Hsueh-Meei Huang

Antioxidants prevent amyloid peptide-induced apoptosis and alteration of calcium homeostasis in cultured cortical neurons

Beta-amyloid ((A)beta) is a peptide of 39-42 amino acids that is the primary component of plaques in Alzheimers disease (AD). The mechanism by which (A)beta expresses its neurotoxic effects may involve induction of reactive oxygen species (ROS) and elevation of intracellular free calcium levels. Cultured cortical cells were utilized to study the alterations in calcium homeostasis underlying the neurotoxic effect of (A)beta. Serum supplement B27 and vitamin E were effective in preventing neuronal death as assessed by lactate dehydrogenase (LDH) release, (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, and number of apoptotic nuclei. In addition, (A)beta-induced cytosolic free calcium ([Ca2+]i) was blocked by antioxidants vitamin E and U83836E, but not by N-methyl-D-aspartic acid (NMDA) receptor antagonist MK-801, or by voltage-gated calcium channel blocker nimodipine. Taken together, the results suggest that NMDA receptor and voltage-gated calcium channels are not involved in (A)beta-induced [Ca2+]i increase. This increase appeared to be the result of extracellular calcium influx by some unknown mechanisms. In addition, antioxidants such as B27 were effective in protecting cultured cortical neurons against (A)beta, and correlated with (A)beta attenuation of early calcium response.

Blockage of amyloid β peptide-induced cytosolic free calcium by fullerenol-1, carboxylate C60 in PC12 cells

Amyloid beta protein (Abeta) alters signal transduction systems, including increases in the cytosolic free calcium ([Ca2+]i) response which have pathophysiological significance in Alzheimers disease (AD). The purposes of this study were to elucidate the mechanism involved in Abetas effect on the Ca2+ signal and to evaluate the effect of fullerenol-1, a water-soluble hydroxyl and superoxide radical scavenger, on the Abeta-induced Ca2+ response. Both Abeta and bradykinin (BK) dose-dependently elevated [Ca2+]i in PC12 cells. Fullerenol-1, at a concentration range between 100 nM and 1 microM, dose-dependently reduced the Abeta-induced [Ca2+]i response, but did not alter the subsequent BK-mediated process. Thapsigargin, an inhibitor of Ca2+-ATPase, released Ca2+ from the internal store and diminished the BK-mediated calcium spike but did not affect the Abeta-induced Ca2+ response. In the absence of extracellular calcium, the Abeta-induced, but not BK-induced, calcium spike was completely abolished. The Ca induced by Abeta did not enter through the voltage-dependent calcium channels or ligand gated calcium channels, because the peak of Abeta-evoked Ca2+ was not significantly altered by various Ca2+ channel blockers or a NMDA receptor antagonist MK801. In addition, neither cholera toxin nor pertussis toxin altered the Abeta-induced Ca response. The results demonstrated that Abeta-stimulated [Ca2+]i increase is due to Ca influx from an extracellular source rather than from the intracellular store. Alteration of the membrane lipid structure and permeability by free radicals generated by Abeta may be a major cause of Ca -influx. Furthermore, fullerenol-1, a novel antioxidant, may provide therapeutic benefits in neurodegenerative diseases such as AD.

Dehydroepiandrosterone inhibits lipopolysaccharide‐induced nitric oxide production in BV‐2 microglia

Levels of dehydroepiandrosterone (DHEA) and its sulfated derivative (DHEAS) decline during aging and reach even lower levels in Alzheimers disease (AD). DHEA is known to exhibit a variety of functional activities in the CNS, including an increase of memory and learning, neurotrophic and neuroprotective effects, and the reduction of risk of age‐related neurodegenerative disorders. However, the influence of DHEA on the immune functions of glial cells is poorly understood. In this study, we investigated the effect of DHEA on activated glia. The production of inducible nitric oxide synthase (iNOS) was studied in lipopolysaccharide (LPS)‐stimulated BV‐2 microglia, as a model of glial activation. The results showed that DHEA but not DHEAS significantly inhibited the production of nitrite in the LPS‐stimulated BV‐2 cell cultures. Pretreatment of BV‐2 cells with DHEA reduced the LPS‐induced iNOS mRNA and protein levels in a dose‐dependent manner. The LPS‐induced iNOS activity in BV‐2 cells was decreased by the exposure of 100 µm DHEA. Moreover, DHEA suppressed iNOS gene expression in LPS‐stimulated BV‐2 cells did not require de novo synthesis of new proteins or destabilize of iNOS mRNA. Since DHEA is biosynthesized by astrocytes and neurons, our findings suggest that it might have an important regulatory function on microglia.

Protective Effect of Nicotinamide on Neuronal Cells under Oxygen and Glucose Deprivation and Hypoxia/Reoxygenation

Nicotinamide (vitamin B3) reduces the infarct volume following focal cerebral ischemia in rats; however, its mechanism of action has not been reported. After cerebral ischemia and/or reperfusion, reactive oxygen species (ROS) and reactive nitrogen species may be generated by inflammatory cells through several cellular pathways, which can lead to intracellular calcium influx and cell damage. Therefore, we investigated the mechanisms of action of nicotinamide in neuroprotection under conditions of hypoxia/reoxygenation. Results showed that nicotinamide significantly protected rat primary cortical cells from hypoxia by reducing lactate dehydrogenase release with 1 h of oxygen-glucose deprivation (OGD) stress. ROS production and calcium influx in neuronal cells during OGD were dose-dependently diminished by up to 10 mM nicotinamide (p < 0.01). This effect was further examined with OGD/reoxygenation (H/R). Cells were stained with the fluorescent dye 4,6-diamidino-2-phenylindole (DAPI) or antibodies against anti-microtubule-associated protein-2 and cleaved caspase-3. Apoptotic cells were studied using Western blotting of cytochrome c and cleaved caspase-3. Results showed that vitamin B3 reduced cell injury, caspase-3 cleavage and nuclear condensation (DAPI staining) in neuronal cells under H/R. In addition, nicotinamide diminished c-fos and zif268 immediate-early gene expressions following OGD. Taken together, these results indicate that the neuroprotective effect of nicotinamide might occur through these mechanisms in this in vitro ischemia/reperfusion model.

Determination of catecholamines in pheochromocytoma cell (PC-12) culture medium by microdialysis–microbore liquid chromatography

An in vitro microdialysis system was constructed for the measurement of catecholamines in pheochromocytoma cell culture medium. The novel microdialysis device is composed of a petri dish, a dialysis membrane and two transmission tubes. The dialysis membrane is located in the space of a petri dish such that it is immersed in the culture medium. Catecholamines contained in the culture medium diffused into a designed dialysis membrane with sufficient recovery (about 60%). Dialysates were collected by a sampling loop and introduced by an on-line injector to a microbore liquid chromatographic system for analysis of catecholamines. This assay yielded a detection limit of 0.2-0.5 pg/injection with acceptable intra- and inter-assay reproducibilities in 5 microl of dialysates. To evaluate the on-line microdialysis system, PC-12 cells were cultured in a petri dish within an incubator. The baseline concentration of dopamine in PC-12 cell culture medium was about 0.29 ng/ml which was elevated to 2.43 ng/ml after treatment with 0.5 mM potassium cyanide. In conclusion, the present microassay provides for the sensitive, direct measurement of catecholamines in culture medium while minimizing pretreatment procedures for sample preparation.

Induction of cytokine genes and IL-1α by chemical hypoxia in PC12 cells

Abstract The role of cytokine in neuronal injury was examined in rat pheochromocytoma (PC12) cells under chemical hypoxia (i.e. KCN) and glucose deprivation. The mRNA levels of interleukin-1alpha (IL-1α), IL-6, and tumor necrosis factor-alpha (TNF-α) were measured by reverse transcription-polymerase chain reaction (RT-PCR) in PC12 cells exposed to 0.5 mM KCN for various time intervals. Cytokine mRNA levels expressed to peak levels 30 minutes after KCN treatment and declined gradually until 240 min. The IL-1α activity reached the highest levels 2 hr after the same KCN treatment. Under parallel conditions, KCN increased cytosolic free calcium concentration ([Ca 2 +] i ) in the absence of glucose. However, IL-1α mRNA induction by KCN was not altered under calcium-free conditions in PC12 cells, indicating its induction was Ca 2 +-independent. However, the phosphatidylcholine (PC)-specific phospholipase C (PLC) inhibitor D609 decreased the KCN-induced IL-1α mRNA and protein in PC12 cells suggests that PC-PLC might play a role in cytokine induction during hypoxia.

Neuroprotective MK801 is associated with nitric oxide synthase during hypoxia/reoxygenation in rat cortical cell cultures

The neuroprotective effect of MK801 against hypoxia and/or reoxygenation‐induced neuronal cell injury and its relationship to neuronal nitric oxide synthetase (nNOS) expression were examined in cultured rat cortical cells. Treatment of cortical neuronal cells with hypoxia (95% N2/5% CO2) for 2 h followed by reoxygenation for 24 h induced a release of lactate dehydrogenase (LDH) into the medium, and reduced the protein level of MAP‐2 as well. MK801 attenuated the release of LDH and the reduction of the MAP‐2 protein by hypoxia, suggesting a neuroprotective role of MK801. MK801 also diminished the number of nuclear condensation by hypoxia/reoxygenation. The NOS inhibitors 7‐nitroindazole (7‐NI) and N (G)‐nitro‐l‐arginine methyl ester (l‐NAME), as well as the Ca2+ channel blocker nimodipine, reduced hypoxia‐induced LDH, suggesting that nitric oxide (NO) and calcium homeostasis contribute to hypoxia and/or the reoxygenation‐induced cell injury. The levels of nNOS immunoactivities and mRNA by RT‐PCR were enhanced by hypoxia with time and, down regulated following 24 h reoxygenation after hypoxia, and were attenuated by MK801. In addition, the reduction of nNOS mRNA levels by hypoxia/reoxygenation was also diminished by MK801. Further delineation of the mechanisms of NO production and nNOS regulation are needed and may lead to additional strategies to protect neuronal cells against hypoxic/reoxygenation insults. J. Cell. Biochem. 84: 367–376, 2002.

Amyloid beta peptide impaired carbachol but not glutamate-mediated phosphoinositide pathways in cultured rat cortical neurons

Signal transduction systems, including cholinergic pathways, which are likely to be of pathophysiological significance are altered in Alzheimers disease (AD). Muscarinic cholinergic receptors are linked to the hydrolysis of phosphoinositide, involving the production of inositol 1,4,5-trisphosphate [Ins (1,4,5)P3] and the mobilization of cytosolic free calcium concentrations ([Ca2+]i). Effects of amyloid peptide (Aβ) on these signals prior to neuronal degeneration were examined in cultured rat cortical cells. Aβ increased the release of lactate dehydrogenase (LDH) in a concentration-dependent manner, however, it was blocked by B27 supplement. Prolonged exposure to a sublethal dose of Aβ 25–35 or 1–42 disrupted carbachol-mediated release of Ins(1,4,5)P3 and [Ca2+]i, which was inhibited in media supplemented with B27 or the antioxidant vitamin E. In order to determine the specificity of the effect of Aβ, various agonists glutamate or KCl but not bradykinin which utilize the phosphoinositide cascade were investigated. Our results indicated that Aβ did not affect the stimulation of glutamate or KCl-mediated production of Ins(1,4,5)P3 or cause elevation in [Ca2+]i. Furthermore, metabotropic agonist trans-1-amino-cyclopentane-1,3,-dicarboxylate (ACPD) elevated calcium level was not inhibited by Aβ pretreatment. Taken together, the results demonstrate that a sublethal dose of Aβ selectively impaired cholinergic receptor-mediated signal transduction pathways, and antioxidant or B27 supplement attenuated this effect of Aβ. Alterations of cholinergic signaling by prolonged exposure to Aβ could be involved in cortical neurodegeneration that occurs in AD. Because functional loss of cholinergic pathways is an important aspect of AD, the differences in susceptibility of these two types of receptors prior to other signs of Aβ action is important and requires further investigation.

Amyloid β peptide enhanced bradykinin-mediated inositol (1,4,5)trisphosphate formation and cytosolic free calcium

Deposition of amyloid beta protein (A beta) and alteration in signal transduction systems may have pathophysiological significance in Alzheimers disease (AD). This study tested the hypothesis that bradykinin (BK) receptor-mediated signal transduction systems in PC12 cells are altered after treatment with A beta at a concentration not toxic to cells. Exposure to varying doses of A beta 25-35 (1-10 microM) for 18 hrs significantly reduced the number of viable cells, while lower concentrations (0.01-0.1 microM) and control peptide in scramble sequence had no effect. In addition, prolonged exposure of PC12 cells to a sublethal dose of A beta 25-35 (0.1 microM) affected the receptor-mediated signal transduction pathways. BK induced both accumulation of Ins(1,4,5)P3 and elevation in cytosolic free calcium concentration ([Ca2+]i) in the control cells. These responses were further enhanced in the cells treated with A beta. Under similar conditions, A beta-treated cells also demonstrated alterations in the number and affinity of BK receptors. Alternatively, extracellular addition of A beta elevated [Ca2+]i rapidly, without detectable alterations in Ins(1,4,5)P3. This rapid elevation was dependent on extracellular calcium, suggesting that A beta induced calcium influx. Taken together, the results demonstrated that treatment with a sublethal dose of A beta peptide for 18 hrs enhanced BK receptor mediated Ins(1,4,5)P3 formation and mobilization of intracellular calcium, associated with a modification in BK receptors. Changes in the balance of these receptor-mediated signals prior to cell injury could be an important underlying mechanism for A beta peptide-induced degenerative alteration in AD.

ATP-stimulated c-fos and zif268 mRNA expression is inhibited by chemical hypoxia in a rat brain-derived type 2 astrocyte cell line, RBA-2.

The stimulus‐transcriptional coupling during ischemia/hypoxia was examined for ATP‐stimulated expression of immediate early genes (IEGs; c‐fos, zif268, c‐myc and nur77) in a rat brain‐derived type 2 astrocyte cell line, RBA‐2. Incubation of cells with 1 mM of extracellular ATP stimulated time‐dependent expression of c‐fos and zif268. ATP induced the largest increases in zif268 mRNA and a lesser one in c‐fos mRNA. ATP also induced a slight increase in nur77 mRNA but was ineffective in inducing c‐myc expression in these cells. Brief exposure of cells to potassium cyanide to simulate chemical hypoxia induced 9‐fold and 7‐fold transient increases in c‐fos and zif268 expression, respectively, but did not affect c‐myc or nur77 expression. When cyanide and ATP were added together, the expression of c‐fos and zif268 expression was inhibited, and the effect was mimicked by simulating chemical hypoxia with sodium azide. To elucidate the mechanism involved, the effect of cyanide on ATP‐stimulated increases in intracellular Ca2+ concentrations, [Ca2+]i, and phospholipase D (PLD) activities were measured. Cyanide induced an increase in [Ca2+]i and further enhanced the ATP‐stimulated increases in [Ca2+]i and PLD activities. Nevertheless, metabolic inhibitor, iodoacetate, blocked the ATP‐induced c‐fos and partially inhibited zif268 expression, and deprivation of cells with glucose also inhibited the ATP‐induced c‐fos expression. Taken together, these results demonstrate that both extracellular ATP and chemical hypoxia induce c‐fos and zif268 expression in RBA‐2 type 2 astrocytes. The chemical hypoxia inhibited ATP‐stimulated c‐fos and zif268 expression is not due to alterations in Ca2+ and PLD signaling, and is at least partially related to metabolic disturbance in these cells. J. Cell. Biochem. 77:323–332, 2000.