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Objective: Apolipoprotein E (APOE) gene polymorphism is correlated closely with resistance to brain damage. This study aims to investigate the effects of APOE4 on oxidative stress damaged cerebral cortical neuron., Materials and Methods: Primary cerebral cortical neurons were isolated from APOE gene knock-out mice (APOE-/- mice). Oxidative stress damaged APOE-/- mouse cerebral cortical neuron model was established. Three experimental designs (experiment 1, 2, 3) were conducted by employing several methods. Lactate dehydrogenase (LDH) and superoxide dismutase (SOD) analysis were employed for neurotoxicity assessment. Flow cytometry and transferase-mediated deoxyuridine-triphosphate-biotin nick end labeling (TUNEL) were used to examine neuron apoptosis. Immunohistochemistry and Nissl staining were used to identify neuron morphology. Western blot was used to detect phosphorylated CaMK II (p-CaMK II) and cleaved caspase 3 expression. Ca2+ levels in neurons were also examined by detecting fluorescence intensity., Results: APOE4 treatment (Vehicle + APOE4) significantly aggravates oxidative stress damaged cerebral cortical neuron by increasing LDH levels and decreasing SOD activities, induces neuron apoptosis compared to Vehicle group (p < 0.05). APOE4 treatment significantly enhanced Ca2+ levels compared to Sham group (p < 0.05), MK801 treatment (Vehicle + APOE4 + MK801) significantly decreased Ca2+ levels compared to the Vehicle+APOE4 group at 12 h and 24 h (p < 0.05). APOE4 triggers CaMK II phosphorylation, caspase 3 activation and neurons apoptosis. Both of MK801 and KN93 inhibit CaMK II phosphorylation, decreases caspase 3 activation, and suppresses neurons apoptosis CONCLUSIONS: APOE triggers Ca2+ overload through NMDAR and CaMK II signaling pathway, both of which cause Ca2+ concentration increasing, CaMK II phosphorylation abnormity, and finally aggravate oxidative stress damaged neurons apoptosis.

In a natural acoustic environment, sound stimuli often occur in a contextual acoustic stream. The aim of the present study was to determine how the frequency tuning of auditory cortical neurons is affected by an acoustic context. A forward masking paradigm was used to determine the frequency receptive fields of rat auditory cortex neurons under quiet and sequential sound conditions. The frequency receptive fields of a cortical neuron were modulated dynamically by a preceding sound stimulus. At a fixed interstimulus interval (ISI), if the preceding sound level was constant, the receptive fields of most neurons were modulated to the greatest extent when the preceding sound frequency was at or near the characteristic frequency of the neuron; if the preceding sound frequency was constant, the modulation was increased with increasing sound stimulus level. When both the frequency and the level of the preceding sound were fixed, the modulation decreased with increasing interstimulus interval. The results indicate that the frequency tuning of auditory cortical neurons is plastic and dynamically modulated in a reverberant acoustical environment, and the degree of modulation depends on both the frequency tuning of the neuron and the contextual acoustical stream., (Copyright (c) 2010 IBRO. Published by Elsevier Ltd. All rights reserved.)

Alpha-secretase (alpha-secretase), cleaves the amyloid precursor protein (APP) within the amyloid-beta (Abeta) sequence, resulting in the release of a secreted fragment of APP (alphaAPPs) and precluding Abeta generation. We investigated the effects of the acetylcholinesterase inhibitor, huperzine A (Hup A), on APP processing and Abeta generation in human neuroblastoma SK-N-SH cells overexpressing wild-type human APP695. Hup A dose-dependently (0-10 microM) increased alphaAPPs release. Therefore, we evaluated two alpha-secretase candidates, a disintegrin and metalloprotease (ADAM) 10 and ADAM17 in Hup A-induced non-amyloidogenic APP metabolism. Hup A enhanced the level of ADAM10, and the inhibitor of tumor necrosis factor-alpha converting enzyme (TACE)/ADAM17 inhibited the Hup A-induced rise in alphaAPPs levels, further suggesting Hup A directed APP metabolism toward the non-amyloidogenic alpha-secretase pathway. Hup A had no effect on Abeta generation in this cell line. The steady-state levels of full-length APP and cell viability were unaffected by Hup A. Alpha-APPs release induced by Hup A treatment was significantly reduced by muscarinic acetylcholine receptor antagonists (particularly by an M1 antagonist), protein kinase C (PKC) inhibitors, GF109203X and calphostin C, and the mitogen-activated kinase kinase (MEK) inhibitors, U0126 and PD98059. Furthermore, Hup A markedly increased the phosphorylation of p44/p42 mitogen-activated protein (MAP) kinase, which was blocked by treatment with U0126 and PD98059. In addition, Hup A inhibited acetylcholinesterase activity by 20% in neuroblastoma cells. Our results indicate that the activation of muscarinic acetylcholine receptors, PKC and MAP kinase may be involved in Hup A-induced alphaAPPs secretion in neuroblastoma cells and suggest multiple pharmacological mechanisms of Hup A regarding the treatment of Alzheimer's disease (AD).

The present study was designed to further explore the potential cause/effect relationship between the expression of both the N141I presenilin (PS)2 mutant familial Alzheimer's disease (FAD) gene and cyclooxgenase (COX) in respect to the mechanism associated with programmed cell death in Alzheimer's disease (AD). We found that expression of mutant N141I PS2 resulting in apoptotic cell death in H4 neuronal cells coincided with >4-fold induction in the expression of the inducible form of COX-2, but not the constitutive COX-1. Moreover, we found that the expression of the N141I PS2 FAD gene strongly promoted (>2-fold) glycogen synthase kinase (GSK)-3beta activity coincidental with a reduction in the level of beta-catenin translocated from the cytoplasmic to the nuclear compartment. Most interestingly, we found that inhibition of COX-2-mediated generation of prostaglandin (PG)-E2 in H4 neuronal cells with the preferential COX-2 inhibitor nimesulide protects against N141I PS2-mediated apoptotic cell death coincidental with an inhibition of GSK-3beta activity and subsequent normalization of beta-catenin cellular distribution. The clinical relevance of this finding was confirmed by the evidence that COX-2 protein and PG-E2 concentrations were selectively increased >2-fold in the cerebral cortex of subjects harboring the N141I PS2 FAD mutation relative to wild-type PS2 AD cases. This study demonstrates for the first time that COX-2 may be a downstream effector of mutant N141I PS2-mediated apoptotic cell death and that inhibition of COX-2 may neuroprotect in AD through modulation of a GSK-3beta-beta-catenin-mediated response. The study provides support for the potential pharmacogenomic identification of N141I PS2 FAD cases that might preferentially benefit from inhibition of COX-2 during the progression of clinical dementia.

The transcription factor, activator protein 1 (AP-1) complexes (c-Jun and c-Fos heterodimers) has been shown to interact with transforming growth factor beta signaling in mammalian cells and Drosophila embryo. Here we show that c-Jun alone is involved in the anti-neuralizing activity of bone morphogenetic protein 4, a transforming growth factor beta superfamily member, in Xenopus neurogenesis. Co-injection of mRNAs encoding c-jun and a dominant negative bone morphogenetic protein receptor completely inhibits dominant negative bone morphogenetic protein receptor-induced neuralization and reverses the epidermal fate in the animal cap. Surprisingly, a dominant negative c-Jun does not induce neural tissue in the animal cap, but it synergizes with dominant negative bone morphogenetic protein receptor for neural induction. Temporal analysis using a dexamethasone-inducible c-Jun shows that exogenous c-Jun activity must be turned on before or at stage 11 to fulfill the anti-neuralizing effect. Neural inhibition by c-Jun does not occur until stage 13 suggesting that c-Jun probably acts by suppressing neural maintenance rather than neural initiation. This is also supported by the fact that c-Jun does not inhibit expression of the neural-initializing gene Zic-r1 but the neural cofactor Sox2, and that ectopic expression of Sox2 attenuates the anti-neuralizing effect of c-Jun. Finally, we display that the c-Jun effect is enhanced by an auto-regulatory loop between c-Jun and bone morphogenetic protein. These studies suggest that c-Jun/AP-1 is a converging point in both the fibroblast growth factor and transforming growth factor beta signaling pathways. Based on our findings, we propose that c-Jun synergizes with bone morphogenetic protein 4 signaling to inhibit neural development in Xenopus ectoderm.

The olfactory bulb, neocortex and archicortex arise from a common pool of progenitors in the dorsal telencephalon. We studied the consequences of supplying excess Notch1 signal in vivo on the cellular and regional destinies of telencephalic precursors using bicistronic replication defective retroviruses. After ventricular injections mid-neurogenesis (E14.5), activated Notch1 retrovirus markedly inhibited the generation of neurons from telencephalic precursors, delayed the emergence of cells from the subventricular zone (SVZ), and produced an augmentation of glial progeny in the neo- and archicortex. However, activated Notch1 had a distinct effect on the progenitors of the olfactory bulb, markedly reducing the numbers of cells of any type that migrated there. To elucidate the mechanism of the cell fate changes elicited by Notch1 signals in the cortical regions, short- and long-term cultures of E14.5 telencephalic progenitors were examined. These studies reveal that activated Notch1 elicits a cessation of proliferation that coincides with an inhibition of the generation of neurons. Later, during gliogenesis, activated Notch1 triggers a rapid cellular proliferation with a significant increase in the generation of cells expressing GFAP. To examine the generation of cells destined for the olfactory bulb, we used stereotaxic injections into the early postnatal anterior subventricular zone (SVZa). We observed that precursors of the olfactory bulb responded to Notch signals by remaining quiescent and failing to give rise to differentiated progeny of any type, unlike cortical precursor cells, which generated glia instead of neurons. These data show that forebrain precursors vary in their response to Notch signals according to spatial and temporal cues, and that Notch signals influence the composition of forebrain regions by modulating the rate of proliferation of neural precursor cells.

Amino acids are demonstrated to be important neurotransmitters mediating the inhibitory transmission from nucleus raphe magnus to spinal nociceptive dorsal horn neurons. In this study, the role of glycine and GABA in the inhibitory processes evoked by stimulation in periaqueductal gray (PAG) of responses of primate spinothalamic tract (STT) neurons to cutaneous mechanical and thermal stimuli was investigated by examining the effects of strychnine and bicuculline, antagonists of glycine and GABAA receptors, respectively, introduced into the dorsal horn through a microdialysis fiber. The inhibitory effects of iontophoretic application of glycine and GABAA agonists on STT cell activity evoked by noxious mechanical stimulation of the skin were selectively blocked by their specific antagonist, strychnine or bicuculline, infused into the dorsal horn. Similarly, intra-spinal application of strychnine or bicuculline resulted in a significant reduction in the PAG stimulation-induced inhibition of responses of STT cells to cutaneous stimuli. This reduction was mainly on the PAG-induced inhibition of the responses to noxious mechanical stimuli. Our results suggest that glycinergic and GABAergic inhibitory interneurons in the spinal cord dorsal horn synapsing on STT cells are activated during stimulation in PAG and contribute to descending antinociceptive actions.

Relationships between leucine-enkephalin fibers and cholinergic neurons in the rat sacral intermediolateral nucleus were examined by light and electron microscopy using double-immunostaining method. Cholinergic neurons in the sacral intermediolateral nucleus were labeled by a rat-mouse monoclonal antibody to choline acetyltransferase and stained bluish green with 5-bromo-4-chloro-3-indolyl-beta-D- galactoside reaction products using beta-galactosidase as a marker. On the same sections, leucine-enkephalin fibers were labeled by a rabbit polyclonal antiserum to leucine-enkephalin and stained brown by diaminobenzidine reaction products using peroxidase as a marker. After embedding in Epon, the sections were examined in light and electron microscopes. In the light microscope, choline acetyltransferase-like immunoreactive cells were seen in the sacral intermediolateral nucleus. In the same region, leucine-enkephalin-like immunoreactive cells. In the electron microscope, 5-bromo-4-chloro-3-indolyl-beta-D-galactoside reaction products were in the form of coarse electron dense deposits in the choline acetyltransferase-like immunoreactive structures and could be distinguished from the much finer grained diaminobenzidine reaction products. Choline acetyltransferase-like immunoreactive neurons received synaptic inputs from leucine-enkephalin fibers-like immunoreactive terminals. These findings suggest that leucine-enkephalin fibers may affect the activity of cholinergic parasympathetic preganglionic neurons.