end-stage, we found only a few endogenous MN in sham and grafted hSOD1(G93A) rats by cresyl violet staining; no choline acetyl transferase-positive nor GFP-positive MN were present in grafted transgenic subjects. In contrast, WT rats analyzed at the same age possessed grafted GFP-positive MN in their spinal cords. These results strongly suggest that the transgenic hSOD1(G93A) environment is detrimental to grafted MN in the long term.