ABSTRACT
Amyloid-ß peptides generated by proteolysis of the ß-amyloid precursor protein (APP) play an important role in the pathogenesis of Alzheimer's disease. The present study aimed to determine whether cytosolic phospholipase A2 α (cPLA2 α) plays a role in elevated APP protein expression induced by aggregated amyloid-ß1-42 (Aß) in cortical neurons and to elucidate its specific role in signal events leading to APP induction. Elevated cPLA2 α and its activity determined by phosphorylation on serine 505 as well as elevated APP protein expression, were detected in primary rat cortical neuronal cultures exposed to Aß for 24 h and in cortical neuron of human amyloid-ß1-42 brain infused mice. Prevention of cPLA2 α up-regulation and its activity by oligonucleotide antisense against cPLA2 α (AS) prevented the elevation of APP protein in cortical neuronal cultures and in mouse neuronal cortex. To determine the role of cPLA2 α in the signals leading to APP induction, increased cPLA2 α expression and activity induced by Aß was prevented by means of AS in neuronal cortical cultures. Under these conditions, the elevated cyclooxygenase-2 and the production of prostaglandin E2 (PGE2 ) were prevented. Addition of PGE2 or cyclic AMP analogue (dbcAMP) to neuronal cultures significantly increased the expression of APP protein, while the presence protein kinase A inhibitor (H-89) attenuated the elevation of APP induced by Aß. Inhibition of elevated cPLA2 α by AS prevented the activation of cAMP response element binding protein (CREB) as detected by its phosphorylated form, its translocation to the nucleus and its DNA binding induced by Aß which coincided with cPLA2 α dependent activation of CREB in the cortex of Aß brain infused mice. Our results show that accumulation of Aß induced elevation of APP protein expression mediated by cPLA2 α, PGE2 release, and CREB activation via protein kinase A pathway.
Subject(s)
Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/biosynthesis , Group IV Phospholipases A2/metabolism , Nerve Degeneration/metabolism , Peptide Fragments/metabolism , Signal Transduction/physiology , Alzheimer Disease/pathology , Animals , Blotting, Western , Cells, Cultured , Disease Models, Animal , Electrophoretic Mobility Shift Assay , Humans , Male , Mice , Mice, Inbred C57BL , Nerve Degeneration/pathology , Neurons/metabolism , Neurons/pathology , Rats , Rats, Sprague-DawleyABSTRACT
Increased cytosolic phospholipase A2α (cPLA2α) immunoreactivity and transcript were observed in Alzheimer's disease (AD) brain associated with amyloid deposits. Thus, the present study examined whether cPLA2α upregulation participate in cortical neuron damage induced by aggregated Aß1-42 and determined its role in the signaling events leading to damage, using an antisense technology. Exposure of primary cortical neurons to 1µM aggregated Aß1-42 for 24h induced up-regulation and activation of cPLA2α and apoptotic cell death of about 30% as detected by: cell count, MTT reduction, caspases-3 and -8 activation, DAPI and TUNEL staining, that were prevented by inhibition of cPLA2α up-regulation and activity in the presence of antisense against cPLA2α (AS). cPLA2α was rapidly activated upon addition of aggregated Aß1-42, as determined by its phosphorylated form on serine 505, and this activity was dependent on NADPH oxidase activity. NOX2- and NOX4-NADPH oxidase upregulation at 24h of aggregated Aß1-42 exposure was not affected by the presence of AS, but superoxide production was reduced, probably due to NOX2 inhibition. cPLA2α upregulation led to activation of neutral sphingomyelinase (N-SMase) as its activity was inhibited in the presence of AS, and could be restored by addition of arachidonic acid. Addition of ceramide analog induced caspase-8 activation leading to caspase-3 activation and apoptotic neuronal death. In conclusion, our results suggest that cPLA2α activity plays a crucial role in the signaling cascade leading to apoptotic neuronal death by aggregated Aß1-42 probably via activation of N-SMase, ceramide production and caspases-3 and -8.