Biomedical subjects

John A Kemp

Publications and source records attributed to John A Kemp.

15 recordsLinked to original sources

PubMed2005

Glutamate- and GABA-based CNS therapeutics.

Glutamate- and GABA-releasing neurons form the basis for neurotransmission in the mammalian central nervous system (CNS). The co-ordination of these excitatory and inhibitory systems, together with intrinsic voltage-gated ion channels and G-protein-coupled receptor modulation, provides the diverse neuronal firing patterns, network activity and synaptic plasticity that are required for the complexity of CNS function. Virtually all of the known molecular components of the gamma-aminobutyric acid (GABA) and glutamate neurotransmitter systems have been considered as potential therapeutic targets. Positive allosteric modulators of GABAA receptors, such as the benzodiazepines, have found wide clinical use, and the N-methyl-D-aspartate receptor antagonists ketamine and memantine have therapeutic utility. In these fundamental neurotransmitter systems, drugs that provide allosteric modulation of ligand-gated ion channels or G-protein-coupled receptors, or seek to selectively target receptor subtypes, appear to hold the greatest promise for the desired balance of efficacy and tolerability. This might also be achieved through targeting transporter subtypes. A large number of compounds based on these strategies are currently in clinical trials for diseases that span a wide range of CNS disorders.

Animals↗

PubMed2005

Evidence for improved performance in cognitive tasks following selective NR2B NMDA receptor antagonist pre-treatment in the rat.

RATIONALE: We previously reported that the NR2B subunit-selective N-methyl-D-aspartate (NMDA) antagonist Ro 63-1908 produced a marked deficit in response control in the five-choice serial reaction time task (5-CSRTT). OBJECTIVES: The present studies were designed to investigate this further by studying the NR2B NMDA antagonists, ifenprodil, traxoprodil (CP101,606), Ro 25-6981 as well as Ro 63-1908 in this test. METHODS: Following training in the 5-CSRTT, separate groups of rats were either tested under (1) standard test conditions [5 s inter-trial interval (ITI), 0.5 s stimulus duration, 100 trials], (2) high (3 s ITI) and low (10 s ITI) event rate of stimulus presentation and (3) a 250-trial protocol in aged 2-year-old rats. In a final study, the effects of traxoprodil were investigated in an operant delayed match to position (DMTP) task, a test of working memory, and compared to dizocilpine and Ro 63-1908. RESULTS: Similar to Ro 63-1908, both traxoprodil (1-10 mg/kg) and Ro 25-6981 (3--30 mg/kg) increased premature responding but also increased response speed with no error trade-off. Conversely, ifenprodil (1--10 mg/kg) slowed response speed and increased omissions with no effect on premature responding. Tested under a variable ITI, Ro 63--1908 (1 mg/kg) increased premature responding at all ITIs, but this change was proportional to controls. At short ITI (3 s), Ro 63-1908 reliably improved performance both in terms of response speed and accuracy (percent correct). In a 250-trial protocol in aged rats, both Ro 63-1908 (0.1-0.3 mg/kg) and, particularly, traxoprodil (1--3 mg/kg) improved performance-increasing response speed and increasing the number of rewards earned during test. Finally, traxoprodil (1--10 mg/kg) improved accuracy and increased response speed in the DMTP task. CONCLUSIONS: The present studies support the view that selective NR2B NMDA antagonists promote impulsive-type responding in the 5-CSRTT; however, under certain test conditions, drugs of this class-notably traxoprodil-may also improve task performance.

Animals↗

PubMed2005

Ionotropic and metabotropic glutamate receptor structure and pharmacology.

RATIONALE: L: -Glutamate is the major excitatory neurotransmitter in the central nervous system (CNS) and mediates its actions via activation of both ionotropic and metabotropic receptor families. The development of selective ligands, including competitive agonists and antagonists and positive and negative allosteric modulators, has enabled investigation of the functional roles of glutamate receptor family members. OBJECTIVE: In this review we describe the subunit structure and composition of the ionotropic and metabotropic glutamate receptors and discuss their pharmacology, particularly with respect to selective tools useful for investigation of their function in the CNS. RESULTS: A large number of ligands are now available that are selective either for glutamate receptor subfamilies or for particular receptor subtypes. Such ligands have enabled considerable advances in the elucidation of the physiological and pathophysiological roles of receptor family members. Furthermore, efficacy in animal models of neurological and psychiatric disorders has supported the progression of several glutamatergic ligands into clinical studies. These include ionotropic glutamate receptor antagonists, which have entered clinical trials for disorders including epilepsy and ischaemic stroke, alpha-amino-3-hydroxy-5-methyl-4-isoazolepropionic acid (AMPA) receptor positive allosteric modulators which are under evaluation as cognitive enhancers, and metabotropic glutamate receptor 2 (mGluR2) agonists which are undergoing clinical evaluation as anxiolytics. Furthermore, preclinical studies have illustrated therapeutic potential for ligands selective for other receptor subtypes in various disorders. These include mGluR1 antagonists in pain, mGluR5 antagonists in anxiety, pain and drug abuse and mGluR5 positive allosteric modulators in schizophrenia. CONCLUSIONS: Selective pharmacological tools have enabled the study of glutamate receptors. However, pharmacological coverage of the family is incomplete and considerable scope remains for the development of novel ligands, particularly those with in vivo utility, and for the their use together with existing tools for the further investigation of the roles of receptor family members in CNS function and as potentially novel therapeutics.

Allosteric Regulation↗

PubMed2004

Pharmacological and genetic evidence indicates that combined inhibition of NR2A and NR2B subunit containing NMDA receptors is required to disrupt prepulse inhibition.

RATIONALE: Glutamate signalling through the N-methyl-D-aspartate (NMDA) receptor is of critical importance for normal central nervous system (CNS) function, as indicated by the marked behavioural disturbances produced by non-subtype selective NMDA antagonists such as dizocilpine (MK-801). OBJECTIVE: The present studies were designed to investigate the involvement of the two major NMDA receptor subunits in the central nervous system, i.e. NR2A and NR2B, on sensorimotor gating in mice. METHODS: These experiments utilised the non-subtype-selective NMDA antagonist dizocilpine, a line of NR2A-KO mice and the selective NR2B antagonist Ro 63-1908, in the study of pre-pulse inhibition of the startle response (PPI). RESULTS: The non-selective NMDA receptor antagonist dizocilpine (0.1-1 mg/kg, IP) robustly disrupted PPI in wild-type mice. Conversely, selective genetic or pharmacological inhibition of either the NMDA NR2A or NR2B receptor subunit containing receptors, respectively, had no effect on PPI. Thus, NR2A KO mice showed normal PPI compared with wild-type littermate controls, and administration of Ro 63-1908 (1-10 mg/kg IP) to wild-type mice did not affect PPI. However, selective inhibition of NR2A and NR2B by administration of Ro 63-1908 to NR2A KO mice significantly disrupted PPI. CONCLUSIONS: These data imply that concomitant inhibition of both NR2A and NR2B subunit-containing NMDA receptors is necessary to disrupt PPI, suggesting that inhibition of NR2A and NR2B-containing NMDA receptors is required to elicit behaviours suggestive of psychomimetic effects in man.

Animals↗

PubMed2004

Antiparkinsonian activity of Ro 25-6981, a NR2B subunit specific NMDA receptor antagonist, in animal models of Parkinson's disease.

N-methyl-D-aspartate (NMDA) receptor antagonists have antiakinetic and antidyskinetic effects in animals models of Parkinson's disease (PD). However, non-selective inhibition of NMDA receptors throughout the central nervous system may result in undesired effects such as ataxia and psychosis. We therefore studied Ro 25-6981, an activity-dependent antagonist of NMDA receptors containing the NR2B subunit which are predominantly expressed in the striatum. Ro 25-6981 induced contraversive rotations in 6-hydroxydopamine (6-OHDA)-lesioned rats without stimulating locomotion in normal rats and reversed parkinsonian symptoms in 1-methyl-4-phenyl-1,2,3,6,-tetrahydropyridine (MPTP)-treated common marmosets. Due to the small number of marmosets, there were no significant differences between Ro 25-6981 and vehicle though there was a significant trend toward differences, as shown by the Page test. Furthermore, Ro 25-6981 potentiated the action of levodopa in both species and attenuated the maximal levodopa response in 6-OHDA-lesioned rats chronically treated with levodopa without reducing the overall response. Ro 25-6981 also potentiated the action of the dopamine receptor agonists apomorphine, A68930 and quinpirole in 6-OHDA-lesioned rats. The present observations suggest a therapeutic potential of NR2B-selective NMDA receptor antagonists in the management of PD.

Animals↗

PubMed2004

Pharmacological manipulation of mGlu2 receptors influences cognitive performance in the rodent.

Atrophy of the medial temporal lobes, including the glutamatergic cortical-hippocampal circuitry, is an early event in Alzheimer's disease (AD) and probably contributes to the characteristic short-term mnemonic decline. Pharmacological strategies directly targeted to ameliorating this functional decline may represent a novel approach for the symptomatic treatment of AD. Presynaptic group II metabotropic glutamate receptors (i.e. mGlu2 and mGlu3) exert a powerful modulatory influence on the function of these pathways, in particular the perforant pathway. Using a combination of mGlu2 receptor knockout mice and the group II agonist LY354740, we show that activation of mGlu2 receptors produces a cognitive impairment, i.e. a delay-dependent deficit in delayed matching and non-matching to position, and impaired spatial learning in a Morris water maze. Conversely, a group II antagonist, LY341495, improved acquisition of spatial learning. LY354740 potently reduced field excitatory postsynaptic potentials in hippocampal slices from wild type but not mGlu2 receptor knockout mice. Taken together, these results suggest that activation of mGlu2 receptors evokes a powerful inhibitory effect on hippocampal synaptic transmission and mGlu2 agonists produce a cognitive deficit consistent with this change. Conversely, mGlu2 receptor antagonists may improve certain aspects of cognition and thus represent a novel approach for the symptomatic treatment of AD.

Amino Acids↗

PubMed2004

Human C-reactive protein increases cerebral infarct size after middle cerebral artery occlusion in adult rats.

Human C-reactive protein (CRP), the classic acute phase plasma protein, increases in concentration after myocardial infarction and stroke. Human CRP binds to ligands exposed in damaged tissue and can then activate complement and its proinflammatory functions. In contrast, rat CRP, which binds to similar ligands, does not activate complement. In the present study, systemic complement depletion with cobra venom factor in adult rats subjected to middle cerebral artery occlusion did not affect cerebral infarct size, indicating that circulating complement does not contribute to injury in this model. However, we have previously reported that administration of human CRP to rats undergoing coronary artery ligation caused a marked increase in size of the resulting myocardial infarction, associated with codeposition of human CRP and rat complement in the infarcts. In the present study, we show that adult rats subjected to middle cerebral artery occlusion and then treated with human CRP similarly developed significantly larger cerebral infarcts compared with control subjects receiving human serum albumin. Human CRP can thus contribute to ischemic tissue damage in the brain as well as in the heart, and inhibition of CRP binding may therefore be a promising target for tissue protective acute therapeutic intervention in stroke as well as in myocardial infarction.

Aging↗

PubMed2003

Identification of critical residues in the amino terminal domain of the human NR2B subunit involved in the RO 25-6981 binding pocket.

N-Methyl-d-aspartate (NMDA) receptors play key roles in both physiological processes, particularly synaptic plasticity, and in neuropathological states such as epilepsy and acute neurodegeneration. R-(R*,S*)-alpha-(4-Hydroxyphenyl)-beta-methyl-4-(phenyl-methyl)-1-piperidine propanol (RO 25-6981), is a high-affinity and selective blocker of NMDA receptors containing the NR2B subunit. Using site-directed mutagenesis, [3H]RO 25-6981 binding, Xenopus oocyte voltage-clamp recordings, and molecular modeling, we have identified several critical residues involved in the RO 25-6981 binding site within the N-terminal LIVBP-like domain of the human NR2B subunit. Two mutations, NR2B(D101A) and NR2B(F176A), resulted in a complete loss of [3H]RO 25-6981 binding and also abolished the high-affinity RO 25-6981-mediated inhibition of NMDA-induced currents. The mutation NR2B(T233A) led to a marked reduction in binding affinity by 13-fold. Mutations F182A, D104A, or K234A had a more moderate influence on the binding affinity (KD values increased by 8-, 7-, and 6-fold, respectively). In a three-dimensional model of the NR2B LIVBP-like domain based on the X-ray crystal structure of the amino-terminal domain of the mGlu1 receptor, the critical residues are located in the central cleft where interaction with RO 25-6981 may stabilize the closed structure of the domain. Our results suggest that the three amino acids Asp-101, Phe-176, and Thr-233 are important molecular determinants for the high-affinity binding of RO 25-6981 to the LIVBP-like domain of human NR2B. A possible binding mode for RO 25-6981 is proposed.

Animals↗

PubMed2003

PS2APP transgenic mice, coexpressing hPS2mut and hAPPswe, show age-related cognitive deficits associated with discrete brain amyloid deposition and inflammation.

Transgenic mice, expressing mutant beta-amyloid precursor proteins (betaAPPs), have lead to a better understanding of the pathophysiological processes in Alzheimer's disease (AD). In many of these models, however, the temporal development of cognitive decline and the relationship to Abeta deposition and inflammation are unclear. We now report a novel transgenic mouse line, PS2APP (PS2N141I x APPswe), which develops a severe cerebral amyloidosis in discrete brain regions, and present a cross-sectional analysis of these mice at 4, 8, 12, and 16 months of age. Each age cohort was investigated for changes in behavior, electrophysiology of synapse efficacy, ELISA-determined Abeta load, histopathology, and in immunoelectron microscopy. Cognitive deficits were first observed at 8 months when Abeta deposits and inflammation were restricted to discrete brain regions, namely the subiculum and frontolateral (motor and orbital) cortex. As early as 5 months, electron microscopy revealed the presence, in these regions, of pre-plaque, immunogold-labeled extracellular fibrillar Abeta. At the same age, increased levels of insoluble Abeta were detected by ELISA, with Abeta1-40 levels exceeding those of Abeta1-42. Further cognitive decline occurred in an age-related manner, and this was accompanied by the spread of amyloidosis to ultimately affect not only neo- and limbic cortices, but also thalamic and pontine nuclei. Dentate gyrus post-tetanic potentiation was significantly attenuated at 17 months, and there were also significant differences in paired-pulse parameters. This systematic cross-sectional study of the behavioral and pathological changes in the PS2APP mouse indicates that it develops age-related cognitive decline associated with severe amyloidosis and inflammation in discrete brain regions and therefore is suitable for testing a range of potential symptomatic and disease-modifying therapies for AD.

Age Factors↗

PubMed2003

The voltage-dependent anion channel is the target for a new class of inhibitors of the mitochondrial permeability transition pore.

The relevance of the mitochondrial permeability transition pore (PTP) in Ca2+ homeostasis and cell death has gained wide attention. Yet, despite detailed functional characterization, the structure of this channel remains elusive. Here we report on a new class of inhibitors of the PTP and on the identification of their molecular target. The most potent among the compounds prepared, Ro 68-3400, inhibited PTP with a potency comparable to that of cyclosporin A. Since Ro 68-3400 has a reactive moiety capable of covalent modification of proteins, [3H]Ro 68-3400 was used as an affinity label for the identification of its protein target. In intact mitochondria isolated from rodent brain and liver and in SH-SY5Y human neuroblastoma cells, [3H]Ro 68-3400 predominantly labeled a protein of approximately 32 kDa. This protein was identified as the isoform 1 of the voltage-dependent anion channel (VDAC). Both functional and affinity labeling experiments indicated that VDAC might correspond to the site for the PTP inhibitor ubiquinone0, whereas other known PTP modulators acted at distinct sites. While Ro 68-3400 represents a new useful tool for the study of the structure and function of VDAC and the PTP, the results obtained provide direct evidence that VDAC1 is a component of this mitochondrial pore.

Animals↗

PubMed2002

Identification and characterization of a novel splice variant of the metabotropic glutamate receptor 5 gene in human hippocampus and cerebellum.

The G-protein coupled metabotropic glutamate receptor mGlu5 plays a pivotal role as a modulator of synaptic plasticity, ion channel activity and excitotoxicity. Two splice variants, hmGlu5a and -5b have been reported previously. During screening of a human brain cDNA library for hmGlu5a, we identified a novel variant (hmGlu5d) generated by alternative splicing at the C-terminal domain. The predicted hmGlu5d protein has a C-terminal 267 amino acid shorter than that of hmGlu5a. The pattern of mRNA expression of mGluR5 variants in human brain were analyzed by RT-PCR and in situ hybridization histochemistry. RT-PCR analysis demonstrated the presence of the hmGlu5d transcript, although at low level, in human whole brain, cerebellum, cerebral cortex and hippocampus. [3H]Quisqualate displayed similar affinity at the hmGlu5 splice variants (K(D) values of 80+/-8 and 54+/-17 nM for hmGlu5a and -5d receptors, respectively). For the five mGlu agonists studied, a similar rank order of potency was observed on both hmGlu5a and -5d receptors: quisqualate>glutamate>DHPG>L-CCGI approximately ACPD. MPEP inhibited the glutamate (2 microM)-induced [Ca(2+)](i) response in hmGlu5a and -5d-HEK293 cells also with similar potency (IC(50) values 25+/-1.5 and 20+/-1.4 nM, respectively). Therefore, the large truncation of the C-terminal tail of mGlu5 does not have any apparent major effect on the potency and efficacy of agonists as measured by the [Ca(2+)](i) responses or by activation of recombinant G-protein coupled inwardly rectifying K(+) (GIRK) channel currents. The only major functional difference is the increased sensitivity of hmGlu5d to protein kinase C (PKC)-mediated desensitization, relative to hmGlu5a.

Aged↗

PubMed2002

Severe impairment of NMDA receptor function in mice carrying targeted point mutations in the glycine binding site results in drug-resistant nonhabituating hyperactivity.

NMDA receptor hypofunction has been implicated in the pathophysiology of schizophrenia, and pharmacological and genetic approaches have been used to model such dysfunction. We previously have described two mouse lines carrying point mutations in the NMDA receptor glycine binding site, Grin1(D481N) and Grin1(K483Q), which exhibit 5- and 86-fold reductions in receptor glycine affinity, respectively. Grin1(D481N) animals exhibit a relatively mild phenotype compatible with a moderate reduction in NMDA receptor function, whereas Grin1(K483Q) animals die shortly after birth. In this study we have characterized compound heterozygote Grin1(D481N/K483Q) mice, which are viable and exhibited biphasic NMDA receptor glycine affinities compatible with the presence of each of the two mutated alleles. Grin1(D481N/K483Q) mice exhibited a marked NMDA receptor hypofunction revealed by deficits in hippocampal long-term potentiation, which were rescued by the glycine site agonist d-serine, which also facilitated NMDA synaptic currents in mutant, but not in wild-type, mice. Analysis of striatal monoamine levels revealed an apparent dopaminergic and serotonergic hyperfunction. Behaviorally, Grin1(D481N/K483Q) mice were insensitive to acute dizocilpine pretreatment and exhibited increased startle response but normal prepulse inhibition. Most strikingly, mutant mice exhibited a sustained, nonhabituating hyperactivity and increased stereotyped behavior that were resistant to suppression by antipsychotics and the benzodiazepine site agonist Zolpidem. They also displayed a disruption of nest building behavior and were unable to perform a cued learning paradigm in the Morris water maze. We speculate that the severity of NMDA receptor hypofunction in these mice may account for their profound behavioral phenotype and insensitivity to antipsychotics.

Animals↗

PubMed2002

Differential regulation of synaptic transmission by mGlu2 and mGlu3 at the perforant path inputs to the dentate gyrus and CA1 revealed in mGlu2 -/- mice.

Group II metabotropic glutamate (mGlu) receptors can act as presynaptic autoinhibitory receptors at perforant path inputs to the hippocampus under conditions of high frequency synaptic activation. We have used mGlu2 -/- mice to examine the relative roles of mGlu2 and mGlu3 in the regulation of perforant path synaptic transmission mediated by both the selective group II receptor agonist, DCG-IV, and by synaptically released glutamate. Field excitatory postsynaptic potentials evoked by stimulation of either the perforant path inputs to the dentate gyrus mid-moleculare or the CA1 stratum lacunosum moleculare were inhibited by DCG-IV with IC(50) values and maximum percentage inhibition of: 169 nM (60%) and 41 nM (72%) in wild-type mice and 273 nM (19%) and 116 nM (49%) in mGlu2 -/- mice, respectively. Activation of presynaptic group II mGlu autoreceptors by synaptically released glutamate, as revealed by a LY341495-mediated increase in the relative amplitude of a test fEPSP evoked after a conditioning burst, was observed in both the dentate gyrus and the stratum lacunosum of wild-type, but not mGlu2 -/- mice. These observations demonstrate that activation of mGlu3 receptors can regulate synaptic transmission at perforant path synapses but suggest that mGlu2 is the major presynaptic group II autoreceptor activated by synaptically released glutamate.

Animals↗

PubMed2002

NMDA receptor pathways as drug targets.

Since the mid 1980s, there has been a great deal of enthusiasm within both academia and industry about the therapeutic potential of drugs targeting the NMDA subtype of glutamate receptors. That early promise is just beginning to translate into approvable drugs. Here we review the reasons for this slow progress and critically assess the future prospects for drugs that act on NMDA receptor pathways, including potential treatments for some major disorders such as stroke and Alzheimer's disease, for which effective therapies are still lacking.

Animals↗

PubMed2002

Role of caspase-3 activation in cerebral ischemia-induced neurodegeneration in adult and neonatal brain.

These studies have addressed the role of caspase-3 activation in neuronal death after cerebral ischemia in different animal models. The authors were unable to show activation of procaspase-3 measured as an induction of DEVDase (Asp-Glu-Val-Asp) activity after focal or transient forebrain ischemia in rats. DEVDase activity could not be induced in the cytosolic fraction of the brain tissue obtained from these animals by exogenous cytochrome c/dATP and Ca2+. However, the addition of granzyme B to these cytosolic fractions resulted in a significant activation of DEVDase, confirming that the conditions were permissive to analyze proteolytic cleavage of the DEVD-AMC (7-amino-4-methyl-coumarin) substrate. Consistent with these findings, zVal-Ala-Asp-fluoromethylketone administered after focal ischemia did not have a neuroprotective effect. In contrast to these findings, a large increase in DEVDase activity was detected in a model of hypoxic-ischemia in postnatal-day-7 rats. Furthermore, in postnatal-day-7 animals treated with MK-801, in which it has been suggested that excessive apoptosis is induced, the authors were unable to detect activation of DEVDase activity but were able to induce it in vitro by the addition of cytochrome c/dATP and Ca2+ to the cytosolic fraction. Analysis of cytochrome c distribution did not provide definitive evidence for selective cytochrome c release in the permanent focal ischemia model, whereas in the transient model a small but consistent amount of cytochrome c was found in the cytosolic fraction. However, in both models the majority of cytochrome c remained associated with the mitochondrial fraction. In conclusion, the authors were unable to substantiate a role of mitochondrially derived cytochrome c and procaspase-3 activation in ischemia-induced cell death in adult brain, but did see a clear induction of caspase-3 in neonatal hypoxia.

Amino Acid Chloromethyl Ketones↗