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1.
Int J Mol Sci ; 22(19)2021 Sep 26.
Artigo em Inglês | MEDLINE | ID: mdl-34638697

RESUMO

BACKGROUND: The prevention of age-related neurodegenerative disorders is an important issue in an aging society. Microglia-mediated neuroinflammation resulting in dopaminergic neuron loss may lead to the pathogenesis of Parkinson's disease (PD). Lipopolysaccharide (LPS), an endotoxin, induces neuroinflammatory microglial activation, contributing to dopaminergic neuron damage. Diosgenin is a phytosteroid sapogenin with a wide spectrum of pharmacological activities, e.g., anti-inflammatory activity. However, the preventive effect of diosgenin on neuroinflammation is not clear. Thus, in this study, we further investigated the neuroprotective effect of diosgenin on LPS-induced neural damage in vitro and in vivo. METHODS: For in vitro experiments, primary mesencephalic neuron-glia cultures and primary microglia cultures isolated from Sprague-Dawley rats were used. Cells were pretreated with diosgenin and then stimulated with LPS. The expression of proinflammatory cytokines or tyrosine hydroxylase (TH) in the cells was analyzed. In vivo, rats were fed a diet containing 0.1% (w/w) diosgenin for 4 weeks before being administered a unilateral substantia nigra (SN) injection of LPS. Four weeks after the LPS injection, the rats were assessed for lesion severity using the amphetamine-induced rotation test and TH immunohistochemistry. RESULTS: Diosgenin pretreatment prevented LPS-induced neurite shortening in TH-positive neurons in mesencephalic neuron-glia cultures. In addition, pretreatment of primary microglia with diosgenin significantly reduced tumor necrosis factor-α (TNF-α) and inducible nitric oxide synthase (iNOS) expression. Moreover, diosgenin pretreatment significantly suppressed LPS-induced extracellular signal-regulated kinase (ERK) activation. In vivo, the intranigral injection of LPS in rats fed a diosgenin-containing diet significantly improved motor dysfunction and reduced TH expression in SN. CONCLUSION: These results support the effectiveness of diosgenin in protecting dopaminergic neurons from LPS-induced neuroinflammation.


Assuntos
Diosgenina/farmacologia , Neurônios Dopaminérgicos/metabolismo , Lipopolissacarídeos/toxicidade , Microglia/metabolismo , Doenças Neurodegenerativas , Fármacos Neuroprotetores/farmacologia , Animais , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Masculino , Neuritos/metabolismo , Doenças Neurodegenerativas/induzido quimicamente , Doenças Neurodegenerativas/metabolismo , Doenças Neurodegenerativas/prevenção & controle , Ratos , Ratos Sprague-Dawley
2.
FASEB J ; 35(11): e21896, 2021 11.
Artigo em Inglês | MEDLINE | ID: mdl-34634154

RESUMO

Surgical intervention with the use of autografts is considered the gold standard to treat peripheral nerve injuries. However, a biomaterial that supports and guides nerve growth would be an attractive alternative to overcome problems with limited availability, morbidity at the site of harvest, and nerve mismatches related to autografts. Native spider silk is a promising material for construction of nerve guidance conduit (NGC), as it enables regeneration of cm-long nerve injuries in sheep, but regulatory requirements for medical devices demand synthetic materials. Here, we use a recombinant spider silk protein (NT2RepCT) and a functionalized variant carrying a peptide derived from vitronectin (VN-NT2RepCT) as substrates for nerve growth support and neurite extension, using a dorsal root ganglion cell line, ND7/23. Two-dimensional coatings were benchmarked against poly-d-lysine and recombinant laminins. Both spider silk coatings performed as the control substrates with regards to proliferation, survival, and neurite growth. Furthermore, NT2RepCT and VN-NT2RepCT spun into continuous fibers in a biomimetic spinning set-up support cell survival, neurite growth, and guidance to an even larger extent than native spider silk. Thus, artificial spider silk is a promising biomaterial for development of NGCs.


Assuntos
Proliferação de Células/efeitos dos fármacos , Regeneração Nervosa/efeitos dos fármacos , Neuritos/metabolismo , Seda/farmacologia , Aranhas/metabolismo , Vitronectina/farmacologia , Animais , Autoenxertos , Materiais Biocompatíveis/farmacologia , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Gânglios Espinais/citologia , Humanos , Laminina/farmacologia , Camundongos , Neuritos/efeitos dos fármacos , Traumatismos dos Nervos Periféricos/cirurgia , Engenharia de Proteínas/métodos , Ratos , Proteínas Recombinantes/farmacologia , Seda/genética , Vitronectina/genética
3.
J Biol Chem ; 297(4): 101159, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34480901

RESUMO

In Alzheimer's disease (AD), deposition of pathological tau and amyloid-ß (Aß) drive synaptic loss and cognitive decline. The injection of misfolded tau aggregates extracted from human AD brains drives templated spreading of tau pathology within WT mouse brain. Here, we assessed the impact of Aß copathology, of deleting loci known to modify AD risk (Ptk2b, Grn, and Tmem106b) and of pharmacological intervention with an Fyn kinase inhibitor on tau spreading after injection of AD tau extracts. The density and spreading of tau inclusions triggered by human tau seed were unaltered in the hippocampus and cortex of APPswe/PSEN1ΔE9 transgenic and AppNL-F/NL-F knock-in mice. In mice with human tau sequence replacing mouse tau, template matching enhanced neuritic tau burden. Human AD brain tau-enriched preparations contained aggregated Aß, and the Aß coinjection caused a redistribution of Aß aggregates in mutant AD model mice. The injection-induced Aß phenotype was spatially distinct from tau accumulation and could be ameliorated by depleting Aß from tau extracts. These data suggest that Aß and tau pathologies propagate by largely independent mechanisms after their initial formation. Altering the activity of the Fyn and Pyk2 (Ptk2b) kinases involved in Aß-oligomer-induced signaling, or deleting expression of the progranulin and TMEM106B lysosomal proteins, did not alter the somatic tau inclusion burden or spreading. However, mouse aging had a prominent effect to increase the accumulation of neuritic tau after injection of human AD tau seeds into WT mice. These studies refine our knowledge of factors capable of modulating tau spreading.


Assuntos
Envelhecimento/metabolismo , Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Córtex Cerebral/metabolismo , Hipocampo/metabolismo , Neuritos/metabolismo , Proteínas tau/metabolismo , Envelhecimento/genética , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/genética , Animais , Camundongos , Camundongos Knockout , Proteínas tau/genética
4.
FASEB J ; 35(9): e21813, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34390512

RESUMO

Cell adhesion is tightly controlled in multicellular organisms, for example, through proteolytic ectodomain shedding of the adhesion-mediating cell surface transmembrane proteins. In the brain, shedding of cell adhesion proteins is required for nervous system development and function, but the shedding of only a few adhesion proteins has been studied in detail in the mammalian brain. One such adhesion protein is the transmembrane protein endoglycan (PODXL2), which belongs to the CD34-family of highly glycosylated sialomucins. Here, we demonstrate that endoglycan is broadly expressed in the developing mouse brains and is proteolytically shed in vitro in mouse neurons and in vivo in mouse brains. Endoglycan shedding in primary neurons was mediated by the transmembrane protease a disintegrin and metalloprotease 10 (ADAM10), but not by its homolog ADAM17. Functionally, endoglycan deficiency reduced the branching of neurites extending from primary neurons in vitro, whereas deletion of ADAM10 had the opposite effect and increased neurite branching. Taken together, our study discovers a function for endoglycan in neurite branching, establishes endoglycan as an ADAM10 substrate and suggests that ADAM10 cleavage of endoglycan may contribute to neurite branching.


Assuntos
Proteína ADAM10/metabolismo , Secretases da Proteína Precursora do Amiloide/metabolismo , Moléculas de Adesão Celular/metabolismo , Desintegrinas/metabolismo , Proteínas de Membrana/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Sialoglicoproteínas/metabolismo , Proteína ADAM17/metabolismo , Animais , Encéfalo/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neurogênese/fisiologia , Proteólise
5.
Cells ; 10(8)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34440636

RESUMO

Neurogenesis and functional brain activity require complex associations of inherently programmed secretory elements that are regulated precisely and temporally. Family with sequence similarity 19 A1 (FAM19A1) is a secreted protein primarily expressed in subsets of terminally differentiated neuronal precursor cells and fully mature neurons in specific brain substructures. Several recent studies have demonstrated the importance of FAM19A1 in brain physiology; however, additional information is needed to support its role in neuronal maturation and function. In this study, dendritic spine morphology in Fam19a1-ablated mice and neurite development during in vitro neurogenesis were examined to understand the putative role of FAM19A1 in neural integrity. Adult Fam19a1-deficient mice showed low dendritic spine density and maturity with reduced dendrite complexity compared to wild-type (WT) littermates. To further explore the effect of FAM19A1 on neuronal maturation, the neurite outgrowth pattern in primary neurons was analyzed in vitro with and without FAM19A1. In response to FAM19A1, WT primary neurons showed reduced neurite complexity, whereas Fam19a1-decifient primary neurons exhibited increased neurite arborization, which was reversed by supplementation with recombinant FAM19A1. Together, these findings suggest that FAM19A1 participates in dendritic spine development and neurite arborization.


Assuntos
Encéfalo/metabolismo , Quimiocinas/metabolismo , Espinhas Dendríticas/metabolismo , Neuritos/metabolismo , Crescimento Neuronal , Fatores Etários , Animais , Encéfalo/patologia , Células Cultivadas , Quimiocinas/genética , Espinhas Dendríticas/patologia , Feminino , Idade Gestacional , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Neuritos/patologia , Gravidez , Transdução de Sinais
6.
Int J Nanomedicine ; 16: 4515-4526, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34239302

RESUMO

Introduction: Neuroregeneration is a major challenge in neuroscience for treating degenerative diseases and for repairing injured nerves. Numerous studies have shown the importance of physical stimulation for neuronal growth and development, and here we report an approach for the physical guidance of neuron orientation and neurite growth using superparamagnetic iron oxide (SPIO) nanoparticles and magnetic fields (MFs). Methods: SPIO nanoparticles were synthesized by classic chemical co-precipitation methods and then characterized by transmission electron microscope, dynamic light scattering, and vibrating sample magnetometer. The cytotoxicity of the prepared SPIO nanoparticles and MF was determined using CCK-8 assay and LIVE/DEAD assay. The immunofluorescence images were captured by a laser scanning confocal microscopy. Cell migration was evaluated using the wound healing assay. Results: The prepared SPIO nanoparticles showed a narrow size distribution, low cytotoxicity, and superparamagnetism. SPIO nanoparticles coated with poly-L-lysine could be internalized by spiral ganglion neurons (SGNs) and showed no cytotoxicity at concentrations less than 300 µg/mL. The neurite extension of SGNs was promoted after internalizing SPIO nanoparticles with or without an external MF, and this might be due to the promotion of growth cone development. It was also confirmed that SPIO can regulate cell migration and can direct neurite outgrowth in SGNs preferentially along the direction imposed by an external MF. Conclusion: Our results provide a fundamental understanding of the regulation of cell behaviors under physical cues and suggest alternative treatments for sensorineural hearing loss caused by the degeneration of SGNs.


Assuntos
Campos Magnéticos , Nanopartículas Magnéticas de Óxido de Ferro , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Gânglio Espiral da Cóclea/citologia , Animais , Ciclo Celular/efeitos dos fármacos , Neurogênese/efeitos dos fármacos
7.
Int J Mol Sci ; 22(13)2021 Jun 25.
Artigo em Inglês | MEDLINE | ID: mdl-34202161

RESUMO

The autonomic nervous system derives from the neural crest (NC) and supplies motor innervation to the smooth muscle of visceral organs, including the lower urinary tract (LUT). During fetal development, sacral NC cells colonize the urogenital sinus to form pelvic ganglia (PG) flanking the bladder neck. The coordinated activity of PG neurons is required for normal urination; however, little is known about the development of PG neuronal diversity. To discover candidate genes involved in PG neurogenesis, the transcriptome profiling of sacral NC and developing PG was performed, and we identified the enrichment of the type 3 serotonin receptor (5-HT3, encoded by Htr3a and Htr3b). We determined that Htr3a is one of the first serotonin receptor genes that is up-regulated in sacral NC progenitors and is maintained in differentiating PG neurons. In vitro cultures showed that the disruption of 5-HT3 signaling alters the differentiation outcomes of sacral NC cells, while the stimulation of 5-HT3 in explanted fetal pelvic ganglia severely diminished neurite arbor outgrowth. Overall, this study provides a valuable resource for the analysis of signaling pathways in PG development, identifies 5-HT3 as a novel regulator of NC lineage diversification and neuronal maturation in the peripheral nervous system, and indicates that the perturbation of 5-HT3 signaling in gestation has the potential to alter bladder function later in life.


Assuntos
Crista Neural/metabolismo , Receptores 5-HT3 de Serotonina/metabolismo , Transdução de Sinais , Sistema Urinário/inervação , Sistema Urinário/metabolismo , Animais , Sistema Nervoso Autônomo , Diferenciação Celular , Biologia Computacional/métodos , Perfilação da Expressão Gênica , Camundongos , Crista Neural/embriologia , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neuritos/metabolismo , Neurogênese , Crescimento Neuronal , Neurônios/metabolismo , Receptores de Serotonina/metabolismo , Receptores 5-HT3 de Serotonina/genética , Transcriptoma , Sistema Urinário/embriologia
8.
Cells ; 10(7)2021 06 22.
Artigo em Inglês | MEDLINE | ID: mdl-34206213

RESUMO

The purpose of this study was to characterize the miRNA profile of purified retinal ganglion cells (RGC) from healthy and diseased rat retina. Diseased retina includes those after a traumatic optic nerve crush (ONC), and after ocular hypertension/glaucoma. Rats were separated into four groups: healthy/intact, 7 days after laser-induced ocular hypertension, 2 days after traumatic ONC, and 7 days after ONC. RGC were purified from rat retina using microbeads conjugated to CD90.1/Thy1. RNA were sequenced using Next Generation Sequencing. Over 100 miRNA were identified that were significantly different in diseased retina compared to healthy retina. Considerable differences were seen in the miRNA expression of RGC 7 days after ONC, whereas after 2 days, few changes were seen. The miRNA profiles of RGC 7 days after ONC and 7 days after ocular hypertension were similar, but discrete miRNA differences were still seen. Candidate mRNA showing different levels of expression after retinal injury were manipulated in RGC cultures using mimics/AntagomiRs. Of the five candidate miRNA identified and subsequently tested for therapeutic efficacy, miR-194 inhibitor and miR-664-2 inhibitor elicited significant RGC neuroprotection, whereas miR-181a mimic and miR-181d-5p mimic elicited significant RGC neuritogenesis.


Assuntos
Glaucoma/genética , Glaucoma/patologia , MicroRNAs/metabolismo , Compressão Nervosa , Nervo Óptico/patologia , Células Ganglionares da Retina/metabolismo , Animais , Feminino , Regulação da Expressão Gênica , Pressão Intraocular/genética , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Neuritos/metabolismo , Neurogênese/genética , Neuroproteção/genética , Hipertensão Ocular/genética , Hipertensão Ocular/fisiopatologia , Ratos Sprague-Dawley
9.
Cells ; 10(7)2021 06 29.
Artigo em Inglês | MEDLINE | ID: mdl-34209640

RESUMO

The age of incidence of spinal cord injury (SCI) and the average age of people living with SCI is continuously increasing. However, SCI is extensively modeled in young adult animals, hampering translation of research to clinical applications. While there has been significant progress in manipulating axon growth after injury, the impact of aging is still unknown. Mitochondria are essential to successful neurite and axon growth, while aging is associated with a decline in mitochondrial functions. Using isolation and culture of adult cortical neurons, we analyzed mitochondrial changes in 2-, 6-, 12- and 18-month-old mice. We observed reduced neurite growth in older neurons. Older neurons also showed dysfunctional respiration, reduced membrane potential, and altered mitochondrial membrane transport proteins; however, mitochondrial DNA (mtDNA) abundance and cellular ATP were increased. Taken together, these data suggest that dysfunctional mitochondria in older neurons may be associated with the age-dependent reduction in neurite growth. Both normal aging and traumatic injury are associated with mitochondrial dysfunction, posing a challenge for an aging SCI population as the two elements can combine to worsen injury outcomes. The results of this study highlight this as an area of great interest in CNS trauma.


Assuntos
Envelhecimento/patologia , Córtex Cerebral/patologia , Mitocôndrias/metabolismo , Neurônios/metabolismo , Neurônios/patologia , Trifosfato de Adenosina/metabolismo , Animais , Respiração Celular , Células Cultivadas , Variações do Número de Cópias de DNA/genética , DNA Mitocondrial/metabolismo , Transporte de Elétrons , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial , Proteínas de Membrana/metabolismo , Camundongos Endogâmicos C57BL , Membranas Mitocondriais/metabolismo , Neuritos/metabolismo , Fosforilação Oxidativa
10.
Int J Mol Sci ; 22(10)2021 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-34069478

RESUMO

Exposure to radiofrequency electromagnetic fields (RF-EMFs) has increased rapidly in children, but information on the effects of RF-EMF exposure to the central nervous system in children is limited. In this study, pups and dams were exposed to whole-body RF-EMF at 4.0 W/kg specific absorption rate (SAR) for 5 h per day for 4 weeks (from postnatal day (P) 1 to P28). The effects of RF-EMF exposure on neurons were evaluated by using both pups' hippocampus and primary cultured hippocampal neurons. The total number of dendritic spines showed statistically significant decreases in the dentate gyrus (DG) but was not altered in the cornu ammonis (CA1) in hippocampal neurons. In particular, the number of mushroom-type dendritic spines showed statistically significant decreases in the CA1 and DG. The expression of glutamate receptors was decreased in mushroom-type dendritic spines in the CA1 and DG of hippocampal neurons following RF-EMF exposure. The expression of brain-derived neurotrophic factor (BDNF) in the CA1 and DG was significantly lower statistically in RF-EMF-exposed mice. The number of post-synaptic density protein 95 (PSD95) puncta gradually increased over time but was significantly decreased statistically at days in vitro (DIV) 5, 7, and 9 following RF-EMF exposure. Decreased BDNF expression was restricted to the soma and was not observed in neurites of hippocampal neurons following RF-EMF exposure. The length of neurite outgrowth and number of branches showed statistically significant decreases, but no changes in the soma size of hippocampal neurons were observed. Further, the memory index showed statistically significant decreases in RF-EMF-exposed mice, suggesting that decreased synaptic density following RF-EMF exposure at early developmental stages may affect memory function. Collectively, these data suggest that hindered neuronal outgrowth following RF-EMF exposure may decrease overall synaptic density during early neurite development of hippocampal neurons.


Assuntos
Campos Eletromagnéticos/efeitos adversos , Neuritos/efeitos da radiação , Ondas de Rádio/efeitos adversos , Animais , Animais Recém-Nascidos/fisiologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Hipocampo/metabolismo , Hipocampo/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos ICR , Neuritos/metabolismo , Neurogênese , Crescimento Neuronal , Neurônios/metabolismo , Neurônios/efeitos da radiação , Sinapses/metabolismo , Sinapses/efeitos da radiação
11.
Brain Behav ; 11(8): e2266, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34156163

RESUMO

AIM: RhoA/Rho kinase pathway is essential for regulating cytoskeletal structure. Although its effect on normal neurite outgrowth has been demonstrated, the role of this pathway in seizure-induced neurite injury has not been revealed. The research examined the phosphorylation level of RhoA/Rho kinase signaling pathway and to clarify the effect of fasudil on RhoA/Rho kinase signaling pathway and neurite outgrowth in kainic acid (KA)-treated Neuro-2A cells and hippocampal neurons. METHOD: Western blotting analysis was used to investigate the expression of key proteins of RhoA/Rho kinase signaling pathway and the depolymerization of actin. After incubated without serum to induce neurite outgrowth, Neuro-2A cells were fixed, and immunofluorescent assay of rhodamine-phalloidin was applied to detect the cellular morphology and neurite length. The influence of KA on neurons was detected in primary hippocampal neurons. Whole-cell patch clamp was conducted in cultured neurons or hippocampal slices to record action potentials. RESULT: KA at the dose of 100-200 µmol/L induced the increase in phosphorylation of Rho-associated coiled-coil-containing protein kinase and decrease in phosphorylation of Lin11, Isl-1 and Mec-3 kinase and cofilin. The effect of 200 µmol/L KA was peaked at 1-2 hours, and then gradually returned to baseline after 8 hours. Pretreatment with Rho kinase inhibitor fasudil reversed KA-induced activation of RhoA/Rho kinase pathway and increase in phosphorylation of slingshot and 14-3-3, which consequently reduced the ratio of G/F-actin. KA treatment induced inhibition of neurite outgrowth and decrease in spines both in Neuro-2a cells and in cultured hippocampal neurons, and pretreatment with fasudil alleviated KA-induced neurite outgrowth inhibition and spine loss. CONCLUSION: These data indicate that inhibiting RhoA/Rho kinase pathway might be a potential treatment for seizure-induced injury.


Assuntos
Neuritos , Quinases Associadas a rho , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , Ácido Caínico/toxicidade , Neuritos/metabolismo , Transdução de Sinais , Quinases Associadas a rho/metabolismo
12.
PLoS Genet ; 17(6): e1009618, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-34115759

RESUMO

Coordination of neurite extension with surrounding glia development is critical for neuronal function, but the underlying molecular mechanisms remain poorly understood. Through a genome-wide mutagenesis screen in C. elegans, we identified dyf-4 and daf-6 as two mutants sharing similar defects in dendrite extension. DAF-6 encodes a glia-specific patched-related membrane protein that plays vital roles in glial morphogenesis. We cloned dyf-4 and found that DYF-4 encodes a glia-secreted protein. Further investigations revealed that DYF-4 interacts with DAF-6 and functions in a same pathway as DAF-6 to regulate sensory compartment formation. Furthermore, we demonstrated that reported glial suppressors of daf-6 could also restore dendrite elongation and ciliogenesis in both dyf-4 and daf-6 mutants. Collectively, our data reveal that DYF-4 is a regulator for DAF-6 which promotes the proper formation of the glial channel and indirectly affects neurite extension and ciliogenesis.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Caenorhabditis elegans/genética , Genoma Helmíntico , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Neurogênese/genética , Animais , Caenorhabditis elegans/citologia , Caenorhabditis elegans/crescimento & desenvolvimento , Caenorhabditis elegans/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Comunicação Celular , Cílios/genética , Cílios/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Mutagênese , Proteínas do Tecido Nervoso/metabolismo , Neuritos/metabolismo , Neuroglia/citologia , Neuroglia/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
13.
Life Sci ; 284: 119747, 2021 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-34171380

RESUMO

BZD9L1 was previously described as a SIRT1/2 inhibitor with anti-cancer activities in colorectal cancer (CRC), either as a standalone chemotherapy or in combination with 5-fluorouracil. BZD9L1 was reported to induce apoptosis in CRC cells; however, the network of intracellular pathways and crosstalk between molecular players mediated by BZD9L1 is not fully understood. This study aimed to uncover the mechanisms involved in BZD9L1-mediated cytotoxicity based on previous and new findings for the prediction and identification of related pathways and key molecular players. BZD9L1-regulated candidate targets (RCTs) were identified using a range of molecular, cell-based and biochemical techniques on the HCT 116 cell line. BZD9L1 regulated major cancer pathways including Notch, p53, cell cycle, NFκB, Myc/MAX, and MAPK/ERK signalling pathways. BZD9L1 also induced reactive oxygen species (ROS), regulated apoptosis-related proteins, and altered cell polarity and adhesion profiles. In silico analyses revealed that most RCTs were interconnected, and were involved in the modulation of catalytic activity, metabolism and transcription regulation, response to cytokines, and apoptosis signalling pathways. These RCTs were implicated in p53-dependent apoptosis pathway. This study provides the first assessment of possible associations of molecular players underlying the cytotoxic activity of BZD9L1, and establishes the links between RCTs and apoptosis through the p53 pathway.


Assuntos
Benzimidazóis/farmacologia , Neoplasias Colorretais/patologia , Sirtuínas/metabolismo , Apoptose/efeitos dos fármacos , Apoptose/genética , Adesão Celular/efeitos dos fármacos , Adesão Celular/genética , Polaridade Celular/efeitos dos fármacos , Polaridade Celular/genética , Neoplasias Colorretais/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ontologia Genética , Células HCT116 , Humanos , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Mapas de Interação de Proteínas/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos , Esferoides Celulares/efeitos dos fármacos , Esferoides Celulares/patologia
14.
Math Biosci ; 338: 108632, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34087317

RESUMO

The processes that determine the establishment of the complex morphology of neurons during development are still poorly understood. Here, we focus on the question how a difference in the length of neurites affects vesicle transport. We performed live imaging experiments and present a lattice-based model to gain a deeper theoretical understanding of intracellular transport in neurons. After a motivation and appropriate scaling of the model we present numerical simulations showing that initial differences in neurite length result in phenomena of biological relevance, i.e. a positive feedback that enhances transport into the longer neurite and oscillation of vesicles concentrations that can be interpreted as cycles of extension and retraction observed in experiments. Thus, our model is a first step towards a better understanding of the interplay between the transport of vesicles and the spatial organization of cells.


Assuntos
Modelos Biológicos , Neuritos , Vesículas Transportadoras , Transporte Biológico , Simulação por Computador , Humanos , Neuritos/metabolismo , Neurônios/citologia , Neurônios/metabolismo , Vesículas Transportadoras/metabolismo
15.
Sci Rep ; 11(1): 12034, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103613

RESUMO

Although it has been reported that mesenchymal stem cells isolated from adult tissues can be induced to overcome their mesenchymal fate and transdifferentiate into neural cells, the findings and their interpretation have been challenged. The main argument against this process is that the cells rapidly adopt neuron-like morphologies through retraction of the cytoplasm rather than active neurite extension. In this study, we examined the sequence of biological events during neural differentiation of human periodontal ligament-derived stem cells (hPDLSCs), human bone marrow-derived stem cells (hBMSCs) and human dental pulp-derived stem cells (hDPSCs) by time-lapse microscopy. We have demonstrated that hPDLSCs, hBMSCs and hDPSCs can directly differentiate into neuron-like cells without passing through a mitotic stage and that they shrink dramatically and change their morphology to that of neuron-like cells through active neurite extension. Furthermore, we observed micronuclei movement and transient cell nuclei lobulation concurrent to in vitro neurogenesis from hBMSCs and hDPSCs. Our results demonstrate that the differentiation of hPDLSCs, hBMSCs and hDPSCs towards a neural lineage occurs through a dedifferentiation step followed by differentiation to neural phenotypes, and therefore we definitively confirm that the rapid acquisition of the neural phenotype is via a differentiation trait.


Assuntos
Células-Tronco Adultas/metabolismo , Células da Medula Óssea/metabolismo , Desdiferenciação Celular , Diferenciação Celular , Neuritos/metabolismo , Ligamento Periodontal/metabolismo , Feminino , Humanos , Masculino
16.
Sci Rep ; 11(1): 13322, 2021 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-34172765

RESUMO

Recently, we found many immune cells including antigen presenting cells neurally hard wired in the T-cell zone of most lymphoid organs like amongst others, lymph nodes in rats, mice and humans. Single immune cells were reached by single neurites and enclosed with a dense neural meshwork. As it is well known that axons are always accompanied by glial cells, we were able to identify Schwann cells in the hilum, medullary and capsule region, like expected. Unexpected was the result, that we found oligodendrocyte-like cells in these regions, myelinating more than one axon. Likewise important was the finding, that one of the standard glial markers used, a polyclonal GFAP antibody equally bound to desmin and therefore marked nearly all stromal cells in cortical, paracortical and medullary cord regions. More detailed analysis showed that these results also appeared in many other non-lymphoid organs. Therefore, polyclonal GFAP antibodies are only conditionally usable for immunohistochemical analysis in peripheral tissues outside the central nervous system. It remains to be elucidated, if the binding of the GFAP antibody to desmin has its reason in a special desmin variant that can give stromal cells glial character.


Assuntos
Desmina/metabolismo , Proteína Glial Fibrilar Ácida/metabolismo , Tecido Linfoide/metabolismo , Neuroglia/metabolismo , Células Estromais/metabolismo , Animais , Células Apresentadoras de Antígenos/metabolismo , Sistema Nervoso Central/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Neuritos/metabolismo , Oligodendroglia/metabolismo , Células de Schwann/metabolismo
17.
J Exp Med ; 218(8)2021 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-34100905

RESUMO

In Alzheimer's disease (AD) models, AD risk variants in the microglial-expressed TREM2 gene decrease Aß plaque-associated microgliosis and increase neuritic dystrophy as well as plaque-associated seeding and spreading of tau aggregates. Whether this Aß-enhanced tau seeding/spreading is due to loss of microglial function or a toxic gain of function in TREM2-deficient microglia is unclear. Depletion of microglia in mice with established brain amyloid has no effect on amyloid but results in less spine and neuronal loss. Microglial repopulation in aged mice improved cognitive and neuronal deficits. In the context of AD pathology, we asked whether microglial removal and repopulation decreased Aß-driven tau seeding and spreading. We show that both TREM2KO and microglial ablation dramatically enhance tau seeding and spreading around plaques. Interestingly, although repopulated microglia clustered around plaques, they had a reduction in disease-associated microglia (DAM) gene expression and elevated tau seeding/spreading. Together, these data suggest that TREM2-dependent activation of the DAM phenotype is essential in delaying Aß-induced pathological tau propagation.


Assuntos
Peptídeos beta-Amiloides/metabolismo , Microglia/metabolismo , Proteínas tau/metabolismo , Animais , Apolipoproteínas E/metabolismo , Homeostase , Macrófagos/metabolismo , Glicoproteínas de Membrana/deficiência , Glicoproteínas de Membrana/metabolismo , Camundongos Knockout , Neuritos/metabolismo , Neuritos/patologia , Fenótipo , Placa Amiloide/patologia , Receptores Imunológicos/deficiência , Receptores Imunológicos/metabolismo
18.
Sci Rep ; 11(1): 10256, 2021 05 13.
Artigo em Inglês | MEDLINE | ID: mdl-33986433

RESUMO

N-type voltage-gated calcium channels (CaV2.2) are predominantly expressed at presynaptic terminals, and their function is regulated by auxiliary α2δ and ß subunits. All four mammalian α2δ subunits enhance calcium currents through CaV1 and CaV2 channels, and this increase is attributed, in part, to increased CaV expression at the plasma membrane. In the present study we provide evidence that α2δ-1, like α2δ-2, is recycled to the plasma membrane through a Rab11a-dependent endosomal recycling pathway. Using a dominant-negative Rab11a mutant, Rab11a(S25N), we show that α2δ-1 increases plasma membrane CaV2.2 expression by increasing the rate and extent of net forward CaV2.2 trafficking in a Rab11a-dependent manner. Dominant-negative Rab11a also reduces the ability of α2δ-1 to increase CaV2.2 expression on the cell-surface of hippocampal neurites. In contrast, α2δ-3 does not enhance rapid forward CaV2.2 trafficking, regardless of whether Rab11a(S25N) is present. In addition, whole-cell CaV2.2 currents are reduced by co-expression of Rab11a(S25N) in the presence of α2δ-1, but not α2δ-3. Taken together these data suggest that α2δ subtypes participate in distinct trafficking pathways which in turn influence the localisation and function of CaV2.2.


Assuntos
Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Encéfalo/metabolismo , Cálcio/metabolismo , Canais de Cálcio/genética , Canais de Cálcio/fisiologia , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo N/genética , Canais de Cálcio Tipo N/metabolismo , Linhagem Celular , Membrana Celular/metabolismo , Ácidos Cicloexanocarboxílicos/metabolismo , Gabapentina/metabolismo , Hipocampo/metabolismo , Neuritos/metabolismo , Neurônios/metabolismo , Terminações Pré-Sinápticas/metabolismo , Cultura Primária de Células , Transporte Proteico , Ratos , Ácido gama-Aminobutírico/metabolismo , Proteínas rab de Ligação ao GTP/genética
19.
Cells ; 10(3)2021 03 23.
Artigo em Inglês | MEDLINE | ID: mdl-33807093

RESUMO

Tropomyosins (Tpms) have been described as master regulators of actin, with Tpm3 products shown to be involved in early developmental processes, and the Tpm3 isoform Tpm3.1 controlling changes in the size of neuronal growth cones and neurite growth. Here, we used primary mouse hippocampal neurons of C57/Bl6 wild type and Bl6Tpm3flox transgenic mice to carry out morphometric analyses in response to the absence of Tpm3 products, as well as to investigate the effect of C-terminal truncation on the ability of Tpm3.1 to modulate neuronal morphogenesis. We found that the knock-out of Tpm3 leads to decreased neurite length and complexity, and that the deletion of two amino acid residues at the C-terminus of Tpm3.1 leads to more detrimental changes in neurite morphology than the deletion of six amino acid residues. We also found that Tpm3.1 that lacks the 6 C-terminal amino acid residues does not associate with stress fibres, does not segregate to the tips of neurites, and does not impact the amount of the filamentous actin pool at the axonal growth cones, as opposed to Tpm3.1, which lacks the two C-terminal amino acid residues. Our study provides further insight into the role of both Tpm3 products and the C-terminus of Tpm3.1, and it forms the basis for future studies that aim to identify the molecular mechanisms underlying Tpm3.1 targeting to different subcellular compartments.


Assuntos
Actinas/metabolismo , Deleção de Genes , Hipocampo/citologia , Neurônios/metabolismo , Tropomiosina/química , Tropomiosina/metabolismo , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos , Animais , Axônios/metabolismo , Células Cultivadas , Cones de Crescimento/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células NIH 3T3 , Neuritos/metabolismo , Isoformas de Proteínas/metabolismo , Relação Estrutura-Atividade
20.
J Biochem ; 170(3): 327-336, 2021 Oct 12.
Artigo em Inglês | MEDLINE | ID: mdl-33822960

RESUMO

Lysophosphatidylethanolamines (LPEs) are bioactive lysophospholipids that have been suggested to play important roles in several biological processes. We performed a quantitative analysis of LPE species and showed their composition in mouse brain. We examined the roles of oleoyl-LPE (18:1 LPE), which is one of the abundant LPE species in brain. In cultured cortical neurons, application of 18:1 LPE-stimulated neurite outgrowth. The effect of 18:1 LPE on neurite outgrowth was inhibited by Gq/11 inhibitor YM-254890, phospholipase C (PLC) inhibitor U73122, protein kinase C (PKC) inhibitor Go6983 or mitogen-activated protein kinase (MAPK) inhibitor U0126. Additionally, 18:1 LPE increased the phosphorylation of MAPK/extracellular signal-regulated kinase 1/2. These results suggest that the action of 18:1 LPE on neurite outgrowth is mediated by the Gq/11/PLC/PKC/MAPK pathway. Moreover, we found that application of 18:1 LPE protects neurons from glutamate-induced excitotoxicity. This effect of 18:1 LPE was suppressed by PKC inhibitor Go6983. These results suggest that 18:1 LPE protects neurons from glutamate toxicity via PKC inhibitor Go6983-sensitive PKC subtype. Collectively, our results demonstrated that 18:1 LPE stimulates neurite outgrowth and protects against glutamate toxicity in cultured cortical neurons. Our findings provide insights into the physiological or pathological roles of 18:1 LPE in the brain.


Assuntos
Encéfalo/efeitos dos fármacos , Ácido Glutâmico/toxicidade , Lisofosfolipídeos/farmacologia , Crescimento Neuronal/efeitos dos fármacos , Neurônios/metabolismo , Animais , Encéfalo/metabolismo , Células Cultivadas , Cromatografia Líquida/métodos , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuritos/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espectrometria de Massas por Ionização por Electrospray/métodos , Fosfolipases Tipo C/metabolismo
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