Delayed post-ischemic administration of CDP-choline increases EAAT2 association to lipid rafts and affords neuroprotection in experimental stroke.
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.
Hurtado
O
O
Departamento de Farmacología, Facultad de Medicina, Universidad Complutense, 28040 Madrid, Spain.
Pradillo
J M
JM
Fernández-López
D
D
Morales
J R
JR
Sobrino
T
T
Castillo
J
J
Alborch
E
E
Moro
M A
MA
Lizasoain
I
I
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
D027342:Excitatory Amino Acid Transporter 2; D018696:Neuroprotective Agents; D018698:Glutamic Acid; D003566:Cytidine Diphosphate Choline