Previous studies found that kainic acid (KA)-induced apoptosis involved the lysosomal enzyme cathepsin B, suggesting a possible mechanism of autophagy in excitotoxicity. The present study was sought to investigate activation and contribution of autophagy to excitotoxic neuronal injury mediated by KA receptors. The formation of autophagosomes was observed with transmission electron microscope after excitotoxin exposure. The contribution of autophagic mechanisms to KA-induced upregulation of microtubule-associated protein 1A/1B light chain 3 (LC3), lysosome- associated membrane protein 2 (LAMP2) and cathepsin B, release of cytochrome c, activation of caspase-3, down-regulation of Bcl-2, upregulation of Bax, p53, puma and apoptotic death of striatal neurons were assessed with co-administration of the autophagy inhibitor 3-methyladenine (3-MA). These studies showed that KA brought about an increase in the formation of autophagosomes and autolysosomes in the cytoplasm of striatal cells. KA-induced increases in the ratio of LC3-II/LC3-I, LAMP2, cathepsin B, release of cytochrome c and activation of caspase-3 were blocked by pre-treatment with 3-MA. 3-MA also reversed KA-induced down-regulation of Bcl-2 and upregulation of Bax protein levels, LC3, p53 and puma mRNA levels in the striatum. KA-induced internucleosomal DNA fragmentation and loss of striatal neurons were robustly inhibited by 3-MA. These results suggest that over-stimulation of KA receptors can activate autophagy. The autophagic mechanism participates in programmed cell death through regulating the mitochondria-mediated apoptotic pathway.

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We have previously reported that prostaglandin A(1) (PGA(1)) reduces infarct size in rodent models of focal ischemia. This study seeks to elucidate the possible molecular mechanisms underlying PGA(1)'s neuroprotective effects against ischemic injury. Rats were subjected to permanent middle cerebral artery occlusion (pMCAO) by intraluminal suture blockade. PGA(1) was injected intracerebroventricularly (icv) immediately after ischemic onset. Western blot analysis was employed to determine alterations in IkappaBalpha, pIKKalpha, and peroxisome proliferator-activated receptor-gamma (PPAR-gamma). Immunohistochemistry was used to confirm the nuclear translocation of nuclear factor-kappaB (NF-kappaB) p65 and the expression of PPAR-gamma. RT-PCR was used to detect levels of c-Myc mRNA. The contribution of PPAR-gamma to PGA(1)'s neuroprotection was evaluated by pretreatment with the PPAR-gamma irreversible antagonist GW9662. A brief increase in pIKKalpha levels and rapid reduction in IkappaBalpha were observed after ischemia. PGA(1) blocked ischemia-induced increases in pIKKalpha levels and reversed the decline in IkappaBalpha levels. Ischemia-induced nuclear translocation of NF-kappaB p65 was attenuated by PGA(1). PGA(1) also repressed the ischemia-induced increase in expression of NF-kappaB target gene c-Myc mRNA. Immunohistochemistry demonstrated an increase in PPAR-gamma immunoreactivity in the nucleus of striatal cells at 3 hr after pMCAO. Western blot analysis revealed that the expression of PPAR-gamma protein significantly increased at 12 hr and peaked at 24 hr. PGA(1) enhanced the ischemia-triggered induction of PPAR-gamma protein. Pretreatment with the irreversible PPAR-gamma antagonist GW9662 attenuated PGA(1)'s neuroprotection against ischemia. These findings suggest that PGA(1)-mediated neuroprotective effect against ischemia appears to be associated with blocking NF-kappaB activation and likely with up-regulating PPAR-gamma expression.

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