title: Delayed post-ischemic administration of CDP-choline increases EAAT2 association to lipid rafts and affords neuroprotection in experimental stroke.
abstract: 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.
authors: Departamento de Farmacología, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain.
authors: O
authors: O
authors: Hurtado
authors: J M
authors: JM
authors: Pradillo
authors: D
authors: D
authors: Fernández-López
authors: J R
authors: JR
authors: Morales
authors: T
authors: T
authors: Sobrino
authors: J
authors: J
authors: Castillo
authors: E
authors: E
authors: Alborch
authors: M A
authors: MA
authors: Moro
authors: I
authors: I
authors: Lizasoain
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods; D003566:Cytidine Diphosphate Choline / Q000008:administration & dosage*; D004195:Disease Models, Animal; D027342:Excitatory Amino Acid Transporter 2 / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D018698:Glutamic Acid; D020244:Infarction, Middle Cerebral Artery / Q000517:prevention & control*; D008297:Male; D021962:Membrane Microdomains / Q000187:drug effects* / Q000378:metabolism; D018696:Neuroprotective Agents / Q000008:administration & dosage*; D051381:Rats; D011916:Rats, Inbred F344; D013997:Time Factors
chemical_list: D027342:Excitatory Amino Acid Transporter 2; D018696:Neuroprotective Agents; D018698:Glutamic Acid; D003566:Cytidine Diphosphate Choline