Effect of the neuroprotective compound SR57746A on nerve growth factor synthesis in cultured astrocytes from neonatal rat cortex

Abstract

The neurotrophic factor promoting activity of the neuroprotective compound SR57746A was evaluated in primary cultures of neonatal rat cortical astrocytes by studying the synthesis of nerve growth factor (NGF) and brain-derived neurotrophic factor (BDNF). A concentration- and time-dependent increase of nerve growth factor mRNA was induced by SR57746A (10 nM-1 microM). In these astrocytes, BDNF mRNA contents were increased to a significant but smaller extent, and beta-actin mRNA showed no variation. SR57746A (1 microM) induced increases of both de novo protein translation after 6 h of incubation and NGF release into the extracellular medium after 6-24 h. These effects were preceded by a transient augmentation of junB, c-fos and c-jun mRNA contents. These increases of AP-1 family mRNA were associated with increased nuclear AP-1 binding activity. The results show that SR57746A can increase the synthesis and release of NGF in rat cortical astrocytes. Such effects may contribute to the drug's previously described neuroprotective effects.

Figure 1

Effects of SR57746A on NGF, BDNF and β-actin mRNA levels in cortical astrocytes. (A) representative autoradiograph, from 6 h incubation experiment, with relative positions of protected oligonucleotides indicated. (B) Time course effect of SR57746A (1 μM); ^*P<0.05, ^**P<0.01, Student's t-test (n=3). (C) concentration effect of SR57746A, after 6 h of incubation, on NGF mRNA contents in astrocytes. ^**P<0.01, Duncan's t-test (n=3).

Figure 2

Effects of SR57746A on NGF protein synthesis. Autoradiography of tris-glycine-PAGE of neo-synthesized proteins from cortical astrocytes, immunoprecipitated by anti-NGF as described in the text. Astrocytes were incubated for 6 h with (+) SR57746A (1 μM), or with (−) 0.1% DMSO. Positions for glycosylated proNGF (42.5 kDa), proNGF (35 kDa) and β-NGF (13.5 kDa) are indicated.

Figure 3

Time-course effect of SR57746A on secreted NGF. Astrocytes were treated with SR57746A (1 μM) for the indicated times. Extracellular NGF was determined by ELISA as described in Methods. Values are the means±s.e.mean of three independent determinations expressed as per cent of control values. ^*P<0.05, ^**P<0.01, Student's t-test (n=4).

Figure 4

Time-course effect of SR57746A (10 nM) on IEG mRNA levels in cortical astrocytes. ^*P<0.05, ^**P<0.01, Student's t-test (n=4).

Figure 5

EMSA for ³²P-labelled AP-1^NGF oligonucleotide. Gel retardation assays were performed with AP-1^NGF oligonucleotide (0.05 ng) and 7 μg of nuclear proteins from cortical (lanes 2–5) astrocytes. Lane 1, AP-1^NGF without nuclear extract; lane 2, AP-1^NGF with nuclear extracts from vehicle-treated cells (C); lane 3, AP-1^NGF with nuclear extracts from cells incubated with SR57746A (10 nM, SR); lane 4, AP-1^NGF with nuclear extracts from vehicle-treated cells and 30 fold molar excess of unlabelled AP-1^NGF (AP1); lane 5, AP-1^NGF with nuclear extracts from vehicle-treated cells and 30 fold molar excess of unlabelled Oct-1 oligonucleotide (Oct).