Sexual dimorphism in a neuronal mechanism of spinal hyperexcitability across rodent and human models of pathological pain.

The prevalence and severity of many chronic pain syndromes differ across sex, and recent studies have identified differences in immune signalling within spinal nociceptive circuits as a potential mediator. Although it has been proposed that sex-specific pain mechanisms converge once they reach neurons within the superficial dorsal horn, direct investigations using rodent and human preclinical pain models have been lacking. Here, we discovered that in the Freund's adjuvant in vivo model of inflammatory pain, where both male and female rats display tactile allodynia, a pathological coupling between KCC2-dependent disinhibition and N-methyl-D-aspartate receptor (NMDAR) potentiation within superficial dorsal horn neurons was observed in male but not female rats. Unlike males, the neuroimmune mediator brain-derived neurotrophic factor (BDNF) failed to downregulate inhibitory signalling elements (KCC2 and STEP61) and upregulate excitatory elements (pFyn, GluN2B and pGluN2B) in female rats, resulting in no effect of ex vivo brain-derived neurotrophic factor on synaptic NMDAR responses in female lamina I neurons. Importantly, this sex difference in spinal pain processing was conserved from rodents to humans. As in rodents, ex vivo spinal treatment with BDNF downregulated markers of disinhibition and upregulated markers of facilitated excitation in superficial dorsal horn neurons from male but not female human organ donors. Ovariectomy in female rats recapitulated the male pathological pain neuronal phenotype, with BDNF driving a coupling between disinhibition and NMDAR potentiation in adult lamina I neurons following the prepubescent elimination of sex hormones in females. This discovery of sexual dimorphism in a central neuronal mechanism of chronic pain across species provides a foundational step towards a better understanding and treatment for pain in both sexes.

STEP inhibition prevents Aß-mediated damage in dendritic complexity and spine density in Alzheimer's disease.

Loss of dendritic spines and decline of cognitive function are hallmarks of patients with Alzheimer's disease (AD). Previous studies have shown that AD pathophysiology involves increased expression of a central nervous system-enriched protein tyrosine phosphatase called STEP (STriatal-Enriched protein tyrosine Phosphatase). STEP opposes the development of synaptic strengthening by dephosphorylating substrates, including GluN2B, Pyk2, and ERK1/2. Genetic reduction of STEP as well as pharmacological inhibition of STEP improve cognitive function and hippocampal memory in the 3×Tg-AD mouse model. Here, we show that the improved cognitive function is accompanied by an increase in synaptic connectivity in cell cultures as well as in the triple transgenic AD mouse model, further highlighting the potential of STEP inhibitors as a therapeutic agent.

Development of a Robust High-Throughput Screening Platform for Inhibitors of the Striatal-Enriched Tyrosine Phosphatase (STEP).

Many human diseases are the result of abnormal expression or activation of protein tyrosine kinases (PTKs) and protein tyrosine phosphatases (PTPs). Not surprisingly, more than 30 tyrosine kinase inhibitors (TKIs) are currently in clinical use and provide unique treatment options for many patients. PTPs on the other hand have long been regarded as "undruggable" and only recently have gained increased attention in drug discovery. Striatal-enriched tyrosine phosphatase (STEP) is a neuron-specific PTP that is overactive in Alzheimer's disease (AD) and other neurodegenerative and neuropsychiatric disorders, including Parkinson's disease, schizophrenia, and fragile X syndrome. An emergent model suggests that the increase in STEP activity interferes with synaptic function and contributes to the characteristic cognitive and behavioral deficits present in these diseases. Prior efforts to generate STEP inhibitors with properties that warrant clinical development have largely failed. To identify novel STEP inhibitor scaffolds, we developed a biophysical, label-free high-throughput screening (HTS) platform based on the protein thermal shift (PTS) technology. In contrast to conventional HTS using STEP enzymatic assays, we found the PTS platform highly robust and capable of identifying true hits with confirmed STEP inhibitory activity and selectivity. This new platform promises to greatly advance STEP drug discovery and should be applicable to other PTP targets.

The activity of the Striatal-enriched protein tyrosine phosphatase in neuronal cells is modulated by adenosine A2A receptor.

We recently demonstrated that a tonic activation of adenosine A2A receptors (A2A Rs) is required for cocaine-induced synaptic depression and increase in the activity of STriatal-Enriched protein tyrosine Phosphatase (STEP). In this study, we elaborated on the relationship between A2A R and STEP using genetic, pharmacological, and cellular tools. We found that the activities of protein tyrosine phosphatases (PTPs), and in particular of STEP, are significantly increased in the striatum and hippocampus of a transgenic rat strain over-expressing the neuronal A2A R (NSEA2A ) with respect to wild-type (WT) rats. Moreover the selective A2A R agonist 4-[2-[[6-Amino-9-(N-ethyl-ß-d-ribofuranuronamidosyl)-9H-purin-2-yl]amino]ethyl]benzenepropanoic acid hydrochloride up-regulates PTPs and STEP activities in WT but not in NSEA2A rats, while the selective A2A R antagonist 4-(-2-[7-amino-2-{2-furyl}{1,2,4}triazolo{2,3-a} {1,3,5}triazin-5-yl-amino]ethyl)phenol restores the tyrosine phosphatase activities in NSEA2A , having no effects in WT rats. In addition, while cocaine induced the activation of PTP and STEP in WT rats, it failed to increase phosphatase activity in NSEA2A rats. A2A Rs modulate STEP activity also in the SH-SY5Y neuroblastoma cell line, where a calcium-dependent calcineurin/PP1 pathway was found to play a major role. In summary, the present study identified a novel interaction between A2A R and STEP that could have important clinical implications, since STEP has emerged as key regulator of signaling pathways involved in neurodegenerative and neuropsychiatric diseases and A2A Rs are considered a promising target for the development of therapeutic strategies for different diseases of the central nervous system. Read the Editorial Highlight for this article on page 270.

Loss of STEP61 couples disinhibition to N-methyl-d-aspartate receptor potentiation in rodent and human spinal pain processing.

Dysregulated excitability within the spinal dorsal horn is a critical mediator of chronic pain. In the rodent nerve injury model of neuropathic pain, BDNF-mediated loss of inhibition (disinhibition) gates the potentiation of excitatory GluN2B N-methyl-d-aspartate receptor (NMDAR) responses at lamina I dorsal horn synapses. However, the centrality of this mechanism across pain states and species, as well as the molecular linker involved, remain unknown. Here, we show that KCC2-dependent disinhibition is coupled to increased GluN2B-mediated synaptic NMDAR responses in a rodent model of inflammatory pain, with an associated downregulation of the tyrosine phosphatase STEP61. The decreased activity of STEP61 is both necessary and sufficient to prime subsequent phosphorylation and potentiation of GluN2B NMDAR by BDNF at lamina I synapses. Blocking disinhibition reversed the downregulation of STEP61 as well as inflammation-mediated behavioural hypersensitivity. For the first time, we characterize GluN2B-mediated NMDAR responses at human lamina I synapses and show that a human ex vivo BDNF model of pathological pain processing downregulates KCC2 and STEP61 and upregulates phosphorylated GluN2B at dorsal horn synapses. Our results demonstrate that STEP61 is the molecular brake that is lost following KCC2-dependent disinhibition and that the decrease in STEP61 activity drives the potentiation of excitatory GluN2B NMDAR responses in rodent and human models of pathological pain. The ex vivo human BDNF model may thus form a translational bridge between rodents and humans for identification and validation of novel molecular pain targets.

Intra-prelimbic cortical inhibition of striatal-enriched tyrosine phosphatase suppresses cocaine seeking in rats.

Cocaine self-administration in rats results in dysfunctional neuroadaptations in the prelimbic (PrL) cortex during early abstinence. Central to these adaptations is decreased phospho-extracellular signal-regulated kinase (p-ERK), which plays a key role in cocaine seeking. Normalizing ERK phosphorylation in the PrL cortex immediately after cocaine self-administration decreases subsequent cocaine seeking. The disturbance in ERK phosphorylation is accompanied by decreased phosphorylation of striatal-enriched protein tyrosine phosphatase (STEP), indicating increased STEP activity. STEP is a well-recognized ERK phosphatase but whether STEP activation during early abstinence mediates the decrease in p-ERK and is involved in relapse is unknown. Here, we show that a single intra-PrL cortical microinfusion of the selective STEP inhibitor, TC-2153, immediately after self-administration suppressed post-abstinence context-induced relapse under extinction conditions and cue-induced reinstatement, but not cocaine prime-induced drug seeking or sucrose seeking. Moreover, an intra-PrL cortical TC-2153 microinfusion immediately after self-administration prevented the cocaine-induced decrease in p-ERK within the PrL cortex during early abstinence. Interestingly, a systemic TC-2153 injection at the same timepoint failed to suppress post-abstinence context-induced relapse or cue-induced reinstatement, but did suppress cocaine prime-induced reinstatement. These data indicate that the STEP-induced ERK dephosphorylation in the PrL cortex during early abstinence is a critical neuroadaptation that promotes relapse to cocaine seeking and that systemic versus intra-PrL cortical inhibition of STEP during early abstinence differentially suppresses cocaine seeking.

STEP61 is a substrate of the E3 ligase parkin and is upregulated in Parkinson's disease.

Parkinson's disease (PD) is characterized by the degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc). The loss of SNc dopaminergic neurons affects the plasticity of striatal neurons and leads to significant motor and cognitive disabilities during the progression of the disease. PARK2 encodes for the E3 ubiquitin ligase parkin and is implicated in genetic and sporadic PD. Mutations in PARK2 are a major contributing factor in the early onset of autosomal-recessive juvenile parkinsonism (AR-JP), although the mechanisms by which a disruption in parkin function contributes to the pathophysiology of PD remain unclear. Here we demonstrate that parkin is an E3 ligase for STEP61 (striatal-enriched protein tyrosine phosphatase), a protein tyrosine phosphatase implicated in several neuropsychiatric disorders. In cellular models, parkin ubiquitinates STEP61 and thereby regulates its level through the proteasome system, whereas clinically relevant parkin mutants fail to do so. STEP61 protein levels are elevated on acute down-regulation of parkin or in PARK2 KO rat striatum. Relevant to PD, STEP61 accumulates in the striatum of human sporadic PD and in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned mice. The increase in STEP61 is associated with a decrease in the phosphorylation of its substrate ERK1/2 and the downstream target of ERK1/2, pCREB [phospho-CREB (cAMP response element-binding protein)]. These results indicate that STEP61 is a novel substrate of parkin, although further studies are necessary to determine whether elevated STEP61 levels directly contribute to the pathophysiology of PD.

Inhibitor of the tyrosine phosphatase STEP reverses cognitive deficits in a mouse model of Alzheimer's disease.

STEP (STriatal-Enriched protein tyrosine Phosphatase) is a neuron-specific phosphatase that regulates N-methyl-D-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking, as well as ERK1/2, p38, Fyn, and Pyk2 activity. STEP is overactive in several neuropsychiatric and neurodegenerative disorders, including Alzheimer's disease (AD). The increase in STEP activity likely disrupts synaptic function and contributes to the cognitive deficits in AD. AD mice lacking STEP have restored levels of glutamate receptors on synaptosomal membranes and improved cognitive function, results that suggest STEP as a novel therapeutic target for AD. Here we describe the first large-scale effort to identify and characterize small-molecule STEP inhibitors. We identified the benzopentathiepin 8-(trifluoromethyl)-1,2,3,4,5-benzopentathiepin-6-amine hydrochloride (known as TC-2153) as an inhibitor of STEP with an IC50 of 24.6 nM. TC-2153 represents a novel class of PTP inhibitors based upon a cyclic polysulfide pharmacophore that forms a reversible covalent bond with the catalytic cysteine in STEP. In cell-based secondary assays, TC-2153 increased tyrosine phosphorylation of STEP substrates ERK1/2, Pyk2, and GluN2B, and exhibited no toxicity in cortical cultures. Validation and specificity experiments performed in wild-type (WT) and STEP knockout (KO) cortical cells and in vivo in WT and STEP KO mice suggest specificity of inhibitors towards STEP compared to highly homologous tyrosine phosphatases. Furthermore, TC-2153 improved cognitive function in several cognitive tasks in 6- and 12-mo-old triple transgenic AD (3xTg-AD) mice, with no change in beta amyloid and phospho-tau levels.

Inhibition of the tyrosine phosphatase STEP61 restores BDNF expression and reverses motor and cognitive deficits in phencyclidine-treated mice.

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.

Down-regulation of BDNF in cell and animal models increases striatal-enriched protein tyrosine phosphatase 61 (STEP61 ) levels.

Brain-derived neurotrophic factor (BDNF) regulates synaptic strengthening and memory consolidation, and altered BDNF expression is implicated in a number of neuropsychiatric and neurodegenerative disorders. BDNF potentiates N-methyl-D-aspartate receptor function through activation of Fyn and ERK1/2. STriatal-Enriched protein tyrosine Phosphatase (STEP) is also implicated in many of the same disorders as BDNF but, in contrast to BDNF, STEP opposes the development of synaptic strengthening. STEP-mediated dephosphorylation of the NMDA receptor subunit GluN2B promotes internalization of GluN2B-containing NMDA receptors, while dephosphorylation of the kinases Fyn, Pyk2, and ERK1/2 leads to their inactivation. Thus, STEP and BDNF have opposing functions. In this study, we demonstrate that manipulation of BDNF expression has a reciprocal effect on STEP61 levels. Reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. Moreover, a newly identified STEP inhibitor reverses the biochemical and motor abnormalities in BDNF(+/-) mice. In contrast, increased BDNF signaling upon treatment with a tropomyosin receptor kinase B agonist results in degradation of STEP61 and a subsequent increase in the tyrosine phosphorylation of STEP substrates in cultured neurons and in mouse frontal cortex. These findings indicate that BDNF-tropomyosin receptor kinase B signaling leads to degradation of STEP61 , while decreased BDNF expression results in increased STEP61 activity. A better understanding of the opposing interaction between STEP and BDNF in normal cognitive functions and in neuropsychiatric disorders will hopefully lead to better therapeutic strategies. Altered expression of BDNF and STEP61 has been implicated in several neurological disorders. BDNF and STEP61 are known to regulate synaptic strengthening, but in opposite directions. Here, we report that reduced BDNF signaling leads to elevation of STEP61 both in BDNF(+/-) mice and after acute BDNF knockdown in cortical cultures. In contrast, activation of TrkB receptor results in the degradation of STEP61 and reverses hyperlocomotor activity in BDNF(+/-) mice. Moreover, inhibition of STEP61 by TC-2153 is sufficient to enhance the Tyr phosphorylation of STEP substrates and also reverses hyperlocomotion in BDNF(+/-) mice. These findings give us a better understanding of the regulation of STEP61 by BDNF in normal cognitive functions and in neuropsychiatric disorders.

Role of Striatal-Enriched Tyrosine Phosphatase in Neuronal Function.

Striatal-enriched protein tyrosine phosphatase (STEP) is a CNS-enriched protein implicated in multiple neurologic and neuropsychiatric disorders. STEP regulates key signaling proteins required for synaptic strengthening as well as NMDA and AMPA receptor trafficking. Both high and low levels of STEP disrupt synaptic function and contribute to learning and behavioral deficits. High levels of STEP are present in human postmortem samples and animal models of Alzheimer's disease, Parkinson's disease, and schizophrenia and in animal models of fragile X syndrome. Low levels of STEP activity are present in additional disorders that include ischemia, Huntington's chorea, alcohol abuse, and stress disorders. Thus the current model of STEP is that optimal levels are required for optimal synaptic function. Here we focus on the role of STEP in Alzheimer's disease and the mechanisms by which STEP activity is increased in this illness. Both genetic lowering of STEP levels and pharmacological inhibition of STEP activity in mouse models of Alzheimer's disease reverse the biochemical and cognitive abnormalities that are present. These findings suggest that STEP is an important point for modulation of proteins required for synaptic plasticity.

Striatal-enriched protein tyrosine phosphatase regulates the PTPα/Fyn signaling pathway.

The tyrosine kinase Fyn has two regulatory tyrosine residues that when phosphorylated either activate (Tyr(420)) or inhibit (Tyr(531)) Fyn activity. Within the central nervous system, two protein tyrosine phosphatases (PTPs) target these regulatory tyrosines in Fyn. PTPα dephosphorylates Tyr(531) and activates Fyn, while STEP (STriatal-Enriched protein tyrosine Phosphatase) dephosphorylates Tyr(420) and inactivates Fyn. Thus, PTPα and STEP have opposing functions in the regulation of Fyn; however, whether there is cross talk between these two PTPs remains unclear. Here, we used molecular techniques in primary neuronal cultures and in vivo to demonstrate that STEP negatively regulates PTPα by directly dephosphorylating PTPα at its regulatory Tyr(789). Dephosphorylation of Tyr(789) prevents the translocation of PTPα to synaptic membranes, blocking its ability to interact with and activate Fyn. Genetic or pharmacologic reduction in STEP61 activity increased the phosphorylation of PTPα at Tyr(789), as well as increased translocation of PTPα to synaptic membranes. Activation of PTPα and Fyn and trafficking of GluN2B to synaptic membranes are necessary for ethanol (EtOH) intake behaviors in rodents. We tested the functional significance of STEP61 in this signaling pathway by EtOH administration to primary cultures as well as in vivo, and demonstrated that the inactivation of STEP61 by EtOH leads to the activation of PTPα, its translocation to synaptic membranes, and the activation of Fyn. These findings indicate a novel mechanism by which STEP61 regulates PTPα and suggest that STEP and PTPα coordinate the regulation of Fyn. STEP61 , PTPα, Fyn, and NMDA receptor (NMDAR) have been implicated in ethanol intake behaviors in the dorsomedial striatum (DMS) in rodents. Here, we report that PTPα is a novel substrate for STEP61. Upon ethanol exposure, STEP61 is phosphorylated and inactivated by protein kinase A (PKA) signaling in the DMS. As a result of STEP61 inhibition, there is an increase in the phosphorylation of PTPα, which translocates to lipid rafts and activates Fyn and subsequent NMDAR signaling. The results demonstrate a synergistic regulation of Fyn-NMDAR signaling by STEP61 and PTPα, which may contribute to the regulation of ethanol-related behaviors. NMDA, N-methyl-D-aspartate; PTPα, receptor-type protein tyrosine phosphatase alpha; STEP, STriatal-Enriched protein tyrosine Phosphatase.

Synthesis of benzopentathiepin analogs and their evaluation as inhibitors of the phosphatase STEP.

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain specific protein tyrosine phosphatase that has been implicated in many neurodegenerative diseases, such as Alzheimer's disease. We recently reported the benzopentathiepin TC-2153 as a potent inhibitor of STEP in vitro, cells and animals. Herein, we report the synthesis and evaluation of TC-2153 analogs in order to define what structural features are important for inhibition and to identify positions tolerant of substitution for further study. The trifluoromethyl substitution is beneficial for inhibitor potency, and the amine is tolerant of acylation, and thus provides a convenient handle for introducing additional functionality such as reporter groups.

Therapeutic implications for striatal-enriched protein tyrosine phosphatase (STEP) in neuropsychiatric disorders.

Striatal-enriched protein tyrosine phosphatase (STEP) is a brain-specific phosphatase that modulates key signaling molecules involved in synaptic plasticity and neuronal function. Targets include extracellular-regulated kinase 1 and 2 (ERK1/2), stress-activated protein kinase p38 (p38), the Src family tyrosine kinase Fyn, N-methyl-D-aspartate receptors (NMDARs), and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs). STEP-mediated dephosphorylation of ERK1/2, p38, and Fyn leads to inactivation of these enzymes, whereas STEP-mediated dephosphorylation of surface NMDARs and AMPARs promotes their endocytosis. Accordingly, the current model of STEP function posits that it opposes long-term potentiation and promotes long-term depression. Phosphorylation, cleavage, dimerization, ubiquitination, and local translation all converge to maintain an appropriate balance of STEP in the central nervous system. Accumulating evidence over the past decade indicates that STEP dysregulation contributes to the pathophysiology of several neuropsychiatric disorders, including Alzheimer's disease, schizophrenia, fragile X syndrome, epileptogenesis, alcohol-induced memory loss, Huntington's disease, drug abuse, stroke/ischemia, and inflammatory pain. This comprehensive review discusses STEP expression and regulation and highlights how disrupted STEP function contributes to the pathophysiology of diverse neuropsychiatric disorders.

Neuroprotective role of a brain-enriched tyrosine phosphatase, STEP, in focal cerebral ischemia.

The striatal-enriched phosphatase (STEP) is a component of the NMDA-receptor-mediated excitotoxic signaling pathway, which plays a key role in ischemic brain injury. Using neuronal cultures and a rat model of ischemic stroke, we show that STEP plays an initial role in neuroprotection, during the insult, by disrupting the p38 MAPK pathway. Degradation of active STEP during reperfusion precedes ischemic brain damage and is associated with secondary activation of p38 MAPK. Application of a cell-permeable STEP-derived peptide that is resistant to degradation and binds to p38 MAPK protects cultured neurons from hypoxia-reoxygenation injury and reduces ischemic brain damage when injected up to 6 h after the insult. Conversely, genetic deletion of STEP in mice leads to sustained p38 MAPK activation and exacerbates brain injury and neurological deficits after ischemia. Administration of the STEP-derived peptide at the onset of reperfusion not only prevents the sustained p38 MAPK activation but also reduces ischemic brain damage in STEP KO mice. The findings indicate a neuroprotective role of STEP and suggest a potential role of the STEP-derived peptide in stroke therapy.

Alterations in STriatal-Enriched protein tyrosine Phosphatase expression, activation, and downstream signaling in early and late stages of the YAC128 Huntington's disease mouse model.

Striatal neurodegeneration and synaptic dysfunction in Huntington's disease are mediated by the mutant huntingtin (mHtt) protein. MHtt disrupts calcium homeostasis and facilitates excitotoxicity, in part by altering NMDA receptor (NMDAR) trafficking and function. Pre-symptomatic (excitotoxin-sensitive) transgenic mice expressing full-length human mHtt with 128 polyglutamine repeats (YAC128 Huntington's disease mice) show increased calpain activity and extrasynaptic NMDAR (Ex-NMDAR) localization and signaling. Furthermore, Ex-NMDAR stimulation facilitates excitotoxicity in wild-type cortical neurons via calpain-mediated cleavage of STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61). The cleavage product, STEP33, cannot dephosphorylate p38 mitogen-activated protein kinase (MAPK), thereby augmenting apoptotic signaling. Here, we show elevated extrasynaptic calpain-mediated cleavage of STEP61 and p38 phosphorylation, as well as STEP61 inactivation and reduced extracellular signal-regulated protein kinase 1/2 phosphorylation (ERK1/2) in the striatum of 6-week-old, excitotoxin-sensitive YAC128 mice. Calpain inhibition reduced basal and NMDA-induced STEP61 cleavage. However, basal p38 phosphorylation was normalized by a peptide disrupting NMDAR-post-synaptic density protein-95 (PSD-95) binding but not by calpain inhibition. In 1-year-old excitotoxin-resistant YAC128 mice, STEP33 levels were not elevated, but STEP61 inactivation and p38 and ERK 1/2 phosphorylation levels were increased. These results show that in YAC128 striatal tissue, enhanced NMDAR-PSD-95 interactions contributes to elevated p38 signaling in early, excitotoxin-sensitive stages, and suggest that STEP61 inactivation enhances MAPK signaling at late, excitotoxin-resistant stages. The YAC128 Huntington's disease mouse model shows early, enhanced susceptibility to NMDA receptor-mediated striatal apoptosis, progressing to late-stage excitotoxicity resistance. This study shows that elevated NMDA receptor-PSD-95 interactions as well as decreased extrasynaptic STriatal-Enriched protein tyrosine Phosphatase 61 (STEP61) activation may contribute to early enhanced apoptotic signaling. In late-stage YAC128 mice, reduced STEP61 levels and activity correlate with elevated MAPK signaling, consistent with excitotoxicity resistance. Solid and dotted arrows indicate conclusions drawn from the current study and other literature, respectively.

Calpain and STriatal-Enriched protein tyrosine phosphatase (STEP) activation contribute to extrasynaptic NMDA receptor localization in a Huntington's disease mouse model.

In Huntington's disease (HD), the mutant huntingtin (mhtt) protein is associated with striatal dysfunction and degeneration. Excitotoxicity and early synaptic defects are attributed, in part, to altered NMDA receptor (NMDAR) trafficking and function. Deleterious extrasynaptic NMDAR localization and signalling are increased early in yeast artificial chromosome mice expressing full-length mhtt with 128 polyglutamine repeats (YAC128 mice). NMDAR trafficking at the plasma membrane is regulated by dephosphorylation of the NMDAR subunit GluN2B tyrosine 1472 (Y1472) residue by STriatal-Enriched protein tyrosine Phosphatase (STEP). NMDAR function is also regulated by calpain cleavage of the GluN2B C-terminus. Activation of both STEP and calpain is calcium-dependent, and disruption of calcium homeostasis occurs early in the HD striatum. Here, we show increased calpain cleavage of GluN2B at both synaptic and extrasynaptic sites, and elevated extrasynaptic total GluN2B expression in the YAC128 striatum. Calpain inhibition significantly reduced extrasynaptic GluN2B expression in the YAC128 but not wild-type striatum. Furthermore, calpain inhibition reduced whole-cell NMDAR current and the surface/internal GluN2B ratio in co-cultured striatal neurons, without affecting synaptic GluN2B localization. Synaptic STEP activity was also significantly higher in the YAC128 striatum, correlating with decreased GluN2B Y1472 phosphorylation. A substrate-trapping STEP protein (TAT-STEP C-S) significantly increased VGLUT1-GluN2B colocalization, as well as increasing synaptic GluN2B expression and Y1472 phosphorylation. Moreover, combined calpain inhibition and STEP inactivation reduced extrasynaptic, while increasing synaptic GluN2B expression in the YAC128 striatum. These results indicate that increased STEP and calpain activation contribute to altered NMDAR localization in an HD mouse model, suggesting new therapeutic targets for HD.

Alcohol inhibition of the NMDA receptor function, long-term potentiation, and fear learning requires striatal-enriched protein tyrosine phosphatase.

Alcohol's deleterious effects on memory are well known. Acute alcohol-induced memory loss is thought to occur via inhibition of NMDA receptor (NMDAR)-dependent long-term potentiation in the hippocampus. We reported previously that ethanol inhibition of NMDAR function and long-term potentiation is correlated with a reduction in the phosphorylation of Tyr(1472) on the NR2B subunit and ethanol's inhibition of the NMDAR field excitatory postsynaptic potential was attenuated by a broad spectrum tyrosine phosphatase inhibitor. These data suggested that ethanol's inhibitory effect may involve protein tyrosine phosphatases. Here we demonstrate that the loss of striatal-enriched protein tyrosine phosphatase (STEP) renders NMDAR function, phosphorylation, and long-term potentiation, as well as fear conditioning, less sensitive to ethanol inhibition. Moreover, the ethanol inhibition was "rescued" when the active STEP protein was reintroduced into the cells. Taken together, our data suggest that STEP contributes to ethanol inhibition of NMDAR function via dephosphorylation of tyrosine sites on NR2B receptors and lend support to the hypothesis that STEP may be required for ethanol's amnesic effects.

Individualized assessments in treatment research: an examination of parent-nominated target problems in the treatment of disruptive behaviors in youth with Tourette syndrome.

Youth with Tourette syndrome (TS) often exhibit disruptive behaviors. Although improvement data on rating scales support the efficacy of structured psychotherapeutic interventions, there is growing interest in personalized outcome assessments. This report examined parent-nominated target problems (PTPs) as an individualized outcome measure in 48 youth with TS and disruptive behaviors, who participated in one of two randomized psychotherapy trials. At baseline, parents described two primary problems to an independent evaluator who generated a structured narrative for each problem. These narratives were reviewed and updated at endpoint. When rated by five treatment-blind judges, the PTP rating demonstrated excellent reliability and good convergent validity with the Disruptive Behavior Rating Scale (DBRS). The PTP rating exhibited comparable treatment effects to the DBRS, and accounted for additional variance in global treatment outcome. The PTP rating serves a reliable, valid, and sensitive personalized assessment in research trials that provides complementary information to standardized rating scales.

Striatal-enriched protein-tyrosine phosphatase (STEP) regulates Pyk2 kinase activity.

Proline-rich tyrosine kinase 2 (Pyk2) is a member of the focal adhesion kinase family and is highly expressed in brain and hematopoietic cells. Pyk2 plays diverse functions in cells, including the regulation of cell adhesion, migration, and cytoskeletal reorganization. In the brain, it is involved in the induction of long term potentiation through regulation of N-methyl-d-aspartate receptor trafficking. This occurs through the phosphorylation and activation of Src family tyrosine kinase members, such as Fyn, that phosphorylate GluN2B at Tyr(1472). Phosphorylation at this site leads to exocytosis of GluN1-GluN2B receptors to synaptic membranes. Pyk2 activity is modulated by phosphorylation at several critical tyrosine sites, including Tyr(402). In this study, we report that Pyk2 is a substrate of striatal-enriched protein-tyrosine phosphatase (STEP). STEP binds to and dephosphorylates Pyk2 at Tyr(402). STEP KO mice showed enhanced phosphorylation of Pyk2 at Tyr(402) and of the Pyk2 substrates paxillin and ASAP1. Functional studies indicated that STEP opposes Pyk2 activation after KCl depolarization of cortical slices and blocks Pyk2 translocation to postsynaptic densities, a key step required for Pyk2 activation and function. This is the first study to identify Pyk2 as a substrate for STEP.