Regulated release of BDNF by cortical oligodendrocytes is mediated through metabotropic glutamate receptors and the PLC pathway

A number of studies suggest that OLGs (oligodendrocytes), the myelinating cells of the central nervous system, are also a source of trophic molecules, such as neurotrophins that may influence survival of proximate neurons. What is less clear is how the release of these molecules may be regulated. The present study investigated the effects of BDNF (brain-derived neurotrophic factor) derived from cortical OLGs on proximate neurons, as well as regulatory mechanisms mediating BDNF release. Initial work determined that BDNF derived from cortical OLGs increased the numbers of VGLUT1 (vesicular glutamate transporter 1)-positive glutamatergic cortical neurons. Furthermore, glutamate acting through metabotropic, and not AMPA/kainate or NMDA (N-methyl-d-aspartate), receptors increased BDNF release. The PLC (phospholipase C) pathway is a key mediator of metabotropic actions to release BDNF in astrocytes and neurons. Treatment of OLGs with the PLC activator m-3M3FBS [N-(3-trifluoromethylphenyl)-2,4,6-trimethylbenzenesulfonamide] induced robust release of BDNF. Moreover, release elicited by the metabotropic receptor agonist ACPD [trans-(1S,3R)-1-aminocyclopentane-1,3-dicarboxylic acid] was inhibited by the PLC antagonist {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122, the IP₃ (inositol triphosphate 3) receptor inhibitor 2-APB (2-aminoethoxydiphenylborane) and the intracellular calcium chelator BAPTA/AM [1,2-bis-(o-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid tetrakis(acetoxymethyl ester)]. Taken together, these results suggest that OLG lineage cells release BDNF, a molecule trophic for proximate neurons. BDNF release is regulated by glutamate acting through mGluRs (metabotropic glutamate receptors) and the PLC pathway. Thus glutamate and BDNF may be molecules that support neuron–OLG interactions in the cortex.     

Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons

Observing and characterizing dynamic cellular processes can yield important information about cellular activity that cannot be gained from static images. Vital fluorescent probes, particularly green fluorescent protein (GFP) have revolutionized cell biology stemming from the ability to label specific intracellular compartments and cellular structures. For example, the live imaging of GFP (and its spectral variants) chimeras have allowed for a dynamic analysis of the cytoskeleton, organelle transport, and membrane dynamics in a multitude of organisms and cell types [1-3]. Although live imaging has become prevalent, this approach still poses many technical challenges, particularly in primary cultured neurons. One challenge is the expression of GFP-tagged proteins in post-mitotic neurons; the other is the ability to capture fluorescent images while minimizing phototoxicity, photobleaching, and maintaining general cell health. Here we provide a protocol that describes a lipid-based transfection method that yields a relatively low transfection rate (~0.5%), however is ideal for the imaging of fully polarized neurons. A low transfection rate is essential so that single axons and dendrites can be characterized as to their orientation to the cell body to confirm directionality of transport, i.e., anterograde v. retrograde. Our approach to imaging GFP expressing neurons relies on a standard wide-field fluorescent microscope outfitted with a CCD camera, image capture software, and a heated imaging chamber. We have imaged a wide variety of organelles or structures, for example, dense-core vesicles, mitochondria, growth cones, and actin without any special optics or excitation requirements other than a fluorescent light source. Additionally, spectrally-distinct, fluorescently labeled proteins, e.g., GFP and dsRed-tagged proteins, can be visualized near simultaneously to characterize co-transport or other coordinated cellular events. The imaging approach described here is flexible for a variety of imaging applications and can be adopted by a laboratory for relatively little cost provided a microscope is available.     

B型酪氨酸激酶受体的轴浆转运

脑源性神经营养因子(brain-derived neurotrophic factor,BDNF)在发育及成熟的中枢神经系统(central nervoussystem,CNS)中起到举足轻重的调节作用,而其中绝大部分作用由其B型酪氨酸激酶受体(tyrosine kinase receptortype B,TrkB)介导,因此TrkB在神经元中的轴浆转运过程显得尤为重要。本文从动力蛋白、潜在调节分子、细胞骨架蛋白等方面对TrkB轴浆转运分子机制的研究进展进行综述,并就其进一步研究提出一系列的问题与展望。     

Retracted: Long non-coding RNA LINC00152 promotes cell growth and invasion of papillary thyroid carcinoma by regulating the miR-497/BDNF axis

Long intergenic non-coding RNA 152 (LINC00152) was reported to be tightly linked to tumorigenesis and progression in multiple cancers. However, its biological role and modulatory mechanism in papillary thyroid carcinoma (PTC) has not been elucidated. In this study, we determined the expression levels of LINC00152 in PTC tissues and cell lines by quantitative real time polymerase chain reaction (qRT-PCR). Cell proliferation, colony formation, migration, and invasion were measured by a Cell Counting Kit-8 assay, colony formation analysis, wound healing, and transwell invasion assay, respectively. A luciferase reporter assay and qRT-PCR were used to determine whether LINC00152 interacts with miR-497 directly. We established a xenograft mouse model to examine the underlying molecular mechanism and effect of LINC00152 on tumor growth in vivo. We found that LINC00152 expression was significantly increased in PTC tissues and derived cell lines. LINC00152 knockdown significantly inhibited proliferation, colony formation, migration, and invasion in vitro, and impaired tumor growth in vivo. We revealed that LINC00152 functioned as a competing endogenous RNA to the miR-497 sponge, downregulating its downstream target brain-derived neurotrophic factor (BDNF), which is an oncogene in thyroid cancer. These findings suggest that LINC00152 is responsible for PTC cell proliferation and invasion and exerts its function by regulating the miR-497/BDNF axis.     

Retrograde Axonal Transport of Dopamine Beta Hydroxylase Antibodies by Neurons in the Trigeminal Ganglion

In this study we describe a population of neurons in the adult rat trigeminal ganglion (TG) that express dopamine beta-hydroxylase (DBH) and tyrosine hydroxylase (TH), and transport anti-DBH from their terminals. We have used NGF and NT3 labeled with biotin and anti-p75^NTR labeled with FITC to examine the transport of neurotrophins and their receptors by these cells. In both the superior cervical ganglion (SCG) and the TG all neurons that transported anti-DBH transported NGF. While 100% of the DBH positive neurons in the TG also transported NT3, approximately 25% of these neurons in the SCG failed to transport NT3. In the SCG virtually all the neurons transported anti-p75^NTR with the neurotrophins while in the TG more than 25% of these neurons failed to transport anti-p75^NTR with the neurotrophins. These findings suggest that DBH positive neurons in the TG depend upon target-derived NGF and NT3 for their noradrenergic phenotype.     

Determination of Amino Acid Sequence in Surfactin,a Crystalline Peptidelipid Surfactant Produced by Bacillus subtilis

Some conditions of autolysis in cultured tobacco cells were examined for temperature, cell culture age and aeration. Cells autolyzed readily at 45°C. Seventy percent of the dry matter, almost 100% of the soluble sugar, 40% of the insoluble sugar and 60% of the total nitrogen in the initial cells were excreted within 5 hr of incubation in water. At lower physiological temperatures, excreted substances were reabsorbed into cells during the early period of incubation under aerobic conditions.

Rapidly growing cells excreted larger amounts of sugar, nitrogen and solid matter than did non-growing cells during autolysis at 30°C.

Plasmolysis was observed in autolyzed cells.

Autolysis was makedly stimulated by anaerobic conditions.     

神经营养因子NGF、BDNF与围绝经期妇女认知功能关系的研究进展

神经营养因子(NTFs)是近几年神经科学研究的热点,研究显示它在神经系统中发挥独特的作用,尤其是神经生长因子(NGF)、脑源性神经营养因子(BDNF)在脑内功能及其表达调控方面具有重要作用。围绝经期妇女随着雌激素水平的降低会产生认知功能的减退,有研究发现去卵巢动物(OVX)雌激素水平降低可以导致某些NGF、BDNF的丢失。通过启动内源性NGF和BDNF的表达而实现对神经元的保护可能为雌激素替代治疗(ERT)脑保护作用的一种机制。本文就近几年的研究进展做一简要综述。     

肌肉注射人神经生长因子基因逆轴突传递修复神经损伤的研究进展

神经生长因子(NGF)促进中枢及外周神经系统神经元细胞存活、分化、轴突再生等重要作用已得到临床的广泛证实。目前临床上主要以局部或肌肉注射NGF蛋白的方式对神经系统的损伤进行治疗。但NGF半衰期短、局部应用副作用大、费用昂贵、难以透过血脑屏障等缺点而限制临床应用。长期以来,科研工作者致力于寻求一种理想的途径或方法以克服这一缺陷。随着基因工程技术的飞速发展,研究人员发现通过骨骼肌肌肉注射途径,以非病毒载体介导外源的NGF基因体内表达并逆轴突传递到神经损伤部位,有望解决这一难题。本文将就NGF及受体的基本结构和特性、逆轴突传递的机制、非病毒载体结合骨骼肌肌肉注射的基因治疗等方面进行总结和阐述。     

Imaging Dendritic Spines of Rat Primary Hippocampal Neurons using Structured Illumination Microscopy

Dendritic spines are protrusions emerging from the dendrite of a neuron and represent the primary postsynaptic targets of excitatory inputs in the brain. Technological advances have identified these structures as key elements in neuron connectivity and synaptic plasticity. The quantitative analysis of spine morphology using light microscopy remains an essential problem due to technical limitations associated with light''s intrinsic refraction limit. Dendritic spines can be readily identified by confocal laser-scanning fluorescence microscopy. However, measuring subtle changes in the shape and size of spines is difficult because spine dimensions other than length are usually smaller than conventional optical resolution fixed by light microscopy''s theoretical resolution limit of 200 nm.Several recently developed super resolution techniques have been used to image cellular structures smaller than the 200 nm, including dendritic spines. These techniques are based on classical far-field operations and therefore allow the use of existing sample preparation methods and to image beyond the surface of a specimen. Described here is a working protocol to apply super resolution structured illumination microscopy (SIM) to the imaging of dendritic spines in primary hippocampal neuron cultures. Possible applications of SIM overlap with those of confocal microscopy. However, the two techniques present different applicability. SIM offers higher effective lateral resolution, while confocal microscopy, due to the usage of a physical pinhole, achieves resolution improvement at the expense of removal of out of focus light. In this protocol, primary neurons are cultured on glass coverslips using a standard protocol, transfected with DNA plasmids encoding fluorescent proteins and imaged using SIM. The whole protocol described herein takes approximately 2 weeks, because dendritic spines are imaged after 16-17 days in vitro, when dendritic development is optimal. After completion of the protocol, dendritic spines can be reconstructed in 3D from series of SIM image stacks using specialized software.     

Expression profiles of BDNF splice variants in cultured DRG neurons stimulated with NGF

Expression of brain-derived neurotrophic factor (BDNF) mRNA is increased in the dorsal root ganglion (DRG) in response to peripheral inflammation. Nerve growth factor (NGF) from inflammatory tissue is thought to induce expression of BDNF. Recently, it was reported that the BDNF gene has eight non-coding exons that are transcribed independently into several splice variants. Expression of these splice variants in DRG neurons stimulated with NGF has not been studied. We examined changes in expression of BDNF splice variants in a rat model of peripheral inflammation and in cultured DRG neurons exposed to NGF. Total BDNF mRNA was increased by inflammation in vivo and by NGF in vitro. Among all splice variants, exon 1-9 showed the greatest increase in expression in both experiments. Our results indicate that exon 1-9 contributes to changes in total BDNF levels and may play an important role in the acute response of DRG to NGF.     

Brain-derived neurotrophic factor (BDNF) induces antagonistic action to Nogo signaling by the upregulation of lateral olfactory tract usher substance (LOTUS) expression

Neurons in the central nervous system (CNS) have limited capacity for axonal regeneration after trauma and neurological disorders due to an endogenous nonpermissive environment for axon regrowth in the CNS. Lateral olfactory tract usher substance (LOTUS) contributes to axonal tract formation in the developing brain and axonal regeneration in the adult brain as an endogenous Nogo receptor-1 (NgR1) antagonist. However, how LOTUS expression is regulated remains unclarified. This study examined molecular mechanism of regulation in LOTUS expression and found that brain-derived neurotrophic factor (BDNF) increased LOTUS expression in cultured hippocampal neurons. Exogenous application of BDNF increased LOTUS expression at both mRNA and protein levels in a dose-dependent manner. We also found that pharmacological inhibition with K252a and gene knockdown by siRNA of tropomyosin-related kinase B (TrkB), BDNF receptor suppressed BDNF-induced increase in LOTUS expression. Further pharmacological analysis of the TrkB signaling pathway revealed that BDNF increased LOTUS expression through mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K) cascades, but not phospholipase C-γ (PLCγ) cascade. Additionally, treatment with c-AMP response element binding protein (CREB) inhibitor partially suppressed BDNF-induced LOTUS expression. Finally, neurite outgrowth assay in cultured hippocampal neurons revealed that BDNF treatment-induced antagonism for NgR1 by up-regulating LOTUS expression. These findings suggest that BDNF may acts as a positive regulator of LOTUS expression through the TrkB signaling, thereby inducing an antagonistic action for NgR1 function by up-regulating LOTUS expression. Also, BDNF may synergistically affect axon regrowth through the upregulation of LOTUS expression.

     

慢病毒载体介导Bdnf基因修饰的骨髓间质干细胞移植治疗脑梗死

为研究经Bdnf基因修饰的骨髓间质干细胞(Mesenchymal stem cells, MSCs)对脑梗死的协同治疗作用, 构建带有大鼠Bdnf基因之慢病毒载体, 并感染大鼠骨髓间质干细胞(Rat mesenchymal stem cells, rMSCs)。运用线栓法制备大鼠大脑中动脉栓塞模型, 经尾静脉注射移植, 对照组注射0.1 mol/L磷酸盐缓冲液(PBS)1 mL, Bdnf-rMSCs和Mock-rMSCs组分别注射Bdnf-rMSCs细胞悬液以及未插入目的基因的空病毒载体感染后的rMSCs细胞悬液各1 mL。各组大鼠分别于术后24 h、移植后2周及2月应用modified Neurological Severity Scores (mNSS)评价神经功能状况。结果显示, 与对照组相比, Mock-rMSCs及Bdnf-rMSCs移植组神经功能改善明显, mNSS评分差异有统计学意义(P<0.001), 而且Bdnf-rMSCs移植组明显优于Mock-rMSCs移植组(P<0.001)。移植后2周及2月, 与对照组相比两移植组梗死区脑组织结构恢复较好, 均可见EGFP阳性细胞在梗死区及其周边区聚集并存活, 并有部分细胞出现神经元样改变。Bdnf- rMSCs移植组中移植细胞大量表达BDNF, 两移植组中均有部分植入细胞表达神经细胞表面标志物。研究表明Bdnf基因修饰的rMSCs经静脉移植后可迁移至脑梗死灶周围, 向神经细胞分化并长期存活。移植后的干细胞可与其分泌的BDNF协同促进脑梗死后神经功能恢复, 这为将来基因工程干细胞移植治疗脑梗死提供了实验依据。     

Decreased Phosphorylation Levels of TrkB Neurotrophin Receptor in the Spinal Cords from Patients with Amyotrophic Lateral Sclerosis

Amyotrophic lateral sclerosis (ALS) is characterized by the selective degeneration of specific populations of cranial and spinal motor neurons. In this study, we examined the expression of the high affinity functional receptor for BDNF, TrkB, and assessed the functional state of TrkB by examining the level of phosphorylation on tyrosine residues in ALS spinal cords. The data showed that TrkB-immunoprecipitates prepared from cell-free lysates of ALS spinal cords by use of an anti-TrkB antibody contained much more TrkB protein than from controls. These TrkB proteins expressed in ALS spinal cords, however, are much less phosphorylated on tyrosine residues than those of controls. Moreover, RT-PCR analysis of TrkB mRNA in ALS spinal cords demonstrated that the expression of Trk B mRNA is also upregulated in ALS spinal cords compared with those of controls. These data strongly suggest that there exists an abnormality in TrkB-mediated intracellular signaling in ALS spinal cords and shed a light on the possibility of the therapeutic intervention by normalizing this intracellular signaling.     

脑源性神经营养因子在大鼠后足切割疼痛中的作用

目的:观察脑源性神经营养因子(BDNF)对大鼠后足切割疼痛的影响。方法:采用纵行切割大鼠后足作为疼痛模型,运用免疫组织化学与免疫荧光双标记方法,观察大鼠后足切割后不同时间点(1-72hr)BDNF在相应节段背根神经节与脊髓内表达的变化。腹腔或鞘内注射BDNF抗体中和内源性BDNF后,以Von Frey尼龙纤维刺激后足行机械痛敏评价。结果:大鼠后足切割后1-24hr内,BDNF在切割侧L42-L5脊髓后角表达明增加,BDNF主要位于后角神经元内与神经末梢,星形胶质细胞与小胶质细胞内未见明显表达;在L42-L5背根神经节,BNDF免疫阳性细胞百分比在切割后1-24hr内也明显增加,增加的主要为大直经神经元;鞘内给予BDNF抗体可明显增加大鼠后足切割后的缩足阈值,而腹腔给予BDNF抗体对大鼠的缩足阈值影响较小。结论:BDNF参与了大鼠后足切割后机械痛敏的过程。     

Brain Source Imaging in Preclinical Rat Models of Focal Epilepsy using High-Resolution EEG Recordings

Electroencephalogram (EEG) has been traditionally used to determine which brain regions are the most likely candidates for resection in patients with focal epilepsy. This methodology relies on the assumption that seizures originate from the same regions of the brain from which interictal epileptiform discharges (IEDs) emerge. Preclinical models are very useful to find correlates between IED locations and the actual regions underlying seizure initiation in focal epilepsy. Rats have been commonly used in preclinical studies of epilepsy¹; hence, there exist a large variety of models for focal epilepsy in this particular species. However, it is challenging to record multichannel EEG and to perform brain source imaging in such a small animal. To overcome this issue, we combine a patented-technology to obtain 32-channel EEG recordings from rodents² and an MRI probabilistic atlas for brain anatomical structures in Wistar rats to perform brain source imaging. In this video, we introduce the procedures to acquire multichannel EEG from Wistar rats with focal cortical dysplasia, and describe the steps both to define the volume conductor model from the MRI atlas and to uniquely determine the IEDs. Finally, we validate the whole methodology by obtaining brain source images of IEDs and compare them with those obtained at different time frames during the seizure onset.     

A Thin-skull Window Technique for Chronic Two-photon In vivo Imaging of Murine Microglia in Models of Neuroinflammation

Traditionally in neuroscience, in vivo two photon imaging of the murine central nervous system has either involved the use of open-skull^1,2 or thinned-skull ³ preparations. While the open-skull technique is very versatile, it is not optimal for studying microglia because it is invasive and can cause microglial activation. Even though the thinned-skull approach is minimally invasive, the repeated re-thinning of skull required for chronic imaging increases the risks of tissue injury and microglial activation and allows for a limited number of imaging sessions. Here we present a chronic thin-skull window method for monitoring murine microglia in vivo over an extended period of time using two-photon microscopy. We demonstrate how to prepare a stable, accessible, thinned-skull cortical window (TSCW) with an apposed glass coverslip that remains translucent over the course of three weeks of intermittent observation. This TSCW preparation is far more immunologically inert with respect to microglial activation than open craniotomy or repeated skull thinning and allows an arbitrary number of imaging sessions during a time period of weeks. We prepare TSCW in CX₃CR₁ GFP/+ mice ⁴ to visualize microglia with enhanced green fluorescent protein to ≤150 μm beneath the pial surface. We also show that this preparation can be used in conjunction with stereotactic brain injections of the HIV-1 neurotoxic protein Tat, adjacent to the TSCW, which is capable of inducing durable microgliosis. Therefore, this method is extremely useful for examining changes in microglial morphology and motility over time in the living brain in models of HIV Associated Neurocognitive Disorder (HAND) and other neurodegenerative diseases with a neuroinflammatory component.     

Acute role of the brain-derived neurotrophic factor (BDNF) on the respiratory neural network activity in mice in vitro

In humans, several pathologies are associated with disturbances of the respiratory control, some of them including alteration in the brain-derived neurotrophic factor (BDNF) signalling pathway. BDNF has long been known as a neurotrophic factor involved in survival, differentiation and maintenance of neuronal populations in the peripheral and central nervous system. More recently BDNF has also been discovered to be a potent neuromodulator with acute effects on neuronal excitability and synaptic plasticity. Animals deleted for the gene encoding BDNF exhibit respiratory alteration suggesting an important but yet undefined role of the neurotrophin in respiratory rhythmogenesis either by a trophic and/or an acute action. The possibility that BDNF might exert an acute regulatory role on the rhythmic activity of the respiratory generator of the pre-B?tzinger complex has been recently examined in newborn mice in vitro. Results obtained, reviewed in the present paper, will help getting insights in respiratory rhythm regulatory mechanisms that involve BDNF signalling.     

Distribution of pluripotent neural crest cells in the embryo and the role of brain-derived neurotrophic factor in the commitment to the primary sensory neuron lineage

Many early migratory neural crest cells are pluripotent in the sense that their progeny are able to generate more than one differentiated phenotype (Sieber-Blum and Cohen, 1980, Dev. Biol. 80:95–106; Baroffio, Dupin, and Le Douarin, 1988, Proc. Natl. Acad. Sci. USA 85:5325–5329; Bronner-Fraser and Fraser, 1988, Nature 335:161–164; Sieber-Blum, 1989a, Science 243:1608–1611; Ito and Sieber-Blum, 1991, Dev. Biol. 148:95–106). At trunk levels, the neural crest contains two classes (Sieber-Blum and Cohen, 1980) and at posterior rhombencephalic levels, three different classes of pluripotent cells (Ito and Sieber-Blum, 1991). We investigated cell differentiation by in vitro clonal analysis to determine when in development the pool of pluripotent neural crest cells becomes exhausted. The data suggest that different classes of pluripotent cells, precursor cells with more restricted developmental potentials, and apparently committed cells, exist at sites of advanced migration (posterior branchial arches) and even at target sites of neural crest cell differentiation [posterior branchial arches, dorsal root ganglia (DRG), sympathetic ganglia (SG), and epidermal ectoderm]. Some putative classes of pluripotent cells persist well into the second half of embryonic development. These observations have implications for our understanding of the mechanisms that control neural crest cell migration and differentiation. They support the idea that cues originating from the microenvironment affect differentiation of pluripotent neural crest cells. One such signal appears to be brain-derived neurotrophic factor (BDNF). In the presence of BDNF, but not nerve growth factor (NGF), there is a significant increase in the number of neural crest cells per colony that express a sensory neuron-specific marker. Because this increase is not accompanied by a corresponding increase in the total number of cells per colony, this suggests that BDNF plays a role in cell type specification. © 1993 John Wiley & Sons, Inc.