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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.
Am J Hum Genet ; 108(11): 2171-2185, 2021 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-34699745

RESUMO

Recent studies indicate that neurodegenerative processes that appear during childhood and adolescence in individuals with Wolfram syndrome (WS) occur in addition to early brain development alteration, which is clinically silent. Underlying pathological mechanisms are still unknown. We have used induced pluripotent stem cell-derived neural cells from individuals affected by WS in order to reveal their phenotypic and molecular correlates. We have observed that a subpopulation of Wolfram neurons displayed aberrant neurite outgrowth associated with altered expression of axon guidance genes. Selective inhibition of the ATF6α arm of the unfolded protein response prevented the altered phenotype, although acute endoplasmic reticulum stress response-which is activated in late Wolfram degenerative processes-was not detected. Among the drugs currently tried in individuals with WS, valproic acid was the one that prevented the pathological phenotypes. These results suggest that early defects in axon guidance may contribute to the loss of neurons in individuals with WS.


Assuntos
Idade de Início , Células-Tronco Pluripotentes Induzidas/citologia , Neuritos , Neurônios/citologia , Síndrome de Wolfram/patologia , Sistemas CRISPR-Cas , Estudos de Casos e Controles , Estresse do Retículo Endoplasmático , Regulação da Expressão Gênica , Humanos , Neuritos/efeitos dos fármacos , Ácido Valproico/farmacologia , Síndrome de Wolfram/genética
3.
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
4.
J Neurosci Methods ; 363: 109341, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34474047

RESUMO

BACKGROUND: Neuronal outgrowth assays using organotypic explant cultures are commonly utilized to study neuroregenerative and -protective effects of drugs such as neurotrophins. While this approach offers higher organized tissue compared to single cell cultures and less experimental effort than in-vivo studies, quantitative evaluation of the neuronal network is often time consuming. Thus, we developed ExplantAnlayzer, a time-saving high-throughput evaluation method, yielding numerous metrics to objectively describe neuronal outgrowth. NEW METHOD: Spiral ganglion explants were cultured in 24-well plates, mechanically fixed in a collagen matrix and immunolabeled against beta-III-tubulin. The explants were imaged using a fluorescent tile-scan microscope and resulting images were stitched. The evaluation was developed as an open-source MATLAB routine and involves several image processing steps, including adaptive thresholding. The neurite network was eventually converted to a graph to track neurites from their terminals back to the explant body. COMPARISON WITH EXISTING METHOD(S): We compared ExplantAnlayzer quantitatively and qualitatively to common existing methods, such as Sholl analyses and manual fiber tracing, using representative explant images. ExplantAnlayzer is able to achieve similar and as detailed results as manual tracing while decreasing manual interaction and required time dramatically. RESULTS: After an initial setup phase, the explant images could be batch-processed altogether. Bright bundles as well as faint fibers were reliably detected. Several metrics describing the outgrowth morphology, including total outgrowth, neurite numbers and length estimations, as well as their growth directions, were computed. CONCLUSIONS: ExplantAnalyzer is a time-saving and objective method for an in-depth evaluation of organotypic explant outgrowth.


Assuntos
Crescimento Neuronal , Neurônios , Células Cultivadas , Fatores de Crescimento Neural , Neuritos
5.
J Neurosci Methods ; 363: 109349, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34480956

RESUMO

BACKGROUND: During development or regeneration, neurons extend processes (i.e., neurites) via mechanisms that can be readily analyzed in culture. However, defining the impact of a drug or genetic manipulation on such mechanisms can be challenging due to the complex arborization and heterogeneous patterns of neurite growth in vitro. New Method: NeuriteNet is a Convolutional Neural Network (CNN) sorting model that uses a novel adaptation of the XRAI saliency map overlay, which is a region-based attribution method. NeuriteNet compares neuronal populations based on differences in neurite growth patterns, sorts them into respective groups, and overlays a saliency map indicating which areas differentiated the image for the sorting procedure. RESULTS: In this study, we demonstrate that NeuriteNet effectively sorts images corresponding to dissociated neurons into control and treatment groups according to known morphological differences. Furthermore, the saliency map overlay highlights the distinguishing features of the neuron when sorting the images into treatment groups. NeuriteNet also identifies novel morphological differences in neurons cultured from control and genetically modified mouse strains. Comparison with Existing Methods: Unlike other neurite analysis platforms, NeuriteNet does not require manual manipulations, such as segmentation of neurites prior to analysis, and is more accurate than experienced researchers for categorizing neurons according to their pattern of neurite growth. CONCLUSIONS: NeuriteNet can be used to effectively screen for morphological differences in a heterogeneous group of neurons and to provide feedback on the key features distinguishing those groups via the saliency map overlay.


Assuntos
Redes Neurais de Computação , Neuritos , Animais , Camundongos , Neurogênese , Neurônios
6.
Proc Natl Acad Sci U S A ; 118(39)2021 09 28.
Artigo em Inglês | MEDLINE | ID: mdl-34531300

RESUMO

The Down syndrome cell adhesion molecule (DSCAM) belongs to the immunoglobulin superfamily (IgSF) and plays important roles in neural development. It has a large ectodomain, including 10 Ig-like domains and 6 fibronectin III (FnIII) domains. Previous data have shown that DSCAM can mediate cell adhesion by forming homophilic dimers between cells and contributes to self-avoidance of neurites or neuronal tiling, which is important for neural network formation. However, the organization and assembly of DSCAM at cell adhesion interfaces has not been fully understood. Here we combine electron microscopy and other biophysical methods to characterize the structure of the DSCAM-mediated cell adhesion and generate three-dimensional views of the adhesion interfaces of DSCAM by electron tomography. The results show that mouse DSCAM forms a regular pattern at the adhesion interfaces. The Ig-like domains contribute to both trans homophilic interactions and cis assembly of the pattern, and the FnIII domains are crucial for the cis pattern formation as well as the interaction with the cell membrane. By contrast, no obvious assembly pattern is observed at the adhesion interfaces mediated by mouse DSCAML1 or Drosophila DSCAMs, suggesting the different structural roles and mechanisms of DSCAMs in mediating cell adhesion and neural network formation.


Assuntos
Moléculas de Adesão Celular/química , Adesão Celular , Síndrome de Down/patologia , Proteínas de Drosophila/química , Neurogênese , Animais , Moléculas de Adesão Celular/genética , Moléculas de Adesão Celular/metabolismo , Membrana Celular/metabolismo , Síndrome de Down/genética , Síndrome de Down/metabolismo , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Camundongos , Neuritos
7.
eNeuro ; 8(5)2021.
Artigo em Inglês | MEDLINE | ID: mdl-34475223

RESUMO

Highlighted Research Paper: Tracking Mitochondrial Density and Positioning along a Growing Neuronal Process in Individual C. elegans Neuron Using a Long-Term Growth and Imaging Microfluidic Device by Sudip Mondal, Jyoti Dubey, Anjali Awasthi, Guruprasad Reddy Sure, Amruta Vasudevan, and Sandhya P. Koushika.


Assuntos
Caenorhabditis elegans , Microfluídica , Animais , Dispositivos Lab-On-A-Chip , Neuritos , Neurônios
8.
Artigo em Inglês | MEDLINE | ID: mdl-34501579

RESUMO

Sciatic nerve injury (SNI) leads to sensory and motor dysfunctions. Nobiletin is a major component of polymethoxylated flavonoid extracted from citrus fruits. The role of nobiletin on sciatic nerve regeneration is still unclear. Thus, the purpose of this study was to investigate whether nobiletin increases DRG neurite elongation and regeneration-related protein expression after SNI. Cytotoxicity of nobiletin was measured in a concentration-dependent manner using the MTT assay. For an in vitro primary cell culture, the sciatic nerve on the middle thigh was crushed by holding twice with forceps. Dorsal root ganglion (DRG) and Schwann cells were cultured 3 days after SNI and harvested 36 h later and 3 days later, respectively. In order to evaluate specific regeneration-related markers and axon growth in the injured sciatic nerve, we applied immunofluorescence staining and Western blot techniques. Nobiletin increased cell viability in human neuroblastoma cells and inhibited cytotoxicity induced by exposure to H2O2. Mean neurite length of DRG neurons was significantly increased in the nobiletin group at a dose of 50 and 100 µM compared to those at other concentrations. GAP-43, a specific marker for axonal regeneration, was enhanced in injury preconditioned Schwann cells with nobiletin treatment and nobiletin significantly upregulated it in injured sciatic nerve at only 3 days post crush (dpc). In addition, nobiletin dramatically facilitated axonal regrowth via activation of the BDNF-ERK1/2 and AKT pathways. These results should provide evidence to distinguish more accurately the biochemical mechanisms regarding nobiletin-activated sciatic nerve regeneration.


Assuntos
Gânglios Espinais , Neuritos , Axônios , Flavonas , Humanos , Peróxido de Hidrogênio , Nervo Isquiático
9.
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
10.
Cells ; 10(9)2021 09 18.
Artigo em Inglês | MEDLINE | ID: mdl-34572121

RESUMO

P-Rex1 is a guanine-nucleotide exchange factor (GEF) that activates Rac-type small G proteins in response to the stimulation of a range of receptors, particularly G protein-coupled receptors (GPCRs), to control cytoskeletal dynamics and other Rac-dependent cell responses. P-Rex1 is mainly expressed in leukocytes and neurons. Whereas its roles in leukocytes have been studied extensively, relatively little is known about its functions in neurons. Here, we used CRISPR/Cas9-mediated P-Rex1 deficiency in neuronal PC12 cells that stably overexpress the GPCR S1PR1, a receptor for sphingosine 1-phosphate (S1P), to investigate the role of P-Rex1 in neuronal GPCR signalling and cell responses. We show that P-Rex1 is required for the S1P-stimulated activation of Rac1 and Akt, basal Rac3 activity, and constitutive cAMP production in PC12-S1PR1 cells. The constitutive cAMP production was not due to increased expression levels of major neuronal adenylyl cyclases, suggesting that P-Rex1 may regulate adenylyl cyclase activity. P-Rex1 was required for maintenance of neurite protrusions and spreading in S1P-stimulated PC12-S1PR1 cells, as well as for cell-cycle progression and proliferation. In summary, we identified novel functional roles of P-Rex1 in neuronal Rac, Akt and cAMP signalling, as well as in neuronal cell-cycle progression and proliferation.


Assuntos
Ciclo Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neuritos/fisiologia , Neurônios/fisiologia , Receptores de Esfingosina-1-Fosfato/metabolismo , Animais , Movimento Celular , Proliferação de Células , Fatores de Troca do Nucleotídeo Guanina/antagonistas & inibidores , Fatores de Troca do Nucleotídeo Guanina/genética , Lisofosfolipídeos/metabolismo , Neurônios/citologia , Células PC12 , Ratos , Transdução de Sinais , Esfingosina/análogos & derivados , Esfingosina/metabolismo , Receptores de Esfingosina-1-Fosfato/antagonistas & inibidores , Receptores de Esfingosina-1-Fosfato/genética , Proteínas rac de Ligação ao GTP/genética , Proteínas rac de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo
11.
J Neuroimaging ; 31(5): 879-892, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34473386

RESUMO

BACKGROUND AND PURPOSE: Identification of changesin brain microstructure following mild traumatic brain injury (mTBI) could be instrumental in understanding the underlying pathophysiology. The purpose of this study was to apply neurite orientation dispersion and density imaging (NODDI) to a rodent model of mTBI to determine whether microstructural changes could be detected immediately following injury. METHODS: Fifteen adult male Wistar rats were scanned on a Bruker 9.4 Tesla small animal MRI using a multi-shell acquisition (30 b = 1000 s/mm2 and 60 b = 2000 s/mm2 ). Nine animals experienced a single closed head controlled cortical impact followed by NODDI from 1 to 4 h post injury. Region of interest analysis focused on the corpus callosum and hippocampus. A mixed analysis of variance (ANOVA) was used to determine statistically significant interactions in neurite density index (NDI), orientation dispersion index (ODI), fractional anisotropy (FA), mean diffusivity (MD), axial diffusivity (AD), and radial diffusivity. Follow up repeated-measures ANOVAs were used to determine individual changes over time. RESULTS: NDI showed a significant increase in the hippocampus and corpus callosum following injury, while ODI showed increases in the corpus callosum. No significant changes were observed in the sham control animals. No changes were found in FA, MD, AD, or RD. Histological analysis revealed increased glial fibrillary acidic protein staining relative to controls in both the hippocampus and corpus callosum, with evidence of activated astrocytes in these regions. CONCLUSIONS: Changes in NODDI metrics were detected as early as 1 h following mTBI. No changes were detected with conventional diffusion tensor imaging (DTI) metrics, suggesting that NODDI provides greater sensitivity to microstructural changes than conventional DTI.


Assuntos
Concussão Encefálica , Imagem de Tensor de Difusão , Animais , Encéfalo , Concussão Encefálica/diagnóstico por imagem , Imagem de Difusão por Ressonância Magnética , Masculino , Neuritos , Ratos , Ratos Wistar , Roedores
12.
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
13.
Chaos ; 31(7): 073140, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-34340345

RESUMO

Defining the morphological disorders causing neurodegenerative diseases is an unresolved problem. In this study, we propose a statistical-physical approach to quantify neurite morphology and evaluate the pathological states induced by Alzheimer's disease (AD). We analyzed the two-dimensional morphologies of neurites of in vitro-cultured human induced-pluripotent stem cell-derived neurons, reprogrammed from both a healthy person and a patient with AD, using discrete chordal Loewner evolution. For the numerically calculated Loewner driving forces, detrended fluctuation analysis was performed, and the morphological characteristics of the neurites were quantified using short-range and long-range scaling exponents. The day in vitro (DIV)-dependent behaviors of the scaling exponents and the associated neurite-type categorizations suggested that differences between healthy and AD neurites can be observed from the early stage (DIV3) of their development. Notably, AD neurites have less long-range autocorrelations than healthy neurites, particularly in the earlier stages (DIV3-10). Immunofluorescence-staining results suggested that these differences precede significant expressions of ß-amyloid and phosphorylated tau, which are known as biological factors causing AD. We expect that these results will lead to a theoretical interpretation of the neurogenerative disease, providing the physical properties of individual neurites with different morphologies.


Assuntos
Doença de Alzheimer , Células-Tronco Pluripotentes Induzidas , Peptídeos beta-Amiloides , Humanos , Neuritos , Neurônios
14.
Int J Mol Sci ; 22(16)2021 Aug 10.
Artigo em Inglês | MEDLINE | ID: mdl-34445294

RESUMO

Coupling of cells to biomaterials is a prerequisite for most biomedical applications; e.g., neuroelectrodes can only stimulate brain tissue in vivo if the electric signal is transferred to neurons attached to the electrodes' surface. Besides, cell survival in vitro also depends on the interaction of cells with the underlying substrate materials; in vitro assays such as multielectrode arrays determine cellular behavior by electrical coupling to the adherent cells. In our study, we investigated the interaction of neurons and glial cells with different electrode materials such as TiN and nanocolumnar TiN surfaces in contrast to gold and ITO substrates. Employing single-cell force spectroscopy, we quantified short-term interaction forces between neuron-like cells (SH-SY5Y cells) and glial cells (U-87 MG cells) for the different materials and contact times. Additionally, results were compared to the spreading dynamics of cells for different culture times as a function of the underlying substrate. The adhesion behavior of glial cells was almost independent of the biomaterial and the maximum growth areas were already seen after one day; however, adhesion dynamics of neurons relied on culture material and time. Neurons spread much better on TiN and nanocolumnar TiN and also formed more neurites after three days in culture. Our designed nanocolumnar TiN offers the possibility for building miniaturized microelectrode arrays for impedance spectroscopy without losing detection sensitivity due to a lowered self-impedance of the electrode. Hence, our results show that this biomaterial promotes adhesion and spreading of neurons and glial cells, which are important for many biomedical applications in vitro and in vivo.


Assuntos
Interfaces Cérebro-Computador , Neuroglia/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Titânio/farmacologia , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Materiais Revestidos Biocompatíveis/química , Materiais Revestidos Biocompatíveis/farmacologia , Matriz Extracelular/química , Ouro/química , Ouro/farmacologia , Humanos , Teste de Materiais , Nanoestruturas/química , Neuritos/efeitos dos fármacos , Neuritos/fisiologia , Neuroglia/fisiologia , Neurônios/fisiologia , Compostos de Estanho/química , Compostos de Estanho/farmacologia , Titânio/química
15.
Transl Vis Sci Technol ; 10(10): 1, 2021 08 12.
Artigo em Inglês | MEDLINE | ID: mdl-34383881

RESUMO

Purpose: Human retinal ganglion cells (hRGC) derived from human pluripotent stem cells are promising candidates to model, protect, and replace degenerating RGCs. Here, we examined intrinsic morphologic and physiologic development of hRGCs. Methods: We used CRISPR-Cas9 to selectively express tdTomato under the RGC-specific promoter, BRN3B. Human pluripotent stem cells were chemically differentiated into hRGCs and cultured up to 7 weeks. We measured soma area, neurite complexity, synaptic protein, axon-related messenger RNA and protein, and voltage-dependent responses. Results: Soma area, neurite complexity, and postsynaptic density protein 95 increased over time. Soma area and neurite complexity increased proportionally week to week, and this relationship was dynamic, strengthening between 2 and 3 weeks and diminishing by 4 weeks. Postsynaptic density 95 localization was dependent on culture duration. After 1 to 2 weeks, postsynaptic density 95 localized within somas but redistributed along neurites after 3 to 4 weeks. Axon initial segment scaffolding protein, Ankyrin G, expression also increased over time, and by 7 weeks, Ankyrin G often localized within putative axons. Voltage-gated inward currents progressively developed, but outward currents matured by 4 weeks. Current-induced spike generation increased over time but limited by depolarization block. Conclusions: Human RGCs develop up to 7 weeks after culture. Thus, the state of hRGC maturation should be accounted for in designing models and treatments for optic neuropathies. Translational Relevance: We characterized hRGC morphologic and physiologic development towards identifying key time points when hRGCs express mechanisms that may be harnessed to enhance the efficacy of neuroprotective and cell replacement therapies.


Assuntos
Células-Tronco Pluripotentes , Células Ganglionares da Retina , Axônios , Diferenciação Celular , Humanos , Neuritos
16.
Exp Gerontol ; 154: 111520, 2021 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-34418483

RESUMO

Neurite orientation dispersion and density imaging (NODDI) is a technique providing more detailed information on the microstructural bases of white matter. Given the previously reported white matter contributions to chronic pain, the present study aims to investigate pain-specific differences in NODDI measures across white matter tracts in a sample of community-dwelling older adults with (n = 29) and without (n = 18) chronic musculoskeletal pain. We further aimed to investigate associations between NODDI measures and clinical and experimental pain measures. As part of the Nepal study, a subset of older adults (>60 years old), underwent multiple laboratory sessions providing self-reported and experimental pain measures and a diffusion weighted neuroimaging sequence. Older adults with chronic musculoskeletal pain had a lower neurite density with less geometric complexity across a number of white matter tracts compared to older pain-free controls (corrected p's < 0.05). Lower neurite density was associated with greater self-reported pain intensity and anatomical pain sites, as well as greater experimental pain sensitivity (p's < 0.05). There were also significant pain-by-sex differences in neurite density and geometric complexity across multiple white matter tracts mainly around the hippocampus (corrected p's < 0.05). Finally, there were no pain differences with respect to extra-cellular water diffusion (corrected p's > 0.05). Our study demonstrates less geometric complexity in neurite density and architecture in chronic musculoskeletal pain, partly in a sex-dependent manner. An increased understanding of neurobiological mechanisms such as those measured by NODDI may contribute to the potential targeting of interventions in our older population suffering from chronic pain.


Assuntos
Neuritos , Substância Branca , Idoso , Encéfalo , Imagem de Difusão por Ressonância Magnética , Imagem de Tensor de Difusão , Feminino , Humanos , Vida Independente , Masculino , Dor , Substância Branca/diagnóstico por imagem
17.
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
18.
Neuroinformatics ; 19(4): 703-717, 2021 10.
Artigo em Inglês | MEDLINE | ID: mdl-34342808

RESUMO

Dendrites shape inputs and integration of depolarization that controls neuronal activity in the nervous system. Neuron pathologies can damage dendrite architecture and cause abnormalities in morphologies after injury. Dendrite regeneration can be quantified by various parameters, including total dendrite length and number of dendrite branches using manual or automated image analysis approaches. However, manual quantification is tedious and time consuming and automated approaches are often trained using wildtype neurons, making them poorly suited for analysis of genetically manipulated or injured dendrite arbors. In this study, we tested how well automated image analysis software performed on class IV Drosophila neurons, which have several hundred individual dendrite branches. We applied each software to automatically quantify features of uninjured neurons and neurons that regenerated new dendrites after injury. Regenerated arbors exhibit defects across multiple features of dendrite morphology, which makes them challenging for automated pipelines to analyze. We compared the performances of three automated pipelines against manual quantification using Simple Neurite Tracer in ImageJ: one that is commercially available (Imaris) and two developed by independent research groups (DeTerm and Tireless Tracing Genie). Out of the three software tested, we determined that Imaris is the most efficient at reconstructing dendrite architecture, but does not accurately measure total dendrite length even after intensive manual editing. Imaris outperforms both DeTerm and Tireless Tracing Genie for counting dendrite branches, and is better able to recreate previous conclusions from this same dataset. This thorough comparison of strengths and weaknesses of each software demonstrates their utility for analyzing regenerated neuron phenotypes in future studies.


Assuntos
Dendritos , Drosophila , Animais , Neuritos , Neurônios , Software
19.
Int J Med Mushrooms ; 23(6): 1-11, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34369729

RESUMO

Neuritin is important in neuritogenesis, neurite arborization, and neurite extension. Lignosus rhinocerotis sclerotia extracts and nerve growth factor (NGF) have been well documented to possess positive neurite stimulatory effects. However, the correlation of neuritin expression with neurite outgrowth of L. rhinocerotis and NGF cotreatment of PC12 cells remains unknown. Thus, the present study investigated neuritin expression in PC12 cells treated with 5 ng/mL of NGF and L. rhinocerotis extracts (20-1280 µg/mL) concurrently for 48 h. The neurite outgrowth score was quantitated, and total protein was harvested for enzyme-linked immunosorbent assay. There was a significant difference (P = 0.051) in neuritin protein abundance in 640 µg/mL of L. rhinocerotis aqueous cotreatment with 5 ng/mL of NGF-treated cells (5 ± 0.39 ng/mL) and 50 ng/mL of NGF-treated PC12 cells (5 ± 0.48 ng/mL) compared to untreated cells (1.9 ± 0.65 ng/ mL), with an average neurite length of 98 ± 3.66, 106 ± 3.00, and 73 ± 4.79 µm, respectively. Expression of microtubule element ß3 tubulin was increased in PC12 cells treated with 50 ng/mL of NGF (3.5 ± 0.21-fold) and also cells cotreated with 640 µg/mL of extract and 5 ng/mL of NGF (4.9 ± 0.29-fold) compared to untreated cells. Upregulation of ß3 tubulin expression in this study confirmed the elongation of PC12 cell processes. Correlation analysis showed that neuritin protein abundance is positively proportional to the average neurite length in PC12 cells cotreated with L. rhinocerotis extract and 5 ng/mL of NGF. This study highlights that neuritin modulation is involved in neurite outgrowth induced by L. rhinocerotis treatment. To our knowledge, this is the first report to show that tiger milk mushroom extracts induce neuritin expression.


Assuntos
Agaricales , Animais , Fator de Crescimento Neural/farmacologia , Neuritos , Crescimento Neuronal , Células PC12 , Polyporaceae , Ratos
20.
Small ; 17(38): e2102062, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34411420

RESUMO

Spiral ganglion neuron (SGN) degeneration can lead to severe hearing loss, and the directional regeneration of SGNs has shown great potential for improving the efficacy of auditory therapy. Here, a novel 3D conductive microstructure with surface topologies is presented by integrating superaligned carbon-nanotube sheets (SA-CNTs) onto Morpho Menelaus butterfly wings for SGN culture. The parallel groove-like topological structures of M. Menelaus wings induce the cultured cells to grow along the direction of its ridges. The excellent conductivity of SA-CNTs significantly improves the efficiency of cellular information conduction. When integrating the SA-CNTs with M. Menelaus wings, the SA-CNTs are aligned in parallel with the M. Menelaus ridges, which further strengthens the consistency of the surface topography in the composite substrate. The SA-CNTs integrated onto butterfly wings provide powerful physical signals and regulate the behavior of SGNs, including cell survival, adhesion, neurite outgrowth, and synapse formation. These features indicate the possibility of directed regeneration after auditory nerve injury.


Assuntos
Borboletas , Gânglio Espiral da Cóclea , Animais , Condutividade Elétrica , Neuritos , Neurônios , Asas de Animais
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