Briefly, amutagenesis kit with the following mutant oligonucleotide primers: 5-CAC GCA CAC GAC CTT CGT CGC-3 (AlaVal) and 5-GAT CCC AAT TCC ACC ACA AGC-3 (ValGly). adenoviruses (AdVs) to deliver and express human wild-type or mutant SOD genes into main neurons as well as differentiated rat pheochromocytoma cells (PC12 cells). We then decided the effect of this overexpression on cell viability. Using an imaging assay (Bindokas et al., 1996), we were able to determine the O2? content of single cells. Our results indicate that expression of mutant SOD induces neural cell death that bears the hallmarks of apoptosis, is usually sensitive to metal chelators, antioxidants, and antiapoptotic brokers, and is associated with abnormalities in free radical production. MATERIALS AND METHODS Human embryonic kidney (HEK) 293 and baby hamster kidney (BHK-21) monolayer cell cultures were produced in DMEM supplemented with 10% fetal bovine serum and 0.01% gentamycin (Life Technologies, Gaithersburg, MD). Rat pheochromocytoma Efaproxiral sodium PC12 Slc4a1 cells were plated on poly-l-lysine-coated glass coverslips at a density of 10,000 cells per coverslip in DMEM Efaproxiral sodium supplemented with 10% bovine calf serum and 10 g/ml penicillin/streptomycin (Sigma, St. Louis, MO). Differentiation of PC12 cells was induced within 24 hr after the addition of DMEM made up Efaproxiral sodium of 100 ng/ml of nerve growth factor (NGF) (Collaborative Biomedical Products, Inc.) and no serum. Seven days later, cells were infected with recombinant AdVs as explained below. Main sympathetic neurons were isolated from superior cervical ganglia of 3- to 5-d-old Holtzman rats by previously explained methods (Jordan et al., 1995). Dissociated cells were managed on coverslips in L-15 medium with 1 g/ml NGF and 5% rat serum (Life Technologies, Grand Island, NY). E17 hippocampal neuronal cultures were isolated according to the method of Efaproxiral sodium Banker (1980) with some modifications (Abele et al., 1990; Scholz and Miller, 1991) and produced on coverslips. Rat cortical astrocytes were cultured following the method of Landis and Weinstein (1983). The astrocytes were used for computer virus infection when they experienced reached confluency in DMEM with 5% horse serum and managed in DMEM with N2.1 in the presence of AraC to prevent cell proliferation. The preparation of AdVs expressing wild-type SOD has been explained previously (Jordan et al., 1995). The SOD cDNA was placed downstream from elongation factor 1- promotor (EF-1) and upstream of cellular heavy chain enhancer (4F2) and the bovine growth hormone polyadenylation site. We also prepared recombinant AdVs expressing SOD that carry a mutation in exon 1 changing alanine to valine at amino acid position 4 (the most common FALS-linked mutation in North America) (Deng et al., 1993) and one with a mutation of valine to glycine at amino acid position 148 in exon 5. Each of the mutations was separately designed into a shuttle vector, pAdKN (Jordan et al., 1995) as follows. Briefly, amutagenesis kit with the following mutant oligonucleotide primers: 5-CAC GCA CAC GAC CTT CGT CGC-3 (AlaVal) and 5-GAT CCC AAT TCC ACC ACA AGC-3 (ValGly). A blunt-endedBHK-21 cells were either mock-infected or infected with AdLacZ, AdSODWT, AdSODA4V, or AdSODV148G viruses at an MOI of 10 and incubated for 72 hr. Cells were scraped, washed with chilly PBS, swollen for 15 min on ice with hypotonic buffer (in mm: 10 HEPES, 10 KCl, and 1 dithiothreitol, pH 7.5) containing protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 500 U/ml aprotinin, and 1 g/ml leupeptin), and then lysed for 15 min with 1.0% Nonidet P-40. Cytoplasmic proteins were obtained by centrifugation at 12,000 rpm for 15 min at 4C. The proteins (50 g) were separated on 12.5% SDS-PAGE. Separated proteins were transferred onto nitrocellulose paper and immunostained with horseradish peroxidase-conjugated anti-SOD polyclonal antibody (The Binding Site, catalog #PP077) using an enhanced chemiluminescence (ECL) Western blotting detection system (Amersham, Arlington Heights, IL). Nitration of proteins was analyzed on the protein extracts prepared from differentiated PC12 cells. Cytoplasmic extracts were Efaproxiral sodium prepared in a similar.