Kuniko Shimazaki

Increase in bcl-2 oncoprotein and the tolerance to ischemia-induced neuronal death in the gerbil hippocampus

We studied the expression of bcl-2 oncoprotein in the gerbil hippocampus after transient ischemia. Immunostaining using monoclonal antibody raised against bcl-2 oncoprotein revealed intense immunoreactivity in the CA1 area following 2 min of ischemia, which induced tolerance to subsequent ischemia and prevented delayed neuronal death (DND). Following ischemia for 5 min, however, bcl-2 oncoprotein immunoreactivity was decreased, reflecting neuronal death in the CA1 area. However, pretreatment with ischemia of 2 min that prevented DND due to subsequent ischemia for 5 min, showed increased immunoreactivity. On the other hand, following 1 min of ischemia which failed to induce tolerance, no increase in the bcl-2 oncoprotein was observed. The results evidenced that expression of bcl-2 oncoprotein in the CA1 area following brief ischemia is closely related to the acquisition of resistance to DND.

Adeno-associated virus vector-mediated bcl-2 gene transfer into post-ischemic gerbil brain in vivo: prospects for gene therapy of ischemia-induced neuronal death.

The proto-oncogene bcl-2 is known as an anti-apoptotic gene that confers the ability to block neuronal cell death after transient ischemia. In order to examine whether the bcl-2 gene can be used for protection of ischemic brain injury, we generated adeno-associated virus (AAV) vectors capable of expressing human bcl-2. Replication-defective AAV vectors were found effectively to transfer and express bcl-2 gene in the gerbil hippocampal neurons. Transduction with AAV bcl-2 5 days before forebrain ischemia prevented the DNA fragmentation in the CA1 neurons that is commonly associated with ischemia-induced cell death. Furthermore, the application of AAV bcl-2 as late as 1 h following an ischemic insult also prevented DNA fragmentation in CA1 neurons. These results suggest that the bcl-2 protein has neuroprotective functions that inhibit ischemic cell death and demonstrate the potential of AAV bcl-2 for use in post-ischemic gene therapy in the brain.

Antagonism of Sphingosine 1-Phosphate Receptor-2 Enhances Migration of Neural Progenitor Cells Toward an Area of Brain Infarction

Background and Purpose— We have previously shown that the sphingosine 1-phosphate (S1P)/S1P receptor-1 (S1P1R) axis contributes to the migration of transplanted neural progenitor cells (NPCs) toward areas of spinal cord injury. In the current study, we examined a strategy to increase endogenous NPC migration toward the injured central nervous system to modify S1PR. Methods— S1P concentration in the ischemic brain was measured in a mouse thrombosis model of the middle cerebral artery. NPC migration in vitro was assessed by a Boyden chamber assay. Endogenous NPC migration toward the insult was evaluated after ventricular administration of the S1P2R antagonist JTE-013. Results— The concentration of S1P in the brain was increased after ischemia and was maximal 14 days after the insult. The increase in S1P in the infarcted brain was primarily caused by accumulation of microglia at the insult. Mouse NPCs mainly expressed S1P1R and S1P2R as S1PRs, and S1P significantly induced the migration of NPCs in vitro through activation of S1P1R. However, an S1P1R agonist failed to have any synergistic effect on S1P-mediated NPC migration, whereas pharmacologic or genetic inhibition of S1P2R by JTE-013 or short hairpin RNA expression enhanced S1P-mediated NPC migration but did not affect proliferation and differentiation. Interestingly, administration of JTE-013 into a brain ventricle significantly enhanced endogenous NPC migration toward the area of ischemia. Conclusions— Our findings suggest that S1P is a chemoattractant for NPCs released from an infarcted area and regulation of S1P2R function further enhances the migration of NPCs toward a brain infarction.

Effects of a spider toxin (JSTX) on hippocampal CA1 neurons in vitro

The effect of a toxin (JSTX) obtained from Nephila clavata (Joro spider) on the CA1 pyramidal neurons of the hippocampus was studied using slice preparations. JSTX blocked the excitatory postsynaptic potentials (EPSPs) in the pyramidal neuron evoked by Schaffer collateral stimulation but was without effect on the antidromic action potentials or on the resting conductance. Depolarization induced by ionophoretic application of glutamate was readily suppressed by JSTX but aspartate-induced depolarization was much less sensitive to the toxin. Among preferential agonists activating 3 receptor subtypes for excitatory amino acids, quisqualate responses were most effectively suppressed by JSTX. Kainate responses were similarly suppressed but in some cells higher concentration of the toxin was needed to block the responses. N-methyl-D-aspartate (NMDA) responses were the least sensitive to JSTX but they were suppressed by +/- 2-amino-5-phosphonovaleric acid (APV). Long term potentiation (LTP) once it had taken place was not completely inhibited by APV. In the presence of JSTX, however, LTP was blocked and tetanic stimuli produced only a short-lived potentiation. In Mg2+ free solution, an orthodromic stimulation evoked repetitive spike responses which were superimposed on the depolarization following the initial spike. APV suppressed the depolarization and associated spikes leaving an orthodromic response which was sensitive to JSTX. The results suggest that JSTX blocks EPSPs in CA1 pyramidal neurons which are mediated by non-NMDA type receptors.

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.

Reduced calcium elevation in hippocampal Ca1 neurons of ischemia-tolerant gerbils

TRANSIENT forebrain ischemia causes selective neuronal death in the hippocampal CA1 neurons. A short sublethal ischemic episode preceding ischemia of longer duration is known to increase tolerance against cell death. The mechanisms of this ischemic tolerance are still poorly understood. Here we show, using Ca2+ imaging, that intracellular calcium ([Ca2+]i) elevation in CA1 neurons after an anoxic-aglycemic episode is markedly inhibited in the ischemia-tolerant gerbil. The hippocampus of gerbils which did not acquire tolerance showed a high [Ca2+]i elevation during the anoxicaglycemic episode, similar to controls. Since hypoxia/ischemia-induced neurodegeneration can be triggered by cytoplasmic Ca2+ overload, the tolerant gerbil may regulate calcium and keep [Ca2+]i below the critical level for initiating neuronal death. NeuroReport 9: 1875–1878

Rescue of amyotrophic lateral sclerosis phenotype in a mouse model by intravenous AAV9-ADAR2 delivery to motor neurons

Amyotrophic lateral sclerosis (ALS) is the most common adult‐onset motor neuron disease, and the lack of effective therapy results in inevitable death within a few years of onset. Failure of GluA2 RNA editing resulting from downregulation of the RNA‐editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) occurs in the majority of ALS cases and causes the death of motor neurons via a Ca2+‐permeable AMPA receptor‐mediated mechanism. Here, we explored the possibility of gene therapy for ALS by upregulating ADAR2 in mouse motor neurons using an adeno‐associated virus serotype 9 (AAV9) vector that provides gene delivery to a wide array of central neurons after peripheral administration. A single intravenous injection of AAV9‐ADAR2 in conditional ADAR2 knockout mice (AR2), which comprise a mechanistic mouse model of sporadic ALS, caused expression of exogenous ADAR2 in the central neurons and effectively prevented progressive motor dysfunction. Notably, AAV9‐ADAR2 rescued the motor neurons of AR2 mice from death by normalizing TDP‐43 expression. This AAV9‐mediated ADAR2 gene delivery may therefore enable the development of a gene therapy for ALS.

Adeno-associated virus vectors for gene transfer to the brain

Gene therapy is a novel method under investigation for the treatment of neurological disorders. Considerable interest has focused on the possibility of using viral vectors to deliver genes to the central nervous system. Adeno-associated virus (AAV) is a potentially useful gene transfer vehicle for neurologic gene therapies. The advantages of AAV vector include the lack of any associated disease with a wild-type virus, the ability to transduce nondividing cells, the possible integration of the gene into the host genome, and the long-term expression of transgenes. The development of novel therapeutic strategies for neurological disorder by using AAV vector has an increasing impact on gene therapy research. This article describes methods that can be used to generate rodent and nonhuman primate models for testing treatment strategies linked to pathophysiological events in the ischemic brain and neurodegenerative disorders such as Parkinsons disease.

Protection Against Aminoglycoside-induced Ototoxicity by Regulated AAV Vector–mediated GDNF Gene Transfer Into the Cochlea

Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s-S2 Tet-on regulation system were directly microinjected into the rat cochleae through the round window at 5 × 1010 genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulated expression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.Since standard aminoglycoside treatment progressively causes hearing disturbance with hair cell degeneration, systemic use of the drugs is limited. Adeno-associated virus (AAV)-based vectors have been of great interest because they mediate stable transgene expression in a variety of postmitotic cells with minimal toxicity. In this study, we investigated the effects of regulated AAV1-mediated glial cell line-derived neurotrophic factor (GDNF) expression in the cochlea on aminoglycoside-induced damage. AAV1-based vectors encoding GDNF or vectors encoding GDNF with an rtTA2s-S2 Tet-on regulation system were directly microinjected into the rat cochleae through the round window at 5 x 10(10) genome copies/body. Seven days after the virus injection, a dose of 333 mg/kg of kanamycin was subcutaneously given twice daily for 12 consecutive days. GDNF expression in the cochlea was confirmed and successfully modulated by the Tet-on system. Monitoring of the auditory brain stem response revealed an improvement of cochlear function after GDNF transduction over the frequencies tested. Damaged spiral ganglion cells and hair cells were significantly reduced by GDNF expression. Our results suggest that AAV1-mediated expression of GDNF using a regulated expression system in the cochlea is a promising strategy to protect the cochlea from aminoglycoside-induced damage.

Development expression of the neural adhesion molecule F3 in the rat brain

We cloned a cDNA encoding rat F3 and analyzed the nucleotide sequences. The results have shown that rat F3 is comprised of 1021 amino acid residues. It shared 99% and 76% identities with mouse and chicken homologs, respectively, at the amino acid sequence level. During postnatal development of the rat brain, cells expressing F3 mRNA appeared in the cortex, hippocampus, superior and inferior colliculi, anterior olfactory nucleus, olfactory bulb and cerebellum, whereas little was observed at postnatal day 1 (P1). Extraordinarily high expression of F3 mRNA was observed in the cerebral cortical neurons of layer 5 at P7. The number of cells with high expression of F3 mRNA expanded to the entire region of the cerebral cortex at P14. The whole cerebrum displayed expression at P90 in which the cortex still showed the highest expression level, although the overall signals were weak in comparison with those at P14. In the hippocampal formation, F3-expressing granule cells of the dentate gyrus were restricted to the outer aspect, then expanded to the inner aspect during development. Finally the granule cells in the entire region of the dentate gyrus transcribed F3 mRNA. We discuss the significance of the expression pattern of F3 mRNA during development.