Anti-apoptotic activity of α-crystallins against Bax-induced apoptosis.

<p>293T cells were transiently co-transfected with pcDNA3-Bax and with either the empty pcDNA3.1, pcDNA3.1-αA- (αA) or pcDNA3.1-αB (αB)-crystallin constructs. (<b>A</b>) TUNEL assay showing that Bax-triggered apoptosis was inhibited in 293T cells overexpressing the α-crystallins, as reflected by the reduced number of TUNEL-positive apoptotic cells. Cell nuclei were counterstained with Hoechst. (<b>B</b>) As assessed by luminescent caspase assay, Bax-induced Caspase-3/-7 activity was inhibited in the presence of αA- (αA) and αB- (αB) crystallins 16 h and 24 h post-transfection. (* p<0.0001 by <i>t</i>-test for Bax <i>versus</i> αA/Bax and for Bax <i>versus</i> αB/Bax at 16 h and 24 h). Data are the mean ± SE of three independent experiments, each performed in triplicates.</p

Interaction of the C-terminal extension domain of αA-crystallin with Bax <i>in vivo</i>.

<p>293T cells transiently transfected with the empty vector (pRluc), full length αA- (αA_wt) or mutant αA- (αA_144-173) crystallin were further treated with 100 nM STS for 3 h before co-immunoprecipitation with anti-Bax antibody. The precipitated samples were then sequentially probed by western blot using anti-αA/αB and anti-Bax antibodies. IP: immunoprecipitated samples (<i>left panels</i>); CE: 20 µg of total proteins from whole cell extract (<i>right panels</i>).</p

Stable expression of α-crystallins in 661W cells.

<p>(<b>A</b>) Schematic representation of the bicistronic pWPI lentiviral vector allowing for pEF1α-driven simultaneous expression of the transgene (αA- or αB-crystallin) and IRES-mediated GFP fluorescent marker. (<b>B</b>) Western blot analysis of αA- and αB-crystallins expressed in lentivirus-transduced 661W cells. Twenty-five micrograms of total proteins from cell extracts were subjected to 12% SDS-PAGE. Myc-tagged αA- and αB-crystallins were expressed in 661W cells transduced with the recombinant lentiviruses pWPI_αA and pWPI_αB, respectively, while no transgene was detected in cells transduced with the empty lentivirus pWPI or in non transduced cells (−). Membranes were further immunoassayed with anti-GFP as a control of transduction efficiency and with anti-ß-actin as a control of equal protein loading. (<b>C</b>) Immunofluorescence analysis with anti-myc showing cytoplasmic expression of αA- and αB-crystallins in 661W cells transduced with the corresponding recombinant lentiviruses. Cell nuclei were counterstained with DAPI and GFP staining was shown as a control of transduction efficiency. pEF1α: EF1α promoter; IRES: internal ribosome entry site from encephalomyocarditis virus.</p

STS-induced apoptosis in 661W cells.

<p>Dose-dependent induction of apoptosis in 661W cells exposed to increasing amounts of STS (25 to 200 nM) for 24 h, as depicted by (<b>A</b>) increased TUNEL-positive apoptotic cells and (<b>B</b>) decreased level of intracellular ATP content. Data are the mean ±SE of at least four independent experiments, each performed in triplicates.</p

STS-induced apoptosis was prevented in 661W cells in the presence of α-Crystallins.

<p>661W cells transduced with the recombinant lentiviruses overexpressing αA-crystallin (pWPI_αA), αB-crystallin (pWPI_αB) or the empty lentivirus (pWPI) were treated with 100 nM STS for 16 h. (<b>A</b>) STS-triggered apoptosis was inhibited in the presence of α-crystallins, as reflected by TUNEL assay using TMR-dUTP. (<b>B</b>) STS-induced caspase activation was decreased in 661W cells overexpressing αA- and αB-crystallins, as measured by colorimetric Caspase-3/-7 assay. (* p<0.005 by <i>t</i>-test for pWPI <i>versus</i> pWPIαA). Data are the mean ± SE of four independent experiments.</p

Interaction of α-crystallins with Bax <i>in vivo</i>.

<p>293T cells transiently transfected with the empty vector (pWPI), myc-tagged αA- (pWPI_aA) or αB- (pWPI_aB) crystallin were further treated with 100 nM STS for 3 h before co-immunoprecipitation with anti-Bax antibody. The precipitated samples were then sequentially probed by western blot using anti-myc and anti-Bax antibodies. IP: immunoprecipitated samples (<i>left panels</i>); CE: 20 µg of total proteins from whole cell extract (<i>right panels</i>).</p

Expression of αA- and αB-crystallins in transiently transfected 293T cells.

<p>(<b>A</b>) Western blot analysis of αA- and αB-crystallin levels 24 h post-transfection. Fifty micrograms of total proteins from cell extracts were subjected to 12% SDS-PAGE and immunoassayed with anti-αA/αB-crystallin to detect the overexpressed α-crystallins and with anti-ß-actin as a control of equal protein loading. (<b>B</b>) Immunofluorescence analysis with anti-αA/αB-crystallin showing cytoplasmic expression of αA- (pcDNA3.1-αA) and αB- (pcDNA3.1-αB) crystallins 24 h post-transfection, while no detection was observed in cells transfected with the empty plasmid (pcDNA3.1).</p

Progression of cell death in inherited RD models.

<p>Depending on the causative genetic insult, the temporal development of retinal degeneration is highly variable in the different animal models. The quantification of dying, TUNEL-positive photoreceptor cells in the outer nuclear layer (ONL) allowed determination of the evolution and the peak of photoreceptor death for each of these animal models (<b>A</b>). The peak was taken as reference point for the ensuing analysis of cell death mechanisms. The bar graph (<b>B</b>) shows a comparison of maximum peak heights for all ten RD models studied. Note the different scales in line graphs. Values are mean ± SEM from at least three different animals. See also <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112142#pone.0112142.s004" target="_blank">Table S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0112142#pone.0112142.s005" target="_blank">S2</a>.</p

Apoptosis in the retina is restricted to the S334ter rat model.

<p>The analysis of BAX expression, mitochondrial cytochrome c release, activation of caspase-9 and -3 shows essentially no positive detection in 9 out of 10 animal models for hereditary retinal degeneration. The notable exception was the S334ter transgenic rat which harbours a mutation in the rhodopsin gene leading to a truncated protein and in which many photoreceptors were positive for apoptosis. In all other animal models, while there were cells displaying clear evidence for apoptosis, their numbers were within the wild-type levels, indicating that this was related to physiological, developmental cell death, which is characteristic for the postnatal rodent retina. Importantly, the numbers of apoptotic cells did not match the numbers of mutation-induced dying cells as evidenced by the TUNEL assay. Scale bar 20 µm.</p

RD animal models used and their genetic defects.

<p>The cartoon illustrates the anatomical localization and metabolic consequences of the causative genetic mutations in the ten different RD animal models used in this study. RD causing mutations in these animal models interfere with the various stages of the phototransduction cascade, from the 11-cis-retinal recycling enzyme RPE65 (<i>Rpe65</i> KO), via the light-sensitive Rhodopsin (<i>Rho</i> KO, P23H, S334ter), cGMP-hydrolyzing phosphodiesterase-6 (PDE6; <i>rd1</i>, <i>rd10</i>, <i>cpfl1</i>), the structural protein Peripherin (<i>Prph2</i>; <i>rd2</i>), to the cyclic-nucleotide-gated (CNG; <i>Cngb1</i> KO, <i>Cnga3</i> KO) channel that allows for Ca²⁺-influx.</p