Jinhe Li

Positive Allosteric Modulation of the α7 Nicotinic Acetylcholine Receptor : Ligand Interactions with Distinct Binding Sites and Evidence for a Prominent Role of the M2-M3 Segment

The α7 nicotinic acetylcholine receptor (nAChR), a homopentameric, rapidly activating and desensitizing ligand-gated ion channel with relatively high degree of calcium permeability, is expressed in the mammalian central nervous system, including regions associated with cognitive processing. Selective agonists targeting the α7 nAChR have shown efficacy in animal models of cognitive dysfunction. Use of positive allosteric modulators selective for the α7 receptor is another strategy that is envisaged in the design of active compounds aiming at improving attention and cognitive dysfunction. The recent discovery of novel positive allosteric modulators such as 1-(5-chloro-2-hydroxyphenyl)-3-(2-chloro-5-trifluoromethylphenyl)urea (NS-1738) and 1-(5-chloro-2,4-dimethoxyphenyl)-3-(5-methylisoxazol-3-yl)urea (PNU-120596) that are selective for the α7 nAChRs but display significant phenotypic differences in their profile of allosteric modulation, suggests that these molecules may act at different sites on the receptor. Taking advantage of the possibility to obtain functional receptors by the fusion of proteins domains from the α7 and the 5-HT3 receptor, we examined the structural determinants required for positive allosteric modulation. This strategy revealed that the extracellular N-terminal domain of α7 plays a critical role in allosteric modulation by NS-1738. In addition, α7-5HT3 chimeras harboring the M2-M3 segment showed that spontaneous activity in response to NS-1738, which confirmed the critical contribution of this small extracellular segment in the receptor gating. In contrast to NS-1738, positive allosteric modulation by PNU-120596 could not be restored in the α7-5HT3 chimeras but was selectively observed in the reverse 5HT3-α7 chimera. All together, these data illustrate the existence of distinct allosteric binding sites with specificity of different profiles of allosteric modulators and open new possibilities to investigate the α7 receptor function.

Role of GSK-3β activation and α7 nAChRs in Aβ1-42-induced tau phosphorylation in PC12 cells

β‐amyloid peptide 1–42 (Aβ1–42) and hyperphosphorylated tau are associated with neurodegeneration in Alzheimer’s disease. Emerging evidence indicates that Aβ1–42 can potentiate hyperphosphorylation of tau in cell lines and in transgenic mice, but the underlying mechanism(s) remains unclear. In this study, Aβ1–42‐induced tau phosphorylation was investigated in differentiated PC12 cells. Treatment of cells with Aβ1–42 increased phosphorylation of tau at serine‐202 as detected by AT8 antibody. This Aβ1–42‐induced tau phosphorylation paralleled phosphorylation of glycogen synthase kinase‐3β (GSK‐3β) at tyrosine‐216 (GSK‐3β‐pY216), which was partially inhibited by the GSK‐3β inhibitor, CHIR98023. Aβ1–42‐induced tau phosphorylation and increase in GSK‐3β‐pY216 phosphorylation were also partially attenuated by α7 nicotinic acetylcholine receptor (α7 nAChR) selective ligands including agonist A‐582941 and antagonists methyllycaconitine and α‐bungarotoxin. The α7 nAChR agonist and the GSK‐3β inhibitor had no additive effect. These observations suggest that α7 nAChR modulation can influence Aβ1–42‐induced tau phosphorylation, possibly involving GSK‐3β. This study provides evidence of nAChR mechanisms underlying Aβ1–42 toxicity and tau phosphorylation, which, if translated in vivo, could provide additional basis for the utility of α7 nAChR ligands in the treatment of Alzheimer’s disease.

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 nAChR-mediated activation of MAP kinase pathways in PC12 cells

The alpha7 nicotinic acetylcholine receptor (alpha7 nAChR) plays a fundamental role in Ca(2+)-dependent activation of signaling pathways that can modulate intracellular events involved in learning and memory. Activation of extracellular signal-regulated kinase-1 and -2 (ERK1/2) are well documented Ca(2+) signaling events, but these have not been well characterized in response to alpha7 nAChR-selective ligands. The present study examined activation of ERK1/2 and explored pathways leading to CREB phosphorylation utilizing alpha7 nAChR-selective ligands in PC12 cells endogenously expressing alpha7 nAChRs. Robust concentration-dependent increase in ERK1/2 phosphorylation was triggered by structurally diverse alpha7 nAChR agonists such as nicotine, choline, GTS-21, SSR-180711A and PNU-282987 in the presence of the positive allosteric modulator (PAM) PNU-120596. This effect was attenuated by selective alpha7 nAChR antagonists or by chelation of extracellular Ca(2+). ERK1/2 phosphorylation was also attenuated by inhibitors of calmodulin-dependent protein kinase II (CaMKII), p38 MAP kinase and mitogen-activated protein kinase kinase1/2 (MEK1/2), indicating the involvement of these kinases upstream of ERK1/2. This was confirmed by direct measurement of p38 MAPK and MEK1/2 phosphorylation. These data suggest that alpha7 nAChR agonist-triggered Ca(2+) transient in PC12 cells induces activation of CaMKII, leading to sequential phosphorylation of p38 MAPK, MEK1/2, ERK1/2 and CREB. Such mechanisms may endow the alpha7 nAChRs with roles in modulating Ca(2+)-dependent intracellular second messenger events implicated in diverse aspects of cognition.

Positive allosteric modulation of α7 neuronal nicotinic acetylcholine receptors: lack of cytotoxicity in PC12 cells and rat primary cortical neurons

Background and purpose:  α7‐Nicotinic acetylcholine receptors (α7 nAChRs) play an important role in cognitive function. Positive allosteric modulators (PAMs) amplify effects of α7 nAChR agonist and could provide an approach for treatment of cognitive deficits in neuropsychiatric diseases. PAMs can either predominantly affect the apparent peak current response (type I) or increase both the apparent peak current response and duration of channel opening, due to prolonged desensitization (type II). The delay of receptor desensitization by type II PAMs raises the possibility of Ca2+‐induced toxicity through prolonged activation of α7 nAChRs. The present study addresses whether type I and II PAMs exhibit different cytotoxicity profiles.

Cerebrospinal fluid cytokine dynamics differ between Alzheimer disease patients and elderly controls.

The time courses of levels of multiple plasma and cerebrospinal fluid (CSF) cytokines in patients with Alzheimer disease (AD) and in age-matched control subjects were compared. Interleukin (IL)-1&bgr;, IL-2, IL-6, IL-8, IL-10, IL-12p70, granulocyte-macrophage colony-stimulating factor, interferon-&ggr;, and tumor necrosis factor alpha levels were measured 7 times over a 24-hour period in plasma and CSF using a lumbar catheter. Baseline plasma and CSF cytokine levels were found to be similar in AD and control subjects. However, the CSF levels of all measured cytokines, except IL-6 and IL-8, diverged over time between AD and control subjects, such that CSF cytokine levels in AD subjects were higher than in controls. This difference was greatest at 24 hours after the insertion of the lumbar catheter. In contrast, no differences in cytokine trajectories were seen in plasma. These data suggest that the neuroinflammatory response to lumbar catheter placement differs between AD and control subjects.

Effect of human cerebrospinal fluid sampling frequency on amyloid-β levels

β‐amyloid peptide (Aβ) is associated with neurodegeneration in Alzheimers disease. Emerging evidence indicates that Aβ levels in cerebrospinal fluid (CSF) may serve as an early clinical biomarker for evaluating pharmacological activity of new drug candidates targeting Aβ production or Aβ clearance. Therefore, it is critical to understand whether intrasubject levels of CSF Aβ are consistent between sampling intervals to determine whether Aβ can be used as a pharmacodynamic biomarker for drug candidates. Previous studies have produced seemingly conflicting observations for the intrasubject stability of CSF Aβ levels; we attempt to reconcile these conflicting observations.

Role of α7 nicotinic acetylcholine receptors in regulating tumor necrosis factor-α (TNF-α) as revealed by subtype selective agonists

Immunological responses to protect against excessive inflammation can be regulated by the central nervous system through the cholinergic anti-inflammatory pathway wherein acetylcholine released from vagus nerves can inhibit inflammatory cytokines. Although a role for the α7 nicotinic acetylcholine receptor (α7 nAChR) in mediating this pathway has been suggested, pharmacological modulation of the pathway by selective agonists remains to be further elucidated. In this study, the role of α7 nAChRs in the regulation of TNF-α release was investigated using high affinity and selective α7 nAChR agonists in mouse peritoneal macrophage and human whole blood in vitro, and in mouse serum in vivo. In mouse peritoneal macrophages, LPS-induced TNF-α release in vitro was inhibited by a selective α7 nAChR agonist, A-833834 (5-[6-(5-Methyl-hexahydro-pyrrolo[3,4-c]pyrrol-2-yl)-pyridazin-3-yl]-1H-indole), and that effect was attenuated by α7 nAChR antagonist methyllycaconitine. The inhibitory effect of A-833834 on LPS-induced TNF-α release was also observed in human whole blood in vitro. I.v. LPS-induced TNF-α release in mouse serum was attenuated following i.p. administration of A-833834. Similarly, i.v. LPS-induced TNF-α release in mouse serum was also attenuated following i.p. administration of A-585539, another α7 nAChR agonist with limited brain penetration, suggesting that these effects are mediated by peripheral α7 nAChRs. A-833834 was also efficacious in suppressing TNF-α release in mouse serum following oral administration in zymosan-induced peritonitis. These studies collectively demonstrate that selectively targeting α7 nAChRs could offer a novel therapeutic modality to treat acute and chronic inflammatory disease states.

Importance of M2–M3 loop in governing properties of genistein at the α7 nicotinic acetylcholine receptor inferred from α7/5-HT3A chimera

Genistein and 5-hydroxyindole (5-HI) potentiate the α7 nicotinic acetylcholine receptor current by primarily increasing peak amplitude, a property of type I α7 positive allosteric modulation. In this study, the effects of these two compounds were investigated at two different α7/5-HT(3) chimeras (chimera 1, comprising of extracellular α7 N-terminus fused to the remainder of 5-HT(3A), and chimera 2 containing an additional α7 encoded M2-M3 loop), and wild-type α7 and 5-HT(3A) receptors. Agonist-evoked responses, examined by expression of the chimeras in Xenopus laevis oocytes or HEK-293 cells, revealed that currents decayed slower and compounds {rank order: N-[(3R)-1-azabicyclo[2.2.2]oct-3-yl]-4-chlorobenzamide hydrochloride (PNU-282987)~2-(1,4-diazabicyclo[3.2.2]nonan-4-yl)-5-phenyl-1,3,4-oxadiazole (NS6784)>acetylcholine>choline} were more potent in chimera 2 than chimera 1 or α7 receptors. In chimera 2, genistein and 5-HI potentiated agonist-evoked responses (EC(50): 4-5 μM for genistein and 300-500 μM for 5-HI) and at higher concentrations evoked current directly consistent with ago-allosteric modulation. At chimera 1 and 5-HT(3A) receptors, neither compound directly evoked any current and 5-HI, only at chimera 1, was able to potentiate agonist-evoked responses. Genistein and 5-HI did not inhibit the binding of the α7 agonist [(3)H](1S,4S)-2,2-dimethyl-5-(6-phenylpyridazin-3-yl)-5-aza-2-azoniabicyclo[2.2.1] heptane ([(3)H]A-585539) to rat brain or chimera 2. In summary, this study supports the role of the M2-M3 loop being critical for the positive allosteric effect of genistein, but not 5-HI, and in agonist-evoked response fine-tuning. The identification of distinct α7 receptor modulatory sites offers unique opportunities for developing CNS therapeutics and understanding its pharmacology.

An integrated multi-study analysis of intra-subject variability in cerebrospinal fluid amyloid-β concentrations collected by lumbar puncture and indwelling lumbar catheter

IntroductionAmyloid-β (Aβ) has been investigated as a diagnostic biomarker and therapeutic drug target. Recent studies found that cerebrospinal fluid (CSF) Aβ fluctuates over time, including as a diurnal pattern, and increases in absolute concentration with serial collection. It is currently unknown what effect differences in CSF collection methodology have on Aβ variability. In this study, we sought to determine the effect of different collection methodologies on the stability of CSF Aβ concentrations over time.MethodsGrouped analysis of CSF Aβ levels from multiple industry and academic groups collected by either lumbar puncture (n=83) or indwelling lumbar catheter (n=178). Participants were either placebo or untreated subjects from clinical drug trials or observational studies. Participants had CSF collected by lumbar puncture or lumbar catheter for quantitation of Aβ concentration by enzyme linked immunosorbent assay. Data from all sponsors was converted to percent of the mean for Aβ40 and Aβ42 for comparison. Repeated measures analysis of variance was performed to assess for factors affecting the linear rise of Aβ concentrations over time.ResultsAnalysis of studies collecting CSF via lumbar catheter revealed tremendous inter-subject variability of Aβ40 and Aβ42 as well as an Aβ diurnal pattern in all of the sponsors’ studies. In contrast, Aβ concentrations from CSF samples collected at two time points by lumbar puncture showed no significant differences. Repeated measures analysis of variance found that only time and draw frequency were significantly associated with the slope of linear rise in Aβ40 and Aβ42 concentrations during the first 6 hours of collection.ConclusionsBased on our findings, we recommend minimizing the frequency of CSF draws in studies measuring Aβ levels and keeping the frequency standardized between experimental groups. The Aβ diurnal pattern was noted in all sponsors’ studies and was not an artifact of study design. Averaging Aβ concentrations at each time point is recommended to minimize the effect of individual variability. Indwelling lumbar catheters are an invaluable research tool for following changes in CSF Aβ over 24-48 hours, but factors affecting Aβ concentration such as linear rise and diurnal variation need to be accounted for in planning study designs.