RESUMEN
Synaptic dysfunction is an early pathogenic event leading to cognitive decline in Huntington's disease (HD). We previously reported that the active ADAM10 level is increased in the HD cortex and striatum, causing excessive proteolysis of the synaptic cell adhesion protein N-Cadherin. Conversely, ADAM10 inhibition is neuroprotective and prevents cognitive decline in HD mice. Although the breakdown of cortico-striatal connection has been historically linked to cognitive deterioration in HD, dendritic spine loss and long-term potentiation (LTP) defects identified in the HD hippocampus are also thought to contribute to the cognitive symptoms of the disease. The aim of this study is to investigate the contribution of ADAM10 to spine pathology and LTP defects of the HD hippocampus. We provide evidence that active ADAM10 is increased in the hippocampus of two mouse models of HD, leading to extensive proteolysis of N-Cadherin, which has a widely recognized role in spine morphology and synaptic plasticity. Importantly, the conditional heterozygous deletion of ADAM10 in the forebrain of HD mice resulted in the recovery of spine loss and ultrastructural synaptic defects in CA1 pyramidal neurons. Meanwhile, normalization of the active ADAM10 level increased the pool of synaptic BDNF protein and activated ERK neuroprotective signaling in the HD hippocampus. We also show that the ADAM10 inhibitor GI254023X restored LTP defects and increased the density of mushroom spines enriched with GluA1-AMPA receptors in HD hippocampal neurons. Notably, we report that administration of the TrkB antagonist ANA12 to HD hippocampal neurons reduced the beneficial effect of GI254023X, indicating that the BDNF receptor TrkB contributes to mediate the neuroprotective activity exerted by ADAM10 inhibition in HD. Collectively, these findings indicate that ADAM10 inhibition coupled with TrkB signaling represents an efficacious strategy to prevent hippocampal synaptic plasticity defects and cognitive dysfunction in HD.
Asunto(s)
Proteína ADAM10 , Secretasas de la Proteína Precursora del Amiloide , Hipocampo , Enfermedad de Huntington , Potenciación a Largo Plazo , Proteínas de la Membrana , Receptor trkB , Transducción de Señal , Animales , Proteína ADAM10/metabolismo , Proteína ADAM10/genética , Enfermedad de Huntington/metabolismo , Enfermedad de Huntington/patología , Ratones , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Secretasas de la Proteína Precursora del Amiloide/antagonistas & inhibidores , Hipocampo/metabolismo , Hipocampo/patología , Receptor trkB/metabolismo , Receptor trkB/antagonistas & inhibidores , Potenciación a Largo Plazo/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Modelos Animales de Enfermedad , Cadherinas/metabolismo , Espinas Dendríticas/metabolismo , Espinas Dendríticas/patología , Neuroprotección , Masculino , Ratones Endogámicos C57BL , Plasticidad Neuronal , Proteínas Tirosina Quinasas/metabolismo , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Proteínas Tirosina Quinasas/genética , Ratones NoqueadosRESUMEN
Synaptic dysfunction and cognitive decline in Huntington's disease (HD) involve hyperactive A disintegrin and metalloproteinase domain-containing protein 10 (ADAM10). To identify the molecular mechanisms through which ADAM10 is associated with synaptic dysfunction in HD, we performed an immunoaffinity purification-mass spectrometry (IP-MS) study of endogenous ADAM10 in the brains of wild-type and HD mice. We found that proteins implicated in synapse organization, synaptic plasticity, and vesicle and organelles trafficking interact with ADAM10, suggesting that it may act as hub protein at the excitatory synapse. Importantly, the ADAM10 interactome is enriched in presynaptic proteins and ADAM10 co-immunoprecipitates with piccolo (PCLO), a key player in the recycling and maintenance of synaptic vesicles. In contrast, reduced ADAM10/PCLO immunoprecipitation occurs in the HD brain, with decreased density of synaptic vesicles in the reserve and docked pools at the HD presynaptic terminal. Conditional heterozygous deletion of ADAM10 in the forebrain of HD mice reduces active ADAM10 to wild-type level and normalizes ADAM10/PCLO complex formation and synaptic vesicle density and distribution. The results indicate that presynaptic ADAM10 and PCLO are a relevant component of HD pathogenesis.
Asunto(s)
Proteína ADAM10/metabolismo , Proteínas del Citoesqueleto/metabolismo , Enfermedad de Huntington/metabolismo , Neuropéptidos/metabolismo , Vesículas Sinápticas/metabolismo , Proteína ADAM10/genética , Animales , Western Blotting , Encéfalo/metabolismo , Encéfalo/patología , Encéfalo/ultraestructura , Humanos , Enfermedad de Huntington/genética , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión , Terminales Presinápticos/metabolismo , Unión Proteica , Mapas de Interacción de Proteínas/genética , Proteómica/métodos , Vesículas Sinápticas/ultraestructura , Sinaptosomas/metabolismo , Espectrometría de Masas en Tándem/métodosRESUMEN
Human mutations and haploinsufficiency of the SHANK family genes are associated with autism spectrum disorders (ASD) and intellectual disability (ID). Complex phenotypes have been also described in all mouse models of Shank mutations and deletions, consistent with the heterogeneity of the human phenotypes. However, the specific role of Shank proteins in synapse and neuronal functions remain to be elucidated. Here, we generated a new mouse model to investigate how simultaneously deletion of Shank1 and Shank3 affects brain development and behavior in mice. Shank1-Shank3 DKO mice showed a low survival rate, a developmental strong reduction in the activation of intracellular signaling pathways involving Akt, S6, ERK1/2, and eEF2 during development and a severe behavioral impairments. Our study suggests that Shank1 and Shank3 proteins are essential to developmentally regulate the activation of Akt and correlated intracellular pathways crucial for mammalian postnatal brain development and synaptic plasticity. Therefore, Akt function might represent a new therapeutic target for enhancing cognitive abilities of syndromic ASD patients.
Asunto(s)
Trastorno del Espectro Autista , Proteínas Proto-Oncogénicas c-akt , Animales , Trastorno del Espectro Autista/genética , Humanos , Ratones , Ratones Noqueados , Proteínas de Microfilamentos , Proteínas del Tejido Nervioso/genética , SinapsisRESUMEN
OBJECTIVES: Evaluate the ability of current ion-releasing materials to remineralise bacteria-driven artificial caries lesions. MATERIALS AND METHODS: Standardised class I cavities were obtained in 60 extracted human molars. Specimens underwent a microbiological cariogenic protocol (28 days) to generate artificial caries lesions and then were randomly divided into four restorative groups: adhesive + composite (negative control); glass ionomer cement (GIC); calcium silicate cement (MTA); and resin-modified calcium silicate cement (RMTA). Microhardness analysis (ΔKHN) was performed on 40 specimens (10/group, t = 30 days, 45 days, 60 days in artificial saliva, AS). Micro-CT scans were acquired (3/group, t = 0 days, 30 days, and 90 days in AS). Confocal microscopy was employed for interfacial ultra-morphology analysis (2/group, t = 0 days and 60 days in AS). Additional specimens were prepared and processed for scanning electron microscopy (SEM) and FTIR (n = 3/group + control) to analyse the ability of the tested materials to induce apatite formation on totally demineralised dentine discs (60 days in AS). Statistical analyses were performed with a significance level of 5%. RESULTS: Adhesive + composite specimens showed the lowest ΔKHN values and the presence of gaps at the interface when assessed through micro-CT even after storage in AS. Conversely, all the tested ion-releasing materials presented an increase in ΔKHN after storage (p < 0.05), while MTA best reduced the demineralised artificial carious lesions gap at the interface. MTA and RMTA also showed apatite deposition on totally demineralised dentine surfaces (SEM and FTIR). CONCLUSIONS: All tested ion-releasing materials expressed mineral precipitation in demineralised dentine. Additionally, calcium silicate-based materials induced apatite precipitation and hardness recovery of artificial carious dentine lesions over time. CLINICAL RELEVANCE: Current ion-releasing materials can induce remineralisation of carious dentine. MTA shows enhanced ability of nucleation/precipitation of hydroxyapatite compared to RMTA and GIC, which may be more appropriate to recover severe mineral-depleted dentine.
Asunto(s)
Caries Dental , Dentina , Humanos , Apatitas , Compuestos de Calcio , Caries Dental/patología , Caries Dental/terapia , Dentina/química , Cementos de Ionómero Vítreo , Hidroxiapatitas , Ensayo de Materiales , Minerales/análisis , Cementos de Resina , Saliva Artificial , SilicatosRESUMEN
BACKGROUND: DNA-RNA compounds have shown promising protection against cell oxidative stress. This study aimed to assess the cytotoxicity, protective, or preventive effect of different experimental formulations on oral epithelia's oxidative stress in vitro. METHODS: Reconstituted human oral epithelia (RHOE) were grown air-lifted in a continuous-flow bioreactor. Mouthwashes and gels containing DNA-RNA compounds and other bioactive molecules were tested on a model of oxidative stress generated by hydrogen peroxide treatment. Epithelia viability was evaluated using a biochemical MTT-based assay and confocal microscopy; structural and ultrastructural morphology was evaluated by light microscopy and TEM. RESULTS: DNA-RNA showed non-cytotoxic activity and effectively protected against oxidative stress, but did not help in its prevention. Gel formulations did not express adequate activity compared to the mouthwashes. Excipients played a fundamental role in enhancing or even decreasing the bioactive molecules' effect. CONCLUSION: A mouthwash formulation with hydrolyzed DNA-RNA effectively protected against oxidative stress without additional enhancement by other bioactive molecules. Active compounds, such as hyaluronic acid, ß-Glucan, allantoin, bisabolol, ruscogenin, and essential oils, showed a protective effect against oxidative stress, which was not synergistic with the one of DNA-RNA. Incorporation of surfactant agents showed a reduced, yet significant, cytotoxic effect.
Asunto(s)
Mucosa Bucal/metabolismo , Antisépticos Bucales/farmacología , Estrés Oxidativo/efectos de los fármacos , Reactores Biológicos/microbiología , ADN/farmacología , Epitelio/efectos de los fármacos , Epitelio/metabolismo , Geles/farmacología , Ingeniería Genética/métodos , Humanos , Mucosa Bucal/efectos de los fármacos , Antisépticos Bucales/metabolismo , ARN/farmacologíaRESUMEN
PURPOSE: To investigate whether the addition of sodium-DNA (Na-DNA) to chlorhexidine (CHX)-containing mouthwash influenced morphology and viability of a reconstituted human oral epithelium (ROE), and protects ROE against oxidative stress. METHODS: Multi-layered 0.5 cm² ROE specimens were positioned inside a continuous flow bioreactor and grown air-lifted for 24 hours. They were treated with phosphate-buffered saline (PBS) (n= 16) or 1 vol% H2O2 for 1 minute (n= 16). Then, they were treated for 5 (n= 8) or 30 minutes (n= 8) with the experimental mouthwash solutions containing: 0.2 wt% CHX, 0.2 wt% CHX + 0.2 wt% Na-DNA, 0.2 wt% Na-DNA, PBS. After 60 minutes washout specimens were subjected to tetrazolium-based viability assay (MTT) confocal laser-scanning microscopy (CLSM), and histological evaluation using optical microscopy and transmission electron microscopy (TEM). RESULTS: ROE treated with Na-DNA for 30 minutes revealed significantly higher viability than PBS, and CHX + Na-DNA showed higher viability after 30-minute treatment than after 5 minutes, suggesting a significant protective activity of Na-DNA. Moreover, the protective effect of Na-DNA on cell viability was higher after the induction of oxidative stress. After treatment with CHX, CLSM revealed cell stress, leading to cell death in the outer layer. On the contrary, specimens treated with Na-DNA showed a much lower number of dead cells compared to PBS, both in the absence or presence of oxidative stress. Histological examination showed that the protective action of Na-DNA formulations reached more in-depth into the epithelium exposed to oxidative stress, due to intercellular spaces opening in the outer epithelium layers, giving way to Na-DNA to the inner parts of the epithelium. It can be concluded that Na-DNA had a topical protective activity when applied for 30 minutes unless the epithelium barrier is damaged, allowing it to act more in-depth. CLINICAL SIGNIFICANCE: Na-DNA showed a clear and protective action against cellular degeneration due to oxidative stress and, partly, to the exposure to CHX. Its addition to chlorhexidine mouthwash or gels could be clinically helpful in contrasting the detrimental activity of CHX on oral tissues, and in the preservation of cell viability, control of inflammation and wound healing.
Asunto(s)
Peróxido de Hidrógeno , Antisépticos Bucales/farmacología , Antisépticos Bucales/toxicidad , Reactores Biológicos , ADN , Humanos , SodioRESUMEN
Intellectual disability affects 2-3% of the world's population and typically begins during childhood, causing impairments in social skills and cognitive abilities. Mutations in the TM4SF2 gene, which encodes the TSPAN7 protein, cause a severe form of intellectual disability, and currently, no therapy is able to ameliorate this cognitive impairment. We previously reported that, in cultured neurons, shRNA-mediated down-regulation of TSPAN7 affects AMPAR trafficking by enhancing PICK1-GluA2 interaction, thereby increasing the intracellular retention of AMPAR. Here, we found that loss of TSPAN7 function in mice causes alterations in hippocampal excitatory synapse structure and functionality as well as cognitive impairment. These changes occurred along with alterations in AMPAR expression levels. We also found that interfering with PICK1-GluA2 binding restored synaptic function in Tm4sf2-/y mice. Moreover, potentiation of AMPAR activity via the administration of the ampakine CX516 reverted the neurological phenotype observed in Tm4sf2-/y mice, suggesting that pharmacological modulation of AMPAR may represent a new approach for treating patients affected by TM4SF2 mutations and intellectual disability.
Asunto(s)
Fármacos actuantes sobre Aminoácidos Excitadores/farmacología , Discapacidad Intelectual/tratamiento farmacológico , Discapacidad Intelectual/metabolismo , Proteínas de la Membrana/deficiencia , Proteínas del Tejido Nervioso/deficiencia , Psicotrópicos/farmacología , Receptores AMPA/metabolismo , Regulación Alostérica , Animales , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular , Modelos Animales de Enfermedad , Expresión Génica/efectos de los fármacos , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/ultraestructura , Discapacidad Intelectual/patología , Masculino , Proteínas de la Membrana/genética , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas del Tejido Nervioso/genética , Proteínas Nucleares/metabolismo , Unión Proteica/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Sinapsis/ultraestructura , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Técnicas de Cultivo de TejidosRESUMEN
Alterations in the balance of inhibitory and excitatory synaptic transmission have been implicated in the pathogenesis of neurological disorders such as epilepsy. Eukaryotic elongation factor 2 kinase (eEF2K) is a highly regulated, ubiquitous kinase involved in the control of protein translation. Here, we show that eEF2K activity negatively regulates GABAergic synaptic transmission. Indeed, loss of eEF2K increases GABAergic synaptic transmission by upregulating the presynaptic protein Synapsin 2b and α5-containing GABAA receptors and thus interferes with the excitation/inhibition balance. This cellular phenotype is accompanied by an increased resistance to epilepsy and an impairment of only a specific hippocampal-dependent fear conditioning. From a clinical perspective, our results identify eEF2K as a potential novel target for antiepileptic drugs, since pharmacological and genetic inhibition of eEF2K can revert the epileptic phenotype in a mouse model of human epilepsy.
Asunto(s)
Quinasa del Factor 2 de Elongación/metabolismo , Epilepsia/enzimología , Neuronas/enzimología , Transmisión Sináptica/fisiología , Animales , Células Cultivadas , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Quinasa del Factor 2 de Elongación/antagonistas & inhibidores , Quinasa del Factor 2 de Elongación/genética , Epilepsia/patología , Miedo/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/enzimología , Hipocampo/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Neuronas/patología , Ratas Sprague-Dawley , Receptores de GABA-A/metabolismo , Sinapsinas/genética , Sinapsinas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Ácido gamma-Aminobutírico/metabolismoRESUMEN
A complex and still not comprehensively resolved panel of transmembrane proteins regulates the outgrowth and the subsequent morphological and functional development of neuronal processes. In order to gain a more detailed description of these events at the molecular level, we have developed a cell surface biotinylation assay to isolate, detect, and quantify neuronal membrane proteins. When we applied our assay to investigate neuron maturation in vitro, we identified 439 differentially expressed proteins, including 20 members of the immunoglobulin superfamily. Among these candidates, we focused on Negr1, a poorly described cell adhesion molecule. We demonstrated that Negr1 controls the development of neurite arborization in vitro and in vivo. Given the tight correlation existing among synaptic cell adhesion molecules, neuron maturation, and a number of neurological disorders, our assay results are a useful tool that can be used to support the understanding of the molecular bases of physiological and pathological brain function.
Asunto(s)
Bioensayo/métodos , Moléculas de Adhesión Celular Neuronal/metabolismo , Membrana Celular/metabolismo , Dendritas/metabolismo , Animales , Biotinilación , Diferenciación Celular , Forma de la Célula , Células Cultivadas , Espinas Dendríticas/metabolismo , Silenciador del Gen , Células HEK293 , Humanos , Proteínas de la Membrana/aislamiento & purificación , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , Neurogénesis , Sinapsis/metabolismoRESUMEN
Huntington's disease (HD) causes selective degeneration of striatal and cortical neurons, resulting in cell mosaicism of coexisting still functional and dysfunctional cells. The impact of non-cell autonomous mechanisms between these cellular states is poorly understood. Here we generated telencephalic organoids with healthy or HD cells, grown separately or as mosaics of the two genotypes. Single-cell RNA sequencing revealed neurodevelopmental abnormalities in the ventral fate acquisition of HD organoids, confirmed by cytoarchitectural and transcriptional defects leading to fewer GABAergic neurons, while dorsal populations showed milder phenotypes mainly in maturation trajectory. Healthy cells in mosaic organoids restored HD cell identity, trajectories, synaptic density, and communication pathways upon cell-cell contact, while showing no significant alterations when grown with HD cells. These findings highlight cell-type-specific alterations in HD and beneficial non-cell autonomous effects of healthy cells, emphasizing the therapeutic potential of modulating cell-cell communication in disease progression and treatment.
Asunto(s)
Enfermedad de Huntington , Organoides , Fenotipo , Telencéfalo , Enfermedad de Huntington/patología , Enfermedad de Huntington/genética , Enfermedad de Huntington/metabolismo , Organoides/patología , Organoides/metabolismo , Animales , Telencéfalo/patología , Telencéfalo/citología , Telencéfalo/metabolismo , Humanos , Ratones , Neuronas GABAérgicas/metabolismo , Neuronas GABAérgicas/patología , Análisis de la Célula Individual , Comunicación Celular , Mosaicismo , Neuronas/metabolismo , Neuronas/patologíaRESUMEN
Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.
Asunto(s)
Diferenciación Celular , Ventrículos Laterales , Factor Inhibidor de Leucemia , Organoides , Células Madre Pluripotentes , Humanos , Organoides/metabolismo , Organoides/citología , Factor Inhibidor de Leucemia/metabolismo , Factor Inhibidor de Leucemia/farmacología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/citología , Ventrículos Laterales/citología , Ventrículos Laterales/metabolismo , Factor de Transcripción STAT3/metabolismo , Neuroglía/metabolismo , Neuroglía/citología , Transducción de SeñalRESUMEN
Oncogenic mutations accumulating in many chromatin-associated proteins have been identified in different tumor types. With a mutation rate from 10 to 57%, ARID1A has been widely considered a tumor suppressor gene. However, whether this role is mainly due to its transcriptional-related activities or its ability to preserve genome integrity is still a matter of intense debate. Here, we show that ARID1A is largely dispensable for preserving enhancer-dependent transcriptional regulation, being ARID1B sufficient and required to compensate for ARID1A loss. We provide in vivo evidence that ARID1A is mainly required to preserve genomic integrity in adult tissues. ARID1A loss primarily results in DNA damage accumulation, interferon type I response activation, and chronic inflammation leading to tumor formation. Our data suggest that in healthy tissues, the increased genomic instability that follows ARID1A mutations and the selective pressure imposed by the microenvironment might result in the emergence of aggressive, possibly immune-resistant, tumors.
Asunto(s)
Neoplasias , Proteínas Nucleares , Humanos , Inestabilidad Genómica , Mutación , Tasa de Mutación , Neoplasias/genética , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Microambiente Tumoral , Animales , RatonesRESUMEN
Several lines of evidence indicate that neuromuscular junction (NMJ) destruction and disassembly is an early phenomenon in amyotrophic lateral sclerosis (ALS). Here we analyzed by confocal and electron microscopy the NMJ structure in the diaphragm of SOD1G93A mice at symptom onset. In these mice, which provide a model for familial ALS, diaphragm denervation (~50%) as well as gastrocnemius denervation (~40%) was found. In addition, the size of the synaptic vesicle pool was reduced and alterations of mitochondria were observed in approximately 40% of the remaining presynaptic terminals. Chronic treatment of SOD1G93A mice with the anabolic steroid nandrolone during the presymptomatic stage preserved the diaphragm muscle mass and features indicative of synaptic activity. These features were represented by the number of vesicles docked within 200 nm from the presynaptic membrane and area of acetylcholine receptor clusters. Structural preservation of mitochondria was documented in presynaptic terminals. However, innervation of diaphragm muscle fibers was only slightly increased in nandrolone-treated SOD1-mutant mice. Altogether the results point out and define fine structural alterations of diaphragm NMJs in the murine model of familial ALS at symptom onset, and indicate that nandrolone may prevent or delay structural alterations in NMJ mitochondria and stimulate presynaptic activity but does not prevent muscle denervation during the disease.
Asunto(s)
Esclerosis Amiotrófica Lateral/patología , Anabolizantes/farmacología , Nandrolona/farmacología , Unión Neuromuscular/ultraestructura , Superóxido Dismutasa/genética , Esclerosis Amiotrófica Lateral/genética , Animales , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones , Ratones Transgénicos , Mitocondrias/ultraestructura , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Fibras Musculares Esqueléticas/fisiología , Mutación , Unión Neuromuscular/efectos de los fármacos , Unión Neuromuscular/genética , Unión Neuromuscular/fisiopatología , Terminales Presinápticos/fisiología , Terminales Presinápticos/ultraestructura , Superóxido Dismutasa/metabolismo , Superóxido Dismutasa-1 , Vesículas Sinápticas/ultraestructuraRESUMEN
Mammalian outer radial glia (oRG) emerge as cortical progenitor cells that directly support the development of an enlarged outer subventricular zone (oSVZ) and, in turn, the expansion of the neocortex. The in vitro generation of oRG is essential to model and investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 pathway using LIF, which is not produced in guided cortical organoids, we developed a cerebral organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The structured oSVZ is composed of progenitor cells expressing specific oRG markers such as GFAP, LIFR, HOPX , which closely matches human oRG in vivo . In this microenvironment, cortical neurons showed faster maturation with enhanced metabolic and functional activity. Incorporation of hPSC-derived brain vascular LIF- producing pericytes in cerebral organoids mimicked the effects of LIF treatment. These data indicate that the cellular complexity of the cortical microenvironment, including cell-types of the brain vasculature, favors the appearance of oRG and provides a platform to routinely study oRG in hPSC-derived brain organoids.
RESUMEN
Human telencephalon is an evolutionarily advanced brain structure associated with many uniquely human behaviors and disorders. However, cell lineages and molecular pathways implicated in human telencephalic development remain largely unknown. We produce human telencephalic organoids from stem cell-derived single neural rosettes and investigate telencephalic development under normal and pathological conditions. We show that single neural rosette-derived organoids contain pallial and subpallial neural progenitors, excitatory and inhibitory neurons, as well as macroglial and periendothelial cells, and exhibit predictable organization and cytoarchitecture. We comprehensively characterize the properties of neurons in SNR-derived organoids and identify transcriptional programs associated with the specification of excitatory and inhibitory neural lineages from a common pool of NPs early in telencephalic development. We also demonstrate that neurons in organoids with a hemizygous deletion of an autism- and intellectual disability-associated gene SHANK3 exhibit intrinsic and excitatory synaptic deficits and impaired expression of several clustered protocadherins. Collectively, this study validates SNR-derived organoids as a reliable model for studying human telencephalic cortico-striatal development and identifies intrinsic, synaptic, and clustered protocadherin expression deficits in human telencephalic tissue with SHANK3 hemizygosity.
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Trastorno Autístico , Trastorno Autístico/genética , Humanos , Proteínas del Tejido Nervioso/metabolismo , Organoides/metabolismo , Protocadherinas , TelencéfaloRESUMEN
Deciphering how the human striatum develops is necessary for understanding the diseases that affect this region. To decode the transcriptional modules that regulate this structure during development, we compiled a catalog of 1116 long intergenic noncoding RNAs (lincRNAs) identified de novo and then profiled 96,789 single cells from the early human fetal striatum. We found that D1 and D2 medium spiny neurons (D1- and D2-MSNs) arise from a common progenitor and that lineage commitment is established during the postmitotic transition, across a pre-MSN phase that exhibits a continuous spectrum of fate determinants. We then uncovered cell type-specific gene regulatory networks that we validated through in silico perturbation. Finally, we identified human-specific lincRNAs that contribute to the phylogenetic divergence of this structure in humans. This work delineates the cellular hierarchies governing MSN lineage commitment.
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Atlas como Asunto , Cuerpo Estriado/citología , Cuerpo Estriado/embriología , Neurogénesis/genética , ARN Largo no Codificante/genética , Análisis de la Célula Individual , Factores de Transcripción/genética , Feto , Neuronas GABAérgicas/metabolismo , Humanos , RNA-Seq , Transcripción GenéticaRESUMEN
Mechanotransduction is the ability of cells to translate mechanical stimuli into biochemical signals that can ultimately influence gene expression, cell morphology and cell fate. Tenocytes are responsible for tendon mechanical adaptation converting mechanical stimuli imposed during mechanical loading, thus affecting extracellular matrix homeostasis. Since we previously demonstrated that MD-Tissue, an injectable collagen-based medical compound containing swine-derived collagen as the main component, is able to affect tenocyte properties, the aim of this study was to analyze whether the effects triggered by MD-Tissue were based on mechanotransduction-related mechanisms. For this purpose, MD-Tissue was used to coat Petri dishes and cytochalasin B was used to deprive tenocytes of mechanical stimulation mediated by the actin cytoskeleton. Cell morphology, migration, collagen turnover pathways and the expression of key mechanosensors were analyzed by morphological and molecular methods. Our findings confirm that MD-Tissue affects collagen turnover pathways and favors cell migration and show that the MD-Tissue-induced effect represents a mechanical input involving the mechanotransduction machinery. Overall, MD-Tissue, acting as a mechanical scaffold, could represent an effective medical device for a novel therapeutic, regenerative and rehabilitative approach to favor tendon healing in tendinopathies.
Asunto(s)
Colágeno/química , Estrés Mecánico , Tenocitos/metabolismo , Citoesqueleto de Actina , Anciano , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Movimiento Celular/efectos de los fármacos , Células Cultivadas , Colágeno Tipo I/metabolismo , Citocalasina B/farmacología , Femenino , Quinasa 1 de Adhesión Focal/genética , Quinasa 1 de Adhesión Focal/metabolismo , Adhesiones Focales/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/genética , Procolágeno-Lisina 2-Oxoglutarato 5-Dioxigenasa/metabolismo , Porcinos , Tenocitos/citología , Tenocitos/efectos de los fármacos , Tenocitos/patología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMEN
Detecting the ultrastructure of brain tissue in human archaeological remains is a rare event that can offer unique insights into the structure of the ancient central nervous system (CNS). Yet ancient brains reported in the literature show only poor preservation of neuronal structures. Using scanning electron microscopy (SEM) and advanced image processing tools, we describe the direct visualization of neuronal tissue in vitrified brain and spinal cord remains which we discovered in a male victim of the AD 79 eruption in Herculaneum. We show exceptionally well preserved ancient neurons from different regions of the human CNS at unprecedented resolution. This tissue typically consists of organic matter, as detected using energy-dispersive X-ray spectroscopy. By means of a self-developed neural image processing network, we also show specific details of the neuronal nanomorphology, like the typical myelin periodicity evidenced in the brain axons. The perfect state of preservation of these structures is due to the unique process of vitrification which occurred at Herculaneum. The discovery of proteins whose genes are expressed in the different region of the human adult brain further agree with the neuronal origin of the unusual archaeological find. The conversion of human tissue into glass is the result of sudden exposure to scorching volcanic ash and the concomitant rapid drop in temperature. The eruptive-induced process of natural vitrification, locking the cellular structure of the CNS, allowed us to study possibly the best known example in archaeology of extraordinarily well-preserved human neuronal tissue from the brain and spinal cord.
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Encéfalo/anatomía & histología , Sistema Nervioso Central/anatomía & histología , Erupciones Volcánicas , Arqueología , Encéfalo/metabolismo , Encéfalo/fisiología , Sistema Nervioso Central/fisiología , Bases de Datos Factuales , Humanos , Procesamiento de Imagen Asistido por Computador , Cinesinas/genética , Masculino , Microscopía Electrónica de Rastreo , Espectrometría por Rayos X , Médula Espinal/anatomía & histología , Médula Espinal/fisiología , Conservación de Tejido , Adulto JovenRESUMEN
Theranostics based on two-photon excitation of therapeutics in the NIR region is an emerging and powerful tool in cancer therapy since this radiation deeply penetrates healthy biological tissues and produces selective cell death. Aggregates of gold nanoparticles coated with glutathione corona functionalized with the dansyl chromophore (a-DG-AuNPs) were synthesized and found efficient nanodevice for applications in photothermal therapy (PTT). Actually the nanoparticle aggregation enhances the quenching of radiative excitation and the consequent conversion into heat. The a-DG-AuNPs are readily internalized in Hep G2 where the chromophore acts as both antenna and transducer of the NIR radiation under two-photons excitation, determining efficient cell ablation via photothermal effect.
Asunto(s)
Terapia por Luz de Baja Intensidad/métodos , Nanopartículas del Metal/administración & dosificación , Neoplasias/terapia , Terapia Fototérmica/métodos , Nanomedicina Teranóstica/métodos , Animales , Línea Celular Tumoral , Terapia Combinada/instrumentación , Terapia Combinada/métodos , Fluorescencia , Glutatión/química , Oro/química , Humanos , Rayos Infrarrojos/uso terapéutico , Rayos Láser , Terapia por Luz de Baja Intensidad/instrumentación , Nanopartículas del Metal/química , Ratones , Neoplasias/patología , Fosfatidilcolinas/química , Fotones/uso terapéutico , Terapia Fototérmica/instrumentación , Nanomedicina Teranóstica/instrumentaciónRESUMEN
A variety of pathophysiological mechanisms are implicated in Huntington's disease (HD). Among them, reduced cholesterol biosynthesis has been detected in the HD mouse brain from pre-symptomatic stages, leading to diminished cholesterol synthesis, particularly in the striatum. In addition, systemic injection of cholesterol-loaded brain-permeable nanoparticles ameliorates synaptic and cognitive function in a transgenic mouse model of HD. To identify an appropriate treatment regimen and gain mechanistic insights into the beneficial activity of exogenous cholesterol in the HD brain, we employed osmotic mini-pumps to infuse three escalating doses of cholesterol directly into the striatum of HD mice in a continuous and rate-controlled manner. All tested doses prevented cognitive decline, while amelioration of disease-related motor defects was dose-dependent. In parallel, we found morphological and functional recovery of synaptic transmission involving both excitatory and inhibitory synapses of striatal medium spiny neurons. The treatment also enhanced endogenous cholesterol biosynthesis and clearance of mutant Huntingtin aggregates. These results indicate that cholesterol infusion to the striatum can exert a dose-dependent, disease-modifying effect and may be therapeutically relevant in HD.