Elevated STEP 61 contributes to PCP-induced reduction of BDNF in cortical neurons

To address the relationship between the expression of STEP 61 and BDNF, to identify underlying mechanisms, and to establish reagents and conditions that could be extended to a PCP mouse model of SZ, we initially made use of cortical cultures. Our previous studies established that PCP treatment of cortical cultures leads to increased expression of STEP 61 [2]. We confirmed that PCP treatment (10 μM, 24 h) led to a significant increase in STEP 61 level and decreased the tyrosine phosphorylation of its substrate pERK1/2 in cortical cultures (p < 0.05, Figure S1A). Similar to previous findings [2], PCP treatment resulted in increased ubiquitination of STEP, suggesting that disruption of STEP degradation contributed to its accumulation (p < 0.01, Figure S1B). We also observed a decrease in the phosphorylation of CREB (p < 0.05, Figure S1A), a downstream target of ERK1/2 that regulates several BDNF transcripts [28, 43, 44]. Consistent with previous studies [39], PCP reduced BDNF protein level in cortical neurons as determined by Western blotting (p < 0.05, Figure S1A) and ELISA (p < 0.01, Figure S1C). ELISA analysis of conditioned medium also showed a decreased secretion of BDNF after PCP treatment (p < 0.05, Fig. 1d). Several BDNF transcripts were measured with quantitative RT-PCR (qRT-PCR). PCP decreased CREB-dependent (Exon I: p < 0.01 and IV: p < 0.001, Figure S1E) and CREB-independent (Exon VI: p < 0.05, Figure S1E) transcripts, consistent with a previous report [45].

Fig. 1 The STEP inhibitor TC-2153 prevents PCP-induced decrease in BDNF signaling. a Cortical cultures were pretreated with vehicle or TC-2153 (1 μM) 1 h prior to PCP treatment (10 μM for 24 h). Cells were lysed and subjected to Western blotting. Proteins were probed with phospho-specific- or pan-antibodies, and phospho-levels were normalized to total protein levels, and then to β-actin as loading control. BDNF levels in cell lysates (b) and culture medium (c) were assayed with ELISA. d mRNA levels of 3 BDNF transcripts (Exon I, IV, and VI) were measured with quantitative real-time PCR. Target expression levels were normalized to GAPDH as internal control. Experimental data were compared to controls and expressed as mean ± SEM. Statistical significance was determined with one-way ANOVA with post hoc Bonferroni’s test (*p < 0.05, **p < 0.01, n = 6 independent batches of cultures for a–d) Full size image

If elevated STEP 61 levels and activity contribute to the decrease in BDNF expression after PCP treatment, then STEP 61 inhibition should prevent the decrease. TC-2153, a recently characterized STEP inhibitor [33], potently inhibits STEP activity by forming a covalent bond with the active site Cys472 required for activity, without changing total or phosphorylation levels of STEP. Treatment with TC-2153 prevented PCP-induced decreases in ERK1/2 and CREB phosphorylation, and prevented the decrease in BDNF protein expression in cortical cultures (Fig. 1a). We confirmed the Western blot data with ELISA, again showing that STEP inhibition restored BDNF protein levels in cells (Fig. 1b) as well as in media (Fig. 1c).

Analysis with qRT-PCR revealed that upon PCP treatment, TC-2153 normalized levels of the two CREB-dependent BDNF transcripts (Exon I and IV, Fig. 1d), but not the CREB-independent BDNF transcript (Exon VI, Fig. 1d). Incubation with TC-2153 alone (1 μM, 1–24 h) induced a transient increase in ERK and CREB phosphorylation levels (p < 0.05 at 1 and 3 h, Figure S2A and S2B), but did not alter STEP 61 (Figure S2C), BDNF protein (Figure S2D and S2E), or mRNA (Exon I, IV and VI; Figure S1F) levels. These results demonstrate that STEP 61 inhibition is sufficient to block PCP-induced decreases in BDNF levels.

As a complementary approach, we used a lentivirus (LV)-based knockdown technique to determine whether reduction of STEP 61 expression would normalize BDNF expression after PCP treatment. shRNA virus targeting STEP 61 was added to cultured neurons at DIV7 for 7 days, followed by PCP treatment (10 μM for 24 h). A two-way ANOVA analysis showed virus infection (LV-STEP 61 shRNA) resulted in a significant decrease in STEP 61 (F(1, 20) = 78.01, p < 0.001, Fig. 2a). PCP induced an increase in STEP 61 levels in control LV-luciferase-treated cultures (p < 0.05) and a decrease in BDNF expression (p < 0.05, Fig. 2a lanes 1 and 2; representative blots in Figure S3A). In contrast, LV-STEP 61 shRNA-treated neurons showed a significant reduction in STEP 61 (p < 0.01) and a concomitant increase in pERK1/2 and pCREB levels (p < 0.05, Fig. 2a lanes 1 and 3). Importantly, PCP failed to induce changes in pERK, pCREB, or BDNF levels in the presence of STEP 61 shRNA virus (p > 0.05, Fig. 2a lanes 3 and 4).

Fig. 2 STEP 61 contributes to the PCP-induced decrease in BDNF expression in cortical cultures. a Cortical neurons were infected with lentivirus containing luciferase vector (LV-Luc) or STEP shRNA (LV-shRNA) for 7 days, followed by control or PCP treatment (10 μM) for 24 h. b Cortical neurons from WT or STEP KO mice were treated with control or PCP (10 μM) for 24 h. c STEP 61 was added back to cortical neurons derived from STEP KO mice by infecting with AAV1/2 STEP 61 (AAV-STEP 61 ) for 7 days, followed by control or PCP treatment. Neurons were lysed after treatment and subjected to Western blotting. STEP 61 and its substrates were probed with phospho-specific- or pan-antibodies, and phospho-levels were normalized to total protein levels, and then to β-actin as loading control. Data were expressed as mean ± SEM, and statistical significance was determined using one-way ANOVA with post hoc Bonferroni’s test (*p < 0.05, **p < 0.01, n = 6 independent batches of cultures for a–c) Full size image

The direct involvement of STEP 61 in the regulation of BDNF by PCP was evaluated in cultures derived from STEP KO mice, combined with rescue experiments, where STEP 61 was added back to the KO cultures. Wild-type (WT) mouse cortical cultures treated with PCP showed similar changes in STEP 61 (p < 0.05), BDNF, pERK1/2, and pCREB (p < 0.05) levels compared to rat cortical cultures (Fig. 2b; representative blots in Figure S3B). Cultures derived from STEP KO mice did not show PCP-induced decreases in BDNF, pERK1/2, and pCREB levels (p > 0.05, Fig. 2b; representative blots in Figure S3B). In the absence of PCP administration, STEP KO cultures had elevated basal levels of pERK1/2 and pCREB (p < 0.05) as previously shown [30]. Reintroduction of STEP 61 into STEP KO cultures led to a significant decrease in basal pERK1/2 and pCREB (p < 0.05, Fig. 2c; representative blots in Figure S3C). After restoring STEP 61 to KO cultures, PCP once again increased STEP 61 (p < 0.05), decreased BDNF level (p < 0.05), and down-regulated ERK1/2 and CREB phosphorylation (p < 0.05, Fig. 2c; representative blots in Figure S3C). AAV1/2-STEP 61 alone did not alter BDNF expression in the absence of PCP administration. Taken together, these results demonstrate that STEP 61 is necessary and sufficient for PCP-induced alterations in BDNF expression in cultures.

Inhibition of STEP in vivo prevents PCP-induced down regulation of BDNF expression

We next examined the effects of TC-2153 on BDNF expression after acute PCP administration in mice (7.5 mg/kg, i.p). PCP administration resulted in elevated STEP 61 in the frontal cortex of mice (Fig. 3a), in agreement with previous findings [2]. We also found increased ubiquitination of STEP 61 upon PCP treatment (p < 0.05, Fig. 3b), suggesting the disruption of STEP 61 degradation might contribute to its accumulation upon PCP administration in mice. There was a decrease in the phosphorylation of ERK1/2 and CREB, as well as a decrease in BDNF levels, as measured by Western blot (Fig. 3a) or ELISA (Fig. 3c). TC-2153 administration alone (10 mg/kg, i.p.) did not change total STEP 61 level (Figure S4A) and, although it evoked a transient increase in pERK1/2 and pCREB levels (Figure S4B and S4C, p < 0.05 at 3 h), it did not alter BDNF protein (Figure S4D and S4E) or mRNAs levels (Figure S4F). However, when given prior to PCP administration, TC-2153 blocked PCP-induced reductions in pERK1/2, pCREB, and BDNF (Fig. 3a,c). We also found a correlated change in Exon IV of BDNF mRNA, but not of Exon I or Exon VI (Fig. 3d). These findings indicate that STEP inhibition with TC-2153 does not change baseline BDNF expression, but is sufficient to prevent PCP-induced reduction of BDNF expression in vivo.

Fig. 3 TC-2153 prevents PCP-induced decreases in BDNF signaling in vivo. a WT (C57BL/6) mice were treated with TC-2153 (10 mg/kg, i.p.) for 3 h, followed by control or PCP administration (7.5 mg/kg, i.p.) for 1 h. Total homogenates from frontal cortex were processed for Western blot analysis. Proteins were probed with phospho-specific- or pan-antibodies, and phospho-levels normalized to total protein levels, and then to β-actin as loading control. b PCP led to increased ubiquitination of STEP 61 in mice. c An ELISA assay was used to measure BDNF levels. d Total RNA was extracted from mouse frontal cortices, and mRNA levels of 3 BDNF transcripts (Exon I, IV, and VI) were measured using quantitative real-time PCR. Target expression levels were normalized to GAPDH as internal control. All data were compared to Veh/Veh, and were expressed as mean ± SEM, and statistical significance was determined using one-way ANOVA with post hoc Bonferroni’s test (for a, c, d) or Student’s t test (for b) (*p < 0.05, **p < 0.01, n = 4 C57BL/6 mice for a, c, d; n = 6 C57BL/6 mice for b) Full size image

A complementary analysis was carried out using STEP KO mice. WT and STEP KO mice were acutely injected with PCP (7.5 mg/kg, i.p.), and biochemical changes were examined in frontal cortex 1 h later. PCP administration in WT mice increased STEP 61 levels, decreased ERK1/2 and CREB phosphorylation (p < 0.05, Figure S5A), and decreased BDNF protein levels (p < 0.05, Figure S5A and S5B). Administration of PCP to STEP KO mice failed to change ERK1/2 and CREB phosphorylation levels (Figure S5A) or reduce BDNF protein levels (Figure S5A and S5B).

STEP inhibition reverses hyperlocomotion in PCP-treated mice

The previous results demonstrate that genetic reduction or pharmacological inhibition of STEP 61 reversed the effects of PCP in the biochemical analyses. Since acute administration of PCP results in hyperactive behavior [46], we next tested whether this behavior could be reversed directly with TC-2153. Acute administration of PCP (7.5 mg/kg, i.p.) led to the expected robust increase in locomotion. Two-way ANOVA with repeated measures analysis demonstrated a significant Time × Treatment interaction [F(51, 544) = 7.85, p < 0.001]. There was a significant attenuation of PCP-induced hyperlocomotion by TC-2153 (p < 0.01, compared to PCP alone) (Fig. 4a). Two-way ANOVA analysis of total distance traveled showed that there was a significant effect of pretreatment (Veh or TC-2153, F(2, 48) = 14.66, p < 0.001) and interaction with treatment (Veh or PCP, F(2, 48) = 16.40, p < 0.001). Bonferroni’s post hoc test also revealed that TC-2153 led to a significant attenuation of PCP-induced hyperactivity compared to the Veh/PCP group (p < 0.01) (Fig. 4b). TC-2153 alone did not alter locomotor activity (data not shown). In addition, pretreatment of Veh or TC-2153 had a significant effect on stereotypic grooming (F(2, 48) = 8.45, p < 0.001), and there was a significant interaction between pretreatment and treatment (Veh or PCP, F(2, 48) = 8.03, p < 0.001). Bonferroni’s post hoc test revealed TC-2153 (p < 0.05) also attenuated PCP-induced stereotypic grooming (Fig. 4c). Consistent with previous findings [46], clozapine also reversed PCP-induced hyperlocomotion and stereotypic grooming (Fig. 4).

Fig. 4 TC-2153 reverses hyperlocomotor activity in acute PCP-injected mice. Male C57BL/6 mice (4–6-month old) were administered with vehicle (Veh), TC-2153 (TC, 10 mg/kg, i.p.), or clozapine (Clz, 2 mg/kg, i.p.) followed by control (Veh/Veh) or PCP challenge (7.5 mg/kg, i.p.) for 1 h. Distance traveled every 5 min during the 90-min test period (a), total distance traveled (b), and total stereotypic counts (c) were recorded and analyzed using Activity Monitor (MED Associates). Data were expressed as mean ± SEM, and statistical significance was determined using two-way ANOVA with repeated measures followed by post hoc Bonferroni’s test (for a) or one-way ANOVA with post hoc Bonferroni’s test (for b, c) (*p < 0.05, **p < 0.01; ***p < 0.001, n = 9 C57BL/6 mice per group for a–c) Full size image

To explore the role of BDNF signaling in the observed effects of PCP in mice, we pretreated mice with a selective TrkB agonist, 7,8-dihydroxyflavone (7,8-DHF) prior to PCP administration. 7,8-DHF has a good bioavailability after peripheral administration [47–49]. We proposed that activation of BDNF/TrkB signaling by 7,8-DHF could attenuate PCP-induced hyperlocomotion. Indeed, 7,8-DHF significantly reduced hyperactivity (p < 0.001, compared to PCP alone) (Fig. 4a, b). In addition, pretreatment of 7,8-DHF also had a significant effect on PCP-induced stereotypic grooming (p < 0.01, compared to PCP alone) (Fig. 4c).

STEP inhibition prevents subchronic PCP-induced cognitive deficits

Subchronic administration of PCP in mice also disrupts cognitive function [2, 35]. We, therefore, examined whether TC-2153 might prevent these effects by using the novel object recognition (NOR) test. TC-2153 (10 mg/kg, i.p) or vehicle was administrated 3 h prior to PCP administration (5 mg/kg, i.p., twice daily) over 5 days, at which point animals were trained in the NOR task and were tested 24 h later for memory retention. A discrimination index was calculated to compare memory retention. Vehicle-treated mice spent more time exploring the novel object, whereas mice injected with PCP did not (treatment effect: (F(2, 23) = 7.001), p < 0.01). In contrast, the PCP/TC-2153-treated group performed similarly to the control group not given PCP (p > 0.05, Fig. 5a). TC-2153 alone did not change recognition memory (data not shown). Similarly, 7,8-DHF (5 mg/kg, i.p.; given 1 h before PCP) reversed PCP-induced cognitive deficits (Fig. 5a).

Fig. 5 TC-2153 prevents PCP-induced decrease of BDNF expression during memory consolidation and memory deficits in the novel object recognition (NOR) test. a Male C57BL/6 mice (4–6-month old) were pretreated with vehicle (Veh) or TC-2153 (TC, 10 mg/kg, i.p.) followed by vehicle or subchronic PCP administration (5 mg/kg, i.p. twice daily) for 5 days, followed by a 1-week drug-free break. A discrimination index was used to evaluate memory retention in different treatment groups. b A second cohort of mice was sacrificed 9 h post-training in the NOR test. BDNF and STEP 61 levels were measured in synaptosomal fraction (P2) from hippocampus. Data were expressed as mean ± SEM. One-way ANOVA with post hoc Bonferroni’s test was performed to determine statistical significance (*p < 0.05, n = 9 C57BL/6 mice per group for a; n = 6 C57BL/6 mice per group for b) Full size image

BDNF signaling is also implicated in memory consolidation in several behavioral paradigms, including the NOR task [50]. We, therefore, analyzed the effects of STEP 61 inhibition on BDNF levels after training in the NOR test. Mice were subjected to subchronic PCP administration (5 mg/kg, i.p., twice daily for 5 days followed by 1 week break) and 9 h post-training, at a time point within the critical period for memory consolidation [50], and hippocampi were collected for biochemical analysis. Subchronic PCP administration reduced BDNF and increased STEP 61 levels at this time point (Fig. 5b). Administration of TC-2153 or 7,8-DHF normalized BDNF levels in PCP-treated mice (TC/PCP vs. Veh/PCP or DHF/PCP vs. Veh/PCP, p < 0.05, Fig. 5b).