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. 2009 Dec;57(16):1754-64.
doi: 10.1002/glia.20888.

Neurotrophin-3 targets the translational initiation machinery in oligodendrocytes

Rochelle P Coelho  1 , Larra M Yuelling, Babette Fuss, Carmen Sato-Bigbee
Affiliations

Neurotrophin-3 targets the translational initiation machinery in oligodendrocytes

Rochelle P Coelho et al. Glia. 2009 Dec.

Abstract

Neurotrophin-3 (NT-3) regulates oligodendrocyte (OLG) differentiation by mechanisms that remain poorly understood. Exposure of OLGs to NT-3 induces a significant increase in the levels of myelin basic protein (MBP). However, we found that this stimulation occurs in the absence of measurable effects on MBP gene promoter activation or mRNA expression, suggesting that NT-3 upregulates MBP protein expression by a posttranscriptional mechanism. Furthermore, NT-3 also causes an increase in the levels of myelin-associated glycoprotein (MAG) and myelin OLG glycoprotein (MOG), raising the possibility of a more general effect on myelin protein synthesis. Surprisingly, (35)S-methionine incorporation into total OLG proteins demonstrated a 50% increase in labeling following only a brief, 15-min treatment with NT-3. Such a remarkably fast response is unlikely due to transcriptional activation, reinforcing the possibility that NT-3 may play a crucial role in regulating protein expression by a posttranscriptional mechanism. In support of this idea, we found that NT-3 stimulates the phosphorylation of essential regulators of the initiation machinery, eukaryotic initiation factor 4E (eIF4E), and its inhibitory binding partner 4E binding protein 1 (4EBP1), two crucial players in controlling cap-dependent protein synthesis. This stimulation involves the activation of pathways mediated by ERK1/2 and PI3K/mTOR, implicating these two kinase systems as modulators of protein synthesis in developing OLGs. Altogether, these observations show for the first time that NT-3 has the capacity of targeting the translational machinery and suggest a potential stimulatory effect of this neurotrophin on myelination by direct action on protein translation in the OLGs.

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Figures

Figure 1
Figure 1. Treatment of OLGs with NT-3 results in increased MBP expression
OLGs were incubated for 3 days in CDM with or without 5, 10, 25, 50 ng/ml NT-3. MBP levels were determined by western blotting using β-actin levels as loading controls. Figures correspond to representative experiments. Results in the bar graph are expressed as percentage of controls (0 ng/ml NT-3) and represent the mean ± SEM from 3 independent experiments performed in triplicate. * p<0.005
Figure 2
Figure 2. NT-3 does not induce significant changes in MBP gene promoter activity
OLGs were transfected with a reporter construct containing the luciferase gene under control of the MBP gene promoter, and incubated for 12, 24 and 48 hrs in CDM in the absence (control) or presence of NT-3 (50 ng/ml). The luciferase activity in the samples is expressed as optical units. The results are the mean ± SEM from 3 independent experiments performed in triplicate.
Figure 3
Figure 3. Treatment of OLG cultures with NT-3 does not have a significant effect on MBP mRNA levels
OLGs cultures were incubated for 12, 24 and 48 hrs in CDM with or without NT-3 (50 ng/ml). MBP mRNA levels were determined by real-time RT-PCR using appropriate primers. Cyclophilin mRNA levels were used for normalization. Bar graphs represent steady-state levels of MBP mRNA relative to controls (not treated with NT-3) which have been set to 1. (A) Total MBP mRNA levels, (B) Exon-2 containing MBP mRNA levels. Results are the mean ± SD from two independent experiments done in duplicates.
Figure 4
Figure 4. Treatment of OLGs with NT-3 results in increased expression of MAG and MOG
OLGs were incubated for 3 days in CDM with or without 5, 10, 25, 50 ng/ml NT-3. MAG and MOG levels were determined by western blotting. Figures correspond to representative experiments. Results are expressed as percentage of controls (0 ng/ml NT-3) and represent the mean ± SEM from two independent experiments performed in triplicate. * p< 0.05, **p<0.005
Figure 5
Figure 5. NT-3 increases 35S- methionine incorporation in OLG proteins
OLGs were incubated for 15 min in F12 medium containing 10 μCi/well 35S- methionine, in the presence or absence of 50 ng/ml NT-3. 35S methionine incorporation into total protein was estimated by scintillation counting as described under “Materials and Methods”. Results are the mean ± SEM from 3 independent experiments done in triplicate. *p<0.005
Figure 6
Figure 6. Treatment of OLGs with NT-3 induces phosphorylation of eIF4E and 4EBP1
Cells were incubated in DMEM/F12 with or without 50 ng/ml NT-3. P-eIF4E and eIF4E (A), or P-4EBP1 and 4EBP1 (B) levels were determined by western blotting. Figures correspond to representative experiments. Results are expressed as percentage of controls (0 time) and represent the mean ± SEM from 3 independent experiments performed in triplicate. *p<0.05, **p<0.005
Figure 7
Figure 7. The stimulation of eIF4E and 4EBP1 phosphorylation by NT-3 involves an ERK-dependent pathway
Cells were incubated for 15 min with or without 50 ng/ml NT-3 in the presence or absence of 10 μM PD98059 (MEKi). P-ERK and ERK (A), P-eIF4E and eIF4E (B), and P-4EBP1and 4EBP1 (C) levels were determined by western blotting. Results are expressed as percentage of controls (0 time for A, DMEM/F12 alone for B and C) and represent the mean ± SEM from 3 independent experiments performed in triplicate. *p<0.05, **p<0.005, ***p<0.0001
Figure 8
Figure 8. The NT-3-dependent induction of eIF4E and 4EBP1 phosphorylation is inhibited by LY294002 and rapamycin
Cells were incubated for 15 min with or without 50 ng/ml NT-3 in the presence or absence of 30 μM LY294002 (PI3K inhibitor) (B and C) or 25 nM rapamycin (mTOR inhibitor) (D and E). P-Akt and Akt (A), P-eIF4E and eIF4E (B and D), and P-4EBP1 and 4EBP1 (C and D) levels were determined by western blotting. Results are expressed as percentage of controls (0 time for A, DMEM/F12 alone for B, C, D and E) and represent the mean ± SEM from 3 independent experiments performed in triplicate. *p<0.05, **p<0.005
Figure 9
Figure 9. Proposed mechanism of NT-3 action as a stimulator of translation initiation in OLGs
Binding of NT-3 to the TrkC receptor activates a classical ERK pathway involving Ras-GTP/Raf, MEK1/2, ERK 1/2 and MNK 1/2 as well as the PI3K/Akt/mTOR cascade leading to the phosphorylation of eIF4E and 4EBP1. Phosphorylation of 4EBP1 releases eIF4E allowing the binding of this factor to eIF4A and eIF4G to form the eIF4F initiation complex and resulting in the initiation of protein translation.
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