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 beta peptide (Aβ) in the brain by mechanisms which remain unclear. One hypothesis suggests that oxidative stress and Aβ aggregation are interrelated processes. Protein kinase C, a major neuronal regulatory protein is activated after oxidative stress and is also altered in the Alzheimer's disease brain. Therefore, we examined the effects of Aβ1−40 peptide on the protein kinase C cascade and cell death in primary neuronal cultures following anoxic conditions. Treatment with Aβ1−40 for 48 h caused a significant increase in the content and activity of Ca2+‐dependent and Ca2+‐independent protein kinase C isoforms. By 72 h various protein kinase C isoforms were down‐regulated. Following 90 min anoxia and 6 h normoxia, a decrease in protein kinase C isoforms was noticed, independent of Aβ1−40 treatment. A combination of Aβ1−40 and 30‐min anoxia enhanced cytotoxicity as noticed by a marked loss in the mitochondrial ability to convert 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyl‐tetrazolium bromide and by enhanced 4′,6‐diamidino‐2‐phenylindole nuclear staining. Phosphorylation of two downstream protein kinase C substrates of apparent molecular mass 80 and 43 kDa, tentatively identified as the mirystoyl alanine‐rich C‐kinase substrate (MARCKS), were gradually elevated up to 72 h upon incubation with Aβ1−40. Anoxia followed by 30 min normoxia enhanced MARCKS phosphorylation in the membrane but not in the cytosolic fraction. In the presence of Aβ1−40, phosphorylation of MARCKS was reduced. After 6 h normoxia, MARCKS phosphorylability was diminished possibly because of protein kinase C down‐regulation. The data suggest that a biphasic modulation of protein kinase C and MARCKS by Aβ1−40 combined with anoxic stress may play a role in Alzheimer's disease pathology.1