Original articleAntisense in vivo knockdown of synaptotagmin I by HVJ–liposome mediated gene transfer attenuates ischemic brain damage in neonatal rats
Introduction
Hypoxic–ischemic brain damage in neonates is a problem of an enormous importance. Synaptic release of the excitatory amino acid glutamate during and after an ischemic event is considered very important to the pathogenesis of brain damage. The extracellular glutamate concentration in vivo increases many fold with hypoxic–ischemic insults. The increased amount of extracellular glutamate excessively activates glutamate-receptors and calcium overload, and may promote ischemic neuronal death. However, it is not clear whether the synaptic release of glutamate at nerve terminals is most responsible for the hypoxic–ischemic brain damage resulting from neuronal death. Furthermore, although the extracellular overflow of glutamate has been documented in neonatal animal models as well as adults, the increases seem to be less extensive in neonates [1], [2], [3].
In a variety of perinatal hypoxic–ischemic models, treatments with glutamate-receptor channel blockers have been tried to protect against neuronal death. Recent studies have shown major problems with glutamate-receptor antagonists [4], [5]. Since modulation of presynaptic release would affect all the synapses, the antisense knockdown of proteins involved in the release of transmitters is expected to minimize treatment-induced disruption of the interaction between multiple chemical transmitter systems. Such an approach seems to be a promising way to develop effective treatments for ischemic brain damage with less side-effects.
Among exocytosis-related proteins that are abundant at nerve terminals, synaptotagmin I is considered to regulate the exocytosis of synaptic vesicles as a major Ca2+ sensor by promoting fusion between the vesicles and plasma membrane via the assembly and clustering of the SNARE complex [6], [7], [8], [9], [10]. It is postulated that the antisense in vivo knockdown of synaptotagmin I would reduce rates of the exocytotic release of transmitters at the synaptic terminals. We have already demonstrated in adult rats that the knockdown of synaptotagmin I prevented amygdaloid seizure-induced damage in the hippocampus [11] and attenuate ischemic damageto the hippocampus [12] and that the nigral injection of antisense oligonucleotides targeting synaptotagmin I successfully disrupted the release of dopamine in the striatum [13]. However, there is no study demonstrating in neonatal animals that antisense in vivo knockdown of synaptotagmin I modulates the exocytotic release of glutamate, thus suppressing the excitotoxic intracellular processes leading to neuronal death. To test the relevance of the ‘transmitter release strategy’ for neuroprotection against ischemic brain damage in neonates, we injected antisense oligodeoxynucleotides (ODNs) against synaptotagmin I into the lateral ventricles using a hemagglutinating virus of Japan (HVJ)–liposome mediated gene transfer technique prior to a transient forebrain ischemia in neonatal rats [14] and examined whether the knockdown of synaptotagmin I in the whole brain could regulate ischemia-induced neuronal death. To achieve a long-lasting downregulation of synaptotagmin I by a single treatment with the antisense ODNs, we used a novel transfection vector (HVJ–liposome) [15], [16], [17]. It has been demonstrated that using this HVJ–liposome method oligodeoxynucleotides can be efficiently delivered into neurons, predominantly in cell nuclei, both in vitro and in vivo [19].
Section snippets
Animal care
Neonatal rats (Wistar strain) used in this study were housed in clear plastic cages with their dams, which were allowed free access to food and water throughout the experiment. The animals were maintained in a temperature-, humidity-, and light-controlled environment with a 12 h light/dark cycle.
All experiments were performed in accordance with the Japanese and International Guidelines on the ethical use of animals, and every effort was made to minimize the number of animals and their suffering.
Results
No animals died before the ischemic manipulation, even though, except in the non-treated group, they received the intraventricular injection. After the ischemic event, 3 of 15 pups (20%) in the non-treated group died. Similarly, the proportion that died during the recovery period was 5 of 17 (29%) in the vehicle group, 6 of 18 (33.3%) in the 0.1 AS group, 4 of 16 (25%) in the 0.5 AS group, and 4 of 16 (25%) in the AS group. These rates did not differ significantly between groups.
As shown in
Discussion
The release of neurotransmitters from a nerve terminal is mediated by the exocytosis of synaptic vesicles. After the release, synaptic vesicles are endocytosed, refilled, and prepared for subsequent rounds of release at nerve terminals. Synaptotagmin I, a synaptic vesicle protein, is a key regulator of Ca2+-dependent exocytosis. There is evidence supporting the involvement of synaptotagmin I in Ca2+-dependent transmitter release: a microinjection of synaptotagmin antibodies or synthetic
Acknowledgement
We are grateful to Dr. Yasufumi Kaneda at Osaka University Medical School for kindly teaching the HVJ–liposome mediated gene transfer technique.
References (25)
- et al.
Brain injury after hypoxia–ischemia in newborn rats: relationship to extracellular levels of excitatory amino acids and cysteine
Brain Res
(1997) - et al.
Clinical trials with neuroprotective drugs in acute ischaemic stroke: are we doing the right thing?
Trends Neurosci
(1999) - et al.
Neuroprotection by the NMDA receptor-associated open-channel blocker memantine in a photothrombotic model of cerebral focal ischemia in neonatal rat
Eur J Phamacol
(1999) - et al.
Mutational analysis of Drosophila synaptotagmin demonstrates its essential role in Ca(2+)-activated neurotransmitter release
Cell
(1993) - et al.
Synaptic function is impaired but not eliminated in C. elegans mutants lacking synaptotagmin
Cell
(1993) - et al.
Synaptotagmin I: a major Ca2+ sensor for transmitter release at a central synapse
Cell
(1994) - et al.
Synaptotagmin I hypothalamic knockdown prevents amygdaloid seizure-induced damage of hippocampal neurons but not of entorhinal neurons
Neurosci Res
(2002) - et al.
Antisense in vivo knockdown of synaptotagmin I and synapsin I by HVJ–liposome mediated gene transfer modulates ischemic injury of hippocampus in opposing ways
Neurosci Res
(2003) - et al.
Nigral injection of antisense oligonucleotides to synaptotagmin I using HVJ–liposome vectors causes disruption of dopamine release in the striatum and impaired skill learning
Brain Res
(2006) - et al.
Pharmacokinetics of antisense oligodeoxyribonucleotides (cyclin B1 and CDC 2 kinase) in the vessel wall in vivo: enhanced therapeutic utility for restenosis by HVJ–liposome delivery
Gene
(1994)
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