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1.
J Cell Sci ; 135(5)2022 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-33912962

RESUMO

Membrane contact sites (MCSs) between the endoplasmic reticulum (ER) and late endosomes/lysosomes (LE/lys) are emerging as critical hubs for diverse cellular events, and changes in their extents are linked to severe neurological diseases. While recent studies show that the synaptotagmin-like mitochondrial-lipid-binding (SMP) domain-containing protein PDZD8 may mediate the formation of ER-LE/lys MCSs, the cellular functions of PDZD8 remain largely elusive. Here, we attempt to investigate the lipid transfer activities of PDZD8 and the extent to which its cellular functions depend on its lipid transfer activities. In accordance with recent studies, we demonstrate that PDZD8 is a protrudin (ZFYVE27)-interacting protein and that PDZD8 acts as a tether at ER-LE/lys MCSs. Furthermore, we discover that the SMP domain of PDZD8 binds glycerophospholipids and ceramides both in vivo and in vitro, and that the SMP domain can transport lipids between membranes in vitro. Functionally, PDZD8 is required for LE/lys positioning and neurite outgrowth, which is dependent on the lipid transfer activity of the SMP domain.


Assuntos
Retículo Endoplasmático , Endossomos , Lipídeos , Lisossomos , Crescimento Neuronal
2.
Int J Mol Sci ; 22(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34360985

RESUMO

Neurodevelopmental disorders can derive from a complex combination of genetic variation and environmental pressures on key developmental processes. Despite this complex aetiology, and the equally complex array of syndromes and conditions diagnosed under the heading of neurodevelopmental disorder, there are parallels in the neuropathology of these conditions that suggest overlapping mechanisms of cellular injury and dysfunction. Neuronal arborisation is a process of dendrite and axon extension that is essential for the connectivity between neurons that underlies normal brain function. Disrupted arborisation and synapse formation are commonly reported in neurodevelopmental disorders. Here, we summarise the evidence for disrupted neuronal arborisation in these conditions, focusing primarily on the cortex and hippocampus. In addition, we explore the developmentally specific mechanisms by which neuronal arborisation is regulated. Finally, we discuss key regulators of neuronal arborisation that could link to neurodevelopmental disease and the potential for pharmacological modification of arborisation and the formation of synaptic connections that may provide therapeutic benefit in the future.


Assuntos
Encéfalo/crescimento & desenvolvimento , Transtornos do Neurodesenvolvimento/patologia , Crescimento Neuronal , Animais , Encéfalo/fisiopatologia , Dendritos/metabolismo , Dendritos/patologia , Humanos , Transtornos do Neurodesenvolvimento/tratamento farmacológico , Transtornos do Neurodesenvolvimento/fisiopatologia , Fármacos Neuroprotetores/uso terapêutico
3.
Int J Mol Sci ; 22(16)2021 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-34445330

RESUMO

We investigated injury-induced heat shock protein 27 (HSP27) expression and its association to axonal outgrowth after injury and different nerve repair models in healthy Wistar and diabetic Goto-Kakizaki rats. By immunohistochemistry, expression of HSP27 in sciatic nerves and DRG and axonal outgrowth (neurofilaments) in sciatic nerves were analyzed after no, immediate, and delayed (7-day delay) nerve repairs (7- or 14-day follow-up). An increased HSP27 expression in nerves and in DRG at the uninjured side was associated with diabetes. HSP27 expression in nerves and in DRG increased substantially after the nerve injuries, being higher at the site where axons and Schwann cells interacted. Regression analysis indicated a positive influence of immediate nerve repair compared to an unrepaired injury, but a shortly delayed nerve repair had no impact on axonal outgrowth. Diabetes was associated with a decreased axonal outgrowth. The increased expression of HSP27 in sciatic nerve and DRG did not influence axonal outgrowth. Injured sciatic nerves should appropriately be repaired in healthy and diabetic rats, but a short delay does not influence axonal outgrowth. HSP27 expression in sciatic nerve or DRG, despite an increase after nerve injury with or without a repair, is not associated with any alteration in axonal outgrowth.


Assuntos
Proteínas de Choque Térmico HSP27/metabolismo , Regeneração Nervosa/fisiologia , Crescimento Neuronal/fisiologia , Traumatismos dos Nervos Periféricos , Animais , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/fisiopatologia , Feminino , Traumatismos dos Nervos Periféricos/metabolismo , Traumatismos dos Nervos Periféricos/fisiopatologia , Ratos , Ratos Wistar , Células de Schwann/metabolismo , Células de Schwann/fisiologia , Nervo Isquiático/metabolismo , Nervo Isquiático/fisiopatologia , Neuropatia Ciática/metabolismo , Neuropatia Ciática/fisiopatologia , Regulação para Cima
4.
Acta Biomater ; 131: 302-313, 2021 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-34271170

RESUMO

Magnetic fiber composites combining superparamagnetic iron oxide nanoparticles (SPIONs) and electrospun fibers have shown promise in tissue engineering fields. Controlled grafting of SPIONs to the fibers post-electrospinning generates biocompatible magnetic composites without altering desired fiber morphology. Here, for the first time, we assess the potential of SPION-grafted scaffolds combined with magnetic fields to promote neurite outgrowth by providing contact guidance from the aligned fibers and mechanical stimulation from the SPIONs in the magnetic field. Neurite outgrowth from primary rat dorsal root ganglia (DRG) was assessed from explants cultured on aligned control and SPION-grafted electrospun fibers as well as on non-grafted fibers with SPIONs dispersed in the culture media. To determine the optimal magnetic field stimulation to promote neurite outgrowth, we generated a static, alternating, and linearly moving magnet and simulated the magnetic flux density at different areas of the scaffold over time. The alternating magnetic field increased neurite length by 40% on control fibers compared to a static magnetic field. Additionally, stimulation with an alternating magnetic field resulted in a 30% increase in neurite length and 62% increase in neurite area on SPION-grafted fibers compared to DRG cultured on PLLA fibers with untethered SPIONs added to the culture media. These findings demonstrate that SPION-grafted fiber composites in combination with magnetic fields are more beneficial for stimulating neurite outgrowth on electrospun fibers than dispersed SPIONs. STATEMENT OF SIGNIFICANCE: Aligned electrospun fibers improve axonal regeneration by acting as a passive guidance cue but do not actively interact with cells, while magnetic nanoparticles can be remotely manipulated to interact with neurons and elicit neurite outgrowth. Here, for the first time, we examine the combination of magnetic fields, magnetic nanoparticles, and aligned electrospun fibers to enhance neurite outgrowth. We show an alternating magnetic field alone increases neurite outgrowth on aligned electrospun fibers. However, combining the alternating field with magnetic nanoparticle-grafted fibers does not affect neurite outgrowth compared to control fibers but improves outgrowth compared to freely dispersed magnetic nanoparticles. This study provides the groundwork for utilizing magnetic electrospun fibers and magnetic fields as a method for promoting axonal growth.


Assuntos
Gânglios Espinais , Tecidos Suporte , Animais , Campos Magnéticos , Nanopartículas Magnéticas de Óxido de Ferro , Neuritos , Crescimento Neuronal , Ratos
5.
J Biochem ; 170(1): 131-138, 2021 Sep 22.
Artigo em Inglês | MEDLINE | ID: mdl-34314486

RESUMO

Understanding the molecular mechanism of neuronal differentiation is important to overcome the incurable diseases caused by nervous system damage. Neurite outgrowth is prerequisite for neuronal differentiation and regeneration, and cAMP response element-binding protein (CREB) is one of the major transcriptional factors positively regulating this process. Neuronal differentiation stimuli activate mammalian target of rapamycin (mTOR) complex 2 (mTORC2)/Akt signalling to phosphorylate CREB; however, the precise molecular mechanism of this event has not been fully understood. In this manuscript, we show that neuronal differentiation stimuli increased a protein level of protein phosphatase 6 (PP6), a member of type 2A Ser/Thr protein phosphatases. PP6 knockdown suppressed mTORC2/Akt/CREB signalling and results in failure of neurite outgrowth. SIN1 is a unique component of mTORC2 that enhances mTORC2 activity towards Akt when it is in dephosphorylated form. We found PP6 knockdown increased SIN1 phosphorylation. These data suggest that PP6 may positively regulate neurite outgrowth by dephosphorylating SIN1 to activate mTORC2/Akt/CREB signalling.


Assuntos
Alvo Mecanístico do Complexo 2 de Rapamicina/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Animais , Células Cultivadas , Humanos , Camundongos , Crescimento Neuronal
6.
Phytother Res ; 35(9): 5318-5329, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34328653

RESUMO

Neurite outgrowth-induced construction of neural circuits and networks is responsible for memory generalization, consolidation, and retrieval. In this study, we found that the traditional Chinese medicine Pseudostellaria heterophylla promoted neurite regrowth and enhanced cognitive function in normal mice. Further, we orally administered Pseudostellaria heterophylla water extracts (PHE) to ICR mice, and detected heterophyllin B (HET-B), an important cyclopeptide, in the plasma and cerebral cortex. We demonstrated that neurites were significantly elongated after coculturing with HET-B for 4 days. Next, the intraperitoneal injection of HET-B on seven consecutive days in 3-month-old ICR mice significantly enhanced the object recognition memory and object location memory than that in control. Immunohistochemical analysis indicated significantly increased ß3-tubulin-positive neurite density, synaptophysin, and postsynaptic density 95 in the perirhinal cortex and hippocampus after administering HET-B. Furthermore, the concentration of neurotransmitters was measured using HPLC analysis; HET-B significantly increased five-levels of HT in the hippocampus, and decreased metabolites of dopamine, dihydroxyphenylacetic acid, and homovanillic acid, in the prefrontal cortex and hippocampus. Taken together, HET-B induces neurite elongation and neurotransmitter regulation and possibly enhances cognitive memory.


Assuntos
Cognição , Crescimento Neuronal , Plasticidade Neuronal , Peptídeos Cíclicos , Animais , Caryophyllaceae/química , Camundongos , Camundongos Endogâmicos ICR , Neuritos/efeitos dos fármacos , Peptídeos Cíclicos/farmacologia
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.
Appl Microbiol Biotechnol ; 105(11): 4649-4662, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-34059940

RESUMO

Alzheimer's disease (AD) is a type of progressive neurodegenerative disease, and amyloid ß-protein 42 (Aß42) serves an important role in the pathological process of development of AD. Paired immunoglobulin-like receptor B (PirB) is a functional receptor for myelin inhibitors of neuron regeneration in the CNS, and it has also been identified to function as a high-affinity receptor for Aß. Here, we used a phage display to identify a specific PirB antagonist peptide 11(PAP11, PFRLQLS), which could reverse Aß42-induced neurotoxicity and promote neurite outgrowth in vitro. Immunofluorescence analysis showed that PAP11 colocalized with PirB on the membrane of cortical neurons. Horseradish peroxidase-streptavidin-biotin assay further proved that PAP11 directly binds to PirB and the dissociation constant (Kd) was 0.128µM. PAP11 functionally antagonized the neurite outgrowth inhibitory effect induced by Aß42 in cortical neurons, and the underlying mechanism was associated with a PirB-ROCK2/CRMP2 signaling pathway. The novel PirB antagonist peptide PAP11 may be a promising candidate therapeutic agent for the treatment of AD and other neurodegenerative diseases. KEY POINTS: • PAP11 was the first PirB antagonist peptide screened by phage display technology. • PAP11 could protect neurons by blocking the binding of Aß42 and PirB. • PAP11 reverse inhibitory effect of neurite outgrowth through ROCK2/CRMP2 pathway.


Assuntos
Peptídeos beta-Amiloides , Glicoproteínas de Membrana/antagonistas & inibidores , Doenças Neurodegenerativas , Crescimento Neuronal , Receptores Imunológicos/antagonistas & inibidores , Células Cultivadas , Humanos , Fragmentos de Peptídeos
9.
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
10.
Aging (Albany NY) ; 13(11): 15620-15637, 2021 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-34106880

RESUMO

Amyloid ß (Aß) plays a major role in the neurodegeneration of Alzheimer's disease (AD). The accumulation of misfolded Aß causes oxidative stress and inflammatory damage leading to apoptotic cell death. Traditional Chinese herbal medicine (CHM) has been widely used in treating neurodegenerative diseases by reducing oxidative stress and neuroinflammation. We examined the neuroprotective effect of formulated CHM Shaoyao Gancao Tang (SG-Tang, made of Paeonia lactiflora and Glycyrrhiza uralensis at 1:1 ratio) in AD cell and mouse models. In Aß-GFP SH-SY5Y cells, SG-Tang reduced Aß aggregation and reactive oxygen species (ROS) production, as well as improved neurite outgrowth. When the Aß-GFP-expressing cells were stimulated with conditioned medium from interferon (IFN)-γ-activated HMC3 microglia, SG-Tang suppressed expressions of inducible nitric oxide synthase (iNOS), NLR family pyrin domain containing 1 (NLRP1) and 3 (NLRP3), tumor necrosis factor (TNF)-α, interleukin (IL)-1ß and IL-6, attenuated caspase-1 activity and ROS production, and promoted neurite outgrowth. In streptozocin-induced hyperglycemic APP/PS1/Tau triple transgenic (3×Tg-AD) mice, SG-Tang also reduced expressions of NLRP1, NLRP3, Aß and Tau in hippocampus and cortex, as well as improved working and spatial memories in Y maze and Morris water maze. Collectively, our results demonstrate the potential of SG-Tang in treating AD by moderating neuroinflammation.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Cognição , Medicamentos de Ervas Chinesas/uso terapêutico , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Neuroproteção , Doença de Alzheimer/fisiopatologia , Peptídeos beta-Amiloides/metabolismo , Animais , Linhagem Celular , Cognição/efeitos dos fármacos , Meios de Cultivo Condicionados/farmacologia , Modelos Animais de Doenças , Medicamentos de Ervas Chinesas/farmacologia , Proteínas de Fluorescência Verde/metabolismo , Humanos , Interferon gama/metabolismo , Memória/efeitos dos fármacos , Transtornos da Memória/complicações , Transtornos da Memória/fisiopatologia , Camundongos Transgênicos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Modelos Biológicos , Crescimento Neuronal/efeitos dos fármacos , Neuroproteção/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Agregados Proteicos/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Aprendizagem Espacial/efeitos dos fármacos , Proteínas tau/metabolismo
11.
Life Sci ; 280: 119715, 2021 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-34116113

RESUMO

AIMS: Resveratrol pretreatment can decrease ischemic cerebral injury and enhance proliferation of neural stem cells via mediation of Sonic Hedgehog signaling. However, it is relatively little known about whether neurorestorative effects of resveratrol are mediated by Shh signaling in ischemic cerebral injury. The present study tests whether the Shh signaling pathway mediates resveratrol to promote neurorestoration of ischemic cerebral injury. MATERIALS AND METHODS: Rats or neurons before middle cerebral artery occlusion/reperfusion (MCAO/R) or oxygen-glucose deprivation/reoxygenation (OGD/R) injury were pretreated with resveratrol. Immunohistochemistry is used to be determined BrdU+/DCX+, BrdU+/Nestin+ and BrdU+/NG2+ cell (markers of new proliferated neural stem/progenitor and oligodendrocyte precursor cell, respectively), BrdU+/MAP2+ and BrdU+/CNPase+ cell (markers of new mature neuron and oligodendrocyte, respectively), BrdU+/TUNEL+ cell (marker of apoptosis for new proliferated cell), SY, NF200, Iba-1 and GFAP (markers of synaptogenesis, axon, microglia and astrocyte, respectively). Shh and Gli-1 mRNAs were detected by RT-PCR assay. Iba-1, GFAP, Shh and Gli-1 proteins were detected by Western blot. KEY FINDINGS: Resveratrol pretreatment significantly reduced neurological deficit scores, promoted proliferation, differentiation, migration and survival of neural stem/progenitor and oligodendrocyte precursor cells, inhibited astrocyte and microglia activation, strengthened synaptophysin and NF200 expression, at the same time, promoted neurite outgrowth of neurons. Meanwhile, expression levels of Shh and Gli-1 proteins were significantly increased and Gli-1 translocated into the nucleus. However, cyclopamine, a Smo inhibitor, canceled the above effects of resveratrol. CONCLUSIONS: It may be mediated, at least partly, by the Shh signaling pathway that resveratrol pretreament promote neurorestoration of ischemic cerebral injury.


Assuntos
Proteínas Hedgehog/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Resveratrol/uso terapêutico , Animais , Infarto da Artéria Cerebral Média/metabolismo , Infarto da Artéria Cerebral Média/patologia , Masculino , Crescimento Neuronal/efeitos dos fármacos , Ratos , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacos
12.
Methods Mol Biol ; 2311: 25-38, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033075

RESUMO

The lack of a convenient, easily maintained, and inexpensive in vitro human neuronal model to study neurodegenerative diseases prompted us to develop a rapid, 1-h differentiated neuronal cell model based on human NT2 cells and C3 transferase. Here, we describe the rapid differentiation of human neuronal NT2 cells, and the differentiation, transduction, and transfection of human SK-N-MC cells and rat PC12 cells to obtain cells with the morphology of differentiated neurons that can express exogenous genes of interest at high level.


Assuntos
Neoplasias das Glândulas Suprarrenais/patologia , Neuroblastoma/metabolismo , Neurogênese , Neurônios/patologia , Feocromocitoma/patologia , Teratocarcinoma/patologia , ADP Ribose Transferases/farmacologia , Neoplasias das Glândulas Suprarrenais/genética , Neoplasias das Glândulas Suprarrenais/metabolismo , Animais , Toxinas Botulínicas/farmacologia , Técnicas de Cultura de Células , Humanos , Neuroblastoma/genética , Neuroblastoma/patologia , Neurogênese/efeitos dos fármacos , Crescimento Neuronal , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Células PC12 , Fenótipo , Feocromocitoma/genética , Feocromocitoma/metabolismo , Ratos , Teratocarcinoma/genética , Teratocarcinoma/metabolismo , Transfecção , Tretinoína/farmacologia
13.
Methods Mol Biol ; 2311: 161-166, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033084

RESUMO

The culturing of neurons results in formation of the layer of neurons with random extensive overlapping outgrowth. To understand specific roles of somas, axons, and dendrites in complex function of neurons and to identify molecular mechanisms of biological processes in these cellular compartments, various methods were developed. We utilized AXon Investigation System (AXIS™) manufactured by Millipore. This device provides an opportunity to orient neuronal outgrowth and spatially isolate neuronal processes from neuronal bodies. AXIS device is a slide-mounted microfluidic system, which consists of four wells. Two of the wells are connected by a channel on each side of the device. Channels are connected by microgrooves (approximately 120). The size of microgrooves (10µm in width and 5µm in height) does not permit passage of cell through while allowing extension of neurites. The microfluidic design also allows for an establishment of a hydrostatic gradient when the volume in one chamber is greater than that in the other (Park et al., Nat Protoc 1:2128-2136, 2006). This feature allows for studying of the effect of specific compounds on selected compartments of neurons.


Assuntos
Técnicas de Cultura de Células/instrumentação , Dispositivos Lab-On-A-Chip , Técnicas Analíticas Microfluídicas/instrumentação , Crescimento Neuronal , Neurônios/fisiologia , Células Cultivadas , Desenho de Equipamento , Feto , Idade Gestacional , Humanos , Pressão Hidrostática , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Neurônios/metabolismo
14.
Methods Mol Biol ; 2311: 167-175, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033085

RESUMO

The use of sensory neurons and assessment of neurite outgrowth in vitro is an important part of understanding neuronal development and plasticity. Cultures of rat dorsal root ganglion (DRG) neurons provide quantitative results very quickly and, when grown on growth promoting or inhibitory substrates, can be utilized to study axonal growth, neurotrophic dependence, and structure and function of growth cones. Since we are interested in axon regeneration and targeting, we have sought to promote neurite outgrowth by refining the techniques of growing DRG neurons in culture. This chapter describes detailed methods for the dissection and purification of DRG neurons and quantitative assessment of neurite on promoting or inhibitory substrates.


Assuntos
Gânglios Espinais/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Técnicas de Cultura de Células , Células Cultivadas , Gânglios Espinais/citologia , Neurônios/fisiologia , Ratos Sprague-Dawley
15.
Methods Mol Biol ; 2311: 195-201, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34033088

RESUMO

The lack of a convenient in vitro human neuronal model to study alcohol-induced neurodegenerative diseases, such as fetal alcohol syndrome (FAS), prompted us to develop human neuronal culture and in vitro human FAS model by incubating cells with physiologically relevant EtOH concentration (50 mM). Here, we describe the detailed method of isolation of human neuronal culture, and ability to analyze neurites extension using Sholl assay. We utilized highly efficient transfection method of neuronal cells to study morphology of neurons with or without EtOH treatment.


Assuntos
Bioensaio , Encéfalo/efeitos dos fármacos , Etanol/toxicidade , Transtornos do Espectro Alcoólico Fetal/patologia , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Animais , Encéfalo/embriologia , Separação Celular , Células Cultivadas , Idade Gestacional , Humanos , Neurônios/patologia , Cultura Primária de Células , Transfecção
16.
Biochem Biophys Res Commun ; 558: 36-43, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-33895549

RESUMO

Down-regulated in renal cell carcinoma 1 (DRR1), a unique stress-induced protein, is highly expressed in the nervous system. This study investigated the roles of DRR1 in the brain by examining its expression pattern at different developmental stages of a rat brain and in cultured primary hippocampal neurons. High expression of DRR1 was observed in all developmental stages of a rat brain and cultured primary hippocampal neurons. We then focused on the role of DRR1 in promoting neurite outgrowth during the early stage of hippocampal neuron development. Results showed that down-regulation of DRR1 suppressed axon outgrowth. Mass spectrometry analysis revealed that tropomodulin-2 (Tmod2) is a novel binding partner of DRR1. Our results showed that both DRR1 and Tmod2 mediate axon formation during the early stage of hippocampal neuron development. Suppression of TMOD2 expression rescued the abnormal axon outgrowth induced by DRR1 knockdown during the early stage of hippocampal neuron development.


Assuntos
Hipocampo/crescimento & desenvolvimento , Hipocampo/metabolismo , Crescimento Neuronal/genética , Crescimento Neuronal/fisiologia , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Células Cultivadas , Regulação para Baixo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Hipocampo/citologia , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/metabolismo , Gravidez , Ligação Proteica , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Tropomodulina/antagonistas & inibidores , Tropomodulina/genética , Tropomodulina/metabolismo , Proteínas Supressoras de Tumor/antagonistas & inibidores
17.
Int J Mol Sci ; 22(8)2021 Apr 16.
Artigo em Inglês | MEDLINE | ID: mdl-33923565

RESUMO

Magnetosomes are membrane-enclosed iron oxide crystals biosynthesized by magnetotactic bacteria. As the biomineralization of bacterial magnetosomes can be genetically controlled, they have become promising nanomaterials for bionanotechnological applications. In the present paper, we explore a novel application of magnetosomes as nanotool for manipulating axonal outgrowth via stretch-growth (SG). SG refers to the process of stimulation of axonal outgrowth through the application of mechanical forces. Thanks to their superior magnetic properties, magnetosomes have been used to magnetize mouse hippocampal neurons in order to stretch axons under the application of magnetic fields. We found that magnetosomes are avidly internalized by cells. They adhere to the cell membrane, are quickly internalized, and slowly degrade after a few days from the internalization process. Our data show that bacterial magnetosomes are more efficient than synthetic iron oxide nanoparticles in stimulating axonal outgrowth via SG.


Assuntos
Axônios/metabolismo , Magnetossomos/metabolismo , Crescimento Neuronal , Animais , Axônios/fisiologia , Axônios/ultraestrutura , Transporte Biológico , Células Cultivadas , Feminino , Hipocampo/citologia , Magnetospirillum/química , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Estresse Mecânico
18.
Biochim Biophys Acta Proteins Proteom ; 1869(8): 140656, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-33857633

RESUMO

Axon guidance is required for the establishment of brain circuits. Whether much of the molecular basis of axon guidance is known from animal models, the molecular machinery coordinating axon growth and pathfinding in humans remains to be elucidated. The use of induced pluripotent stem cells (iPSC) from human donors has revolutionized in vitro studies of the human brain. iPSC can be differentiated into neuronal stem cells which can be used to generate neural tissue-like cultures, known as neurospheres, that reproduce, in many aspects, the cell types and molecules present in the brain. Here, we analyzed quantitative changes in the proteome of neurospheres during differentiation. Relative quantification was performed at early time points during differentiation using iTRAQ-based labeling and LC-MS/MS analysis. We identified 6438 proteins, from which 433 were downregulated and 479 were upregulated during differentiation. We show that human neurospheres have a molecular profile that correlates to the fetal brain. During differentiation, upregulated pathways are related to neuronal development and differentiation, cell adhesion, and axonal guidance whereas cell proliferation pathways were downregulated. We developed a functional assay to check for neurite outgrowth in neurospheres and confirmed that neurite outgrowth potential is increased after 10 days of differentiation and is enhanced by increasing cyclic AMP levels. The proteins identified here represent a resource to monitor neurosphere differentiation and coupled to the neurite outgrowth assay can be used to functionally explore neurological disorders using human neurospheres as a model.


Assuntos
Axônios/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Neurais/metabolismo , Axônios/patologia , Encéfalo/metabolismo , Proliferação de Células/fisiologia , Cromatografia Líquida/métodos , Humanos , Células-Tronco Neurais/fisiologia , Neurogênese/fisiologia , Crescimento Neuronal/fisiologia , Neurônios/metabolismo , Proteômica/métodos , Espectrometria de Massas em Tandem/métodos
19.
Chem Biol Interact ; 341: 109454, 2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-33798505

RESUMO

Doxycycline has been used as antibiotic since the 1960s. Recently, studies have shown that doxycycline is neuroprotective in models of neurodegenerative diseases and brain injuries, mainly due to anti-inflammatory and anti-apoptotic effects. However, it is not known if doxycycline has neurotrophic potential, which is relevant, considering the role of axonal degeneration at the early stages of neurodegeneration in Alzheimer's disease, Amyotrophic Lateral Sclerosis and Parkinson's disease as well as in normal aging. Axons are preceded by the formation of neurites, the hallmark of the neuronal differentiation induced by neurotrophins like NGF. Therefore, the modulation of neurotrophin receptors aimed at formation and regeneration of axons has been proposed as a strategy to delay the progression of neurodegeneration and has gained relevance as new techniques for early diagnosis arise. Based on these premises, we investigated the potential of doxycycline to mimic the effects of Nerve Growth Factor (NGF) with focus on the signaling pathways and neuronal modulators of neurite initiation, growth and branching. We used PC12 cells, a neuronal model widely employed to study the neurotrophic pathways and mechanisms induced by NGF. Results showed that doxycycline induced neurite outgrowth via activation of the trkA receptor and the downstream signaling pathways, PI3K/Akt and MAPK/ERK, without inducing the expression of NGF. Doxycycline also increased the expression of GAP-43, synapsin I and NF200, proteins involved in axonal and synaptic plasticity. Altogether, these data demonstrate, for the first time, the neurotrophic potential of doxycycline, which might be useful to restore the neuronal connectivity lost at the initial phase of neurodegeneration.


Assuntos
Antibacterianos/farmacologia , Doxiciclina/farmacologia , Fator de Crescimento Neural/metabolismo , Animais , Carbazóis/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Proteína GAP-43/metabolismo , Alcaloides Indólicos/farmacologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Fator de Crescimento Neural/farmacologia , Proteínas de Neurofilamentos/metabolismo , Crescimento Neuronal/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Células PC12 , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Receptor trkA/antagonistas & inibidores , Receptor trkA/metabolismo , Transdução de Sinais/efeitos dos fármacos , Sinapsinas/metabolismo
20.
Toxins (Basel) ; 13(4)2021 04 07.
Artigo em Inglês | MEDLINE | ID: mdl-33916905

RESUMO

The CRISPR-Cas13 system based on a bacterial enzyme has been explored as a powerful new method for RNA manipulation. Due to the high efficiency and specificity of RNA editing/interference achieved by this system, it is currently being developed as a new therapeutic tool for the treatment of neurological and other diseases. However, the safety of this new generation of RNA therapies is still unclear. In this study, we constructed a vector expressing CRISPR-Cas13 under a constitutive neuron-specific promoter. CRISPR-Cas13 from Leptotrichia wadei was expressed in primary cultures of mouse cortical neurons. We found that the presence of CRISPR-Cas13 impedes the development of cultured neurons. These results show a neurotoxic action of Cas13 and call for more studies to test for and possibly mitigate the toxic effects of Cas13 enzymes in order to improve CRISPR-Cas13-based tools for RNA targeting.


Assuntos
Proteínas Associadas a CRISPR/metabolismo , Sistemas CRISPR-Cas , Córtex Cerebral/enzimologia , Leptotrichia/enzimologia , Crescimento Neuronal , Neurônios/enzimologia , Animais , Proteínas Associadas a CRISPR/genética , Células Cultivadas , Córtex Cerebral/patologia , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Leptotrichia/genética , Camundongos , Neurônios/patologia
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