Tetsuro Mohri

Toxic effect of a β-amyloid peptide (β22–35) on the hippocampal neuron and its prevention

A synthetic truncated beta-amyloid peptide, beta 22-35, was shown to have a cytotoxic effect on cultured neurons from the rat hippocampus in serum-free medium. The peptide formed aggregates and typical amyloid fibrils resembling those of the beta-amyloid protein (AP) in neutral buffer solution and showed characteristic staining with Congo red and thioflavin-S. The neurotoxicity of beta 22-35 was suppressed by addition of calf serum, dibutyryl cAMP or insulin to culture medium, but not by addition of NGF or substance P. beta 22-35 had no effect on the glial cells. These results suggest that the AP can induce neurotoxicity in the hippocampal cells in vitro and the toxicity may involve a disorder in the intracellular signal transduction.

Activation of the mitogen-activated protein kinase cascade through nitric oxide synthesis as a mechanism of neuritogenic effect of genipin in PC12h cells.

Prominent neurite outgrowth induced by genipin, a plant‐derived iridoid, was substantially inhibited by addition of NG‐nitro‐l‐arginine methyl ester (l‐NAME), a nitric oxide (NO) synthase (NOS) inhibitor, and carboxy‐PTIO, an NO scavenger, in PC12h cells. Increases of the NADPH‐diaphorase activity and neuronal and inducible NOS proteins in cells preceded the neurite outgrowth after addition of genipin to medium. NO donors could induce the neurite outgrowth dose‐dependently in the cells. On the other hand, an inhibitor of soluble guanylate cyclase (SGC), which is known to be a stimulatory target of NO, abolished greatly the genipin‐induced neurite outgrowth. Addition of extracellular signal‐regulated kinase (ERK) kinase inhibitors could almost completely abolish the neurite induction. l‐NAME remarkably depressed genipin‐stimulated phosphorylation of ERK‐1 and ‐2. A neuritogenic effect of nerve growth factor (NGF) in PC12h cells was also remarkably inhibited by the NOS inhibitor, NO scavenger and SGC inhibitor. These findings suggest that induced NO production followed by cyclic GMP‐mediated stimulation of the mitogen‐activated protein kinase (MAPK) cascade is implicated in the neuritogenesis by genipin and NGF in PC12h cells.

Effect of lipid peroxidation on membrane-bound Ca2+-ATPase activity of the intestinal brush-border membranes

We have studied lipid peroxidation and Ca2+-ATPase activity of the porcine intestinal brush-border membranes using a oxygen-radical-generating system consisting of dithiothreitol (DTT)/Fe2+ and tert-butyl hydroperoxide (t-BuOOH). The rates of lipid peroxidation were measured by formation of thiobarbituric acid-reactive substances (TBAR) and conjugated diene. Incubation of the membranes with DTT/Fe2+ in the absence and presence of t-BuOOH resulted in a slight (about 20%) and a marked (about 50%) inhibition of Ca2+-ATPase activity, respectively. The degree of inhibition was dependent on the hydroperoxide concentration. Addition of thiourea effectively protected Ca2+-ATPase activity but catalase and superoxide dismutase showed a slight and no effect on protection of the ATPase activity, respectively. Results of kinetic studies on the ATPase activity with varying ATP and Ca2+ concentrations revealed that the decrease in the enzyme activity by treatment with these oxidizing agents is mainly due to decrease of the Vmax value. Modification of SH groups in the membrane proteins by thiol group reagents such as N-ethylmaleimide, monoiodoacetate and monoiodacetamide did not induce the inhibition of Ca2+-ATPase activity. From these results, it is suggested that inhibition of the ATPase activity of the membranes by treatment with DTT/Fe2+ in the presence and absence of t-BuOOH is dependent on lipid peroxidation and that oxidative modification of SH groups may not be directly involved to the loss of the ATPase activity. In addition, results of the fluorescence anisotropy measurements of pyrene-labeled membranes suggested that change in the Ca2+-ATPase activity is partly related to a decrease in the membrane lipid fluidity.

Protection by ethanol of cortical neurons from N-methyl-d-aspartate-induced neurotoxicity is associated with blocking calcium influx

Effect of ethanol on N-methyl-D-aspartate (NMDA)-induced neurotoxicity in rat dissociated cortical cells (8-12 day cultures) was studied. Treatment of cells with NMDA (50 and 500 microM) for 15 min caused cytotoxic effects on the cells, as examined by microscopic observations and lactate dehydrogenase release from cells 18 h after the treatment. Ca2+ is essential for these effects in medium during treatment. Presence of ethanol (50-300 mM) simultaneously with NMDA protected cells from the cytotoxicity depending on the concentration of ethanol. Calcium accumulation in cells on addition of NMDA, as monitored by fluorescence ratio (F405/F485) of Indo-1-preloaded cortical cells, was also decreased depending on the concentration of added ethanol. APV (200 microM) and ketamine (100 microM) blocked both the cytotoxicity and cellular calcium accumulation due to NMDA. These results suggest that ethanol effects its protection of neurons from NMDA-induced cytotoxicity by blocking the receptor-mediated calcium influx.

A change in the lipid fluidity of the porcine intestinal brush-border membranes by lipid peroxidation. Studies using pyrene and fluorescent stearic acid derivatives

The effect of lipid peroxidation on the lipid fluidity of porcine intestinal brush-border membranes was examined by measuring the rotational mobility and the accessibility to fluorescence quenchers (CH3COOT1, CuSO4 and KI) of pyrene or n-(9-anthroyloxy)stearic acid (n = 2 or 12) in the membranes. The harmonic mean of the rotational relaxation times of pyrene increased and the rate constants, kq, of the quenching reaction of pyrene and 2-(9-anthroyloxy)stearic acid incorporated in the membrane lipids decreased upon lipid peroxidation, indicating reduction of the lipid fluidity of the membranes by lipid peroxidation. In addition, the kq value of the reaction of 2-(9-anthroyloxy)stearic acid in the membranes with Cu2+ decreased in proportion to the amount of the products of lipid peroxidation. On the other hand, the kq value of the reaction of 12-(9-anthroyloxy)stearic acid with Cu2+ or I- was unaffected by lipid peroxidation. Based on these results, a localized change in the lipid fluidity of the membranes in association with lipid peroxidation has been discussed.

Propentofylline protects β-amyloid protein-induced apoptosis in cultured rat hippocampal neurons

Abstract β-Amyloid protein 1–42 (β42) can induce apoptosis in the cultured hippocampal neurons, suggesting that it plays an important role in causing neurodegeneration in Alzheimers disease. Recently, propentofylline, a synthetic xanthine derivative, has been reported to depress ischemic degeneration of hippocampal neurons in gerbils. The present study investigated whether or not propentofylline affected the β42-induced apoptosis of hippocampal neurons, and if so, which type of signaling machinery works in the neuroprotective action of propentofylline. Addition of propentofylline markedly attenuated the β42-induced cell death of rat hippocampal neurons. The amyloid protein certainly induced apoptosis in the cultured hippocampal cells revealed by nuclear condensation, caspase-3 activation and an increase of Bax. Intriguingly, propentofylline blocked both the apoptotic features induced by β42 and further induced an anti-apoptotic protein, Bcl-2, during a short time of incubation. The neuroprotective action of propentofylline was comparably replaced with dibutyryl cAMP (dbcAMP) and was completely suppressed by a low concentration of specific protein kinase A (PKA) inhibitor. Taken altogether, the data strongly suggest that the protection of propentofylline on the β42-induced neurotoxicity is caused by enhancing anti-apoptotic action through cAMP–PKA system. Propentofylline as a therapeutic agent to Alzheimers disease is discussed.

Increase of the molecular rigidity of the protein conformation in the intestinal brush-border membranes by lipid peroxidation

The effect of lipid peroxidation on the protein conformation of the porcine intestinal brush-border membranes was studied using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM). By a kinetic analysis of the reaction of the membranes with DACM, it was shown that the reaction rate of the SH groups (SHf) of the membrane proteins, whose reaction with the dye is very fast, decreases in proportion to the extent of thiobarbituric acid-reactive substance formation. The difference in the rate of the reaction of the SHf groups for DACM between the control and peroxidized membranes completely disappeared after denaturation of the proteins by treatment with guanidine hydrochloride. The reaction of DACM with the SHf groups of the control membranes accelerated when the temperature was increased with an apparent transition temperature between 25 degrees C and 30 degrees C. On the other hand, no transition was observed in the peroxidized membranes over the temperature range 20-43 degrees C. These results suggest that the conformation around the SHf groups of the proteins in the peroxidized membranes is apparently different from that in the control membranes. A modification of the conformation around the SH groups in the membrane proteins associated with lipid peroxidation was further demonstrated by finding that the quenching efficiency of the fluorescence of the DACM-labeled membranes by Tl+ was markedly decreased after lipid peroxidation. Based on these results, changes in the protein conformation of the porcine intestinal brush-border membranes by lipid peroxidation are discussed.

Extracellular pH modulates N-methyl-D-aspartate receptor-mediated neurotoxicity and calcium accumulation in rat cortical cultures

The effect of extracellular pH (pHo) on the excitotoxicity of N-methyl-D-aspartate (NMDA) in cultured rat cortical cells was studied. Treatment of cells with 500 microM NMDA for 15 min at various pHs in a range from 6.5 to 8.0 progressively enhanced staining with Trypan blue and release of lactate dehydrogenase with increased pH after 18 h of culture following treatment. The cytotoxic effect of high concentration of K+ (40 mM) or veratridine (10 microM) was also directly related to the increase in pHo. Free calcium accumulation in cells on addition of NMDA increased parallel to pHo. Changes in intracellular pH were estimated to be minor compared with extracellular changes. Specific NMDA antagonists could block both the NMDA- and membrane depolarization-induced neurotoxicity and calcium accumulation completely. These results suggest that the proton concentration outside of cells attenuates NMDA-induced neurotoxicity by blocking calcium accumulation.

Effects of α-tocopherol on the lipid peroxidation and fluidity of porcine intestinal brush-border membranes

Abstract The effect of α-tocopherol on the lipid fluidity of porcine intestinal brush-border membranes was studied using pyrene as a fluorescent probe. Addition of α-tocopherol to the medium decreased fluorescence intensity and lifetime, but increased the fluorescence polarization of pyrene-labeled membranes. β-, γ-, and δ-Tocopherols gave no appreciable effect on the fluorescence intensity and polarization of the complex. The apparent dissociation constant (3.1 ± 0.12 μ M) of the interaction of α-tocopherol with the membranes, estimated from the change in the fluorescence intensity with varying concentrations of α-tocopherol, was in good agreement with the concentration required to cause the half-maximal inhibition of lipid peroxidation of the membranes performed by incubation with 100 μM ascorbic acid and 10 μM Fe 2+ . Decrease of the slope in the thermal Perrin plot of the polarization of pyrene-labeled membranes by α-tocopherol suggests that the movement of pyrene molecules in the membranes is restricted by binding of the tocopherol. This interpretation was confirmed by an increased harmonic mean of the rotational relaxation time of the dye molecules in the membranes from 10.9 ± 0.16 to 18.5 ± 0.51 μ s after addition of 25 μM α-tocopherol to the medium. The perturbation of lipid phase in the membranes induced by α-tocopherol was also suggested from a decreased quenching rate constant of pyrene fluorescence in the membranes for Tl + . Based on these results, the effect of α-tocopherol on the lipid fluidity of the membranes is discussed.

Fundamental role of nitric oxide in neuritogenesis of PC12h cells.

We investigated the neuritogenic action of nitric oxide (NO)‐generating agents and their mechanisms of action in a subclone of rat pheochromocytoma, PC12h cells. NO donors such as sodium nitroprusside (SNP, 0.05–1 μM), NOR1 (5–100 μM), NOR2 (5–20 μM), NOR3 (5–20 μM), NOR4 (5–100 μM), or S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP, 10–100 μM) significantly induced neurite outgrowth. NOR4‐induced neurite outgrowth was accompanied by expression of neurofilament 200 kDa subunit (NF200) protein, an axonal marker, and was significantly inhibited by an NO scavenger, a soluble GC inhibitor, and a PKG inhibitor: 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazole‐1‐oxyl‐3‐oxide (carboxy‐PTIO, 20–100 μM), 1H‐[1,2,4]oxadiazolo[4,3‐a] quinoxalin‐1‐one (ODQ, 100 μM) and KT5823 (0.2–1 μM), respectively. The intracellular cGMP concentration of cells was markedly increased by treatment with NOR4 (100 μM). A mitogen‐activated protein kinase (MAPK) kinase inhibitor, PD98059 (10–50 μM), abolished the NOR4‐induced neurite outgrowth. In agreement with this observation, NOR4 did phosphorylate extracellular signal‐regulated kinase (ERK) 1 and 2, substrates of MAPK kinase. A membrane‐permeable cGMP analog, 8‐Br‐cGMP (1 mM) also induced significant neurite outgrowth. The 8‐Br‐cGMP‐induced neurite outgrowth was almost completely inhibited by both KT5823 (0.5 μM) and PD98059 (50 μM). Moreover, sustained ERK phosphorylation was observed in the 8‐Br‐cGMP‐treated PC12h cells. These results suggest that NO itself has the ability to induce neurite outgrowth and that NO‐induced ERK activation involves the NO‐cGMP‐PKG signaling pathway in PC12h cells.