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1.
PLoS One ; 15(7): e0234529, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32673338

RESUMO

Morphometry characterization is an important procedure in describing neuronal cultures and identifying phenotypic differences. This task usually requires labor-intensive measurements and the classification of numerous neurites from large numbers of neurons in culture. To automate these measurements, we wrote AutoNeuriteJ, an imageJ/Fiji plugin that measures and classifies neurites from a very large number of neurons. We showed that AutoNeuriteJ is able to detect variations of neuritic growth induced by several compounds known to affect the neuronal growth. In these experiments measurement of more than 5000 mouse neurons per conditions was obtained within a few hours. Moreover, by analyzing mouse neurons deficient for the microtubule associated protein 6 (MAP6) and wild type neurons we illustrate that AutoNeuriteJ is capable to detect subtle phenotypic difference in axonal length. Overall the use of AutoNeuriteJ will provide rapid, unbiased and accurate measurement of neuron morphologies.


Assuntos
Processamento de Imagem Assistida por Computador/métodos , Neuritos/metabolismo , Neurônios/fisiologia , Animais , Axônios/fisiologia , Proliferação de Células , Células Cultivadas , Hipocampo/fisiologia , Camundongos , Camundongos Knockout , Proteínas Associadas aos Microtúbulos/genética , Neurogênese/fisiologia , Software
2.
PLoS One ; 15(4): e0230814, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32251425

RESUMO

Microtubules are a major cytoskeletal component of neurites, and the regulation of microtubule stability is essential for neurite morphogenesis. ßPix (ARHGEF7) is a guanine nucleotide exchange factor for the small GTPases Rac1 and Cdc42, which modulate the organization of actin filaments and microtubules. ßPix is expressed as alternatively spliced variants, including the ubiquitous isoform ßPix-a and the neuronal isoforms ßPix-b and ßPix-d, but the function of the neuronal isoforms remains unclear. Here, we reveal the novel role of ßPix neuronal isoforms in regulating tubulin acetylation and neurite outgrowth. At DIV4, hippocampal neurons cultured from ßPix neuronal isoform knockout (ßPix-NIKO) mice exhibit defects in neurite morphology and tubulin acetylation, a type of tubulin modification which often labels stable microtubules. Treating ßPix-NIKO neurons with paclitaxel, which stabilizes the microtubules, or reintroducing either neuronal ßPix isoform to the KO neurons overcomes the impairment in neurite morphology and tubulin acetylation, suggesting that neuronal ßPix isoforms may promote microtubule stabilization during neurite development. ßPix-NIKO neurons also exhibit lower phosphorylation levels for Stathmin1, a microtubule-destabilizing protein, at Ser16. Expressing either ßPix neuronal isoform in the ßPix-NIKO neurons restores Stathmin1 phosphorylation levels, with ßPix-d having a greater effect than ßPix-b. Furthermore, we find that the recovery of neurite length and Stathmin1 phosphorylation via ßPix-d expression requires PAK kinase activity. Taken together, our study demonstrates that ßPix-d regulates the phosphorylation of Stathmin1 in a PAK-dependent manner and that neuronal ßPix isoforms promote tubulin acetylation and neurite morphogenesis during neuronal development.


Assuntos
Crescimento Neuronal/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Transdução de Sinais/fisiologia , Estatmina/metabolismo , Tubulina (Proteína)/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Acetilação , Citoesqueleto de Actina/metabolismo , Animais , Proteínas de Ciclo Celular/metabolismo , Células Cultivadas , Feminino , Hipocampo/metabolismo , Hipocampo/fisiologia , Masculino , Camundongos , Camundongos Knockout , Microtúbulos/metabolismo , Neuritos/metabolismo , Neuritos/fisiologia , Neurônios/metabolismo , Neurônios/fisiologia , Fosforilação/fisiologia , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/fisiologia
3.
Biomed Res Int ; 2020: 6734048, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32149119

RESUMO

Retinoic acid- (RA-) triggered neuroblastoma cell lines are widely used cell modules of neuronal differentiation in neurodegenerative disease studies, but the gene regulatory mechanism underlying differentiation is unclear now. In this study, system biological analysis was performed on public microarray data from three neuroblastoma cell lines (SK-N-SH, SH-SY5Y-A, and SH-SY5Y-E) to explore the potential molecular processes of all-trans retinoic acid- (ATRA-) triggered differentiation. RT-qPCR, functional genomics analysis, western blotting, chromatin immunoprecipitation (ChIP), and homologous sequence analysis were further performed to validate the gene regulation processes and identify the RA response element in a specific gene. The potential disturbed biological pathways (111 functional GO terms in 14 interactive functional groups) and gene regulatory network (10 regulators and 71 regulated genes) in neuroblastoma differentiation were obtained. 15 of the 71 regulated genes are neuronal projection-related. Among them, NTRK2 is the only one that was dramatically upregulated in the RT-qPCR test that we performed on ATRA-treated SH-SY5Y-A cells. We further found that the overexpression of the NTRK2 gene can trigger differentiation-like changes in SH-SY5Y-A cells. Functional genomic analysis and western blotting assay suggested that, in neuroblastoma cells, ATRA may directly regulate the NTRK2 gene by activating the RA receptor (RAR) that binds in its promoter region. A novel RA response DNA element in the NTRK2 gene was then identified by bioinformatics analysis and chromatin immunoprecipitation (ChIP) assay. The novel element is sequence conservation and position variation among different species. Our study systematically provided the potential regulatory information of ATRA-triggered neuroblastoma differentiation, and in the NTRK2 gene, we identified a novel RA response DNA element, which may contribute to the differentiation in a human-specific manner.


Assuntos
Regulação da Expressão Gênica , Glicoproteínas de Membrana/genética , Neuroblastoma/genética , Neuroblastoma/metabolismo , Receptor trkB/genética , Tretinoína/metabolismo , Sítios de Ligação , Diferenciação Celular , Linhagem Celular Tumoral , Redes Reguladoras de Genes , Humanos , Glicoproteínas de Membrana/metabolismo , Neuritos/metabolismo , Receptor trkB/metabolismo , Receptores do Ácido Retinoico/metabolismo , Transcriptoma
4.
PLoS Pathog ; 16(2): e1008343, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-32069324

RESUMO

Neurotropic viral infections continue to pose a serious threat to human and animal wellbeing. Host responses combatting the invading virus in these infections often cause irreversible damage to the nervous system, resulting in poor prognosis. Rabies is the most lethal neurotropic virus, which specifically infects neurons and spreads through the host nervous system by retrograde axonal transport. The key pathogenic mechanisms associated with rabies infection and axonal transmission in neurons remains unclear. Here we studied the pathogenesis of different field isolates of lyssavirus including rabies using ex-vivo model systems generated with mouse primary neurons derived from the peripheral and central nervous systems. In this study, we show that neurons activate selective and compartmentalized degeneration of their axons and dendrites in response to infection with different field strains of lyssavirus. We further show that this axonal degeneration is mediated by the loss of NAD and calpain-mediated digestion of key structural proteins such as MAP2 and neurofilament. We then analysed the role of SARM1 gene in rabies infection, which has been shown to mediate axonal self-destruction during injury. We show that SARM1 is required for the accelerated execution of rabies induced axonal degeneration and the deletion of SARM1 gene significantly delayed axonal degeneration in rabies infected neurons. Using a microfluidic-based ex-vivo neuronal model, we show that SARM1-mediated axonal degeneration impedes the spread of rabies virus among interconnected neurons. However, this neuronal defense mechanism also results in the pathological loss of axons and dendrites. This study therefore identifies a potential host-directed mechanism behind neurological dysfunction in rabies infection. This study also implicates a novel role of SARM1 mediated axonal degeneration in neurotropic viral infection.


Assuntos
Proteínas do Domínio Armadillo/metabolismo , Axônios/metabolismo , Proteínas do Citoesqueleto/metabolismo , Raiva/fisiopatologia , Animais , Proteínas do Domínio Armadillo/genética , Proteínas do Domínio Armadillo/fisiologia , Transporte Axonal/fisiologia , Axônios/fisiologia , Proteínas do Citoesqueleto/genética , Proteínas do Citoesqueleto/fisiologia , Modelos Animais de Doenças , Gânglios Espinais/virologia , Lyssavirus/patogenicidade , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuritos/metabolismo , Neuritos/virologia , Neurônios/metabolismo , Neurônios/virologia , Raiva/metabolismo , Vírus da Raiva/metabolismo , Vírus da Raiva/patogenicidade
5.
PLoS One ; 15(1): e0221851, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31961897

RESUMO

BACKGROUND: There is currently no effective treatment for promoting regeneration of injured nerves in patients who have sustained injury to the central nervous system such as spinal cord injury. Chondroitinase ABC is an enzyme, which promotes neurite outgrowth and regeneration. It has shown considerable promise as a therapy for these conditions. The aim of the study is to determine if targeting chondroitinase ABC expression to the neuronal axon can further enhance its ability to promote axon outgrowth. Long-distance axon regeneration has not yet been achieved, and would be a significant step in attaining functional recovery following spinal cord injury. METHODOLOGY/PRINCIPAL FINDINGS: To investigate this, neuronal cultures were transfected with constructs encoding axon-targeted chondroitinase, non-targeted chondroitinase or GFP, and the effects on neuron outgrowth and sprouting determined on substrates either permissive or inhibitory to neuron regeneration. The mechanisms underlying the observed effects were also explored. Targeting chondroitinase to the neuronal axon markedly enhances its ability to promote neurite outgrowth. The increase in neurite length is associated with an upregulation of ß-integrin staining at the axonal cell surface. Staining for phosphofocal adhesion kinase, is also increased, indicating that the ß-integrins are in an activated state. Expression of chondroitinase within the neurons also resulted in a decrease in expression of PTEN and RhoA, molecules which present a block to neurite outgrowth, thus identifying two of the pathways by which ChABC promotes neurite outgrowth. CONCLUSIONS / SIGNIFICANCE: The novel finding that targeting ChABC to the axon significantly enhances its ability to promote neurite extension, suggests that this may be an effective way of promoting long-distance axon regeneration following spinal cord injury. It could also potentially improve its efficacy in the treatment of other pathologies, where it has been shown to promote recovery, such as myocardial infarction, stroke and Parkinson's disease.


Assuntos
Condroitina ABC Liase/genética , Regeneração Nervosa/genética , Crescimento Neuronal/genética , Traumatismos da Medula Espinal/genética , Animais , Axônios/metabolismo , Condroitina ABC Liase/antagonistas & inibidores , Regulação da Expressão Gênica/genética , Humanos , Neuritos/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , PTEN Fosfo-Hidrolase/genética , Recuperação de Função Fisiológica/genética , Traumatismos da Medula Espinal/fisiopatologia , Traumatismos da Medula Espinal/terapia , Proteína rhoA de Ligação ao GTP/genética
6.
Cell ; 180(2): 323-339.e19, 2020 01 23.
Artigo em Inglês | MEDLINE | ID: mdl-31928845

RESUMO

Teneurins are ancient metazoan cell adhesion receptors that control brain development and neuronal wiring in higher animals. The extracellular C terminus binds the adhesion GPCR Latrophilin, forming a trans-cellular complex with synaptogenic functions. However, Teneurins, Latrophilins, and FLRT proteins are also expressed during murine cortical cell migration at earlier developmental stages. Here, we present crystal structures of Teneurin-Latrophilin complexes that reveal how the lectin and olfactomedin domains of Latrophilin bind across a spiraling beta-barrel domain of Teneurin, the YD shell. We couple structure-based protein engineering to biophysical analysis, cell migration assays, and in utero electroporation experiments to probe the importance of the interaction in cortical neuron migration. We show that binding of Latrophilins to Teneurins and FLRTs directs the migration of neurons using a contact repulsion-dependent mechanism. The effect is observed with cell bodies and small neurites rather than their processes. The results exemplify how a structure-encoded synaptogenic protein complex is also used for repulsive cell guidance.


Assuntos
Proteínas do Tecido Nervoso/ultraestrutura , Receptores de Peptídeos/metabolismo , Tenascina/metabolismo , Animais , Adesão Celular/fisiologia , Cristalografia por Raios X/métodos , Células HEK293 , Humanos , Células K562 , Glicoproteínas de Membrana/metabolismo , Glicoproteínas de Membrana/ultraestrutura , Proteínas de Membrana/metabolismo , Proteínas de Membrana/ultraestrutura , Camundongos , Camundongos Endogâmicos C57BL/embriologia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neurogênese/fisiologia , Neurônios/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/metabolismo , Complexo Glicoproteico GPIb-IX de Plaquetas/ultraestrutura , Ligação Proteica/fisiologia , Proteínas/metabolismo , Proteínas/ultraestrutura , Receptores de Superfície Celular/metabolismo , Receptores de Peptídeos/ultraestrutura , Sinapses/metabolismo , Tenascina/ultraestrutura
7.
J Nat Med ; 74(1): 212-218, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31707550

RESUMO

Blood-brain barrier (BBB)-permeable components in the methanolic extract of Nelumbo nucifera flowers showed accelerative effects on neurite outgrowth in PC-12 cells. Among the constituents isolated from N. nucifera flowers in our previous study, aporphine-type alkaloids, lirinidine, asimilobine, N-methylasimilobine, and pronuciferine, showed accelerative effects. Lirinidine, N-methylasimilobine, and an alkaloid-rich diethyl ether fraction at low concentrations increased the expression of mRNAs coding for TrkA, Vav3, and Rac1. In addition, good permeability of asimilobine and N-methylasimilobine was confirmed using an in vitro BBB model. Asimilobine and N-methylasimilobine are considered to be suitable as seed compounds of drugs for Alzheimer's disease, because of their activity and BBB permeability.


Assuntos
Alcaloides/farmacologia , Aporfinas/farmacologia , Barreira Hematoencefálica/efeitos dos fármacos , Nelumbo/química , Neuritos/metabolismo , Doença de Alzheimer/tratamento farmacológico , Animais , Linhagem Celular Tumoral , Flores/química , Metanol , Crescimento Neuronal/efeitos dos fármacos , Células PC12 , Extratos Vegetais/farmacologia , Ratos , Compostos de Espiro/farmacologia
8.
Am J Physiol Gastrointest Liver Physiol ; 318(1): G53-G65, 2020 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-31682159

RESUMO

Detection of nutritional and noxious food components in the gut is a crucial component of gastrointestinal function. Contents in the gut lumen interact with enteroendocrine cells dispersed throughout the gut epithelium. Enteroendocrine cells release many different hormones, neuropeptides, and neurotransmitters that communicate either directly or indirectly with the central nervous system and the enteric nervous system, a network of neurons and glia located within the gut wall. Several populations of enteric neurons extend processes that innervate the gastrointestinal lamina propria; however, how these processes develop and begin to transmit information from the mucosa is not fully understood. In this study, we found that Tuj1-immunoreactive neurites begin to project out of the myenteric plexus at embryonic day (E)13.5 in the mouse small intestine, even before the formation of villi. Using live calcium imaging, we discovered that neurites were capable of transmitting electrical information from stimulated villi to the plexus by E15.5. In unpeeled gut preparations where all layers were left intact, we also mimicked the basolateral release of 5-HT from enteroendocrine cells, which triggered responses in myenteric cell bodies at postnatal day (P)0. Altogether, our results show that enteric neurons extend neurites out of the myenteric plexus early during mouse enteric nervous system development, innervating the gastrointestinal mucosa, even before villus formation in mice of either sex. Neurites are already able to conduct electrical information at E15.5, and responses to 5-HT develop postnatally.NEW & NOTEWORTHY How enteric neurons project into the gut mucosa and begin to communicate with the epithelium during development is not known. Our study shows that enteric neurites project into the lamina propria as early as E13.5 in the mouse, before development of the submucous plexus and before formation of intestinal villi. These neurites are capable of transmitting electrical signals back to their cell bodies by E15.5 and respond to serotonin applied to neurite terminals by birth.


Assuntos
Mucosa Intestinal/inervação , Intestino Delgado/inervação , Microvilosidades/fisiologia , Plexo Mientérico/crescimento & desenvolvimento , Neuritos/fisiologia , Neurogênese , Animais , Células Enteroendócrinas/metabolismo , Células Enteroendócrinas/fisiologia , Potenciais Evocados , Feminino , Idade Gestacional , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Plexo Mientérico/efeitos dos fármacos , Plexo Mientérico/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Serotonina/farmacologia , Tubulina (Proteína)/metabolismo
9.
Histochem Cell Biol ; 153(3): 177-184, 2020 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-31879799

RESUMO

Zonisamide, an anti-epileptic and anti-Parkinson's disease drug, displays neurotrophic activity on cultured motor neurons and facilitates axonal regeneration after peripheral nerve injury in mice, but its underlying mechanisms remain unclear. In this study, zonisamide enhanced neurite outgrowth from cultured adult rat dorsal root ganglion (DRG) neurons in a concentration-dependent manner (1 µM < 10 µM < 100 µM), and its activity was significantly attenuated by co-treatment with a phosphatidyl inositol-3'-phosphate-kinase (PI3K) inhibitor LY294002 or a mitogen-activated protein kinase (MAPK) inhibitor U0126. In agreement with these findings, 100 µM zonisamide for 1 h induced phosphorylation of AKT and ERK1/2, key molecules of PI3K and MAPK signaling pathways, respectively in mouse neuroblastoma × rat DRG neuron hybrid cells ND7/23. In contrast, zonisamide failed to promote proliferation or migration of immortalized Fischer rat Schwann cells 1 (IFRS1). These findings suggest that the beneficial effects of zonisamide on peripheral nerve regeneration may be attributable to its direct actions on neurons through PI3K and MAPK pathways, rather than the stimulation of Schwann cells.


Assuntos
Anticonvulsivantes/farmacologia , Gânglios Espinais/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Zonisamida/farmacologia , Animais , Movimento Celular , Proliferação de Células , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Gânglios Espinais/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Ratos , Ratos Wistar , Células de Schwann/citologia , Células de Schwann/metabolismo , Relação Estrutura-Atividade
10.
Cell Rep ; 29(13): 4646-4656.e4, 2019 12 24.
Artigo em Inglês | MEDLINE | ID: mdl-31875567

RESUMO

Stem cell-derived neurons are generally obtained in mass cultures that lack both spatial organization and any meaningful connectivity. We implement a microfluidic system for long-term culture of human neurons with patterned projections and synaptic terminals. Co-culture of human midbrain dopaminergic and striatal medium spiny neurons on the microchip establishes an orchestrated nigro-striatal circuitry with functional dopaminergic synapses. We use this platform to dissect the mitochondrial dysfunctions associated with a genetic form of Parkinson's disease (PD) with OPA1 mutations. Remarkably, we find that axons of OPA1 mutant dopaminergic neurons exhibit a significant reduction of mitochondrial mass. This defect causes a significant loss of dopaminergic synapses, which worsens in long-term cultures. Therefore, PD-associated depletion of mitochondria at synapses might precede loss of neuronal connectivity and neurodegeneration. In vitro reconstitution of human circuitries by microfluidic technology offers a powerful system to study brain networks by establishing ordered neuronal compartments and correct synapse identity.


Assuntos
Neurônios Dopaminérgicos/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Dispositivos Lab-On-A-Chip , Mitocôndrias/metabolismo , Neostriado/metabolismo , Substância Negra/metabolismo , Sinapses/metabolismo , Axônios/metabolismo , Células Cultivadas , GTP Fosfo-Hidrolases/genética , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Mutação/genética , Rede Nervosa/metabolismo , Neuritos/metabolismo , Doença de Parkinson/metabolismo
11.
PLoS One ; 14(10): e0224022, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31671109

RESUMO

Neurotrophins and their mimetics are potential treatments for hearing disorders because of their trophic effects on spiral ganglion neurons (SGNs) whose connections to hair cells may be compromised in many forms of hearing loss. Studies in noise or ototoxin-exposed animals have shown that local delivery of NT-3 or BDNF has beneficial effects on SGNs and hearing. We evaluated several TrkB or TrkC monoclonal antibody agonists and small molecules, along with BDNF and NT-3, in rat cochlea ex vivo models. The TrkB agonists BDNF and a monoclonal antibody, M3, had the greatest effects on SGN survival, neurite outgrowth and branching. In organotypic cochlear explants, BDNF and M3 enhanced synapse formation between SGNs and inner hair cells and restored these connections after excitotoxin-induced synaptopathy. Loss of these synapses has recently been implicated in hidden hearing loss, a condition characterized by difficulty hearing speech in the presence of background noise. The unique profile of M3 revealed here warrants further investigation, and the broad activity profile of BDNF observed underpins its continued development as a hearing loss therapeutic.


Assuntos
Anticorpos Monoclonais/imunologia , Fator Neurotrófico Derivado do Encéfalo/farmacologia , Cóclea/citologia , Perda Auditiva/patologia , Neuritos/metabolismo , Receptor trkA/agonistas , Sinapses/metabolismo , Animais , Linhagem Celular , Sobrevivência Celular , Modelos Animais de Doenças , Perda Auditiva/imunologia , Humanos , Neuritos/efeitos dos fármacos , Neuritos/imunologia , Ratos , Receptor trkA/imunologia , Sinapses/efeitos dos fármacos , Sinapses/imunologia
12.
Mol Med Rep ; 20(5): 4059-4066, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31702028

RESUMO

The present study aimed to explore the role of the PTEN/Akt/mTOR signaling pathway in the neurite outgrowth and apoptosis of cortical neurons. Cortical neurons were seeded on or adjacent to chondroitin sulfate proteoglycans. The length, number and crossing behavior of the neurites were calculated. Immunohistochemical staining and TUNEL data were analyzed. Neurites treated with PTEN inhibitor exhibited significant enhancements in elongation, initiation and crossing abilities when they encountered chondroitin sulfate proteoglycans in vitro. These effects disappeared when the PTEN/Akt/mTOR signaling pathway was blocked. Neurons exhibited significant enhancements in survival ability following PTEN inhibition. The present study demonstrated that PTEN inhibition can promote axonal elongation and initiation in cerebral cortical neurons, as well as the ability to cross the chondroitin sulfate proteoglycan border. In addition, PTEN inhibition is useful for protecting the neuron from apoptosis. The PTEN/Akt/mTOR signaling pathway is an important signaling pathway.


Assuntos
Apoptose , Neuritos/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Serina-Treonina Quinases TOR/metabolismo , Animais , Inibidores Enzimáticos/farmacologia , PTEN Fosfo-Hidrolase/antagonistas & inibidores , Ratos , Ratos Wistar
13.
Invest Ophthalmol Vis Sci ; 60(13): 4408-4415, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31639827

RESUMO

Purpose: We study the density and excitatory response of neurites, and Schwann cells (SCs) in fresh and cryopreserved stromal lenticules derived from small incision lenticule extraction (SMILE). Methods: Human stromal lenticules (n = 23) were immunostained for ß III-tubulin and imaged using spinning disk confocal laser microscopy, followed by three-dimensional reconstruction, to reveal neurite distribution. The lenticule neurite density (LND) was assessed using a validated neurite tracing and length measurement method with NeuronJ. LND was compared among groups of different lenticule thickness (71-165 µm) obtained from -3 to >-6 diopters (D) corrections. SCs were identified by marker expression and the laser effect on SC-neurite interaction was examined under transmission electron microscopy (TEM). Fresh porcine SMILE-lenticules (n = 18) were used for LND comparison among storage conditions and functional excitatory calcium response assay. Results: Using a validated neurite length measurement method, we found an inverse correlation of LND with lenticule thickness. Higher LND was found in thinner lenticules obtained from lower power of correction (r = -0.8925, P < 0.0001), whereas total lenticule neurite lengths did not alter significantly with regards to lenticule thickness. SCs were identified by GAP43 and p75NTR expression and were closely associated with lenticule neurites under TEM. In porcine lenticules, LND and excitatory calcium response were reduced after cold and cryogenic storage, when compared to fresh lenticules. Conclusions: The stromal neurites showed variations in density related to SMILE lenticule thickness and cryopreservation. With the presence of SC support and excitatory response, these neurite residues could retain minimal functionality that might serve as a potential advantage in the event of lenticule implantation.


Assuntos
Substância Própria/cirurgia , Cirurgia da Córnea a Laser/métodos , Neuritos/metabolismo , Adulto , Cálcio/metabolismo , Substância Própria/metabolismo , Criopreservação , Feminino , Proteína GAP-43/metabolismo , Humanos , Processamento de Imagem Assistida por Computador , Imageamento Tridimensional , Masculino , Microscopia Confocal , Microscopia Eletrônica de Transmissão , Microcirurgia , Miopia/cirurgia , Proteínas do Tecido Nervoso/metabolismo , Neuritos/patologia , Receptores de Fator de Crescimento Neural/metabolismo , Células de Schwann/metabolismo
14.
Eur J Pharmacol ; 865: 172737, 2019 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-31622594

RESUMO

Strong evidence has shown that long non-coding RNAs (lncRNAs) play important roles in genetic modulations in human CNS. In this study, we utilized an in vitro model of human induced pluripotent stem cells (hiPSCs)-derived neurons, and hypothesized that lncRNA of bladder cancer associated transcript 1 (BLACAT1) had a functional role in anesthesia-induced cytotoxicity in human lineage neural cells. To test that, HiPSCs were induced toward neural cells and then treated with ketamine in vitro. We demonstrated that, ketamine induced neuronal apoptosis, neurite degeneration, and upregulated BLACAT1 gene expression. Inversely, lentiviral-induced BLACAT1 downregulation rescued ketamine-induced neural cytotoxicity in hiPSCs-derived neurons. In addition, BLACAT1 downregulation was demonstrated to prevent ketamine-induced mitochondrial dysregulations by suppressing ROS and caspase 3/7 activities in hiPSCs-derived neurons. Thus, we discovered a new mechanism that inhibition of LncRNA BLACAT1 could rescue anesthesia-induced neural cytotoxicity in human induced pluripotent stem cells derived neurons.


Assuntos
Citotoxinas/toxicidade , Regulação para Baixo , Neurônios/citologia , Neurônios/efeitos dos fármacos , Células-Tronco Pluripotentes/citologia , RNA Longo não Codificante/genética , Anestesia/efeitos adversos , Apoptose/efeitos dos fármacos , Apoptose/genética , Humanos , Ketamina/toxicidade , Neuritos/efeitos dos fármacos , Neuritos/metabolismo
15.
Int J Nanomedicine ; 14: 7683-7694, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31571871

RESUMO

Background: Nerve regeneration is important for the treatment of degenerative diseases and neurons injured by accidents. Nerve growth factor (NGF) has been previously conjugated to materials for promotion of neurogenesis. Materials and methods: Photoreactive gelatin was prepared by chemical coupling of gelatin with azidobenzoic acid (P-gel), and then NGF was immobilized on substrates in the presence or absence of micropatterned photomasks. UV irradiation induced crosslinking reactions of P-gel with itself, NGF, and the plate for immobilization. Results: By adjustment of the P-gel concentration, the nanometer-order height of micropatterns was controlled. NGF was quantitatively immobilized with increasing amounts of P-gel. Immobilized NGF induced neurite outgrowth of PC12 cells, a cell line derived from a pheochromocytoma of the rat adrenal medulla, at the same level as soluble NGF. The immobilized NGF showed higher thermal stability than the soluble NGF and was repeatedly used without loss of biological activity. The 3D structure (height of the formed micropattern) regulated the behavior of neurite guidance. As a result, the orientation of neurites was regulated by the stripe pattern width. Conclusion: The micropattern-immobilized NGF nanolayer biochemically and topologically regulated neurite formation.


Assuntos
Proteínas Imobilizadas/farmacologia , Microtecnologia/métodos , Nanopartículas/química , Fator de Crescimento Neural/farmacologia , Neuritos/metabolismo , Animais , Humanos , Neuritos/efeitos dos fármacos , Neuritos/ultraestrutura , Células PC12 , Estabilidade Proteica/efeitos dos fármacos , Ratos , Solubilidade , Suínos , Temperatura
16.
EMBO Rep ; 20(11): e47732, 2019 11 05.
Artigo em Inglês | MEDLINE | ID: mdl-31486213

RESUMO

Crosstalk between the actin and microtubule cytoskeletons underlies cellular morphogenesis. Interactions between actin filaments and microtubules are particularly important for establishing the complex polarized morphology of neurons. Here, we characterized the neuronal function of growth arrest-specific 2-like 1 (Gas2L1), a protein that can directly bind to actin, microtubules and microtubule plus-end-tracking end binding proteins. We found that Gas2L1 promotes axon branching, but restricts axon elongation in cultured rat hippocampal neurons. Using pull-down experiments and in vitro reconstitution assays, in which purified Gas2L1 was combined with actin and dynamic microtubules, we demonstrated that Gas2L1 is autoinhibited. This autoinhibition is relieved by simultaneous binding to actin filaments and microtubules. In neurons, Gas2L1 primarily localizes to the actin cytoskeleton and functions as an actin stabilizer. The microtubule-binding tail region of Gas2L1 directs its actin-stabilizing activity towards the axon. We propose that Gas2L1 acts as an actin regulator, the function of which is spatially modulated by microtubules.


Assuntos
Actinas/metabolismo , Axônios/metabolismo , Proteínas dos Microfilamentos/metabolismo , Microtúbulos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Animais , Biomarcadores , Células COS , Chlorocebus aethiops , Feminino , Células HEK293 , Hipocampo/metabolismo , Humanos , Masculino , Imagem Molecular , Neuritos/metabolismo , Ligação Proteica , Estabilidade Proteica , Transporte Proteico , Células Piramidais/citologia , Células Piramidais/metabolismo , Ratos
17.
Molecules ; 24(18)2019 Sep 04.
Artigo em Inglês | MEDLINE | ID: mdl-31487775

RESUMO

Neuroinflammatory microenvironment, regulating neurite regrowth and neuronal survival, plays a critical role in Alzheimer's disease (AD). During neuroinflammation, microglia are activated, inducing the release of inflammatory or anti-inflammatory factors depending on their polarization into classical M1 microglia or alternative M2 phenotype. Therefore, optimizing brain microenvironment by small molecule-targeted microglia polarization and promoting neurite regeneration might be a potential therapeutic strategy for AD. In this study, we found platycodigenin, a naturally occurring triterpenoid, promoted M2 polarization and inhibited M1 polarization in lipopolysaccharide (LPS)-stimulated BV2 and primary microglia. Platycodigenin downregulated pro-inflammatory molecules such as interleukin (IL)-1ß, tumor necrosis factor (TNF)-α, IL-6 and nitric oxide (NO), while upregulated anti-inflammatory cytokine IL-10. Further investigation confirmed that platycodigenin inhibited cyclooxygenase-2 (Cox2) positive M1 but increased Ym1/2 positive M2 microglial polarization in primary microglia. In addition, platycodigenin significantly decreased LPS-induced the hyperphosphorylation of mitogen-activated protein kinase (MAPK) p38 and nuclear factor-κB (NF-κB) p65 subunits. Furthermore, the inactivation of peroxisome proliferators-activated receptor γ (PPARγ) induced by LPS was completely ameliorated by platycodigenin. Platycodigenin also promoted neurite regeneration and neuronal survival after Aß treatment in primary cortical neurons. Taken together, our study for the first time clarified that platycodigenin effectively ameliorated LPS-induced inflammation and Aß-induced neurite atrophy and neuronal death.


Assuntos
Microglia/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neuritos/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Saponinas/farmacologia , Plasticidade Celular/efeitos dos fármacos , Plasticidade Celular/imunologia , Citocinas/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Microglia/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Óxido Nítrico/metabolismo , PPAR gama/metabolismo , Transdução de Sinais/efeitos dos fármacos
18.
Biol Pharm Bull ; 42(9): 1545-1553, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31474714

RESUMO

The aim of the present study is to construct and characterize a novel three-dimensional culture system for mouse neurons using the functional polymer, FP001. Stereoscopically extended neurites were found in primary mouse cortical neurons cultured in the FP001-containing medium. Neurons cultured with FP001 were distributed throughout the medium of the observation range whereas neurons cultured without FP001 were distributed only on the bottom of the dish. These results demonstrated that neurons can be three-dimensionally cultured using the FP001-containing medium. The mRNA expression of the glutamatergic neuronal marker vesicular glutamate transporter 1 in neurons cultured in the FP001-containing medium were higher than that in neurons cultured in the FP001-free medium. Expression of the matured neuronal marker, microtubule-associated protein 2 (MAP2) a,b, and the synapse formation marker, Synapsin I, in neurons cultured with FP001 was also higher than that in neurons cultured without FP001. The expression pattern of MAP2a,b in neurons cultured with FP001, but not that in neurons cultured without FP001, was similar to that in the embryonic cerebral cortex. Exposure to glutamate significantly increased 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction activity in neurons cultured with FP001 compared to that in neurons cultured without FP001. These results suggested that glutamatergic neurotransmission in neurons three-dimensionally cultured in the FP001-containing medium may be upregulated compared to neurons two-dimensionally cultured in the FP001-free medium. Thus, neurons with the properties close to those in the embryonic brain could be obtained by three-dimensionally culturing neurons using FP001, compared to two-dimensional culture with a conventional adhesion method.


Assuntos
Técnicas de Cultura de Células/métodos , Córtex Cerebral/citologia , Meios de Cultura/química , Neurônios/citologia , Polissacarídeos Bacterianos/química , Animais , Córtex Cerebral/embriologia , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Proteínas Associadas aos Microtúbulos/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Sinapsinas/metabolismo
19.
DNA Repair (Amst) ; 83: 102696, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31546172

RESUMO

Mutations in the CSA and CSB genes are causative of Cockayne syndrome neurological disorder. Since the identification of indispensable functions of these two proteins in transcription-coupled repair and restoring RNA synthesis following DNA damage, the paradoxical less severe clinical symptoms reported in some CS-A patients have been puzzling. In this study we compared the effects of a CSA or a CSB defect at the levels of the cell and the intact organism. We showed that CSA-deficient zebrafish embryos exhibited modest hypersensitive to UV damage than CSB depletion. We found that loss of CSA can effectively release aggregation of mutant crystallin proteins in vitro. We described the opposite effect of CSA and CSB on neuritogenesis and elucidated the differentiated gene expression pathways regulated by these two proteins. Our data demonstrate convergent and divergent roles for CSA and CSB in DNA repair and transcription regulation and provide potential explanations for the observed differences between CS-A and CS-B patients.


Assuntos
Síndrome de Cockayne/genética , DNA Helicases/metabolismo , Enzimas Reparadoras do DNA/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular Tumoral , DNA Helicases/genética , Reparo do DNA , Enzimas Reparadoras do DNA/genética , Humanos , Neuritos/metabolismo , Proteínas de Ligação a Poli-ADP-Ribose/genética , Fatores de Transcrição/genética , Peixe-Zebra
20.
Proc Natl Acad Sci U S A ; 116(40): 20104-20114, 2019 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-31527246

RESUMO

Viral cancers show oncogene addiction to viral oncoproteins, which are required for survival and proliferation of the dedifferentiated cancer cell. Human Merkel cell carcinomas (MCCs) that harbor a clonally integrated Merkel cell polyomavirus (MCV) genome have low mutation burden and require viral T antigen expression for tumor growth. Here, we showed that MCV+ MCC cells cocultured with keratinocytes undergo neuron-like differentiation with neurite outgrowth, secretory vesicle accumulation, and the generation of sodium-dependent action potentials, hallmarks of a neuronal cell lineage. Cocultured keratinocytes are essential for induction of the neuronal phenotype. Keratinocyte-conditioned medium was insufficient to induce this phenotype. Single-cell RNA sequencing revealed that T antigen knockdown inhibited cell cycle gene expression and reduced expression of key Merkel cell lineage/MCC marker genes, including HES6, SOX2, ATOH1, and KRT20 Of these, T antigen knockdown directly inhibited Sox2 and Atoh1 expression. MCV large T up-regulated Sox2 through its retinoblastoma protein-inhibition domain, which in turn activated Atoh1 expression. The knockdown of Sox2 in MCV+ MCCs mimicked T antigen knockdown by inducing MCC cell growth arrest and neuron-like differentiation. These results show Sox2-dependent conversion of an undifferentiated, aggressive cancer cell to a differentiated neuron-like phenotype and suggest that the ontology of MCC arises from a neuronal cell precursor.


Assuntos
Antígenos Virais de Tumores/genética , Carcinoma de Célula de Merkel/etiologia , Carcinoma de Célula de Merkel/metabolismo , Poliomavírus das Células de Merkel/genética , Fenótipo , Infecções por Polyomavirus/complicações , Fatores de Transcrição SOXB1/genética , Antígenos Virais de Tumores/imunologia , Antígenos Virais de Tumores/metabolismo , Carcinoma de Célula de Merkel/patologia , Ciclo Celular/genética , Linhagem Celular Tumoral , Linhagem da Célula/genética , Transformação Celular Viral , Técnicas de Silenciamento de Genes , Humanos , Queratinócitos , Células de Merkel/metabolismo , Poliomavírus das Células de Merkel/imunologia , Neuritos/metabolismo , Neurônios/metabolismo , Infecções por Polyomavirus/imunologia , Infecções por Polyomavirus/virologia , Fatores de Transcrição SOXB1/metabolismo , Infecções Tumorais por Vírus/complicações , Infecções Tumorais por Vírus/imunologia , Infecções Tumorais por Vírus/virologia
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