Journal: The Journal of Neuroscience
Article Title: Toxic Role of K + Channel Oxidation in Mammalian Brain
doi: 10.1523/JNEUROSCI.6153-11.2012
Figure Lengend Snippet: In a C. elegans model of AD, a K+ channel resistant to oxidative stress protects the neurons from apoptotic death. A, Anterior region of transgenic worm (strain FDX(ses25)) expressing Pgcy-5::GFP and Pflp-6::Aβ1–42 constructs, probed with anti-Aβ1–42 monoclonal antibody 6E10 and DNA stain DAPI. Shown is a single optical section (1 μm) from a deconvolution series. Left, GFP (green) + DAPI (blue) signal; middle, Aβ (red) + DAPI; right, fused epifluorescence images overlain on DIC image. Scale bar, 20 μm. B, Chemotaxis to biotin in parental worms (N2, hollow) and in transgenic worms expressing human Aβ1–42 in the ASE neurons of N2 (FDX(ses19), light gray), wild-type KVS-1 (FDX(ses21), dark gray), and C113S-KVS-1 (FDX(ses20), black), at the indicated time points. n = 4 experiments. C, Viability of cultured, age-synchronized, ASE right (ASER) neurons in N2 worms (hollow triangles) and in transgenic worms expressing human Aβ1–42 in the ASE neurons of N2 (FDX(ses25), filled triangles), wild-type KVS-1 (FDX(ses27), hollow squares), and C113S-KVS-1 (FDX(ses26), filled squares), at the indicated time points. The ASER neurons were marked with the gcy-5::GFP reporter, which drives GFP expression only in the ASER neuron. The disappearance of GFP fluorescence was used as a measure of a neuron's viability. A single experiment started with 300–500 fluorescent cells. Data were calculated as 100*(number of fluorescent cells at day X divided by number of fluorescent cells at day 1). n = 3 experiments. D, Chemotaxis to biotin in 9-d-old N2 worms and in transgenic worms expressing human Aβ1–42 in the ASE neurons of N2 (FDX(ses19)), wild-type KVS-1 (FDX(ses21)), and C113S-KVS-1 (FDX(ses20)) grown in the absence (hollow) or presence (gray) of 1 μg/ml Q-VD-OPh. Worms were maintained at 20°C in liquid cultures (S medium). Q-VD-OPh was administered daily. Prior to the experiment, worms were placed in ice for 15 min to settle, the medium was gently aspirated, and then they were washed in M9 buffer and collected in 50 ml Falcon tubes. The difference between the strains used in these experiments and in behavioral assays resides in the different markers used for the transformation (see Materials and Methods).
Article Snippet: Stained worms were imaged with a Zeiss Axiophot epifluorescence microscope equipped with digital deconvolution capacity (Intelligent Imaging Innovations).
Techniques: Transgenic Assay, Expressing, Construct, Staining, Chemotaxis Assay, Cell Culture, Fluorescence, Transformation Assay
