Role of MAPK in neuroprotection by cart in ischemic stroke

BACKGROUND AND SIGNIFICANCE: Our previous work indicated that CART (cocaine and amphetamine regulated transcript) plays an important role in estrogen-mediated neuroprotection in ischemic stroke (Xu Y et al. 2006). Specifically, we previously demonstrated that CART protects primary cultured cortical neurons from cell death induced by oxygen-glucose deprivation (OGD) and decreases infarct size after ischemic stroke induced in mice by middle cerebral artery occlusion (MCAO). The aims of this study are: 1) to further investigate the neuroprotective role of CART in vitro and in vivo using small interfering RNA (siRNA) to downregulate endogenous CART, and 2) to explore a potential mechanism of neuroprotection by CART. CART and its mechanism of action may serve as a novel therapeutic strategy for neuroprotection in ischemic stroke. METHODS: Five double-stranded RNA fragments corresponding to selected mouse CART mRNA sites were produced using plasmid siRNA expression vector under control of U6 promoter. The vector also co-expresses the coral GFP marker (cGFP) under CMV promoter, which is used to track transfection efficiency. Cortical neurons and HEK293T cells were transfected with CART siRNA or control vector, and interference efficiency was assayed by RT-PCR and ELISA. HEK293 cells were also co-transfected with CART cDNA, since they do not express CART. Finally, CART or vehicle, CART siRNA or control vector in vehicle (cationic polymer polyethylenimine, PEI) were injected into mouse lateral ventricles in vivo 24 hours prior to MCAO. Brains were removed, stained with 2,3,5- triphenyltetrazolium chloride (TTC) for infarct size measurement. Neuronal cell death in vitro was induced by OGD, and assayed by propidium iodide (PI)/Calcein staining, and survival assayed by MTT. The phosphorylation of ERK1/2 was measured by western blot with anti-ERK anti-p(44/42)ERK antibodies. RESULTS: CART expression was effectively suppressed by siCART. Selected siRNA reduced CART RNA and protein levels in vitro by approximately 80% and 90%, respectively. CART administration reduced infarct size at 24 hrs after MCAO from 39·6% to 22·4% (n=6 per group, p<0.05), while administration of siCART increased infarct size to 48·4% compared to saline-injected mice (23.5·4%, n=6 per group, p<0.05). Neuronal survival by MTT after 2-hr OGD was higher in CART-treated cultures (49±6%, n=3, each in triplicate) compared to saline-treated cultures (35±%, n=3 in triplicate, p<0.05), while survival was significantly reduced in cultures treated with siCART (21±4%, n=3 in triplicate) compared to saline-treated cultures. Similar results were obtained using PI/Calcien staining. Finally, CART increased ERK phosphorylation in cultured neurons after OGD, while siCART inhibited ERK activation after OGD. CONCLUSIONS: The findings suggest that CART is an important endogenous neuroprotectant against ischemic neuronal cell death in vitro and in vivo, and that mechanism of neuroprotection is in part linked to ERK activation. CART may serve as a novel neuroprotectant against stroke-related brain damage.