AMPAR receptor inhibitors suppress proliferation of human small cell lung cancer cell lines.

BACKGROUND: Small cell lung cancer (SCLC) is a neuroendocrine tumor with poor prognosis. Neuroendocrine tumors possess characteristics of both nerve cells and hormone-secreting cells; therefore, targeting the neuronal properties of these tumors may lead to the development of new therapeutic options. Among the endogenous signaling pathways in the nervous system, targeting the glutamate pathway may be a useful strategy for glioblastoma treatment. Perampanel, an antagonist of the synaptic glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor (AMPAR), has been reported to be effective in patients with glioblastoma. In this study, we aimed to investigate the antitumor effects of AMPAR antagonists in human SCLC cell lines. METHODS: We performed to examine the expression of AMPAR using Western blot and immunohistochemical analysis. The antitumor effects of AMPAR antagonists on human SCLC cell lines were investigated in vitro and in vivo. We also analyzed the signaling pathway of AMPAR antagonists in SCLC cell lines. Statistical analysis was performed by the GraphPad Prism 6 software. RESULTS: We first examined the expression of endogenous AMPAR in six human SCLC cell lines, detecting AMPAR proteins in all of them. Next, we tested the anti-proliferative effect of two AMPAR antagonists, talampanel and cyanquixaline, using SCLC cells in vitro and in vivo. Both AMPAR antagonists inhibited cell proliferation and mitogen-activated protein kinase (MAPK) phosphorylation in SCLC cells in vitro. Further, we observed reduced proliferation of implanted cell lines in an in vivo setting, assessed by Ki-67 immunohistochemistry. Additionally, using immunohistochemical analysis we confirmed AMPAR protein expression in human SCLC samples. CONCLUSION: AMPAR may be a potential therapeutic target for SCLC.

[11C]K-2 image with positron emission tomography represents cell surface AMPA receptors.

The glutamate α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors (AMPARs) is an important molecule in neurotransmission. We have recently developed the first positron emission tomography (PET) tracer [11C]K-2 to visualize and quantify AMPARs in the living human brain. After injection, [11C]K-2 is hydrolyzed at the terminal amide (and is thus metabolized to a major metabolite, [11C]K-2OH) within 10 min, representing the PET image in rodents and humans. Here, we found that K-2OH did not penetrate the cell membrane but slowly passed through the blood brain barrier (BBB) with paracellular transport. Furthermore, major efflux transporters in the BBB did not carry K-2OH. Logan graphical analysis exhibited reversible binding kinetics of this radiotracer in healthy individuals; these results demonstrated that the PET image of this tracer represents cell surface AMPARs with passive penetration of [11C]K-2OH through the BBB, resulting in reversible binding kinetics. Thus, PET images with this tracer depict the physiologically crucial fraction of AMPARs.

CRMP2-binding compound, edonerpic maleate, accelerates motor function recovery from brain damage.

Brain damage such as stroke is a devastating neurological condition that may severely compromise patient quality of life. No effective medication-mediated intervention to accelerate rehabilitation has been established. We found that a small compound, edonerpic maleate, facilitated experience-driven synaptic glutamate AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic-acid) receptor delivery and resulted in the acceleration of motor function recovery after motor cortex cryoinjury in mice in a training-dependent manner through cortical reorganization. Edonerpic bound to collapsin-response-mediator-protein 2 (CRMP2) and failed to augment recovery in CRMP2-deficient mice. Edonerpic maleate enhanced motor function recovery from internal capsule hemorrhage in nonhuman primates. Thus, edonerpic maleate, a neural plasticity enhancer, could be a clinically potent small compound with which to accelerate rehabilitation after brain damage.

Social isolation suppresses actin dynamics and synaptic plasticity through ADF/cofilin inactivation in the developing rat barrel cortex.

Exposure to a stressful environment early in life can cause psychiatric disorders by disrupting circuit formation. Actin plays central roles in regulating neuronal structure and protein trafficking. We have recently reported that neonatal isolation inactivated ADF/cofilin, the actin depolymerizing factor, resulted in a reduced actin dynamics at spines and an attenuation of synaptic α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor delivery in the juvenile rat medial prefrontal cortex (mPFC), leading to altered social behaviours. Here, we investigated the impact of neonatal social isolation in the developing rat barrel cortex. Similar to the mPFC study, we detected an increase in stable actin fraction in spines and this resulted in a decreased synaptic AMPA receptor delivery. Thus, we conclude that early life social isolation affects multiple cortical areas with common molecular changes.

Optical inactivation of synaptic AMPA receptors erases fear memory.

The synaptic delivery of neurotransmitter receptors, such as GluA1 AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid) receptors, mediates important processes in cognitive function, including memory acquisition and retention. Understanding the roles of these receptors has been hampered by the lack of a method to inactivate them in vivo with high spatiotemporal precision. We developed a technique to inactivate synaptic GluA1 AMPA receptors in vivo using chromophore-assisted light inactivation (CALI). We raised a monoclonal antibody specific for the extracellular domain of GluA1 that induced effective CALI when conjugated with a photosensitizer (eosin). Mice that had been injected in the CA1 hippocampal region with the antibody conjugate underwent a fear memory task. Exposing the hippocampus to green light using an implanted cannula erased acquired fear memory in the animals by inactivation of synaptic GluA1. Our optical technique for inactivating synaptic proteins will enable elucidation of their physiological roles in cognition.

Neonatal isolation augments social dominance by altering actin dynamics in the medial prefrontal cortex.

Social separation early in life can lead to the development of impaired interpersonal relationships and profound social disorders. However, the underlying cellular and molecular mechanisms involved are largely unknown. Here, we found that isolation of neonatal rats induced glucocorticoid-dependent social dominance over nonisolated control rats in juveniles from the same litter. Furthermore, neonatal isolation inactivated the actin-depolymerizing factor (ADF)/cofilin in the juvenile medial prefrontal cortex (mPFC). Isolation-induced inactivation of ADF/cofilin increased stable actin fractions at dendritic spines in the juvenile mPFC, decreasing glutamate synaptic AMPA receptors. Expression of constitutively active ADF/cofilin in the mPFC rescued the effect of isolation on social dominance. Thus, neonatal isolation affects spines in the mPFC by reducing actin dynamics, leading to altered social behavior later in life.

Estrous Cycle-Dependent Phasic Changes in the Stoichiometry of Hippocampal Synaptic AMPA Receptors in Rats.

Cognitive function can be affected by the estrous cycle. However, the effect of the estrous cycle on synaptic functions is poorly understood. Here we show that in female rats, inhibitory-avoidance (IA) task (hippocampus-dependent contextual fear-learning task) drives GluA2-lacking Ca2+-permeable AMPA receptors (CP-AMPARs) into the hippocampal CA3-CA1 synapses during all periods of the estrous cycle except the proestrous period, when estrogen levels are high. In addition, IA task failed to drive CP-AMPARs into the CA3-CA1 synapses of ovariectomized rats only when estrogen was present. Thus, changes in the stoichiometry of AMPA receptors during learning depend on estrogen levels. Furthermore, the induction of long-term potentiation (LTP) after IA task was prevented during the proestrous period, while intact LTP is still expressed after IA task during other period of the estrous cycle. Consistent with this finding, rats conditioned by IA training failed to acquire hippocampus-dependent Y-maze task during the proestrous period. On the other hand, during other estrous period, rats were able to learn Y-maze task after IA conditioning. These results suggest that high estrogen levels prevent the IA learning-induced delivery of CP-AMPARs into hippocampal CA3-CA1 synapses and limit synaptic plasticity after IA task, thus preventing the acquisition of additional learning.

Effects of GABA and bicuculline on the electrical activity of rat olfactory placode neurons derived at E13.5 and cultured for 1 week on multi-electrode dishes.

The present study was performed to record the electrical activity of olfactory placode neurons and to check the effect of GABA and bicuculline on it. Olfactory placodes obtained at day 13.5 of gestation were cultured for 1 week on multi-electrode dishes. Olfactory placode neurons showed spontaneous firing, with firing rates of 0.77 +/- 0.05 Hz (0.03-3.82 Hz, n = 12), but there was no bursting activity. Perfusion with 10 microM GABA almost immediately inhibited 8 of 11 firing activities (we could not test it in 1 activity). In contrast, perfusion with 10 microM bicuculline induced facilitation in 5 of 12 activities and did not induce any change in 7 other activities. Statistical analysis by chi(2)-test showed a significant difference in the response of neurons to the two drugs. Fisher's exact probability test showed that the inhibitory effect of GABA was significant (p<0.05) whereas neither the facilitatory effect nor the lack of effect of bicuculline was significant (p>0.1). These results suggest that cultured olfactory placode neurons, even in a probably immature stage, respond to GABA with inhibition, as generally observed at mature stages.

Nicotine inhibition of pulsatile GnRH secretion is mediated by GABAA receptor system in the cultured rat embryonic olfactory placode.

In past work, we suggested that nicotine inhibition of in vivo pulsatile LH release is not mediated by opiate receptors known to be involved in the inhibition of LH release. In the present study, we examined whether nicotine inhibits the pulsatile gonadotropin-releasing hormone (GnRH) release, and whether this inhibition of GnRH release by nicotine is mediated by the GABA receptor system, by checking in vitro pulsatile GnRH release from cultured GnRH neurons obtained from olfactory placodes of rat embryos at E13.5. The mean interpulse interval of pulsatile GnRH release into the medium was 34.2+/-2.0 min in the control period and increased to 95.3+/-19.0 min (n=6) in the period of nicotine treatment at a concentration of 500 nM, showing an inhibitory effect of nicotine on pulsatile GnRH release. The GABA(A) receptor antagonist bicuculline used alone at a concentration of 20 microM caused no significant changes in the pulsatile GnRH release, but when used in combination with 500 nM of nicotine, bicuculline blocked the nicotine inhibition of GnRH release. In a separate experiment, nicotine treatment at a concentration of 500 nM significantly increased GABA release. These results suggest that, in the cultured embryonic olfactory placode, nicotine stimulates GABA release, which then inhibits GnRH release through GABA(A) receptor system.

Role of gamma-aminobutyric acid neurons in the release of gonadotropin-releasing hormone in cultured rat embryonic olfactory placodes.

We recently established a primary cell culture system of gonadotropin-releasing hormone (GnRH) neurons originating from olfactory placodes of rat embryos at E13.5 and showed that cultured olfactory placodes released GnRH into the medium in a pulsatile fashion with an interpulse interval of about 30 min. Since the reported presence of gamma-aminobutyric acid (GABA) neurons in the culture of rat olfactory placode raises questions as to the role played by these GABA neurons in the GnRH pulse generation, we immunostained GnRH neurons and GABA neurons in this culture system to examine the interrelationship between both types of neurons, and determined the effects of GABA and the GABA(A) receptor antagonist, bicuculline, on GnRH release. The immunohistochemical study showed that GnRH neurons received fiber terminals from GABA neurons. GnRH neurons in culture released GnRH into the medium at intervals of 30-40 min, confirming our previous study. Treatment with 20 microM GABA prolonged the interpulse interval and decreased the amplitude of GnRH pulses. Bicuculline administered at 20 microM did not affect either parameter, but 50 microM bicuculline elevated the mean GnRH level, although it did not affect either the interpulse interval or the amplitude of GnRH pulses. In addition, 50 microM bicuculline increased the mean trough levels of GnRH pulses, although 20 microM bicuculline did not. In light of the in vivo studies performed previously, we suggest that the GnRH pulse generator, which probably consists of a small population of GnRH neurons in the culture, does not involve GABA neurons to generate the pulsatile GnRH release, although it may be responsive to the inhibitory transmitter GABA. We also found that there may be another population of GnRH neurons in the culture whose activity is strongly suppressed by the tonic inhibition of GABA neurons. Although it is speculative, these latter GnRH neurons may be responsible for the surge of GnRH release.