ABSTRACT
Pharmacological approaches to induce N-methyl-d-aspartate receptor (NMDAR) hypofunction have been intensively used to understand the aetiology and pathophysiology of schizophrenia. Yet, the precise cellular and molecular mechanisms that relate to brain network dysfunction remain largely unknown. Here, we used a set of complementary approaches to assess the functional network abnormalities present in male mice that underwent a 7-day subchronic phencyclidine (PCP 10 mg/kg, subcutaneously, once daily) treatment. Our data revealed that pharmacological intervention with PCP affected cognitive performance and auditory evoked gamma oscillations in the prefrontal cortex (PFC) mimicking endophenotypes of some schizophrenia patients. We further assessed PFC cellular function and identified altered neuronal intrinsic membrane properties, reduced parvalbumin (PV) immunostaining and diminished inhibition onto L5 PFC pyramidal cells. A decrease in the strength of optogenetically-evoked glutamatergic current at the ventral hippocampus to PFC synapse was also demonstrated, along with a weaker shunt of excitatory transmission by local PFC interneurons. On a macrocircuit level, functional ultrasound measurements indicated compromised functional connectivity within several brain regions particularly involving PFC and frontostriatal circuits. Herein, we reproduced a panel of schizophrenia endophenotypes induced by subchronic PCP application in mice. We further recapitulated electrophysiological signatures associated with schizophrenia and provided an anatomical reference to critical elements in the brain circuitry. Together, our findings contribute to a better understanding of the physiological underpinnings of deficits induced by subchronic NMDAR antagonist regimes and provide a test system for characterization of pharmacological compounds.
Subject(s)
Disease Models, Animal , Phencyclidine , Prefrontal Cortex , Receptors, N-Methyl-D-Aspartate , Animals , Prefrontal Cortex/drug effects , Prefrontal Cortex/metabolism , Male , Phencyclidine/pharmacology , Receptors, N-Methyl-D-Aspartate/metabolism , Mice , Schizophrenia/chemically induced , Schizophrenia/physiopathology , Schizophrenia/metabolism , Mice, Inbred C57BL , Parvalbumins/metabolism , Adaptation, Physiological/drug effects , Adaptation, Physiological/physiology , Pyramidal Cells/drug effects , Pyramidal Cells/physiology , Gamma Rhythm/drug effects , Gamma Rhythm/physiology , Hippocampus/drug effects , Hippocampus/metabolism , Excitatory Amino Acid Antagonists/pharmacologyABSTRACT
Dopamine D2/3 receptor agonists are less likely to trigger dyskinesias than L-dopa while still offering relief from the motor symptoms of Parkinson's disease (PD). However, these drugs can cause serious impulse control problems and gambling disorders. Adjunctive therapies capable of blocking these side effects without impacting the antiparkinsonian effect would be clinically useful. G-protein-coupled receptor 52 (GPR52) is an orphan Gs-protein-coupled receptor that is coexpressed with striatal D2 receptors. Activating GPR52 attenuates behaviors associated with increased striatal dopamine release without altering basal function. Iatrogenic gambling disorder may be mediated, at least partly, by striatal dopamine signaling. We therefore investigated whether 2 potent small-molecule GPR52 agonists (BD442618, BD502657) could block the increase in preference for uncertain outcomes caused by acute d-amphetamine and chronic ropinirole, without altering baseline choice patterns. In the rat betting task (rBT), subjects choose between a guaranteed reward (the "wager") versus the 50:50 chance of double the wager or nothing. Although wager size varies across trial blocks, both options are constantly matched for expected value. The effects of BD442618 on the rBT were acutely assessed alone or in combination with d-amphetamine and subsequently in combination with chronic ropinirole. The latter experiment was then repeated with BD502657. BD442618 did not alter baseline decision making but attenuated the increase in preference for uncertainty caused by both acute amphetamine and chronic ropinirole administration. Similarly, BD502657 abrogated chronic ropinirole's effects. These data provide the first evidence that GPR52 agonists may be useful in treating iatrogenic gambling disorder or other conditions hallmarked by hyperdopaminergic states. (PsycInfo Database Record (c) 2021 APA, all rights reserved).
Subject(s)
Indoles/pharmacology , Receptors, Dopamine D2 , Receptors, G-Protein-Coupled/agonists , Uncertainty , Animals , Dextroamphetamine/administration & dosage , Dextroamphetamine/pharmacology , Dopamine/metabolism , Dopamine Agonists/administration & dosage , Dopamine Agonists/pharmacology , Indoles/administration & dosage , Male , Rats , Rats, Long-Evans , Receptors, Dopamine D2/metabolismABSTRACT
Protein tyrosine phosphatase non-receptor type 5 (PTPN5, STEP) is a brain specific phosphatase that regulates synaptic function and plasticity by modulation of N-methyl-d-aspartate receptor (NMDAR) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) trafficking. Dysregulation of STEP has been linked to neurodegenerative and neuropsychiatric diseases, highlighting this enzyme as an attractive therapeutic target for drug discovery. Selective targeting of STEP with small molecules has been hampered by high conservation of the active site among protein tyrosine phosphatases. We report the discovery of the first small molecule allosteric activator for STEP that binds to the phosphatase domain. Allosteric binding is confirmed by both X-ray and 15N NMR experiments, and specificity has been demonstrated by an enzymatic test cascade. Molecular dynamics simulations indicate stimulation of enzymatic activity by a long-range allosteric mechanism. To allow the scientific community to make use of this tool, we offer to provide the compound in the course of an open innovation initiative.
Subject(s)
Protein Tyrosine Phosphatases, Non-Receptor/chemistry , Small Molecule Libraries/chemistry , Allosteric Regulation , Allosteric Site , Animals , Catalytic Domain , Crystallography, X-Ray , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/metabolism , Humans , Mice , Molecular Dynamics Simulation , Nuclear Magnetic Resonance, Biomolecular , Protein Binding , Protein Isoforms/chemistry , Protein Isoforms/metabolism , Protein Tyrosine Phosphatases, Non-Receptor/metabolism , Small Molecule Libraries/metabolismABSTRACT
The identification and optimization of a novel series of centrally efficacious gamma secretase modulators (GSMs) offering an alternative to the privileged aryl imidazole motif is described. Chiral bicyclic tetrahydroindazolyl amine substituted triazolopyridines were identified as structurally distinct novel series of GSMs. Representative compound BI-1408 ((R)-42) was demonstrated to be centrally efficacious in rats at a 30â¯mg/kg oral dose.
Subject(s)
Amyloid Precursor Protein Secretases/metabolism , Imidazoles/chemistry , Administration, Oral , Amyloid Precursor Protein Secretases/chemistry , Amyloid beta-Peptides/antagonists & inhibitors , Amyloid beta-Peptides/metabolism , Animals , Bridged Bicyclo Compounds/chemistry , Cells, Cultured , Drug Design , Drug Evaluation, Preclinical , Female , Half-Life , Imidazoles/chemical synthesis , Imidazoles/pharmacokinetics , Inhibitory Concentration 50 , Neurons/cytology , Neurons/drug effects , Neurons/metabolism , Rats , Rats, Wistar , Structure-Activity RelationshipABSTRACT
Aversively established contextual fear memory manifests itself in robust freezing behavior, often lasting several weeks or months. Therefore, this approach is amenable to investigate the underlying neural circuitries by lesion or inactivation of specific brain regions or to test efficacy of substances that disrupt either the ability to acquire the association or to retrieve memories. In contrast, investigation of memory enhancement using this technique is time intensive since the non-treated control group naturally forgets the learned association only weeks after acquisition. Pharmacological interventions have been used to overcome this time span by disrupting memory at any time point, however, limiting it a mechanistic model of reversal of impairments instead of studying memory enhancement. Here, we investigated several parameters of the cued and contextual fear conditioning (CFC) protocol such that, while memory acquisition is established, loss of fear association occurs within a shorter time frame, allowing studies of memory enhancement in the context of natural forgetting. We found that three predictive tone-cues, each separated from a 0.3 mA foot shock by an interstimulus interval of 2 s and a pre-exposure to the context enables the investigation of enhanced contextual memory 7 d post training without the necessity of inducing pharmacological lesions.
ABSTRACT
The persistently active protein kinase Mζ (PKMζ) has been found to be involved in the formation and maintenance of long-term memory. Most of the studies investigating PKMζ, however, have used either putatively unselective inhibitors or conventional knock-out animal models in which compensatory mechanisms may occur. Here, we overexpressed an active form of PKMζ in rat hippocampus, a structure highly involved in memory formation, and embedded in several neural networks. We investigated PKMζ's influence on synaptic plasticity using electrophysiological recordings of basal transmission, paired pulse facilitation, and LTP and combined this with behavioral cognitive experiments addressing formation and retention of both contextual memory during aversive conditioning and spatial memory during spontaneous exploration. We demonstrate that hippocampal slices overexpressing PKMζ show enhanced basal transmission, suggesting a potential role of PKMζ in postsynaptic AMPAR trafficking. Moreover, the PKMζ-overexpressing slices augmented LTP and this effect was not abolished by protein-synthesis blockers, indicating that PKMζ induces enhanced LTP formation in a protein-synthesis-independent manner. In addition, we found selectively enhanced long-term memory for contextual but not cued fear memory, underlining the theory of the hippocampus' involvement in the contextual aspect of aversive reinforced tasks. Memory for spatial orientation during spontaneous exploration remained unaltered, suggesting that PKMζ may not affect the neural circuits underlying spontaneous tasks that are different from aversive tasks. In this study, using an overexpression strategy as opposed to an inhibitor-based approach, we demonstrate an important modulatory role of PKMζ in synaptic plasticity and selective memory processing. SIGNIFICANCE STATEMENT: Most of the literature investigating protein kinase Mζ (PKMζ) used inhibitors with selectivity that has been called into question or conventional knock-out animal models in which compensatory mechanisms may occur. To avoid these issues, some studies have been done using viral overexpression of PKMζ in different brain structures to show cognitive enhancement. However, electrophysiological experiments were exclusively done in knock-out models or inhibitory studies to show depletion of LTP. There was no study showing the effect of PKMζ overexpression in the hippocampus on behavior and LTP experiments. To our knowledge, this is the first study to combine these aspects with the result of enhanced memory for contextual fear memory and to show enhanced LTP in hippocampal slices overexpressing PKMζ.
Subject(s)
Fear/physiology , Hippocampus/enzymology , Hippocampus/physiology , Long-Term Potentiation/genetics , Memory/physiology , Protein Kinase C/genetics , Protein Kinase C/physiology , Animals , Conditioning, Operant , Cues , Gene Expression Regulation, Enzymologic/genetics , Gene Expression Regulation, Enzymologic/physiology , Genetic Vectors , Male , Neuronal Plasticity/physiology , Orientation/physiology , Protein Kinase C/biosynthesis , Protein Synthesis Inhibitors/pharmacology , Rats , Rats, Wistar , Receptors, AMPA/antagonists & inhibitors , Spatial Memory/physiology , Synaptic Transmission/genetics , Synaptic Transmission/physiologyABSTRACT
Kinase inhibitors represent a relatively new class of drugs that offer novel therapies targeting specific -malfunctioning kinase-mediated signaling pathways in oncology and potentially inflammation. As the ATP binding sites of the â¼500 human kinases are structurally conserved and because most current drugs target the ATP binding site, there is a need to profile all the kinases that a drug may bind and/or inhibit. We have developed a chemical proteomics method that affinity purifies kinases from cell or tissue lysates using kinase inhibitors immobilized on self-assembling monolayers. The method can be applied to assess the selectivity of a given kinase inhibitor and thus to guide its preclinical or clinical development.
Subject(s)
Chromatography, Affinity , Enzyme Inhibitors/chemistry , Phosphotransferases/isolation & purification , Proteomics/methods , Adenosine Triphosphate/metabolism , Binding Sites/drug effects , Enzyme Inhibitors/metabolism , Enzyme Inhibitors/pharmacology , Phosphotransferases/antagonists & inhibitors , Phosphotransferases/metabolism , Protein Binding/drug effectsABSTRACT
The NMDA (N-methyl-D-aspartate)-receptor is fundamentally involved in cognitive functions. Recent studies demonstrated a functional interaction between the metabotropic glutamate receptor 5 (mGlu(5) receptor) and the NMDA-receptor in neurons. In rat hippocampal slices, it was shown that activation of mGlu(5) receptor by a positive modulator in the presence of a subthreshold agonist concentration potentiated NMDA-receptor mediated currents and phosphorylation of intracellular signalling proteins. In the present study, we investigated the functional interaction of mGlu(5) receptor and NMDA-receptor by the selective mGlu(5) receptor positive modulator ADX-47273 in-vitro and in-vivo. In rat primary neurons, this compound potentiated Ca(2+) mobilization in the presence of a subthreshold concentration of the mGluR(1/5) agonist DHPG (0.3 microM) with an EC(50) of 0.28+/-0.05 microM. NMDA-induced Ca(2+)-mobilization in primary neurons could be potentiated when neurons were pre-stimulated with 1 microM ADX-47273 in the presence of 0.3 microM DHPG. The specific mGlu(5) receptor antagonist MPEP and the Src-family kinase inhibitor PP2 blocked this potentiation demonstrating the functional interaction of the NMDA-receptor and mGlu(5) receptor in neurons. Furthermore, ADX-47273 elicited an enhancement of NMDA-receptor dependent long-term potentiation in rat hippocampal slices that could be reversed by MPEP. After intraperitoneal administration to rats, ADX-47273 showed a dose-dependent reduction of NMDA-receptor antagonist (ketamine) induced hyperlocomotion, supporting the mechanistic interaction of the NMDA-receptor and mGlu(5) receptor in-vivo. In conclusion, these findings further support the idea of a functional interaction between the mGlu(5) receptor and NMDA-receptor, which may provide a pharmacological strategy for addressing CNS diseases with cognitive impairments linked to NMDA-receptor hypofunction.
Subject(s)
Excitatory Amino Acid Agonists/pharmacology , Receptors, Metabotropic Glutamate/metabolism , Receptors, N-Methyl-D-Aspartate/metabolism , Allosteric Regulation/drug effects , Allosteric Regulation/physiology , Animals , Calcium/metabolism , Cognition/drug effects , Cognition/physiology , Dose-Response Relationship, Drug , Excitatory Amino Acid Agonists/administration & dosage , Hippocampus/cytology , Hippocampus/drug effects , Injections, Intraperitoneal , Male , Methoxyhydroxyphenylglycol/administration & dosage , Methoxyhydroxyphenylglycol/analogs & derivatives , Methoxyhydroxyphenylglycol/pharmacology , Neurons/drug effects , Neurons/metabolism , Oxadiazoles/administration & dosage , Oxadiazoles/pharmacology , Phosphorylation , Piperidines/administration & dosage , Piperidines/pharmacology , Pyridines/administration & dosage , Pyridines/pharmacology , Rats , Rats, Wistar , Receptor, Metabotropic Glutamate 5 , Receptors, Metabotropic Glutamate/agonists , src-Family Kinases/metabolismABSTRACT
Gamma-secretase is a unique intramembrane-cleaving protease complex, which cleaves the Alzheimer's disease-associated beta-amyloid precursor protein (APP) and a number of other type I membrane proteins. Human gamma-secretase consists of the catalytic subunit presenilin (PS) (PS1 or PS2), the substrate receptor nicastrin, APH-1 (APH-1a or APH-1b), and PEN-2. To facilitate in-depth biochemical analysis of gamma-secretase, we developed a fast and convenient multistep purification procedure for the endogenous enzyme. The enzyme was purified from HEK293 cells in an active form and had a molecular mass of approximately 500 kDa. Purified gamma-secretase was capable of producing the major amyloid-beta peptide (Abeta) species, such as Abeta40 and Abeta42, from a recombinant APP substrate in physiological ratios. Abeta generation could be modulated by pharmacological gamma-secretase modulators. Moreover, the Abeta42/Abeta40 ratio was strongly increased by purified PS1 L166P, an aggressive familial Alzheimer's disease mutant. Tandem mass spectrometry analysis revealed the consistent coisolation of several proteins with the known gamma-secretase core subunits. Among these were the previously described gamma-secretase interactors CD147 and TMP21 as well as other known interactors of these. Interestingly, the Niemann-Pick type C1 protein, a cholesterol transporter previously implicated in gamma-secretase-mediated processing of APP, was identified as a major copurifying protein. Affinity capture experiments using a biotinylated transition-state analogue inhibitor of gamma-secretase showed that these proteins are absent from active gamma-secretase complexes. Taken together, we provide an effective procedure for isolating endogenous gamma-secretase in considerably high grade, thus aiding further characterization of this pivotal enzyme. In addition, we provide evidence that the copurifying proteins identified are unlikely to be part of the active gamma-secretase enzyme.
Subject(s)
Amyloid Precursor Protein Secretases/isolation & purification , Amyloid Precursor Protein Secretases/metabolism , Enzyme Inhibitors/pharmacology , Amyloid Precursor Protein Secretases/antagonists & inhibitors , Amyloid Precursor Protein Secretases/chemistry , Biotinylation/drug effects , Cell Line , Chromatography, Affinity , Chromatography, Liquid , Electrophoresis, Polyacrylamide Gel , Humans , Molecular Weight , Multiprotein Complexes/metabolism , Mutant Proteins/metabolism , Protein Binding/drug effects , Protein Subunits/metabolism , Substrate Specificity/drug effects , Tandem Mass SpectrometryABSTRACT
We describe a chemical proteomics approach to profile the interaction of small molecules with hundreds of endogenously expressed protein kinases and purine-binding proteins. This subproteome is captured by immobilized nonselective kinase inhibitors (kinobeads), and the bound proteins are quantified in parallel by mass spectrometry using isobaric tags for relative and absolute quantification (iTRAQ). By measuring the competition with the affinity matrix, we assess the binding of drugs to their targets in cell lysates and in cells. By mapping drug-induced changes in the phosphorylation state of the captured proteome, we also analyze signaling pathways downstream of target kinases. Quantitative profiling of the drugs imatinib (Gleevec), dasatinib (Sprycel) and bosutinib in K562 cells confirms known targets including ABL and SRC family kinases and identifies the receptor tyrosine kinase DDR1 and the oxidoreductase NQO2 as novel targets of imatinib. The data suggest that our approach is a valuable tool for drug discovery.
Subject(s)
Protein Kinase Inhibitors/pharmacology , Proteomics/methods , Proto-Oncogene Proteins c-abl/antagonists & inhibitors , Benzamides , Cell Extracts , Chromatography, Affinity , Discoidin Domain Receptor 1 , Enzymes, Immobilized/antagonists & inhibitors , HeLa Cells , Humans , Imatinib Mesylate , Inhibitory Concentration 50 , K562 Cells , Pharmaceutical Preparations , Phosphorylation/drug effects , Piperazines/pharmacology , Pyrimidines/pharmacology , Quinone Reductases/antagonists & inhibitors , Receptor Protein-Tyrosine Kinases/antagonists & inhibitors , Signal Transduction/drug effectsABSTRACT
PDZ domains are small globular domains that recognize the last 4-7 amino acids at the C-terminus of target proteins. The specificity of the PDZ-ligand recognition is due to side chain-side chain interactions, as well as the positioning of an alpha-helix involved in ligand binding. We have used computer-aided protein design to produce mutant versions of a Class I PDZ domain that bind to novel Class I and Class II target sequences both in vitro and in vivo, thus providing an alternative to primary antibodies in western blotting, affinity chromatography and pull-down experiments. Our results suggest that by combining different backbone templates with computer-aided protein design, PDZ domains could be engineered to specifically recognize a large number of proteins.