The Role Of BDNF And Its Precusor In Glioma

Objective1.There are two forms of BDNF (proBDNF and mature BDNF) in human central nervous system (CNS). BDNF is first synthesized as a precursor of BDNF (proBDNF) and is subsequently cleaved either intracellularly or extracellularly by a series of proteases to release mature homodimeric proteins (mature BDNF). ProBDNF and mature BDNF play opposing roles in neuronal function. Their diverse actions are mediated through two different transmembrane receptor signaling systems:p75NTR and TrkB. ProBDNF binds to both p75NTR and the co-receptor sortilin with a high affinity, which has opposite role in cell apoptosis, long-term depression, synaptic retraction and neurite collapse.2. BDNF signal transduction can be "hijacked" by malignant cells for their own use. BDNF, in conjunction with its primary receptor TrkB is often overexpressed in a variety of human cancers, where it may allow tumor expansion and contribute to malignant transformation by autocrine or paracrine mechanisms. However, histological data in previous studies were unable to distinguish mature BDNF from proBDNF due to the lack of specific antibodies. Gliomas represent about40%of intracranial tumors, which biological characteristics and the origins suggest that microenvironment of CNS contributes to glioma development. High grade glioma is incurable with a short survival time and poor prognosis. BDNF gene is most abundantly expressed in24cell lines derived from human malignant gliomas. Upregulation of BDNF and TrkB was observed in human malignant tumor.3. The increased expression of p75neurotrophin receptor (NTR) is a characteristic of high-grade glioma, but the potential significance of increased p75NTR in this tumor is not fully understood. Because proBDNF is a potent ligand of p75NTR which is highly expressed in high grade glioma, it is suggested that proBDNF may have an impact on glioma. ProBDNF-p75NTR pathway and mature BDNF-TrkB pathway may play opposite role in the biological behavior of gliomas.4. The antibodies used in this study can distinguish proBDNF from mature BDNF. For the first time our study systemically investigated the expression of proBDNF and its receptors p75NTR and sortilin in human glioma. We also distinguished proBDNF from mature BDNF and evaluated the ratio of proBDNF to mature BDNF in different grade gliomas by specific antibodies for the first time. To prove proBDNF and mature BDNF play opposite in glioma, we explored the effect of proBDNF and mature BDNF on C6glioma cell growth, differentiation, invasity and possible mechanism in vitro.Methods1. The expression of proBDNF was assayed in different grades human gliomas (n=52) and controls (n=13) by immunochemistry, quantitative real-time PCR and Western blot methods. ProBDNF/mature BDNF and their receptors-p75NTR/sortilin and TrkB were examined in the same samples. Different subcellular localization of proBDNF/p75NTR, proBDNF/sortilin and mature BDNF/TrkB in gliomas was assayed by immunofluorescence double labelling. The relationship between proBNDF/mature BDNF and their receptors expression and malignancy grade of glioma was analyzed. The ratio of proBDNF to mature BDNF in different grade gliomas and control samples was determined for the first time.2. Since proBDNF and its main receptors p75NTR/sortilin were expressed in C6cells, we investigated the roles of endogenous proBDNF and exogenous proBDNF on C6glioma cells differentiation, growth, apoptosis and migration in vitro by using C6glioma cells as a model. C6cells were treated individualy as follows:(1) with different concentration of recombinant proBDNF protein (1,3,10,30ng/mL);(2) with different concentration of recombinant mature BDNF protein (1,3,10,30ng/mL);(3) with specific proBDNF antibodies (1,3,10μg/mL) to neutralize endogenous proBDNF;(4) with specific mature BDNF antibodies(1,3,10μg/mL) to neutralize endogenous mature BDNF;(5) with p75NTR-ECD-Fc (5μg/mL) to block p75NTR in C6cells. The C6cell morphology and GFAP expression were assayed after treatments. MTT assay was performed to investigate the effect of proBDNF/mature BDNF on C6cell growth and viability. DAPI nuclear staining method was performed to investigate the effect of proBDNF/mature BDNF on C6cells apoptosis. C6cells motility and invasion were examined by scrach assay and Boyden chamber assay.Results1. The expression of proBDNF, p75NTR and sortilin was significantly increased in high grade glioma. ProBDNF/p75NTR and proBDNF/sortilin were colocalized in glioma cells and had different subcellur distribution. ProBDNF was present in both cytoplasm and nuclei of tumor cells. Correlation analysis showed that proBDNF, p75NTR and sortilin were positively related to the malignancy grade of glioma.2. ProBDNF and mature BDNF co-existed in human glioma cells. The ratio of proBDNF to mature BDNF exhibited a significant decrease of in high grade gliomas compared with control and were negatively correlated with tumor grade.3. Results in vitro showed that:(1) Recombinant proBDNF for24h revealed a major morphological transformation and induced an increase of GFAP of C6cells through p75NTR, while GFAP expression was significantly decreased in the presence of proBDNF antibodies. Recombinant proBDNF increased apoptosis, decreased cell growth and migration in vitro via p75NTR. In contrast, proBDNF antibodies decreased apoptosis, increased cell growth and migration.(2) The biological role of mature BDNF is contrast to that of proBDNF in C6glioma cells. Recombinant mature BDNF decreased apoptosis, increased cell viability and migration. Mature BDNF antibodies increased apoptosis, decreased cell growth and migration.ConclusionsProBDNF and mature BDNF co-exist in glioma cells. Our data in vivo and in vitro indicates that proBDNF, which should be a functional protein, may play an inhibitory role in glioma, while mature BDNF is the main form to promote the growth of glioma. These findings in vivo and in vitro suggest that proBDNF/p75/sortilin pathway may be a balancing signal to tumor growth by mature BDNF/TrkB pathway. The relative expression levels of proBDNF and mature BDNF may determine the balance of these two signaling pathways in the malignancy and prognosis of glioma, which might provide therapeutic targets for clinical intervention.