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Biomedical subjects

Zhe-Yu Chen

Publications and source records attributed to Zhe-Yu Chen.

At least 19 recordsLinked to original sources

Single-cell characterization of retrograde signaling by brain-derived neurotrophic factor.

Brain-derived neurotrophic factor (BDNF) is a key regulator of hippocampal synaptic plasticity in the developing and adult nervous system. It can be released from pyramidal neuron dendrites in an activity-dependent manner and has therefore been suggested to serve as a signal that provides the retrograde intercellular communication necessary for Hebbian plasticity and hippocampal-dependent learning. Although much has been learned about BDNF function by field stimulation of hippocampal neurons, it is not known whether moderate action potential-independent depolarization of single cells is capable of releasing sufficient BDNF to influence transmission at individual synapses. In this study, we show directly at the single-cell level that such modulation can occur. By using K-252a, anti-BDNF antibody, and interruption of regulated release, we confirm a model in which postsynaptic depolarization elicits calcium-dependent release of BDNF that diffuses retrogradely and enhances presynaptic transmitter release.

Animals↗

Genetic variant BDNF (Val66Met) polymorphism alters anxiety-related behavior.

A common single-nucleotide polymorphism in the brain-derived neurotrophic factor (BDNF) gene, a methionine (Met) substitution for valine (Val) at codon 66 (Val66Met), is associated with alterations in brain anatomy and memory, but its relevance to clinical disorders is unclear. We generated a variant BDNF mouse (BDNF(Met/Met)) that reproduces the phenotypic hallmarks in humans with the variant allele. BDNF(Met) was expressed in brain at normal levels, but its secretion from neurons was defective. When placed in stressful settings, BDNF(Met/Met) mice exhibited increased anxiety-related behaviors that were not normalized by the antidepressant, fluoxetine. A variant BDNF may thus play a key role in genetic predispositions to anxiety and depressive disorders.

Alleles↗

Cell survival through Trk neurotrophin receptors is differentially regulated by ubiquitination.

Specificity of neurotrophin factor signaling is dictated through the action of Trk receptor tyrosine kinases. Once activated, Trk receptors are internalized and targeted for degradation. However, the mechanisms implicated in this process are incompletely understood. Here we report that the Trk receptors are multimonoubiquitinated in response to neurotrophins. We have identified an E3 ubiquitin ligase, Nedd4-2, that associates with the TrkA receptor and is phosphorylated upon NGF binding. The binding of Nedd4-2 to TrkA through a PPXY motif leads to the ubiquitination and downregulation of TrkA. Activated TrkA receptor levels and the survival of NGF-dependent sensory neurons, but not BDNF-dependent sensory neurons, are directly influenced by Nedd4-2 expression. Unexpectedly, Nedd4-2 does not bind or ubiquitinate related TrkB receptors, due to the lack of a consensus PPXY motif. Our results indicate that Trk neurotrophin receptors are differentially regulated by ubiquitination to modulate the survival of neurons.

Amino Acid Sequence↗

Modified techniques for adult-to-adult living donor liver transplantation.

BACKGROUND: Because of critical organ shortage, transplant professionals have utilized living donor liver transplantation (LDLT) in recent years. We summarized our experience in adult-to- adult LDLT with grafts of right liver lobe by a modified technique. METHODS: From January 2002 to August 2005, 24 adult patients underwent living donor liver transplantation with grafts of the right liver lobe at West China Hospital, Sichuan University, China. Twenty-two patients underwent modified procedures designed to improve the reconstruction of the right hepatic vein and the tributaries of the middle hepatic vein by interposing a great saphenous vein (GSV) graft and the anastomosis of the hepatic arteries and bile ducts. RESULTS: No severe complications and death occurred in all donors. In the first 2 patients, (patients 1 and 2), operative procedure was not modified. One patient suffered from "small-for-size syndrome" and the other died of sepsis with progressive deterioration of graft function. In the rest 22 patients (patients 3 to 24), however, the procedure of venous reconstruction was modified, and better results were obtained. Complications occurred in 7 recipients including acute rejection (2 patients), hepatic artery thrombosis (1), bile leakage (1), intestinal bleeding (1), left subphrenic abscess (1), and pulmonary infection (1). One patient with pulmonary infection died of multiple organ failure (MOF). The 22 patients underwent direct anastomosis of the right hepatic vein to the inferior vena cava (IVC), 9 direct anastomosis plus the reconstruction of the right inferior hepatic vein, and 10 direct anastomosis plus the reconstruction of the tributaries of the middle hepatic vein by interposing a GSV graft to provide sufficient venous outflow. Trifurcation of the portal vein was met in 3 patients. Venoplasty or separate anastomosis was performed. The ratio of graft to recipient body weight ranged from 0.72% to 1.17%. Among these patients, 19 had the ratio <1.0% and 4 <0.8%, and the ratio of graft weight to recipient standard liver volume was between 31.86% and 62.48%. Among these patients, 10 had the ratio <50% and 2 <40%. No "small-for-size syndrome" occurred in the 22 recipients who were subjected to modified procedures. CONCLUSIONS: With the modified surgical techniques for the reconstruction of the hepatic vein to obtain an adequate outflow and provide a sufficient functioning liver mass, living donor liver graft in adults using the right lobe can be safe to prevent the "small-for-size syndrome".

Adult↗

A novel endocytic recycling signal distinguishes biological responses of Trk neurotrophin receptors.

Endocytic trafficking of signaling receptors to alternate intracellular pathways has been shown to lead to diverse biological consequences. In this study, we report that two neurotrophin receptors (tropomyosin-related kinase TrkA and TrkB) traverse divergent endocytic pathways after binding to their respective ligands (nerve growth factor and brain-derived neurotrophic factor). We provide evidence that TrkA receptors in neurosecretory cells and neurons predominantly recycle back to the cell surface in a ligand-dependent manner. We have identified a specific sequence in the TrkA juxtamembrane region, which is distinct from that in TrkB receptors, and is both necessary and sufficient for rapid recycling of internalized receptors. Conversely, TrkB receptors are predominantly sorted to the degradative pathway. Transplantation of the TrkA recycling sequence into TrkB receptors reroutes the TrkB receptor to the recycling pathway. Finally, we link these divergent trafficking pathways to alternate biological responses. On prolonged neurotrophin treatment, TrkA receptors produce prolonged activation of phosphatidylinositol 3-kinase/Akt signaling as well as survival responses, compared with TrkB receptors. These results indicate that TrkA receptors, which predominantly recycle in signal-dependent manner, have unique biological properties dictated by its specific endocytic trafficking itinerary.

Amino Acid Sequence↗

Sortilin controls intracellular sorting of brain-derived neurotrophic factor to the regulated secretory pathway.

Brain-derived neurotrophic factor (BDNF), after activity-dependent secretion from neurons, modulates critical nervous system functions. Recently, a variant in the human bdnf gene, resulting in a valine to methionine substitution in the prodomain, has been shown to lead to defective regulated secretion from neurons and memory impairment. Here, we report a novel function for a Vps10p domain protein, sortilin, in controlling BDNF sorting to the regulated secretory pathway. Sortilin interacts specifically with BDNF in a region encompassing the methionine substitution and colocalizes with BDNF in secretory granules in neurons. A truncated form of sortilin causes BDNF missorting to the constitutive secretory pathway without affecting neurotrophin-4 (NT-4) secretion. In addition, sortilin small interfering RNA introduced into primary neurons also led to BDNF missorting from the regulated to the constitutive secretory pathway. Together, these data suggest a mechanism to understand the defect associated with variant BDNF and provide a framework, based on divergent presynaptic regulation of sorting to secretory pathways, to explain how two ligands for tropomyosin-related kinase B, BDNF and NT-4, can mediate diverse biological responses.

Adaptor Proteins, Vesicular Transport↗

ProBDNF induces neuronal apoptosis via activation of a receptor complex of p75NTR and sortilin.

Brain-derived neurotrophic factor (BDNF) is best characterized for critical roles in neuronal survival, differentiation, and synaptic modulation mediated by the TrkB receptor tyrosine kinase. Developmentally regulated death signaling by BDNF has also been demonstrated via activation of p75NTR. Because recent studies suggest that proNGF, the precursor form of NGF, is more active than mature NGF in inducing apoptosis after binding to p75NTR and a coreceptor, sortilin, we asked whether the precursor of BDNF (proBDNF) is also a proapoptotic ligand in the nervous system. proBDNF is secreted by cultured neurons, and recombinant proBDNF binds to sortilin. In sympathetic neurons coexpressing sortilin and p75NTR, we found that proBDNF is an apoptotic ligand that induces death at subnanomolar concentrations. In contrast, mature BDNF, but not proBDNF, is effective in inducing TrkB phosphorylation. proBDNF effects are dependent on cellular coexpression of both p75NTR and sortilin, because neurons deficient in p75NTR are resistant to proBDNF-induced apoptosis, and competitive antagonists of sortilin block sympathetic neuron death. Moreover, addition of preformed complexes of soluble sortilin and proBDNF failed to induce apoptosis of cells coexpressing both sortilin and p75NTR, suggesting that interaction of proBDNF with both receptors on the cell surface is required to initiate cell death. Together with our past findings, these data suggest that the neurotrophin family is capable of modulating diverse biological processes via differential processing of the proneurotrophins.

Adaptor Proteins, Vesicular Transport↗

Transactivation of Trk neurotrophin receptors by G-protein-coupled receptor ligands occurs on intracellular membranes.

Neurotrophins, such as NGF and BDNF, activate Trk receptor tyrosine kinases through receptor dimerization at the cell surface followed by autophosphorylation and intracellular signaling. It has been shown that activation of Trk receptor tyrosine kinases can also occur via a G-protein-coupled receptor (GPCR) mechanism, without involvement of neurotrophins. Two GPCR ligands, adenosine and pituitary adenylate cyclase-activating polypeptide (PACAP), can activate Trk receptor activity to increase the survival of neural cells through stimulation of Akt activity. To investigate the mechanism of Trk receptor transactivation, we have examined the localization of Trk receptors in PC12 cells and primary neurons after treatment with adenosine agonists and PACAP. In contrast to neurotrophin treatment, Trk receptors were sensitive to transcriptional and translational inhibitors, and they were found predominantly in intracellular locations particularly associated with Golgi membranes. Biotinylation and immunostaining experiments confirm that most of the transactivated Trk receptors are found in intracellular membranes. These results indicate that there are alternative modes of activating Trk receptor tyrosine kinases in the absence of neurotrophin binding at the cell surface and that receptor signaling may occur and persist inside of neuronal cells.

Adenosine↗

Variant brain-derived neurotrophic factor (BDNF) (Met66) alters the intracellular trafficking and activity-dependent secretion of wild-type BDNF in neurosecretory cells and cortical neurons.

Brain-derived neurotrophic factor (BDNF) plays a critical role in nervous system and cardiovascular development and function. Recently, a common single nucleotide polymorphism in the bdnf gene, resulting in a valine to methionine substitution in the prodomain (BDNF(Met)), has been shown to lead to memory impairment and susceptibility to neuropsychiatric disorders in humans heterozygous for the variant BDNF. When expressed by itself in hippocampal neurons, less BDNF(Met) is secreted in an activity-dependent manner. The nature of the cellular defect when both BDNF(Met) and wild-type BDNF (BDNF(Val)) are present in the same cell is not known. Given that this is the predominant expression profile in humans, we examined the effect of coexpressed BDNF(Met) on BDNF(Val) intracellular trafficking and processing. Our data indicate that abnormal trafficking of BDNF(Met) occurred only in neuronal and neurosecretory cells and that BDNF(Met) could alter the intracellular distribution and activity-dependent secretion of BDNF(Val). We determined that, when coexpressed in the same cell, approximately 70% of the variant BDNF forms BDNF(Val).BDNF(Met) heterodimers, which are inefficiently sorted into secretory granules resulting in a quantitative decreased secretion. Finally, we determined the form of BDNF secreted in an activity-dependent manner and observed no differences in the forms of BDNF(Met) or the BDNF(Val).BDNF(Met) heterodimer compared with BDNF(Val). Together, these findings indicate that components of the regulated secretory machinery interacts specifically with a signal in the BDNF prodomain and that perturbations in BDNF trafficking may lead to selective impairment in CNS function.

Amino Acid Substitution↗

Protective effect of liposome-mediated glial cell line-derived neurotrophic factor gene transfer in vivo on motoneurons following spinal cord injury in rats.

OBJECTIVE: To investigate the effect of liposome-mediated glial cell line-derived neurotrophic factor (GDNF) gene transfer in vivo on spinal cord motoneurons after spinal cord injury (SCI) in adult rats. METHODS: Sixty male Sprague-Dawley rats were divided equally into two groups: GDNF group and control group. The SCI model was established according to the method of Nystrom, and then the DC-Chol liposomes and recombinant plasmid pEGFP-GDNF cDNA complexes were injected into the injured spinal cord. The expression of GDNF cDNA 1 week after injection was detected by RT-PCR and fluorescence microscope. We observed the remaining motoneurons in the anterior horn and the changes of cholinesterase (CHE) and acid phosphatase (ACP) activity using Nissl and enzyme histochemistry staining. The locomotion function of hind limbs of rats was evaluated using inclined plane test and BBB locomotor scale. RESULTS: RT-PCR and fluorescence observation confirmed the presence of expression of GDNF cDNA 1 week and 4 weeks after injection. At 1, 2, 4 weeks after SCI, the number of motoneurons in the anterior horn in GDNF group (20.4+/-3.2, 21.7+/-3.6, 22.5+/-3.4) was more than that in control group (16.8+/-2.8, 17.3+/-2.7, 18.2+/-3.2, P<0.05). At 1, 2 weeks after SCI, the mean gray of the CHE-stained spinal motoneurons in GDNF group (74.2+/-25.8, 98.7+/-31.6) was less than that in control group (98.5+/-32.2, 134.6+/-45.2, P<0.01), and the mean gray of ACP in GDNF group (84.5+/-32.6, 79.5+/-28.4) was more than that in control group (61.2+/-24.9, 52.6+/-19.9, P<0.01). The locomotion functional scales in GDNF group were higher than that in control group within 1 to 4 weeks after SCI (P<0.05). CONCLUSIONS: GDNF gene transfer in vivo can protect motoneurons from death and degeneration induced by incomplete spinal cord injury as well as enhance locomotion functional restoration of hind limbs. These results suggest that liposome-mediated delivery of GDNF cDNA might be a practical method for treating traumatic spinal cord injury.

Animals↗

[Effect of emodin on motility signal transduction in colonic smooth muscle cells in rats with multiple organ dysfunction syndrome].

OBJECTIVE: To observe the effect of emodin on motility signal transduction and calcium ion in colonic smooth muscle cells (SMC) in rats with bacterial peritonitis caused multiple organ dysfunction syndrome (MODS). METHODS: Observation was conducted in colon of MODS model rats on (1) effects of emodin on the contraction of muscular strip and cells of colonic smooth muscle, and influences of specific myoglobulin light chain kinase inhibitor (ML-7) and selective proteinkinase C inhibitor (Calphostin C) on these effects; and (2) effect of emodin on calcium ion in SMC. RESULTS: Emodin could directly contract the muscular strip and cells of smooth muscle; ML-7 and Calphostine could inhibit these contractile action to some extent. Under MODS condition, emodin could still increase the intracellular calcium ion concentration; this effect could be inhibited by heparin (inosamine triphosphate receptor inhibitor IP3 and ryanodine receptor inhibitor in MODS model but the calcium chelator EGTA and nifedipine (the specific cell membrane voltage dependent calcium channel blocker) showed no influence on it. CONCLUSION: Emodin could directly contract the colonic smooth muscle in MODS model rats, which is mediated by raise the signal path MLCK of calcium ion and the PKCa path for increase calcium sensibility. The mechanism of increasing calcium ion is mainly through IP3 and RyR the two calcium ion channel receptor in the sarcoplasm.

Animals↗

Olfactory ensheathing cells genetically modified to secrete GDNF to promote spinal cord repair.

Olfactory ensheathing cell (OEC) transplantation has emerged as a very promising therapy for spinal cord repair. In this study, we tested the ability of genetically modified OECs to secrete high levels of glial cell line-derived neurotrophic factor (GDNF) to promote spinal cord repair. The GDNF gene was transduced into OECs using a retroviral-based system. The engineered OECs were first characterized by their ability to express and secrete biologically active GDNF in vitro. After implantation into the spinal cord of adult rats with complete spinal cord transection, OEC survival and GDNF production were examined. The locomotor functions of animals were assessed and axon regeneration was evaluated at the morphological level. To our knowledge, we report for the first time that the genetically modified OECs are capable of producing GDNF in vivo to significantly improve recovery after spinal cord injury (SCI). This work combined the outgrowth-promoting property of OECs with the neuroprotective effects of the additionally overexpressed neurotrophic factors and opens new avenues for the treatment of SCI.

Animals↗

Identification of the key amino acids of glial cell line-derived neurotrophic factor family receptor alpha1 involved in its biological function.

Glial cell line-derived neurotrophic factor (GDNF) plays a critical role in neurodevelopment and survival of midbrain dopaminergic and spinal motor neurons in vitro and in vivo. The biological actions of GDNF are mediated by a two-receptor complex consisting of a glycosylphosphatidylinositol-linked cell surface molecule, the GDNF family receptor alpha1 (GFRalpha1), and receptor protein tyrosine kinase Ret. Although structural analysis of GDNF has been extensively examined, less is known about the structural basis of GFRalpha1 function. In this study, based on evolutionary trace method and relative solvent accessibility prediction of residues, a set of trace residues that are solvent-accessible was selected for site-directed mutagenesis. A series of GFRalpha1 mutations was made, and PC12 cell lines stably expressing different GFRalpha1 mutants were generated. According to the survival and differentiation responses of these stable PC12 cells upon GDNF stimulation and the GDNF-GFRalpha1-Ret interaction assay, residues 152NN153, Arg259, and 316SNS318 in the GFRalpha1 central region were found to be critical for GFRalpha1 binding to GDNF and eliciting downstream signal transduction. The single mutation R259A in the GFRalpha1 molecule simultaneously lost its binding ability to GDNF and Ret. However N152A/N153A or S316A/N317A/S318A mutation in the GFRalpha1 molecule still retained the ability to bind with Ret. These findings suggest that distinct structural elements in GFRalpha1 may be involved in binding to GDNF and Ret.

Amino Acid Substitution↗

A structure-function analysis of glial cell-line-derived neurotrophic factor receptor alpha1.

The GFRalpha1 cDNA was amplified by RT-PCR from fetal rat hippocampus. The soluble recombinant GFRalpha1 and its mutants were obtained from an Escherichia coli expression system. The biological activity of soluble GFRalpha1 and its mutants were evaluated in PC12 cells. The results suggest that the central domain of GFRalpha1 is a crucial determinant for ligand binding. This established a solid basis for further study to find the key amino acid mediating the binding of GDNF and GFRalpha1.

Amino Acid Sequence↗

[Deducing functional epitopes for GDNF proteins and its specific GFRalpha co-receptors using phylogenetic approach].

Glial cell line-derived neurotrophic factor (GDNF) has received much attention as potential therapeutic agent for the treatment of neurodegenerative diseases. It will be very important to discover the molecular mechanism of this factor and its specific GFRalpha co-receptor. Based on the principle of molecular evolution that site-specific functional importance is relevant to the pressure it undergoes under natural selection, evolutionary trace method was used to identify the functional epitopes in GDNF and GFRalpha families. Some trace residues had been proved to be important in ligand-receptor binding, especially in rat GFRalpha1, where alanine scanning mutagenesis confirmed that sites N(152)N(153), R(259), S(316)N(317)S(318) and Q(247)D(248)S(249) were critical for GFRalpha1 binding to GDNF or Ret and thus affected the formation of GDNF-GFRalpha1-Ret complex.

Animals↗

Cationic liposome-mediated GDNF gene transfer after spinal cord injury.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to protect cranial and spinal motoneurons, which suggests potential uses of GDNF in the treatment of spinal cord injury (SCI) and motor neuron disease. We examined neuroprotective effect of cationic liposome-mediated GDNF gene transfer in vivo on axonal regeneration and locomotor function recovery after SCI in adult rats. The mixture of DC-Chol liposomes and recombinant plasmid pEGFP-GDNF cDNA was injected after SCI. RT-PCR confirmed the increased expression of GDNF mRNA in the injected areas at 7 days after injection. The expression of EGFP-GDNF was observed in the cells around the injection locus by fluorescence microscope at least 4 weeks after injection. Four weeks after GDNF gene transfer, regeneration of the corticospinal tracts was assessed using anterograde tract tracing. There are more HRP labeling of corticospinal tract axons across the lesion in GDNF group compared with control group. In GDNF group, the maximum distance these labeled axons extended varied in different animals and ranged from 5 mm to approximately 9 mm from the lesion. In control group, no HRP labeled axons extended caudal to the lesion. The locomotion function of hindlimbs of rats was evaluated using inclined plane test and BBB locomotor scores. The locomotion functional scores in GDNF group were higher than that in control group within 1-4 weeks after SCI (p < 0.05). These data demonstrate that in vivo transfer of GDNF cDNA can promote axonal regeneration and enhance locomotion functional recovery, suggesting that cationic liposome-mediated delivery of GDNF cDNA may be a practical gene transfer method for traumatic SCI treatment.

Animals↗

Etiology and management of hemmorrhage in spontaneous liver rupture: a report of 70 cases.

AIM: To analyze the causes and management of hemorrhage in spontaneous liver rupture. METHODS: Seventy cases of spontaneous liver rupture were retrospectively analyzed for causes of hemorrhage and therapeutic effects of surgical approaches. RESULTS: It was demonstrated that the causes of spontaneous liver rupture were primary liver cancer in 60 cases (85.7 %), cirrhosis in 3 cases (4.3 %), liver angioma in 2 cases (2.9 %), liver adenoma in 4 cases (5.7 %),and secondary liver cancer in 1 case (1.4 %). Hemostasis was achieved with surgical approaches in 68 cases (97.1 %) and non-surgical approaches in 2 cases (2.9 %). Surgical interventions included suture, ligation of hepatic artery, hepatic artery chemoembolization and partial hepatic resection. CONCLUSION: The results suggest that surgical intervention is still the main therapeutic method and the best procedure that should be selected according to causes of disease and patient's condition and history.

Adenoma↗

Cloning, Expression and Tumor Suppression of Human Endostatin.

Human endostatin cDNA was cloned from total RNA of normal Chinese liver cell line L02 by RT-PCR. Endostatin DNA sequence encoded 184 amino acid residues. Five base pairs and 3 amino acid residues are different from that reported, it may be due to interspecies difference. The endostatin cDNA was inserted into the pET-28a(+) containing T7 promoter. The recombinant plasmid was transformed the E.coli BL21(DE3). Recombinant human endostatin was highly expressed as inclusion body when the expression strain BL-ENDO was induced with 1 mmol/L IPTG. Result of SDS-PAGE analysis revealed that recombinant human endostatin was accounted for up to 25% of soluble protein in E.coli. Purified and refolded recombinant human endostatin was active in inhibiting tumor growth and metastasis.

Journal Article↗