Significance and goal: CDP-choline (citicoline) stroke clinical trials in Europe and Japan showed significant improvement, while US studies provided ambiguous results. The route of administration (oral in USA vs iv in non-USA) and 24-hr time window may have hindered its effectiveness in the USA trials. It has now been realized that oral administration in USA trials was inappropriate and new Phase III trials will soon be undertaken. Our studies show that CDP-choline treatment delayed by 3-hr did not offer any neuroprotection. CDP-choline mode of action has not been clearly identified, and understanding its mechanism(s) should lead to more effective treatment of ischemic brain injury. The efficacy of CDP-choline in stroke therapy might still be achieved. Rationale: Phospholipid degradation is a significant promoter of neuronal death after transient cerebral ischemia. CDP-choline neuroprotection is thought to be due to increased phosphatidyl-choline (PtdCho) synthesis in the injured brain, although the evidence is limited. We showed in gerbil transient forebrain ischemia that CDP-choline: (a) provided significant neuroprotection (b) significantly restored PtdCho, cardioliPin and sphingomyelin, (c) attenuated arachidonic acid release and metabolism, and (d) increased glutathione levels. Our preliminary results show that CDP-choline affects activation of membrane and mitochondrial phospholipase A2 (PLA2) that is raM-Ca 2+ dependent (characteristic of secretory PLA2; sPLA2), supporting the hypothesis and Aim 1. In vitro, CDP-choline and its components (cytidine and choline) did not inhibit PLA2 activity, and thus as such CDP-choline is not a """"""""direct PLA2 inhibitor"""""""". Instead CDP-choline in vivo likely affects PLA2 activation. Hypothesis: CDP-choline attenuates phospholipid hydrolysis by preventing activation of PLA2. To test this hypothesis in transient cerebral ischemia, we propose the following specific aims:
Aim 1 : Determine whether CDP-choline inhibits PLA2 activation and protein expression. Since this aim is central to our hypothesis, it will be tested both in gerbil transient forebrain ischemia and transient focal cerebral ischemia of spontaneously hypertensive rat.
Aim 2 : Determine whether CDP-choline alters cytidine triphosphate phosphocholine cytidylyltransferase (PCCT) and sphingomyelinase activities mediated through PLA2. PCCT makes endogenous CDP-choline and is the rate-limiting enzyme in PtdCho synthesis. Exogenous CDP-choline is hydrolyzed, absorbed as cytidine and choline, and has to be re-synthesized by PCCT. PtdCho hydrolysis by PLA2 results in lyso-PtdCho and arachidonic acid. Lyso-PtdCho inhibits PCCT activity resulting in impaired PtdCho synthesis. Arachidonic acid activates neutral sphingomyelinase, resulting in membrane disintegration. PCCT activity was stimulated by exogenous CDP-choline.
Aim 2 will be tested in gerbil transient forebrain ischemia.
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