Bax-Induced Apoptosis in Leber's Congenital Amaurosis: A Dual Role in Rod and Cone Degeneration

Pathogenesis in the Rpe65−/− mouse model of Leber's congenital amaurosis (LCA) is characterized by a slow and progressive degeneration of the rod photoreceptors. On the opposite, cones degenerate rapidly at early ages. Retinal degeneration in Rpe65−/− mice, showing a null mutation in the gene encoding the retinal pigment epithelium 65-kDa protein (Rpe65), was previously reported to depend on continuous activation of a residual transduction cascade by unliganded opsin. However, the mechanisms of apoptotic signals triggered by abnormal phototransduction remain elusive. We previously reported that activation of a Bcl-2-dependent pathway was associated with apoptosis of rod photoreceptors in Rpe65−/− mice during the course of the disease. In this study we first assessed whether activation of Bcl-2-mediated apoptotic pathway was dependent on constitutive activation of the visual cascade through opsin apoprotein. We then challenged the direct role of pro-apoptotic Bax protein in triggering apoptosis of rod and cone photoreceptors

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

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

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

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

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

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

New functions and signaling mechanisms for the class of adhesion G protein-coupled receptors.

The class of adhesion G protein-coupled receptors (aGPCRs), with 33 human homologs, is the second largest family of GPCRs. In addition to a seven-transmembrane α-helix-a structural feature of all GPCRs-the class of aGPCRs is characterized by the presence of a large N-terminal extracellular region. In addition, all aGPCRs but one (GPR123) contain a GPCR autoproteolysis-inducing (GAIN) domain that mediates autoproteolytic cleavage at the GPCR autoproteolysis site motif to generate N- and a C-terminal fragments (NTF and CTF, respectively) during protein maturation. Subsequently, the NTF and CTF are associated noncovalently as a heterodimer at the plasma membrane. While the biological function of the GAIN domain-mediated autocleavage is not fully understood, mounting evidence suggests that the NTF and CTF possess distinct biological activities in addition to their function as a receptor unit. We discuss recent advances in understanding the biological functions, signaling mechanisms, and disease associations of the aGPCRs