Antisense in vivo knockdown of synaptotagmin I and synapsin I by HVJ-liposome mediated gene transfer modulates ischemic injury of hippocampus in opposing ways
Introduction
The synaptic release of excitatory amino acid glutamate following an ischemic insult is thought to be an important factor in the development of neuronal death. Surgical removal of glutamatergic afferent fibers to the hippocampus prevented the ischemia-induced damage of CA1 pyramidal neurons in the hippocampus (Wieloch et al., 1985; Onodera et al., 1986; Jorgensen et al., 1987; Buchan and Pulsinelli, 1990). Furthermore, AMPA receptor antagonist but not NMDA receptor antagonist markedly attenuated the ischemic cell death of CA1 pyramidal neurons, even when administrated hours after an ischemic insult (Sheardown et al., 1990, Nellgard and Wieloch, 1992; Sheardown et al., 1993). The neuroprotection afforded by surgical transection of glutamate containing afferents to the CA1 neurons and postischemic pharmacological blockade of glutamate receptors strongly supports the notion that a major determinant of the glutamate receptor-mediated excitotoxicity in ischemic brain injury may be excitatory transmitter glutamate released from synaptic terminals in a stimulus-dependent manner after ischemic insult (Benveniste et al., 1989). However, it is not fully understood whether the synaptic release of glutamate at nerve terminals is most responsible for the excitotoxic intracellular processes leading to cell death of neurons.
Wahlestedt et al. have demonstrated that antisense-induced in vivo knockdown of NMDA receptor subunit NR1 markedly attenuated ischemic brain damege (Wahlestedt et al., 1993). However, recent studies have shown that the major problems with antagonists of glutamate receptors are psychomimetic effects, sedation and catatonia (De Keyser et al., 1999), probably due to drug-induced imbalance between dopaminergic and glutamatergic system. Since modulation of presynaptic release would affect all the synapses, an antisense knockdown of transmitter-release relating protein is expected to minimize treatment-induced derangement in the interaction between multiple chemical transmitter systems. Therefore, an antisense in vivo knockdown of transmitter-release relating protein seems to be a promising strategy to develop the effective therapies for ischemic brain injury with the less side-effect.
Among exocytosis-relating proteins that are abundant at nerve terminals, synapsin I as a protein related to synaptic vesicle trafficking is considered to control rates of synaptic vesicle exocytosis by participating in regulation of the vesicle life cycle via dissociation-reassociation cycle of synapsin with vesicle depended on phosphorylation (Hilfiker et al., 1998; Hosaka et al., 1999; Chi et al., 2001; Murthy, 2001). Synaptotagmin I as a major Ca2+ sensor is considered to regulate the synaptic vesicle exocytosis by promoting fusion between synaptic vesicle and plasma membrane via the assembly and clustering of SNARE complex (Bommert et al., 1993; Littleton et al., 1993; Nonet et al., 1993; Geppert et al., 1994; Littleton et al., 2001). According to the possible role of these proteins in transmitter release, it has been postulated that both antisense in vivo knockdown of synapsin I and that of synaptotagmin I reduces rates of exocytotic release of transmitter at overall synaptic terminals. To achieve a long-lasting downregulation of synapsin I or synaptotagmin I by a single treatment with the antisense oligodeoxynucleotides (ODNs), we used a novel transfection vector (HVJ-liposome)(Kaneda et al., 1989; Tomita et al., 1992; Morishita et al., 1994).
To test the relevance of the ‘transmitter-release strategy’ for neuroprotective therapies to regulate ischemic brain injury, we injected the antisense ODNs against synaptotagmin I or synapsin I into the lateralventricle by using HVJ (hemagglutinating virus of Japan)-liposome mediated gene transfer technique prior to transient forebrain ischemia and examined whether the antisense-induced knockdown of synaptotagmin I or synapsin I in the whole brain could regulate ischemia-induced damage of neurons in the hippocampus.
Section snippets
Preparation of HVJ-liposome containing oligodeoxynucleotides
Detailed preparation of anionic HVJ-liposome containing ODNs (ODNs–HVJ-liposome) has been described elsewhere (Yamada et al., 1996;Saeki and Kaneda, 1998). Briefly, three kinds of lipid (phosphatidylserine, phosphatidylcholine, and cholesterol) dissolved in chloroform (1 mg/ml) were mixed in a weight ratio of 1:4.8:2. Ten milligrams of lipid mixture was transferred into a glass tube and dried as a thin lipid film by rotary evaporator filled with nitrogen gas at 40 °C. The lipid thin film
Antisense-induced downregulation of synapsin I and synaptotagmin I protein levels in the hippocampus, neocortex and striatum
We first used quantitative Western blot analysis to examine the antisense-induced downregulation of synapsin I or synaptotagmin I protein levels in the hippocampus, neocortex and striatum following a single intraventricular injection of HVJ-liposomes containing antisense ODNs or reversed antisense ODNs.
Fig. 1A shows representative blots obtaind from the hippocampus of non-treated control rats (NT) and treated rats (syn-AS or syn-R) that received an intraventricular injection of syn-AS or syn-R
Discussion
Western blot analysis revealed that a single intraventricular injection of stm-AS–HVJ-liposome complex and syn-AS–HVJ-liposome complex caused a 30% inhibition of synaptotagmin I expression and 56% inhibition of synapsin I expression in the hippocampus for more than a week, respectively (see Fig. 1, Fig. 2). The blocking effect of this intraventricular injection of antisense ODNs by HVJ-liposome method on the biosynthesis of synaptotagmin I or synapsin I and the long-lasting action of antisense
Acknowledgements
This study was supported by the grant-in-aid for Scientific Research (no. 13670665) from the Ministry of Education, Science and Culture, Japan, and the Health Sciences Research Grants (no. H12-brain-018) from the Ministry of Health, Labor and Welfare, Japan. This investigation was also supported in part by the Grant for Scientific Research from Kitasato University Graduate School of Medical Science, Japan. We thank Dr. K. Akagawa for kindly giving us antibody against syntaxin IA.
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