Overexpression of Ca2+-permeable AMPA receptor promotes delayed cell death of hippocampal CA1 neurons following transient forebrain ischemia.

To examine the role of Ca(2+) entry through AMPA receptors in the pathogenesis of the ischemia-induced cell death of hippocampal neurons, we delivered cDNA of Q/R site-unedited form (GluR2Q) of AMPA receptor subunit GluR2 in the hippocampus by using an HVJ-liposome-mediated gene transfer technique. Two days prior to transient forebrain ischemia, we injected an HVJ-liposome containing cDNA of the GluR2Q-myc fusion gene into a rat unilateral hippocampus. In the absence of ischemic insult, overexpression of Ca(2+)-permeable GluR2Q did not cause any neurodegeneration in the cDNA-injected hippocampus. In ischemic rats, overexpression of Ca(2+)-permeable GluR2Q markedly promoted ischemic cell death of CA1 pyramidal neurons, while complete rescue of CA1 pyramidal neurons from ischemic damage occurred in the hippocampal hemisphere opposite the GluR2Q expression. Overexpression of the Q/R-site edited form (GluR2R) of subunit GluR2 did not affect the ischemia-induced damage of CA1 pyramidal neurons. From these results, we suggest that the Ca(2+)-permeability of AMPA receptors does not have a direct contribution to glutamate receptor-mediated neurotoxicity but has a promotive action in the evolution of ischemia-induced neurodegeneration of vulnerable neurons.

Antisense in vivo knockdown of synaptotagmin I and synapsin I by HVJ-liposome mediated gene transfer modulates ischemic injury of hippocampus in opposing ways.

Neurotransmitter release during and after ischemic event is thought to be involved in excitotoxicity as a pathogenesis for the ischemic brain damage, which is mediated by excessive activation of glutamate receptors and attendant calcium overload. To ascertain the role of transmitter release from nerve terminals in promoting the ischemic neurodegeneration, we delivered antisense oligodeoxynucleotides (ODNs) to synaptotagmin I or synapsin I into the rat brain by using HVJ-liposome gene transfer technique. The antisense ODNs were injected into the lateralventricle in rats 4 days prior to transient forebrain ischemia of 20 min. With a single antisense treatment, long-lasting downregulation of the transmitter release relating protein levels at overall synaptic terminals was achieved. The antisense in vivo knockdown of synaptotagmin I prevented almost completely the ischemic damage of hippocampal CA1 neurons, while the in vivo knockdown of synapsin I markedly promoted the ischemic damage of CA1 pyramidal neurons and extended the injury to relatively resistant CA2/CA3 region. The modulation of ischemic hippocampal damage by the in vivo knockdown of synaptotagmin I or synapsin I suggests that transmitter release from terminals plays an important role in the evolution of ischemic brain damage and therefore the transmitter release strategy by the use of antisense ODNs-HVJ-liposome complex is reliable for neuroprotective therapies.

Synaptotagmin I hypothalamic knockdown prevents amygdaloid seizure-induced damage of hippocampal neurons but not of entorhinal neurons.

We have previously demonstrated that an acute pharmacological interruption of the afferent inputs from the hypothalamus to the hippocampus resulted in the blockade of the genesis and spread of intra-amygdala kainate-induced seizure activity in the hippocampus. This finding suggests that a sustained interruption of the hypothalamic stimulative influences may completely prevent amygdaloid seizure-induced hippocampal neuron damage. To test this assumption, we delivered antisense oligodeoxynucleotides (ODNs) against synaptotagmin I, a regulatory protein of the transmitter release machinery, into the hypothalamus by using a Hemagglutinating virus of Japan (HVJ)-liposome-mediated gene transfer technique. Four days prior to the induction of status epilepticus by intra-amygdala injection of kainate, the synaptotagmin I antisense was injected into the supramammillary nucleus (SuM) of the hypothalamus to chronically suppress the stimulative influences to the hippocampus via the reduction of transmitter release. The synaptotagmin I hypothalamic knockdown resulted in the almost complete prevention of seizure-induced damage of hippocampal neurons but not of entorhinal neurons following the kainate-induced amygdaloid seizures. This result suggests that the hypothalamic stimulative influences to the hippocampus have a major contribution to the amygdaloid seizure-induced hippocampal sclerosis, probably via disinhibition mechanism.