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1.
Mol Psychiatry ; 26(10): 5766-5788, 2021 10.
Artículo en Inglés | MEDLINE | ID: mdl-32647257

RESUMEN

A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of ß-amyloid-(Aß)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar Aß deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.


Asunto(s)
Enfermedad de Alzheimer , Síndrome de Down , Enfermedad de Alzheimer/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Péptidos beta-Amiloides/metabolismo , Ácido Aspártico Endopeptidasas/genética , Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/metabolismo , Síndrome de Down/genética , Genes Supresores , Humanos , Organoides/metabolismo , Trisomía
2.
Proc Natl Acad Sci U S A ; 115(30): 7813-7818, 2018 07 24.
Artículo en Inglés | MEDLINE | ID: mdl-29991596

RESUMEN

α-Synuclein (α-Syn) aggregation, proceeding from oligomers to fibrils, is one central hallmark of neurodegeneration in synucleinopathies. α-Syn oligomers are toxic by triggering neurodegenerative processes in in vitro and in vivo models. However, the precise contribution of α-Syn oligomers to neurite pathology in human neurons and the underlying mechanisms remain unclear. Here, we demonstrate the formation of oligomeric α-Syn intermediates and reduced axonal mitochondrial transport in human neurons derived from induced pluripotent stem cells (iPSC) from a Parkinson's disease patient carrying an α-Syn gene duplication. We further show that increased levels of α-Syn oligomers disrupt axonal integrity in human neurons. We apply an α-Syn oligomerization model by expressing α-Syn oligomer-forming mutants (E46K and E57K) and wild-type α-Syn in human iPSC-derived neurons. Pronounced α-Syn oligomerization led to impaired anterograde axonal transport of mitochondria, which can be restored by the inhibition of α-Syn oligomer formation. Furthermore, α-Syn oligomers were associated with a subcellular relocation of transport-regulating proteins Miro1, KLC1, and Tau as well as reduced ATP levels, underlying axonal transport deficits. Consequently, reduced axonal density and structural synaptic degeneration were observed in human neurons in the presence of high levels of α-Syn oligomers. Together, increased dosage of α-Syn resulting in α-Syn oligomerization causes axonal transport disruption and energy deficits, leading to synapse loss in human neurons. This study identifies α-Syn oligomers as the critical species triggering early axonal dysfunction in synucleinopathies.


Asunto(s)
Transporte Axonal , Axones/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Modelos Biológicos , Enfermedades Neurodegenerativas/metabolismo , Multimerización de Proteína , Axones/patología , Línea Celular , Metabolismo Energético/genética , Humanos , Células Madre Pluripotentes Inducidas/patología , Cinesinas , Proteínas Asociadas a Microtúbulos/genética , Proteínas Asociadas a Microtúbulos/metabolismo , Mitocondrias/genética , Mitocondrias/metabolismo , Mitocondrias/patología , Proteínas Mitocondriales/genética , Proteínas Mitocondriales/metabolismo , Mutación Missense , Enfermedades Neurodegenerativas/genética , Enfermedades Neurodegenerativas/patología , alfa-Sinucleína , Proteínas de Unión al GTP rho/genética , Proteínas de Unión al GTP rho/metabolismo , Proteínas tau/genética , Proteínas tau/metabolismo
3.
Mol Ther ; 25(2): 342-355, 2017 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-28153087

RESUMEN

Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 enables us to generate targeted sequence changes in the genomes of cells and organisms. However, off-target effects have been a persistent problem hampering the development of therapeutics based on CRISPR/Cas9 and potentially confounding research results. Efforts to improve Cas9 specificity, like the development of RNA-guided FokI-nucleases (RFNs), usually come at the cost of editing efficiency and/or genome targetability. To overcome these limitations, we engineered improved chimeras of RFNs that enable higher cleavage efficiency and provide broader genome targetability, while retaining high fidelity for genome editing in human cells. Furthermore, we developed a new RFN ortholog derived from Staphylococcus aureus Cas9 and characterize its utility for efficient genome engineering. Finally, we demonstrate the feasibility of RFN orthologs to functionally hetero-dimerize to modify endogenous genes, unveiling a new dimension of RFN target design opportunities.


Asunto(s)
Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Edición Génica , Ingeniería de Proteínas , ARN Guía de Kinetoplastida , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Sitios de Unión , Proteína 9 Asociada a CRISPR , Sistemas CRISPR-Cas , Desoxirribonucleasas de Localización Especificada Tipo II/química , Desoxirribonucleasas de Localización Especificada Tipo II/genética , Endonucleasas/metabolismo , Variación Genética , Humanos , Modelos Biológicos , Mutación , Células Madre Pluripotentes/metabolismo , Unión Proteica , Multimerización de Proteína , Factores de Transcripción del Factor Regulador X/química , Factores de Transcripción del Factor Regulador X/genética
4.
Ann Neurol ; 79(5): 826-840, 2016 May.
Artículo en Inglés | MEDLINE | ID: mdl-26971897

RESUMEN

OBJECTIVE: Mutations in the spastic paraplegia gene 11 (SPG11), encoding spatacsin, cause the most frequent form of autosomal-recessive complex hereditary spastic paraplegia (HSP) and juvenile-onset amyotrophic lateral sclerosis (ALS5). When SPG11 is mutated, patients frequently present with spastic paraparesis, a thin corpus callosum, and cognitive impairment. We previously delineated a neurodegenerative phenotype in neurons of these patients. In the current study, we recapitulated early developmental phenotypes of SPG11 and outlined their cellular and molecular mechanisms in patient-specific induced pluripotent stem cell (iPSC)-derived cortical neural progenitor cells (NPCs). METHODS: We generated and characterized iPSC-derived NPCs and neurons from 3 SPG11 patients and 2 age-matched controls. RESULTS: Gene expression profiling of SPG11-NPCs revealed widespread transcriptional alterations in neurodevelopmental pathways. These include changes in cell-cycle, neurogenesis, cortical development pathways, in addition to autophagic deficits. More important, the GSK3ß-signaling pathway was found to be dysregulated in SPG11-NPCs. Impaired proliferation of SPG11-NPCs resulted in a significant diminution in the number of neural cells. The decrease in mitotically active SPG11-NPCs was rescued by GSK3 modulation. INTERPRETATION: This iPSC-derived NPC model provides the first evidence for an early neurodevelopmental phenotype in SPG11, with GSK3ß as a potential novel target to reverse the disease phenotype. Ann Neurol 2016;79:826-840.

6.
Hum Mol Genet ; 23(18): 4859-74, 2014 Sep 15.
Artículo en Inglés | MEDLINE | ID: mdl-24794856

RESUMEN

Hereditary spastic paraplegias are a group of inherited motor neuron diseases characterized by progressive paraparesis and spasticity. Mutations in the spastic paraplegia gene SPG11, encoding spatacsin, cause an autosomal-recessive disease trait; however, the precise knowledge about the role of spatacsin in neurons is very limited. We for the first time analyzed the expression and function of spatacsin in human forebrain neurons derived from human pluripotent stem cells including lines from two SPG11 patients and two controls. SPG11 patients'-derived neurons exhibited downregulation of specific axonal-related genes, decreased neurite complexity and accumulation of membranous bodies within axonal processes. Altogether, these data point towards axonal pathologies in human neurons with SPG11 mutations. To further corroborate spatacsin function, we investigated human pluripotent stem cell-derived neurons and mouse cortical neurons. In these cells, spatacsin was located in axons and dendrites. It colocalized with cytoskeletal and synaptic vesicle (SV) markers and was present in synaptosomes. Knockdown of spatacsin in mouse cortical neurons evidenced that the loss of function of spatacsin leads to axonal instability by downregulation of acetylated tubulin. Finally, time-lapse assays performed in SPG11 patients'-derived neurons and spatacsin-silenced mouse neurons highlighted a reduction in the anterograde vesicle trafficking indicative of impaired axonal transport. By employing SPG11 patient-derived forebrain neurons and mouse cortical neurons, this study provides the first evidence that SPG11 is implicated in axonal maintenance and cargo trafficking. Understanding the cellular functions of spatacsin will allow deciphering mechanisms of motor cortex dysfunction in autosomal-recessive hereditary spastic paraplegia.


Asunto(s)
Axones/metabolismo , Neuronas/metabolismo , Prosencéfalo/citología , Proteínas/metabolismo , Paraplejía Espástica Hereditaria/patología , Animales , Células Cultivadas , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Ratones Endogámicos C57BL , Neuronas/citología , Neuronas/patología , Células Madre Pluripotentes/metabolismo , Prosencéfalo/metabolismo , Proteínas/genética , Paraplejía Espástica Hereditaria/genética , Tubulina (Proteína)/metabolismo
7.
Hum Mol Genet ; 23(10): 2527-41, 2014 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-24381312

RESUMEN

The hereditary spastic paraplegias (HSPs) are a heterogeneous group of motorneuron diseases characterized by progressive spasticity and paresis of the lower limbs. Mutations in Spastic Gait 4 (SPG4), encoding spastin, are the most frequent cause of HSP. To understand how mutations in SPG4 affect human neurons, we generated human induced pluripotent stem cells (hiPSCs) from fibroblasts of two patients carrying a c.1684C>T nonsense mutation and from two controls. These SPG4 and control hiPSCs were able to differentiate into neurons and glia at comparable efficiency. All known spastin isoforms were reduced in SPG4 neuronal cells. The complexity of SPG4 neurites was decreased, which was paralleled by an imbalance of axonal transport with less retrograde movement. Prominent neurite swellings with disrupted microtubules were present in SPG4 neurons at an ultrastructural level. While some of these swellings contain acetylated and detyrosinated tubulin, these tubulin modifications were unchanged in total cell lysates of SPG4 neurons. Upregulation of another microtubule-severing protein, p60 katanin, may partially compensate for microtubuli dynamics in SPG4 neurons. Overexpression of the M1 or M87 spastin isoforms restored neurite length, branching, numbers of primary neurites and reduced swellings in SPG4 neuronal cells. We conclude that neurite complexity and maintenance in HSP patient-derived neurons are critically sensitive to spastin gene dosage. Our data show that elevation of single spastin isoform levels is sufficient to restore neurite complexity and reduce neurite swellings in patient cells. Furthermore, our human model offers an ideal platform for pharmacological screenings with the goal to restore physiological spastin levels in SPG4 patients.


Asunto(s)
Adenosina Trifosfatasas/genética , Dosificación de Gen , Paraplejía Espástica Hereditaria/genética , Adenosina Trifosfatasas/metabolismo , Adulto , Transporte Axonal , Forma de la Célula , Células Cultivadas , Femenino , Expresión Génica , Terapia Genética , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Masculino , Microtúbulos/metabolismo , Persona de Mediana Edad , Neuritas/metabolismo , Neuritas/patología , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Paraplejía Espástica Hereditaria/patología , Paraplejía Espástica Hereditaria/terapia , Espastina
8.
Hum Mol Genet ; 21(16): 3655-67, 2012 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-22641814

RESUMEN

Spontaneous neural activity promotes axon growth in many types of developing neurons, including motoneurons. In motoneurons from a mouse model of spinal muscular atrophy (SMA), defects in axonal growth and presynaptic function correlate with a reduced frequency of spontaneous Ca(2+) transients in axons which are mediated by N-type Ca(2+) channels. To characterize the mechanisms that initiate spontaneous Ca(2+) transients, we investigated the role of voltage-gated sodium channels (VGSCs). We found that low concentrations of the VGSC inhibitors tetrodotoxin (TTX) and saxitoxin (STX) reduce the rate of axon growth in cultured embryonic mouse motoneurons without affecting their survival. STX was 5- to 10-fold more potent than TTX and Ca(2+) imaging confirmed that low concentrations of STX strongly reduce the frequency of spontaneous Ca(2+) transients in somatic and axonal regions. These findings suggest that the Na(V)1.9, a VGSC that opens at low thresholds, could act upstream of spontaneous Ca(2+) transients. qPCR from cultured and laser-microdissected spinal cord motoneurons revealed abundant expression of Na(V)1.9. Na(V)1.9 protein is preferentially localized in axons and growth cones. Suppression of Na(V)1.9 expression reduced axon elongation. Motoneurons from Na(V)1.9(-/-) mice showed the reduced axon growth in combination with reduced spontaneous Ca(2+) transients in the soma and axon terminals. Thus, Na(V)1.9 function appears to be essential for activity-dependent axon growth, acting upstream of spontaneous Ca(2+) elevation through voltage-gated calcium channels (VGCCs). Na(V)1.9 activation could therefore serve as a target for modulating axonal regeneration in motoneuron diseases such as SMA in which presynaptic activity of VGCCs is reduced.


Asunto(s)
Axones/metabolismo , Calcio/metabolismo , Neuronas Motoras/metabolismo , Canal de Sodio Activado por Voltaje NAV1.9/metabolismo , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Conos de Crecimiento/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Neuronas Motoras/efectos de los fármacos , Atrofia Muscular Espinal/metabolismo , Canal de Sodio Activado por Voltaje NAV1.9/genética , Conejos , Saxitoxina/farmacología , Bloqueadores de los Canales de Sodio/farmacología , Médula Espinal/citología , Médula Espinal/metabolismo , Tetrodotoxina/farmacología , Canales de Sodio Activados por Voltaje/metabolismo
9.
Cancer Gene Ther ; 31(9): 1357-1379, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39039193

RESUMEN

Trk (NTRK) receptor and NTRK gene fusions are oncogenic drivers of a wide variety of tumors. Although Trk receptors are typically activated at the cell surface, signaling of constitutive active Trk and diverse intracellular NTRK fusion oncogenes is barely investigated. Here, we show that a high intracellular abundance is sufficient for neurotrophin-independent, constitutive activation of TrkB kinase domains. In HEK293 cells, constitutive active TrkB kinase and an intracellular NTRK2-fusion oncogene (SQSTM1-NTRK2) reduced actin filopodia dynamics, phosphorylated FAK, and altered the cell morphology. Atypical cellular responses could be mimicked with the intracellular kinase domain, which did not activate the Trk-associated MAPK/ERK pathway. In glioblastoma-like U87MG cells, expression of TrkB or SQSTM1-NTRK2 reduced cell motility and caused drastic changes in the transcriptome. Clinically approved Trk inhibitors or mutating Y705 in the kinase domain, blocked the cellular effects and transcriptome changes. Atypical signaling was also seen for TrkA and TrkC. Moreover, hallmarks of atypical pTrk kinase were found in biopsies of Nestin-positive glioblastoma. Therefore, we suggest Western blot-like immunoassay screening of NTRK-related (brain) tumor biopsies to identify patients with atypical panTrk or phosphoTrk signals. Such patients could be candidates for treatment with NTRK inhibitors such as Larotrectinhib or Entrectinhib.


Asunto(s)
Proteínas de Fusión Oncogénica , Receptor trkB , Humanos , Receptor trkB/metabolismo , Receptor trkB/genética , Proteínas de Fusión Oncogénica/genética , Proteínas de Fusión Oncogénica/metabolismo , Línea Celular Tumoral , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/genética , Células HEK293 , Transducción de Señal , Glioblastoma/genética , Glioblastoma/metabolismo , Glioblastoma/patología , Movimiento Celular/genética
10.
Cell Stem Cell ; 30(10): 1331-1350.e11, 2023 10 05.
Artículo en Inglés | MEDLINE | ID: mdl-37802038

RESUMEN

Mesial temporal lobe epilepsy (MTLE) is the most common focal epilepsy. One-third of patients have drug-refractory seizures and are left with suboptimal therapeutic options such as brain tissue-destructive surgery. Here, we report the development and characterization of a cell therapy alternative for drug-resistant MTLE, which is derived from a human embryonic stem cell line and comprises cryopreserved, post-mitotic, medial ganglionic eminence (MGE) pallial-type GABAergic interneurons. Single-dose intrahippocampal delivery of the interneurons in a mouse model of chronic MTLE resulted in consistent mesiotemporal seizure suppression, with most animals becoming seizure-free and surviving longer. The grafted interneurons dispersed locally, functionally integrated, persisted long term, and significantly reduced dentate granule cell dispersion, a pathological hallmark of MTLE. These disease-modifying effects were dose-dependent, with a broad therapeutic range. No adverse effects were observed. These findings support an ongoing phase 1/2 clinical trial (NCT05135091) for drug-resistant MTLE.


Asunto(s)
Epilepsia del Lóbulo Temporal , Hipocampo , Ratones , Animales , Humanos , Hipocampo/patología , Epilepsia del Lóbulo Temporal/patología , Epilepsia del Lóbulo Temporal/cirugía , Convulsiones/patología , Convulsiones/cirugía , Interneuronas/fisiología , Encéfalo/patología
11.
EBioMedicine ; 94: 104692, 2023 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-37451904

RESUMEN

BACKGROUND: People with Down syndrome (DS) show clinical signs of accelerated ageing. Causative mechanisms remain unknown and hypotheses range from the (essentially untreatable) amplified-chromosomal-instability explanation, to potential actions of individual supernumerary chromosome-21 genes. The latter explanation could open a route to therapeutic amelioration if the specific over-acting genes could be identified and their action toned-down. METHODS: Biological age was estimated through patterns of sugar molecules attached to plasma immunoglobulin-G (IgG-glycans, an established "biological-ageing-clock") in n = 246 individuals with DS from three European populations, clinically characterised for the presence of co-morbidities, and compared to n = 256 age-, sex- and demography-matched healthy controls. Isogenic human induced pluripotent stem cell (hiPSCs) models of full and partial trisomy-21 with CRISPR-Cas9 gene editing and two kinase inhibitors were studied prior and after differentiation to cerebral organoids. FINDINGS: Biological age in adults with DS is (on average) 18.4-19.1 years older than in chronological-age-matched controls independent of co-morbidities, and this shift remains constant throughout lifespan. Changes are detectable from early childhood, and do not require a supernumerary chromosome, but are seen in segmental duplication of only 31 genes, along with increased DNA damage and decreased levels of LaminB1 in nucleated blood cells. We demonstrate that these cell-autonomous phenotypes can be gene-dose-modelled and pharmacologically corrected in hiPSCs and derived cerebral organoids. Using isogenic hiPSC models we show that chromosome-21 gene DYRK1A overdose is sufficient and necessary to cause excess unrepaired DNA damage. INTERPRETATION: Explanation of hitherto observed accelerated ageing in DS as a developmental progeroid syndrome driven by DYRK1A overdose provides a target for early pharmacological preventative intervention strategies. FUNDING: Main funding came from the "Research Cooperability" Program of the Croatian Science Foundation funded by the European Union from the European Social Fund under the Operational Programme Efficient Human Resources 2014-2020, Project PZS-2019-02-4277, and the Wellcome Trust Grants 098330/Z/12/Z and 217199/Z/19/Z (UK). All other funding is described in details in the "Acknowledgements".


Asunto(s)
Síndrome de Down , Células Madre Pluripotentes Inducidas , Adulto , Humanos , Envejecimiento , Diferenciación Celular , Síndrome de Down/genética , Quinasas DyrK
12.
Hum Mol Genet ; 19(10): 1951-66, 2010 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-20167579

RESUMEN

Axonal transport and translation of beta-actin mRNA plays an important role for axonal growth and presynaptic differentiation in many neurons including hippocampal, cortical and spinal motor neurons. Several beta-actin mRNA-binding and transport proteins have been identified, including ZBP1, ZBP2 and hnRNP-R. hnRNP-R has been found as an interaction partner of the survival motor neuron protein that is deficient in spinal muscular atrophy. Little is known about the function of hnRNP-R in axonal beta-actin translocation. hnRNP-R and beta-actin mRNA are colocalized in axons. Recombinant hnRNP-R interacts directly with the 3'-UTR of beta-actin mRNA. We studied the role of hnRNP-R in motor neurons by knockdown in zebrafish embryos and isolated mouse motor neurons. Suppression of hnRNP-R in developing zebrafish embryos results in reduced axon growth in spinal motor neurons, without any alteration in motor neuron survival. ShRNA-mediated knockdown in isolated embryonic mouse motor neurons reduces beta-actin mRNA translocation to the axonal growth cone, which is paralleled by reduced axon elongation. Dendrite growth and neuronal survival were not affected by hnRNP-R depletion in these neurons. The loss of beta-actin mRNA in axonal growth cones of hnRNP-R-depleted motor neurons resembles that observed in Smn-deficient motor neurons, a model for the human disease spinal muscular atrophy. In particular, hnRNP-R-depleted motor neurons also exhibit defects in presynaptic clustering of voltage-gated calcium channels. Our data suggest that hnRNP-R-mediated axonal beta-actin mRNA translocation plays an essential physiological role for axon growth and presynaptic differentiation.


Asunto(s)
Actinas/metabolismo , Axones/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/metabolismo , Neuronas Motoras/metabolismo , Transporte de ARN , Columna Vertebral/metabolismo , Proteínas de Pez Cebra/metabolismo , Regiones no Traducidas 3'/genética , Actinas/genética , Animales , Axones/patología , Canales de Calcio Tipo N/metabolismo , Separación Celular , Embrión no Mamífero/patología , Técnicas de Silenciamiento del Gen , Conos de Crecimiento/metabolismo , Ribonucleoproteínas Nucleares Heterogéneas/genética , Ratones , Neuronas Motoras/patología , Unión Proteica , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/metabolismo , Pez Cebra/metabolismo
13.
Histochem Cell Biol ; 138(5): 737-48, 2012 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-22810847

RESUMEN

Reduced levels of the SMN (survival of motoneuron) protein cause spinal muscular atrophy, the main form of motoneuron disease in children and young adults. In cultured motoneurons, reduced SMN levels lead to disturbed axon growth that correlates with reduced actin mRNA and protein in growth cones, indicating that anterograde transport and local translation of ß-actin mRNA are altered in this disease. However, it is not fully understood how local translation of the ß-actin mRNA is regulated in SMN-deficient motoneurons. Here, we established a lentiviral GFP-based reporter construct to monitor local translation of ß-actin mRNA. Time-lapse imaging of fluorescence recovery after photobleaching (FRAP) in living motoneurons revealed that ß-actin is locally translated in the growth cones of embryonic motoneurons. Interestingly, local translation of the ß-actin reporter construct was differentially regulated by various Laminin isoforms, indicating that Laminins provide extracellular cues for the regulation of local translation in growth cones. Notably, local translation of ß-actin mRNA was deregulated in motoneurons from a mouse model for the most severe form of SMA (Smn(-/-);SMN2). Taken together our findings suggest that local translation of ß-actin in growth cones of motoneurons is regulated by Laminin signalling and that this signalling is disturbed in SMA.


Asunto(s)
Actinas/biosíntesis , Axones/metabolismo , Laminina/metabolismo , Neuronas Motoras/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Conos de Crecimiento/metabolismo , Humanos , Ratones , Ratones Transgénicos , Fotoblanqueo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Transducción de Señal/genética , Atrofias Musculares Espinales de la Infancia/genética , Atrofias Musculares Espinales de la Infancia/metabolismo , Proteína 1 para la Supervivencia de la Neurona Motora/análisis , Proteína 1 para la Supervivencia de la Neurona Motora/genética , Imagen de Lapso de Tiempo
14.
J Clin Endocrinol Metab ; 105(3)2020 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-31628846

RESUMEN

CONTEXT: Kallmann syndrome (KS) is a rare, genetically heterogeneous Mendelian disorder. Structural defects in KS patients have helped define the genetic architecture of gonadotropin-releasing hormone (GnRH) neuronal development in this condition. OBJECTIVE: Examine the functional role a novel structural defect affecting a long noncoding RNA (lncRNA), RMST, found in a KS patient. DESIGN: Whole genome sequencing, induced pluripotent stem cells and derived neural crest cells (NCC) from the KS patient were contrasted with controls. SETTING: The Harvard Reproductive Sciences Center, Massachusetts General Hospital Center for Genomic Medicine, and Singapore Genome Institute. PATIENT: A KS patient with a unique translocation, t(7;12)(q22;q24). INTERVENTIONS/MAIN OUTCOME MEASURE/RESULTS: A novel translocation was detected affecting the lncRNA, RMST, on chromosome 12 in the absence of any other KS mutations. Compared with controls, the patient's induced pluripotent stem cells and NCC provided functional information regarding RMST. Whereas RMST expression increased during NCC differentiation in controls, it was substantially reduced in the KS patient's NCC coincident with abrogated NCC morphological development and abnormal expression of several "downstream" genes essential for GnRH ontogeny (SOX2, PAX3, CHD7, TUBB3, and MKRN3). Additionally, an intronic single nucleotide polymorphism in RMST was significantly implicated in a genome-wide association study associated with age of menarche. CONCLUSIONS: A novel deletion in RMST implicates the loss of function of a lncRNA as a unique cause of KS and suggests it plays a critical role in the ontogeny of GnRH neurons and puberty.


Asunto(s)
Hormona Liberadora de Gonadotropina/metabolismo , Síndrome de Kallmann/genética , Síndrome de Kallmann/patología , ARN Largo no Codificante/genética , Translocación Genética , Adulto , Cromosomas Humanos Par 12/genética , Cromosomas Humanos Par 7/genética , Estudio de Asociación del Genoma Completo , Hormona Liberadora de Gonadotropina/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Masculino , Cresta Neural/metabolismo , Cresta Neural/patología , Pronóstico
15.
Mol Neurobiol ; 53(6): 4210-4225, 2016 08.
Artículo en Inglés | MEDLINE | ID: mdl-26215835

RESUMEN

The transforming growth factor-ß (TGF-ß) family member activin A exerts multiple neurotrophic and protective effects in the brain. Activin also modulates cognitive functions and affective behavior and is a presumed target of antidepressant therapy. Despite its important role in the injured and intact brain, the mechanisms underlying activin effects in the CNS are still largely unknown. Our goal was to identify the first target genes of activin signaling in the hippocampus in vivo. Electroconvulsive seizures, a rodent model of electroconvulsive therapy in humans, were applied to C57BL/6J mice to elicit a strong increase in activin A signaling. Chromatin immunoprecipitation experiments with hippocampal lysates subsequently revealed that binding of SMAD2/3, the intracellular effectors of activin signaling, was significantly enriched at the Pmepa1 gene, which encodes a negative feedback regulator of TGF-ß signaling in cancer cells, and at the Kdm6b gene, which encodes an epigenetic regulator promoting transcriptional plasticity. Underlining the significance of these findings, activin treatment also induced PMEPA1 and KDM6B expression in human forebrain neurons generated from embryonic stem cells suggesting interspecies conservation of activin effects in mammalian neurons. Importantly, physiological stimuli such as provided by environmental enrichment proved already sufficient to engender a rapid and significant induction of activin signaling concomitant with an upregulation of Pmepa1 and Kdm6b expression. Taken together, our study identified the first target genes of activin signaling in the brain. With the induction of Kdm6b expression, activin is likely to gain impact on a presumed epigenetic regulator of activity-dependent neuronal plasticity.


Asunto(s)
Activinas/metabolismo , Conducta Animal , Fenómenos Electrofisiológicos , Histona Demetilasas con Dominio de Jumonji/metabolismo , Proteínas de la Membrana/metabolismo , Transducción de Señal , Animales , Secuencia de Bases , Línea Celular , Electrochoque , Femenino , Hipocampo/metabolismo , Humanos , Masculino , Ratones Endogámicos C57BL , Neuronas/metabolismo , Proteínas Smad/metabolismo , Regulación hacia Arriba
16.
Stem Cell Reports ; 5(3): 305-13, 2015 Sep 08.
Artículo en Inglés | MEDLINE | ID: mdl-26321143

RESUMEN

Human pluripotent stem cells (hPSCs) offer the opportunity to generate neuronal cells, including nociceptors. Using a chemical-based approach, we generated nociceptive sensory neurons from HUES6 embryonic stem cells and retrovirally reprogrammed induced hPSCs derived from fibroblasts. The nociceptive neurons expressed respective markers and showed tetrodotoxin-sensitive (TTXs) and -resistant (TTXr) voltage-gated sodium currents in patch-clamp experiments. In contrast to their counterparts from rodent dorsal root ganglia, TTXr currents of hPSC-derived nociceptors unexpectedly displayed a significantly more hyperpolarized voltage dependence of activation and fast inactivation. This apparent discrepancy is most likely due to a substantial expression of the developmentally important sodium channel NAV1.5. In view of the obstacles to recapitulate neuropathic pain in animal models, our data advance hPSC-derived nociceptors as a better model to study developmental and pathogenetic processes in human nociceptive neurons and to develop more specific small molecules to attenuate pain.


Asunto(s)
Diferenciación Celular , Células Madre Pluripotentes Inducidas/metabolismo , Canal de Sodio Activado por Voltaje NAV1.5/metabolismo , Nociceptores/metabolismo , Animales , Línea Celular , Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Células Madre Embrionarias Humanas/citología , Células Madre Embrionarias Humanas/metabolismo , Humanos , Células Madre Pluripotentes Inducidas/citología , Activación del Canal Iónico , Nociceptores/citología , Ratas , Tetrodotoxina
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