Satoshi Ono

JNK activation is associated with intracellular β-amyloid accumulation

Abstract c-Jun has been implicated in the pathogenesis of Alzheimer’s disease (AD), but the upstream cascade leading to c-Jun activation in AD is not known. Activation of c-Jun N-terminal kinase (JNK) is obviously a candidate for the upstream event. We tested this possibility focusing on PS1-linked AD. First, we observed that JNK is actually activated in cerebral neurons of PS1-linked AD patients, using immunohistochemistry and Western blot analyses with anti-activated JNK antibodies. We analyzed the relationship between β-amyloid (βA) and JNK activation by using aged transgenic mice overexpressing mutant (M146L) PS1 and human AD brains. The mice showed no neuronal loss but a very few diffuse βA deposits, corresponding to the early stage of PS1-linked AD brain. Some neurons were reactive for anti-βA antibodies in the cerebral cortex. Interestingly, JNK activation was observed in neurons showing intracellular βA immunoreactivity in transgenic mice. Association between intracellular βA and JNK activation was confirmed in cortical neurons of sporadic and PS1-linked AD patients. Furthermore, introduction of βA peptides into the primary culture cortical neurons induced JNK activation and cell death. Collectively, these results suggested that intracellular βA accumulation might trigger JNK activation leading to neuronal death.

Grafting neural stem cells improved the impaired spatial recognition in ischemic rats

To determine the possible therapeutic potential of neural stem cells (NSCs) introduced into the damaged central nervous system, we grafted adult hippocampus-derived NSCs into the hippocampus of rats with transient global ischemia. Transient four-vessel occlusion yielded 90-95% losses of pyramidal neurons in the hippocampal CA1 region. In this region, 1-3% of the grafted cells survived; and 3-9% of them expressed NeuN, a neuronal marker. Rats with more than 120 NeuN-positive cells showed partial improvement of impaired spatial learning in a water maze test. These results suggest that NSCs grafted in the ischemic brain are able to differentiate into neurons and to improve spatial recognition.

Involvement of L-Type-Like Amino Acid Transporters in S-Nitrosocysteine-Stimulated Noradrenaline Release in the Rat Hippocampus

Abstract: Nitrogen oxides, such as nitric oxide, have been shown to regulate neuronal functions, including neurotransmitter release. We investigated the effect of S‐nitroso‐l‐cysteine (SNC) on noradrenaline (NA) release in the rat hippocampus in vivo and in vitro. SNC stimulated [3H]NA release from prelabeled hippocampal slices in a dose‐dependent manner. SNC stimulated endogenous NA release within 30 min to almost five times the basal level in vivo (microdialysis in freely moving rats). In a Na+‐containing Tyrodes buffer, SNC‐stimulated [3H]NA release was inhibited 30% by the coaddition of l‐leucine. In the Na+‐free, choline‐containing buffer, SNC‐stimulated [3H]NA release, which was similar to that in the Na+‐containing buffer, was inhibited markedly by l‐leucine, l‐alanine, l‐methionine, l‐phenylalanine, and l‐tyrosine. The effects of the other amino acids examined were smaller or very limited. The effect of l‐leucine was stronger than that of d‐leucine. A specific inhibitor of the L‐type amino acid transporter, 2‐aminobicyclo[2.2.1]‐heptane‐2‐carboxylate (BCH), inhibited the effects of SNC on [3H]NA release in the Na+‐free buffer. Uptake of l‐[3H]leucine into the slices in the Na+‐free buffer was inhibited by SNC, BCH, and l‐phenylalanine, but not by l‐lysine. The effect of SNC on cyclic GMP accumulation was not inhibited by l‐leucine, although SNC stimulated cyclic GMP accumulation at concentrations up to 25 µM, much less than the concentration that stimulates NA release. These findings suggest that SNC is incorporated into rat hippocampus via the L‐type‐like amino acid transporter, at least in Na+‐free conditions, and that SNC stimulates NA release in vivo and in vitro in a cyclic GMP‐independent manner.

NO donors stimulate noradrenaline release from rat hippocampus in a calmodulin-dependent manner in the presence of L-cysteine

Nitrogen oxides (NO) such as nitric oxide have been suggested to potentiate neurotransmitter release in a variety of neuronal cells. In this study, we showed that NO donors stimulate the release of noradrenaline (NA) from rat hippocampus both in vivo and in vitro. Co‐addition of NO donors (sodium nitroprusside [SNP] or S‐nitroso‐N‐acetylpenicillamine [SNAP]) and thiol compounds (dithiothreitol [DTT] or L‐cysteine) stimulated [3H]NA release from prelabeled hippocampal slices. Microdialysis in freely moving rats was used to ascertain the role of NO in control of NA release from the hippocampus in vivo. Co‐addition of SNAP and L‐cysteine stimulated endogenous NA release within 30 min. The concentration of NA peaked between 30–60 min to almost 3 times basal level. Another thiol compound, glutathione, had no effect on [3H]NA release in the presence of SNP or SNAP. In the presence of SNAP, the effect of L‐cysteine was much higher than that of the D‐isomer, although SNAP did not show stereospecificity. The effect of SNAP/L‐cysteine was rapid and the maximal increase in [3H]NA release was attained 0–1 min after application, which was similar in time course to the effect of KCl. Unlike the release by KCl, SNAP/L‐cysteine‐stimulated NA release was independent of extracellular CaCl2. However, pretreatment with the calmodulin antagonists W‐7 or trifluoperazine significantly reduced the SNAP/L‐cysteine‐stimulated [3H]NA release. Formation of nitric oxide and activation of guanylate cyclase by nitric oxide were not responsible for SNAP/L‐cysteine‐stimulated NA release. These findings suggest that NO donors stimulate NA release from the hippocampus in the presence of thiol compounds such as L‐cysteine in vivo and in vitro in a calmodulin‐dependent, Ca2+‐ and cyclic GMP‐independent manner. The physiological roles of thiol compounds such as L‐cysteine or glutathione as intermediates of NO are discussed.

Effects of R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino) ethoxy]ethanol hydrochloride (T-588), a novel cognitive enhancer, on noradrenaline release in rat cerebral cortical slices

We investigated the effects of R(-)-1-(benzo[b]thiophen-5-yl)-2-[2-(N,N-diethylamino)ethoxy]ethan ol hydrochloride (T-588), a novel cognitive enhancer, on noradrenaline (NA) release from rat cerebral cortical slices in vitro. Addition of T-588 in an assay mixture stimulated [3H]NA release from prelabeled slices in the presence or absence of extracellular CaCl2, and in the presence of the Ca2+/calmodulin antagonists N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide and trifluoperazine. T-588 stimulated NA release with a time lag of about 1 min, and the high level of release was maintained for at least 10 min, whereas maximal KCl-evoked NA release was observed within 1 min after the addition of KCl, and the effect declined subsequently. The effect of T-588 was reversible (pretreatment with T-588 showed no effect on NA release after two washes by centrifugation). We also compared the effects of T-588 and N-ethylmaleimide (NEM), a sulfhydryl alkylating agent known to stimulate neurotransmitter release in several types of cells. The addition of NEM stimulated NA release irreversibly from the slices in a Ca2+-independent manner, and the effect of NEM, but not that of T-588, was inhibited by the simultaneous addition of dithiothreitol, a sulfhydryl group reducing agent. The addition of T-588, which stimulated NA release by itself, inhibited the NA release by 0.6 mM NEM, although the effect of T-588 was additive in the presence of 0.2 mM NEM. These findings suggest that T-588 stimulates NA release from rat cerebral cortical slices in a Ca2+- and calmodulin-independent manner, possibly via an NEM-sensitive factor(s), although the mechanism of the effects of T-588 seems to be different from that of NEM.