RESUMEN
Human brain organoid models that recapitulate the physiology and complexity of the human brain have a great potential for in vitro disease modeling, in particular for neurodegenerative diseases, such as Parkinson disease. In the present study, we compare single-cell RNA-sequencing data of human midbrain organoids to the developing human embryonic midbrain. We demonstrate that the in vitro model is comparable to its in vivo equivalents in terms of developmental path and cellular composition. Moreover, we investigate the potential of midbrain organoids for modeling early developmental changes in Parkinson disease. Therefore, we compare the single-cell RNA-sequencing data of healthy-individual-derived midbrain organoids to their isogenic LRRK2-p.Gly2019Ser-mutant counterparts. We show that the LRRK2 p.Gly2019Ser variant alters neurodevelopment, resulting in an untimely and incomplete differentiation with reduced cellular variability. Finally, we present four candidate genes, APP, DNAJC6, GATA3, and PTN, that might contribute to the LRRK2-p.Gly2019Ser-associated transcriptome changes that occur during early neurodevelopment.
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Sustitución de Aminoácidos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/genética , Neurogénesis/genética , Organoides/metabolismo , Enfermedad de Parkinson/genética , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Diferenciación Celular , Citocinas/genética , Citocinas/metabolismo , Embrión de Mamíferos , Factor de Transcripción GATA3/genética , Factor de Transcripción GATA3/metabolismo , Regulación del Desarrollo de la Expresión Génica , Glicina/química , Glicina/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Humanos , Proteína 2 Quinasa Serina-Treonina Rica en Repeticiones de Leucina/metabolismo , Mesencéfalo , Modelos Biológicos , Mutación , Organoides/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Análisis de Secuencia de ARN , Serina/química , Serina/metabolismo , Análisis de la Célula Individual/métodos , TranscriptomaRESUMEN
Parkinson's disease (PD) is a complex, progressive neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta in the midbrain. Despite extensive research efforts, the molecular and cellular changes that precede neurodegeneration in PD are poorly understood. To address this, here we describe the use of patient specific human midbrain organoids harboring the SNCA triplication to investigate mechanisms underlying dopaminergic degeneration. Our midbrain organoid model recapitulates key pathological hallmarks of PD, including the aggregation of α-synuclein and the progressive loss of dopaminergic neurons. We found that these pathological hallmarks are associated with an increase in senescence associated cellular phenotypes in astrocytes including nuclear lamina defects, the presence of senescence associated heterochromatin foci, and the upregulation of cell cycle arrest genes. These results suggest a role of pathological α-synuclein in inducing astrosenescence which may, in turn, increase the vulnerability of dopaminergic neurons to degeneration.
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Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Astrocitos/metabolismo , Enfermedades Neurodegenerativas/metabolismo , Mesencéfalo/metabolismo , Mesencéfalo/patología , Neuronas Dopaminérgicas/metabolismo , Organoides/metabolismo , Organoides/patología , Sustancia Negra/metabolismoRESUMEN
In the mouse neocortex, neural progenitor cells generate both differentiating neurons and daughter cells that maintain progenitor fate. Here, we show that the TRIM-NHL protein TRIM32 regulates protein degradation and microRNA activity to control the balance between those two daughter cell types. In both horizontally and vertically dividing progenitors, TRIM32 becomes polarized in mitosis and is concentrated in one of the two daughter cells. TRIM32 overexpression induces neuronal differentiation while inhibition of TRIM32 causes both daughter cells to retain progenitor cell fate. TRIM32 ubiquitinates and degrades the transcription factor c-Myc but also binds Argonaute-1 and thereby increases the activity of specific microRNAs. We show that Let-7 is one of the TRIM32 targets and is required and sufficient for neuronal differentiation. TRIM32 is the mouse ortholog of Drosophila Brat and Mei-P26 and might be part of a protein family that regulates the balance between differentiation and proliferation in stem cell lineages.
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Diferenciación Celular , Proliferación Celular , MicroARNs/metabolismo , Neuronas/metabolismo , Células Madre/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Ratones , Neurogénesis , Neuronas/citología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Células Madre/citologíaRESUMEN
Idiopathic Parkinson's disease is characterized by a progressive loss of dopaminergic neurons, but the exact disease aetiology remains largely unknown. To date, Parkinson's disease research has mainly focused on nigral dopaminergic neurons, although recent studies suggest disease-related changes also in non-neuronal cells and in midbrain regions beyond the substantia nigra. While there is some evidence for glial involvement in Parkinson's disease, the molecular mechanisms remain poorly understood. The aim of this study was to characterize the contribution of all cell types of the midbrain to Parkinson's disease pathology by single-nuclei RNA sequencing and to assess the cell type-specific risk for Parkinson's disease using the latest genome-wide association study. We profiled >41 000 single-nuclei transcriptomes of post-mortem midbrain from six idiopathic Parkinson's disease patients and five age-/sex-matched controls. To validate our findings in a spatial context, we utilized immunolabelling of the same tissues. Moreover, we analysed Parkinson's disease-associated risk enrichment in genes with cell type-specific expression patterns. We discovered a neuronal cell cluster characterized by CADPS2 overexpression and low TH levels, which was exclusively present in idiopathic Parkinson's disease midbrains. Validation analyses in laser-microdissected neurons suggest that this cluster represents dysfunctional dopaminergic neurons. With regard to glial cells, we observed an increase in nigral microglia in Parkinson's disease patients. Moreover, nigral idiopathic Parkinson's disease microglia were more amoeboid, indicating an activated state. We also discovered a reduction in idiopathic Parkinson's disease oligodendrocyte numbers with the remaining cells being characterized by a stress-induced upregulation of S100B. Parkinson's disease risk variants were associated with glia- and neuron-specific gene expression patterns in idiopathic Parkinson's disease cases. Furthermore, astrocytes and microglia presented idiopathic Parkinson's disease-specific cell proliferation and dysregulation of genes related to unfolded protein response and cytokine signalling. While reactive patient astrocytes showed CD44 overexpression, idiopathic Parkinson's disease microglia revealed a pro-inflammatory trajectory characterized by elevated levels of IL1B, GPNMB and HSP90AA1. Taken together, we generated the first single-nuclei RNA sequencing dataset from the idiopathic Parkinson's disease midbrain, which highlights a disease-specific neuronal cell cluster as well as 'pan-glial' activation as a central mechanism in the pathology of the movement disorder. This finding warrants further research into inflammatory signalling and immunomodulatory treatments in Parkinson's disease.
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Enfermedad de Parkinson , Neuronas Dopaminérgicas/metabolismo , Estudio de Asociación del Genoma Completo , Humanos , Glicoproteínas de Membrana/metabolismo , Mesencéfalo , Microglía/metabolismo , Enfermedad de Parkinson/metabolismo , Sustancia Negra/metabolismoRESUMEN
The human brain is a complex, three-dimensional structure. To better recapitulate brain complexity, recent efforts have focused on the development of human-specific midbrain organoids. Human iPSC-derived midbrain organoids consist of differentiated and functional neurons, which contain active synapses, as well as astrocytes and oligodendrocytes. However, the absence of microglia, with their ability to remodel neuronal networks and phagocytose apoptotic cells and debris, represents a major disadvantage for the current midbrain organoid systems. Additionally, neuroinflammation-related disease modeling is not possible in the absence of microglia. So far, no studies about the effects of human iPSC-derived microglia on midbrain organoid neural cells have been published. Here we describe an approach to derive microglia from human iPSCs and integrate them into iPSC-derived midbrain organoids. Using single nuclear RNA Sequencing, we provide a detailed characterization of microglia in midbrain organoids as well as the influence of their presence on the other cells of the organoids. Furthermore, we describe the effects that microglia have on cell death and oxidative stress-related gene expression. Finally, we show that microglia in midbrain organoids affect synaptic remodeling and increase neuronal excitability. Altogether, we show a more suitable system to further investigate brain development, as well as neurodegenerative diseases and neuroinflammation.
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Células Madre Pluripotentes Inducidas , Organoides , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mesencéfalo , Microglía/metabolismo , Neurogénesis/genética , Organoides/metabolismoRESUMEN
Mitochondrial Rho GTPase 1 (Miro1) protein is a well-known adaptor for mitochondrial transport and also regulates mitochondrial quality control and function. Furthermore, Miro1 was associated with mitochondrial-endoplasmic reticulum (ER) contact sites (MERCs), which are key regulators of cellular calcium homeostasis and the initiation of autophagy. Impairments of these mechanisms were linked to neurodegeneration in Parkinson's disease (PD). We recently revealed that PD fibroblasts harboring Miro1 mutations displayed dysregulations in MERC organization and abundance, affecting mitochondrial homeostasis and clearance. We hypothesize that mutant Miro1 impairs the function of MERCs and mitochondrial dynamics, altering neuronal homeostasis and integrity in PD. PD skin fibroblasts harboring the Miro1-R272Q mutation were differentiated into patient-derived neurons. Live-cell imaging and immunocytochemistry were used to study mitophagy and the organization and function of MERCs. Markers of autophagy or mitochondrial function were assessed by western blotting. Quantification of organelle juxtapositions revealed an increased number of MERCs in patient-derived neurons. Live-cell imaging results showed alterations of mitochondrial dynamics and increased sensitivity to calcium stress, as well as reduced mitochondrial clearance. Finally, western blot analysis indicated a blockage of the autophagy flux in Miro1-mutant neurons. Miro1-mutant neurons display altered ER-mitochondrial tethering compared with control neurons. This alteration likely interferes with proper MERC function, contributing to a defective autophagic flux and cytosolic calcium handling capacity. Moreover, mutant Miro1 affects mitochondrial dynamics in neurons, which may result in disrupted mitochondrial turnover and altered mitochondrial movement.
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Retículo Endoplásmico/genética , Mitocondrias/genética , Proteínas Mitocondriales/genética , Enfermedad de Parkinson/genética , Proteínas de Unión al GTP rho/genética , Calcio/metabolismo , Diferenciación Celular/genética , Citosol/metabolismo , Homeostasis/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Dinámicas Mitocondriales/genética , Mitofagia/genética , Neuronas/metabolismo , Neuronas/patología , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patologíaRESUMEN
BACKGROUND: The etiology of Parkinson's disease (PD) is only partially understood despite the fact that environmental causes, risk factors, and specific gene mutations are contributors to the disease. Biallelic mutations in the phosphatase and tensin homolog (PTEN)-induced putative kinase 1 (PINK1) gene involved in mitochondrial homeostasis, vesicle trafficking, and autophagy are sufficient to cause PD. OBJECTIVES: We sought to evaluate the difference between controls' and PINK1 patients' derived neurons in their transition from neuroepithelial stem cells to neurons, allowing us to identify potential pathways to target with repurposed compounds. METHODS: Using two-dimensional and three-dimensional models of patients' derived neurons we recapitulated PD-related phenotypes. We introduced the usage of midbrain organoids for testing compounds. Using Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated protein 9 (Cas9), we corrected the point mutations of three patients' derived cells. We evaluated the effect of the selected compound in a mouse model. RESULTS: PD patient-derived cells presented differences in their energetic profile, imbalanced proliferation, apoptosis, mitophagy, and a reduced differentiation efficiency to tyrosine hydroxylase positive (TH+) neurons compared to controls' cells. Correction of a patient's point mutation ameliorated the metabolic properties and neuronal firing rates as well as reversing the differentiation phenotype, and reducing the increased astrocytic levels. Treatment with 2-hydroxypropyl-ß-cyclodextrin increased the autophagy and mitophagy capacity of neurons concomitant with an improved dopaminergic differentiation of patient-specific neurons in midbrain organoids and ameliorated neurotoxicity in a mouse model. CONCLUSION: We show that treatment with a repurposed compound is sufficient for restoring the impaired dopaminergic differentiation of PD patient-derived cells. © 2021 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
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Enfermedad de Parkinson , 2-Hidroxipropil-beta-Ciclodextrina/metabolismo , Animales , Encéfalo/metabolismo , Neuronas Dopaminérgicas/metabolismo , Humanos , Ratones , Neuronas/metabolismo , Organoides/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , FenotipoRESUMEN
Alpha-synuclein (aSyn) is a central player in Parkinson's disease (PD) but the precise molecular mechanisms underlying its pathogenicity remain unclear. It has recently been suggested that nuclear aSyn may modulate gene expression, possibly via interactions with DNA. However, the biological behavior of aSyn in the nucleus and the factors affecting its transcriptional role are not known. Here, we investigated the mechanisms underlying aSyn-mediated transcription deregulation by assessing its effects in the nucleus and the impact of phosphorylation in these dynamics. We found that aSyn induced severe transcriptional deregulation, including the downregulation of important cell cycle-related genes. Importantly, transcriptional deregulation was concomitant with reduced binding of aSyn to DNA. By forcing the nuclear presence of aSyn in the nucleus (aSyn-NLS), we found the accumulation of high molecular weight aSyn species altered gene expression and reduced toxicity when compared with the wild-type or exclusively cytosolic protein. Interestingly, nuclear localization of aSyn, and the effect on gene expression and cytotoxicity, was also modulated by phosphorylation on serine 129. Thus, we hypothesize that the role of aSyn on gene expression and, ultimately, toxicity, may be modulated by the phosphorylation status and nuclear presence of different aSyn species. Our findings shed new light onto the subcellular dynamics of aSyn and unveil an intricate interplay between subcellular location, phosphorylation and toxicity, opening novel avenues for the design of future strategies for therapeutic intervention in PD and other synucleinopathies.
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alfa-Sinucleína/metabolismo , alfa-Sinucleína/fisiología , Animales , Línea Celular , Núcleo Celular , Proteínas de Unión al ADN , Regulación hacia Abajo , Expresión Génica , Regulación de la Expresión Génica/fisiología , Humanos , Ratones , Señales de Localización Nuclear/fisiología , Enfermedad de Parkinson/patología , Fosforilación , Cultivo Primario de Células , RatasRESUMEN
BACKGROUND: VPS35 is part of the retromer complex and is responsible for the trafficking and recycling of proteins implicated in autophagy and lysosomal degradation, but also takes part in the degradation of mitochondrial proteins via mitochondria-derived vesicles. The p.D620N mutation of VPS35 causes an autosomal-dominant form of Parkinson's disease (PD), clinically representing typical PD. OBJECTIVE: Most of the studies on p.D620N VPS35 were performed on human tumor cell lines, rodent models overexpressing mutant VPS35, or in patient-derived fibroblasts. Here, based on identified target proteins, we investigated the implication of mutant VPS35 in autophagy, lysosomal degradation, and mitochondrial function in induced pluripotent stem cell-derived neurons from a patient harboring the p.D620N mutation. METHODS: We reprogrammed fibroblasts from a PD patient carrying the p.D620N mutation in the VPS35 gene and from two healthy donors in induced pluripotent stem cells. These were subsequently differentiated into neuronal precursor cells to finally generate midbrain dopaminergic neurons. RESULTS: We observed a decreased autophagic flux and lysosomal mass associated with an accumulation of α-synuclein in patient-derived neurons compared to controls. Moreover, patient-derived neurons presented a mitochondrial dysfunction with decreased membrane potential, impaired mitochondrial respiration, and increased production of reactive oxygen species associated with a defect in mitochondrial quality control via mitophagy. CONCLUSION: We describe for the first time the impact of the p.D620N VPS35 mutation on autophago-lysosome pathway and mitochondrial function in stem cell-derived neurons from an affected p.D620N carrier and define neuronal phenotypes for future pharmacological interventions. © 2020 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.
Asunto(s)
Enfermedad de Parkinson , Proteínas de Transporte Vesicular , Neuronas Dopaminérgicas/metabolismo , Humanos , Mitocondrias/metabolismo , Mutación/genética , Enfermedad de Parkinson/metabolismo , Transporte de Proteínas , Proteínas de Transporte Vesicular/genética , alfa-Sinucleína/metabolismoRESUMEN
In this study, we have aimed at developing a novel electrochemical sensing approach capable of detecting dopamine, the main biomarker in Parkinson's disease, within the highly complex cell culture matrix of human midbrain organoids in a non-invasive and label-free manner. With its ability to generate organotypic structures in vitro, induced pluripotent stem cell technology has provided the basis for the development of advanced patient-derived disease models. These include models of the human midbrain, the affected region in the neurodegenerative disorder Parkinson's disease. Up to now, however, the analysis of so-called human midbrain organoids has relied on time-consuming and invasive strategies, incapable of monitoring organoid development. Using a redox-cycling approach in combination with a 3-mercaptopropionic acid self-assembled monolayer modification enabled the increase of sensor selectivity and sensitivity towards dopamine, while simultaneously reducing matrix-mediated interferences. In this work, we demonstrate the ability to detect and monitor even small differences in dopamine release between healthy and Parkinson`s disease-specific midbrain organoids over prolonged cultivation periods, which was additionally verified using liquid chromatography-multiple reaction monitoring mass spectrometry. Furthermore, the detection of a phenotypic rescue in midbrain organoids carrying a pathogenic mutation in leucine-rich repeat kinase 2, upon treatment with the leucine-rich repeat kinase 2 inhibitor II underlines the practical implementability of our sensing approach for drug screening applications as well as personalized disease modelling.
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Organoides , Enfermedad de Parkinson , Evaluación Preclínica de Medicamentos , Humanos , Mesencéfalo , Neurotransmisores , Organoides/metabolismo , Oxidación-Reducción , Enfermedad de Parkinson/metabolismoRESUMEN
Mammalian target of rapamycin (mTOR) signaling plays essential roles in brain development. Hyperactive mTOR is an essential pathological mechanism in autism spectrum disorder (ASD). Here, we show that tripartite motif protein 32 (TRIM32), as a maintainer of mTOR activity through promoting the proteasomal degradation of G protein signaling protein 10 (RGS10), regulates the proliferation of medial/lateral ganglionic eminence (M/LGE) progenitors. Deficiency of TRIM32 results in an impaired generation of GABAergic interneurons and autism-like behaviors in mice, concomitant with an elevated autophagy, which can be rescued by treatment embryonically with 3BDO, an mTOR activator. Transplantation of M/LGE progenitors or treatment postnatally with clonazepam, an agonist of the GABAA receptor, rescues the hyperexcitability and the autistic behaviors of TRIM32-/- mice, indicating a causal contribution of GABAergic disinhibition. Thus, the present study suggests a novel mechanism for ASD etiology in that TRIM32 deficiency-caused hypoactive mTOR, which is linked to an elevated autophagy, leads to autism-like behaviors via impairing generation of GABAergic interneurons. TRIM32-/- mouse is a novel autism model mouse.
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Trastorno Autístico/genética , Proliferación Celular/genética , Neuronas GABAérgicas/metabolismo , Interneuronas/metabolismo , Células-Madre Neurales/metabolismo , Neurogénesis/genética , Serina-Treonina Quinasas TOR/metabolismo , Ubiquitina-Proteína Ligasas/genética , Animales , Trastorno Autístico/metabolismo , Autofagia/efectos de los fármacos , Autofagia/genética , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Clonazepam/farmacología , Agonistas de Receptores de GABA-A/farmacología , Neuronas GABAérgicas/efectos de los fármacos , Interneuronas/efectos de los fármacos , Ratones , Ratones Noqueados , Células-Madre Neurales/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteínas RGS/metabolismoRESUMEN
Methods to assess neuronal receptor functions are needed in toxicology and for drug development. Human-based test systems that allow studies on glutamate signalling are still scarce. To address this issue, we developed and characterized pluripotent stem cell (PSC)-based neural cultures capable of forming a functional network. Starting from a stably proliferating neuroepithelial stem cell (NESC) population, we generate "mixed cortical cultures" (MCC) within 24 days. Characterization by immunocytochemistry, gene expression profiling and functional tests (multi-electrode arrays) showed that MCC contain various functional neurotransmitter receptors, and in particular, the N-methyl-D-aspartate subtype of ionotropic glutamate receptors (NMDA-R). As this important receptor is found neither on conventional neural cell lines nor on most stem cell-derived neurons, we focused here on the characterization of rapid glutamate-triggered Ca2+ signalling. Changes of the intracellular free calcium ion concentration ([Ca2+]i) were measured by fluorescent imaging as the main endpoint, and a method to evaluate and quantify signals in hundreds of cells at the same time was developed. We observed responses to glutamate in the low µM range. MCC responded to kainate and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), and a subpopulation of 50% had functional NMDA-R. The receptor was modulated by Mg2+, Zn2+ and Pb2+ in the expected ways, and various toxicologically relevant agonists (quinolinic acid, ibotenic acid, domoic acid) triggered [Ca2+]i responses in MCC. Antagonists, such as phencyclidine, ketamine and dextromethorphan, were also readily identified. Thus, the MCC developed here may fill an important gap in the panel of test systems available to characterize the effects of chemicals on neurotransmitter receptors.
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N-Metilaspartato/metabolismo , Receptores de Glutamato/metabolismo , Animales , Calcio , Células Cultivadas , Agonistas de Aminoácidos Excitadores , Ácido Glutámico , Humanos , Ácido Kaínico/análogos & derivados , Células-Madre Neurales , Neuronas , Receptores AMPA , Receptores de N-Metil-D-Aspartato , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol PropiónicoRESUMEN
Molecularly imprinted polymer (MIP)-based electrochemical sensors for the protein α-synuclein (a marker for Parkinson's disease) were developed using a peptide epitope from the protein. MIPs doped with various concentrations and species of transition metal dichalcogenides (TMDs) to enhance conductivity were electropolymerized with and without template molecules. The current during the electropolymerization was compared with that associated with the electrochemical response (at 0.24~0.29 V vs. ref. electrode) to target peptide molecules in the finished sensor. We found that this relationship can aid in the rational design of conductive MIPs for the recognition of biomarkers in biological fluids. The sensing range and limit of detection of TMD-doped imprinted poly(AN-co-MSAN)-coated electrodes were 0.001-100 pg/mL and 0.5 fg/mL (SNR = 3), respectively. To show the potential applicability of the MIP electrochemical sensor, cell culture medium from PD patient-specific midbrain organoids generated from induced pluripotent stem cells was analyzed. α-Synuclein levels were found to be significantly reduced in the organoids from PD patients, compared to those generated from age-matched controls. The relative standard deviation and recovery are less than 5% and 95-115%, respectively. Preparation of TMD-doped α-synuclein (SNCA) peptide-imprinted poly(AN-co-MSAN)-coated electrodes.
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Disulfuros/química , Polímeros Impresos Molecularmente/química , Molibdeno/química , Sulfuros/química , Compuestos de Tungsteno/química , alfa-Sinucleína/análisis , Técnicas Electroquímicas/métodos , Humanos , Límite de Detección , Mesencéfalo/química , Organoides/química , Enfermedad de Parkinson/diagnóstico , Fragmentos de Péptidos/química , alfa-Sinucleína/químicaRESUMEN
Human stem cell-derived organoids have great potential for modelling physiological and pathological processes. They recapitulate in vitro the organization and function of a respective organ or part of an organ. Human midbrain organoids (hMOs) have been described to contain midbrain-specific dopaminergic neurons that release the neurotransmitter dopamine. However, the human midbrain contains also additional neuronal cell types, which are functionally interacting with each other. Here, we analysed hMOs at high-resolution by means of single-cell RNA sequencing (scRNA-seq), imaging and electrophysiology to unravel cell heterogeneity. Our findings demonstrate that hMOs show essential neuronal functional properties as spontaneous electrophysiological activity of different neuronal subtypes, including dopaminergic, GABAergic, glutamatergic and serotonergic neurons. Recapitulating these in vivo features makes hMOs an excellent tool for in vitro disease phenotyping and drug discovery.
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Neuronas Dopaminérgicas/metabolismo , Organoides/metabolismo , Análisis de Secuencia de ARN/métodos , Transcriptoma/fisiología , Diferenciación Celular , HumanosRESUMEN
It is well known that adult neurogenesis occurs in two distinct regions, the subgranular zone of the dentate gyrus and the subventricular zone along the walls of the lateral ventricles. Until now, the contribution of these newly born neurons to behavior and cognition is still uncertain. The current study tested the functional impacts of diminished hippocampal neurogenesis on emotional and cognitive functions in transgenic Gfap-tk mice. Our results showed that anxiety-related behavior evaluated both in the elevated plus maze as well as in the open field, social interaction in the sociability test, and spatial working memory in the spontaneous alternation test were not affected. On the other hand, recognition and emotional memory in the object recognition test and contextual fear conditioning, and hippocampal long-term potentiation were impaired in transgenic mice. Furthermore, we evaluated whether environmental enrichment together with physical exercise could improve or even restore the level of adult neurogenesis, as well as the behavioral functions. Our results clearly demonstrated that environmental enrichment together with physical exercise successfully elevated the overall number of progenitor cells and young neurons in the dentate gyrus of transgenic mice. Furthermore, it led to a significant improvement in object recognition memory and contextual fear conditioning, and reverted impairments in hippocampal long-term potentiation. Thus, our results confirm the importance of adult neurogenesis for learning and memory processes and for hippocampal circuitry in general. Environmental enrichment and physical exercise beneficially influenced adult neurogenesis after it had been disrupted and most importantly recovered cognitive functions and long-term potentiation. © 2016 Wiley Periodicals, Inc.
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Trastornos del Conocimiento/terapia , Ambiente , Hipocampo/fisiopatología , Potenciación a Largo Plazo/fisiología , Actividad Motora/fisiología , Neurogénesis/fisiología , Animales , Ansiedad/patología , Ansiedad/fisiopatología , Ansiedad/terapia , Trastornos del Conocimiento/patología , Trastornos del Conocimiento/fisiopatología , Condicionamiento Psicológico/fisiología , Modelos Animales de Enfermedad , Terapia por Ejercicio , Miedo/fisiología , Hipocampo/patología , Vivienda para Animales , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neuronas/patología , Neuronas/fisiología , Reconocimiento en Psicología/fisiología , Conducta SocialRESUMEN
Polymerase basic protein 1 (PB1) is the catalytic core of the influenza A virus (IAV) RNA polymerase complex essential for viral transcription and replication. Understanding the intrinsic mechanisms which block PB1 function could stimulate development of new anti-influenza therapeutics. Affinity purification coupled with mass spectrometry (AP-MS) was used to identify host factors interacting with PB1. Among PB1 interactors, the E3 ubiquitin ligase TRIM32 interacts with PB1 proteins derived from multiple IAV strains. TRIM32 senses IAV infection by interacting with PB1 and translocates with PB1 to the nucleus following influenza infection. Ectopic TRIM32 expression attenuates IAV infection. Conversely, RNAi depletion and knockout of TRIM32 increase susceptibility of tracheal and lung epithelial cells to IAV infection. Reconstitution of trim32-/- mouse embryonic fibroblasts with TRIM32, but not a catalytically inactive mutant, restores viral restriction. Furthermore, TRIM32 directly ubiquitinates PB1, leading to PB1 protein degradation and subsequent reduction of polymerase activity. Thus, TRIM32 is an intrinsic IAV restriction factor which senses and targets the PB1 polymerase for ubiquitination and protein degradation. TRIM32 represents a model of intrinsic immunity, in which a host protein directly senses and counters viral infection in a species specific fashion by directly limiting viral replication.
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Interacciones Huésped-Parásitos/fisiología , Virus de la Influenza A/metabolismo , Gripe Humana/metabolismo , Factores de Transcripción/metabolismo , Proteínas Virales/metabolismo , Animales , Línea Celular , Técnicas de Inactivación de Genes , Humanos , Immunoblotting , Inmunohistoquímica , Inmunoprecipitación , Gripe Humana/inmunología , Espectrometría de Masas , Ratones , ARN Interferente Pequeño , Reacción en Cadena en Tiempo Real de la Polimerasa , Factores de Transcripción/inmunología , Transfección , Proteínas de Motivos Tripartitos , Ubiquitina-Proteína Ligasas/inmunología , Ubiquitina-Proteína Ligasas/metabolismo , UbiquitinaciónRESUMEN
Adult neural stem cells with the ability to generate neurons and glia cells are active throughout life in both the dentate gyrus (DG) and the subventricular zone (SVZ). Differentiation of adult neural stem cells is induced by cell fate determinants like the transcription factor Prox1. Evidence has been provided for a function of Prox1 as an inducer of neuronal differentiation within the DG. We now show that within the SVZ Prox1 induces differentiation into oligodendrocytes. Moreover, we find that loss of Prox1 expression in vivo reduces cell migration into the corpus callosum, where the few Prox1 deficient SVZ-derived remaining cells fail to differentiate into oligodendrocytes. Thus, our work uncovers a novel function of Prox1 as a fate determinant for oligodendrocytes in the adult mammalian brain. These data indicate that the neurogenic and oligodendrogliogenic lineages in the two adult neurogenic niches exhibit a distinct requirement for Prox1, being important for neurogenesis in the DG but being indispensable for oligodendrogliogenesis in the SVZ. Stem Cells 2016;34:2115-2129.
Asunto(s)
Células Madre Adultas/citología , Células Madre Adultas/metabolismo , Proteínas de Homeodominio/metabolismo , Ventrículos Laterales/citología , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Oligodendroglía/citología , Proteínas Supresoras de Tumor/metabolismo , Animales , Tipificación del Cuerpo/genética , Diferenciación Celular/genética , Linaje de la Célula/genética , Movimiento Celular/genética , Células Cultivadas , Elementos de Facilitación Genéticos/genética , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Ratones , Neurogénesis/genética , Bulbo Olfatorio/citología , Bulbo Olfatorio/metabolismo , Factor de Transcripción 2 de los Oligodendrocitos/genética , Factor de Transcripción 2 de los Oligodendrocitos/metabolismo , Oligodendroglía/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Receptores Notch/genética , Receptores Notch/metabolismoRESUMEN
In neural stem cells (NSCs), the balance between stem cell maintenance and neuronal differentiation depends on cell-fate determinants such as TRIM32. Previously, we have shown that TRIM32 associates with the RNA-induced silencing complex and increases the activity of microRNAs such as Let-7a. However, the exact mechanism of microRNA regulation by TRIM32 during neuronal differentiation has yet to be elucidated. Here, we used a mass spectrometry approach to identify novel protein-protein interaction partners of TRIM32 during neuronal differentiation. We found that TRIM32 associates with proteins involved in neurogenesis and RNA-related processes, such as the RNA helicase DDX6, which has been implicated in microRNA regulation. We demonstrate, that DDX6 colocalizes with TRIM32 in NSCs and neurons and that it increases the activity of Let-7a. Furthermore, we provide evidence that DDX6 is necessary and sufficient for neuronal differentiation and that it functions in cooperation with TRIM32.
Asunto(s)
Diferenciación Celular/genética , ARN Helicasas DEAD-box/genética , MicroARNs/genética , Células-Madre Neurales/metabolismo , Proteínas Proto-Oncogénicas/genética , Ubiquitina-Proteína Ligasas/genética , Animales , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Línea Celular Tumoral , Células Cultivadas , ARN Helicasas DEAD-box/metabolismo , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Immunoblotting , Ratones , Ratones Noqueados , MicroARNs/metabolismo , Microscopía Fluorescente , Células 3T3 NIH , Neurogénesis/genética , Unión Proteica , Mapas de Interacción de Proteínas/genética , Proteínas Proto-Oncogénicas/metabolismo , Interferencia de ARN , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
Parkinson's disease is a slowly progressing neurodegenerative disorder caused by loss of dopaminergic neurons in the substantia nigra (SN), leading to severe impairment in motor and non-motor functions. Endogenous subventricular zone (SVZ) neural stem cells constantly give birth to new cells that might serve as a possible source for regeneration in the adult brain. However, neurodegeneration is accompanied by neuroinflammation and dopamine depletion, potentially compromising regeneration. We therefore employed in vivo imaging methods to study striatal deafferentation (N-ω-fluoropropyl-2ß-carbomethoxy-3ß-(4-[(123) I]iodophenyl)nortropane single photon emission computed tomography, DaTscan(™) ) and neuroinflammation in the SN and striatum (N,N-diethyl-2-(2-(4-(2-[(18) F]fluoroethoxy)phenyl)-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)acetamide positron emission tomography, [(18) F]DPA-714 PET) in the intranigral 6-hydroxydopamine Parkinson's disease mouse model. Additionally, we transduced cells in the SVZ with a lentivirus encoding firefly luciferase and followed migration of progenitor cells in the SVZ-olfactory bulb axis via bioluminescence imaging under disease and control conditions. We found that activation of microglia in the SN is an acute process accompanying the degeneration of dopaminergic cell bodies in the SN. Dopaminergic deafferentation of the striatum does not influence the generation of doublecortin-positive neuroblasts in the SVZ, but generates chronic astrogliosis in the nigrostriatal system.