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1.
Sun Y  Lim Y  Li F  Liu S  Lu JJ  Haberberger R  Zhong JH  Zhou XF 《PloS one》2012,7(4):e35883

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.  相似文献   

2.
Mature brain‐derived neurotrophic factor (mBDNF) plays a vital role in the nervous system, whereas proBDNF elicits neurodegeneration and neuronal apoptosis. Although current enzyme‐linked immunosorbent assay (ELISA) has been widely used to measure BDNF levels, it cannot differentiate mBDNF from proBDNF. As the function of proBDNF differs from mBDNF, it is necessary to establish an ELISA assay specific for the detection of mBDNF. Therefore, we aimed to establish a new mBDNF‐specific sandwich ELISA. In this study, we have screened and found a combination of antibodies for a sandwich ELISA. A monoclonal antibody and sheep anti‐BDNF were chosen as capture and detection antibody for sandwich ELISA respectively. The new ELISA showed no cross‐reactivity to human recombinant NT‐3, NT‐4, nerve growth factor and negligible cross‐reactivity (0.99–4.99%) for proBDNF compared to commercial ELISA kits (33.18–91.09%). The application of the new mBDNF ELISA was shown through the measurement of mBDNF levels in different brain regions of rats and in the brain of β‐site amyloid precursor protein cleaving enzyme 1 (BACE1)?/? and WT mice and compared to western blot. Overall, this new ELISA will be useful for the measurement of mBDNF levels with high specificity.

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3.
4.
Increasing evidence suggests that n-hexane induces nerve injury via neuronal apoptosis induced by its active metabolite 2,5-hexanedione (HD). However, the underlying mechanism remains unknown. Studies have confirmed that pro-nerve growth factor (proNGF), a precursor of mature nerve growth factor (mNGF), might activate apoptotic signaling by binding to p75 neurotrophin receptor (p75NTR) in neurons. Therefore, we studied the mechanism of the proNGF/p75NTR pathway in HD-induced neuronal apoptosis. Sprague–Dawley (SD) rats were injected with 400 mg/kg HD once a day for 5 weeks, and VSC4.1 cells were treated with 10, 20, and 40 mM HD in vitro. Results showed that HD effectively induced neuronal apoptosis. Moreover, it up-regulated proNGF and p75NTR levels, activated c-Jun N-terminal kinase (JNK) and c-Jun, and disrupted the balance between B-cell lymphoma-2 (Bcl-2) and Bcl-2-associated X protein (Bax). Our findings revealed that the proNGF/p75NTR signaling pathway was involved in HD-induced neuronal apoptosis; it can serve as a theoretical basis for further exploration of the neurotoxic mechanisms of HD.  相似文献   

5.
6.
Accumulating evidence underscores the importance of ligand-receptor dynamics in shaping cellular signaling. In the nervous system, growth factor-activated Trk receptor trafficking serves to convey biochemical signaling that underlies fundamental neural functions. Focus has been placed on axonal trafficking but little is known about growth factor-activated Trk dynamics in the neuronal soma, particularly at the molecular scale, due in large part to technical hurdles in observing individual growth factor-Trk complexes for long periods of time inside live cells. Quantum dots (QDs) are intensely fluorescent nanoparticles that have been used to study the dynamics of ligand-receptor complexes at the plasma membrane but the value of QDs for investigating ligand-receptor intracellular dynamics has not been well exploited. The current study establishes that QD conjugated brain-derived neurotrophic factor (QD-BDNF) binds to TrkB receptors with high specificity, activates TrkB downstream signaling, and allows single QD tracking capability for long recording durations deep within the soma of live neurons. QD-BDNF complexes undergo internalization, recycling, and intracellular trafficking in the neuronal soma. These trafficking events exhibit little time-synchrony and diverse heterogeneity in underlying dynamics that include phases of sustained rapid motor transport without pause as well as immobility of surprisingly long-lasting duration (several minutes). Moreover, the trajectories formed by dynamic individual BDNF complexes show no apparent end destination; BDNF complexes can be found meandering over long distances of several microns throughout the expanse of the neuronal soma in a circuitous fashion. The complex, heterogeneous nature of neuronal soma trafficking dynamics contrasts the reported linear nature of axonal transport data and calls for models that surpass our generally limited notions of nuclear-directed transport in the soma. QD-ligand probes are poised to provide understanding of how the molecular mechanisms underlying intracellular ligand-receptor trafficking shape cell signaling under conditions of both healthy and dysfunctional neurological disease models.  相似文献   

7.
The FGF receptors (FGFRs) control a multitude of cellular processes both during development and in the adult through the initiation of signaling cascades that regulate proliferation, survival, and differentiation. Although FGFR tyrosine phosphorylation and the recruitment of Src homology 2 domain proteins have been widely described, we have previously shown that FGFR is also phosphorylated on Ser779 in response to ligand and binds the 14-3-3 family of phosphoserine/threonine-binding adaptor/scaffold proteins. However, whether this receptor phosphoserine mode of signaling is able to regulate specific signaling pathways and biological responses is unclear. Using PC12 pheochromocytoma cells and primary mouse bone marrow stromal cells as models for growth factor-regulated neuronal differentiation, we show that Ser779 in the cytoplasmic domains of FGFR1 and FGFR2 is required for the sustained activation of Ras and ERK but not for other FGFR phosphotyrosine pathways. The regulation of Ras and ERK signaling by Ser779 was critical not only for neuronal differentiation but also for cell survival under limiting growth factor concentrations. PKCϵ can phosphorylate Ser779 in vitro, whereas overexpression of PKCϵ results in constitutive Ser779 phosphorylation and enhanced PC12 cell differentiation. Furthermore, siRNA knockdown of PKCϵ reduces both growth factor-induced Ser779 phosphorylation and neuronal differentiation. Our findings show that in addition to FGFR tyrosine phosphorylation, the phosphorylation of a conserved serine residue, Ser779, can quantitatively control Ras/MAPK signaling to promote specific cellular responses.  相似文献   

8.
CIN85/CMS family of adaptor molecules   总被引:7,自引:0,他引:7  
Dikic I 《FEBS letters》2002,529(1):110-115
CIN85 and CMS belong to a family of ubiquitously expressed adaptor molecules containing three SH3 domains, a proline-rich region and a coiled-coil domain. By binding to numerous proteins they assemble multimeric complexes implicated in cell-specific signals controlling T-cell activation, kidney glomeruli function or apoptosis in neuronal cells. CIN85/CMS also associate with accessory endocytic proteins, components of the actin cytoskeleton as well as other adaptor proteins involved in receptor tyrosine kinase (RTK) signaling. These interactions enable CIN85/CMS to function within a network of signaling pathways that co-ordinate critical steps involved in downregulation and degradation of RTKs.  相似文献   

9.
Mice lacking epidermal growth factor receptor (EGFR) develop a neurodegeneration of unknown etiology affecting exclusively the frontal cortex and olfactory bulbs. Here, we show that EGFR signaling controls cortical degeneration by regulating cortical astrocyte apoptosis. Whereas EGFR(-/-) midbrain astrocytes are unaffected, mutant cortical astrocytes display increased apoptosis mediated by an Akt-caspase-dependent mechanism and are unable to support neuronal survival. The expression of many neurotrophic factors is unaltered in EGFR(-/-) cortical astrocytes suggesting that neuronal loss occurs as a consequence of increased astrocyte apoptosis rather than impaired secretion of trophic factors. Neuron-specific expression of activated Ras can compensate for the deficiency of EGFR(-/-) cortical astrocytes and prevent neuronal death. These results identify two functionally distinct astrocyte populations, which differentially depend on EGFR signaling for their survival and also for their ability to support neuronal survival. These spatial differences in astrocyte composition provide a mechanism for the region-specific neurodegeneration in EGFR(-/-) mice.  相似文献   

10.
ProNGF and p75NTR are upregulated and induce cell death following status epilepticus (SE) in rats. However, less is known about the proneurotrophin response to SE in mice, a more genetically tractable species where mechanisms can be more readily dissected. We evaluated the temporal- and cell-specific induction of the proneurotrophins and their receptors, including p75NTR, sortilin, and sorCS2, following mild SE induced with kainic acid (KA) or severe SE induced by pilocarpine. We found that mature NGF, p75NTR, and proBDNF were upregulated following SE, while proNGF was not altered, indicating potential mechanistic differences between rats and mice. ProBDNF was localized to mossy fibers and microglia following SE. p75NTR was transiently induced primarily in axons and axon terminals following SE, as well as in neuron and astrocyte cell bodies. ProBDNF and p75NTR increased independently of cell death and their localization was different depending on the severity of SE. We also examined the expression of proneurotrophin co-receptors, sortilin and sorCS2. Following severe SE, sorCS2, but not sortilin, was elevated in neurons and astrocytes. These data indicate that important differences exist between rat and mouse in the proneurotrophin response following SE. Moreover, the proBDNF and p75NTR increase after seizures in the absence of significant cell death suggests that proneurotrophin signaling may play other roles following SE.  相似文献   

11.
神经营养因子是一类分泌性多肽类生长因子,可促进中枢和外周神经元的生长、存活以及分化,但其前体分子却具有不同的生物学活性,也有着不同的受体以及细胞内信号通路。本文对近年来关于脑源性神经营养因子前体蛋白的研究予以综述,着重讨论其在神经损伤与情绪障碍和神经退行性变疾病模型中的作用。  相似文献   

12.
Vascular endothelial growth factor (VEGF), a specific pro-angiogenic peptide, has shown neuroprotective effects in the Parkinson’s disease (PD) models, but the underlying mechanisms remain elusive. In this study, the neuroprotective properties of VEGF on 1-methyl-4-phenylpyridinium ion (MPP+)-induced neurotoxicity in primary cerebellar granule neurons were investigated. Pretreatment of VEGF prevented MPP+-induced neuronal apoptosis in a concentration- and time-dependent manner. And this prevention was blocked by PTK787/ZK222584, a VEGF receptor-2 specific inhibitor. Both inhibition of the Akt pathway and activation of the extracellular signal-regulated kinase (ERK) pathway contribute to MPP+-induced neuronal apoptosis. VEGF reversed the inhibition of phosphoinositide 3-kinase (PI3-K)/Akt pathway caused by MPP+, but further enhanced the activation of ERK induced by MPP+. Interestingly, VEGF and PD98059 (an ERK kinase inhibitor) play a synergistic role in protecting neurons from MPP+-induced toxicity. Collectively, these findings suggest that the PI3-K/Akt and ERK pathways activated by VEGF play opposite roles in MPP+-induced neuronal apoptosis. This finding offers not only a new and clinically significant modality as to how VEGF exerts its neuroprotective effects but also a novel therapeutic strategy for PD by differentially regulating PD-associated signaling pathways.  相似文献   

13.
Activation of astrocytes in central nervous system inflammation leads to a disturbance of crosstalk between astrocytes and neurons, and that this may contribute to the death of neurons. CDK11p58 is a member of the large family of p34cdc2-related kinases. It specifically expresses in G2/M phase of the cell cycle and is closely related to cell cycle arrest and apoptosis. Here, we show that astrocyte-conditioned medium stimulated by lipopolysaccharide upregulates CDK11p58 expression and meanwhile causes neuronal apoptosis. CDK11p58 knockdown in PC12 cells represses neuronal apoptosis. CDK11p58 overexpression in PC12 cells promotes neuronal apoptosis. AKT signaling pathway is involved in CDK11p58-induced neuronal apoptosis process.  相似文献   

14.
Diabetic peripheral neuropathy (DPN) is one of the most common and troublesome complications of diabetes mellitus. It has been demonstrated that nerve growth factor (NGF) exerts a pivotal role in the regulation of neuronal growth and the promotion of DPN recovery. However, the exact molecular mechanisms are not well understood. Recent studies have indicated that as a novel therapeutic target, endoplasmic reticulum (ER) stress participates in the onset and progression of DPN. In the present study, it has been demonstrated that NGF prevents the sciatic nerve from degeneration and demyelination in DPN rats. Thus, RSC 96 cells, which retain the characteristic features of Schwann cells (SCs), were cultured in medium containing 30 mM glucose (high glucose, HG) to mimic SCs in DPN mice. The 50-ng/ml dose of NGF was identified to be the optimal concentration for treating an excessive ER stress level under HG conditions for 24 h. We found that NGF treatment significantly inhibits HG-induced ER stress and subsequently suppresses ER-related apoptosis. Further, NGF administration also activates the upstream signaling pathway of ER stress, PI3K/Akt/GSK3β signaling and ERK1/2 signaling. Co-treatment with the PI3K inhibitor LY294002 or ERK1/2 inhibitor U0126 significantly reverses the protective role of NGF on HG-induced excessive ER stress and subsequent apoptosis. These observations suggest that the neuroprotective role of NGF in DPN is mediated by the inhibition of excessive ER stress via the activation of the PI3K/Akt/GSK3β and ERK1/2 signaling pathways.  相似文献   

15.
Treatment with mature brain-derived neurotrophic factor (mBDNF) promotes functional recovery after ischemia in animal trials but the possible role of its precursor protein proBDNF and its receptors or the factors responsible for the conversion of proBDNF to mBDNF in ischemic stroke are not known. The main aim of this study was to characterize the time-dependent expression of genes and/or proteins related to BDNF processing and signaling after ischemia as well as the sensorimotor behavioral dysfunction in a photothrombotic ischemic model in rats. Characterization of different genes and proteins related to BDNF processing and signaling was performed using qPCR, immunoblotting and enzyme-linked immunosorbent assays. We showed in this study that some sensory and motor functional deficiencies appeared in the ischemic group at day 1 and persisted until day 14. Most changes in gene expression of BDNF and its processing enzymes occurred within the first 24 h in the ipsilateral cortex, but not in the contralateral cortex. At the protein level, proBDNF expression was increased at 6 h, mBDNF expression was increased between 15 h and 1 day while p75 receptor protein expression was increased between 6 h and 3 days in the ipsilateral cortex, but not in the contralateral cortex. Therefore, cerebral ischemia in rats led to the up-regulation of genes and/or proteins of BDNF, proBDNF and their processing enzymes and receptors in a time-dependent manner. We propose that the balance between BDNF and proBDNF and their associated proteins may play an important role in the pathogenesis and recovery from ischemia.  相似文献   

16.
Calcium signaling in neurons as in other cell types mediates changes in gene expression, cell growth, development, survival, and cell death. However, neuronal Ca2+ signaling processes have become adapted to modulate the function of other important pathways including axon outgrowth and changes in synaptic strength. Ca2+ plays a key role as the trigger for fast neurotransmitter release. The ubiquitous Ca2+ sensor calmodulin is involved in various aspects of neuronal regulation. The mechanisms by which changes in intracellular Ca2+ concentration in neurons can bring about such diverse responses has, however, become a topic of widespread interest that has recently focused on the roles of specialized neuronal Ca2+ sensors. In this article, we summarize synaptotagmins in neurotransmitter release, the neuronal roles of calmodulin, and the functional significance of the NCS and the CaBP/calneuron protein families of neuronal Ca2+ sensors.Calcium signaling in many cell types can mediate changes in gene expression, cell growth, development, survival, and cell death. However, neuronal calcium signaling processes have become adapted to modulate the function of important pathways in the brain, including neuronal survival, axon outgrowth, and changes in synaptic strength. Changes in the concentration of intracellular free Ca2+ ([Ca2+]i) are essential for the transmission of information through the nervous system as the trigger for neurotransmitter release at synapses. In addition, alterations in [Ca2+]i can lead to a wide range of different physiological changes that can modify neuronal functions over time scales of milliseconds through tens of minutes to days or longer (Berridge 1998). Many of these processes have been shown to be dependent upon the particular route of Ca2+ entry into the cell. It has long been known that the physiological outcome from a change in [Ca2+]i depends on its location, amplitude, and duration. The importance of location becomes even more pronounced in neurons because of their complex and extended morphologies. [Ca2+]i also regulates neuronal development and neuronal survival (Spitzer 2006). In addition, modifications to Ca2+ signaling pathways have been suggested to underlie various neuropathological disorders (Braunewell 2005; Berridge 2010).Highly localized Ca2+ elevations (Augustine et al. 2003) formed following Ca2+ entry though voltage-gated Ca2+ channels (VGCCs) lead to synaptic vesicle fusion with the presynaptic membrane and thereby allow neurotransmitter release within less than a millisecond. Differently localized and timed Ca2+ signals can, for example, result in changes to the properties of the VGCCs (Catterall and Few 2008) or lead to changes in gene expression (Bito et al. 1997). Postsynaptic Ca2+ signals arising from activation of NMDA receptors give rise to two important processes in synaptic plasticity, long term potentiation (LTP) and long term depression (LTD). LTP and LTD are examples of the way synaptic transmission can change synaptic efficacy and are thought to be important in modulating learning and memory. Importantly, the Ca2+ signals that bring about either LTP or LTD differ only in their timing and duration. LTP is triggered by Ca2+ signals on the micromolar scale for shorter durations, whereas LTD is triggered by changes in [Ca2+]i on the nanomolar scale for longer durations (Yang et al. 1999). Specific Ca2+ signals are likely to be decoded by different Ca2+ sensor proteins. These are proteins that undergo a conformational change on Ca2+ binding and then interact with and regulate various target proteins. Among those Ca2+ sensors that are important for neuronal function are the synaptotagmins that control neurotransmitter release (Chapman 2008), the ubiquitous EF-hand containing sensor calmodulin that has many neuronal roles, and the more recently discovered neuronal EF-hand containing proteins, including the neuronal calcium sensor (NCS) protein (Burgoyne 2007) and the calcium-binding protein (CaBP)/calneuron (Haeseleer et al. 2002) families. We will briefly review synaptotagmins and the neuronal functions of calmodulin but concentrate on the NCS and CaBP families of Ca2+ sensors.  相似文献   

17.
Nerve growth factor (NGF), the prototypical neurotrophic factor, is involved in the maintenance and growth of specific neuronal populations, whereas its precursor, proNGF, is involved in neuronal apoptosis. Binding of NGF or proNGF to TrkA, p75NTR, and VP10p receptors triggers complex intracellular signaling pathways that can be modulated by endogenous small‐molecule ligands. Here, we show by isothermal titration calorimetry and NMR that ATP binds to the intrinsically disordered pro‐peptide of proNGF with a micromolar dissociation constant. We demonstrate that Mg2+, known to play a physiological role in neurons, modulates the ATP/proNGF interaction. An integrative structural biophysics analysis by small angle X‐ray scattering and hydrogen‐deuterium exchange mass spectrometry unveils that ATP binding induces a conformational rearrangement of the flexible pro‐peptide domain of proNGF. This suggests that ATP may act as an allosteric modulator of the overall proNGF conformation, whose likely distinct biological activity may ultimately affect its physiological homeostasis.  相似文献   

18.
A distinct group of receptors including DCC, UNC5, RET and Ptc1 is known to function in ligand-dependent neuronal growth and differentiation or axon guidance. Acting as "dependence receptors", they may also regulate neuronal cell survival by inducing apoptosis in the absence of cognate ligand. Receptor-initiated apoptosis requires proteolytic (caspase) cleavage and exposure of a pro-apoptotic region in the cytoplasmic domains of the receptors. In contrast, classical apoptosis induced by growth factor or cytokine deprivation involves loss of survival signaling without receptor cleavage. DCC, UNC5, RET and Ptc1 are downregulated or mutated in diverse cancers, and show properties characteristic of tumor suppressors, consistent with their ability to promote neuronal cell death. Dysfunctional dependence receptors have been linked to the loss of specific neurons in certain inherited and neurodegenerative diseases. Dependence receptor-initiated apoptosis represents a novel paradigm for the controlled removal of specific cells during neural development and elimination of malignant cells that have strayed beyond regions of ligand availability.  相似文献   

19.
Brain-derived neurotropic factor (BDNF) plays an important role in mechanisms of depression. Precursor protein of this factor (proBDNF) can initiate apoptosis in the brain, while the mature form of BDNF is involved in neurogenesis. It is known that chronic alcoholization leads to the activation of apoptotic processes, neurodegeneration, brain injury, and cognitive dysfunction. In this work, we have studied the influence of long-term ethanol exposure on the proBDNF and BDNF protein levels, as well as on the expression of genes that encode these proteins in the brain structures of ASC mice with genetic predisposition to depressive-like behavior and in mice from parental nondepressive CBA strain. It was shown that chronic alcoholization results in a reduction of the BDNF level in the hippocampus and an increase in the amount of TrkB and p75 receptors in the frontal cortex of nondepressive CBA mice. At the same time, the long-term alcoholization of depressive ASC mice results in an increase of the proBDNF level in the frontal cortex and a reduction in the p75 protein level in the hippocampus. It has also been shown that, in depressive ASC mice, proBDNF and BDNF levels are significantly lower in the hippocampus and the frontal cortex compared with nondepressive CBA strain. However, no significant differences in the expression of genes encoding the studied proteins were observed. Thus, changes in the expression patterns of proBDNF, BDNF, and their receptors under the influence of alcoholization in the depressive ASC strain and nondepressive CBA strain mice are different.  相似文献   

20.
Sterile α motif and HD domain-containing protein 1 (SAMHD1) is a mammalian dNTP hydrolase (dNTPase) that regulates intracellular dNTP balance. We have previously reported that SAMHD1 mRNA and protein levels are significantly downregulated in CD4+ T-cells of patients with cutaneous T-cell lymphoma (CTCL), a disease characterized by infiltration of neoplastic CD4+ T-lymphocytes into the skin. However, functional significance of SAMHD1 in CTCL development and progression remains unknown. Here we investigate the mechanism by which SAMHD1 induces apoptosis in CTCL-derived CD4+ T-cells. We stably expressed exogenous SAMHD1 in the CTCL-derived HuT78 T-cell line containing a very low level of endogenous SAMHD1 protein. We found that low-level exogenous expression of SAMHD1 led to a significant reduction in HuT78 cell growth, proliferation, and colony formation. Exogenous SAMHD1 expression in HuT78 cells also resulted in increased spontaneous and Fas ligand (Fas-L)-induced apoptosis levels via activation of the extrinsic pathway, including caspase-8, ?3 and ?7. Additionally, increased SAMHD1 significantly reduced the protein and mRNA expression of the short isoform of cFLIP (cFLIPS), an important negative regulator of Fas-L-mediated apoptotic signaling. Our results indicate that exogenous SAMHD1 expression inhibits HuT78 cell growth and proliferation in part by increasing apoptosis. These findings implicate that SAMHD1 acts as an inhibitor in CTCL cell growth, suggesting that downregulation of SAMHD1 expression in neoplastic T-cells can facilitate uncontrolled cell proliferation.  相似文献   

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