Pamela S. Puttfarcken

Kainic acid-induced lipid peroxidation : protection with butylated hydroxytoluene and U78517F in primary cultures of cerebellar granule cells

The generation of free radicals in the progression of kainic acid (KA)-mediated neuronal death has been implicated in both in vitro and in vivo studies. In the present study, the association between KA-induced neurodegeneration and the appearance of lipid peroxidation products was investigated and compared to three well characterized free radical generating (FRG) systems: 200 microM ferrous ammonium sulfate (FAS), 20 microM copper (Cu2+), and 0.01 U/ml xanthine oxidase/2.3 mM purine/2.4 microM transferrin (XO). KA caused a dose-dependent increase in conjugated diene and lipid hydroperoxide formation as did the FRG systems. The antioxidant, butylated hydroxytoluene (BHT), decreased both FRG system- and KA-induced lipid peroxidation by approximately 60-70%. Unlike BHT, the potency of the lipid peroxidation inhibitor, U78517F, depended upon the system utilized to induce free radical generation. U78517F was most potent in attenuating FAS-induced lipid peroxidation (100 nM), followed by KA (1.5 microM), and then Cu2+ and XO (> 2 microM). Results were confirmed by measurement of cytolysis through the release of lactic dehydrogenase (LDH). These data provide further evidence that the generation of free radicals, subsequently leading to membrane disruption, is central to the mechanism of KA-elicited neuronal death in cultures of cerebellar granule cells.

Reduced nicotinic receptor-mediated antinociception following in vivo antisense knock-down in rat.

Pharmacological activation of neuronal nicotinic acetylcholine receptors can produce non-opioid antinociception in rodents. However, multiple nAChR subtypes exist, the most abundant of which contain alpha4 and beta2 subunits. The purpose of the present study was to investigate the role of alpha4-containing nAChRs in mediating nicotinic antinociception using an in vivo antisense strategy. Both i.c.v. infusion and repeated bolus injections into the cerebral aqueduct of an antisense oligonucleotide against the alpha4 subunit significantly attenuated the antinociceptive effects of the nAChR agonist A-85380 in the paw withdrawal test of acute thermal pain. Rats treated with a scrambled oligonucleotide displayed a full antinociceptive response to A-85380, while discontinuing antisense treatment restored the antinociceptive effects of the nicotinic agonist. Double immunohistochemical labeling revealed near-complete overlap of expression of the serotonin marker tryptophan hydroxylase and the alpha4 nAChR subunit in the dorsal raphe nucleus. The expression of alpha4-containing nAChRs by serotonergic neurons in the dorsal raphe offered a means to address nonspecific alpha4 knock-down, i.e., oligonucleotide-induced neurotoxicity. Immunohistochemical detection of alpha4 expression was reduced by nearly 50% in the dorsal raphe of antisense-treated rats as compared to either saline or missense-treated controls. In contrast, the expression of tryptophan hydroxylase, as well as, the alpha7 nAChR subunit in antisense-infused rats was similar to that observed in saline- and missense-treated controls. The results of these studies suggest that alpha4-containing nAChRs, possibly expressed by serotonergic neurons, are involved in nicotinic-mediated analgesia. However, these data do not eliminate the possibility that other nicotinic subunit combinations may also play a role in antinociception produced by nAChR activation.

Preclinical Characterization of A‐582941: A Novel α7 Neuronal Nicotinic Receptor Agonist with Broad Spectrum Cognition‐Enhancing Properties

Among the diverse sets of nicotinic acetylcholine receptors (nAChRs), the α7 subtype is highly expressed in the hippocampus and cortex and is thought to play important roles in a variety of cognitive processes. In this review, we describe the properties of a novel biaryl diamine α7 nAChR agonist, A‐582941. A‐582941 was found to exhibit high‐affinity binding and partial agonism at α7 nAChRs, with acceptable pharmacokinetic properties and excellent distribution to the central nervous system (CNS). In vitro and in vivo studies indicated that A‐582941 activates signaling pathways known to be involved in cognitive function such as ERK1/2 and CREB phosphorylation. A‐582941 enhanced cognitive performance in behavioral models that capture domains of working memory, short‐term recognition memory, memory consolidation, and sensory gating deficit. A‐582941 exhibited a benign secondary pharmacodynamic and tolerability profile as assessed in a battery of assays of cardiovascular, gastrointestinal, and CNS function. The studies summarized in this review collectively provide preclinical validation that α7 nAChR agonism offers a mechanism with potential to improve cognitive deficits associated with various neurodegenerative and psychiatric disorders.

High content screen microscopy analysis of Aβ1–42-induced neurite outgrowth reduction in rat primary cortical neurons: Neuroprotective effects of α7 neuronal nicotinic acetylcholine receptor ligands

beta-Amyloid peptide 1-42 (A beta(1-42)) is generated from amyloid precursor protein (APP) and associated with neurodegeneration in Alzheimers disease (AD). A beta(1-42) has been shown to be cytotoxic when incubated with cultured neurons. However, APP transgenic mice over-expressing A beta(1-42) do not show substantial loss of neurons, despite deficits in learning and memory. It is thus emerging that A beta(1-42)-induced memory deficits may involve subtler neuronal alternations leading to synaptic deficits, prior to frank neurodegeneration in AD brains. In this study, high content screen (HCS) microscopy, an advanced high-throughput cellular image processing and analysis technique, was utilized in establishing an in vitro model of A beta(1-42)-induced neurotoxicity utilizing rat neonatal primary cortical cells. Neurite outgrowth was found to be significantly reduced by A beta(1-42) (300 nM to 30 microM), but not by the scrambled control peptide control, in a time- and concentration-dependent manner. In contrast, no reduction in the total number of neurons was observed. The A beta(1-42)-induced reduction of neurite outgrowth was attenuated by the NMDA receptor antagonist memantine and the alpha 7 nicotinic acetylcholine receptor (nAChR) selective agonist PNU-282987. Interestingly, the alpha 7 nAChR antagonist methyllycaconitine also significantly prevented reduction in A beta(1-42)-induced neurite outgrowth. The observed neuroprotective effects could arise either from interference of A beta(1-42) interactions with alpha 7 nAChRs or by modification of receptor-mediated signaling pathways. Our studies demonstrate that reduction of neurite outgrowth may serve as a model representing A beta(1-42)-mediated neuritic and synaptic toxicity, which, in combination of HCS, provides a high-throughput cell-based assay that can be used to evaluate compounds with neuroprotective properties in neurons.

Different nicotinic acetylcholine receptor subtypes mediating striatal and prefrontal cortical [3H]dopamine release

Different nicotinic acetylcholine receptor subtypes appear to modulate dopamine release from the striatum and prefrontal cortex. In this study a combination of subtype-selective antagonists and agonists were used to extensively characterize the nAChRs involved in dopamine release from slice preparations of these two brain regions. alpha-conotoxin-MII inhibited nicotine-evoked [3H]dopamine (DA) release from striatum by 45%, but did not affect cortical dopamine release. Neither methyllycaconitine, alpha-bungarotoxin, nor alpha-conotoxin-ImI affected nicotine-evoked [3H]DA release from either striatum or prefrontal cortex. MG 624, a novel selective nAChR antagonist, inhibited cortical [3H]DA by 53%, but had no effect on striatal release. Compared to nicotine, (+/-)-UB-165 showed less efficacy with respect to dopamine release from striatum, and had no effect on cortical dopamine release. (+/-)-UB-165-evoked striatal dopamine release was completely blocked by mecamylamine, partially blocked (up to 55%) by alpha-conotoxin-MII, and unaffected by methyllycaconitine or alpha-conotoxin-ImI. alpha4beta2* and alpha6beta2beta3* nAChRs appear to play a role in striatal dopamine release, whereas alpha4beta2* nAChRs modulate release from prefrontal cortex. alpha7* nAChRs do not appear to play a role in nAChR-mediated dopamine release from either brain region.

Selective α7 nicotinic acetylcholine receptor activation regulates glycogen synthase kinase3β and decreases tau phosphorylation in vivo

The alpha7 nicotinic acetylcholine receptor (nAChR) plays an important role in cognitive processes and has generated recent interest as a potential drug target for treating neurodegenerative disorders such as Alzheimers disease (AD). The property of Ca(2+) permeation associated with alpha7 nAChR agonism may lead to Ca(2+)-dependent intracellular signaling that contribute to the procognitive and neuroprotective effects that have been described with this pharmacology. In this study, we investigated whether alpha7 nAChR agonism leads to increased phosphorylation of the inhibitory regulating amino acid residue Ser-9 on GSK3beta, a major kinase responsible for tau hyperphosphorylation in AD neuropathology. Immunohistochemical analysis revealed that the selective alpha7 agonist A-582941 increased S(9)-GSK3beta phosphorylation in mouse cingulate cortex and hippocampus that was not observed in alpha7 nAChR knock-out mice. A-582941 steady state exposure through continuous (2 wk) infusion also increased S(9)-GSK3beta phosphorylation in the hippocampus of Tg2576 (APP), as well as wild-type mice. Moreover, A-582941 continuous infusion decreased phosphorylation of tau in hippocampal CA3 Mossy fibers and spinal motoneurons in a hypothermia-induced tau hyperphosphorylation mouse model and AD double transgenic APP/tau mouse line, respectively. These studies demonstrate that inactivation of GSK3beta may be associated with alpha7 nAChR-induced signaling leading to attenuated tau hyperphosphorylation, raising the intriguing possibility that alpha7 nAChR agonism may have disease modifying benefit in the treatment of tauopathies, in particular AD.

A-995662 [(R)-8-(4-methyl-5-(4-(trifluoromethyl)phenyl)oxazol-2-ylamino)-1,2,3,4-tetrahydronaphthalen-2-ol], a novel, selective TRPV1 receptor antagonist, reduces spinal release of glutamate and CGRP in a rat knee joint pain model.

&NA; The TRPV1 antagonist A‐995662 demonstrates analgesic efficacy in monoiodoacetate‐induced osteoarthritic (OA) pain in rat, and repeated dosing results in increased in vivo potency and a prolonged duration of action. To identify possible mechanism(s) underlying these observations, release of neuropeptides and the neurotransmitter glutamate from isolated spinal cord was measured. In OA rats, basal release of glutamate, bradykinin and calcitonin gene‐related peptide (CGRP) was significantly elevated compared to naïve levels, whereas substance P (SP) levels were not changed. In vitro studies showed that capsaicin‐evoked TRPV1‐dependent CGRP release was 54.7 ± 7.7% higher in OA, relative to levels measured for naïve rats, suggesting that TRPV1 activity was higher under OA conditions. The efficacy of A‐995662 in OA corresponded with its ability to inhibit glutamate and CGRP release from the spinal cord. A single, fully efficacious dose of A‐995662, 100 &mgr;mol/kg, reduced spinal glutamate and CGRP release, while a single sub‐efficacious dose of A‐995662 (25 &mgr;mol/kg) was ineffective. Multiple dosing with A‐995662 increased the potency and duration of efficacy in OA rats. Changes in efficacy did not correlate with plasma concentrations of A‐995662, but were accompanied with reductions in spinal glutamate release. These findings suggest that repeated dosing of TRPV1 antagonists enhances therapeutic potency and duration of action against OA pain, at least in part, by the sustained reduction in release of glutamate and CGRP from the spinal cord.

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.

TRPV1b overexpression negatively regulates TRPV1 responsiveness to capsaicin, heat and low pH in HEK293 cells

Transient receptor potential channel type V (TRPV) 1 is a non‐selective cation channel that can be activated by capsaicin, endogenous vanilloids, heat and protons. The human TRPV1 splice variant, TRPV1b, lacking exon 7, was cloned from human dorsal root ganglia (DRG) RNA. The expression profile and relative abundance of TRPV1b and TRPV1 in 35 different human tissues were determined by quantitative RT‐PCR using isoform‐specific probes. TRPV1b was most abundant in fetal brain, adult cerebellum and DRG. Functional studies using electrophysiological techniques showed that recombinant TRPV1b was not activated by capsaicin (1 µm), protons (pH 5.0) or heat (50°C). However, recombinant TRPV1b did form multimeric complexes and was detected on the plasma membrane of cells, demonstrating that the lack of channel function was not due to defects in complex formation or cell surface expression. These results demonstrate that exon 7, which encodes the third ankyrin domain and 44 amino acids thereafter, is required for normal channel function of human TRPV1. Moreover, when co‐expressed with TRPV1, TRPV1b formed complexes with TRPV1, and inhibited TRPV1 channel function in response to capsaicin, acidic pH, heat and endogenous vanilloids, dose‐dependently. Taken together, these data support the hypothesis that TRPV1b is a naturally existing inhibitory modulator of TRPV1.

Assessment of nicotinic acetylcholine receptor-mediated release of [3H]-norepinephrine from rat brain slices using a new 96-well format assay

The study of the modulatory effects of nicotinic acetylcholine receptor (nAChR) agonists on neurotransmitter release from tissue slices has been hampered by laborious and limiting superfusion techniques. A new methodology was developed utilizing 96-well filter plates. This new method produced comparable results to previously published data, yet expanded throughput to permit more complete pharmacological characterization. Rat brain slices, preloaded with [(3)H]-norepinephrine ([(3)H]-NE), were distributed onto 96-well filter plates. Following a 5 min preincubation, the slices were incubated for 5 min with nicotinic agonists or antagonists. (-)-Nicotine (NIC) and 1,1-dimethyl-4-phenylpiperazine (DMPP) evoked release of [(3)H]-NE from a number of brain regions and spinal cord, with the highest response seen in the hippocampus. Concentration-response curves revealed a rank order of potency of (+/-)-epibatidine>>anatoxin-a>A-85380>DMPP=NIC=(-)-cytisine in the hippocampus, thalamus, and frontal cortex. EC(50) values were approximately 0.005, 0.2, 1, 5, 5 and 5 microM, respectively. Concentration-inhibition curves of nicotine evoked [(3)H]-NE release from hippocampal and thalamic slices resulted in a rank order of potency of mecamylamine>hexamethonium>d-tubocurare>DHbetaE. Schild analysis revealed apparent noncompetitive antagonism of [(3)H]-NE release from hippocampus by mecamylamine, hexamethonium, and DHbetaE. In contrast, DHbetaE antagonism of [(3)H]-dopamine release from striatal slices using a similar methodology was competitive.