Ann E. Kingston

DHPG-induced LTD in area CA1 of juvenile rat hippocampus; characterisation and sensitivity to novel mGlu receptor antagonists.

We have used extracellular microelectrode recording to characterise a form of long-term depression (LTD) of synaptic transmission that can be induced by metabotropic glutamate (mGlu) receptor activation in the CA1 region of the young (12-18 day old) rat hippocampus. Activation of group I mGlu receptors by the specific agonist 3,5-dihydroxyphenylglyine (DHPG) induced LTD of field excitatory postsynaptic potentials (fEPSPs). The mGlu5 selective agonist 2-chloro-5-hydroxyphenylglycine was also capable of inducing LTD. In contrast, the group II specific agonist DCG-IV had no effect on synaptic transmission, whilst the group III receptor agonist (S)-2-amino-4-phosphonobutyrate elicited a depression that reversed fully upon agonist washout. DHPG-induced LTD could still be generated after prior saturation of electrically-induced NMDA receptor-dependent LTD. DHPG-induced LTD was reversed by tetanic stimulation comprising 100 shocks delivered at 100 Hz. A novel mGlu receptor antagonist, (RS)-2-amino-2-(3-cis and trans-carboxycyclobutyl-3-(9-thioxanthyl)propionic acid) (LY393053) that potently inhibits mGlu1 and mGlu5 receptors, prevented the induction of DHPG-induced LTD. Like other mGlu receptor antagonists, LY393053 also reversed pre-established DHPG-induced LTD. In contrast, a potent mGlu1 selective antagonist (S)-2-methyl-4-carboxyphenylglycine (LY367385) did not prevent the induction of DHPG-induced LTD. In conclusion, DHPG, probably via activation of mGlu5 receptors, is able to induce a robust form of LTD in the CA1 region of the young rat hippocampus that is mechanistically distinct from NMDA receptor-dependent homosynaptic LTD.

(S)-3,4-DCPG, a potent and selective mGlu8a receptor agonist, activates metabotropic glutamate receptors on primary afferent terminals in the neonatal rat spinal cord

(S)-3,4-Dicarboxyphenylglycine (DCPG) has been tested on cloned human mGlu1-8 receptors individually expressed in AV12-664 cells co-expressing a rat glutamate/aspartate transporter and shown to be a potent and selective mGlu8a receptor agonist (EC(50) value 31+/-2 nM, n=3) with weaker effects on the other cloned mGlu receptors (EC(50) or IC(50) values >3.5 microM on mGlu1-7). Electrophysiological characterisation on the neonatal rat spinal cord preparation revealed that (S)-3,4-DCPG depressed the fast component of the dorsal root-evoked ventral root potential (fDR-VRP) giving a biphasic concentration-response curve showing EC(50) values of 1.3+/-0.2 microM (n=17) and 391+/-81 microM (n=17) for the higher and lower affinity components, respectively. The receptor mediating the high-affinity component was antagonised by 200 microM (S)-alpha-methyl-2-amino-4-phosphonobutyrate (MAP4, K(D) value 5.4+/-1.5 microM (n=3)), a group III metabotropic glutamate (mGlu) receptor antagonist. The alpha-methyl substituted analogue of (S)-3,4-DCPG, (RS)-3,4-MDCPG (100 microM), antagonised the effects of (S)-3,4-DCPG (K(D) value 5.0+/-0.4 microM, n=3) in a similar manner to MAP4. (S)-3,4-DCPG-induced depressions of the fDR-VRP in the low-affinity range of the concentration-response curve were potentiated by 200 microM (S)-alpha-ethylglutamate (EGLU), a group II mGlu receptor antagonist, and were relatively unaffected by MAP4 (200 microM). However, depressions of the fDR-VRP mediated by the AMPA selective antagonist (R)-3,4-DCPG were not potentiated by EGLU, suggesting that the low-affinity component of the concentration-response curve for (S)-3,4-DCPG is not due to antagonism of postsynaptic AMPA receptors. It is suggested that the receptor responsible for mediating the high-affinity component is mGlu8. The receptor responsible for mediating the low-affinity effect of (S)-3,4-DCPG has yet to be identified but it is unlikely to be one of the known mGlu receptors present on primary afferent terminals or an ionotropic glutamate receptor of the AMPA or NMDA subtype.

(+)-2-Methyl-4-carboxyphenylglycine (LY367385) selectively antagonises metabotropic glutamate mGluR1 receptors

Abstract The synthesis of three novel 4-carboxyphenylglycine derivatives is described. 2-Methyl substituents increase the antagonist potency compared to (S)-4CPG at mGluR1 receptors. Resolution of compound 1 showed that the activity resided in the (+)-isomer LY367385.

A 71-kD heat shock protein (hsp) from Mycobacterium tuberculosis has modulatory effects on experimental rat arthritis

The effects of a mycobacterial 71‐kD hsp antigen have been investigated for its ability to modulate arthritis in rats. Subcutaneous injection (base of tail) of increasing amounts of hsp71 from Mycobacterium tuberculosis (MTB) produced dose‐dependent differential inhibitory effects on induction of arthritis by MTB and CP20961 in rats. As little as 1 μg of the hsp71 produced a reduction in MTB arthritis, whereas complete protection was observed when 50 μg were administered. When 71‐kD‐treated rats were challenged with CP20961, all developed reduced symptoms of arthritis compared with control rats, but in this model no complete protection was observed over the dose range studied. The effects of 71‐kD pretreatment on collagen II arthritis were not significant, but in general symptoms of arthritis were milder than in the control group. The same pattern of results was observed previously when hsp65 was used in the different models. These results show that the modulatory effects of hsp on adjuvant arthritis are not restricted to the hsp65 series, but are also mediated by a member of the hsp70 family.

Neuroprotection by metabotropic glutamate receptor agonists: LY354740, LY379268 and LY389795

In rat cortical neuronal cultures, metabotropic glutamate (mGlu) receptor agonists: LY354740 (+)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate); LY379268 (−)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate, and LY389795 (−)-2-thia-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate, were neuroprotective against toxicity induced by N-methyl-d-aspartic acid (NMDA), kainic acid and staurosporine as measured by release of lactate dehydrogenase (LDH) activity into culture supernatants and DNA fragmentation by oligonucleosome formation. The potencies of the agonists were at least 100 times greater in reducing nucleosome formation than LDH release indicating a differential effect on neurons dying by apoptosis than by necrosis. In vivo studies showed that LY354740 was able to mediate a partial protection against apoptosis in CA1 hippocampal cells under ischaemic conditions where substantial CA1 cell loss occurred. The effects of the agonists in vitro were: (a) reversed by mGlu receptor antagonist LY341495, (b) enhanced by the presence of glial cells, (c) abrogated by RNA and protein synthesis inhibitors, and (d) unaltered by inhibition of endogenous adenosine activity. These results suggest that group II mGlu receptor agonists may represent a novel therapeutic strategy for the treatment of neurodegenerative diseases.

Pharmacological and Pharmacokinetic Properties of a Structurally Novel, Potent, and Selective Metabotropic Glutamate 2/3 Receptor Agonist: In Vitro Characterization of Agonist (–)-(1R,4S,5S,6S)-4-Amino-2-sulfonylbicyclo[3.1.0]-hexane-4,6-dicarboxylic Acid (LY404039)

Group II metabotropic glutamate (mGlu) receptor agonists, including (1S,2S,5R,6S)-2-aminobicyclo[3.1.0]hexane-2,6-dicarboxylate monohydrate (LY354740) and (–)-2-oxa-4-aminobicyclo[3.1.0]hexane-4,6-dicarboxylate (LY379268), have demonstrated efficacy in animal models of anxiety and schizophrenia, and LY354740 decreased anxiety in human subjects. Herein, we report the in vitro pharmacological profile and pharmacokinetic properties of another potent, selective, and structurally novel mGlu2/3 receptor agonist, (–)-(1R,4S,5S,6S)-4-amino-2-sulfonylbicyclo[3.1.0]hexane-4,6-dicarboxylic acid (LY404039) and provide comparisons with LY354740. Similar to LY354740, LY404039 is a nanomolar potent agonist at recombinant human mGlu2 and mGlu3 receptors (Ki = 149 and 92, respectively) and in rat neurons expressing native mGlu2/3 receptors (Ki = 88). LY404039 is highly selective for mGlu2/3 receptors, showing more than 100-fold selectivity for these receptors, versus ionotropic glutamate receptors, glutamate transporters, and other receptors targeted by known anxiolytic and antipsychotic medications. Functionally, LY404039 potently inhibited forskolin-stimulated cAMP formation in cells expressing human mGlu2 and mGlu3 receptors. Electrophysiological studies indicated that LY404039 suppressed electrically evoked excitatory activity in the striatum, and serotonin-induced l-glutamate release in the prefrontal cortex; effects reversed by LY341495. These characteristics suggest LY404039 modulates glutamatergic activity in limbic and forebrain areas relevant to psychiatric disorders; and that, similar to LY354740, it works through a mechanism that may be devoid of negative side effects associated with current antipsychotics and anxiolytics. Interestingly, despite the slightly lower potency (∼2–5-fold) of LY404039 versus LY354740 in binding, functional, and electrophysiological assays, LY404039 demonstrated higher plasma exposure and better oral bioavailability in pharmacokinetic experiments. Collectively, the current data indicate that LY404039 may be valuable in the treatment of neuropsychiatric disorders, including anxiety and psychosis.

Functional Metabotropic Glutamate Receptors on Nuclei from Brain and Primary Cultured Striatal Neurons ROLE OF TRANSPORTERS IN DELIVERING LIGAND

G-protein-coupled receptors are well known for converting an extracellular signal into an intracellular response. Here we showed that the metabotropic glutamate receptor 5 (mGlu5) plays a dynamic intracellular role in signal transduction. Activation of endogenously expressed mGlu5 on striatal nuclear membranes leads to rapid, sustained calcium (Ca2+) responses within the nucleoplasm that can be blocked by receptor-specific antagonists. Extracellular ligands such as glutamate and quisqualate reach nuclear receptors via both sodium-dependent transporters and cystine glutamate exchangers. Inhibition of either transport system blocks radiolabeled agonist uptake as well as agonist-induced nuclear Ca2+ changes. Impermeable antagonists like LY393053 and LY367366 not only blocked [3H]quisqualate binding but also prevented nontransported agonists such as (RS)-3,5-dihydroxyphenylglycine from inducing intracellular Ca2+ changes in heterologous cells. In contrast, neither LY compound prevented quisqualate or glutamate from activating intracellular receptors leading to Ca2+ responses. Inasmuch as Ca2+ can enter the nucleoplasm via the nuclear pore complex or from the nuclear lumen, the presence of nuclear mGlu5 receptors appeared to amplify the latter process generating a faster nuclear response in heterologous cells. In isolated striatal nuclei, nuclear receptor activation results in the de novo appearance of phosphorylated CREB protein. Thus, activation of nuclear mGlu5 receptors initiates a signaling cascade that is known to alter gene transcription and regulate many paradigms of synaptic plasticity. These studies demonstrated that mGlu5 receptors play a dynamic role in signaling both on and off the plasma membrane.

Neuroprotective Actions of Novel and Potent Ligands of Group I and Group II Metabotropic Glutamate Receptors

ABSTRACT: The role of group I metabotropic glutamate (mGlu) receptors in neurodegeneration is controversial because of the contradictory effects of mGlu1/5 agonists in in vitro models of neuronal cell death. In this study, novel and selective antagonists of mGlu1 and mGlu5: LY367385 and LY367366 were found to show consistent neuroprotective effects against N‐methyl‐d‐aspartate (NMDA)‐induced excitotoxicity in vitro and in vivo. Furthermore, intraventricular administration of LY367385 reduced hippocampal cell death in gerbils subjected to transient global ischemia.

Defective Tumor Necrosis Factor-α-dependent Control of Astrocyte Glutamate Release in a Transgenic Mouse Model of Alzheimer Disease

The cytokine tumor necrosis factor-α (TNFα) induces Ca2+-dependent glutamate release from astrocytes via the downstream action of prostaglandin (PG) E2. By this process, astrocytes may participate in intercellular communication and neuromodulation. Acute inflammation in vitro, induced by adding reactive microglia to astrocyte cultures, enhances TNFα production and amplifies glutamate release, switching the pathway into a neurodamaging cascade (Bezzi, P., Domercq, M., Brambilla, L., Galli, R., Schols, D., De Clercq, E., Vescovi, A., Bagetta, G., Kollias, G., Meldolesi, J., and Volterra, A. (2001) Nat. Neurosci. 4, 702–710). Because glial inflammation is a component of Alzheimer disease (AD) and TNFα is overexpressed in AD brains, we investigated possible alterations of the cytokine-dependent pathway in PDAPP mice, a transgenic model of AD. Glutamate release was measured in acute hippocampal and cerebellar slices from mice at early (4-month-old) and late (12-month-old) disease stages in comparison with age-matched controls. Surprisingly, TNFα-evoked glutamate release, normal in 4-month-old PDAPP mice, was dramatically reduced in the hippocampus of 12-month-old animals. This defect correlated with the presence of numerous β-amyloid deposits and hypertrophic astrocytes. In contrast, release was normal in cerebellum, a region devoid of β-amyloid deposition and astrocytosis. The Ca2+-dependent process by which TNFα evokes glutamate release in acute slices is distinct from synaptic release and displays properties identical to those observed in cultured astrocytes, notably PG dependence. However, prostaglandin E2 induced normal glutamate release responses in 12-month-old PDAPP mice, suggesting that the pathology-associated defect involves the TNFα-dependent control of secretion rather than the secretory process itself. Reduced expression of DENN/MADD, a mediator of TNFα-PG coupling, might account for the defect. Alteration of this neuromodulatory astrocytic pathway is described here for the first time in relation to Alzheimer disease.

Up-regulation of astrocyte metabotropic glutamate receptor 5 by amyloid-β peptide

The effects of amyloid-beta peptide (Aβ) on astrocyte responses to activation of mGlu5 receptors have been investigated using calcium imaging. Pre-incubation with Aβ1-40 peptide for up to 72 h produced a time- and concentration-dependent 2-4 fold enhancement in the magnitude of the intracellular calcium mobilization response to the group I metabotropic glutamate receptor agonist (S)-3,5-dihydroxyphenylglycine (DHPG). In contrast, pre-treatment with Aβ1-40 did not alter the calcium responses induced by other G protein coupled- or ion channel-receptors. Aβ 1-40-enhanced DHPG responses were blocked by the mGlu5 antagonist MPEP but not by inhibitors of voltage dependent calcium channels or by the AMPA/KA receptor antagonist CNQX. Up-regulation of mGlu5 coupled responses was associated with significant increases in astrocyte mGlu5 receptor-mRNA and-protein expression after preincubation with Aβ . The changes observed in vitro were consistent with results obtained from human Alzheimers disease (AD) patients.Immunostaining for mGlu5 receptors was increased on astrocytes which were colocalized with Aβ plaques in hippocampal tissue from AD patients compared to age-matched controls. These results suggest that modulation of mGlu5 receptors in astrocytes could be an important mechanism in determining the progression of pathology in AD.