Brain-derived neurotrophic factor enhances the basal rate of protein synthesis by increasing active eukaryotic elongation factor 2 levels and promoting translation elongation in cortical neurons

Abstract

The constitutive and activity-dependent components of protein synthesis are both critical for neural function. Although the mechanisms controlling extracellularly induced protein synthesis are becoming clear, less is understood about the molecular networks that regulate the basal translation rate. Here we describe the effects of chronic treatment with various neurotrophic factors and cytokines on the basal rate of protein synthesis in primary cortical neurons. Among the examined factors, brain-derived neurotrophic factor (BDNF) showed the strongest effect. The rate of protein synthesis increased in the cortical tissues of BDNF transgenic mice, whereas it decreased in BDNF knock-out mice. BDNF specifically increased the level of the active, unphosphorylated form of eukaryotic elongation factor 2 (eEF2). The levels of active eEF2 increased and decreased in BDNF transgenic and BDNF knock-out mice, respectively. BDNF decreased kinase activity and increased phosphatase activity against eEF2 in vitro. Additionally, BDNF shortened the ribosomal transit time, an index of translation elongation. In agreement with these results, overexpression of eEF2 enhanced protein synthesis. Taken together, our results demonstrate that the increased level of active eEF2 induced by chronic BDNF stimulation enhances translational elongation processes and increases the total rate of protein synthesis in neurons.

FIGURE 1.

Chronic effects of various neurotrophic factors and cytokines on protein synthesis in cortical neurons. Neurons were treated without or with BDNF (50 ng/ml), NT-4 (50 ng/ml), NT-3 (50 ng/ml), insulin (10 μg/ml), nerve growth factor (NGF) (50 ng/ml), cilliary neurotrophic factor (CNTF) (10 ng/ml), glial cell line derived neurotrophic factor (GDNF) (10 ng/ml), human growth factor (HGF) (20 ng/ml), interleukin (IL)-1α (5 ng/ml), IL-1β (5 ng/ml), IL-2 (2.5 ng/ml), IL-6 (1 ng/ml), or interferon (IFN)-γ (5 × 10⁵ unit/ml) for 5 days. The incorporation of [³⁵S]methionine into newly synthesized proteins was analyzed. Bars represent the mean ± S.D. (n = 4). *, p < 0.05 (t test). Similar results were obtained in three or four independent experiments.

FIGURE 2.

Protein synthesis rate in the cortices of BDNF mutant mice. The incorporation of [³⁵S]methionine into newly synthesized proteins was analyzed in the homogenates of cortical tissue from wild-type (wt) and BDNF mutant (+/−, heterozygous; −/−, homozygous) mice (A) or wild-type and transgenic (Tg) mice (B). Bars represent the mean ± S.D.; n = 10, *, p < 0.05 (analysis of variance) (A) and, n = 6, *, p < 0.05 (t test) (B).

FIGURE 3.

Effects of chronic BDNF treatment on the levels of translation factors. Western blotting analysis was performed to examine the protein levels of initiation factors (eIFs), elongation factors (eEFs), and release factors (eRFs) in neurons following BDNF treatment (50 ng/ml) for 5 days. Bands were analyzed using densitometry. Only the increase in the level of eEF2 is significant. n = 4–9 for each protein. GAPDH, glyceraldehyde-3-phosphate dehydrogenase; NSE, neuron-specific enolase.

FIGURE 4.

Effect of chronic BDNF treatment on the levels and phosphorylation status of eEF2. Conventional and quantitative RT-PCR was performed to examine eEF2 mRNA levels in neurons following BDNF treatment for 5 days (A). A single band of predicted size (236 bp) was observed (n = 4 in each group). Quantitative RT-PCR indicates there is no difference between control and BDNF-treated neurons (n = 4, p = 0.931 (t test)). Neurons were pulse labeled with [³⁵S]methionine for 1 h, and then washed. eEF2 was immunoprecipitated after 1 and 6 h and analyzed in neurons at 4 days in culture and quantified by Image analyzer (BAS 5000, Fuji Film). Bars represent mean ± S.D. (n = 3). *, p < 0.01 (t test) (B). Western blotting (WB) (D and E) and densitometric analysis (D and E) were performed to examine the total and phosphorylated (inactive) protein levels of eEF2 in neurons following BDNF treatment (50 ng/ml) for 5 days. Bars represent the mean ± S.D. (n = 8). *, p < 0.001 (t test). The phosphorylation ratio of eEF2 is presented in panel F.

FIGURE 5.

The levels and phosphorylation status of eEF2 in the cortices of BDNF mutant mice. Western blotting (A) and densitometric analysis (B) were performed to examine the total and phosphorylated (inactive) protein levels of eEF2 in cortical tissues of wild-type (wt), BDNF mutant (KO(−/−)), and BDNF transgenic (Tg) mice. Bars represent the mean ± S.D.; n = 4, *, p < 0.05 (analysis of variance). The phosphorylation ratio of eEF2 was calculated and is shown in panel C. KO, knock-out.

FIGURE 6.

Effects of chronic BDNF treatment on the levels and activities of eEF2K and PP2A. Western blotting (A) and densitometric analysis (B) were performed to examine the protein levels of eEF2K and PP2A in neurons treated with BDNF (50 ng/ml) for 5 days. Bars represent the mean ± S.D. (n = 6); *, p < 0.01, and **, p < 0.005 (t test). eEF2K kinase activity and PP2A phosphatase activity were measured (C). Bars represent the mean ± S.D. (n = 4 (eEF2K) and n = 8 (PP2A)); ***, p < 0.001 (t test).

FIGURE 7.

Effects of chronic BDNF treatment on the ribosomal transit time in cortical neurons. A, a representative example of the transit times from control neurons or neurons treated with BDNF (50 ng/ml) for 5 days. Open squares (control) and diamonds (BDNF) represent the total synthesized protein. Filled diamonds (control) and circles (BDNF) represent completed peptides at each time point. Solid lines for total (nascent plus completed) protein and dashed lines for completed peptides were obtained using linear regression analysis. To calculate the transit time, the difference between the lines along the time axis was measured and doubled. Quantitative data are shown in panel B. Bars represent mean ± S.D. (n = 8; *, p < 0.01, t test).

FIGURE 8.

Overexpression of eEF2 in cortical neurons. cDNA encoding rat eEF2 or EGFP (as a control) was transfected into primary cortical neurons. A, expression and phosphorylation of eEF2 were analyzed by Western blotting and quantified using densitometry. Actin was used as a standard. Bars represent the mean ± S.D. (n = 4; *, p < 0.05, t test). B, the incorporation of [³⁵S]methionine into newly synthesized protein was determined using sister transfectants to the cells used to determine the level of eEF2 expression. Bars represent the mean ± S.D. (n = 12; *, p < 0.01, t test).