RESUMO
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.
Assuntos
Dor Crônica , Simportadores , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Feminino , Humanos , Masculino , Neurônios/metabolismo , Ratos , Caracteres SexuaisRESUMO
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.
Assuntos
Descoberta de Drogas , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Ensaios de Triagem em Larga Escala/métodos , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Humanos , Estrutura MolecularRESUMO
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.
Assuntos
Doença de Alzheimer , Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/genética , Animais , Modelos Animais de Doenças , Hipocampo , Memória , Camundongos , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Autism spectrum disorders (ASDs) are highly prevalent childhood illnesses characterized by impairments in communication, social behavior, and repetitive behaviors. Studies have found aberrant synaptic plasticity and neuronal connectivity during the early stages of brain development and have suggested that these contribute to an increased risk for ASD. STEP is a protein tyrosine phosphatase that regulates synaptic plasticity and is implicated in several cognitive disorders. Here we test the hypothesis that STEP may contribute to some of the aberrant behaviors present in the VPA-induced mouse model of ASD. In utero VPA exposure of pregnant dams results in autistic-like behavior in the pups, which is associated with a significant increase in the STEP expression in the prefrontal cortex. The elevated STEP protein levels are correlated with increased dephosphorylation of STEP substrates GluN2B, Pyk2 and ERK, suggesting upregulated STEP activity. Moreover, pharmacological inhibition of STEP rescues the sociability, repetitive and abnormal anxiety phenotypes commonly associated with ASD. These data suggest that STEP may play a role in the VPA model of ASD and STEP inhibition may have a potential therapeutic benefit in this model.
Assuntos
Transtorno do Espectro Autista/fisiopatologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Transtorno do Espectro Autista/tratamento farmacológico , Transtorno Autístico/tratamento farmacológico , Comportamento Animal , Modelos Animais de Doenças , Comportamento Exploratório/fisiologia , Feminino , Inibição Psicológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Plasticidade Neuronal/fisiologia , Córtex Pré-Frontal , Gravidez , Efeitos Tardios da Exposição Pré-Natal/induzido quimicamente , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Fosfatases não Receptoras/fisiologia , Comportamento Social , Comportamento Estereotipado/fisiologia , Ácido Valproico/efeitos adversosRESUMO
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.
Assuntos
Neurônios/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Receptor A2A de Adenosina/metabolismo , Animais , Linhagem Celular , Cocaína/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Inibidores da Captação de Dopamina/farmacologia , Feminino , Humanos , Masculino , Ratos , Ratos Sprague-Dawley , Ratos TransgênicosRESUMO
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.
Assuntos
Neuralgia/genética , Proteínas Tirosina Fosfatases não Receptoras/genética , Receptores de N-Metil-D-Aspartato/metabolismo , Adolescente , Adulto , Idoso , Animais , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Neuralgia/fisiopatologia , Fosforilação , Ratos , Receptores de N-Metil-D-Aspartato/genética , Sinapses/metabolismo , Adulto JovemRESUMO
Cognitive disabilities that occur with age represent a growing and expensive health problem. Age-associated memory deficits are observed across many species, but the underlying molecular mechanisms remain to be fully identified. Here, we report elevations in the levels and activity of the striatal-enriched phosphatase (STEP) in the hippocampus of aged memory-impaired mice and rats, in aged rhesus monkeys, and in people diagnosed with amnestic mild cognitive impairment (aMCI). The accumulation of STEP with aging is related to dysfunction of the ubiquitin-proteasome system that normally leads to the degradation of STEP. Higher level of active STEP is linked to enhanced dephosphorylation of its substrates GluN2B and ERK1/2, CREB inactivation, and a decrease in total levels of GluN2B and brain-derived neurotrophic factor (BDNF). These molecular events are reversed in aged STEP knockout and heterozygous mice, which perform similarly to young control mice in the Morris water maze (MWM) and Y-maze tasks. In addition, administration of the STEP inhibitor TC-2153 to old rats significantly improved performance in a delayed alternation T-maze memory task. In contrast, viral-mediated STEP overexpression in the hippocampus is sufficient to induce memory impairment in the MWM and Y-maze tests, and these cognitive deficits are reversed by STEP inhibition. In old LOU/C/Jall rats, a model of healthy aging with preserved memory capacities, levels of STEP and GluN2B are stable, and phosphorylation of GluN2B and ERK1/2 is unaltered. Altogether, these data suggest that elevated levels of STEP that appear with advancing age in several species contribute to the cognitive declines associated with aging.
Assuntos
Hipocampo/metabolismo , Transtornos da Memória/fisiopatologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Tirosina/metabolismo , Idoso de 80 Anos ou mais , Animais , Estudos de Casos e Controles , Feminino , Humanos , Macaca mulatta , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosforilação , Ratos , Ratos Sprague-DawleyRESUMO
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.
Assuntos
Benzotiepinas/farmacologia , Cocaína/administração & dosagem , Inibidores da Captação de Dopamina/administração & dosagem , Comportamento de Procura de Droga/efeitos dos fármacos , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Animais , MAP Quinases Reguladas por Sinal Extracelular , Masculino , Fosfoproteínas , Córtex Pré-Frontal , Ratos , Ratos Sprague-Dawley , AutoadministraçãoRESUMO
NMDA receptor signaling is critical for the development of synaptic plasticity, learning, and memory, and dysregulation of NMDAR signaling is implicated in a number of neurological disorders including schizophrenia (SZ). Previous work has demonstrated that the STriatal-Enriched protein tyrosine Phosphatase 61 kDa (STEP61) is elevated in human SZ postmortem cortical samples and after administration of psychotomimetics to cultures or mice. Here, we report that activation of synaptic NMDAR by bicuculline or D-serine results in the ubiquitination and proteasomal degradation of STEP61, and increased surface localization of GluN1/GluN2B receptors. Moreover, bicuculline or D-serine treatments rescue the motor and cognitive deficits in MK-801-treated mice and reduce STEP61 in mouse frontal cortex. These results suggest that STEP61 may contribute to the therapeutic effects of D-serine.
Assuntos
Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Proteólise , Receptores de N-Metil-D-Aspartato/metabolismo , Sinapses/metabolismo , Ubiquitinação , Animais , Bicuculina/farmacologia , Células Cultivadas , Proteína 4 Homóloga a Disks-Large/metabolismo , Lipoilação , Masculino , Camundongos Endogâmicos C57BL , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Proteólise/efeitos dos fármacos , Ratos , Especificidade por Substrato/efeitos dos fármacos , Sinapses/efeitos dos fármacos , Ubiquitinação/efeitos dos fármacosRESUMO
Fragile X syndrome (FXS) is the leading cause of inherited intellectual disability, with additional symptoms including attention deficit and hyperactivity, anxiety, impulsivity, and repetitive movements or actions. The majority of FXS cases are attributed to a CGG expansion that leads to transcriptional silencing and diminished expression of fragile X mental retardation protein (FMRP). FMRP, an RNA binding protein, regulates the synthesis of dendritically-translated mRNAs by stalling ribosomal translation. Loss of FMRP leads to increased translation of some of these mRNAs, including the CNS-specific tyrosine phosphatase STEP (STriatal-Enriched protein tyrosine Phosphatase). Genetic reduction of STEP in Fmr1 KO mice have diminished audiogenic seizures and a reversal of social and non-social anxiety-related abnormalities. This study investigates whether a newly discovered STEP inhibitor (TC-2153) could attenuate the behavioral and synaptic abnormalities in Fmr1 KO mice. TC-2153 reversed audiogenic seizure incidences, reduced hyperactivity, normalized anxiety states, and increased sociability in Fmr1 KO mice. Moreover, TC-2153 reduced dendritic spine density and improved synaptic aberrations in Fmr1 KO neuronal cultures as well as in vivo. TC-2153 also reversed the mGluR-mediated exaggerated LTD in brain slices derived from Fmr1 KO mice. These studies suggest that STEP inhibition may have therapeutic benefit in FXS.
Assuntos
Potenciais Pós-Sinápticos Excitadores/genética , Proteína do X Frágil da Deficiência Intelectual/metabolismo , Síndrome do Cromossomo X Frágil/complicações , Síndrome do Cromossomo X Frágil/patologia , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Sinapses/patologia , Adaptação Ocular/efeitos dos fármacos , Adaptação Ocular/genética , Animais , Animais Recém-Nascidos , Ansiedade/tratamento farmacológico , Ansiedade/etiologia , Benzotiepinas/farmacologia , Comportamento de Escolha/efeitos dos fármacos , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/genética , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Epilepsia Reflexa/tratamento farmacológico , Epilepsia Reflexa/etiologia , Potenciais Pós-Sinápticos Excitadores/efeitos dos fármacos , Comportamento Exploratório/efeitos dos fármacos , Proteína do X Frágil da Deficiência Intelectual/genética , Síndrome do Cromossomo X Frágil/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hipocampo/patologia , Hipocampo/ultraestrutura , Camundongos , Camundongos TransgênicosRESUMO
Excessive activity of striatal-enriched protein tyrosine phosphatase (STEP) in the brain has been detected in numerous neuropsychiatric disorders including Alzheimer's disease. Notably, knockdown of STEP in an Alzheimer mouse model effected an increase in the phosphorylation levels of downstream STEP substrates and a significant reversal in the observed cognitive and memory deficits. These data point to the promising potential of STEP as a target for drug discovery in Alzheimer's treatment. We previously reported a substrate-based approach to the development of low molecular weight STEP inhibitors with Ki values as low as 7.8 µM. Herein, we disclose the first X-ray crystal structures of inhibitors bound to STEP and the surprising finding that they occupy noncoincident binding sites. Moreover, we utilize this structural information to optimize the inhibitor structure to achieve a Ki of 110 nM, with 15-60-fold selectivity across a series of phosphatases.
Assuntos
Organofosfonatos/química , Proteínas Tirosina Fosfatases não Receptoras/antagonistas & inibidores , Sulfonamidas/química , Doença de Alzheimer/tratamento farmacológico , Animais , Domínio Catalítico , Cristalografia por Raios X , Descoberta de Drogas , Estabilidade de Medicamentos , Fosfatases de Especificidade Dupla/antagonistas & inibidores , Microssomos Hepáticos/metabolismo , Organofosfonatos/síntese química , Organofosfonatos/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/química , Ratos , Sulfonamidas/síntese química , Sulfonamidas/metabolismoRESUMO
Inbred mouse strains have been used preferentially for behavioral testing over outbred counterparts, even though outbred mice reflect the genetic diversity in the human population better. Here, we compare the sociability of widely available outbred CD1 mice with the commonly used inbred C57BL/6J (C57) mice in the one-chamber social interaction test and the three-chamber sociability test. In the one-chamber task, intra-strain pairs of juvenile, non-littermate, male CD1 or C57 mice display a series of social and aggressive behaviors. While CD1 and C57 pairs spend equal amount of time socializing, CD1 pairs spend significantly more time engaged in aggressive behaviors than C57 mice. In the three-chamber task, sociability of C57 mice was less dependent on acclimation paradigms than CD1 mice. Following acclimation to all three chambers, both groups of age-matched male mice spent more time in the chamber containing a stranger mouse than in the empty chamber, suggesting that CD1 mice are sociable like C57 mice. However, the observed power suggests that it is easier to achieve statistical significance with C57 than CD1 mice. Because the stranger mouse could be considered as a novel object, we assessed for a novelty effect by adding an object. CD1 mice spend more time in the chamber with a stranger mouse than that a novel object, suggesting that their preference is social in nature. Thus, outbred CD1 mice are as appropriate as inbred C57 mice for studying social behavior using either the single or the three-chamber test using a specific acclimation paradigm.
Assuntos
Transtorno Autístico/fisiopatologia , Comportamento Animal/fisiologia , Comportamento Exploratório/fisiologia , Comportamento Social , Animais , Modelos Animais de Doenças , Masculino , Camundongos Endogâmicos , Análise e Desempenho de TarefasRESUMO
Dysregulation of protein tyrosine phosphorylation has been implicated in a number of human diseases, including cancer, diabetes, and neurodegenerative diseases. As a result of their essential role in regulating protein tyrosine phosphorylation levels, protein tyrosine phosphatases (PTPs) have emerged as important yet challenging therapeutic targets. Here we report on the development and application of a glutathione-responsive motif to facilitate the efficient intracellular delivery of a novel class of selenosulfide phosphatase inhibitors for the selective active site directed inhibition of the targeted PTP by selenosulfide exchange with the active site cysteine. The strategy leverages the large difference in extracellular and intracellular glutathione levels to deliver selenosulfide phosphatase inhibitors to cells. As an initial exploration of the prodrug platform and the corresponding selenosulfide covalent inhibitor class, potent and selective inhibitors were developed for two therapeutically relevant PTP targets: the Mycobacterium tuberculosis virulence factor mPTPA and the CNS-specific tyrosine phosphatase, striatal-enriched protein tyrosine phosphatase (STEP). The lead selenosulfide inhibitors enable potent and selective inhibition of their respective targets over a panel of human PTPs and a representative cysteine protease. Kinetic parameters of the inhibitors were characterized, including reversibility of inhibition and rapid rate of GSH exchange at intracellular GSH concentrations. Additionally, active site covalent inhibitor-labeling with an mPTPA inhibitor was rigorously confirmed by mass spectrometry, and cellular activity was demonstrated with a STEP prodrug inhibitor in cortical neurons.
RESUMO
In chronic pain states, the neurotrophin brain-derived neurotrophic factor (BDNF) transforms the output of lamina I spinal neurons by decreasing synaptic inhibition. Pain hypersensitivity also depends on N-methyl-D-aspartate receptors (NMDARs) and Src-family kinases, but the locus of NMDAR dysregulation remains unknown. Here, we show that NMDAR-mediated currents at lamina I synapses are potentiated in a peripheral nerve injury model of neuropathic pain. We find that BDNF mediates NMDAR potentiation through activation of TrkB and phosphorylation of the GluN2B subunit by the Src-family kinase Fyn. Surprisingly, we find that Cl--dependent disinhibition is necessary and sufficient to prime potentiation of synaptic NMDARs by BDNF. Thus, we propose that spinal pain amplification is mediated by a feedforward mechanism whereby loss of inhibition gates the increase in synaptic excitation within individual lamina I neurons. Given that neither disinhibition alone nor BDNF-TrkB signaling is sufficient to potentiate NMDARs, we have discovered a form of molecular coincidence detection in lamina I neurons.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/genética , Neuralgia/genética , Traumatismos dos Nervos Periféricos/metabolismo , Proteínas Proto-Oncogênicas c-fyn/genética , Receptor trkB/genética , Receptores de N-Metil-D-Aspartato/genética , Animais , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Humanos , Neuralgia/metabolismo , Neuralgia/fisiopatologia , Neurônios/metabolismo , Neurônios/patologia , Traumatismos dos Nervos Periféricos/genética , Traumatismos dos Nervos Periféricos/fisiopatologia , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Ratos , Receptores de N-Metil-D-Aspartato/metabolismo , Nervos Espinhais/metabolismo , Nervos Espinhais/fisiopatologia , Sinapses/genética , Sinapses/patologia , Quinases da Família src/genéticaRESUMO
N-methyl-D-aspartate receptors (NMDARs) are necessary for the induction of synaptic plasticity and for the consolidation of learning and memory. NMDAR function is tightly regulated by functionally opposed families of kinases and phosphatases. Herein we show that the striatal-enriched protein tyrosine phosphatase (STEP) is recruited by Gαq-coupled receptors, including the M1 muscarinic acetylcholine receptor (M1R), and opposes the Src tyrosine kinase-mediated increase in the function of NMDARs composed of GluN2A. STEP activation by M1R stimulation requires IP3Rs and can depress NMDA-evoked currents with modest intracellular Ca2+ buffering. Src recruitment by M1R stimulation requires coincident NMDAR activation and can augment NMDA-evoked currents with high intracellular Ca2+ buffering. Our findings suggest that Src and STEP recruitment is contingent on differing intracellular Ca2+ dynamics that dictate whether NMDAR function is augmented or depressed following M1R stimulation.
Assuntos
Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Quinases da Família src/metabolismo , Animais , Animais Geneticamente Modificados , Região CA1 Hipocampal/citologia , Região CA1 Hipocampal/metabolismo , Plasticidade Neuronal , Proteínas Tirosina Fosfatases não Receptoras/genética , Ratos , Ratos Wistar , Receptor Muscarínico M1/metabolismoRESUMO
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.
Assuntos
Encefalopatias/metabolismo , Encéfalo/metabolismo , Neurônios/metabolismo , Proteínas Tirosina Fosfatases não Receptoras/metabolismo , Animais , Humanos , Camundongos , Fosforilação , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Regulação para Baixo/fisiologia , Neurônios/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Animais , Benzotiepinas/farmacologia , Encéfalo/citologia , Fator Neurotrófico Derivado do Encéfalo/genética , Células Cultivadas , Inibidores de Cisteína Proteinase/farmacologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Embrião de Mamíferos , Feminino , Flavonas/farmacologia , Leupeptinas/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Atividade Motora/efeitos dos fármacos , Atividade Motora/genética , Neurônios/efeitos dos fármacos , Proteínas Tirosina Fosfatases/genética , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Fatores de TempoRESUMO
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.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/metabolismo , Transtornos Cognitivos/tratamento farmacológico , Fenciclidina/uso terapêutico , Proteínas Tirosina Fosfatases/metabolismo , Animais , Benzotiepinas/farmacologia , Fator Neurotrófico Derivado do Encéfalo/análise , Proteína de Ligação a CREB/antagonistas & inibidores , Proteína de Ligação a CREB/genética , Proteína de Ligação a CREB/metabolismo , Células Cultivadas , Transtornos Cognitivos/metabolismo , Transtornos Cognitivos/patologia , Regulação para Baixo/efeitos dos fármacos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Atividade Motora/efeitos dos fármacos , Neurônios/citologia , Neurônios/metabolismo , Fenciclidina/farmacologia , Fosforilação/efeitos dos fármacos , Proteínas Tirosina Fosfatases/antagonistas & inibidores , Proteínas Tirosina Fosfatases/genética , Interferência de RNA , Receptores de N-Metil-D-Aspartato/antagonistas & inibidores , Receptores de N-Metil-D-Aspartato/metabolismo , UbiquitinaçãoRESUMO
The information from nociceptors is processed in the dorsal horn of the spinal cord by complex circuits involving excitatory and inhibitory interneurons. It is well documented that GluN2B and ERK1/2 phosphorylation contributes to central sensitization. Striatal-enriched protein tyrosine phosphatase (STEP) dephosphorylates GluN2B and ERK1/2, promoting internalization of GluN2B and inactivation of ERK1/2. The activity of STEP was modulated by genetic (STEP knockout mice) and pharmacological (recently synthesized STEP inhibitor, TC-2153) approaches. STEP(61) protein levels in the lumbar spinal cord were determined in male and female mice of different ages. Inflammatory pain was induced by complete Freund's adjuvant injection. Behavioral tests, immunoblotting, and electrophysiology were used to analyze the effect of STEP on nociception. Our results show that both genetic deletion and pharmacological inhibition of STEP induced thermal hyperalgesia and mechanical allodynia, which were accompanied by increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Striatal-enriched protein tyrosine phosphatase heterozygous and knockout mice presented a similar phenotype. Furthermore, electrophysiological experiments showed that TC-2153 increased C fiber-evoked spinal field potentials. Interestingly, we found that STEP(61) protein levels in the lumbar spinal cord inversely correlated with thermal hyperalgesia associated with age and female gender in mice. Consistently, STEP knockout mice failed to show age-related thermal hyperalgesia, although gender-related differences were preserved. Moreover, in a model of inflammatory pain, hyperalgesia was associated with increased phosphorylation-mediated STEP(61) inactivation and increased pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204)levels in the lumbar spinal cord. Collectively, the present results underscore an important role of spinal STEP activity in the modulation of nociception.
Assuntos
Nociceptividade/fisiologia , Limiar da Dor/efeitos dos fármacos , Dor/patologia , Dor/fisiopatologia , Proteínas Tirosina Fosfatases não Receptoras/deficiência , Animais , Benzotiepinas/farmacologia , Benzotiepinas/uso terapêutico , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/uso terapêutico , Potenciais Evocados/efeitos dos fármacos , Potenciais Evocados/genética , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Hiperalgesia/tratamento farmacológico , Hiperalgesia/genética , Hiperalgesia/metabolismo , Hiperalgesia/patologia , Inflamação/induzido quimicamente , Inflamação/complicações , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Fibras Nervosas Amielínicas/efeitos dos fármacos , Fibras Nervosas Amielínicas/fisiologia , Nociceptividade/efeitos dos fármacos , Dor/etiologia , Proteínas Tirosina Fosfatases não Receptoras/genética , Ratos , Ratos Sprague-Dawley , Transdução de SinaisRESUMO
Brain-derived neurotrophic factor (BDNF) promotes synaptic strengthening through the regulation of kinase and phosphatase activity. Conversely, striatal-enriched protein tyrosine phosphatase (STEP) opposes synaptic strengthening through inactivation or internalization of signaling molecules. Here, we investigated whether BDNF regulates STEP levels/activity. BDNF induced a reduction of STEP61 levels in primary cortical neurons, an effect that was prevented by inhibition of tyrosine kinases, phospholipase C gamma, or the ubiquitin-proteasome system (UPS). The levels of pGluN2B(Tyr1472) and pERK1/2(Thr202/Tyr204), two STEP substrates, increased in BDNF-treated cultures, and blockade of the UPS prevented STEP61 degradation and reduced BDNF-induced GluN2B and ERK1/2 phosphorylation. Moreover, brief or sustained cell depolarization reduced STEP61 levels in cortical neurons by different mechanisms. BDNF also promoted UPS-mediated STEP61 degradation in cultured striatal and hippocampal neurons. In contrast, nerve growth factor and neurotrophin-3 had no effect on STEP61 levels. Our results thus indicate that STEP61 degradation is an important event in BDNF-mediated effects.