BDNF Induces Striatal-Enriched Protein Tyrosine Phosphatase 61 Degradation Through the Proteasome

Abstract

Brain-derived neurotrophic factor (BDNF) promotes synaptic strengthening through the regulation of kinase and phosphatase activity. Conversely, striatal-enriched protein tyrosine phosphatase (STEP) opposes synaptic strengthening through inactivation or internalization of signaling molecules. Here, we investigated whether BDNF regulates STEP levels/activity. BDNF induced a reduction of STEP61 levels in primary cortical neurons, an effect that was prevented by inhibition of tyrosine kinases, phospholipase C gamma, or the ubiquitin-proteasome system (UPS). The levels of pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204), two STEP substrates, increased in BDNF-treated cultures, and blockade of the UPS prevented STEP61 degradation and reduced BDNF-induced GluN2B and ERK1/2 phosphorylation. Moreover, brief or sustained cell depolarization reduced STEP61 levels in cortical neurons by different mechanisms. BDNF also promoted UPS-mediated STEP61 degradation in cultured striatal and hippocampal neurons. In contrast, nerve growth factor and neurotrophin-3 had no effect on STEP61 levels. Our results thus indicate that STEP61 degradation is an important event in BDNF-mediated effects.

Keywords: Depolarization; ERK1/2; GluN2B; NGF; NT-3; PLCγ; STEP33.

Fig. 1

Effect of BDNF on STEP₆₁ levels in primary cortical neurons. a The expression of TrkB was analyzed by Western blot of protein extracts obtained from mouse primary cortical cultures. Mouse adult tissue served as positive control. Primary cortical cultures were incubated with 10 ng/ml BDNF b for 24 h or c during different time periods up to 6 h, and STEP₆₁ levels were examined by Western blot. d STEP₆₁ and pTrkB^Tyr816 levels were analyzed in cortical cultures treated for 60 min with or without the tyrosine kinase inhibitor K252a (200 nM; K) and then incubated in the presence or absence of BDNF (10 ng/ml; B) for additional 15 min. Representative immunoblots are shown. Values obtained by densitometric analysis of Western blot data are expressed as percentage of control (Ctr) cultures incubated in the absence of BDNF and are shown as mean ±SEM of three to seven experiments performed in duplicate in independent cultures. Data were analyzed by one-way ANOVA with Dunnett’s (c), or Bonferroni’s (d) post hoc test. **p<0.01, and ***p<0.001 compared with Ctr cultures; ###p<0.001 compared with cultures incubated with BDNF alone. e STEP and MAP2 were analyzed by immunocytochemistry in untreated (Ctr) and BDNF-treated (10 ng/ml, 15 min) cortical cultures. High magnification insets are shown. Arrows denote loss of STEP immunoreactivity in a dendrite

Fig. 2

PLCγ mediates the degradation of STEP₆₁ by BDNF in primary cortical cultures. Mouse primary cortical cultures were treated for 60 min with or without a the MAPK inhibitor PD98059 (25 μM; PD), b the PI-3 K inhibitor wortmannin (50 nM; W), or c the PLC inhibitor U73122 (5 μM; U) and then incubated in the presence or absence of BDNF (10 ng/ml; B) for additional 15 min. STEP₆₁ and a pERK1/2^{Thr202/Tyr204}, b pAkt^Ser473, or c pPLCγ^Tyr783 were examined by Western blot. Representative immunoblots are shown. Values obtained by densitometric analysis of Western blot data are expressed as percentage of control (Ctr) cultures and are shown as mean±SEM of three experiments performed in duplicate in independent cultures. Data were analyzed by one-way ANOVA with Bonferroni’s post hoc test. *p<0.05 and **p<0.01 compared with Ctr cultures; ##p<0.01 compared with cultures incubated with BDNF alone

Fig. 3

BDNF promotes STEP₆₁ ubiquitination and degradation through the proteasome. a STEP₆₁ levels were analyzed by Western blot of protein extracts obtained from mouse primary cortical cultures treated for 60 min with or without the proteasome inhibitor MG-132 (10 μM; MG) and then incubated in the presence or absence of BDNF (10 ng/ml; B) for additional 15 min. b The levels of STEP₆₁-ubiquitin conjugates were determined in protein extracts from control cultures and cultures exposed for 15 min to BDNF (10 ng/ml) in the presence of MG-132 (10 μM) and subjected to ubiquitin (Ub) pulldown using Agarose-TUBE2 and immunoblotted (IB) with anti-STEP and anti-ubiquitin antibodies. Representative immunoblots are shown. Values obtained by densitometric analysis of Western blot data are expressed as percentage of control (Ctr) cultures and are shown as mean±SEM of four to seven experiments performed in duplicate in independent cultures. Data were analyzed by one-way ANOVA with Bonferroni’s post hoc test. ***p<0.001 compared with Ctr cultures; ##p<0.01 compared with cultures incubated with BDNF alone

Fig. 4

Effect of BDNF-induced STEP₆₁ degradation on GluN2B^Tyr1472 and ERK1/2^{Thr202/Tyr204} phosphorylation levels in primary cortical cultures. a STEP₆₁, b pGluN2B^Tyr1472, and c pERK1/2^{Thr202/Tyr204} levels were analyzed by Western blot of protein extracts obtained from mouse primary cortical cultures treated for 60 min in the presence or absence of the proteasome inhibitor MG-132 (10 μM; MG) and then incubated with or without 10 ng/ml BDNF (B) for additional 60 min. Representative immunoblots are shown. Values obtained by densitometric analysis of Western blot data are expressed as percentage of control (Ctr) cultures and shown as mean±SEM of four to eight experiments performed in duplicate in independent cultures. Data were analyzed by one-way ANOVA with Bonferroni’s post hoc test. *p<0.05, **p<0.01, and ***p<0.001 compared with Ctr cultures; ##p<0.01 and ###p<0.001 compared with cultures incubated with BDNF alone

Fig. 5

BDNF induces STEP₆₁ degradation in primary striatal and hippocampal cultures through the proteasome. a The expression of TrkB was analyzed by Western blot of protein extracts obtained from mouse primary striatal and hippocampal cultures at DIV 8. Mouse adult striatal and hippocampal tissue served as positive control. b The expression of STEP was analyzed by Western blot of protein extracts obtained from mouse striatal and hippocampal adult tissue and cultured neurons at DIV 8. Representative immunoblots are shown. STEP₆₁ levels were analyzed by Western blot of protein extracts obtained from primary c striatal and d hippocampal cultures treated for 60 min with or without the proteasome inhibitor MG-132 (10 μM; MG) and then incubated in the presence or absence of BDNF (10 ng/ml; B) for additional 15 min. Representative immunoblots are shown. Values obtained by densitometric analysis of Western blot data are expressed as percentage of control (Ctr) cultures and are shown as mean±SEM of three to six experiments performed in duplicate in independent cultures. Data were analyzed by one-way ANOVA with Bonferroni’s post hoc test. ***p<0.001 compared with Ctr cultures; #p<0.05 and ###p<0.001 compared with cultures incubated with BDNF alone

Fig. 6

NGF and NT-3 have no effect on STEP₆₁ levels in primary neurons. a The expression of TrkA and TrkC was analyzed by Western blot of protein extracts obtained from mouse primary cortical, striatal, and hippocampal cultures. Mouse adult tissue served as positive control. b Mouse primary cortical, striatal, and hippocampal cultures were incubated in the presence or absence of NGF, NT-3, or BDNF (10 ng/ml) for 15 min and the levels of STEP₆₁, pPLCγ^Ser783, pAkt^Ser473, and pERK1/2^{Thr202/Tyr204} were examined by Western blot. c The expression of TrkA, TrkB, and TrkC was analyzed by Western blot of protein extracts obtained from rat primary cortical cultures. Mouse adult cortical tissue served as positive control. d Rat primary cortical neurons were incubated in the presence or absence of NGF, NT-3, or BDNF (10 ng/ml) for 15 min and the levels of STEP₆₁, pPLCγ^Ser783, pAkt^Ser473, and pERK1/2^{Thr202/Tyr204} were examined by Western blot. Representative immunoblots are shown

Fig. 7

STEP₆₁ levels after cell depolarization. a STEP₆₁ and pPLCγ^Ser783 levels were analyzed by Western blot of protein extracts obtained from mouse primary cortical cultures incubated with or without ANA-12 (100 μM; ANA) or MG-132 (10 μM; MG) for 60 min and then stimulated with 50 mM KCl for 5 min. b The levels of STEP and spectrin breakdown products (SBDPs) at 145–150 kDa were analyzed by Western blot of protein extracts obtained from mouse primary cortical cultures incubated with 50 mM KCl for 60 min. Representative immunoblots are shown. Values obtained by densitometric analysis of Western blot data are expressed as percentage of control (Ctr) cultures and shown as mean±SEM of three to five experiments performed in duplicate in independent cultures. Data were analyzed by one-way ANOVA with Bonferroni’s post hoc test (a) and Student’s t test (b). ***p<0.001 compared with Ctr cultures; #p<0.05 compared with cultures incubated with KCl alone