RESUMO
Zika virus (ZIKV) infection is a global public health concern linked to adult neurological disorders and congenital diseases in newborns. Host lipid metabolism, including lipid droplet (LD) biogenesis, has been associated with viral replication and pathogenesis of different viruses. However, the mechanisms of LD formation and their roles in ZIKV infection in neural cells are still unclear. Here, we demonstrate that ZIKV regulates the expression of pathways associated with lipid metabolism, including the upregulation and activation of lipogenesis-associated transcription factors and decreased expression of lipolysis-associated proteins, leading to significant LD accumulation in human neuroblastoma SH-SY5Y cells and in neural stem cells (NSCs). Pharmacological inhibition of DGAT-1 decreased LD accumulation and ZIKV replication in vitro in human cells and in an in vivo mouse model of infection. In accordance with the role of LDs in the regulation of inflammation and innate immunity, we show that blocking LD formation has major roles in inflammatory cytokine production in the brain. Moreover, we observed that inhibition of DGAT-1 inhibited the weight loss and mortality induced by ZIKV infection in vivo. Our results reveal that LD biogenesis triggered by ZIKV infection is a crucial step for ZIKV replication and pathogenesis in neural cells. Therefore, targeting lipid metabolism and LD biogenesis may represent potential strategies for anti-ZIKV treatment development.
Assuntos
Neuroblastoma , Infecção por Zika virus , Zika virus , Animais , Humanos , Camundongos , Gotículas Lipídicas , Replicação ViralRESUMO
The Zika virus (ZIKV) caused neurological abnormalities in more than 3500 Brazilian newborns between 2015 and 2020. Data have pointed to oxidative stress in astrocytes as well as to dysregulations in neural cell proliferation and cell cycle as important events accounting for the cell death and neurological complications observed in Congenital Zika Syndrome. Copper imbalance has been shown to induce similar alterations in other pathologies, and disturbances in copper homeostasis have already been described in viral infections. Here, we investigated copper homeostasis imbalance as a factor that could contribute to the cytotoxic effects of ZIKV infection in astrocytes. Human induced pluripotent stem cell-derived astrocytes were infected with ZIKV; changes in the gene expression of copper homeostasis proteins were analyzed. The effect of the administration of CuCl2 or a copper chelator on oxidative stress, cell viability and percentage of infection were also studied. ZIKV infection leads to a downregulation of one of the transporters mediating copper release, ATP7B protein. We also observed the activation of mechanisms that counteract high copper levels, including the synthesis of copper chaperones and the reduction of the copper importer protein CTR1. Finally, we show that chelator-mediated copper sequestration in ZIKV-infected astrocytes reduces the levels of reactive oxygen species and improves cell viability, but does not change the overall percentage of infected cells. In summary, our results show that copper homeostasis imbalance plays a role in the pathology of ZIKV in astrocytes, indicating that it may also be a factor accounting for the developmental abnormalities in the central nervous system following viral infection. Evaluating micronutrient levels and the use of copper chelators in pregnant women susceptible to ZIKV infection may be promising strategies to manage novel cases of congenital ZIKV syndrome.
Assuntos
Células-Tronco Pluripotentes Induzidas , Infecção por Zika virus , Zika virus , Humanos , Recém-Nascido , Feminino , Gravidez , Infecção por Zika virus/metabolismo , Astrócitos/metabolismo , Cobre/farmacologia , Cobre/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Estresse Oxidativo , Morte Celular , Quelantes/metabolismo , Quelantes/farmacologiaRESUMO
Astrogliosis comprises a variety of changes in astrocytes that occur in a context-specific manner, triggered by temporally diverse signaling events that vary with the nature and severity of brain insults. However, most mechanisms underlying astrogliosis were described using animals, which fail to reproduce some aspects of human astroglial signaling. Here, we report an in vitro model to study astrogliosis using human-induced pluripotent stem cells (iPSC)-derived astrocytes which replicate temporally intertwined aspects of reactive astrocytes in vivo. We analyzed the time course of astrogliosis by measuring nuclear translocation of NF-kB, production of cytokines, changes in morphology and function of iPSC-derived astrocytes exposed to TNF-α. We observed NF-kB p65 subunit nuclear translocation and increased gene expression of IL-1ß, IL-6, and TNF-α in the first hours following TNF-α stimulation. After 24 hr, conditioned media from iPSC-derived astrocytes exposed to TNF-α exhibited increased secretion of inflammation-related cytokines. After 5 days, TNF-α-stimulated cells presented a typical phenotype of astrogliosis such as increased immunolabeling of Vimentin and GFAP and nuclei with elongated shape and shrinkage. Moreover, ~50% decrease in aspartate uptake was observed during the time course of astrogliosis with no evident cell damage, suggesting astroglial dysfunction. Together, our results indicate that human iPSC-derived astrocytes reproduce canonical events associated with astrogliosis in a time dependent fashion. The approach described here may contribute to a better understanding of mechanisms governing human astrogliosis with potential applicability as a platform to uncover novel biomarkers and drug targets to prevent or mitigate astrogliosis associated with human brain disorders.
Assuntos
Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Encefalopatias/metabolismo , Citocinas/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Gliose/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Filamentos Intermediários/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Vimentina/metabolismoRESUMO
Reactive oxygen species (ROS) and oxygen (O2) have been implicated in neurogenesis and self-renewal of neural progenitor cells (NPCs). On the other hand, oxidative unbalance, either by an impairment of antioxidant defenses or by an intensified production of ROS, is increasingly related to risk factors of neurodevelopmental disorders, such as schizophrenia. In this scenario, human induced pluripotent stem cells (hiPSCs) emerged as an interesting platform for the study of cellular and molecular aspects of this mental disorder, by complementing other experimental models, with exclusive advantages such as the recapitulation of brain development. Herein we discuss the role of O2/ROS signaling for neuronal differentiation and how its unbalance could be related to neurodevelopmental disorders, such as schizophrenia. Identifying the role of O2/ROS in neurogenesis as well as tackling oxidative stress and its disturbances in schizophrenic patients' derived cells will provide an interesting opportunity for the study of neural stem cells differentiation and neurodevelopmental disorders.
Assuntos
Células-Tronco Pluripotentes Induzidas/metabolismo , Neurogênese , Oxigênio/metabolismo , Esquizofrenia/metabolismo , Epigênese Genética , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Mitocôndrias/metabolismo , Modelos Neurológicos , Neurônios/metabolismo , Neurônios/patologia , Estresse Oxidativo , Espécies Reativas de Oxigênio/metabolismo , Esquizofrenia/genética , Esquizofrenia/fisiopatologia , Transdução de SinaisRESUMO
Psychedelic compounds have gained renewed interest for their potential therapeutic applications, but their metabolism and effects on complex biological systems remain poorly understood. Here, we present a systematic characterization of Lysergic Acid Diethylamide (LSD) metabolites in the model organism Caenorhabditis elegans using state-of-the-art analytical techniques. By employing ultra-high performance liquid chromatography coupled with high-resolution tandem mass spectrometry, we putatively identified a range of LSD metabolites, shedding light on their metabolic pathways and offering insights into their pharmacokinetics. Our study demonstrates the suitability of Caenorhabditis elegans as a valuable model system for investigating the metabolism of psychedelic compounds and provides a foundation for further research on the therapeutic potential of LSD.
Assuntos
Caenorhabditis elegans , Alucinógenos , Animais , Cromatografia Líquida de Alta Pressão , Dietilamida do Ácido Lisérgico , Espectrometria de Massas em TandemRESUMO
Sphingosine 1-phosphate (S1P) is a bioactive signaling lysophospholipid. Effects of S1P on proliferation, survival, migration, and differentiation have already been described; however, its role as a mediator of interactions between neurons and glial cells has been poorly explored. Here we describe effects of S1P, via the activation of its receptors in astrocytes, on the differentiation of neural progenitor cells (NPC) derived from either embryonic stem cells or the developing cerebral cortex. S1P added directly to NPC induced their differentiation, but S1P-primed astrocytes were able to promote even more pronounced changes in maturation, neurite outgrowth, and arborization in NPC. An increase in laminin by astrocytes was observed after S1P treatment. The effects of S1P-primed astrocytes on neural precursor cells were abrogated by antibodies against laminin. Together, our data indicate that S1P-treated astrocytes are able to induce neuronal differentiation of NPC by increasing the levels of laminin. These results implicate S1P signaling pathways as new targets for understanding neuroglial interactions within the central nervous system.
Assuntos
Astrócitos/efeitos dos fármacos , Lisofosfolipídeos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Esfingosina/análogos & derivados , Animais , Western Blotting , Diferenciação Celular/efeitos dos fármacos , Córtex Cerebral/citologia , Córtex Cerebral/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados , Imuno-Histoquímica , Laminina/metabolismo , Metabolismo dos Lipídeos/efeitos dos fármacos , Camundongos , Neuritos/efeitos dos fármacos , Reação em Cadeia da Polimerase em Tempo Real , Esfingosina/farmacologiaRESUMO
Lysophosphatidic acid (LPA) plays important roles in many biological processes, such as brain development, oncogenesis and immune functions, via its specific receptors. We previously demonstrated that LPA-primed astrocytes induce neuronal commitment of cerebral cortical progenitors (Spohr et al. 2008). In the present study, we analyzed neurite outgrowth induced by LPA-treated astrocytes and the molecular mechanism underlying this event. LPA-primed astrocytes increase neuronal differentiation, arborization and neurite outgrowth of developing cortical neurons. Treatment of astrocytes with epidermal growth factor (EGF) ligands yielded similar results, suggesting that members of the EGF family might mediate LPA-induced neuritogenesis. Furthermore, treatment of astrocytes with LPA or EGF ligands led to an increase in the levels of the extracellular matrix molecule, laminin (LN), thus enhancing astrocyte permissiveness to neurite outgrowth. This event was reversed by pharmacological inhibitors of the MAPK signaling pathway and of the EGF receptor. Our data reveal an important role of astrocytes and EGF receptor ligands pathway as mediators of bioactive lipids action in brain development, and implicate the LN and MAPK pathway in this process.
Assuntos
Astrócitos/efeitos dos fármacos , Axônios/efeitos dos fármacos , Córtex Cerebral/citologia , Receptores ErbB/fisiologia , Proteínas da Matriz Extracelular/fisiologia , Lisofosfolipídeos/farmacologia , Transdução de Sinais/efeitos dos fármacos , Células-Tronco/efeitos dos fármacos , Animais , Western Blotting , Contagem de Células , Diferenciação Celular/efeitos dos fármacos , Células Cultivadas , Córtex Cerebral/efeitos dos fármacos , Técnicas de Cocultura , Meios de Cultivo Condicionados , Ensaio de Imunoadsorção Enzimática , Receptores ErbB/antagonistas & inibidores , Receptores ErbB/efeitos dos fármacos , Feminino , Imuno-Histoquímica , Laminina/fisiologia , Masculino , Camundongos , Proteínas Quinases Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Neuritos/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologiaRESUMO
Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.
Assuntos
Axônios/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Neurais/metabolismo , Axônios/patologia , Encéfalo/metabolismo , Proliferação de Células/fisiologia , Cromatografia Líquida/métodos , Humanos , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodosRESUMO
Mitochondrial dysfunction is a central component in the pathophysiology of multiple neuropsychiatric and degenerative disorders. Evaluating mitochondrial function in human-derived neural cells can help characterize dysregulation in oxidative metabolism associated with the onset of brain disorders, and may also help define targeted therapies. Astrocytes play a number of different key roles in the brain, being implicated in neurogenesis, synaptogenesis, blood-brain-barrier permeability, and homeostasis, and, consequently, the malfunctioning of astrocytes is related to many neuropathologies. Here we describe protocols for generating induced pluripotent stem cell (iPSC)-derived astrocytes and evaluating multiple aspects of mitochondrial function. We use a high-resolution respirometry assay that measures real-time variations in mitochondrial oxygen flow, allowing the evaluation of cellular respiration in the context of an intact intracellular microenvironment, something not possible with permeabilized cells or isolated mitochondria, where the cellular microenvironment is disrupted. Given that an impairment in the mitochondrial regulation of intracellular calcium homeostasis is involved in many pathologic stresses, we also describe a protocol to evaluate mitochondrial calcium dynamics in human neural cells, by fluorimetry. Lastly, we outline a mitochondrial function assay that allows for the measurement of the enzymatic activity of mitochondrial hexokinase (mt-HK), an enzyme that is functionally coupled to oxidative phosphorylation and is involved in redox homeostasis, particularly in the brain. In all, these protocols allow a detailed characterization of mitochondrial function in human neural cells. High-resolution respirometry, calcium dynamics, and mt-HK activity assays provide data regarding the functional status of mitochondria, which may reflect mitochondrial stress or dysfunction. © 2020 Wiley Periodicals LLC. Basic Protocol 1: Generation of iPSC-derived human astrocytes Basic Protocol 2: Measuring real-time oxygen flux in human iPSC-derived astrocytes using a high-resolution OROBOROS Oxygraph 2k (O2k) Basic Protocol 3: Measuring mitochondrial calcium dynamics fluorometrically in permeabilized human neural cells Basic Protocol 4: Measuring OXPHOS-dependent activity of mitochondrial hexokinase in permeabilized human neural cells using a spectrophotometer.
Assuntos
Astrócitos/metabolismo , Metabolismo Energético , Células-Tronco Pluripotentes Induzidas/metabolismo , Mitocôndrias/metabolismo , Células-Tronco Neurais/metabolismo , Sinalização do Cálcio , Respiração Celular , Células Cultivadas , Hexoquinase/metabolismo , Humanos , Consumo de Oxigênio , Espectrometria de Fluorescência , Fatores de TempoRESUMO
Zika virus (ZIKV) has been extensively studied since it was linked to congenital malformations, and recent research has revealed that astrocytes are targets of ZIKV. However, the consequences of ZIKV infection, especially to this cell type, remain largely unknown, particularly considering integrative studies aiming to understand the crosstalk among key cellular mechanisms and fates involved in the neurotoxicity of the virus. Here, the consequences of ZIKV infection in iPSC-derived astrocytes are presented. Our results show ROS imbalance, mitochondrial defects and DNA breakage, which have been previously linked to neurological disorders. We have also detected glial reactivity, also present in mice and in post-mortem brains from infected neonates from the Northeast of Brazil. Given the role of glia in the developing brain, these findings may help to explain the observed effects in congenital Zika syndrome related to neuronal loss and motor deficit.
Assuntos
Astrócitos/metabolismo , Astrócitos/virologia , Infecção por Zika virus/metabolismo , Animais , Encéfalo/metabolismo , Dano ao DNA/fisiologia , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Pluripotentes Induzidas/virologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Mitocôndrias/fisiologia , Mitocôndrias/virologia , Neurônios/metabolismo , Estresse Oxidativo/fisiologia , Zika virus/metabolismo , Infecção por Zika virus/fisiopatologia , Infecção por Zika virus/virologiaRESUMO
Astrocytes play a critical role in the development and homeostasis of the central nervous system (CNS). Astrocyte dysfunction results in several neurological and degenerative diseases. However, a major challenge to our understanding of astrocyte physiology and pathology is the restriction of studies to animal models, human post-mortem brain tissues, or samples obtained from invasive surgical procedures. Here, we report a protocol to generate human functional astrocytes from cerebral organoids derived from human pluripotent stem cells. The cellular isolation of cerebral organoids yielded cells that were morphologically and functionally like astrocytes. Immunolabelling and proteomic assays revealed that human organoid-derived astrocytes express the main astrocytic molecular markers, including glutamate transporters, specific enzymes and cytoskeletal proteins. We found that organoid-derived astrocytes strongly supported neuronal survival and neurite outgrowth and responded to ATP through transient calcium wave elevations, which are hallmarks of astrocyte physiology. Additionally, these astrocytes presented similar functional pathways to those isolated from adult human cortex by surgical procedures. This is the first study to provide proteomic and functional analyses of astrocytes isolated from human cerebral organoids. The isolation of these astrocytes holds great potential for the investigation of developmental and evolutionary features of the human brain and provides a useful approach to drug screening and neurodegenerative disease modelling.
Assuntos
Astrócitos/citologia , Córtex Cerebral/citologia , Crescimento Neuronal , Organoides/citologia , Animais , Astrócitos/metabolismo , Sinalização do Cálcio , Células Cultivadas , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/metabolismo , Ácido Glutâmico/metabolismo , Humanos , Camundongos , Células-Tronco Pluripotentes/citologia , Células-Tronco Pluripotentes/metabolismo , Proteoma/genética , Proteoma/metabolismoRESUMO
Schizophrenia has been considered a devastating clinical syndrome rather than a single disease. Nevertheless, the mechanisms behind the onset of schizophrenia have been only partially elucidated. Several studies propose that levels of trace elements are abnormal in schizophrenia; however, conflicting data generated from different biological sources prevent conclusions being drawn. In this work, we used synchrotron radiation X-ray microfluorescence spectroscopy to compare trace element levels in neural progenitor cells (NPCs) derived from two clones of induced pluripotent stem cell lines of a clozapine-resistant schizophrenic patient and two controls. Our data reveal the presence of elevated levels of potassium and zinc in schizophrenic NPCs. Neural cells treated with valproate, an adjunctive medication for schizophrenia, brought potassium and zinc content back to control levels. These results expand the understanding of atomic element imbalance related to schizophrenia and may provide novel insights for the screening of drugs to treat mental disorders.
Assuntos
Antipsicóticos/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Potássio/metabolismo , Esquizofrenia/tratamento farmacológico , Ácido Valproico/farmacologia , Zinco/metabolismo , Antipsicóticos/uso terapêutico , Linhagem Celular , Clozapina/uso terapêutico , Resistência a Medicamentos , Células-Tronco Embrionárias/efeitos dos fármacos , Células-Tronco Embrionárias/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/metabolismo , Células-Tronco Neurais/metabolismo , Esquizofrenia/metabolismoRESUMO
Lysophosphatidic acid (LPA) is one of the main membrane-derived lysophospholipids, inducing diverse cellular responses like cell proliferation, cell death inhibition, and cytoskeletal rearrangement, and thus is important in many biological processes. In the central nervous system (CNS), post-mitotic neurons release LPA extracellularly whereas astrocytes do not. Astrocytes play a key role in brain development and pathology, producing various cytokines, chemokines, growth factors, and extracellular matrix (ECM) components that act as molecular coordinators of neuron-glia communication. However, many molecular mechanisms underlying these events remain unclear-in particular, how the multifaceted interplay between the signaling pathways regulated by lysophospholipids is integrated in the complex nature of the CNS. Previously we showed that LPA-primed astrocytes induce neuronal commitment by activating LPA1-LPA2 receptors. Further, we revealed that these events were mediated by modulation and organization of laminin levels by astrocytes, through the induction of the epidermal growth factor receptor (EGFR) signaling pathway and the activation of the mitogen-activated protein (MAP) kinase (MAPK) cascade in response to LPA (Spohr et al., 2008, 2011). In the present work, we aimed to answer whether LPA affects astrocytic production and rearrangement of fibronectin, and to investigate the mechanisms involved in neuronal differentiation and maturation of cortical neurons induced by LPA-primed astrocytes. We show that PKA activation is required for LPA-primed astrocytes to induce neurite outgrowth and neuronal maturation and to rearrange and enhance the production of fibronectin and laminin. We propose a potential mechanism by which neurons and astrocytes communicate, as well as how such interactions drive cellular events such as neurite outgrowth, cell fate commitment, and maturation.
RESUMO
Currently, stem cell research faces a major bottleneck related to the low efficiency of methods to produce large quantities of human embryonic stem cells (ESC) for use in clinical trials. Most culture media currently employed for human ESC cultivation contain animal compounds, and cells are grown in static flasks. Besides the immediate contamination with nonhuman compounds, cell expansion in flasks tends to be laborious and nonefficient. Here we cultured human ESC in stirred microcarrier (MC) systems using an animal/human-component-free medium, to overcome both issues. The method developed to culture cells on suspended beads combined the use of polymeric MCs in stirred vessels with an optimized culture medium free of supplements of animal and human origin. This approach generated approximately 160 million cells within 6 days, which were shown to remain pluripotent. The process developed herein provides a step forward toward therapy due to the economic advantages in the production of human ESC and to their consequent low immunogenic potential.
Assuntos
Reatores Biológicos , Células-Tronco Embrionárias/citologia , Animais , Sequência de Bases , Linhagem Celular , Meios de Cultura , Primers do DNA , Humanos , Microscopia Eletrônica de Varredura , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Schizophrenia has been defined as a neurodevelopmental disease that causes changes in the process of thoughts, perceptions, and emotions, usually leading to a mental deterioration and affective blunting. Studies have shown altered cell respiration and oxidative stress response in schizophrenia; however, most of the knowledge has been acquired from postmortem brain analyses or from nonneural cells. Here we describe that neural cells, derived from induced pluripotent stem cells generated from skin fibroblasts of a schizophrenic patient, presented a twofold increase in extramitochondrial oxygen consumption as well as elevated levels of reactive oxygen species (ROS), when compared to controls. This difference in ROS levels was reverted by the mood stabilizer valproic acid. Our model shows evidence that metabolic changes occurring during neurogenesis are associated with schizophrenia, contributing to a better understanding of the development of the disease and highlighting potential targets for treatment and drug screening.
Assuntos
Células-Tronco Pluripotentes Induzidas/citologia , Espécies Reativas de Oxigênio/metabolismo , Células Cultivadas , Feminino , Fibroblastos/citologia , Expressão Gênica/efeitos dos fármacos , Humanos , Pessoa de Meia-Idade , Células-Tronco Neurais/citologia , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Neurogênese , Esquizofrenia/metabolismo , Esquizofrenia/patologia , Pele/citologia , Ácido Valproico/farmacologiaRESUMO
Strategies aimed at improving spinal cord regeneration after trauma are still challenging neurologists and neuroscientists throughout the world. Many cell-based therapies have been tested, with limited success in terms of functional outcome. In this study, we investigated the effects of human dental pulp cells (HDPCs) in a mouse model of compressive spinal cord injury (SCI). These cells present some advantages, such as the ease of the extraction process, and expression of trophic factors and embryonic markers from both ecto-mesenchymal and mesenchymal components. Young adult female C57/BL6 mice were subjected to laminectomy at T9 and compression of the spinal cord with a vascular clip for 1 min. The cells were transplanted 7 days or 28 days after the lesion, in order to compare the recovery when treatment is applied in a subacute or chronic phase. We performed quantitative analyses of white-matter preservation, trophic-factor expression and quantification, and ultrastructural and functional analysis. Our results for the HDPC-transplanted animals showed better white-matter preservation than the DMEM groups, higher levels of trophic-factor expression in the tissue, better tissue organization, and the presence of many axons being myelinated by either Schwann cells or oligodendrocytes, in addition to the presence of some healthy-appearing intact neurons with synapse contacts on their cell bodies. We also demonstrated that HDPCs were able to express some glial markers such as GFAP and S-100. The functional analysis also showed locomotor improvement in these animals. Based on these findings, we propose that HDPCs may be feasible candidates for therapeutic intervention after SCI and central nervous system disorders in humans.
Assuntos
Transplante de Células/métodos , Polpa Dentária/transplante , Fibras Nervosas Mielinizadas/patologia , Recuperação de Função Fisiológica/fisiologia , Compressão da Medula Espinal/terapia , Medula Espinal/patologia , Animais , Axônios/patologia , Polpa Dentária/citologia , Feminino , Humanos , Camundongos , Modelos Animais , Atividade Motora/fisiologia , Neuroglia/patologia , Neurônios/patologia , Medula Espinal/fisiopatologia , Compressão da Medula Espinal/patologia , Compressão da Medula Espinal/fisiopatologia , Resultado do TratamentoRESUMO
We tested the effects of mouse embryonic stem cells (mES) grafts in mice spinal cord injury (SCI). Young adult female C57/Bl6 mice were subjected to laminectomy at T9 and 1-minute compression of the spinal cord with a vascular clip. Four groups were analyzed: laminectomy (Sham), injured (SCI), vehicle (DMEM), and mES-treated (EST). mES pre-differentiated with retinoic acid were injected (8 x 10(5) cells/2 microl) into the lesion epicenter, 10 min after SCI. Basso mouse scale (BMS) and Global mobility test (GMT) were assessed weekly up to 8 weeks, when morphological analyses were performed. GMT analysis showed that EST animals moved faster (10.73+/-0.9076, +/-SEM) than SCI (5.581+/-0.2905) and DMEM (5.705+/-0.2848), but slower than Sham animals (15.80+/-0.3887, p<0.001). By BMS, EST animals reached the final phase of locomotor recovery (3.872+/-0.7112, p<0.01), while animals of the SCI and DMEM groups improved to an intermediate phase (2.037+/-0.3994 and 2.111+/-0.3889, respectively). White matter area and number of myelinated nerve fibers were greater in EST (46.80+/-1.24 and 279.4+/-16.33, respectively) than the SCI group (39.97+/-0.925 and 81.39+/-8.078, p<0.05, respectively). EST group also presented better G-ratio values when compared with SCI group (p<0.001). Immunohistochemical revealed the differentiation of transplanted cells into astrocytes, oligodendrocytes, and Schwann cells, indicating an integration of transplanted cells with host tissue. Ultrastructural analysis showed, in the EST group, better tissue preservation and more remyelination by oligodendrocytes and Schwann cells than the other groups. Our results indicate that acute transplantation of predifferentiated mES into the injured spinal cord increased the spared white matter and number of nerve fibers, improving locomotor function.
Assuntos
Células-Tronco Embrionárias/fisiologia , Recuperação de Função Fisiológica/fisiologia , Compressão da Medula Espinal/fisiopatologia , Compressão da Medula Espinal/cirurgia , Transplante de Células-Tronco , Análise de Variância , Animais , Comportamento Animal , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Modelos Animais de Doenças , Embrião de Mamíferos , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Microscopia Eletrônica de Transmissão/métodos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Atividade Motora/fisiologia , Proteína Básica da Mielina/metabolismo , Fibras Nervosas Mielinizadas/patologia , Proteínas do Tecido Nervoso/metabolismo , Neuroglia/fisiologia , Neuroglia/ultraestrutura , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Células de Schwann/fisiologia , Células de Schwann/ultraestrutura , Compressão da Medula Espinal/patologiaRESUMO
Lysophosphatidic acid (LPA) is a simple phospholipid derived from cell membranes that has extracellular signaling properties mediated by at least five G protein-coupled receptors referred to as LPA(1)-LPA(5). In the nervous system, receptor-mediated LPA signaling has been demonstrated to influence a range of cellular processes; however, an unaddressed aspect of LPA signaling is its potential to produce specific secondary effects, whereby LPA receptor-expressing cells exposed to, or "primed," by LPA may then act on other cells via distinct, yet LPA-initiated, mechanisms. In the present study, we examined cerebral cortical astrocytes as possible indirect mediators of the effects of LPA on developing cortical neurons. Cultured astrocytes express at least four LPA receptor subtypes, known as LPA(1)-LPA(4). Cerebral cortical astrocytes primed by LPA exposure were found to increase neuronal differentiation of cortical progenitor cells. Treatment of unprimed astrocyte-progenitor cocultures with conditioned medium derived from LPA-primed astrocytes yielded similar results, suggesting the involvement of an astrocyte-derived soluble factor induced by LPA. At least two LPA receptor subtypes are involved in LPA priming, since the priming effect was lost in astrocytes derived from LPA receptor double-null mice (LPA(1)((-/-))/LPA(2)((-/-))). Moreover, the loss of LPA-dependent differentiation in receptor double-null astrocytes could be rescued by retrovirally transduced expression of a single deleted receptor. These data demonstrate that receptor-mediated LPA signaling in astrocytes can induce LPA-dependent, indirect effects on neuronal differentiation.