Ruud Zwart

Sazetidine-A Is a Potent and Selective Agonist at Native and Recombinant α4β2 Nicotinic Acetylcholine Receptors

Sazetidine-A has been recently proposed to be a “silent desensitizer” of α4β2 nicotinic acetylcholine receptors (nAChRs), implying that it desensitizes α4β2 nAChRs without first activating them. This unusual pharmacological property of sazetidine-A makes it, potentially, an excellent research tool to distinguish between the role of activation and desensitization of α4β2 nAChRs in mediating the central nervous system effects of nicotine itself, as well as those of new nicotinic drugs. We were surprised to find that sazetidine-A potently and efficaciously stimulated nAChR-mediated dopamine release from rat striatal slices, which is mediated by α4β2* and α6β2* subtypes of nAChR. The agonist effects on native striatal nAChRs prompted us to re-examine the effects of sazetidine-A on recombinant α4β2 nAChRs in more detail. We expressed the two alternative stoichiometries of α4β2 nAChR in Xenopus laevis oocytes and investigated the agonist properties of sazetidine-A on both α4(2)β2(3) and α4(3)β2(2) nAChRs. We found that sazetidine-A potently activated both stoichiometries of α4β2 nAChR: it was a full agonist on α4(2)β2(3) nAChRs, whereas it had an efficacy of only 6% on α4(3)β2(2) nAChRs. In contrast to what has been published before, we therefore conclude that sazetidine-A is an agonist of native and recombinant α4β2 nAChRs but shows differential efficacy on α4β2 nAChRs subtypes.

The nicotinic α4β2 receptor selective agonist, TC-2559, increases dopamine neuronal activity in the ventral tegmental area of rat midbrain slices

Abstract The ability of α4β2 nicotinic acetylcholine receptors to modulate dopaminergic (DA) cell activity in the ventral tegmental area (VTA) in rat midbrain slices was assessed using a selective α4β2 receptor agonist, TC-2559 ((E)-N-methyl-4-[3-(5-ethoxypyridin)y1]-3-buten-1-amine). The selectivity of TC-2559 was characterized across 6 recombinant human nicotinic receptors (α4β2, α2β4, α4β4, α3β4, α3β2 and α7) stably expressed in mammalian cell lines. Using a fluorescent imaging plate reader and fluo-3 to monitor changes in intracellular calcium, TC-2559 was found to be at least 69 fold more potent on α4β2 than on other heteromeric subtypes, with an efficacy of 33%. No activity on the homomeric α7 subtype was detected. TC-2559 also showed selectivity for α4β2 over the α4β4 and α7 subtypes expressed in Xenopus oocytes. When bath applied to VTA slices, TC-2559 increased the firing of DA cells in a dose-dependent manner, in the same concentration range that activates α4β2 receptors in recombinant cell lines or oocytes. The effect of TC-2559 was blocked by 2 μM dihydro-β-erythroidine (an α4β2-preferring antagonist), but not by 10 nM methyllycaconitine (an α7 antagonist). Glutamate receptor antagonists (6-cyano-7-nitroquinoxaline-2,3-dione and D(−)-2-amino-5-phosphonopentanoic acid) did not reduce TC-2559-induced responses, suggesting that the increase in DA cell firing induced by TC-2559 is caused by direct postsynaptic depolarisation via the activation of α4β2 receptors and not by enhancement of glutamate release.

Expression and functional characterisation of a human chimeric nicotinic receptor with α6β4 properties

Abstract Despite being cloned several years ago, the expression of functional nicotinic acetylcholine receptors containing the human α6 subunit in recombinant mammalian cell lines has yet to be demonstrated. The resulting lack of selective ligands has hindered the evaluation of the role of α6-containing nicotinic receptors. We report that functional channels were recorded following co-transfection of human embryonic kidney (HEK-293) cells with a chimeric α6/α4 subunit and the β4 nicotinic receptor subunit. They displayed an agonist rank order potency of epibatidine≫1,1-dimethyl-4-phenylpiperazinium (DMPP)≥cytisine>acetylcholine>nicotine measured in a fluorescent imaging plate reader assay. Nicotine, cytisine, DMPP and epibatidine displayed partial agonist properties whilst α-conotoxin MII and methyllycaconitine blocked the functional responses elicited by acetylcholine stimulation. Co-transfection of the α6/α4 chimera with the β2 nicotinic receptor subunit did not result in functional receptors. The human α6/α4β4 chimeric nicotinic receptor expressed in HEK-293 cells may provide a valuable tool for the generation of subtype specific ligands.

Chaperone protein 14-3-3 and protein kinase A increase the relative abundance of low agonist sensitivity human α4β2 nicotinic acetylcholine receptors in Xenopus oocytes

α4 and β2 nicotinic acetylcholine (nACh) receptor subunits expressed heterologously in Xenopus oocytes assemble into a mixture of receptors with high and low agonist sensitivity whose relative abundance is influenced by the heteropentamer subunit ratio. We have found that inhibition of protein kinase A by KT5720 decreased maximal [3H]cytisine binding and acetylcholine (ACh)‐induced current responses, and increased the relative proportion of α4β2 receptors with high agonist sensitivity. Mutation of serine 467, a putative protein kinase A substrate in a chaperone protein binding motif within the large cytoplasmic domain of the α4 subunit, to alanine or asparate decreased or increased, respectively, maximal [3H]cytisine binding and ACh response amplitude. Expression of α4S467A mutant subunits decreased steady levels of α4 and the relative proportion of α4β2 receptors with low agonist sensitivity, whilst expression of α4S467D increased steady levels of α4 and α4β2 receptors with low agonist sensitivity. Difopein, an inhibitor of chaperone 14‐3‐3 proteins, decreased [3H]cytisine binding and ACh responses and increased the proportion of α4β2 with high sensitivity to activation by ACh. Thus, post‐translational modification affecting steady‐state levels of α4 subunits provides a possible means for physiologically relevant, chaperone‐mediated variation in the relative proportion of high and low agonist sensitivity α4β2 nACh receptors.

PSAB-OFP, a selective α7 nicotinic receptor agonist, is also a potent agonist of the 5-HT3 receptor

5-Hydroxytryptamine 3 (5-HT(3)) and alpha 7 nicotinic receptors share high sequence homology and pharmacological cross-reactivity. An assessment of the potential role of alpha 7 receptors in many neurophysiological processes, and hence their therapeutic value, requires the development of selective alpha 7 receptor agonists. We used a recently reported selective alpha 7 receptor agonist, (R)-(-)-5Phenylspiro[1-azabicyclo[2.2.2] octane-3,2-(3H)furo[2,3-b]pyridine (PSAB-OFP) and confirmed its activity on human recombinant alpha 7 receptors. However, PSAB-OFP also displayed high affinity binding to 5-HT(3) receptors. To assess the functional activity of PSAB-OFP on 5-HT(3) receptors we studied recombinant human 5-HT(3) receptors expressed in Xenopus oocytes, as well as native mouse 5-HT(3) receptors expressed in N1E-115 neuroblastoma cells, using whole-cell patch clamp and Ca(2+) imaging. Our results show that PSAB-OFP is an equipotent, partial agonist of both alpha 7 and 5-HT(3) receptors. We conclude that it will be necessary to identify the determinant of this overlapping pharmacology in order to develop more selective alpha 7 receptor ligands.

α7 and β2 Nicotinic Acetylcholine Receptor Subunits Form Heteromeric Receptor Complexes that Are Expressed in the Human Cortex and Display Distinct Pharmacological Properties.

The existence of α7β2 nicotinic acetylcholine receptors (nAChRs) has recently been demonstrated in both the rodent and human brain. Since α7-containing nAChRs are promising drug targets for schizophrenia and Alzheimer’s disease, it is critical to determine whether α7β2 nAChRs are present in the human brain, in which brain areas, and whether they differ functionally from α7 nAChR homomers. We used α-bungarotoxin to affinity purify α7-containing nAChRs from surgically excised human temporal cortex, and found that α7 subunits co-purify with β2 subunits, indicating the presence of α7β2 nAChRs in the human brain. We validated these results by demonstrating co-purification of β2 from wild-type, but not α7 or β2 knock-out mice. The pharmacology and kinetics of human α7β2 nAChRs differed significantly from that of α7 homomers in response to nAChR agonists when expressed in Xenopus oocytes and HEK293 cells. Notably, α7β2 heteromers expressed in HEK293 cells display markedly slower rise and decay phases. These results demonstrate that α7 subunits in the human brain form heteromeric complexes with β2 subunits, and that human α7β2 nAChR heteromers respond to nAChR agonists with a unique pharmacology and kinetic profile. α7β2 nAChRs thus represent an alternative mechanism for the reported clinical efficacy of α7 nAChR ligands.

Forebrain-selective AMPA-receptor antagonism guided by TARP γ-8 as an antiepileptic mechanism

Pharmacological manipulation of specific neural circuits to optimize therapeutic index is an unrealized goal in neurology and psychiatry. AMPA receptors are important for excitatory synaptic transmission, and their antagonists are antiepileptic. Although efficacious, AMPA-receptor antagonists, including perampanel (Fycompa), the only approved antagonist for epilepsy, induce dizziness and motor impairment. We hypothesized that blockade of forebrain AMPA receptors without blocking cerebellar AMPA receptors would be antiepileptic and devoid of motor impairment. Taking advantage of an AMPA receptor auxiliary protein, TARP γ-8, which is selectively expressed in the forebrain and modulates the pharmacological properties of AMPA receptors, we discovered that LY3130481 selectively antagonized recombinant and native AMPA receptors containing γ-8, but not γ-2 (cerebellum) or other TARP members. Two amino acid residues unique to γ-8 determined this selectivity. We also observed antagonism of AMPA receptors expressed in hippocampal, but not cerebellar, tissue from an patient with epilepsy. Corresponding to this selective activity, LY3130481 prevented multiple seizure types in rats and mice and without motor side effects. These findings demonstrate the first rationally discovered molecule targeting specific neural circuitries for therapeutic advantage.

The subtype-selective nicotinic acetylcholine receptor positive allosteric potentiator 2087101 differentially facilitates neurotransmission in the brain

Positive allosteric modulators of centrally expressed nicotinic acetylcholine receptors have therapeutic potentials in areas of cognition, motor function and reward. Several chemical classes of allosteric modulators that are selective for alpha7 nicotinic receptors have been characterised, but potentiators for the most widely expressed alpha4beta2 nicotinic receptor subtype are few and less defined, owing probably to the difficulty to achieve selectivity over other heteromeric receptor subtypes. 2087101 (2-amino-5-keto)thiazole) is a potent potentiator of both alpha7 and alpha4beta2 receptors and it has selectivity against the alpha3beta4 subtype, which may be responsible for the undesirable peripheral side effects. To further characterise its ability to differentiate between native nicotinic receptors, we examined the effects of 2087101 on alpha7, alpha4beta2* and alpha3beta4* receptor-mediated responses in the rat brain in electrophysiological and neurochemical experiments. 2087101 significantly potentiated agonist-induced, alpha7 and non-alpha7 receptor-mediated, GABAergic postsynaptic currents in cultured hippocampal neurones, but not the nicotine-stimulated [(3)H]noradrenaline release from hippocampal slices, which was primarily mediated by alpha3beta4* receptors, confirming its selectivity for alpha7 and alpha4beta2* receptors in native systems. 2087101 also significantly enhanced nicotine-stimulated firing increase in dopamine neurones of the ventral tegmental area, an effect that was dihydro-beta-erythroidine-sensitive and thereby mediated by alpha4beta2* nicotinic receptors. 2087101 can therefore enhance native nicotinic activities mediated by alpha7 and alpha4beta2*, but not alpha3beta4* receptors, showing its unique ability to discriminate between native heteromeric nicotinic receptor subtypes and its therapeutic potential for treating brain disorders by concurrent modulation of both alpha7 and alpha4beta2* nicotinic receptors.

Electrophysiological Characterization of Human and Mouse Sodium-Dependent Citrate Transporters (NaCT/SLC13A5) Reveal Species Differences with Respect to Substrate Sensitivity and Cation Dependence

The citric acid cycle intermediate citrate plays a crucial role in metabolic processes such as fatty acid synthesis, glucose metabolism, and β-oxidation. Citrate is imported from the circulation across the plasma membrane into liver cells mainly by the sodium-dependent citrate transporter (NaCT; SLC13A5). Deletion of NaCT from mice led to metabolic changes similar to caloric restriction; therefore, NaCT has been proposed as an attractive therapeutic target for the treatment of obesity and type 2 diabetes. In this study, we expressed mouse and human NaCT into Xenopus oocytes and examined some basic functional properties of those transporters. Interestingly, striking differences were found between mouse and human NaCT with respect to their sensitivities to citric acid cycle intermediates as substrates for these transporters. Mouse NaCT had at least 20- to 800-fold higher affinity for these intermediates than human NaCT. Mouse NaCT is fully active at physiologic plasma levels of citrate, but its human counterpart is not. Replacement of extracellular sodium by other monovalent cations revealed that human NaCT was markedly less dependent on extracellular sodium than mouse NaCT. The low sensitivity of human NaCT for citrate raises questions about the translatability of this target from the mouse to the human situation and raises doubts about the validity of this transporter as a therapeutic target for the treatment of metabolic diseases in humans.

Proteomic analysis of postsynaptic proteins in regions of the human neocortex

The postsynaptic proteome of excitatory synapses comprises ~1,000 highly conserved proteins that control the behavioral repertoire, and mutations disrupting their function cause >130 brain diseases. Here, we document the composition of postsynaptic proteomes in human neocortical regions and integrate it with genetic, functional and structural magnetic resonance imaging, positron emission tomography imaging, and behavioral data. Neocortical regions show signatures of expression of individual proteins, protein complexes, biochemical and metabolic pathways. We characterized the compositional signatures in brain regions involved with language, emotion and memory functions. Integrating large-scale GWAS with regional proteome data identifies the same cortical region for smoking behavior as found with fMRI data. The neocortical postsynaptic proteome data resource can be used to link genetics to brain imaging and behavior, and to study the role of postsynaptic proteins in localization of brain functions.The protein composition of excitatory synapses differs in the areas of the human neocortex controlling language, emotion and other behaviors. This neocortical postsynaptic proteome data resource can be used to link genetics to brain imaging and behavior.