In trisomy 16 mouse, increased gene dosage of SOD-1, APP, glutamate R5 receptor,etc. activates prooxidant processes. Thus, primary cultures of trisomy 16 mouse brain may serve as a model to study aging and oxidative stress-induced degenerative diseases. The objectives of this project are: 1. to study various stress response genes that are regulated by reactive oxygen species (ROS), i.e. metallothionein (MT) isoforms, heat shock protein, thioredoxin peroxidases, and transcription factor AP-1. 2. to analyze the effect of ROS on the transcriptional regulation of MT-I/II and on the antioxidant- and metal-response element- binding factors In trisomy 16 primary cultures, protein oxidation was 50 % higher than in control cells. Upon exposure to exogenous H2O2 (0.01 mM) or kainic acid (0.05 mM) a 2.5- to 3-fold increase of protein carbonyl groups was found in trisomy 16 but not in control cells. This finding suggests that the removal of endogenously formed ROS may be impaired in trisomy 16 cells. MT-I/II immunoblots were increased by 2 folds in trisomy 16 but not control cells, whereas, heat shock protein and the thioredoxin peroxidase isoforms (I, II, III, IV,V) remained unchanged. It remains to be established whether the basal MT-I/II level was increased due to a compensatory upregulation of MT protein expression or to a decrease in the rate of MT-I/II protein degradation. When control cells were exposed to 0.001mM H2O2, MT-I/II was increased within 1h, in contrast, to HSP-70 and thioredoxin peroxidase III that were increased 48 h after exposure to 0.1 mM H2O2 suggesting that these proteins are regulated differently by ROS. Comparative studies with neuronal and astrocyte primary cultures showed that a 15 min exposure to 0.01 mM H2O2 elicited maximal AP-1 DNA-binding activity earlier(30 min) in trisomy 16 than in control cells. Whereas, in astrocyte cultures 0.01 mM H2O2 elicited a small and similar increase of AP-1 DNA-binding activity in trisomy 16 and control cells. This finding indicates that astrocytes may be more ROS-resistant than neurons.
