Daniel B. Timmermann

Broad-Spectrum Efficacy across Cognitive Domains by α7 Nicotinic Acetylcholine Receptor Agonism Correlates with Activation of ERK1/2 and CREB Phosphorylation Pathways

The α7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and may represent a drug target for treating cognitive deficits in neurodegenerative and psychiatric disorders. In the present study, we used a novel α7 nAChR-selective agonist, 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941) to interrogate cognitive efficacy, as well as examine potential cellular mechanisms of cognition. Exhibiting high affinity to native rat (K i = 10.8 nm) and human (K i = 16.7 nm) α7 nAChRs, A-582941 enhanced cognitive performance in behavioral assays including the monkey delayed matching-to-sample, rat social recognition, and mouse inhibitory avoidance models that capture domains of working memory, short-term recognition memory, and long-term memory consolidation, respectively. In addition, A-582941 normalized sensory gating deficits induced by the α7 nAChR antagonist methyllycaconitine in rats, and in DBA/2 mice that exhibit a natural sensory gating deficit. Examination of signaling pathways known to be involved in cognitive function revealed that α7 nAChR agonism increased extracellular-signal regulated kinase 1/2 (ERK1/2) phosphorylation in PC12 cells. Furthermore, increases in ERK1/2 and cAMP response element-binding protein (CREB) phosphorylation were observed in mouse cingulate cortex and/or hippocampus after acute A-582941 administration producing plasma concentrations in the range of α7 binding affinities and behavioral efficacious doses. The MEK inhibitor SL327 completely blocked α7 agonist-evoked ERK1/2 phosphorylation. Our results demonstrate that α7 nAChR agonism can lead to broad-spectrum efficacy in animal models at doses that enhance ERK1/2 and CREB phosphorylation/activation and may represent a mechanism that offers potential to improve cognitive deficits associated with neurodegenerative and psychiatric diseases, such as Alzheimers disease and schizophrenia.

An Allosteric Modulator of the α7 Nicotinic Acetylcholine Receptor Possessing Cognition-Enhancing Properties in Vivo

Augmentation of nicotinic α7 receptor function is considered to be a potential therapeutic strategy aimed at ameliorating cognitive and mnemonic dysfunction in relation to debilitating pathological conditions, such as Alzheimers disease and schizophrenia. In the present report, a novel positive allosteric modulator of the α7 nicotinic acetylcholine receptor (nAChR), 1-(5-chloro-2-hydroxy-phenyl)-3-(2-chloro-5-trifluoromethyl-phenyl)-urea (NS1738), is described. NS1738 was unable to displace or affect radioligand binding to the agonist binding site of nicotinic receptors, and it was devoid of effect when applied alone in electrophysiological paradigms. However, when applied in the presence of acetylcholine (ACh), NS1738 produced a marked increase in the current flowing through α7 nAChRs, as determined in both oocyte electrophysiology and patch-clamp recordings from mammalian cells. NS1738 acted by increasing the peak amplitude of ACh-evoked currents at all concentrations; thus, it increased the maximal efficacy of ACh. Oocyte experiments indicated an increase in ACh potency as well. NS1738 had only marginal effects on the desensitization kinetics of α7 nAChRs, as determined from patch-clamp studies of both transfected cells and cultured hippocampal neurons. NS1738 was modestly brain-penetrant, and it was demonstrated to counteract a (–)-scopolamine-induced deficit in acquisition of a water-maze learning task in rats. Moreover, NS1738 improved performance in the rat social recognition test to the same extent as (–)-nicotine, demonstrating that NS1738 is capable of producing cognitive enhancement in vivo. These data support the notion that α7 nAChR allosteric modulation may constitute a novel pharmacological principle for the treatment of cognitive dysfunction.

The β Subunit Determines the Ion Selectivity of the GABAA Receptor

The γ-aminobutyric acid, type A (GABAA) receptor is a chloride-conducting receptor composed of α, β, and γ subunits assembled in a pentameric structure forming a central pore. Each subunit has a large extracellular agonist binding domain and four transmembrane domains (M1–M4), with the second transmembrane (M2) domain lining the pore. Mutation of five amino acids in the M1–M2 loop of the β3subunit to the corresponding amino acids of the α7nicotinic acetylcholine subunit rendered the GABAA receptor cation-selective upon co-expression with wild type α2 and γ2 subunits. Similar mutations in the α2 or γ2 subunits did not lead to such a change in ion selectivity. This suggests a unique role for the β3subunit in determining the ion selectivity of the GABAAreceptor. The pharmacology of the mutated GABAA receptor is similar to that of the wild type receptor, with respect to muscimol binding, Zn2+ and bicuculline sensitivity, flumazenil binding, and potentiation of GABA-evoked currents by diazepam. There was, however, an increase in GABA sensitivity (EC50 = 1.3 μm) compared with the wild type receptor (EC50 = 6.4 μm) and a loss of desensitization to GABA of the mutant receptor.

Augmentation of cognitive function by NS9283, a stoichiometry-dependent positive allosteric modulator of α2- and α4-containing nicotinic acetylcholine receptors

Positive allosteric modulation of α4β2 nicotinic acetylcholine (nACh) receptors could add a new dimension to the pharmacology and therapeutic approach to these receptors. The novel modulator NS9283 was therefore tested extensively.

Distribution of high-voltage-activated calcium channels in cultured γ-aminobutyric acidergic neurons from mouse cerebral cortex

The localization of voltage‐gated calcium channel (VGCC) α1 subunits in cultured GABAergic mouse cortical neurons was examined by immunocytochemical methods. Cav1.2 and Cav1.3 subunits of L‐type VGCCs were found in cell bodies and dendrites of GABA‐immunopositive neurons. Likewise, the Cav2.3 subunit of R‐type VGCCs was expressed in a somatodendritic pattern. Cav2.2 subunits of N‐type channels were found exclusively in small varicosities that were identified as presynaptic nerve terminals based on their expression of synaptic marker proteins. Two splice variants of the Cav2.1 subunit of P/Q‐type VGCCs showed widely differing expression patterns. The rbA isoform displayed a purely somatodendritic staining pattern, whereas the BI isoform was confined to axon‐like fibers and nerve terminals. The nerve terminals of these cultured GABAergic neurons express Cav2.2 either alone or in combination with Cav2.1 (BI isoform) but never express Cav2.1 alone. The functional association between VGCCs and the neurotransmitter release machinery was probed using the FM1‐43 dye‐labeling technique. N‐type VGCCs were found to be tightly coupled to exocytosis in these cultured cortical neurons, and P‐type VGCCs were also important in a fraction of the cells. The predominant role of N‐type VGCCs in neurotransmitter release and the specific localization of the BI isoform of Cav2.1 in the nerve terminals of these neurons distinguish them from previously studied central neurons. The complementary localization patterns observed for two different isoforms of the Cav2.1 subunits provide direct evidence for alternative splicing as a means of generating functional diversity among neuronal calcium channels.

Role of channel activation in cognitive enhancement mediated by α7 nicotinic acetylcholine receptors

Background and purpose:  Several agonists of the α7 nicotinic acetylcholine receptor (nAChR) have been developed for treatment of cognitive deficits. However, agonist efficacy in vivo is difficult to reconcile with rapid α7 nAChR desensitization in vitro; and furthermore, the correlation between in vitro receptor efficacy and in vivo behavioural efficacy is not well delineated. The possibility that agonists of this receptor actually function in vivo as inhibitors via desensitization has not been finally resolved.

In Vitro Pharmacological Characterization of a Novel Selective α7 Neuronal Nicotinic Acetylcholine Receptor Agonist ABT-107

Enhancement of α7 nicotinic acetylcholine receptor (nAChR) activity is considered a therapeutic approach for ameliorating cognitive deficits present in Alzheimers disease and schizophrenia. In this study, we describe the in vitro profile of a novel selective α7 nAChR agonist, 5-(6-[(3R)-1-azabicyclo[2,2,2]oct-3-yloxy]pyridazin-3-yl)-1H-indole (ABT-107). ABT-107 displayed high affinity binding to α7 nAChRs [rat or human cortex, [3H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1]heptane (A-585539), Ki = 0.2–0.6 nM or [3H]methyllycaconitine (MLA), 7 nM] that was at least 100-fold selective versus non-α7 nAChRs and other receptors. Functionally, ABT-107 did not evoke detectible currents in Xenopus oocytes expressing human or nonhuman α3β4, chimeric (α6/α3)β4, or 5-HT3A receptors, and weak or negligible Ca2+ responses in human neuroblastoma IMR-32 cells (α3* function) and human α4β2 and α4β4 nAChRs expressed in human embryonic kidney 293 cells. ABT-107 potently evoked human and rat α7 nAChR current responses in oocytes (EC50, 50–90 nM total charge, ∼80% normalized to acetylcholine) that were enhanced by the positive allosteric modulator (PAM) 4-[5-(4-chloro-phenyl)-2-methyl-3-propionyl-pyrrol-1-yl]-benzenesulfonamide (A-867744). In rat hippocampus, ABT-107 alone evoked α7-like currents, which were inhibited by the α7 antagonist MLA. In dentate gyrus granule cells, ABT-107 enhanced spontaneous inhibitory postsynaptic current activity when coapplied with A-867744. In the presence of an α7 PAM [A-867744 or N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-120596)], the addition of ABT-107 elicited MLA-sensitive α7 nAChR-mediated Ca2+ signals in IMR-32 cells and rat cortical cultures and enhanced extracellular signal-regulated kinase phosphorylation in differentiated PC-12 cells. ABT-107 was also effective in protecting rat cortical cultures against glutamate-induced toxicity. In summary, ABT-107 is a selective high affinity α7 nAChR agonist suitable for characterizing the roles of this subtype in pharmacological studies.

α7 nicotinic acetylcholine receptor activation ameliorates scopolamine-induced behavioural changes in a modified continuous Y-maze task in mice

The alpha7 (alpha7) nicotinic acetylcholine receptor may represent a drug target for the treatment of disorders associated with working memory/attentional dysfunction. We investigated the effects of three distinct alpha7 nicotinic acetylcholine receptor agonists: 2-methyl-5-(6-phenyl-pyridazin-3-yl)-octahydro-pyrrolo[3,4-c]pyrrole (A-582941; 0.01-0.1 mg/kg), 4-bromophenyl 1,4-diazabicyclo(3.2.2) nonane-4-carboxylate (SSR180711; 0.3-3 mg/kg) and N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide (PNU-282987; 1-10 mg/kg), on scopolamine-induced deficits in a modified Y-maze procedure. Mice were forced to choose one of two visually distinct arms, and were confined there for a 5 min exploration period before being allowed to explore both arms for a 2 min test session, immediately thereafter. The time spent in each arm, entries and total distance travelled were recorded using an automated system. Characterisation experiments showed that scopolamine-treated (1 mg/kg) mice spent less time exploring the unfamiliar arm, when compared with vehicle-treated animals. Combination experiments showed that all three alpha7 agonists ameliorated scopolamine-induced changes in unfamiliar arm exploration. In conclusion, the present data support the idea that alpha7 nicotinic acetylcholine receptors may represent an interesting target for the treatment of conditions associated with attentional/working memory dysfunction.

11C-NS14492 as a Novel PET Radioligand for Imaging Cerebral α7 Nicotinic Acetylcholine Receptors: In Vivo Evaluation and Drug Occupancy Measurements

Small-molecule α7 nicotinic acetylcholine receptor (α7nAChR) agonists are currently validated for use as treatment for cognitive disturbances in schizophrenia and in Alzheimer disease. A suitable radiolabeled α7nAChR PET tracer would be important for in vivo quantification of α7nAChR binding in humans and to measure α7nAChR occupancy of α7nAChR drug candidates. Here, we present the radiosynthesis and in vivo evaluation of 11C-NS14492 as a selective α7nAChR PET radioligand. Methods: The high-affinity α7nAChR-selective partial agonist NS14492 was radiolabeled by methylation of its desmethyl precursor using 11C-methyl triflate. Female Danish Landrace pigs were studied at baseline and after intravenous administration of blocking doses of either the α7nAChR partial agonist SSR180711 or the unlabeled NS14492. 11C-NS14492 was given as an intravenous bolus injection, and the pigs were scanned for 90 min both at baseline and in the blocked conditions. Arterial blood was collected during the scanning, plasma was counted, and parent compound fraction was determined with radio–high-performance liquid chromatography. PET data were quantified with a graphical analysis with arterial input; 11C-NS14492 regional distribution volumes were calculated, and α7nAChR occupancy was determined using an occupancy plot. Results: 11C-NS14492 had a high uptake in the pig brain, with the highest binding in the cerebral cortex and thalamus in accordance with α7nAChR distribution. Pretreatment with NS14492 and SSR180711 consistently decreased distribution volumes of 11C-NS14492 in all examined regions, in a dose-dependent manner, supporting the finding that the radioligand binds selectively to α7nAChR in vivo. Conclusion: We report here that 11C-NS14492 is the first, to our knowledge, PET radioligand for α7nAChR showing a dose-dependent decline in cerebral binding after receptor blockade. This compound is considered a promising PET tracer for in vivo measurements of α7nAChR binding in the human brain.

Alpha-7 nicotinic acetylcholine receptor agonists selectively activate limbic regions of the rat forebrain: An effect similar to antipsychotics

It is considered that activation of nicotinic α7 receptors (α7 nAChR) is useful for the treatment of cognitive disturbances in schizophrenia and Alzheimers disease. Recently, selective α7 nAChR agonists have been discovered and are used to validate the α7 nAChR as a drug target for the treatment of cognitive disturbances in schizophrenia. One important feature shared by all known antipsychotics is their capacity to induce expression of the neuronal immediate‐early gene c‐fos in the limbic forebrain. Using two novel and selective α7 nAChR agonists, PNU‐282987 and SSR180711, we investigated their ability to induce c‐Fos expression in the limbic forebrain with particular emphasis on the same regions reported to be activated by antipsychotics. Both α7 nAChR agonists increased c‐Fos dose‐dependently in the prefrontal cortex and the shell of nucleus accumbens, while leaving the core of nucleus accumbens and the dorsolateral striatum unaffected. The accumbal and cortical effect of SSR180711 was blocked completely by pre‐administration of the α7 nAChR antagonist methyllycaconitine. Also, SSR180711 displayed no c‐Fos‐inducing effect in α7 nAChR knock‐out mice. In conclusion, these results show that selective pharmacologic stimulation of α7 nAChR function results in activation of forebrain regions similar to conventional antipsychotics.