Astrocytic GluN2A and GluN2B Oppose the Synaptotoxic Effects of Amyloid-ß1-40 in Hippocampal Cells.

Early-stage Alzheimer's disease (AD) is characterized by synaptic dysfunction, a phenomenon in which soluble oligomers of amyloid-beta (Aß) and N-methyl-D-aspartate receptor (NMDAR) are implicated. Here, we demonstrated that astrocytes express NMDARs and therefore have the potential to modulate the synaptotoxic actions of Aß. We found that specific pharmacological antagonism of two of the major NMDAR subunits, GluN2A and GluN2B, exacerbates Aß-induced synaptotoxicity suggesting, for the first time, that astrocytic GluN2A and GluN2B mediate synaptoprotection. From the perspective of the pathogenic mechanisms of Alzheimer's disease, in which Aß and NMDAR play significant roles, these observations are striking since neuronal GluN2A and GluN2B are well known modulators of neurodegeneration. We did initial studies to understand the basis for the differential effects of astrocytic and neuronal GluN2A and GluN2B in the promotion of synapse survival, and identified a neurotrophin produced by astrocytes, nerve growth factor ß (ß-NGF), as a likely mediator of the synaptoprotective effects of astrocytic GluN2A and GluN2B activation. The results presented suggest that astrocytes may be suitable druggable targets for the prevention and/or delay of the synaptic loss that occurs during early stages of AD.

N-acetyl-serotonin protects HepG2 cells from oxidative stress injury induced by hydrogen peroxide.

Oxidative stress plays an important role in the pathogenesis of liver diseases. N-Acetyl-serotonin (NAS) has been reported to protect against oxidative damage, though the mechanisms by which NAS protects hepatocytes from oxidative stress remain unknown. To determine whether pretreatment with NAS could reduce hydrogen peroxide- (H2O2-) induced oxidative stress in HepG2 cells by inhibiting the mitochondrial apoptosis pathway, we investigated the H2O2-induced oxidative damage to HepG2 cells with or without NAS using MTT, Hoechst 33342, rhodamine 123, Terminal dUTP Nick End Labeling Assay (TUNEL), dihydrodichlorofluorescein (H2DCF), Annexin V and propidium iodide (PI) double staining, immunocytochemistry, and western blot. H2O2 produced dramatic injuries in HepG2 cells, represented by classical morphological changes of apoptosis, increased levels of malondialdehyde (MDA) and intracellular reactive oxygen species (ROS), decreased activity of superoxide dismutase (SOD), and increased activities of caspase-9 and caspase-3, release of cytochrome c (Cyt-C) and apoptosis-inducing factor (AIF) from mitochondria, and loss of membrane potential (ΔΨm). NAS significantly inhibited H2O2-induced changes, indicating that it protected against H2O2-induced oxidative damage by reducing MDA levels and increasing SOD activity and that it protected the HepG2 cells from apoptosis through regulating the mitochondrial apoptosis pathway, involving inhibition of mitochondrial hyperpolarization, release of mitochondrial apoptogenic factors, and caspase activity.

Immunolocalization of CaMKII and NR2B in hippocampal subregions of rat during postnatal development.

Although the expression of CaMKII and synaptic-associated proteins has been widely studied, the temporospatial distribution of CaMKII and NMDAR subunits in different hippocampal subregions during postnatal development still lacks detailed information. In this study, we used immunofluorescent staining to assess CaMKII and NR2B expressions and the relationship between them in CA1, CA3, and DG of rat hippocampus on postnatal (P) days: P0, P4, P7, P10, P14, P21, P28, and P56. The results showed that from P0 to P56, CaMKII expression increased gradually, while NR2B expression decreased gradually, and the time points of their expression peak differed in CA1, CA3, and DG during postnatal development. Although the expression of CaMKII was negatively correlated with NR2B in CA1 and DG, the coexpression of CaMKII with NR2B existed in CA1, CA3, and DG during postnatal development. Interestingly, after P21, CaMKII expression and the coexpression of CaMKII with NR2B became obvious in the Stratum lucidum of CA3. The specific temporospatial distribution pattern of CaMKII with NR2B might be related to the different physiological functions during postnatal development. Discovery of the alteration of the relationship between expression of CaMKII and NMDAR subunits may be helpful for understanding mechanisms and therapy of neurodegenerative diseases.

Immunoreactivity of Ki-67/ß-tubulin and immunocolocalization with active caspase-3 in rat dentate gyrus during postnatal development.

This study was based on our previous report that the expression of active caspase-3 kept at a high level in dentate gyrus during postnatal development, which is not related to an apoptotic event. We addressed the hypothesis that the active caspase-3 expression may be related to a nonapoptotic role in the regulation of the cell cycle and differentiation or other physiological functions. To confirm this hypothesis, through a temporal investigation from postnatal day (P) 0, 4, 7, 10, 14, 21, 28, to 56, based on immunofluorescent method, we dual labeled active caspase-3 with Ki-67 or ß-tubulin in the dentate gyrus. Our results showed a minority of active caspase-3 positive cells were colabeled with the proliferation marker Ki-67 in stratum moleculare (MOL), granular cell layer (GCL), subgranular zone (SGZ) and polymorphic stratum (POLY) from P0 to P14, and the colabeled cells decreased gradually with age. From P21 to P56, the colocalization of the two proteins was mainly focused on SGZ. There was a positive correlation between the positive cells of active caspase-3 with that of Ki-67. In addition, an extensive colocalization between active caspase-3 and ß-tubulin was observed at all the age groups. There was a strong positive correlation between the intensity of active caspase-3 in GCL with that of ß-tubulin in MOL, GCL and POLY of dentate gyrus and the stratum lucidum of CA3. Our data raised the possibility of a nonapoptotic role of active caspase-3 in dentate gyrus, which may be partly associated with cellular proliferation and differentiation, and also may be related to neurite outgrowth, axonal transport, or dendrite elongation of granular cells during postnatal development.