Glutamate transport is the only mechanism for maintaining extracellular
glutamate concentrations below excitotoxic levels. Among
glutamate transporters, EAAT2 is responsible for up to 90% of all
glutamate transport and has been reported to be associated to
lipid rafts. In this context, we have recently shown that
CDP-choline induces EAAT2 translocation to the membrane. Since
CDP-choline preserves membrane stability by recovering levels of
sphingomyelin, a
glycosphingolipid present in
lipid rafts, we have decided to investigate whether
CDP-choline increases association of EAAT2 transporter to
lipid rafts.
Flotillin-1 was used as a marker of
lipid rafts due to its known association to these microdomains. After gradient centrifugation, we have found that
flotillin-1 appears mainly in fractions 2 and 3 and that EAAT2
protein is predominantly found colocalised with
flotillin-1 in fraction 2. We have also demonstrated that
CDP-choline increased EAAT2 levels in fraction 2 at both times examined (3 and 6 h after 1 g/kg
CDP-choline administration). In agreement with this, [(3)H]
glutamate uptake was also increased in
flotillin-associated vesicles obtained from brain homogenates of animals treated with
CDP-choline. Exposure to
middle cerebral artery occlusion also increased EAAT2 levels in
lipid rafts, an effect which was further enhanced in those animals receiving 2 g/kg
CDP-choline 4 h after the occlusion.
Infarct volume measured at 48 h after
ischemia showed a reduction in the group treated with
CDP-choline 4 h after occlusion. In summary, we have demonstrated that
CDP-choline redistributes EAAT2 to
lipid raft microdomains and improves
glutamate uptake. This effect is also found after experimental
stroke, when
CDP-choline is administered 4 h after the ischemic occlusion. Since we have also shown that this delayed post-ischemic administration of
CDP-choline induces a potent neuroprotection, our data provides a novel target for neuroprotection in
stroke.