Inhibition of the tyrosine phosphatase STEP61 restores BDNF expression and reverses motor and cognitive deficits in phencyclidine-treated mice
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Brain-derived neurotrophic factor (BDNF) and STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61) have opposing functions in the brain, with BDNF supporting and STEP61 opposing synaptic strengthening. BDNF and STEP61 also exhibit an inverse pattern of expression in a number of brain disorders, including schizophrenia (SZ). NMDAR antagonists such as phencyclidine (PCP) elicit SZ-like symptoms in rodent models and unaffected individuals, and exacerbate psychotic episodes in SZ. Here we characterize the regulation of BDNF expression by STEP61, utilizing PCP-treated cortical culture and PCP-treated mice. PCP-treated cortical neurons showed both an increase in STEP61 levels and a decrease in BDNF expression. The reduction in BDNF expression was prevented by STEP61 knockdown or use of the STEP inhibitor, TC-2153. The PCP-induced increase in STEP61 expression was associated with the inhibition of CREB-dependent BDNF transcription. Similarly, both genetic and pharmacologic inhibition of STEP prevented the PCP-induced reduction in BDNF expression in vivo and normalized PCP-induced hyperlocomotion and cognitive deficits. These results suggest a mechanism by which STEP61 regulates BDNF expression, with implications for cognitive functioning in CNS disorders.
KeywordsBDNF Phencyclidine Schizophrenia STEP inhibitor Ubiquitination
Adeno-associated virus of mixed serotype ½
Brain-derived neurotrophic factor
Days in vitro
Extracellular-signal regulated kinase
Novel object recognition
Proline-rich tyrosine kinase 2
- RIPA buffer
Radio-immunoprecipitation assay buffer
Sodium dodecyl sulfate
Standard error of the mean
Short hairpin RNA
Single nucleotide polymorphism
STriatal-Enriched protein tyrosine Phosphatase, 61 kDa
Brain-derived neurotrophic factor (BDNF) and STriatal-Enriched protein tyrosine Phosphatase; STEP, PTPN5) are both implicated in the pathophysiology of schizophrenia (SZ) [1, 2]. BDNF expression is reduced in animal models of SZ , and postmortem studies show significant decreases in BDNF and its high-affinity receptor TrkB in the cortex and hippocampus of SZ patients [4, 5]. Moreover, significant reductions in BDNF levels are found in the plasma of SZ patients during their first psychotic episode  and are reversed by neuroleptic treatment . BDNF heterozygote and TrkB receptor conditional knock out (KO) mice display SZ-related behaviors that include increased locomotor activity, deficits in sensorimotor gating, and impaired cognitive functions [8, 9]. In addition, the association of a single nucleotide polymorphism (SNP) of the BDNF gene (rs6265; Val66Met) has been found in studies predominantly in Caucasian samples, although contrary reports also exist .
The STEP-family of tyrosine phosphatases is alternatively spliced from a single gene to produce several members of which STEP61 is a membrane-associated isoform enriched at post-synaptic compartments and the endoplasmic reticulum [11, 12]. STEP61 is the only isoform expressed in cortex . Substrates of STEP include the GluN2B subunit of the NMDA receptor , the GluA2 subunit of the AMPA receptor , and the kinases ERK1/2, Fyn, and Pyk2 [16, 17, 18]. Dephosphorylation of the glutamate receptors results in internalization of GluN1/GluN2B and GluA1/GluA2, while dephosphorylation of regulatory tyrosines of the kinases leads to their inactivation. The current model of STEP function is that it normally opposes the development of synaptic strengthening .
STEP61 is elevated in human postmortem samples from SZ patients and in psychotomimetic mouse models . STEP KO mice are resistant to the locomotor, and cognitive effects of psychotomimetics and neuroleptic treatment of mice result in STEP61 inactivation . Moreover, a case–control study found nominal association between PTPN5 SNP rs4075664 and SZ in all the samples examined and a significant association of two additional SNPs (rs2278732 and rs4757710) in male samples from an Israeli Jewish cohort . These studies indicate that BDNF signaling is generally low, while STEP61 signaling is generally high in SZ patients and in animal models of SZ.
There is crosstalk between BDNF expression and N-methyl-D-aspartate receptor (NMDAR) signaling [21, 22, 23], and BDNF potentiates NMDAR function through activation of ERK1/2 and Fyn [24, 25]. On the other hand, NMDAR signaling is known to increase activity-dependent transcription and secretion of BDNF [26, 27, 28, 29]. Notably, both ERK1/2 and Fyn are tyrosine dephosphorylated and inactivated by STEP [16, 17, 30]. Mice null for STEP shows increased tyrosine phosphorylation of these substrates [30, 31, 32] and increased localization of NMDAR at synaptic membranes . Moreover, pharmacological inhibition of STEP61 by a recently discovered inhibitor, TC-2153, also resulted in increased tyrosine phosphorylation of STEP substrates, showed relative specificity to STEP compared to other PTPs, increased the distribution of NMDAR at synaptic membranes, and reversed cognitive deficits in a mouse model of Alzheimer’s disease .
Noncompetitive NMDAR antagonists, such as the psychotomimetics phencyclidine (PCP), ketamine, and MK-801, are used to model SZ-like symptoms in humans, rodents, and nonhuman primates [34, 35, 36], supporting aspects of the glutamate hypothesis of SZ [37, 38]. A previous study showed that PCP treatment led to the accumulation of STEP61 , while a second study found decreased BDNF expression upon PCP treatment in cultures . However, it remains unclear whether elevated STEP61 contributes to the reduction of BDNF and whether the regulation of BDNF by STEP61 has functional consequence in vivo. Here we examined the relationship of STEP61 activity and BDNF expression, and the functional consequences of their disruption in PCP-treated cortical culture and a mouse model of SZ. STEP61 expression was increased, while BDNF levels were decreased upon PCP administration both in cultures and in mice. Genetic and pharmacological techniques to decrease STEP61 activity in these models normalized BDNF expression and rescued motor and cognitive deficits. These findings suggest that STEP61 regulates BDNF expression and contributes to the observed balance between BDNF and STEP61 signaling that may explain aspects of the pathophysiology of SZ.
Materials and methods
Antibodies and reagents
Antibodies are listed in Supplementary Table 1. PCP was purchased from Sigma (Ronkonkoma, NY); the proteasome inhibitors MG-132 and lactacystin were obtained from Calbiochem (San Diego, CA, USA). The tyrosine kinase inhibitor K252a, the TrkB agonist 7,8-DHF, and the neuroleptic clozapine were purchased from Tocris Biosciences (Ellisville, MO, USA). TC-2153 was synthesized as previously described .
Primary cortical cultures
All experimental procedures were approved by the Yale University Institutional Animal Care and Use Committee and were in strict accordance with the NIH Guide for the Care and Use of Laboratory Animals. Primary cortical neurons were isolated from rat or mouse E18 embryos as described . Neuronal cultures were maintained in a Neurobasal medium with B27 supplement (Invitrogen, San Diego, CA, USA) for 12–14 days. Cultures were then treated with PCP (10 μM) for 24 h, while in some experiments, the STEP inhibitor (TC-2153, 1 μM) was added 1 h prior to PCP and was present throughout the 24-h PCP treatment. Neurons were lysed in 1× RIPA buffer (Pierce Biotechnology, Rockford, IL, USA) with complete phosphatase and protease inhibitors (Roche, Indianapolis, IN, USA). Lysates were spun at 1000×g for 10 min to isolate homogenates.
Lentivirus-based STEP shRNA (LV-STEP) was validated as described [31, 40, 41]. Rat cortical cultures were infected with LV-STEP or a luciferase control at DIV 7 for 7 days. A recombinant adeno-associated virus of mixed serotype 1/2 (AAV1/2) was custom made (GeneDetect LTD, Auckland, New Zealand), and validated as described . At DIV 5, STEP KO mouse cortical neurons were infected with AAV1/2-STEP61 or AAV1/2-control vector for 7 days prior to PCP exposure.
BDNF levels in rat cortical neuronal cultures (lysates and conditioned media) or frontal cortical tissues were quantified using an Emax ImmunoAssay ELISA kit (Promega, Madison, WI, USA) following the manufacturer’s instructions. Briefly, cells were lysed in RIPA buffer with protease and phosphatase inhibitors. Lysates were centrifuged at 14,000×g for 20 min, and the supernatants were added to microplates pre-coated with a mouse anti-BDNF monoclonal antibody and incubated for 2 h at room temperature (RT). After washes, a rabbit anti-BDNF polyclonal antibody was added for another 2 h at RT. After extensive washes, an anti-IgY HRP conjugate was incubated for 1 h, followed by 3,3′,5,5′-tetramethylbenzidine (TMB) solution. The reaction was stopped with 1 N HCl, and optical density reading was obtained using an ELx800 plate reader (Biotek, Winooski, VT, USA) at 450 nm. To measure BDNF secreted into media, media (1 ml) was collected after treatments and concentrated 10× by using Amicon Ultra Filters with a 3 kDa cutoff (Millipore, Billerica, MA, USA). A standard curve was used to determine BDNF levels in lysates, which were expressed as pg/μg in lysates or pg/ml in media.
Total RNA extraction and quantitative reverse transcription (RT)-PCR
Total RNA was extracted from cortical cultures using RNeasy kit (Qiagen, Valencia, CA, USA) and from cortical tissues using RNeasy lipid tissue kit (Qiagen), following the manufacturer’s instructions. RNA quantification was done by a nanodrop spectrophotometer (Thermo Scientific, Wilmington, DE, USA), and 1 μg of total RNA was used in reverse transcription reactions to obtain complimentary DNA (cDNA) using a Quanti Tech reverse transcription kit (Qiagen). Real-time quantitative PCR was performed in 96-well plates using a StepOnePlus Real-Time PCR Systems (v2.3, Applied Biosystems) kit and Syber Green PCR master mix (Applied Biosystems, Carlsbad, CA, USA). Levels of target mRNAs were normalized to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA. The forward primers used for BDNF: Exon I (5′-CTCAAAGGGAAACGTGTCTCT-3′), Exon IV (5′-TGCGAGTATTACCTCCGCCAT-3′), Exon VI (5′-TTGGGGCAGACGAGAAAGCGC-3′) and a common reverse primer: (5′-TCACGTGCTCAAAAGTGTCAG-3′), GAPDH forward primer: (5′-TCCATGACAACTTTGGCATTGTGG-3′), and reverse primer: (5′-GTTGCTGTTGAAGTCGCAGGAGAC-3′) were synthesized at the Yale Keck Center as described .
Drug administration for biochemical analysis
Male C57BL/6J mice (3–4-month old) were obtained from the Jackson Laboratory (Bar Harbor, Maine). Mice were injected with vehicle (2 % DMSO in saline) or TC-2153 (10 mg/kg, i.p.) followed by PCP administration (7.5 mg/kg, i.p. for 1 h). The effective doses of TC-2153 and PCP were chosen based on previous findings [2, 33]. Frontal cortices were collected after PCP administration and processed by differential centrifugation to get crude synaptosomal membrane fractions (P2) as described . Briefly, brain tissue was homogenized in TEVP (in mM): 10 Tris–HCl, pH 7.4, 1 EDTA, 1 EGTA, and 320 sucrose with protease and phosphatase inhibitors (Roche, Indianapolis, IN, USA). Homogenates were spun at 1000×g for 10 min to obtain supernatant (S1), and S1 was further spun at 12,000×g for 15 min to obtain the crude synaptosomal membrane fraction (P2). P2 was solubilized in the TEVP buffer with brief sonication and saved for later analysis.
Measurement of ubiquitinated STEP after PCP treatment
Ubiquitinated proteins were pulled down as described . Briefly, lysates of PCP-treated (10 μM, 24 h) neurons or P2 fractions from PCP-administrated (7.5 mg/kg, i.p. for 1 h) mouse frontal cortices were incubated with agarose-TUBE2 beads (Tandem Ubiquitin Binding Entity, LifeSensors, Malvern, PA, USA) overnight at 4 °C. Ubiquitinated STEP species were visualized using anti-STEP antibody.
Samples were resolved on 8 or 12 % SDS-PAGE, transferred onto nitrocellulose membranes (Bio-Rad, Richmond, CA, USA), and incubated with primary antibodies (Supplementary Table 1) overnight at 4 °C, and peroxidase-conjugated secondary antibodies (1:5000; Pierce) for 2 h at RT. Immunoreactivity was developed with a Chemiluminescent Substrate kit (Pierce) and visualized by a G:BOX with the GeneSnap software (Syngene, Cambridge, UK). All densitometric bands were quantified with the Genetools program (Syngene).
Male WT C57BL/6 mice (3–5-month old) were used for behavioral tests. Mice were injected i.p. with vehicle (2 % DMSO in saline), TC-2153 (10 mg/kg in 2 % DMSO), 7,8-DHF (5 mg/kg in 2 % DMSO), or clozapine (2 mg/kg in 2 % DMSO). Thirty min after drug pretreatment, mice were placed in an activity chamber (MED Associates, St. Albans, VT, USA) for 30 min of baseline recording, followed by Veh or PCP injection (7.5 mg/kg, i.p.), and monitored for an additional 1 h. Horizontal activity and stereotypies were measured using the Activity Monitor version 5 software (MED Associates, St. Albans, VT, USA).
Novel object recognition (NOR)
Male C57BL/6 mice were administrated PCP (5 mg/kg, i.p. twice daily) for 5 days, followed by a 1-week break. Some groups of mice were administrated i.p. with vehicle (Veh, 3 h prior to the first PCP injection, daily), TC-2153 (10 mg/kg, 3 h prior to the first PCP injection, daily), or 7,8-DHF (5 mg/kg, 1 h prior to the first PCP injection, daily). Mice were off treatment during the 1-week break. We used a three-day paradigm for the NOR test as described . On the first day, mice were habituated to an open-field box for 10 min. On the second day, mice were injected i.p. with Veh (2 % DMSO in saline, 3 h prior to training), TC-2153 (10 mg/kg, 3 h prior to training), or 7,8-DHF (5 mg/kg, 1 h prior to training). Mice were returned to the same box with two identical objects and allowed to explore both for a total of 30 s. A cohort of mice was sacrificed for biochemical analysis 9 h after the training session, and another cohort was run through the behavioral test. Twenty-four hours after training (3rd day), mice were subjected to another trial (3 min), this time with one familiar and one novel object. The location of objects was counter balanced to minimize bias. Mouse activity was recorded throughout the NOR test with an ANY-maze video system (Stoelting, Wood Dale, IL, USA). Time spent with the novel or familiar object was used to calculate a discrimination index (DI) = (novel time − familiar time)/(novel time + familiar time).
Data are expressed as mean ± SEM, and statistical analysis were performed with IBM SPSS Statistics 19 and Prism 5.0 (GraphPad Software, La Jolla, CA, USA). Significance (p < 0.05) was determined by Student’s t test, one-way analysis of variance (ANOVA,) or two-way ANOVA with Bonferroni’s post hoc test. For locomotion, data were analyzed with two-way ANOVA with repeated measures, with Bonferroni’s post hoc or paired t test.
Elevated STEP61 contributes to PCP-induced reduction of BDNF in cortical neurons
If elevated STEP61 levels and activity contribute to the decrease in BDNF expression after PCP treatment, then STEP61 inhibition should prevent the decrease. TC-2153, a recently characterized STEP inhibitor , 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 STEP61 (Figure S2C), BDNF protein (Figure S2D and S2E), or mRNA (Exon I, IV and VI; Figure S1F) levels. These results demonstrate that STEP61 inhibition is sufficient to block PCP-induced decreases in BDNF levels.
The direct involvement of STEP61 in the regulation of BDNF by PCP was evaluated in cultures derived from STEP KO mice, combined with rescue experiments, where STEP61 was added back to the KO cultures. Wild-type (WT) mouse cortical cultures treated with PCP showed similar changes in STEP61 (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 . Reintroduction of STEP61 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 STEP61 to KO cultures, PCP once again increased STEP61 (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-STEP61 alone did not alter BDNF expression in the absence of PCP administration. Taken together, these results demonstrate that STEP61 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
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 STEP61 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
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, 48, 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
BDNF signaling is also implicated in memory consolidation in several behavioral paradigms, including the NOR task . We, therefore, analyzed the effects of STEP61 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 , and hippocampi were collected for biochemical analysis. Subchronic PCP administration reduced BDNF and increased STEP61 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).
We also treated STEP KO mice with the same protocol and found that these mice did not show a reduction in BDNF upon PCP challenge (Figure S6). These findings, together with the prior results, indicate that lowering STEP61 activity directly with TC-2153 prevents PCP-induced reduction in BDNF and has beneficial effects on cognitive function.
One of the more interesting aspects of recent studies on the neurobiology of STEP is the finding that STEP activity is disrupted in multiple disorders. Both high [2, 32, 51, 52, 53] and low [54, 55, 56] levels of STEP activity are implicated in a growing number of neuropsychiatric and neurodegenerative disorders. Dysfunctional BDNF/TrkB signaling is implicated in many of the same neurodegenerative and neuropsychiatric disorders , but in an opposite direction. The present study provides a possible explanation by demonstrating the regulation of BDNF expression by STEP61 activity. These results suggest that STEP61 contributes to the regulation of BDNF expression patterns, with implications for cognitive functioning in CNS disorders .
The results help clarify the mechanisms involved in the regulation of STEP61 and BDNF by PCP. PCP is a noncompetitive NMDAR antagonist that recapitulates in rodents some of the behavioral symptoms observed in humans affected by SZ, including disrupted sensorimotor gating, impaired cognitive functions, and increased social isolation and stereotypic behaviors [2, 38]. In general, PCP rodent models are considered to have good predictable validity as they recapitulate many endophenotypes present in SZ [57, 58]. Previous findings show that acute and subchronic PCP treatments result in increased STEP61 levels, dephosphorylation of the STEP61 substrate GluN2B at Tyr1472, and internalization of NMDAR from synaptosomal membranes . PCP increases STEP61 activity  and decreases BDNF gene transcription and protein expression [39, 45]; however, whether these observations were causally related was not fully appreciated. The present study demonstrates that either genetic or pharmacological reduction of STEP activity reversed PCP-induced disruption of BDNF expression and reversed cognitive and motor abnormalities in PCP-treatment mice. In addition, small molecule TrkB agonists (BDNF mimetics) are emerging as new therapeutic agents because of their superior pharmaceutical properties. Indeed, 7,8-DHF and its analogs confer neuroprotection and improve cognitive functions in a variety of rodent models of neuropsychiatric and neurodegenerative disorders [33, 47, 48, 49, 59]. However, the efficacy of 7,8-DHF in PCP animal models has not been evaluated. Here, we showed direct activation of BDNF/TrkB signaling by 7,8-DHF achieved comparable efficacy to STEP inhibition by TC-2153, consistent with the hypothesis that STEP and BDNF signaling have opposing functions.
NMDA receptor antagonists, including PCP, induce hyperlocomotion in humans and animal models of SZ [34, 35, 36]. Here we found that, in addition to reversing subchronic PCP-induced cognitive deficits, STEP inhibition also attenuated acute PCP-induced hyperactivity. Previous findings have demonstrated that neuroleptics block the cognitive and hyperlocomotion induced by PCP . This result is explained in part by the finding that neuroleptics function through dopamine D2 receptor blockade and activation of protein kinase A (PKA). PKA phosphorylates STEP at the inhibitory Ser221 within the STEP substrate-binding domain, which prevents STEP from binding to and dephosphorylating its substrates [2, 16]. Because both clozapine and TC-2153 are effective in reducing PCP-induced deficits, future studies are necessary to determine whether combining neuroleptics and TC-2153 might allow the use of lower dosages of each to reduce PCP-induced effects. At the present time, TC-2153 is a useful tool for laboratory studies, and additional efforts are underway to discover novel STEP inhibitor platforms that might prove useful therapeutically.
The results also show that STEP61 modulates BDNF expression pattern by regulating BDNF transcription, possibly through ERK1/2-CREB signaling. Distinct promoters regulate the temporal and spatial expression of BDNF transcripts. In particular, transcriptions of Exon I and IV of the BDNF gene are regulated by CREB in a neuronal activity-dependent manner through NMDAR or voltage-gated calcium channels [28, 29, 44]. STEP gates ERK1/2 activity by direct dephosphorylation of a regulatory Tyr residue within the activation loop , and STEP KO mice display elevated basal pERK1/2 and pCREB levels . At the same time, STEP dampens NMDAR signaling by dephosphorylation at Tyr1472, thus facilitating its internalization. Consistent with these findings, we demonstrate that direct inhibition of STEP61 by TC-2153 restored BDNF expression, possibly through Exon IV transcription, which was reestablished upon STEP inhibition both in vitro and in vivo. Importantly, STEP61 inhibition did not rescue the expression of BDNF Exon VI, a CREB-independent transcript. We found a different pattern of regulation of Exon I and Exon IV in cultures and in vivo, which may be due to different responses of each promoter to distinct temporal responses in the cell and in vivo models. In accordance with this hypothesis, it has been shown that distinct neuronal activity results in changes in selective transcripts (either Exon I or Exon IV), despite similar changes of CREB phosphorylation levels [60, 61].
STEP inhibition by TC-2153 did not alter BDNF expression in the absence of PCP treatment. TC-2153 administration in neuronal cultures and in mice did lead to a transient increase in pERK1/2 and pS133 CREB levels. However, it is possible that more sustained activation of ERK1/2/CREB or phosphorylation of CREB by additional kinases at other sites is required to stimulate target transcription including BDNF [62, 63, 64]. Moreover, modification of CREB by post-translational changes other than phosphorylation may be needed for full activation . Thus, STEP61 inhibition may be required but is not sufficient to alter BDNF transcription under baseline conditions. In agreement with this, STEP KO mice did not show increased BDNF levels.
The ubiquitin proteasome system (UPS) is a major degradation pathway that regulates STEP61 levels , and dysfunction of this pathway in Alzheimer’s disease patients and animal models results in the accumulation of STEP61 [32, 51]. Recent studies have shown that disruptions of the UPS contribute to the accumulation of STEP61 in MK-801-treated neuronal cultures and mice  and Parkinson’s disease patients . Here we demonstrate that disruption of STEP61 ubiquitination also may contribute to its accumulation in PCP-treated cultures and in mice. Consistent with this finding in SZ, several studies now report alterations of several UPS components both in pyramidal neurons from SZ patients  and after PCP administration to rats . Moreover, NMDAR signaling (inhibited by PCP) is essential for the activation and translocation of the proteasome into spines , and the blockade of NMDARs leads to a disruption of the UPS and accumulation of synaptic proteins .
In summary, our data indicate that increased STEP61 levels lead to the reduction of BDNF levels in a PCP mouse model of SZ. Inhibition of STEP61 could have beneficial effects by restoring BDNF expression. A better understanding of the interaction between STEP and BDNF will extend our understanding of the molecular basis of a number of neuropsychiatric disorders characterized by disruptions in BDNF or STEP61 expression patterns.
We thank Dr. Marilee Ogren for helpful discussion, and Drs. Thomas Lanz and Thao Nguyen (Pfizer Research and Development, Cambridge, MA, USA) for providing lentivirus-STEP shRNA. This work was supported by the National Institute of Health grants MH091037 and MH52711 (P.J.L.), MH090963 (A.C.N.), and GM054051 (J.A.E.).
Compliance with ethical standards
Conflict of interest
The authors declare no conflict of interest.
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