Volume 6, Issue 3, Pages 336-341 (September 2002) A Single-Subunit NADH-Quinone Oxidoreductase Renders Resistance to Mammalian Nerve Cells against Complex I Inhibition  Byoung Boo Seo, Eiko Nakamaru-Ogiso, Terence R. Flotte, Takao Yagi, Akemi Matsuno-Yagi  Molecular Therapy  Volume 6, Issue 3, Pages 336-341 (September 2002) DOI: 10.1006/mthe.2002.0674 Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 1 Expression and mitochondrial localization of the Ndi1 protein in PC12 and MN9D cells infected with the rAAV-NDI1 particles. Cells (1 × 105) in 1 ml of growth medium were infected with 9 × 108 IU of rAAV-NDI1. After culturing the cells in the same medium for 24 hours, the expressed Ndi1 protein was located by immunostaining with double-labeling using affinity-purified rabbit antibody to the Ndi1 protein (FITC, green) and antibody to the cytochrome oxidase subunit IV (rhodamine, red). In the overlapping images (rightmost panel), cells with various levels of Ndi1 protein expression are observed in different colors. Those cells with high levels of expression appear greenish and those with low levels appear reddish. Noninfected control cells reacted only with anti-cytochrome oxidase antibody. Molecular Therapy 2002 6, 336-341DOI: (10.1006/mthe.2002.0674) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 2 Effect of the concentration of rAAV-NDI1 particles on the number of overexpressing cells. MN9D cells (1 × 105) in 1 ml of growth medium were infected with rAAV-NDI1 at the concentrations indicated on the abscissa. Cells were cultured in the same medium for 2 days and immunostained with antibody to Ndi1P as in Fig. 1, and then those expressing high levels of Ndi1P were counted. Each sample was counted in triplicate. Molecular Therapy 2002 6, 336-341DOI: (10.1006/mthe.2002.0674) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 3 Respiratory activities of NDI1-transduced cells and noninfected control cells. PC12 cells were infected with rAAV-NDI1 as in Fig. 1 and were cultured in galactose medium in the presence of 0.1 μm rotenone to eliminate nontransduced cells. The cells were harvested by trypsinization and treated with 50–150 μg of digitonin until more than 90% of cells were stained by trypan blue. Oxygen consumption was measured polarographically in 0.6 ml of a buffer containing 20 mM Hepes, pH 7.1, 250 mM sucrose, and 10 mM MgCl2 by using a Clark-type electrode in a water-jacketed chamber maintained at 37°C. Upper trace, nontransduced control cells with 5.6 × 107 cells/ml. Lower trace, NDI1-transduced cells with 4.8 × 107 cells/ml. Where indicated, 5 mM glutamate (Glu), 5 mM malate (Mal), 5 μM rotenone (Rot), 0.5 mM flavone, 5 mM succinate (Succ), and 5 μM antimycin (AntA) were added. Molecular Therapy 2002 6, 336-341DOI: (10.1006/mthe.2002.0674) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 4 Effects of complex I inhibitor on cell growth of the NDI1-transduced cells. Infection of the cells with rAAV-NDI1 and selection of NDI1-transduced cells were carried out as given in Fig. 3. (A) PC12 cells and MN9D cells expressing Ndi1 protein (circles) or noninfected control cells (triangles) were placed in galactose medium in the presence or absence of 0.1 μM rotenone. Under these conditions, the sole energy source is oxidative phosphorylation. Viable cells were counted after Trypan blue staining on the days indicated. The number of cells surviving in the presence of rotenone is presented as the percent of no-rotenone samples. (B) PC12 cells expressing Ndi1 protein (circles) or noninfected control cells (triangles) were cultured in the presence of pyridaben (1 μM), fenpyroximate (1 μM), or capsaicin (250 μM). (Left) Galactose medium was used as in (A). (Right) The medium contained 25 mM glucose to allow cells to grow under glycolysis. Data were collected in triplicate. Molecular Therapy 2002 6, 336-341DOI: (10.1006/mthe.2002.0674) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 5 Morphological differentiation of NDI1-transduced cells. MN9D cells stably expressing Ndi1 were prepared as described in Fig. 3. The nontransduced control cells (top) and NDI1-transduced MN9D cells (bottom) were placed in 1 ml of DMEM containing 10% FBS by seeding 0.1% gelatin-coated 6-well plates with approximately 1 × 105 cells per well and were cultured in the absence (left) or presence (right) of 2 μM retinoic acid for 7 days. Neurite extension was observed by phase contrast microscopy. Molecular Therapy 2002 6, 336-341DOI: (10.1006/mthe.2002.0674) Copyright © 2002 American Society for Gene Therapy Terms and Conditions

FIG. 6 Expression of Ndi1 in MN9D cells with neurite outgrowth. (A, B) MN9D cells stably expressing Ndi1 protein were prepared as given in Fig. 3. These cells were cultured in 1 ml of DMEM containing 1% FBS on 0.1% gelatin-coated dishes with approximately 0.5 × 105 cells in the presence of 2 μM retinoic acid for 5 days. After neurite formation was observed, cells were immunostained using antibody to the Ndi1 protein. (C, D) MN9D cells with approximately 0.5 × 105 cells were first treated with 2 μM retinoic acid for 5 days under the same conditions as in (A, B) to allow neurite extension, and then infected with 9 × 108 IU of rAAV-NDI1. After culturing the infected cells for 2 days in galactose medium plus 0.1 μM rotenone, immunostaining of the Ndi1 protein was performed as in (A, B). In all images, the expressed Ndi1 protein is observed both in cell bodies and neurites. Molecular Therapy 2002 6, 336-341DOI: (10.1006/mthe.2002.0674) Copyright © 2002 American Society for Gene Therapy Terms and Conditions