Time- and concentration-dependent inhibition of TrkB expression by VPA. (A) SH-SY5Y cells were incubated in the presence of 1 mM VPA for the indicated periods of time. Zero time samples were treated with vehicle and used as control. (B) SH-SY5Y cells were incubated for 24 hours in the presence of either vehicle or VPA at the indicated concentrations. Cell lysates were analyzed for TrkB expression. Values are the mean ± S.E.M. of four experiments. *P < 0.05; ***P < 0.001 vs. control (vehicle) by analysis of variance (ANOVA) followed by Newman-Keuls post hoc test.

VPA reduces plasma membrane expression of TrkB. SH-SY5Y cells were treated with either vehicle or VPA (1 mM) for 24 hours. Cells were then exposed to the cell impermeant biotinylating agent sulfosuccinimidyl-6-(biotin-amido)hexanoate and solubilized proteins were precipitated by using streptavidin-conjugated agarose beads. The precipitate (surface protein) and total extract (cell lysate) were analyzed for TrkB by Western blot (A). TrkB levels in the surface protein preparation were normalized to pan-cadherin levels, a plasma membrane (membr) marker. Densitometric values are expressed as percent of control (vehicle) and are the mean ± S.E.M. of four experiments. (B) TrkB expression was analyzed by immunofluorescence (green color) in nonpermeabilized cells with an antibody directed against an extracellular domain of the receptor. Nuclei were stained in blue with 4′,6-diamidino-2phenylindole (DAPI). Values are the mean ± S.E.M. of four experiments. Scale bar, 25 µm. ***P < 0.001 vs. control (vehicle) by Student’s t test.

Inhibition of BDNF-induced TrkB activation and signaling by VPA. (A–D) SH-SY5Y cells were incubated for 24 hours with either vehicle or 1 mM VPA and then exposed for 5 minutes to either vehicle or 1 nM BDNF. Cell lysates were analyzed for the expression of phospho-Tyr706/707-TrkB (pTrkB), total TrkB-FL (A), phospho-Tyr783-PLCγ1 (pPLCγ1), total PLCγ1 (B), phospho-Thr308-Akt (pAkt), total Akt (C), phospho-Ser9-GSK-3β (pGSK-3β), and total GSK-3β (D). Values are the mean ± S.E.M. of four experiments. (E and F) SH-SY5Y cells were incubated for 24 hours with either vehicle or 1 mM VPA and then exposed for 5 minutes to either vehicle, 0.05 nM BDNF (BDNF 0.05), or 0.1 nM BDNF (BDNF 0.1). Cell lysates were analyzed for phospho-ERK1/2 (pERK1/2), total ERK1/2, phospho-Ser235/236-S6 ribosomal protein (pS6rp), and total S6 ribosomal protein (S6rp). Values are the mean ± S.E.M. of three experiments. (G and H) LAN-1 (G) and Kelly (H) cells were incubated for 24 hours with either vehicle or 1 mM VPA and then exposed for 5 minutes to either vehicle or 1 nM BDNF. Values are the mean ± S.E.M. of three experiments. *P < 0.05; ***P < 0.001 vs. control (vehicle); ^#P < 0.05; ^###P < 0.001 vs. BDNF + vehicle by ANOVA followed by Newman-Keuls post hoc test.

VPA curtails BDNF-induced proneurotrophic and antiapoptotic responses. (A) SH-SY5Y cells were preincubated for 24 hours with either vehicle or 1 mM VPA and then exposed for 48 hours to either vehicle or 1.0 nM BDNF. Cells were fixed and stained with an antiactin antibody (red color). Nuclei were stained in blue with DAPI. For each sample, the number of neurite-bearing cells was determined and calculated as percent of total cells. Values are the mean ± S.E.M. of three separate experiments. (B–E) Cells were preincubated for 24 hours with either vehicle, VPA 0.6 mM (VPA 0.6), or VPA 1.0 mM (VPA 1.0) and then exposed to either vehicle or 1 nM BDNF for 48 hours. Cell lysates were analyzed for the expression of MAP2 (B), synapsyn-1 (synap-1) (C), GluR1 (D), and postsynaptic density 95 (PSD95) (E). Values are the mean ± S.E.M. of four experiments. (F) SH-SY5Y cells were pretreated for 24 hours with either vehicle or 1 mM VPA and then incubated in serum-free medium in the absence and in the presence of 1 nM BDNF for 48 hours. Cells were fixed and analyzed by immunofluorescence for cleaved caspase 3 expression (green color). Cell nuclei were stained with DAPI. For each sample, the number of cleaved caspase 3-positive cells was determined and calculated as percent of total cells. Values are the mean ± S.E.M. of three experiments. (G) SH-SY5Y cells were treated as indicated in (F) and cell lysates were analyzed for cleaved PARP (cleav PARP) and uncleaved PARP (PARP). Values are the mean ± S.E.M. of three experiments. *P < 0.05; ***P < 0.001 vs. control (vehicle); ^#P < 0.05; ^###P < 0.001 vs. BDNF alone by ANOVA followed by Newman-Keuls post hoc test.

HDAC inhibitors and HDAC1 knockdown mimic the inhibitory effect of VPA on TrkB expression and signaling. (A–C) SH-SY5Y cells were incubated for 24 hours with either vehicle, 1 mM sodium butyrate, 300 nM trichostatin A (TSA), 10 µM MC1568 (A), 1 µM entinostat (B), or 40 nM romidepsin (C). Cell lysates were then analyzed for TrkB expression. Values are the mean ± S.E.M. of four experiments. *P < 0.05; ***P < 0.001 vs. the respective control (vehicle) by Student’s t test. (D) SH-SY5Y cells were incubated for 24 hours with either vehicle or 1 µM entinostat and then TrkB expression was analyzed by immunofluorescence (green color) in nonpermeabilized cells stained with an antibody directed against an extracellular domain of the receptor. Nuclei were stained in blue with DAPI. Images are representative of four independent experiments. Scale bar, 25 µm. (E and F) SH-SY5Y cells were incubated for 24 hours with either vehicle, 4 µM PCI-34051, 5 µM tubacin (E), or 1 mM valpromide (VPM) (F). Values are the mean ± S.E.M. of four experiments. (G and H) SH-SY5Y cells were preincubated for 24 hours with either vehicle, 300 nM TSA, 1 mM VPM (G), 1 mM sodium butyrate, or 10 µM MC1568 (H) and then exposed for 5 minutes to 1 nM BDNF. Values are the mean ± S.E.M. of three experiments. (I and J) SH-SY5Y cells were preincubated for 24 hours with either vehicle, 0.5 µM entinostat (ent 0.5), 1 µM entinostat (ent 1) (I), 20 nM romidepsin (romi 20), or 40 nM romidepsin (romi 40) (J) and then exposed for 5 minutes to 1 nM BDNF. Values are the mean ± S.E.M. of four experiments. ***P < 0.001 vs. control; ^#P < 0.05; ^###P < 0.001 vs. BDNF + vehicle by ANOVA followed by Newman-Keuls post hoc test. (K and L) SH-SY5Y cells were transfected with either control siRNA or HDAC1 siRNA duplexes and cell lysates were analyzed for HDAC1, H3 acetyl-Lys9/Lys14 (Ac-H3), total H3 (K), and TrkB expression (L). Each lane was loaded with a sample of an independent transfection. Values are the mean ± S.E.M. of three experiments. *P < 0.05; ***P < 0.001 vs. control siRNA by Student’s t test.

Involvement of RUNX3 in VPA-induced downregulation of TrkB. (A) SH-SY5Y cells were incubated with 1 mM VPA for the indicated periods of time. Zero time samples were treated with vehicle and used as control. (B) Cells were treated for 24 hours with the indicated concentrations of VPA. Cell lysates were analyzed for the protein levels of RUNX3 and H3-acetyl-Lys9/Lys14 (Ac-H3). Values are the mean ± S.E.M. of four experiments. *P < 0.05; ***P < 0.001 vs. control (vehicle) by ANOVA followed by Newman-Keuls post hoc test. (C) SH-SY5Y cells were incubated with either vehicle or 1 mM VPA for 24 hours. Nuclear extracts were prepared and analyzed for RUNX3 and HDAC1 levels. Values are the mean ± S.E.M. of four experiments. ***P < 0.001 by Student’s t test. (D) SH-SY5Y cells transfected with either control siRNA or RUNX3 siRNA were incubated for 24 hours with either vehicle, 0.6 mM VPA (VPA 0.6), or 1 mM VPA (VPA 1.0). Cell lysates were then analyzed for RUNX3 and TrkB expression. Values are the mean ± S.E.M. of three experiments. ***P < 0.001 vs. control siRNA + vehicle; ^#P < 0.05; ^###P < 0.001 vs. the corresponding value in control siRNA-treated group by Student’s t test. (E) SH-SY5Y cells were treated for 24 hours with either vehicle or 1 mM VPA. Cell lysates were analyzed for EZH2 protein levels. Values are the mean ± S.E.M. of four experiments. ***P < 0.001 vs. control (vehicle) by Student’s t test. (F) Cells were transfected with either control siRNA or HDAC1 siRNA duplexes and cell lysates were analyzed for HDAC1, EZH2, RUNX3, and TrkB protein levels. Values are the mean ± S.E.M. of three experiments. *P < 0.05; ***P < 0.001 vs. the corresponding value in control siRNA-treated group by Student’s t test. (G) Cells were treated for 24 hours with either vehicle, 1 µM DZNep (DZNep 1), or 5 µM DZNep (DZNep 5). Cell lysates were then analyzed for EZH2, RUNX3, and TrkB expression. Values are the mean ± S.E.M. of four experiments. *P < 0.05; ***P < 0.001 vs. control (vehicle) by Student’s t test.