Hidenobu Tanaka

Late Calcium EDTA Rescues Hippocampal CA1 Neurons from Global Ischemia-Induced Death

Transient global ischemia induces a delayed rise in intracellular Zn2+, which may be mediated via glutamate receptor 2 (GluR2)-lacking AMPA receptors (AMPARs), and selective, delayed death of hippocampal CA1 neurons. The molecular mechanisms underlying Zn2+ toxicity in vivo are not well delineated. Here we show the striking finding that intraventricular injection of the high-affinity Zn2+ chelator calcium EDTA (CaEDTA) at 30 min before ischemia (early CaEDTA) or at 48-60 hr (late CaEDTA), but not 3-6 hr, after ischemia, afforded robust protection of CA1 neurons in ∼50% (late CaEDTA) to 75% (early CaEDTA) of animals. We also show that Zn2+ acts via temporally distinct mechanisms to promote neuronal death. Early CaEDTA attenuated ischemia-induced GluR2 mRNA and protein downregulation (and, by inference, formation of Zn2+-permeable AMPARs), the delayed rise in Zn2+, and neuronal death. These findings suggest that Zn2+ acts at step(s) upstream from GluR2 gene downregulation and implicate Zn2+ in transcriptional regulation and/or GluR2 mRNA stability. Early CaEDTA also blocked mitochondrial release of cytochrome c and Smac/DIABLO (second mitochondria-derived activator of caspases/direct inhibitor of apoptosis protein-binding protein with low pI), caspase-3 activity (but not procaspase-3 cleavage), p75NTR induction, and DNA fragmentation. These findings indicate that CaEDTA preserves the functional integrity of the mitochondrial outer membrane and arrests the caspase death cascade. Late injection of CaEDTA at a time when GluR2 is downregulated and caspase is activated inhibited the delayed rise in Zn2+, p75NTR induction, DNA fragmentation, and cell death. The finding of neuroprotection by late CaEDTA administration has striking implications for intervention in the delayed neuronal death associated with global ischemia.

Ischemic Preconditioning: Neuronal Survival in the Face of Caspase-3 Activation

Apoptosis is an evolutionarily conserved process critical to tissue development and tissue homeostasis in eukaryotic organisms and, when dysregulated, causes inappropriate cell death. Global ischemia is a neuronal insult that induces delayed cell death with many features of apoptosis. Ischemic preconditioning affords robust protection of CA1 neurons against a subsequent severe ischemic challenge. The molecular mechanisms underlying ischemic tolerance are unclear. Here we show that ischemia induces pronounced caspase-3 activity in naive neurons that die and in preconditioned neurons that survive. Preconditioning intervenes downstream of proteolytic processing and activation of caspase-3 (a protease implicated in the execution of apoptosis) and upstream of the caspase-3 target caspase-activated DNase (CAD, a deoxyribonuclease that catalyzes DNA fragmentation) to arrest neuronal death. We further show that global ischemia promotes expression of the pro-survival inhibitor-of-apoptosis (IAP) family member cIAP, but unleashes Smac/DIABLO (second mitochondria-derived activator of caspases/direct IAP-binding protein with low pI), a factor that neutralizes the protective actions of IAPs and promotes neuronal death. Preconditioning blocks the mitochondrial release of Smac/DIABLO, but not the ischemia-induced upregulation of IAPs. In the absence of Smac/DIABLO, cIAP halts the caspase death cascade and arrests neuronal death. These findings suggest that preconditioning preserves the integrity of the mitochondrial membrane, enabling neurons to survive in the face of caspase activation.

Reaction of astrocytes in the gerbil hippocampus following transient ischemia : immunohistochemical observations with antibodies against glial fibrillary acidic protein, glutamine synthetase, and S-100 protein

An immunohistochemical method was used to study the distribution and changes with time of the astrocytic reaction in the gerbil hippocampus following transient ischemia. Three markers were investigated with specific antibodies to glial fibrillary acidic protein (GFAP), glutamine synthetase (GS), and S-100 protein. On Day 2 after ischemia, and more prominently on Day 3, reactive astrocytes were intensely stained for GFAP in the hippocampal formation, especially in the CA1 region and dentate gyrus. This response by astrocytes preceded CA1 pyramidal cell degeneration, which became apparent on Day 5. On Day 5, immunoreactive cells were not stained as intensely as on Day 3, but cells in the CA1 region and dentate gyrus were still more intensely stained than those in normal animals. GS and S-100 showed similar changes in distribution after ischemia, although the change in GS was less prominent: the hilus of the dentate gyrus was most intensely stained. Both immunoreactivities seemed to increase rather transiently on Day 2 or 3 and to decrease to the initial level on Day 5. The fact that reactive astrocytes appeared in CA1 before the onset of visible neural degeneration indicates that signals from indisposed neurons may be transmitted to astrocytes for their quick functioning. It is also suggested that degenerative changes occur in the dentate gyrus and may be involved in the delayed neural death of CA1 pyramidal cells. These observations indicate that astrocytes play a role in the neural degeneration induced by ischemia and that several types of astrocytes seem to react differently.

Disturbance of hippocampal long-term potentiation after transient ischemia in GFAP deficient mice

GFAP (glial fibrillary acidic protein) is an intermediate filament protein found exclusively in the astrocytes of the central nervous system. We studied the role of GFAP in the neuronal degeneration in the hippocampus after transient ischemia using knockout mice. Wild‐type C57 Black/6 (GFAP+/+) mice and mutant (GFAP−/−) mice were subjected to occlusion of both carotid arteries for 5–15 min. Hippocampal slices were prepared 3 days after reperfusion and the field excitatory postsynaptic potentials (fEPSP) in the CA1 were recorded. High frequency stimulation induced robust long‐term potentiation (LTP) in GFAP−/−, as in GFAP+/+ mice. After ischemia, however, the LTP in GFAP−/− was significantly depressed. Similarly, paired pulse facilitation (PPF) displayed little difference between GFAP+/+ and GFAP−/−, but after ischemia, the PPF in GFAP−/− showed a depression. Histological study revealed that loss of CA1 and CA3 pyramidal neurons after ischemia was marked in GFAP−/−. MAP2 (dendritic) immunostaining in the post‐ischemic hippocampus showed little difference but NF200 (axonal) immunoreactivity was reduced in GFAP−/−. S100β (glial) immunoreactivity was similar in the post‐ischemic hippocampus of the GFAP+/+ and GFAP−/−, indicating that reactive astrocytosis did not require GFAP. Our results suggest that GFAP has an important role in astrocyte‐neural interactions and that ischemic insult impairs LTP and accelerates neuronal death.

The AMPAR subunit GluR2: still front and center-stage11Published on the World Wide Web on 30 October 2000.

Abnormal influx of Ca(2+) through AMPA-type glutamate receptors (AMPARs) is thought to contribute to the neuronal death associated with a number of brain disorders. AMPARs exist as both Ca(2+)-impermeable and Ca(2+)-permeable channels. AMPARs are encoded by four genes designated GluR1 (GluR-A) through GluR4 (GluR-D). The presence of the GluR2 subunit renders heteromeric AMPA receptor assemblies Ca(2+)-impermeable. Molecular diversity of AMPARs under physiological and pathological conditions is generated by differential spatio-temporal patterns of GluR expression, by alternative RNA splicing and editing and by targeting and trafficking of receptor subunits at dendritic spines. The GluR2 gene is under transcriptional control by the RE1 element specific transcription factor, a gene silencing factor which renders it neuron-specific. GluR2 transcripts are edited by ADAR2 (double-stranded RNA-specific editase 1). AMPAR targeting and trafficking to spines are regulated by synaptic activity and are critical to synaptic plasticity. Recent studies involving animal models of transient forebrain ischemia and epilepsy show that GluR2 mRNA and GluR2 subunit expression are downregulated in vulnerable neurons prior to cell death. Ca(2+) imaging and electrical recording from individual pyramidal neurons in hippocampal slices reveal changes in AMPAR functional properties after ischemia. In slices from post-ischemia animals, CA1 neurons with robust action potentials exhibit greatly enhanced AMPA-elicited rises in intracellular Ca(2+). Excitatory postsynaptic currents in post-ischemic CA1 exhibit an enhanced Ca(2+)-dependent component that appears to be mediated by Ca(2+)-permeable AMPARs. These studies provide evidence for Ca(2+) influx through AMPARs in neurons destined to die. To examine whether acute GluR2 downregulation, even in the absence of a neurological insult, can induce neuronal death, we performed knockdown experiments in rats and gerbils with antisense oligonucleotides targeted to GluR2 mRNA. GluR2 antisense oligonucleotide induced neuronal cell death of pyramidal neurons and enhanced pathogenicity of brief ischemic episodes. These observations provide evidence for Ca(2+) influx through AMPARs in neurons destined to die and implicate Ca(2+)-permeable AMPARs in the pathogenesis of ischemia-induced neuronal death.