Influence of astrocyte-conditioned medium on the formation of synapses in neural stem cells: the role of neurotrophin proteins

Abstract

To discuss whether neurotrophin proteins, brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3), and neural growth factor (NGF), in the astrocyte-conditioned medium (ACM) are involved in the synapse formation in neural stem cells (NSCs). (1) Cells derived from a pheochromocytoma of the rat adrenal medulla of the line PC12 were induced by amyloid-beta protein (Abeta)1-40 for 0, 4, 6, 12, and 24 h respectively. Then part of these PC12 cells underwent flow cytometry to examine the apoptotic rates. Different cells were added into Falcon Cell Culture Insert: Group A containing astrocytes isolated from Wistar rat, Group B with PC12 cells and astrocytes, Groups C1-C5 containing astrocytes and PC12 cells induced by Abeta(1-40) for 0, 4, 6, 12, and 24 h respectively, Group D1-5 with PC12 cells induced by Abeta(1-40) for 0, 4, 6, 12, and 24 h respectively, and Group E containing astrocytes induced by Abeta(1-40) for 6 h. Flow cytometry was used to detect the apoptotic rates of different groups. Double-antibody sandwich ELISA was used to detect the levels of BDNF, NT-3, and NGF. (2) The different kinds of the astrocyte-conditioned medium as described above were mixed with DMEM/F12 medium according to the proportion of 1:3 and then divided into 13 groups: Group I (Group A + NSCs), Group II (Group B + NSCs), Group III-VII (Groups C1-C5 + NSCs), Group VIII (NSCs without ASM), Group IX-XIII (Groups D1-D5 + Mscs), and Group XIV (Group E + NSCs). The expression of synaptophysin and growth-associated protein-43 (GAP-43 protein) were detected by co-focal laser scanning microscopy. The number of mature synapse was observed by transmission electron microscope(TEM). Flow cytometry showed that the apoptotic rates of the PC12 cells were low 0, 2, and 4 h after Abeta(1-40) induction, with the peak 6 h after induction (P < 0.05). The BDNF total protein level in the ACM of Group C3 was the highest (A = 1.53 +/- 0.25) (P < 0.05). The expression levels of synaptophysin (A = 33.39 +/- 2.71) and GAP-43 (A = 49.18 +/- 6.45), and the mature synapse number of NSCs (4.70 +/- 0.52 synapse/field of vision) of Group V were the highest in comparison with the other groups (all P < 0.05). After incubation of astrocytes with Abeta(1-40)-induced PC12 cells(Abeta-PC12), the ACM induces the synapse formation in the NSCs. The BDNF in the ACM is probably involved in this process.