Lentivirus Tau (P301S) expression in adult amyloid precursor protein (APP)-transgenic mice leads to tangle formation.

AIMS: In this study, we aimed to investigate the interaction between amyloid- and Tau-associated pathologies to gain further insights into the development of Alzheimer's disease. We examined the formation of neurofibrillary tangles (NFT) in adult mouse brain without the prior overexpression of Tau at embryonic or early post-natal stages to dissociate any developmental mechanisms. METHODS: Lentivirus technology was used to examine the effects of overexpressing mutant Tau-P301S in the adult mouse brains of both wild-type and amyloid precursor protein (APP)-transgenic mice. RESULTS: We find that injection of lentivirus Tau-P301S into the hippocampus of adult wild-type mice increases levels of hyperphosphorylated Tau, as early as 3 months post injection. However, no NFT are found even after 13 months of Tau expression. In contrast, the overexpression of Tau-P301S in adult APP-transgenic animals results in the formation of Gallyas-stained NFT. CONCLUSIONS: Our current findings are the first to show that overexpression of Tau-P301S in adult mice overexpressing APP, but not wild-type mice, leads to enhanced Tau-related pathology.

Regulation of mglu(7) receptors by proteins that interact with the intracellular C-terminus.

The metabotropic glutamate type 7 (mglu(7)) receptor is a widely distributed, mainly presynaptic Group III mglu receptor that can regulate glutamate release. Recently, largely as a result of the identification of specific proteins that interact with the C-terminal domain of this receptor, considerable progress has been made towards understanding some of the mechanisms that underlie the regulation, signal transduction pathways and targeting of mglu(7) receptors. This has led to the proposal that there are three distinct functionally relevant domains present in the intracellular C-terminus of this receptor: (1) a proximal intracellular signalling domain that interacts with G-protein betagamma-subunits and the Ca(2+) sensor Ca(2+)-calmodulin, and is phosphorylated by protein kinase; (2) a central domain thought to provide a signal for axonal targeting; and (3) an extreme PDZ-binding motif that interacts with the protein kinase C interacting protein, PICK1.

PICK1 interacts with and regulates PKC phosphorylation of mGLUR7.

The G-protein-coupled metabotropic glutamate receptor subtype 7a (mGluR7a) is a member of group III metabotropic glutamate receptors that plays an important role as a presynaptic receptor in regulating transmitter release at glutamatergic synapses. Here we report that the protein interacting with C-kinase (PICK1) binds to the C terminus (ct) of mGluR7a. In the yeast two-hybrid system, the extreme ct of mGluR7a was shown to interact with the PSD-95/Discs large/ZO-1 (PDZ) domain of PICK1. Pull-down assays indicated that PICK1 was retained by a glutathione S-transferase fusion of ct-mGluR7a. Furthermore, recombinant and native PICK1/mGluR7a complexes were coimmunoprecipitated from COS-7 cells and rat brain tissue, respectively. Confocal microscopy showed that both PICK1 and mGluR7a displayed synaptic colocalization in cultured hippocampal neurons. PICK1 has previously been shown to bind protein kinase C alpha-subunit (PKCalpha), and mGluR7a is known to be phosphorylated by PKC. We show a relationship between these three proteins using recombinant PICK1, mGluR7, and PKCalpha, where they were co-immunoprecipitated as a complex from COS-7 cells. In addition, PICK1 caused a reduction in PKCalpha-evoked phosphorylation of mGluR7a in in vitro phosphorylation assays. These results suggest a role for PICK1 in modulating PKCalpha-evoked phosphorylation of mGluR7a.

PDZ proteins interacting with C-terminal GluR2/3 are involved in a PKC-dependent regulation of AMPA receptors at hippocampal synapses.

We investigated the role of PDZ proteins (GRIP, ABP, and PICK1) interacting with the C-terminal GluR2 by infusing a ct-GluR2 peptide ("pep2-SVKI") into CA1 pyramidal neurons in hippocampal slices using whole-cell recordings. Pep2-SVKI, but not a control or PICK1 selective peptide, caused AMPAR-mediated EPSC amplitude to increase in approximately one-third of control neurons and in most neurons following the prior induction of LTD. Pep2-SVKI also blocked LTD; however, this occurred in all neurons. A PKC inhibitor prevented these effects of pep2-SVKI on synaptic transmission and LTD. We propose a model in which the maintenance of LTD involves the binding of AMPARs to PDZ proteins to prevent their reinsertion. We also present evidence that PKC regulates AMPAR reinsertion during dedepression.

Characterisation and partial purification of the GABA(B) receptor from the rat cerebellum using the novel antagonist [3H]CGP 62349.

The novel GABA(B) receptor antagonist [3H]CGP 62349 binds rat cerebellar synaptosomal membranes with high affinity at a single population of sites (K(d) = 0.9 nM, B(max) = 760 fmol/mg protein). Solubilisation with 1% Triton X-100/0.5 M NaCl/10% glycerol resulted in a marked increase in [3H]CGP 62349 binding (K(d) = 0.5 nM, B(max) = 1285 fmol/mg protein). Competition of [3HCGP 35348 = CGP 36742. The GABA(A) ligand isoguvacine did not displace [3H]CGP 62349 binding. Partial purification of [3H]CGP 62349 binding sites was obtained by sucrose density centrifugation and a predominant protein in the peak binding fraction was recognised by an anti-GABA(B) receptor antibody and had a molecular weight similar to the recombinant expressed GABA(B)R1a. These results demonstrate that [3H]CGP 62349 provides a useful additional tool for further characterisation of the pharmacology and biochemistry of the native GABA(B) receptor.

The protein kinase C alpha binding protein PICK1 interacts with short but not long form alternative splice variants of AMPA receptor subunits.

Here we report an interaction between AMPA receptor subunits and a single PDZ domain-containing protein called PICK1 which is known to bind protein kinase C alpha (PKC alpha). The interaction occurs within the last ten amino acid residues containing a novel PDZ binding motif (E S V/I K I) of the short C-terminal alternative splice variants of AMPA receptor subunits. No interaction occurs with the corresponding long splice variants which do not contain the E S V/I K I motif. The PDZ domain of PICK1 is required for the interaction and the mutation of a single amino acid in this region (Lys-27 to Glu) prevents interaction between PICK1 and GluR2 in the yeast two-hybrid assay. A similar mutation has been reported to prevent the binding of PICK1 to PKC alpha indicating that the same domain of PICK1 binds both PKC alpha and GluRs. Flag-tagged PICK1 is retained by a glutathione S-transferase (GST) fusion of the C-terminal of GluR2 (GST-ct-GluR2; short splice variant) but not by GST-ct-GluR1 (long splice variant). Recombinant full length GluR2 is coimmunoprecipitated with flag-PICK1 using an anti-flag antibody and flag-PICK1 is coimmunoprecipitated with an N-terminal directed anti-GluR2 antibody. Transient expression of both proteins in COS cells reveals colocalization and an altered pattern of distribution for each protein from when they are expressed individually. This novel interaction provides a possible regulatory mechanism to specifically modulate distinct splice variants and may be involved in targeting the phosphorylation of short form GluRs by PKC alpha.

Different effects of phospholipase A2 on agonist binding to hippocampal, cortical and recombinant homomeric alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptors.

Secretory phospholipase A2 (sPLA2) has been reported to modulate agonist and antagonist binding to alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Here we report that in hippocampal membranes sPLA2 increases the affinity of [3H]AMPA binding whereas in cortical membranes the affinity of binding is decreased. To test whether these results are due to the interaction of sPLA2 with different AMPA receptor subunits we used membranes from BHK cells expressing homomeric GluR1, GluR2 or GluR4 receptors. Pretreatment with the enzyme had no effect on [3H]AMPA binding to any of the homomeric receptors. We interpret these data to suggest that sPLA2 does not act directly at the AMPA receptor. Instead, sPLA2 may interact with a receptor-associated protein present in brain but not in BHK cells and this interaction may exert different effects in cortex and hippocampus.

The regulation of AMPA receptor-binding sites.

A wide variety of mechanisms have been identified that can regulate the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA)-receptor complex. Modulation has been shown to occur at the nucleic acid level via RNA editing and alternative splicing. At the posttranslational level, processes such as phosphorylation, glycosylation, chemical modification of reactive groups on the receptor proteins, interaction with a putative receptor-associated modulatory protein, and changes in the lipid environment have been reported to regulate receptor binding and function. In this review, we discuss general aspects of the cell biology, pharmacology, and function of AMPA receptors. In particular, we focus on some factors shown to modulate agonist binding and discuss possible molecular mechanisms underlying the regulation observed.

High-affinity binding sites for 125I-labelled pancreatic secretory phospholipase A2 in rat brain.

Porcine pancreatic secretory phospholipase A2 (ppsPLA2) has been shown to modulate agonist and antagonist binding to alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptors and to effect neurotransmission in the central nervous system (CNS). To further elucidate the mechanism of action of ppsPLA2 in the CNS, the binding profile of 125I-labelled ppsPLA2 to rat whole-brain membranes was assessed. Two classes of calcium-dependent binding sites were detected using unlabelled ppsPLA2 as a displacer with IC50 values of 3 and 217 nM. Similar values were obtained for [125I]ppsPLA2 binding to membranes prepared from isolated cortical and hippocampal rat brain regions. [125I]ppsPLA2 binding displayed bell-shaped concentration-dependence curves to Ca2+, Zn2 + and pH. Binding was not inhibited by AMPA, the false substrate, oleoyloxyethyl phosphocholine (OOPC), or by BSA-galactose or wheat germ agglutinin. [125I]ppsPLA2 binding was reduced by treatment of the rat brain membranes with mercaptoethanol and proteinase K treatment or by their pre-incubation at 95 degrees C. These results show a different binding profile to the previously characterised snake venom sPLA2 N-type receptors and suggest the existence of novel class of sPLA2 N-type binding sites.

Prevention of internal thoracic artery spasm.

Perioperative spasm of the internal thoracic artery is a common experience in coronary artery bypass grafting. We describe a simple method of harvesting the internal thoracic artery with improved ease of dissection that helps to prevent the arterial spasm.

Pharmacology and regional distribution of the binding of 6-[3H]nitro-7-sulphamoylbenzo[f]-quinoxaline-2,3-dione to rat brain.

6-Nitro-7-sulphamoylbenzo[f]quinoxaline-2,3-dione (NBQX) is a competitive antagonist selective for alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors. Here we report the pharmacological characteristics and anatomical distribution of [3H]NBQX binding to rat brain. The association rate of [3H]NBQX to rat cerebrocortical membranes was rapid, with peak binding occurring within 10 min at 0 degree C. The off-rate was also rapid, with near-complete dissociation of the radioligand within 5 min of addition of 1 mM unlabelled L-glutamate. [3H]NBQX bound to a single class of sites with KD and Bmax values of 47 nM and 2.6 pmol mg-1 of protein, respectively. The rank order of inhibition of [3H]NBQX binding by AMPA receptor ligands was NBQX > > 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) > or = (S)-5-fluorowillardiine > or = AMPA > > L-glutamate. The chaotrope KSCN had no effect on the IC50 value of unlabelled NBQX displacement of [3H]NBQX binding. The kainate receptor-selective ligands NS102 and kainate were only very weak displacers. It is interesting that NBQX and CNQX displaced significantly more [3H]NBQX than any of the agonists tested. Autoradiographic analysis of the binding of [3H]NBQX to coronal sections showed a distribution compatible with that of [3H]AMPA binding. These data indicate that [3H]NBQX provides a useful novel tool to characterise the antagonist binding properties of AMPA receptors.

Regulation of glutamate release by presynaptic kainate receptors in the hippocampus.

Most reported actions of kainate are mediated by AMPA (alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate) receptors. Here we report that, unlike AMPA which stimulates, kainate elicits a dose-dependent decrease in L-glutamate release from rat hippocampal synaptosomes and also depresses glutamatergic synaptic transmission. Brief exposure to kainate inhibited Ca(2+)-dependent [3H]L-glutamate release by up to 80%. Inhibition was reversed by kainate antagonists but not by the AMPA-selective non-competitive antagonist 1-(4-aminophenyl)-4-methyl-7,8-methylenedioxy-5H-2,3-benzodiazepine (GYKI 52466). A corresponding reversible kainate-evoked depression of NMDA (N-methyl-D-aspartate) receptor-mediated excitatory postsynaptic currents (e.p.s.cs) was observed when AMPA receptors were blocked by GYKI 52466. The synaptic depression was preceded by a brief period of enhanced release and a small inward current was also observed. The effects of kainate were unaffected by metabotropic glutamate (mGlu), GABAA, GABAB, glycine and adenosine receptor antagonists. These results indicate that glutamate release can be modulated directly by kainate autoreceptors.

Characterisation of the interaction between guanyl nucleotides and AMPA receptors in rat brain.

Guanyl nucleotides inhibit the binding of the AMPA receptor agonists [3H]fluorowillardiine and [3H]AMPA and the competitive antagonist [3H]CNQX to rat brain cerebrocortical membranes. The rank order of inhibition for each of the radioligands tested was GTP > GDP > GMP. The nucleotides CTP and ATP showed no effect. GTP inhibition was unaffected by the presence or absence of NaCl and MgCl2. Pre-treatment of the membranes with GTP, and its removal before addition of radioligand, did not inhibit binding. Quantitative autoradiography demonstrated that GTP inhibition occurred throughout the brain. These results are consistent with guanyl nucleotides acting at an extracellular site present on all AMPA receptor subunits, occupation of which inhibits agonist and antagonist binding.

Phospholipase A2 down-regulates the affinity of [3H]AMPA binding to rat cortical membranes.

The effects of exogenous phospholipase A2 on the binding of alpha-[3H]amino-3-hydroxy-5-methylisoxazole-4-propionate ([3H]AMPA) to rat cortical membranes in the presence of the chaotrope potassium thiocyanate were assessed. Pretreatment of membranes with secretory phospholipase A2 (sPLA2) elicited a concentration-dependent decrease in specific [3H]AMPA binding due mainly to a decrease in affinity (KD). This observation, together with protease inhibitor and western blot evidence, suggest that the sPLA2 effect is not due to proteolysis. The sPLA2-evoked decrease was temperature and calcium dependent. Inclusion of the specific inhibitor oleoyloxyethyl phosphocholine or preincubation of the enzyme with reducing agents to degrade its secondary structure significantly reduced the sPLA2 inhibition. These results suggest that the effects of sPLA2 arise from an enzymatic action rather than a competitive interaction at the AMPA binding site. However, arachidonic acid, a major metabolite of sPLA2 action, did not cause a similar decrease in the affinity of [3H]AMPA binding. In contrast to the effects on [3H]AMPA binding, sPLA2 caused an increase in [3H]CNQX binding, which is in accordance with the functionality of the AMPA receptor complex. These results suggest that sPLA2 may play a role in the physiological and pathophysiological regulation of AMPA receptors.

Cyclothiazide unmasks AMPA-evoked stimulation of [3H]-L-glutamate release from rat hippocampal synaptosomes.

The effect of alpha-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) on Ca(2+)-sensitive, tetrodotoxin (TTX)-insensitive K(+)-stimulated [3H]-L-glutamate release from rat hippocampal synaptosomes was determined. AMPA in the presence, but not in the absence of cyclothiazide, a drug which blocks AMPA receptor desensitization, elicited a dose-dependent increase in K(+)-stimulated [3H]-L-glutamate release but had no effect on basal release. The AMPA/cyclothiazide stimulation was blocked by CNQX and by GYKI 52466, an antagonist at the cyclothiazide site. These results indicate that AMPA receptors are present on presynaptic terminals and suggest that they may play a role in the regulation of neurotransmitter release.

Modulation of alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) binding sites by nitric oxide.

Nitric oxide release is reported to be involved in physiological processes associated with altered sensitivity of the alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) class of glutamate receptor. A series of compounds liberating nitric oxide were therefore tested for their ability to modulate in vitro the characteristics of [3H]AMPA binding to sections of rat brain. Pretreatment of forebrain or cerebellar sections with sodium nitroprusside (1 mM), S-nitroso-N-acetylpenicillamine (SNAP, 200 microM), glyceryl trinitrate (1 microM), or isosorbide dinitrate (0.5 mM) all increased the binding of 3 nM [3H]AMPA by 15-30%. These actions were reproduced by 8-bromo-cyclic GMP (200 microM) in the cerebellum but not in the forebrain. In a similar manner, the effect of SNAP was attenuated by an inhibitor of cyclic GMP-dependent protein kinase in the cerebellum but not in the forebrain. The elevated [3H]AMPA binding observed after pretreatment with SNAP was caused by an increase in binding affinity, but the capacity of the sites was unchanged. Autoradiographic analysis showed that forebrain binding was enhanced in the cerebral cortex and hippocampus but not in the striatum. Nitric oxide therefore appears to be able to increase the affinity of AMPA binding sites via two distinct mechanisms in different brain areas. This action may contribute to synaptic plasticity associated with nitric oxide release.