Journal:

Article Title: Inhibitors of Cytochrome c Release with Therapeutic Potential for Huntington's Disease

doi: 10.1523/JNEUROSCI.1867-08.2008

Figure Lengend Snippet: Methazolamide inhibits the release of mitochondrial apoptogenic factors and forestalls the activation of caspase-9 and -3. A, Cell death is induced in mutant-htt striatal cells by shifting them to the nonpermissive temperature of 37°C in SDM. Test cell cultures contain 100 μM methazolamide, whereas controls are devoid of these drugs. After 5 h, the cells are stained with Mitotracker and then fixed and stained with antibodies to cytochrome c. Mitochondrial cytochrome c is demonstrated as having a punctate pattern that colocalizes with Mitotracker. Cells in which cytochrome c is released demonstrate a more diffuse and decreased intensity of cytochrome c staining (arrows). Methazolamide retains intensity and punctate characteristics of the cytochrome c staining (Scale bar, 5 μm). B, Mutant-htt striatal cells are treated with 100 μM methazolamide (or processed in the absence of methazolamide) for 18 h. Subsequently, they were extracted; either cytosolic components or whole cell lysates were obtained. The samples, each of which contains 50 μg of protein, were analyzed by Western blot using antibodies either to cytochrome c, Smac with cytosolic components, or to caspase-9, or caspase-3 with whole cell lysates. β-Actin was used as a loading control. This blot is representative of three independent experiments. Densitometry was performed to quantify the intensity of the bands from the three independent experiments (*p < 0.05). C, D, Mutant-htt striatal cells were treated with 100 μM methazolamide for 5 and 18 h and compared with untreated controls. Cells were lysed and their complement of caspase-9 and -3 activities were evaluated by a fluorogenic assay. The results are the mean ±SD of three independent experiments (*p < 0.05, **p < 0.001). Experimental controls include cells treated with neither a cell death stimulus nor a test drugs (white bars). Others receive a death stimulus but are again not treated with a test drug (black bars). In addition to these controls, there are measurements of cell death in the presence of methazolamide either with or without a death stimulus (gray bars).

Article Snippet: Caspase-3-like substrate Ac-DEVD-AFC ( N -acetyl-DEVD-7-amino-4-trifluoromethyl coumarin) was from BD Pharmingen, caspase-9-like substrate Ac-LEHD-AFC (7-amino-4-trifluoromethylcoumarin) was from Calbiochem.

Techniques: Activation Assay, Mutagenesis, Staining, Western Blot