The tyrosine phosphatase STEP: implications in schizophrenia and the molecular mechanism underlying antipsychotic medications.
ABSTRACT Glutamatergic signaling through N-methyl-D-aspartate receptors (NMDARs) is required for synaptic plasticity. Disruptions in glutamatergic signaling are proposed to contribute to the behavioral and cognitive deficits observed in schizophrenia (SZ). One possible source of compromised glutamatergic function in SZ is decreased surface expression of GluN2B-containing NMDARs. STEP(61) is a brain-enriched protein tyrosine phosphatase that dephosphorylates a regulatory tyrosine on GluN2B, thereby promoting its internalization. Here, we report that STEP(61) levels are significantly higher in the postmortem anterior cingulate cortex and dorsolateral prefrontal cortex of SZ patients, as well as in mice treated with the psychotomimetics MK-801 and phencyclidine (PCP). Accumulation of STEP(61) after MK-801 treatment is due to a disruption in the ubiquitin proteasome system that normally degrades STEP(61). STEP knockout mice are less sensitive to both the locomotor and cognitive effects of acute and chronic administration of PCP, supporting the functional relevance of increased STEP(61) levels in SZ. In addition, chronic treatment of mice with both typical and atypical antipsychotic medications results in a protein kinase A-mediated phosphorylation and inactivation of STEP(61) and, consequently, increased surface expression of GluN1/GluN2B receptors. Taken together, our findings suggest that STEP(61) accumulation may contribute to the pathophysiology of SZ. Moreover, we show a mechanistic link between neuroleptic treatment, STEP(61) inactivation and increased surface expression of NMDARs, consistent with the glutamate hypothesis of SZ.
- SourceAvailable from: Deepa Venkitaramani[show abstract] [hide abstract]
ABSTRACT: Amyloid beta (Abeta) is involved in the etiology of Alzheimer's disease (AD) and may contribute to cognitive deficits by increasing internalization of ionotropic glutamate receptors. Striatal-enriched protein tyrosine phosphatase 61 (STEP(61)), which is targeted in part to the postsynaptic terminal, has been implicated in this process. Here we show that STEP(61) levels are progressively increased in the cortex of Tg2576 mice over the first year, as well as in prefrontal cortex of human AD brains. The increased STEP(61) was associated with greater STEP activity, dephosphorylation of phospho-tyr(1472) of the NR2B subunit, and decreased NR1 and NR2B subunits on neuronal membranes. Treatment with Abeta-enriched medium also increased STEP(61) levels and decreased NR1/NR2B abundance in mouse cortical cultures as determined by biotinylation experiments. In STEP knock-out cultures, Abeta treatment failed to induce NMDA receptor internalization. The mechanism for the increase in STEP(61) levels appears to involve the ubiquitin proteasome system. Blocking the proteasome resulted in elevated levels of STEP(61). Moreover, STEP(61)-ubiquitin conjugates were increased in wild-type cortical slices upon Abeta treatment as well as in 12 month Tg2576 cortex. These findings reveal a novel mechanism by which Abeta-mediated accumulation of STEP(61) results in increased internalization of NR1/NR2B receptor that may contribute to the cognitive deficits in AD.Journal of Neuroscience 04/2010; 30(17):5948-57. · 6.91 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The use of in vivo receptor imaging by positron emission tomography (PET) and single photon emission tomography (SPET) has permitted exploration of targets for antipsychotic drug action in living patients. Early PET and SPET studies focused on striatal D2 dopamine receptors. There is broad agreement that unwanted extrapyramidal (parkinsonian) side effects of antipsychotic drugs result from high striatal dopamine D2/D3 receptor blockade by these drugs. The dopamine hypothesis of antipsychotic drug action suggests that clinical response is directly related to the level of striatal D2/D3 receptor occupancy of antipsychotic drugs. This may be true for classical antipsychotic drugs, but recent evidence suggests that novel, atypical antipsychotic drugs produce efficacy in association with modest and transient striatal D2/D3 receptor occupancy levels. Furthermore, atypical antipsychotic drugs appear to show preferential occupancy of limbic cortical dopamine D2 receptors. Cortical dopamine D2/D2-like receptors may be a common site of action for all antipsychotic drugs. Data from receptor challenge paradigms has highlighted the need to explore the neurotransmitter systems involved in regulating or stabilising dopamine transmission, either via dopamine autoreceptors or non-dopaminergic pathways. These may be promising targets for drug development. In vivo PET and SPET imaging has produced unique data contributing to the design of better, less toxic drugs for schizophrenia.Nuclear Medicine Communications 08/2001; 22(7):829-33. · 1.38 Impact Factor
- [show abstract] [hide abstract]
ABSTRACT: The cause of schizophrenia is unknown, but it has a significant genetic component. Pharmacologic studies, studies of gene expression in man, and studies of mouse mutants suggest involvement of glutamate and dopamine neurotransmitter systems. However, so far, strong association has not been found between schizophrenia and variants of the genes encoding components of these systems. Here, we report the results of a genomewide scan of schizophrenia families in Iceland; these results support previous work, done in five populations, showing that schizophrenia maps to chromosome 8p. Extensive fine-mapping of the 8p locus and haplotype-association analysis, supplemented by a transmission/disequilibrium test, identifies neuregulin 1 (NRG1) as a candidate gene for schizophrenia. NRG1 is expressed at central nervous system synapses and has a clear role in the expression and activation of neurotransmitter receptors, including glutamate receptors. Mutant mice heterozygous for either NRG1 or its receptor, ErbB4, show a behavioral phenotype that overlaps with mouse models for schizophrenia. Furthermore, NRG1 hypomorphs have fewer functional NMDA receptors than wild-type mice. We also demonstrate that the behavioral phenotypes of the NRG1 hypomorphs are partially reversible with clozapine, an atypical antipsychotic drug used to treat schizophrenia.The American Journal of Human Genetics 11/2002; 71(4):877-92. · 11.20 Impact Factor
The tyrosine phosphatase STEP: implications in
schizophrenia and the molecular mechanism
underlying antipsychotic medications
NC Carty1, J Xu1, P Kurup1, J Brouillette1, SM Goebel-Goody1, DR Austin2, P Yuan2, G Chen2, PR Correa3, V Haroutunian4,
C Pittenger1,3,5and PJ Lombroso1,3,6
Glutamatergic signaling through N-methyl-D-aspartate receptors (NMDARs) is required for synaptic plasticity. Disruptions in
glutamatergic signaling are proposed to contribute to the behavioral and cognitive deficits observed in schizophrenia (SZ). One
possible source of compromised glutamatergic function in SZ is decreased surface expression of GluN2B-containing NMDARs.
STEP61is a brain-enriched protein tyrosine phosphatase that dephosphorylates a regulatory tyrosine on GluN2B, thereby
promoting its internalization. Here, we report that STEP61levels are significantly higher in the postmortem anterior cingulate
cortex and dorsolateral prefrontal cortex of SZ patients, as well as in mice treated with the psychotomimetics MK-801 and
phencyclidine (PCP). Accumulation of STEP61after MK-801 treatment is due to a disruption in the ubiquitin proteasome system
that normally degrades STEP61. STEP knockout mice are less sensitive to both the locomotor and cognitive effects of acute and
chronic administration of PCP, supporting the functional relevance of increased STEP61levels in SZ. In addition, chronic
treatment of mice with both typical and atypical antipsychotic medications results in a protein kinase A-mediated
phosphorylationand inactivationofSTEP61and, consequently,increasedsurfaceexpressionofGluN1/GluN2B receptors.Taken
together, our findings suggest that STEP61accumulation may contribute to the pathophysiology of SZ. Moreover, we show a
mechanistic link between neuroleptic treatment, STEP61inactivation and increased surface expression of NMDARs, consistent
with the glutamate hypothesis of SZ.
Translational Psychiatry (2012) 2, e137; doi:10.1038/tp.2012.63; published online 10 July 2012
Disrupted glutamatergic signaling, and specifically hypo-
function of N-methyl-D-aspartate receptors (NMDARs), is
proposed as a contributing factor in the etiology of schizo-
phrenia (SZ).1–3Several neuropathological studies show
abnormal glutamate receptor densities in the prefrontal
cortex, thalamus and temporal lobe, as well as decreased
receptor function in SZ brains.4,5Noncompetitive NMDAR
antagonists such as phencyclidine (PCP) and ketamine
elicit SZ-like symptoms in normal individuals and exacer-
bate psychotic episodes in SZ patients;2,3,6,7and adminis-
tration of these psychotomimetics, or the more selective
NMDAR antagonist MK-801, is used to model SZ in rodents
and primates. These drugs produce cognitive deficits and
behavioral phenotypes reminiscent of SZ symptoms, as
well as induce biochemical modifications and disrupted
glutamatergic transmission thought to be present in SZ
brains.2,8–18Altogether, these findings link aberrant NMDAR
function to behavioral deficits in SZ;19–21however, the
mechanism(s) underlying NMDAR hypofunction remain
The brain-enriched protein tyrosine phosphatase STEP
is a critical regulator of NMDAR function.22The STEP gene
is alternatively spliced to produce several isoforms with
domains that regulate their localization, substrate specificity
and activity.23–25STEP61 is associated with postsynaptic
densities in the striatum, cortex, hippocampus and related
GluN2B,29,30the AMPA receptor subunit GluA2,31Fyn,32
Pyk2,33,34and the MAPK proteins ERK1/2 and p38.35,36
STEP61-mediated dephosphorylation of GluN2B and GluA2
promotes their internalization,29,30while dephosphorylation of
the kinases inactivates them.32,33,35Thus, elevated STEP61
reduces synaptic expression of GluN2B-containing NMDARs
via two mechanisms: (1) dephosphorylation of GluN2B
Tyr1472(refs 29, 30, 37) and (2) dephosphorylation and inacti-
vation of Fyn Tyr420(ref. 32), a kinase that phosphorylates
GluN2B at Tyr1472(ref. 38). A model has therefore emerged
whereby STEP opposes synaptic strengthening by negatively
regulating kinase activity and NMDAR surface expression.22
Dopamine signaling also regulates STEP61 activity and
consequently NMDAR trafficking. D1 receptor stimulation
Received 14 Feburary 2012; revised 13 June 2012; accepted 14 June 2012
1Child Study Center, Yale University School of Medicine, New Haven, CT, USA;2Laboratory of Molecular Pathophysiology, Mood and Anxiety Disorders Research
Program, National Institute of Mental Health, Intramural Research Program, National Institutes of Health, Bethesda, MD, USA;3Department of Psychiatry, Yale
Yale University School of Medicine, New Haven, CT, USA and6Department of Neurobiology, Yale University School of Medicine, New Haven, CT, USA
Correspondence: Dr PJ Lombroso, Child Study Center, Yale University School of Medicine, P.O. Box 207900, New Haven, CT 06520-7900, USA.
Keywords: glutamate hypothesis; neuroleptics; NMDA receptor trafficking; schizophrenia; STEP; tyrosine phosphatase
Citation: Transl Psychiatry (2012) 2, e137, doi:10.1038/tp.2012.63
& 2012 Macmillan Publishers Limited All rights reserved 2158-3188/12
activates protein kinase A and leads to the phosphorylation
of STEP61at a regulatory serine (Ser221) within the sub-
strate-binding KIM domain, thereby preventing STEP61from
interacting with and dephosphorylating its substrates.39,40
When STEP61 is phosphorylated at this site, or in STEP
knockout (KO) mice, the tyrosine phosphorylation of STEP61
substrates and surfaceexpression
containing receptors are increased.33,37,39,41Protein kinase
A also activates dopamine and cAMP-regulated phospho-
protein-32 that inhibits PP1-mediated dephosphorylation of
Because of the relationship between STEP61, dopamine
signaling and NMDAR function, we hypothesized that
dysregulation of STEP61might contribute to the pathophysiol-
ogy of SZ. We find elevated STEP61levels in postmortem
anterior cingulate cortex and dorsolateral prefrontal cortex
(DLPFC) of two different cohorts of SZ patients, as well in
frontal cortex of mice treated with psychotomimetics. We also
demonstrate that antipsychotics inactivate STEP61, leading to
increased NMDAR phosphorylation and surface expression.
These results suggest that the inactivation of STEP61may
contribute to the beneficial effects of medications used to
Materials and methods
Postmortem brain tissue. Postmortem anterior cingulate
cortex from SZ patients and nonpsychotic controls was
obtained from the Stanley Foundation Brain Bank. A second
cohort of postmortem samples was obtained from the
Mount Sinai Brain Bank and consisted of DLPFC. Subject
and tissue parameters for both cohorts are shown in
Supplementary Tables S1 and S2. Tissue collection44,45
and sample preparation were performed as described.46
Samples were stored at ?801C until processing by quanti-
tative immunoblotting. Lactate dehydrogenase was used for
Primary cortical cultures and stimulation. All procedures
were approved by the Yale University Institutional Animal
Care and Use Committee and strictly adhered to the NIH
Guide for the Care and Use of Laboratory Animals. Primary
cortical neurons were isolated from rat E18 embryos.30
Neurons (14–21 DIV) were treated with MK-801 (50mM;
Tocris, Minneapolis, MN, USA) or PCP (100mM; Sigma,
Ronkonkoma, NY, USA) for the time points indicated. The
D2 antagonist sulpiride (25–50mM; Sigma) or D1 agonist
SKF-82958 (25–50mM; Sigma) were applied to neurons
for 15min. In some cases, neurons were pretreated
for 30min before MK-801 application with anisomycin
(40mM; EMD Biosciences, Billerica, MA, USA), actinomycin
D (25mM; Sigma), LY294001 (10mM; Tocris) or U0126
(10mM; EMD Biosciences). After treatments, cells were
lysed in 1? RIPA buffer supplemented with NaF (5mM),
Na3VO4 (2mM), MG-132 (10mM, EMD Biosciences) and
complete protease inhibitor cocktail (Roche, Indianapolis, IN,
USA), and spun at 1000g for 10min, and supernatants were
subjected to SDS-PAGE and western blotting.
Ubiquitinated protein pull-down. MK-801-treated cultured
neurons or cortical tissue were homogenized as described.30
Lysates were incubated with 20ml of Agarose-TUBE2
(Tandem Ubiquitin Binding Entity, LifeSensors, Malvern,
PA, USA) beads overnight at 41C, bound proteins eluted
and processed by western blots.
Surface biotinylation and phosphatase activity. After
stimulations, cortical cultures were labeled with EZ-Link
Sulfo-NHS-SS-Biotin (Pierce, Rockford, IL, USA) as des-
cribed.31Neurons were lysed and incubated with NeutrAvidin
Biotin-binding Protein immobilized to agarose beads. For
phosphatase activity, the GST-GluN2B C-terminal was
phosphorylated by Fyn, mixed with immunoprecipitated
STEP and a phospho-specific antibody was used to assess
phosphorylation of GluN2B at Tyr1472(ref. 30).
Subcellular fractionations and western blot analyses.
Subcellular fractionation was performed and synaptosomal
fractions (P2) were prepared for western blot analysis or all
experiments where glutamate receptor subunits or STEP
substrates were investigated in vivo from cortical tissue.31
Antibodies used are shown in Supplementary Table S3.
Bands were visualized with a G:BOX with a GeneSnap
image program (Syngene, Fredrick, MD, USA) and quantified
with Image J 1.33 (NIH). Levels of phosphoproteins were
normalized first to total protein levels and then normalized
again with glyceraldehyde-3-phosphate dehydrogenase.
In vivo treatments. Male wild-type (WT) C57BL/6 mice
(6–8 months) received subchronic injections haloperidol
(2mgkg?1), clozapine (5mgkg?1) or risperidone (2mgkg?1)
administered intraperitoneal (i.p.) daily for 3 weeks. These
drugs were dissolved in 100mM acetic acid and titrated to pH
6.5 and diluted in 0.9% normal saline to desired concentration.
MK-801 (0.6mgkg?1, i.p.) and PCP (5mgkg?1, i.p.) were
dissolved in 0.9% saline and administered to male WT mice.
Control animals received 0.9% saline injections. Mice receiving
subchronic antipsychotic treatment were killed 24h following
the last injection or at indicated time points following MK-801
Frontal cortex (anterior to motor cortex) was dissected out
and synaptosomal fractions (P2) were prepared.31
Behavioral assessments. For locomotor activity, male WT
and STEP KO mice (n¼8–10; 6–10 months) were assessed
for 2 days in an open field.37On day 1, mice were drug naı ¨ve;
on day 2, they were administered PCP (2.5–20mgkg?1
dose) or vehicle by i.p. injection. Separate mice were used
for each dose of PCP. Total distance traveled in 60min was
determined by AnyMaze software (Stoelting, Wood Dale,
IL, USA). For object recognition, PCP was administered sub-
chronically (5mgkg?1, i.p. twice daily for 7 days)47,48to male
WT and STEP KO mice (n¼10–11; 6–8 months). At 1 week
after the last injection, preference to a novel object after
a 2-hr delay was determined as described previously.37
Data analysis. All data were presented as means±s.e.m.,
and Pp0.05 was considered significant. For the postmortem
data, ANCOVA (IBM SPSS Statistics v19, Poughkeepsie,
Role of STEP in schizophrenia
NC Carty et al
NY, USA) was used to determine differences between
groups (Control and SZ) using sex, age and postmortem
interval (PMI) as covariates. For all other biochemical
analyses, either Student’s unpaired two-tailed t-test or one-
way analysis of variances were performed with Fisher’s post-
hoc tests. For locomotor activity, a repeated-measures
analysis of variance with Fisher’s post-hoc was used. For
object recognition, a one-sample t-test was used as
previously described37to determine whether a group spent
more time with the novel object compared with the chance
value of 15sec.
STEP61is elevated in the cortex of SZ patients. We first
examined STEP61 expression in brain homogenates pre-
pared from anterior cingulate cortex of a well-characterized
group of SZ patients from the Stanley Foundation, compared
with age-, sex- and PMI-matched control patients (SZ: n¼12;
controls: n¼12; Supplementary Table S1).46STEP61was
significantly elevated in SZ subjects compared with controls
using sex, age and PMI as covariates (Po0.05; Figures 1a
and b), with no interactions between those covariates and the
main effect. As most SZ patients from the Stanley Foundation
were under antipsychotic medication at the time of death
(Supplementary Table S1), we wanted to confirm our findings
in a second SZ cohort from the Mount Sinai Brain Bank where
(Supplementary Table S2). DLPFC homogenates from SZ
(n¼14) and controls (n¼12) were assessed for STEP61
levels. We again observed a significant increase in STEP61in
the DLPFC of SZ subjects using sex, age and PMI as
covariates (Po0.05; Figures 1c and d), and there was no
influence of the covariates on the main effect.
MK-801 increases STEP61activity and promotes NMDAR
internalization. Noncompetitive NMDAR antagonists have
psychotomimetic properties in humans and produce SZ-like
effects in animal models. We therefore determined whether
treatment with NMDAR antagonists would mimic the
increase in STEP61observed in human SZ tissue. We first
examined STEP61 after stimulating cortical cultures with
the NMDAR antagonist MK-801. STEP61levels were signi-
ficantly increased in cultured neuronal homogenates at
all time points examined (10, 30 and 60min; Po0.01;
Figure 2a). MK-801 treatment concomitantly decreased
STEP61phosphorylation (Ser221) (Po0.01), suggesting an
increase in STEP61activity. Consistent with this expectation,
the tyrosine phosphorylation of STEP61-regulated sites on
GluN2B (Tyr1472), Pyk2 (Tyr402) and ERK1/2 (Tyr204/187)
were significantly decreased (Po0.01; Figure 2b). Total
levels of GluN2B, Pyk2 and ERK1/2 did not change following
MK-801 treatment in neuronal homogenates (data not
shown), suggesting that the overall phosphorylation state
and not the total level of these proteins were affected by
To more accurately determine whether MK-801 increased
STEP61levels and altered STEP61substrates in vivo at the
synapse, WT mice were injected acutely with MK-801, and
synaptosomal (P2) fractions were prepared from frontal
cortex at various time points post injection. Consistent with
the cultured neuron findings, STEP61levels were increased,
while phosphorylation of STEP61was decreased 10min post
GluN2B, Pyk2 and ERK1/2 was also significantly decreased
(Po0.05; Figure 2d).
Increased STEP61 activity has been shown in other
contexts to promote internalization of NMDARs.29,30,37Thus,
we examined GluN2B levels in P2 fractions following MK-801
treatment. There was a trend for GluN2B levels to decrease
after 10minpost injection (?27.3±12.6;P¼0.09),consistent
with increased internalization of GluN2B NMDARs. In most
cases, substrate phosphorylation and GluN2B levels returned
to baseline at later time points in vivo in P2 fractions, in
contrast to the cultured neuron findings (although ERK1/2
phosphorylation remained low even 60min post injection).
These results likely reflect pharmacokinetic complexities
STEP61was measuredinprotein homogenatesfromtheanteriorcingulatecortexof
controls and patients with SZ (n¼12) (a, b) or the DLPFC of controls (n¼12) and
patients with SZ (n¼14) (c, d). (a, c) Representative western blots of subjects
demonstrating an overall increase in STEP61expression in SZ patients compared
with controls. The top band is pSTEP61 while the bottom band is the
nonphosphorylated, active form of STEP61, which was quantified. Lactate
dehydrogenase (LDH) levels were not significantly different between control and
SZ samples, and were used for normalization. (b, d) analysis of covariance model
with sex, age and PMI as covariates was used to demonstrate that STEP61was
significantly higher in SZ subjects (*Po0.05).
STEP61levels are increased in human postmortem brains with SZ.
Role of STEP in schizophrenia
NC Carty et al
in vivo not captured in culture. Similar results were observed
in the hippocampus (data not shown).
To further confirm that STEP61activity, and not just its
quantity, was increased by MK-801, we conducted a
phosphatase assay using a phospho–GluN2B fusion protein
as a substrate of STEP61immunoprecipitated from MK-801-
treated and control cultured neurons. Immunoprecipitated
GluN2B phosphorylation compared with control (Po0.01;
As a more direct approach to determine whether surface
levels of NMDARs were altered by MK-801, we investigated
NMDAR surface expression using a surface biotinylation
assay in cultured neurons. MK-801 significantly reduced
GluN2B and GluN1 surface expression (Po0.05), but not
GluN2A (P¼0.28) or GABAAb2/3 (P¼0.79) that are not
STEP61 substrates (Figure 3b). As observed previously
(Figure 2), MK-801 significantly increased STEP61in total
neuronal homogenates and concomitantly decreased phos-
phorylation of GluN2B (Po0.05) and ERK1/2 (Po0.01;
Figure 3c). Total levels of NMDARs, ERK1/2 and GABAAb2/
3 remained unchanged (P40.05; Figure 3c). Taken together,
our findings indicate that MK-801 increased STEP61levels
and activity, decreased the tyrosine phosphorylation of
STEP61substrates and reduced the surface expression of
with MK-801 (50mM) for 10, 30 or 60min. (a) MK-801 treatment significantly increased total levels of STEP61, while also significantly reducing STEP61phosphorylation at
Ser221(pSTEP) (one-way analysis of variance (ANOVA); *Po0.01 different from control, Tukey’s post-hoc; n¼4). (b) Administration of MK-801 significantly reduced the
Tyr phosphorylation of STEP61substrates at sites that STEP is known to dephosphorylate, including GluN2B at Tyr1472(pGluN2B), Pyk2 at Tyr402(pPyk2) and ERK1/2 at
Tyr204/187(pERK1/2) (one-way ANOVA; *Po0.01 different from control, Tukey’s post-hoc; n¼4). (c, d) WT mice were acutely injected with MK-801 (0.6mgkg?1, i.p.) or
saline (control). Frontal cortex was dissected at 10, 30 or 60min post injection. (c) MK-801 treatment in vivo significantly increased STEP61levels and attenuated STEP61
each phosphoprotein was normalized to the total protein level for that protein and then to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data represent the mean
percentage of control±s.e.m.
Role of STEP in schizophrenia
NC Carty et al
Accumulation of STEP61 occurs via blockade of the
ubiquitin proteasome system. We tested several mecha-
nisms that might explain the MK-801-induced increase in
STEP61. Translation of STEP is regulated in part by the
PI3K-Akt and ERK pathways.31,49
cultures were pretreated with either the PI3K inhibitor
(LY294001) or MEK inhibitor (U0126) before MK-801
administration (Figure 3d). Neither inhibitor blocked the
MK-801-induced increase in STEP61, demonstrating that
the PI3K-Akt and ERK pathways were not involved. Likewise,
pretreatment with inhibitors of transcription (actinomycin D)
and translation (anisomycin) failed to block the elevation
in STEP61 levels (Figure 3d). These results indicate that
the observed increase in STEP61levels following MK-801
treatment was not due to increased transcription or
STEP61 is normally ubiquitinated and degraded by the
ubiquitin proteasome system (UPS), and disruption of the
treatedwith MK-801 (50mM) for 2h, and the activity of immunoprecipitated STEP61from control and treated neuronswas measured against phospho–GluN2B fusion protein.
Phosphorylation of GluN2B at Tyr1472(pGluN2B) was significantly lower in MK-801-treated immunoprecipitates than in controls (Student’s t-test; **Po0.01; n¼4). (b, c)
Surface proteins were biotinylated after MK-801 treatment. (b) MK-801 treatment significantly decreased surface levels of GluN2B and the obligatory subunit GluN1, but not
of GluN2B at Tyr1472and ERK1/2 at Tyr204/187(pERK1/2) was observed again alongside increased STEP61levels. For (b, c), *Po0.05 and **Po0.01 (Student’s t-test;
n¼4). (d) Cortical cultures were pretreated with various inhibitors for 20–30min before applying MK-801; none prevented the MK-801-induced increase in STEP61or the
to the total protein level for that protein and then to glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Data were normalized to control and represent the mean
normalized value±s.e.m. (e) Ubiquitinated proteins were isolated from control and MK-801-treated cultures, and probed with anti-STEP and anti-Ub antibodies. An increase
(n¼4). (f) Ubiquitinated proteins were also more abundant in mouse cortical lysates isolated following an acute injection of MK-801 (0.6mgkg?1, i.p.) (n¼4).
The MK-801-induced accumulation of STEP61occurs via blockade of the UPS and promotes internalization of NMDARs. (a) Primary cortical cultures were
Role of STEP in schizophrenia
NC Carty et al
UPS increases STEP61levels.30The UPS can be activated
by NMDAR signaling, and blocking NMDARs leads to rapid
accumulation of several proteins normally degraded by the
UPS.50,51To determine whether disruptions in the UPS
mediate the MK-801-induced increase in STEP61, we
measured polyubiquitinated STEP61following MK-801 treat-
ment in cultured neurons, as well as in brain homogenates
from MK-801-treated WT mice. Polyubiquitinated proteins
were enriched and probed with an anti-STEP antibody or
anti-glutamate receptor-interacting protein 1 antibody as a
positive control (Figures 3e and f). Acute injections of
10min¼113±1.8%; 30min¼ þ117±0.6%; 60min¼126±
2.6% (Pso0.01), suggesting that blocking NMDARs with
MK-801 disrupts the UPS and leads to the accumulation of
phosphorylation of its substrates. Our findings thus far
demonstrate that MK-801 increased both STEP61expression
and activity in synaptosomal fractions. As increased STEP61
activity leads to loss of GluN2B-containing NMDARs from
neuronal membrane surfaces, we next investigated whether
antipsychotic medications exert their beneficial effects by
inactivating STEP61. We first examined the phosphorylation
of STEP61 and its substrates in cultured neurons treated
with the D1R agonist, SKF-82958, or the D2R antagonist,
sulpiride. Both treatments significantly increased the phos-
phorylation of STEP61, and tyrosine phosphorylation of
GluN2B and ERK1/2 at the two doses examined (Pso0.05;
Figures 4a and b), consistent with an inactivation of STEP61
and activation of its substrates. No significant changes were
observed in total levels of GluN2B, ERK1/2 or STEP61in
cultured neuronal homogenates (data not shown).
To more closely recapitulate the clinical use of antipsycho-
tics, WT mice were subchronically treated with three different
antipsychotic medications (haloperidol, clozapine or risper-
idone) for 21 days, and phosphorylation of STEP61and its
substrates were examined in synaptosomal (P2) fractions
from frontal cortex (Figures 4c–h).
(Po0.05) and concomitantly enhanced the tyrosine phos-
2: Po0.005; Pyk2: Po0.05; Figures 4c–f). The atypical
antipsychotics, clozapine and risperidone, similarly increased
STEP61 phosphorylation (Po0.05 and Po0.005, respec-
tively; Figure 4c), as well as the tyrosine phosphorylation of
Pyk2 and ERK1/2 (Pso0.005; Figures 4e and f).
Consistent with increased surface expression of GluN2B,
clozapine and risperidone significantly enhanced GluN2B
levels in the P2 fraction (Pso0.005) and a similar trend was
results using SKF-82958 and sulpiride, subchronic treatment
of haloperidol did not alter total STEP61levels in P2 fractions
(þ0.8±5.4%; P40.05), while clozapine and risperidone
did (þ135.1±30.5% and
Po0.05). As a positive control, antipsychotic treatment
significantly increased the phosphorylation of dopamine and
Po0.05; Figure 4h), which is in agreement with prior
studies.43Comparable results were observed for these
measures in the striatum and hippocampus (data not shown).
No significant changes were detected in GluN2A (P¼0.907)
or GABAAb 2/3 (P¼0.431) in P2 fractions (data not shown).
Altogether, these findings indicate that different classes of
neuroleptics increase STEP61phosphorylation, increase the
tyrosine phosphorylation of STEP61substrates and enhance
NMDAR surface expression.
abnormalities elicited by the psychotomimetic PCP. If
increased activity of STEP61and consequent downstream
changes are mechanistically important for the psycho-
tomimetic effects of NMDAR antagonists, then STEP KO
mice should be less sensitive behaviorally than WT controls
to these effects. Consistent with previous reports,52,53we
found that even low doses of acute MK-801 injections
(0.2mgkg?1) resulted in convulsive jumping and wild running
fits that severely affected motor coordination and prevented
accurate baseline locomotor measurements and analysis of
object recognition (data not shown). Therefore, we utilized
another well-characterized psychotomimetic and NMDAR
antagonist, PCP, for behavioral analyses. To confirm that
PCP altered STEP61and its substrates in a similar manner
as MK-801 (Figure 2), we administered PCP acutely to both
in cortical neurons and in vivo (Supplementary Figure 1).
In cortical neurons, PCP significantly decreased STEP61
phosphorylation (Po0.01) and increased STEP61 levels
decreased the tyrosine phosphorylation of GluN2B, Pyk2
and ERK1/2 (Pso0.05) (Supplementary Figure 1a).
WT mice were subsequently injected with an acute dose of
PCP (5mgkg?1; i.p.) and synaptosomal (P2) fractions
prepared from the frontal cortex 60-min post injection
(Supplementary Figure 1b). STEP61 phosphorylation was
significantly decreased in P2 fractions (Po0.01), while total
STEP61levels were increased (Po0.05). Tyrosine phosphor-
ylation of GluN2B, Pyk2 and ERK1/2 was significantly
reduced following an acute injection of PCP (Pso0.05).
These findings indicate that PCP, like MK-801, increases
STEP61 activity and levels, leading to decreased tyrosine
phosphorylation of STEP61substrates.
We next investigated the effect of PCP on behavior in WT
versus STEP KO mice. As expected, WT mice became
increasingly hyperactive with acute injections of low and
intermediated doses of PCP (2.5–15mgkg?1) and were
sedated by the highest dose (20mgkg?1; Figure 5a). There
was no difference between genotypes in baseline locomotor
activity (data not shown). While there was a within-genotype
dose effect for both genotypes (Po0.001), STEP KO mice
were significantly less hyperactive than WT mice in response
to PCP (significant dose by genotype interaction; Po0.02).
These results confirm our hypothesis that STEP KO mice are
less sensitive behaviorally to PCP-induced hyperactivity.
Subchronic administration of PCP induces long-term
cognitive impairments and mimics aspects of the positive
and negative symptomatology of SZ in both rodents and
primates.54–59To assess whether STEP KO mice were less
mice were treated for 7 days with PCP (5mgkg?1, i.p. twice
KO miceare lesssensitive tobehavioral
Role of STEP in schizophrenia
NC Carty et al
daily) andtestedin a novelobjectrecognition task (Figure 5b).
No genotypic differences were observed in exploration time of
two identical objects during the acquisition phase (data not
significantly more time with the novel object than chance
(15s) during the 24-hr delay retention trial (Po0.05,
Figure 5b). After PCP treatment, however, WT mice did not
KOs treated with PCP were protected from this impairment
and continued to show a clear preference for the novel object
despite PCP treatment (Po0.05). Taken together, the
behavioral findings indicate that STEP KOs are less sensitive
to PCP-induced hyperactivity and cognitive impairment
compared with WT mice.
To determine the molecular alterations induced by sub-
chronic PCP treatment that could account for these long-term
behavioral effects, we analyzed whether there were changes
in STEP61 or its substrates that occurred in a sustained
fashion 24h after the last of the subchronic PCP injections.
No significant differences were observed for STEP61phos-
(þ6.0±6.9%, PX0.05) in PCP- versus saline-injected WT
mice. In line with previous studies,60we found that ERK1/2, a
direct substrate of STEP61,35was activated 24h after the last
PCP injection in WT mice but not in STEP KOs (Figure 6).
Altogether, these findings suggest that STEP61is transiently
activated after the first PCP injection and that other proteins
downstream of STEP61are altered more sustainably. We also
were treated with the DA D1R agonist SKF-82958 (25 or 50mM) or D2R antagonist sulpiride (25 or 50mM). Both drugs at either concentration significantly increased
phosphorylation of STEP61at Ser221(pSTEP), GluN2B at Tyr1472(pGluN2B) and pERK1/2 at Tyr204/187(pERK1/2) (one-way analysis of variance; *Po0.05 and **Po0.01
different from control, Tukey’s post-hoc; n¼4). (c–h) WT mice were injected with saline (SAL; control), haloperidol (HAL; 2mgkg?1), clozapine (CLZ; 5mgkg?1) and
risperidone (RIS; 2mgkg?1) daily for 3 weeks, and synaptosomal (P2) fractions were prepared from frontal cortex. Phosphorylation of (c) STEP61, (d) GluN2B, (e) Pyk2 and
(f) ERK1/2 were significantly increased by neuroleptics (Student’s t-test, saline versus drug; *Po0.05 and **Po0.005; n¼4–6) (g) Total GluN2B in P2 fractions was
also significantly increased following neuroleptic treatment (Student’s t-test, saline versus drug; **Po0.005; n¼4–6). (h) Increased phosphorylation of dopamine and
cAMP-regulated phosphoprotein-32 at Thr34(pDARPP-32) following neuroleptic treatment was used as a positive control (Student’s t-test, saline versus drug; *Po0.05;
n¼4–6). In all panels, phosphoproteins were first normalized to the total protein and then to GADPDH. Data represent the mean percentage of control±s.e.m.
Role of STEP in schizophrenia
NC Carty et al
observed that the level of Tau, a direct substrate of ERK1/2
that is required for synaptic plasticity,61was decreased in WT
mice after subchronic PCP treatment, but not in STEP KOs
(Figure 6). Furthermore, PSD-95 levels were significantly
increased in STEP KOs, and not in WT mice, after subchronic
PCP treatment (Figure 6). These findings demonstrate that
the expression of signaling molecules required for synaptic
plasticity and cognitive function are differentially affected by
subchronic PCP treatment in WT and STEP KO mice,
providing a potential explanation for why STEP KOs are less
sensitive behaviorally to PCP.
Several converging lines of evidence point to reduced
NMDAR function as an integral part of SZ pathophysiology.
Abnormalities in NMDAR density are observed in postmortem
activation patterns during cognitive tasks.4,5,62–65Moreover,
positron emission tomography scans suggest that hippocam-
pal NMDAR binding is significantly reduced in untreated, but
not antipsychotic-treated, SZ patients.66Genetic association
studies have identified several candidate genes implicated in
NMDAR stability or trafficking, including neuregulin 1,67
disrupted-in-schizophrenia 168and calcineurin.69Given that
NMDARs have an integral role in cognitive function and
synaptic plasticity,70these studies suggest that cognitive
impairments in SZ are mediated, at least in part, by NMDAR
We present data establishing a link between high levels of
STEP61and aberrant NMDAR signaling in SZ. STEP61levels
are increased in two independent patient populations and two
different brain regions (DLPFC and anterior cingulate cortex).
It has been suggested that medication status might affect
gene expression in SZ.71,72Given that STEP61 levels
are elevated in SZ patient populations both on (Stanley
total levels of Tau and PSD-95 in WT and STEP KO mice. (a, b) WT and STEP KO
an equal volume of saline in control mice, and tissue was extracted 24h following
the last injection. PCP treatment significantly increased the phosphorylation of
ERK1/2 and decreased protein levels of Tau in cortical synaptosomal (P2) fractions
of WT mice, but not STEP KOs (*Po0.05, **Po0.01; one-sample t-test).
Conversely, PSD-95 was significantly increased after subchronic PCP injections in
P2 fractions of STEP KO mice and not WT mice (*Po0.05; one-sample t-test). All
protein levels were normalized to glyceraldehyde-3-phosphate dehydrogenase
(GAPDH), and pERK1/2 was normalized to the total ERK1/2 levels. Data represent
the mean percentage of control±s.e.m.
cognitive deficits. (a) WT (n¼10) and STEP KO (n¼8) were acutely injected with
A repeated-measures analysis of variance indicated a main effect of dose
(Po0.001) and a significant dose by genotype interaction (Po0.02). Tukey’s post-
hoc indicated that WT was significantly different from STEP KO at the PCP doses
indicated (*Po0.05). (b) Exploration time of the novel and familiar objects during
the 24-h delay object recognition retention trial (n¼12 for all groups). Both saline-
treated WT and STEP KO mice spent significantly more time with the novel object
than the chance value of 15s (one-sample t-test; *Po0.05). WT mice treated
subchronically with PCP (5mgkg?1, i.p. twice daily for 7 consecutive days) did not
spend more time than chance with the novel object. In contrast, STEP KOs spent
significantly more time with the novel object (one-sample t-test; *Po0.05).
STEP KO mice are less sensitive to PCP-induced behavioral and
Role of STEP in schizophrenia
NC Carty et al
Foundation cohort; Supplementary Table S1) or off (Mt. Sinai
cohort; Supplementary Table S2) antipsychotic medication, it
seems unlikely that antipsychotic treatment was responsible
for the observed elevation of STEP61in human postmortem
We propose that increased STEP61levels promote the
dephosphorylation of STEP61substrates, including GluN2B,
leading to internalization and hypofunction of NMDARs. In
support of this, acute treatment with the psychotomimetics
MK-801 and PCP increase STEP61 levels and activity,
decrease tyrosine phosphorylation of STEP61 substrates
and reduce the surface expression of GluN2B-containing
significant changes in STEP61levels or phosphorylation after
subchronic PCP treatment, both short- and long-term
changes in NMDAR binding are found in mouse brain
following chronic PCP treatment.18Further studies will be
needed to determine whether acute disruption of STEP61
activity and its downstream substrates (for example, GluN2B,
ERK1/2 and Fyn) are sufficient to lead to longer-term PCP-
induced changes in synaptic plasticity.
Recent evidence supports a link between NMDAR-
mediated signaling and the UPS. UPS activation occurs
and degradation of glutamate receptor-interacting protein 1
is NMDAR-dependent.51Moreover, Ca2þ
NMDARs and L-type voltage-gated Ca2þchannels activates
CaMKII, a necessary component for proteasomal trafficking
into dendritic spines.73We found that MK-801 treatment
impairs the UPS and led to an increase in STEP61. Previous
studies establish that disruption of the UPS by b-amyloid
promotes an elevation of STEP61.30However, it is likely that
impairment of the UPS leads to accumulation of additional
proteins as well. The fact that STEP KOs are less sensitive
behaviorally to the acute and chronic effects of PCP suggest
that STEP61accumulation is an important contributor to the
observed findings. Although we provide evidence for UPS
involvement in the psychotomimetic-induced increase in
STEP61expression in cultured neurons and mice, whether a
similar mechanism occurs in humans with SZ remains to be
We tested the causal importance of the changes in STEP61
expression and its substrates by examining the behavioral
effectsofPCP in WTand STEP KOmice. AcuteuseofPCP in
humans induces positive symptoms of SZ, while PCP abuse
can lead to persistent SZ-like symptoms.2,74,75Unlike other
psychotomimetic drugs, PCP elicits positive and negative
symptoms, and cognitive deficits in humans.76,77Our data are
consistent with STEP KOs being less sensitive than WT to
PCP-induced hyperactivity and cognitive deficits. As STEP
KOs exhibit increased cortical NMDAR surface expression
and ERK1/2 phosphorylation,37,41it is possible that the
enhanced tyrosine phosphorylation of STEP61 substrates
and increased NMDAR surface expression contribute to the
findings that STEP KOs are less sensitive to the behavioral
effects of PCP. Moreover, the activation and expression of
signaling molecules required for synaptic plasticity and
cognitive function, including ERK1/2, Tau and PSD-95, are
differentially affected by subchronic PCP treatment in WT and
STEP KO mice, pointing to potential molecular underpinnings
that may explain why STEP KOs are less sensitive behavio-
rally to PCP than WT.
Another important finding was that neuroleptic treatments
regulate STEP61phosphorylation. Rodent and human studies
demonstrate that first-generation neuroleptics are more
effective at treating positive symptoms in SZ, compared with
their efficacy on negative symptoms or cognitive deficits.78,79
Second-generation medications, including clozapine and
risperidone, appear to be more effective at reducing the
cognitive and behavioral deficits induced by PCP, ketamine
and MK-801.12,47,48While the underlying mechanism(s)
mediating the beneficial effects of neuroleptics are still under
debate, our data suggest that neuroleptics function, at least in
part, through the inactivation of STEP61.
Administration of D2 antagonists to cortical cultures led to
the phosphorylation of STEP61, a modification that prevents
STEP61 from interacting with its substrates. Subchronic
haloperidol treatment in mice led to a similar increase in
STEP61 phosphorylation with no change in total STEP61
levels. Subchronic risperidone and clozapine treatment
significantly increased the phosphorylation of STEP61. How-
ever, both of these neuroleptics also increased STEP61levels
in synaptosomal (P2) fractions. While the mechanism by
which these two drugs increase STEP61expression remains
to be determined, it is possible that subchronic neuroleptic
treatment promotes trafficking of STEP61to the synapse as a
homeostatic mechanism. As STEP61 phosphorylation is
increased by antipsychotics to a greater magnitude than its
expression level, it appears that increased STEP61in the P2
fraction is insufficient to reverse the decreased STEP61
activity and enhanced NMDAR surface expression elicited
by neuroleptic treatment.
In summary, our results implicate STEP61as one likely
mediator of NMDAR hypofunction in SZ. Increased STEP61
levels are present both in postmortem SZ brains and mice
exposed to psychotomimetics, and genetically eliminating
STEP61results in progeny less sensitive behaviorally to these
drugs. One possible explanation for increased STEP61levels
is impaired UPS function in SZ. It is likely that other
mechanisms affect STEP61expression, such as mutations
in specific UPS enzymes or disruption of microRNAs that
regulate STEP61expression. The present findings establish
that neuroleptics used to treat SZ act, at least in part, via
STEP61inactivation. Dysregulation of STEP61represents a
previously unappreciated pathophysiological contributor to
SZ and implicates STEP61as a novel pharmacological target
Conflict of interest
is now at Johnson & Johnson Pharmaceutical Research and
Development; the work under his supervision was initiated
while he was at the NIMH.
Acknowledgements. We thank Ms Veronica Galvin and Mr Evan Wilson-
Wallis for technical assistance, as well as laboratory members for helpful
discussions and critical reading of the manuscript. The work was funded by NIH
Role of STEP in schizophrenia
NC Carty et al
Institutes of Health (IRP-NIMH-NIH).
Author contributions: NCC performed animal injections, tissue proces-
sing and biochemistry; NCC performed the majority of behavioral tests with
assistance from PRC and CP; PK and JB assisted in tissue and cell culture
preparation and biochemistry; JX assisted in cell culture preparation, stimulations
and biochemistry: DRA and PY performed the postmortem analyses with
supervision from GC; SMG-G performed statistical analyses; NCC, SMG-G, CP
and PJL wrote the manuscript. All authors contributed to the experimental design.
schizophrenia. Am J Psychiatry 2001; 158: 1367–1377.
2. Krystal JH, Anand A, Moghaddam B. Effects of NMDA receptor antagonists: implications
for the pathophysiology of schizophrenia. Arch Gen Psychiatry 2002; 59: 663–664.
3. Jentsch JD, Roth RH. The neuropsychopharmacology of phencyclidine: from NMDA
receptor hypofunction to the dopamine hypothesis of schizophrenia. Neuropsycho-
pharmacology 1999; 20: 201–225.
4. Beneyto M, Kristiansen LV, Oni-Orisan A, McCullumsmith RE, Meador-Woodruff JH.
Abnormal glutamate receptor expression in the medial temporal lobe in schizophrenia
and mood disorders. Neuropsychopharmacology 2007; 32: 1888–1902.
5. Meador-Woodruff JH, Clinton SM, Beneyto M, McCullumsmith RE. Molecular
abnormalities of the glutamate synapse in the thalamus in schizophrenia. Ann N Y Acad
Sci 2003; 1003: 75–93.
6. Stone JM, Erlandsson K, Arstad E, Squassante L, Teneggi V, Bressan RA et al.
Relationship between ketamine-induced psychotic symptoms and NMDA receptor
occupancy: a [(123)I]CNS-1261 SPET study. Psychopharmacology 2008; 197: 401–408.
7. Krystal JH, Karper LP, Seibyl JP, Freeman GK, Delaney R, Bremner JD et al.
Subanesthetic effects of the noncompetitive NMDA antagonist, ketamine, in humans.
Psychotomimetic, perceptual, cognitive, and neuroendocrine responses. Arch Gen
Psychiatry 1994; 51: 199–214.
8. Jentsch JD, Elsworth JD, Taylor JR, Redmond Jr DE, Roth RH. Dysregulation of
mesoprefrontal dopamine neurons induced by acute and repeated phencyclidine
administration in the nonhuman primate: implications for schizophrenia. Adv Pharmacol
1998; 42: 810–814.
9. Stoet G, Snyder LH. Effects of the NMDA antagonist ketamine on task-switching
performance: evidence for specific impairments of executive control. Neuropsycho-
pharmacology 2006; 31: 1675–1681.
10. Vales K, Bubenikova-Valesova V, Klement D, Stuchlik A. Analysis of sensitivity to MK-801
treatment in a novel active allothetic place avoidance task and in the working memory
version ofthe Morris water maze reveals differences between Long-Evansand Wistarrats.
Neurosci Res 2006; 55: 383–388.
11. Zhang WN, Bast T, Feldon J. Microinfusion of the non-competitive N-methyl-D-aspartate
receptor antagonist MK-801 (dizocilpine) into the dorsal hippocampus of wistar rats does
not affect latent inhibition and prepulse inhibition, but increases startle reaction and
locomotor activity. Neuroscience 2000; 101: 589–599.
12. Ishii D, Matsuzawa D, Kanahara N, Matsuda S, Sutoh C, Ohtsuka H et al. Effects of
aripiprazole on MK-801-induced prepulse inhibition deficits and mitogen-activated protein
kinase signal transduction pathway. Neurosci Lett 2010; 471: 53–57.
13. Bast T, Zhang W, Feldon J, White IM. Effects of MK801 and neuroleptics on
prepulse inhibition: re-examination in two strains of rats. Pharmacol Biochem Behav
2000; 67: 647–658.
14. Harris LW, Sharp T, Gartlon J, Jones DN, Harrison PJ. Long-term behavioural, molecular
and morphological effects of neonatal NMDA receptor antagonism. Eur J Neurosci 2003;
15. Jentsch JD, Elsworth JD, Redmond Jr DE, Roth RH. Phencyclidine increases forebrain
monoamine metabolism in rats and monkeys: modulation by the isomers of HA966.
J Neurosci 1997; 17: 1769–1775.
16. Moghaddam B, Adams B, Verma A, Daly D. Activation of glutamatergic neurotransmission
by ketamine: a novel step in the pathway from NMDA receptor blockade to dopaminergic
and cognitive disruptions associated with the prefrontal cortex. J Neurosci 1997; 17:
17. Bubenikova-Valesova V, Horacek J, Vrajova M, Hoschl C. Models of schizophrenia in
humans and animals based on inhibition of NMDA receptors. Neurosci Biobehav Rev
2008; 32: 1014–1023.
18. Newell KA, Zavitsanou K, Huang XF. Short and long term changes in NMDA receptor
binding in mouse brain following chronic phencyclidine treatment. J Neural Transm 2007;
plasticity to failures of self-monitoring. Schizophr Bull 2009; 35: 509–527.
20. Olney JW, Farber NB. Glutamate receptor dysfunction and schizophrenia. Arch Gen
Psychiatry 1995; 52: 998–1007.
21. Abi-Saab WM, D’Souza DC, Moghaddam B, Krystal JH. The NMDA antagonist model for
schizophrenia: promise and pitfalls. Pharmacopsychiatry 1998; 31(Suppl 2): 104–109.
22. Goebel-Goody SM, Baum M, Paspalas CD, Fernandez SM, Carty NC, Kurup P et al.
Therapeutic implications for striatal-enriched protein tyrosine phosphatase (STEP) in
neuropsychiatric disorders. Pharmacol Rev 2012; 64: 65–87.
23. Sharma E, Zhao F, Bult A, Lombroso PJ. Identification of two alternatively spliced
transcripts of STEP: a subfamily of brain-enriched protein tyrosine phosphatases. Brain
Res Mol Brain Res 1995; 32: 87–93.
24. Bult A, Zhao F, Dirkx Jr R, Raghunathan A, Solimena M, Lombroso PJ. STEP: a family of
brain-enriched PTPs. Alternative splicing produces transmembrane, cytosolic and
truncated isoforms. Eur J Cell Biol 1997; 72: 337–344.
25. BultA,ZhaoF,DirkxJrR,SharmaE,LukacsiE,SolimenaM etal.STEP61:amember ofa
family of brain-enriched PTPs is localized to the endoplasmic reticulum. J Neurosci 1996;
26. Lombroso PJ, Naegele JR, Sharma E, Lerner M. A protein tyrosine phosphatase
expressed within dopaminoceptive neurons of the basal ganglia and related structures.
J Neurosci 1993; 13: 3064–3074.
27. Boulanger LM, Lombroso PJ, Raghunathan A, During MJ, Wahle P, Naegele JR. Cellular
and molecular characterization of a brain-enriched protein tyrosine phosphatase.
J Neurosci 1995; 15: 1532–1544.
28. Goebel-Goody SM, Davies KD, Alvestad Linger RM, Freund RK, Browning MD. Phospho-
regulation of synaptic and extrasynaptic N-methyl-D-aspartate receptors in adult
hippocampal slices. Neuroscience 2009; 158: 1446–1459.
29. Snyder EM, Nong Y, Almeida CG, Paul S, Moran T, Choi EY et al. Regulation of NMDA
receptor trafficking by amyloid-beta. Nat Neurosci 2005; 8: 1051–1058.
30. Kurup P, Zhang Y, Xu J, Venkitaramani DV, Haroutunian V, Greengard P et al. Abeta-
mediated NMDA receptor endocytosis in Alzheimer’s disease involves ubiquitination of the
tyrosine phosphatase STEP61. J Neurosci 2010; 30: 5948–5957.
31. Zhang Y, Venkitaramani DV, Gladding CM, Zhang Y, Kurup P, Molnar E et al. The tyrosine
phosphatase STEP mediates AMPA receptor endocytosis after metabotropic glutamate
receptor stimulation. J Neurosci 2008; 28: 10561–10566.
32. Nguyen TH, Liu J, Lombroso PJ. Striatal enriched phosphatase 61 dephosphorylates
Fyn at phosphotyrosine 420. J Biol Chem 2002; 277: 24274–24279.
33. Venkitaramani DV, Moura PJ, Picciotto MR, Lombroso PJ. Striatal-enriched protein
tyrosine phosphatase (STEP) knockout mice have enhanced hippocampal memory.
Eur J Neurosci 2011; 33: 2288–2298.
34. Xu J, Kurup P, Bartos JA, Patriarchi T, Hell JW, Lombroso PJ. STriatal-enriched
protein tyrosine phosphatase (STEP) regulates Pyk2 activity. J Biol Chem 2012; 287:
35. Paul S, Nairn AC, Wang P, Lombroso PJ. NMDA-mediated activation of the tyrosine
phosphatase STEP regulates the duration of ERK signaling. Nat Neurosci 2003; 6: 34–42.
36. Xu J, Kurup P, Zhang Y, Goebel-Goody SM, Wu PH, Hawasli AH et al. Extrasynaptic
NMDA receptors couple preferentially to excitotoxicity via calpain-mediated cleavage of
STEP. J Neurosci 2009; 29: 9330–9343.
37. Zhang Y, Kurup P, Xu J, Carty N, Fernandez SM, Nygaard HB et al. Genetic reduction of
striatal-enriched tyrosine phosphatase (STEP) reverses cognitive and cellular deficits in an
Alzheimer’s disease mouse model. Proc Natl Acad Sci USA 2010; 107: 19014–19019.
38. Nakazawa T, Komai S, Tezuka T, Hisatsune C, Umemori H, Semba K et al.
Characterization of Fyn-mediated tyrosine phosphorylation sites on GluR epsilon 2
(NR2B) subunit of the N-methyl-D-aspartate receptor. J Biol Chem 2001; 276: 693–699.
39. Paul S, Snyder GL, Yokakura H, Picciotto MR, Nairn AC, Lombroso PJ. The dopamine/D1
STEP via a PKA-dependent pathway. J Neurosci 2000; 20: 5630–5638.
40. Valjent E, Pascoli V, Svenningsson P, Paul S, Enslen H, Corvol JC et al. Regulation of a
protein phosphatase cascade allows convergent dopamine and glutamate signals to
activate ERK in the striatum. Proc Natl Acad Sci USA 2005; 102: 491–496.
41. Venkitaramani DV, Paul S, Zhang Y, Kurup P, Ding L, Tressler L et al. Knockout of striatal
enriched protein tyrosine phosphatase in mice results in increased ERK1/2
phosphorylation. Synapse 2009; 63: 69–81.
42. Flores-Hernandez J, Cepeda C, Hernandez-Echeagaray E, Calvert CR, Jokel ES,
striatal neurons: role of D1 receptors and DARPP-32. J Neurophysiol 2002; 88:
43. BateupHS, SvenningssonP, Kuroiwa M, Gong S,Nishi A, HeintzN etal. Cell type-specific
regulation of DARPP-32 phosphorylation by psychostimulant and antipsychotic drugs.
Nat Neurosci 2008; 11: 932–939.
44. Jarskog LF, Gilmore JH, Selinger ES, Lieberman JA. Cortical bcl-2 protein expression and
apoptotic regulation in schizophrenia. Biol Psychiatry 2000; 48: 641–650.
45. Torrey EF, Webster M, Knable M, Johnston N, Yolken RH. The stanley foundation brain
collection and neuropathology consortium. Schizophr Res 2000; 44: 151–155.
46. Yuan P, Zhou R, Wang Y, Li X, Li J, Chen G et al. Altered levels of extracellular signal-
regulated kinase signaling proteins in postmortem frontal cortex of individuals with mood
disorders and schizophrenia. J Affect Disord 2010; 124: 164–169.
cognitive deficit in the novel object recognition task in the rat. Behav Brain Res 2007; 184:
48. Beraki S, Kuzmin A, Tai F, Ogren SO. Repeated low dose of phencyclidine administration
impairs spatial learning in mice: blockade by clozapine but not by haloperidol.
Eur Neuropsychopharmacol 2008; 18: 486–497.
Role of STEP in schizophrenia
NC Carty et al
49. Hu Y, Zhang Y, Venkitaramani DV, Lombroso PJ. Translation of striatal-enriched protein
tyrosine phosphatase (STEP) after beta1-adrenergic receptor stimulation. J Neurochem
2007; 103: 531–541.
50. Colledge M, Snyder EM, Crozier RA, Soderling JA, Jin Y, Langeberg LK et al.
Ubiquitination regulates PSD-95 degradation and AMPA receptor surface expression.
Neuron 2003; 40: 595–607.
51. Guo L, Wang Y. Glutamate stimulates glutamate receptor interacting protein 1 degradation
by ubiquitin-proteasome system to regulate surface expression of GluR2. Neuroscience
2007; 145: 100–109.
52. Deutsch SI, Hitri A. Measurement of an explosive behavior in the mouse, induced by MK-
801, a PCP analogue. Clin Neuropharmacol 1993; 16: 251–257.
53. Deutsch SI, Rosse RB, Riggs RL, Koetzner L, Mastropaolo J. The competitive NMDA
antagonist CPP blocks MK-801-elicited popping behavior in mice. Neuropsycho-
pharmacology 1996; 15: 329–331.
54. Elsworth JD, Jentsch JD, Morrow BA, Redmond Jr DE, Roth RH. Clozapine normalizes
prefrontal cortex dopamine transmission in monkeys subchronically exposed to
phencyclidine. Neuropsychopharmacology 2008; 33: 491–496.
55. Jentsch JD, Redmond Jr DE, Elsworth JD, Taylor JR, Youngren KD, Roth RH. Enduring
cognitive deficits and cortical dopamine dysfunction in monkeys after long-term
administration of phencyclidine. Science 1997; 277: 953–955.
56. Jentsch JD, Tran A, Le D, Youngren KD, Roth RH. Subchronic phencyclidine
administration reduces mesoprefrontal dopamine utilization and impairs prefrontal
cortical-dependent cognition in the rat. Neuropsychopharmacology 1997; 17: 92–99.
57. Hashimoto K, Fujita Y, Shimizu E, Iyo M. Phencyclidine-induced cognitive deficits in mice
are improved by subsequent subchronic administration of clozapine, but not haloperidol.
Eur J Pharmacol 2005; 519: 114–117.
58. Noda Y, Yamada K, Furukawa H, Nabeshima T. Enhancement of immobility in a forced
swimming test by subacute or repeated treatment with phencyclidine: a new model of
schizophrenia. Br J Pharmacol 1995; 116: 2531–2537.
59. Nabeshima T, Hiramatsu M, Furukawa H, Kameyama T. Effects of acute and chronic
administrations of phencyclidine on the levels of serotonin and 5-hydroxyindoleacetic acid
in discrete brain areas of mouse. Life Sci 1985; 36: 939–946.
of mitogen-activated protein kinases following chronic administration of phencyclidine in
rat brain. Neuropsychopharmacology 2001; 24: 267–277.
61. Chen Q, Zhou Z, Zhang L, Wang Y, Zhang YW, Zhong M et al. Tau protein is involved in
morphological plasticity in hippocampal neurons in response to BDNF. Neurochem Int
2012; 60: 233–242.
62. Gao XM, Sakai K, Roberts RC, Conley RR, Dean B, Tamminga CA. Ionotropic glutamate
receptors and expression of N-methyl-D-aspartate receptor subunits in subregions of
human hippocampus: effects of schizophrenia. Am J Psychiatry 2000; 157: 1141–1149.
64. Meador-Woodruff JH, Hogg Jr AJ, Smith RE. Striatal ionotropic glutamate receptor
expression in schizophrenia, bipolar disorder, and major depressive disorder. Brain Res
Bull 2001; 55: 631–640.
65. Meador-Woodruff JH, Watson SJ. Postmortem studies in schizophrenic brain. J Psychiatr
Res 1997; 31: 157–158.
66. Pilowsky LS. Probing targets for antipsychotic drug action with PET and SPET receptor
imaging. Nucl Med Commun 2001; 22: 829–833.
Neuregulin 1 and susceptibility to schizophrenia. Am J Hum Genet 2002; 71: 877–892.
68. Johnstone M, Thomson PA, Hall J, McIntosh AM, Lawrie SM, Porteous DJ. DISC1 in
schizophrenia: genetic mouse models and human genomic imaging. Schizophr Bull 2011;
69. Miyakawa T, Leiter LM, Gerber DJ, Gainetdinov RR, Sotnikova TD, Zeng H et al.
Conditional calcineurin knockout mice exhibit multiple abnormal behaviors related to
schizophrenia. Proc Natl Acad Sci USA 2003; 100: 8987–8992.
70. Bliss TV, Collingridge GL. A synaptic model of memory: long-term potentiation in the
hippocampus. Nature 1993; 361: 31–39.
71. Fatemi SH, Folsom TD, Reutiman TJ, Novak J, Engel RH. Comparative gene expression
study of the chronic exposure to clozapine and haloperidol in rat frontal cortex. Schizophr
Res 2012; 134: 211–218.
72. Girgenti MJ, Nisenbaum LK, Bymaster F, Terwilliger R, Duman RS, Newton SS.
Antipsychotic-induced gene regulation in multiple brain regions. J Neurochem 2010; 113:
73. Bingol B, Wang CF, Arnott D, Cheng D, Peng J, Sheng M. Autophosphorylated
CaMKIIalpha acts as a scaffold to recruit proteasomes to dendritic spines. Cell 2010; 140:
74. Cohen BD, Rosenbaum G, Luby ED, Gottlieb JS. Comparison of phencyclidine
hydrochloride (Sernyl) with other drugs. Simulation of schizophrenic performance with
(Amytal) sodium; II. Symbolic and sequential thinking. Arch Gen Psychiatry 1962; 6:
75. Javitt DC, Zukin SR. Recent advances in the phencyclidine model of schizophrenia.
Am J Psychiatry 1991; 148: 1301–1308.
76. Ellison G, Keys A, Noguchi K. Long-term changes in brain following continuous
phencyclidine administration: an autoradiographic study using flunitrazepam, ketanserin,
mazindol, quinuclidinyl benzilate, piperidyl-3,4-3H(N)-TCP, and AMPA receptor ligands.
Pharmacol Toxicol 1999; 84: 9–17.
77. Ellison GD, Keys AS. Persisting changes in brain glucose uptake following neurotoxic
doses of phencyclidine which mirror the acute effects of the drug. Psychopharmacology
1996; 126: 271–274.
78. Molteni R, Calabrese F, Racagni G, Fumagalli F, Riva MA. Antipsychotic drug actions on
gene modulation and signaling mechanisms. Pharmacol Ther 2009; 124: 74–85.
79. Shin JK, Malone DT, Crosby IT, Capuano B. Schizophrenia: a systematic review of the
disease state, current therapeutics and their molecular mechanisms of action. Curr Med
Chem 2011; 18: 1380–1404.
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Role of STEP in schizophrenia
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