Biphasic modulation of protein kinase C and enhanced cell toxicity by amyloid beta peptide and anoxia in neuronal cultures

A major feature of Alzheimer's disease is the deposition of the amyloid bet a peptide (AP) in the brain by mechanisms which remain unclear. One hypothe sis suggests that oxidative stress and AP aggregation are interrelated proc esses. Protein kinase C, a major neuronal regulatory protein is activated a fter oxidative stress and is also altered in the Alzheimer's disease brain. Therefore, we examined the effects of A beta (1-40) peptide on the protein kinase C cascade and cell death in primary neuronal cultures following ano xic conditions. Treatment with A beta (1-40) for 48 h caused a significant increase in the content and activity of Ca2+-dependent and Ca2+-independent protein kinase C isoforms. By 72h Various protein kinase C isoforms were d own-regulated. Following 90 min anoxia and 6 h normoxia, a decrease in prot ein kinase C isoforms was noticed, independent of A beta (1-40) treatment. A combination of A beta (1-40) and 30-min anoxia enhanced cytotoxicity as n oticed by a marked loss in the mitochondrial ability to convert 3-(4,5-dime thylthiazol-2-yl)-2,5-diphenyl bromide and by enhanced 4',6-diamidino-2-phe nylindole nuclear staining. Phosphorylation of two downstream protein kinas e C substrates of apparent molecular mass 80 and 43 kDa, tentatively identi fied as the mirystoyl alanine-rich C-kinase substrate (MARCKS), were gradua lly elevated up to 72 h upon incubation with A beta (1-40). Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of A beta (1-40), phosphorylati on of MARCKS was reduced. After 6h normoxia, MARCKS phosphorylability was d iminished possibly because of protein kinase C down-regulation. The data su ggest that a biphasic modulation of protein kinase C and MARCKS by A beta ( 1-40) combined with anoxic stress may play a role in Alzheimer's disease pa thology.(1).