RESUMO
Three-dimensional (3D) in vitro culture systems using human induced pluripotent stem cells (hiPSCs) are useful tools to model neurodegenerative disease biology in physiologically relevant microenvironments. Though many successful biomaterials-based 3D model systems have been established for other neurogenerative diseases, such as Alzheimer's disease, relatively few exist for Parkinson's disease (PD) research. We employed tissue engineering approaches to construct a 3D silk scaffold-based platform for the culture of hiPSC-dopaminergic (DA) neurons derived from healthy individuals and PD patients harboring LRRK2 G2019S or GBA N370S mutations. We then compared results from protein, gene expression, and metabolic analyses obtained from two-dimensional (2D) and 3D culture systems. The 3D platform enabled the formation of dense dopamine neuronal network architectures and developed biological profiles both similar and distinct from 2D culture systems in healthy and PD disease lines. PD cultures developed in 3D platforms showed elevated levels of α-synuclein and alterations in purine metabolite profiles. Furthermore, computational network analysis of transcriptomic networks nominated several novel molecular interactions occurring in neurons from patients with mutations in LRRK2 and GBA. We conclude that the brain-like 3D system presented here is a realistic platform to interrogate molecular mechanisms underlying PD biology.
Assuntos
Neurônios Dopaminérgicos/patologia , Doença de Parkinson/patologia , Bioengenharia , Técnicas de Cultura de Células em Três Dimensões , Células Cultivadas , Neurônios Dopaminérgicos/citologia , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/patologia , Neurogênese , Seda/química , Alicerces Teciduais/químicaRESUMO
3-dimensional (3D) laboratory tissue cultures have emerged as an alternative to traditional 2-dimensional (2D) culture systems that do not recapitulate native cell behavior. The discrepancy between in vivo and in vitro tissue-cell-molecular responses impedes understanding of human physiology in general and creates roadblocks for the discovery of therapeutic solutions. Two parallel approaches have emerged for the design of 3D culture systems. The first is biomedical engineering methodology, including bioengineered materials, bioprinting, microfluidics and bioreactors, used alone or in combination, to mimic the microenvironments of native tissues. The second approach is organoid technology, in which stem cells are exposed to chemical and/or biological cues to activate differentiation programs that are reminiscent of human (prenatal) development. This review article describes recent technological advances in engineering 3D cultures that more closely resemble the human brain. The contributions of in vitro 3D tissue culture systems to new insights in neurophysiology, neurological diseases and regenerative medicine are highlighted. Perspectives on designing improved tissue models of the human brain are offered, focusing on an integrative approach merging biomedical engineering tools with organoid biology.
RESUMO
Cognitive decline is a debilitating feature of most neurodegenerative diseases of the central nervous system, including Alzheimer's disease. The causes leading to such impairment are only poorly understood and effective treatments are slow to emerge. Here we show that cognitive capacities in the neurodegenerating brain are constrained by an epigenetic blockade of gene transcription that is potentially reversible. This blockade is mediated by histone deacetylase 2, which is increased by Alzheimer's-disease-related neurotoxic insults in vitro, in two mouse models of neurodegeneration and in patients with Alzheimer's disease. Histone deacetylase 2 associates with and reduces the histone acetylation of genes important for learning and memory, which show a concomitant decrease in expression. Importantly, reversing the build-up of histone deacetylase 2 by short-hairpin-RNA-mediated knockdown unlocks the repression of these genes, reinstates structural and synaptic plasticity, and abolishes neurodegeneration-associated memory impairments. These findings advocate for the development of selective inhibitors of histone deacetylase 2 and suggest that cognitive capacities following neurodegeneration are not entirely lost, but merely impaired by this epigenetic blockade.
Assuntos
Encéfalo/fisiopatologia , Epigênese Genética , Histona Desacetilase 2/genética , Transtornos da Memória/genética , Transtornos da Memória/fisiopatologia , Doenças Neurodegenerativas/genética , Doenças Neurodegenerativas/fisiopatologia , Acetilação/efeitos dos fármacos , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/toxicidade , Animais , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Modelos Animais de Doenças , Epigênese Genética/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Histona Desacetilase 2/deficiência , Histona Desacetilase 2/metabolismo , Histonas/metabolismo , Humanos , Peróxido de Hidrogênio/toxicidade , Transtornos da Memória/complicações , Camundongos , Doenças Neurodegenerativas/complicações , Plasticidade Neuronal/efeitos dos fármacos , Plasticidade Neuronal/genética , Fragmentos de Peptídeos/toxicidade , Fosforilação/efeitos dos fármacos , Regiões Promotoras Genéticas/efeitos dos fármacos , Regiões Promotoras Genéticas/genética , RNA Polimerase II/metabolismo , Receptores de Glucocorticoides/metabolismoRESUMO
Aire induces the expression of a large set of autoantigen genes in the thymus, driving immunological tolerance in maturing T cells. To determine the full spectrum of molecular mechanisms underlying the Aire transactivation function, we screened an AIRE-dependent gene-expression system with a genome-scale lentiviral shRNA library, targeting factors associated with chromatin architecture/function, transcription, and mRNA processing. Fifty-one functional allies were identified, with a preponderance of factors that impact transcriptional elongation compared with initiation, in particular members of the positive transcription elongation factor b (P-TEFb) involved in the release of "paused" RNA polymerases (CCNT2 and HEXIM1); mRNA processing and polyadenylation factors were also highlighted (HNRNPL/F, SFRS1, SFRS3, and CLP1). Aire's functional allies were validated on transfected and endogenous target genes, including the generation of lentigenic knockdown (KD) mice. We uncovered the effect of the splicing factor Hnrnpl on Aire-induced transcription. Transcripts sensitive to the P-TEFb inhibitor flavopiridol were reduced by Hnrnpl knockdown in thymic epithelial cells, independently of their dependence on Aire, therefore indicating a general effect of Hnrnpl on RNA elongation. This conclusion was substantiated by demonstration of HNRNPL interactions with P-TEFb components (CDK9, CCNT2, HEXIM1, and the small 7SK RNA). Aire-containing complexes include 7SK RNA, the latter interaction disrupted by HNRNPL knockdown, suggesting that HNRNPL may partake in delivering inactive P-TEFb to Aire. Thus, these results indicate that mRNA processing factors cooperate with Aire to release stalled polymerases and to activate ectopic expression of autoantigen genes in the thymus.
Assuntos
Ribonucleoproteínas Nucleares Heterogêneas/fisiologia , Interferência de RNA , Fatores de Transcrição/genética , Transcrição Gênica/fisiologia , Animais , Linhagem Celular , Técnicas de Silenciamento de Genes , Ribonucleoproteínas Nucleares Heterogêneas/genética , Humanos , Camundongos , Fatores de Transcrição/fisiologia , Proteína AIRERESUMO
A new series of potent inhibitors of cellular lipid uptake from HDL particles mediated by scavenger receptor, class B, type I (SR-BI) was identified. The series was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR) that measured the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is characterized by a linear peptidomimetic scaffold with two adjacent amide groups, as well as an aryl-substituted heterocycle. Analogs of the initial hit were rapidly prepared via Ugi 4-component reaction, and select enantiopure compounds were prepared via a stepwise sequence. Structure-activity relationship (SAR) studies suggest an oxygenated arene is preferred at the western end of the molecule, as well as highly lipophilic substituents on the central and eastern nitrogens. Compound 5e, with (R)-stereochemistry at the central carbon, was designated as probe ML279. Mechanistic studies indicate that ML279 stabilizes the interaction of HDL particles with SR-BI, and its effect is reversible. It shows good potency (IC50=17 nM), is non-toxic, plasma stable, and has improved solubility over our alternative probe ML278.
Assuntos
Alanina/análogos & derivados , Antígenos CD36/antagonistas & inibidores , Furanos/química , Compostos Heterocíclicos/química , Tetrazóis/química , Alanina/síntese química , Alanina/química , Alanina/metabolismo , Animais , Antígenos CD36/genética , Antígenos CD36/metabolismo , Células CHO , Cricetinae , Cricetulus , Avaliação Pré-Clínica de Medicamentos , Lipoproteínas HDL/metabolismo , Ligação Proteica , Relação Estrutura-Atividade , Tetrazóis/síntese química , Tetrazóis/metabolismoRESUMO
We report a new series of 8-membered benzo-fused lactams that inhibit cellular lipid uptake from HDL particles mediated by Scavenger Receptor, Class B, Type I (SR-BI). The series was identified via a high-throughput screen of the National Institutes of Health Molecular Libraries Small Molecule Repository (NIH MLSMR), measuring the transfer of the fluorescent lipid DiI from HDL particles to CHO cells overexpressing SR-BI. The series is part of a previously reported diversity-oriented synthesis (DOS) library prepared via a build-couple-pair approach. Detailed structure-activity relationship (SAR) studies were performed with a selection of the original library, as well as additional analogs prepared via solution phase synthesis. These studies demonstrate that the orientation of the substituents on the aliphatic ring have a critical effect on activity. Additionally, a lipophilic group is required at the western end of the molecule, and a northern hydroxyl group and a southern sulfonamide substituent also proved to be optimal. Compound 2p was found to possess a superior combination of potency (av IC50=0.10µM) and solubility (79µM in PBS), and it was designated as probe ML312.
Assuntos
Antígenos CD36/antagonistas & inibidores , Lactamas/farmacologia , Metabolismo dos Lipídeos , Animais , Antígenos CD36/fisiologia , Humanos , Lactamas/química , Relação Estrutura-AtividadeRESUMO
Chromatin modifications, especially histone-tail acetylation, have been implicated in memory formation. Increased histone-tail acetylation induced by inhibitors of histone deacetylases (HDACis) facilitates learning and memory in wild-type mice as well as in mouse models of neurodegeneration. Harnessing the therapeutic potential of HDACis requires knowledge of the specific HDAC family member(s) linked to cognitive enhancement. Here we show that neuron-specific overexpression of HDAC2, but not that of HDAC1, decreased dendritic spine density, synapse number, synaptic plasticity and memory formation. Conversely, Hdac2 deficiency resulted in increased synapse number and memory facilitation, similar to chronic treatment with HDACis in mice. Notably, reduced synapse number and learning impairment of HDAC2-overexpressing mice were ameliorated by chronic treatment with HDACis. Correspondingly, treatment with HDACis failed to further facilitate memory formation in Hdac2-deficient mice. Furthermore, analysis of promoter occupancy revealed an association of HDAC2 with the promoters of genes implicated in synaptic plasticity and memory formation. Taken together, our results suggest that HDAC2 functions in modulating synaptic plasticity and long-lasting changes of neural circuits, which in turn negatively regulates learning and memory. These observations encourage the development and testing of HDAC2-selective inhibitors for human diseases associated with memory impairment.
Assuntos
Sinapses Elétricas/fisiologia , Histona Desacetilases/metabolismo , Memória/fisiologia , Proteínas Repressoras/metabolismo , Animais , Butiratos/farmacologia , Espinhas Dendríticas/fisiologia , Feminino , Regulação da Expressão Gênica , Hipocampo/metabolismo , Histona Desacetilase 1 , Histona Desacetilase 2 , Inibidores de Histona Desacetilases , Histona Desacetilases/deficiência , Histona Desacetilases/genética , Ácidos Hidroxâmicos/farmacologia , Aprendizagem/efeitos dos fármacos , Masculino , Memória/efeitos dos fármacos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neurônios/metabolismo , Regiões Promotoras Genéticas/genética , Proteínas Repressoras/antagonistas & inibidores , Proteínas Repressoras/genética , Sódio/farmacologia , VorinostatRESUMO
The HDL receptor, scavenger receptor, class B, type I (SR-BI), is a homooligomeric cell surface glycoprotein that controls HDL structure and metabolism by mediating the cellular selective uptake of lipids, mainly cholesteryl esters, from HDL. The mechanism underlying SR-BI-mediated lipid transfer, which differs from classic receptor-mediated endocytosis, involves a two-step process (binding followed by lipid transport) that is poorly understood. Our previous structure/activity analysis of the small-molecule inhibitor blocker of lipid transport 1 (BLT-1), which potently (IC(50) â¼ 50 nM) blocks SR-BI-mediated lipid transport, established that the sulfur in BLT-1's thiosemicarbazone moiety was essential for activity. Here we show that BLT-1 is an irreversible inhibitor of SR-BI, raising the possibility that cysteine(s) in SR-BI interact with BLT-1. Mass spectrometric analysis of purified SR-BI showed two of its six exoplasmic cysteines have free thiol groups (Cys251 and Cys384). Converting Cys384 (but not Cys251) to serine resulted in complete BLT-1 insensitivity, establishing that the unique molecular target of BLT-1 inhibition of cellular SR-BI dependent lipid transport is SR-BI itself. The C384S substitution reduced the receptor's intrinsic lipid uptake activity by approximately 60% without dramatically altering its surface expression, homooligomerization, or HDL binding. Thus, a small-molecule screening approach identified a key residue in SR-BI involved in lipid transport, providing a powerful springboard into the analyses of the structure and mechanism of SR-BI, and highlighting the power of this approach for such analyses.
Assuntos
Receptores Depuradores Classe B/química , Substituição de Aminoácidos , Animais , Sítios de Ligação , Transporte Biológico Ativo , Células COS , Chlorocebus aethiops , Ciclopentanos/farmacologia , Cisteína/química , Humanos , Técnicas In Vitro , Metabolismo dos Lipídeos , Lipoproteínas HDL/metabolismo , Espectrometria de Massas , Camundongos , Modelos Moleculares , Mutagênese Sítio-Dirigida , Proteínas Mutantes/antagonistas & inibidores , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Receptores Depuradores Classe B/antagonistas & inibidores , Receptores Depuradores Classe B/genética , Receptores Depuradores Classe B/metabolismo , Tiossemicarbazonas/farmacologiaRESUMO
Traumatic Brain injury-induced disturbances in mitochondrial fission-and-fusion dynamics have been linked to the onset and propagation of neuroinflammation and neurodegeneration. However, cell-type-specific contributions and crosstalk between neurons, microglia, and astrocytes in mitochondria-driven neurodegeneration after brain injury remain undefined. We developed a human three-dimensional in vitro triculture tissue model of a contusion injury composed of neurons, microglia, and astrocytes and examined the contributions of mitochondrial dysregulation to neuroinflammation and progression of injury-induced neurodegeneration. Pharmacological studies presented here suggest that fragmented mitochondria released by microglia are a key contributor to secondary neuronal damage progression after contusion injury, a pathway that requires astrocyte-microglia crosstalk. Controlling mitochondrial dysfunction thus offers an exciting option for developing therapies for TBI patients.
Assuntos
Lesões Encefálicas Traumáticas , Contusões , Humanos , Doenças Neuroinflamatórias , Inflamação/metabolismo , Encéfalo/metabolismo , Lesões Encefálicas Traumáticas/metabolismo , Contusões/metabolismo , Mitocôndrias/metabolismo , Microglia/metabolismo , Astrócitos/metabolismoRESUMO
Studies of underlying neurodegenerative processes in Parkinson's Disease (PD) have traditionally utilized cell cultures grown on two-dimensional (2D) surfaces. Biomimetic three-dimensional (3D) cell culture platforms have been developed to better emulate features of the brain's natural microenvironment. We here use our bioengineered brain-like tissue model, composed of a silk-hydrogel composite, to study the 3D microenvironment's contributions on the development and performance of dopaminergic-like neurons (DLNs). Compared with 2D culture, SH-SY5Y cells differentiated in 3D microenvironments were enriched for DLNs concomitant with a reduction in proliferative capacity during the neurodevelopmental process. Additionally, the 3D DLN cultures were more sensitive to oxidative stresses elicited by the PD-related neurotoxin 1-methyl-4-phenylpyridinium (MPP). MPP induced transcriptomic profile changes specific to 3D-differentiated DLN cultures, replicating the dysfunction of neuronal signaling pathways and mitochondrial dynamics implicated in PD. Overall, this physiologically-relevant 3D platform resembles a useful tool for studying dopamine neuron biology and interrogating molecular mechanisms underlying neurodegeneration in PD.
Assuntos
Neuroblastoma , Doença de Parkinson , Humanos , Dopamina , Doença de Parkinson/metabolismo , Linhagem Celular Tumoral , Neurônios Dopaminérgicos , Fenótipo , Apoptose , Microambiente TumoralRESUMO
Scavenger receptor class B, type I (SR-BI), is a high-density lipoprotein (HDL) receptor, which also binds low-density lipoprotein (LDL), and mediates the cellular selective uptake of cholesteryl esters from lipoproteins. SR-BI also is a coreceptor for hepatitis C virus and a signaling receptor that regulates cell metabolism. Many investigators have reported that lipoproteins bind to SR-BI via a single class of independent (not interacting), high-affinity binding sites (one site model). We have reinvestigated the ligand concentration dependence of (125)I-HDL binding to SR-BI and SR-BI-mediated specific uptake of [(3)H]CE from [(3)H]CE-HDL using an expanded range of ligand concentrations (<1 µg of protein/mL, lower than previously reported). Scatchard and nonlinear least-squares model fitting analyses of the binding and uptake data were both inconsistent with a single class of independent binding sites binding univalent lipoprotein ligands. The data are best fit by models in which SR-BI has either two independent classes of binding sites or one class of sites exhibiting negative cooperativity due to either classic allostery or ensemble effects ("lattice model"). Similar results were observed for LDL. Application of the "infinite dilution" dissociation rate method established that the binding of (125)I-HDL to SR-BI at 4 °C exhibits negative cooperativity. The unexpected complexity of the interactions of lipoproteins with SR-BI should be taken into account when interpreting the results of experiments that explore the mechanism(s) by which SR-BI mediates ligand binding, lipid transport, and cell signaling.
Assuntos
Lipoproteínas HDL/química , Lipoproteínas HDL/metabolismo , Receptores de Lipoproteínas/química , Receptores de Lipoproteínas/metabolismo , Receptores Depuradores Classe B/química , Receptores Depuradores Classe B/metabolismo , Animais , Sítios de Ligação , Células CHO , LDL-Colesterol/metabolismo , Cricetinae , Cricetulus , Humanos , LigantesRESUMO
Bioengineered 3D tunable neuronal constructs are a versatile platform for studying neuronal network functions, offering numerous advantages over existing technologies and providing for the discovery of new biological insights. Functional neural networks can be evaluated using calcium imaging and quantitatively described using network science. This protocol includes instructions for fabricating protein-based composite scaffolds, 3D in vitro culture of embryonic mouse cortical neurons, virally induced expression of GCaMP6f, wide-field calcium imaging, and computational analysis with open-source software and custom MATLAB code. For complete details on the use and execution of this protocol, please refer to Dingle et al. (2020).
Assuntos
Córtex Cerebral/metabolismo , Colágeno/química , Rede Nervosa/metabolismo , Redes Neurais de Computação , Neurônios/metabolismo , Seda/química , Alicerces Teciduais/química , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Córtex Cerebral/citologia , Camundongos , Rede Nervosa/citologia , Neurônios/citologiaRESUMO
Traumatic brain injury (TBI) survivors suffer long term from mental illness, neurodegeneration, and neuroinflammation. Studies of 3D tissue models have provided new insights into the pathobiology of many brain diseases. Here, a 3D in vitro contusion model is developed consisting of mouse cortical neurons grown on a silk scaffold embedded in collagen and used outcomes from an in vivo model for benchmarking. Molecular, cellular, and network events are characterized in response to controlled cortical impact (CCI). In this model, CCI induces degradation of neural network structure and function and release of glutamate, which are associated with the expression of programmed necrosis marker phosphorylated Mixed Lineage Kinase Domain Like Pseudokinase (pMLKL). Neurodegeneration is observed first in the directly impacted area and it subsequently spreads over time in 3D space. CCI reduces phosphorylated protein kinase B (pAKT) and Glycogen synthase kinase 3 beta (GSK3ß) in neurons in vitro and in vivo, but discordant responses are observed in phosphprylated ribosomal S6 kinase (pS6) and phosphorylated Tau (pTau) expression. In summary, the 3D brain-like culture system mimicked many aspects of in vivo responses to CCI, providing evidence that the model can be used to study the molecular, cellular, and functional sequelae of TBI, opening up new possibilities for discovery of therapeutics.
Assuntos
Lesões Encefálicas Traumáticas , Modelos Animais de Doenças , Animais , Encéfalo , Camundongos , Neurônios , Técnicas de Cultura de TecidosRESUMO
Three-dimensional (3D) in vitro cultures recapitulate key features of the brain including morphology, cell-cell and cell-extracellular matrix interactions, gradients of factors, and mechanical properties. However, there remains a need for experimental and computational tools to investigate network functions in these 3D models. To address this need, we present an experimental system based on 3D scaffold-based cortical neuron cultures in which we expressed the genetically encoded calcium indicator GCaMP6f to record neuronal activity at the millimeter-scale. Functional neural network descriptors were computed with graph-theory-based network analysis methods, showing the formation of functional networks at 3 weeks of culture. Changes to the functional network properties upon perturbations to glutamatergic neurotransmission or GABAergic neurotransmission were quantitatively characterized. The results illustrate the applicability of our 3D experimental system for the study of brain network development, function, and disruption in a biomimetic microenvironment.
RESUMO
The prevalence of dementia and other neurodegenerative diseases continues to rise as age demographics in the population shift, inspiring the development of long-term tissue culture systems with which to study chronic brain disease. Here, it is investigated whether a 3D bioengineered neural tissue model derived from human induced pluripotent stem cells (hiPSCs) can remain stable and functional for multiple years in culture. Silk-based scaffolds are seeded with neurons and glial cells derived from hiPSCs supplied by human donors who are either healthy or have been diagnosed with Alzheimer's disease. Cell retention and markers of stress remain stable for over 2 years. Diseased samples display decreased spontaneous electrical activity and a subset displays sporadic-like indicators of increased pathological ß-amyloid and tau markers characteristic of Alzheimer's disease with concomitant increases in oxidative stress. It can be concluded that the long-term stability of the platform is suited to study chronic brain disease including neurodegeneration.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Seda/química , Alicerces Teciduais/química , Proteínas tau/metabolismo , Doença de Alzheimer/patologia , Humanos , Células-Tronco Pluripotentes Induzidas/patologiaRESUMO
The replication of the complex structure and three dimensional (3-D) interconnectivity of neurons in the brain is a great challenge. A few 3-D neuronal patterning approaches have been developed to mimic the cell distribution in the brain but none have demonstrated the relationship between 3-D neuron patterning and network connectivity. Here, we used photolithographic crosslinking to fabricate in vitro 3-D neuronal structures with distinct sizes, shapes or interconnectivities, i.e., milli-blocks, micro-stripes, separated micro-blocks and connected micro-blocks, which have spatial confinement from "Z" dimension to "XYZ" dimension. During a 4-week culture period, the 3-D neuronal system has shown high cell viability, axonal, dendritic, synaptic growth and neural network activity of cortical neurons. We further studied the calcium oscillation of neurons in different 3-D patterns and used signal processing both in Fast Fourier Transform (FFT) and time domain (TD) to model the fluorescent signal variation. We observed that the firing frequency decreased as the spatial confinement in 3-D system increased. Besides, the neuronal synchronization significantly decreased by irregularly connecting micro-blocks, indicating that network connectivity can be adjusted by changing the linking conditions of 3-D gels. Earlier works showed the importance of 3-D culture over 2-D in terms of cell growth. Here, we showed that not only 3-D geometry over 2-D culture matters, but also the spatial organization of cells in 3-D dictates the neuronal firing frequency and synchronicity.
Assuntos
Técnicas de Cultura de Células/métodos , Rede Nervosa/fisiologia , Neurônios/fisiologia , Potenciais de Ação , Animais , Materiais Biocompatíveis/química , Sinalização do Cálcio , Células Cultivadas , Córtex Cerebral/citologia , Córtex Cerebral/fisiologia , Hidrogéis/química , Camundongos , Rede Nervosa/citologia , Neurônios/citologia , Alicerces Teciduais/químicaRESUMO
The cornea is a valuable tissue for studying peripheral sensory nerve structure and regeneration due to its avascularity, transparency, and dense innervation. Somatosensory innervation of the cornea serves to identify changes in environmental stimuli at the ocular surface, thereby promoting barrier function to protect the eye against injury or infection. Due to regulatory demands to screen ocular safety of potential chemical exposure, a need remains to develop functional human tissue models to predict ocular damage and pain using in vitro-based systems to increase throughput and minimize animal use. In this review, we summarize the anatomical and functional roles of corneal innervation in propagation of sensory input, corneal neuropathies associated with pain, and the status of current in vivo and in vitro models. Emphasis is placed on tissue engineering approaches to study the human corneal pain response in vitro with integration of proper cell types, controlled microenvironment, and high-throughput readouts to predict pain induction. Further developments in this field will aid in defining molecular signatures to distinguish acute and chronic pain triggers based on the immune response and epithelial, stromal, and neuronal interactions that occur at the ocular surface that lead to functional outcomes in the brain depending on severity and persistence of the stimulus.
Assuntos
Córnea/fisiologia , Doenças da Córnea/fisiopatologia , Dor Ocular/fisiopatologia , Neuralgia/fisiopatologia , Animais , Humanos , Modelos TeóricosRESUMO
HIV Nef has a number of important biological effects, including the down-modulation of several immunological important molecules (CD4, major histocompatibility complex [MHC] class I). Down-modulation of CD4 seems to be via clathrin-dependent endocytosis, whereas down-modulation of MHC class I remains unexplained. Several mutant proteins, including mutations in the small GTPase Arf6, have been used to probe membrane traffic pathways. One such mutant has recently been used to propose that Nef acts through Arf6 to activate the endocytosis of MHC class I. Here, we show that MHC class I down-modulation is unaffected by other Arf6 mutants that provide more specific perturbations in the GDP-GTP cycling of Arf6. Inhibition of phosphatidylinositol-3-phosphate kinase, an upstream activator of Arf6, also had no effect on the internalization step, but its activity is required to direct MHC class I to the trans-Golgi network. We conclude that the apparent Arf6 dependency of Nef-mediated MHC class I down-modulation is due to nonspecific perturbations in membrane traffic.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Clatrina/metabolismo , Genes MHC Classe I/fisiologia , Genes nef/fisiologia , HIV/metabolismo , Fator 6 de Ribosilação do ADP , Células Cultivadas , Clonagem Molecular , Regulação para Baixo/fisiologia , Endocitose/fisiologia , Ativação Enzimática/fisiologia , Citometria de Fluxo , Complexo de Golgi/metabolismo , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Humanos , Células Jurkat , Mutação , Fosfatidilinositol 3-Quinases/metabolismo , Transporte Proteico/fisiologiaRESUMO
The scavenger receptor class B, type I (SR-BI) mediates the cellular selective uptake of cholesteryl esters and other lipids from high-density lipoproteins (HDL) and low-density lipoproteins (LDL). This process, unlike classical receptor-mediated endocytosis, does not result in lipoprotein degradation. Instead, the lipid depleted particles are released into the medium. Here we show that selective lipid uptake mediated by murine SR-BI can be uncoupled from the endocytosis of HDL or LDL particles. We found that blocking selective lipid uptake by incubating cells with the small chemical inhibitors BLT-1 or BLT-4 did not affect endocytosis of HDL. Similarly, blocking endocytosis by hyperosmotic sucrose or K+ depletion did not prevent selective lipid uptake from HDL or LDL. These findings suggest that mSR-BI-mediated selective uptake occurs at the cell surface upon the association of lipoproteins with mSR-BI and does not require endocytosis of HDL or LDL particles.
Assuntos
Endocitose/efeitos dos fármacos , Metabolismo dos Lipídeos , Naftalenos/farmacologia , Receptores Imunológicos/metabolismo , Ureia/análogos & derivados , Ureia/farmacologia , Animais , Antígenos CD36 , Células CHO , Cricetinae , Endocitose/fisiologia , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Camundongos , Pressão Osmótica/efeitos dos fármacos , Potássio/metabolismo , Receptores Depuradores , Receptores Depuradores Classe B , Sacarose/farmacologiaRESUMO
A bio-acoustic levitational assembly method for engineering of multilayered, 3D brainlike constructs is presented. Acoustic radiation forces are used to levitate neuroprogenitors derived from human embryonic stem cells in 3D multilayered fibrin tissue constructs. The neuro-progenitor cells are subsequently differentiated in neural cells, resulting in a 3D neuronal construct with inter and intralayer neurite elongations.