Nobuyoshi Nishiyama

3-Hydroxykynurenine, an endogenous oxidative stress generator, causes neuronal cell death with apoptotic features and region selectivity

Abstract: 3‐Hydroxykynurenine (3‐HK) is a potential endogenous neurotoxin whose increased levels have been described in several neurodegenerative disorders. Here, we characterized in vitro neurotoxicity of 3‐HK. Of the tested kynurenine pathway metabolites, only 3‐HK, and to a lesser extent 3‐hydroxyanthranilic acid, were toxic to primary cultured striatal neurons. 3‐HK toxicity was inhibited by various antioxidants, indicating that the generation of reactive oxygen species is essential to the toxicity. 3‐HK‐induced neuronal cell death showed several features of apoptosis, as determined by the blockade by macromolecule synthesis inhibitors, and by the observation of cell body shrinkage with nuclear chromatin condensation and fragmentation. In addition, 3‐HK toxicity was dependent on its cellular uptake via transporters for large neutral amino acids, because uptake inhibition blocked the toxicity. Cortical and striatal neurons were much more vulnerable to 3‐HK toxicity than cerebellar neurons, which may be attributable to the differences in transporter activities of these neurons. These results indicate that 3‐HK, depending on transporter‐mediated cellular uptake and on intracellular generation of oxidative stress, induces neuronal cell death with brain region selectivity and with apoptotic features, which may be relevant to pathology of neurodegenerative disorders.

Mossy fiber Zn2+ spillover modulates heterosynaptic N-methyl-D-aspartate receptor activity in hippocampal CA3 circuits.

Although Zn2+ is contained in large amounts in the synaptic terminals of hippocampal mossy fibers (MFs), its physiological role in synaptic transmission is poorly understood. By using the newly developed high-sensitivity Zn2+ indicator ZnAF-2, the spatiotemporal dynamics of Zn2+ was monitored in rat hippocampal slices. When high-frequency stimulation was delivered to the MFs, the concentration of extracellular Zn2+ was immediately elevated in the stratum lucidum, followed by a mild increase in the stratum radiatum adjacent to the stratum lucidum, but not in the distal area of stratum radiatum. The Zn2+ increase was insensitive to a non–N-methyl-d-aspartate (NMDA) receptor antagonist but was efficiently attenuated by tetrodotoxin or Ca2+-free medium, suggesting that Zn2+ is released by MF synaptic terminals in an activity-dependent manner, and thereafter diffuses extracellularly into the neighboring stratum radiatum. Electrophysiological analyses revealed that NMDA receptor–mediated synaptic responses in CA3 proximal stratum radiatum were inhibited in the immediate aftermath of MF activation and that this inhibition was no longer observed in the presence of a Zn2+-chelating agent. Thus, Zn2+ serves as a spatiotemporal mediator in imprinting the history of MF activity in contiguous hippocampal networks. We predict herein a novel form of metaplasticity, i.e., an experience-dependent non-Hebbian modulation of synaptic plasticity.

Autosomal dominant guanosine triphosphate cyclohydrolase I deficiency (Segawa disease).

Autosomal dominant guanosine triphosphate cyclohydrolase I (GCH‐I) deficiency (Segawa disease) is a dopa‐responsive dystonia caused by mutation of the GCH‐I gene located on 14q22.1‐q22.2. Neurohistochemical examination revealed a decrease of the tyrosine hydroxylase protein as well as its activity in the striatum and decrease of dopamine content, particularly in its ventral portion rich in D1 receptors (striatal direct pathways). Neuroimaging, clinical neurophysiological, and biochemical studies showed preservation of the structure and function of the terminal of the nigrostriatal DA neuron. Clinical neurophysiological studies showed no progressive decrement of DA activities. As the enzymatic activity of pteridine metabolism is highest in the early developmental course, it may modulate dopamine receptors maturing early in the developmental course. Its product, tetrahydrobiopterin, has higher affinity to tyrosine hydroxylase among hydroxylases. Thus, partial deficiency of tetrahydrobiopterin caused by heterozygous mutation of the GCH‐I gene decreases dopamine activity rather selectively. This affects the DA receptors that mature early and demonstrates characteristic symptoms age‐dependently along with the developmental decrement of the tyrosine hydroxylase activities at the terminals and the maturational processes of the projecting neurons of the basal ganglia. A difference in the ratio of mutant/wild‐type GCH‐I mRNA that depends on the locus of mutation may explain intrafamilial and interfamilial variation of phenotype. Ann Neurol 2003;54 (suppl 6):S32–S45

Generation and Characterization of Mutant Mice Lacking Ryanodine Receptor Type 3

The ryanodine receptor type 3 (RyR-3) functions as a Ca2+-induced Ca2+ release (CICR) channel and is distributed in a wide variety of cell types including skeletal muscle and smooth muscle cells, neurons, and certain non-excitable cells. However, the physiological roles of RyR-3 are totally unclear. To gain an insight into the function of RyR-3 in vivo, we have generated mice lacking RyR-3 by means of the gene targeting technique. The mutant mice thus obtained showed apparently normal growth and reproduction. Although Ca2+-induced Ca2+ release from intracellular Ca2+ stores of the mutant skeletal muscle differed in Ca2+ sensitivity from that of wild-type muscle, excitation-contraction coupling of the mutant muscle seemed to be normal. Moreover, we could not find any significant disturbance in the smooth muscle and lymphocytes from the mutant mice. On the other hand, the mutant mice showed increased locomotor activity, which was about 2-fold greater than that of the control mice. These results indicate that the loss of RyR-3 causes no gross abnormalities and suggest that the lack of RyR-3-mediated Ca2+ signaling results in abnormalities of certain neurons in the central nervous system.

Neurotrophic factor gene expression in astrocytes during development and following injury.

Astrocyte cultures were utilized to examine regulation of the expression of several trophic factor genes. Regulation by the beta-adrenergic receptor was demonstrated by exposure of striatal and cortical astrocytes to isoproterenol, which resulted in increased content of mRNAs for nerve growth factor (NGF), brain-derived neurotrophic factor, and proenkephalin (PE), as well as NGF and Met-enkephalin. Developmental regulation was analyzed by preparing cortical astrocytes from animals of four different ages--embryonic day 20, postnatal days 3 and 8, and adult. Because both the PE and NGF genes showed developmental downregulation, we asked whether we could prepare reactive astrocytes from lesioned adult brain and see expression turned back on. Astrocytes prepared from 6-hydroxydopamine-lesioned rat striatum or MPTP-lesioned mouse striatum contained increased GFAP and NGF mRNA. Comparable changes in GFAP and NGF could be achieved by treatment of control cultures with interferon-gamma or interleukin-1 beta. These results suggest that locus coeruleus neurons could control astrocyte synthesis of neurotrophic factors through release of of norepinephrine, but that in injured brain other factors, such as cytokines, may become equally important.

Effect of basic fibroblast growth factor on neurons cultured from various regions of postnatal rat brain

Neurons from various brain regions of postnatal (15 days after birth) and fetal (16 days gestation) rats were cultured in the presence of basic fibroblast growth factor (bFGF). bFGF increased the survival of neurons from postnatal septum, striatum, midbrain, and hippocampus. Fetal neurons derived from cerebral cortex, septum, striatum, midbrain, thalamus, and colliculus were far more dependent on bFGF for survival in comparison with postnatal neurons. In contrast, cerebellum neurons of postnatal and fetal rat brain did not respond to bFGF. The increase of postnatal and fetal neuronal survival with bFGF treatment (0.01-10 ng/ml) was dose-dependent and reached 2-4-fold and 5-10-fold more than the control, respectively. Fetal cortical neurons showed almost complete dependence on bFGF since almost all neurons died in control cultures. Nerve growth factor was slightly effective only on postnatal septal and striatal neurons, being ineffective on the other neurons tested. These results indicate that bFGF can function as a neurotrophic factor not only on fetal but also on postnatal neurons of the central nervous system, and that bFGF has great potential for application in vivo.

Docosahexaenoic acid improves long-term potentiation attenuated by phospholipase A2 inhibitor in rat hippocampal slices

We investigated the possible involvement of phospholipase A2 (PLA2) and its products in long‐term potentiation (LTP) in the CA1 neurotransmission of rat hippocampal slices. Inhibitors of Ca2+‐independent PLA2 (iPLA2) prevented the induction of LTP without affecting the maintenance phase of LTP whereas Ca2+‐dependent PLA2 inhibitors were virtually ineffective, which suggests a pivotal role of iPLA2 in the initiation of LTP. We then investigated the effect of docosahexaenoic acid (DHA) and arachidonic acid (AA) on BEL (bromoenol lactone, an iPLA2‐inhibitor) ‐impaired LTP, and found that either DHA or AA abolished the effect of BEL. However, DHA did not restore BEL‐attenuated LTP when applied after the tetanus. DHA per se affected neither the induction nor maintenance of LTP. Linoleic acid had no effects, either. These results suggest that DHA is crucial for the induction of LTP and that endogenously released DHA during tetanus is sufficient to trigger the formation of LTP.

Activin exerts a neurotrophic effect on cultured hippocampal neurons.

Activin is a member of the transforming growth factor (TGF)-beta superfamily, which comprises a growing list of multifunctional proteins that serve as regulators of cell proliferation and differentiation. Recently, activin was shown to regulate the neurotransmitter phenotype in peripheral neurons. It is also a potent survival factor for neurogenic clonal cell lines, retinal neurons and midbrain dopaminergic neurons. We have studied the effect of activin on hippocampal cells which show abundant expression of activin receptors or binding sites. Exposure of primary cultures of rat hippocampal neurons to activin supported neuronal survival. This neurotrophic action of activin was blocked by treatment with the tyrosine kinase inhibitor genistein or the protein kinase C inhibitor calphostin C. However, the Ca2+/calmodulin kinase inhibitor KN-62 had no effect. Nicardipine, a blocker of the L-type Ca2+ channel, also inhibited the neurotrophic effect of activin. Furthermore, activin potentiated the depolarization-induced elevation in intracellular Ca2+ concentration ([Ca2+]i). The neurotrophic effect and the potentiation of depolarization-induced increase of [Ca2+]i caused by activin were completely abolished by the protein synthesis inhibitor cycloheximide. These results suggest that activin supports neuronal survival by increasing the expression of voltage-dependent Ca2+ channel through the action of a tyrosine kinase and of protein kinase C, but not of Ca2+/calmodulin kinase.

Phospholipase A2 mediates ischemic injury in the hippocampus: a regional difference of neuronal vulnerability.

Although it is well known that the hippocampal CA1 subfield is highly vulnerable to ischemic injury, cellular mechanisms leading to this neuronal degeneration are not fully understood. Using organotypic cultures of rat hippocampal slices, we determined whether phospholipase A2 (PLA2) is activated in response to ischemic conditions (OGD; oxygen and glucose deprivation). The PLA2 activity in the pyramidal cell layer increased immediately following a 35‐min exposure to OGD, which was likely to be mediated by selective activation of cytosolic Ca2+‐dependent PLA2 subtype (cPLA2). This enhancement lasted for at least 24 h. Interestingly, no apparent increase was detected in the dentate gyrus. Twenty‐four hours after the OGD exposure, neuronal death was detected mainly in the CA1 region of hippocampal slices. To examine whether the PLA2 activation is causally or protectively involved in the ischemic injury, we investigated the effect of pharmacological blockade of PLA2 on the OGD‐induced neuronal death. The PLA2 inhibitor bromophenacyl bromide efficiently prevented the cell death in a concentration‐dependent manner. Similar results were obtained for the selective cPLA2 inhibitor AACOCF3. However, the Ca2+‐independent PLA2 inhibitor bromoenol lactone and the secretory PLA2 inhibitor LY311727 were virtually ineffective. These results suggest that cPLA2 plays a causative role in the neuronal death following OGD exposure. Thus, the present study may provide novel therapeutic targets for the development of neuroprotective agents.

ANTI‐AGEING EFFECT OF AGED GARLIC EXTRACT IN THE INBRED BRAIN ATROPHY MOUSE MODEL

1. The effects of chronically administered aged garlic extract (AGE) on the age‐related changes in a novel strain of senescence accelerated mouse (SAM) characterized by age‐related brain atrophy (SAMP10) were investigated.