Original article
Antisense in vivo knockdown of synaptotagmin I by HVJ–liposome mediated gene transfer attenuates ischemic brain damage in neonatal rats

https://doi.org/10.1016/j.braindev.2007.08.002Get rights and content

Abstract

Synaptic release of the excitatory amino acid glutamate is considered as an important mechanism in the pathogenesis of ischemic brain damage in neonates. Synaptotagmin I is one of exocytosis-related proteins at nerve terminals and considered to accelerate the exocytosis of synaptic vesicles by promoting fusion between the vesicles and plasma membrane. To test the possibility that antisense in vivo knockdown of synaptotagmin I modulates the exocytotic release of glutamate, thus suppressing the excitotoxic intracellular processes leading to neuronal death following ischemia in the neonatal brain, we injected antisense oligodeoxynucleotides (ODNs) targeting synaptotagmin I (0.3 (AS), 0.15 (0.5 AS), or 0.03 μg (0.1 AS), or vehicle) into the lateral ventricles of 7-day-old rats by using a hemagglutinating virus of Japan (HVJ)–liposome mediated gene transfer technique. At 10 days of age, these rats were subjected to an electrical coagulation of the right external and internal carotid arteries, then the insertion of a solid nylon thread into the right common carotid artery toward the ascending aorta up to 10–12 mm from the upper edge of the sternocleidomastoid muscle. Cerebral ischemia was induced by clamping the left external and internal carotid arteries with a clip, and ended by removing the clip 2 h later. Twenty-four hours after the end of ischemia, the extent of ischemic brain damage was neuropathologically and quantitatively evaluated in the neocortex and striatum. While the relative volume of damage in the cerebral cortex and striatum of the vehicle group was extended to 40% and 13.7%, respectively, that in the AS group was significantly reduced to 4.8% and 0.6%. In the 0.5 AS group, the relative volume of ischemic damage in the cerebral cortex and striatum was reduced to 20.5% and 15.4%, respectively, and the difference between the 0.5 AS group and vehicle group was statistically significant in the neocortex, but not in the striatum. These results indicated that antisense in vivo knockdown of synaptotagmin I successfully attenuated ischemic brain damage in neonatal rats and that the effect was dose-dependent. It was also suggested that this treatment was more effective in the neocortex than in the striatum 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)

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