Expression of Rho^P23H-EGFP in transgenic <i>Xenopus</i>.

<p>(<b>a</b>) Schematic drawing (<i>left</i>) of the <i>Xenopus</i> rod photoreceptor. The inner (IS) and outer (OS) segments, the nucleus (N), the prominent endoplasmic reticulum (ER), Golgi (G), connecting cilium (CC) and mitochondria (M) located apically in the IS are shown. The OS contains numerous stacks of disk membranes. (<b>b</b>) A segment of OS is shown in an electron micrograph. Scale bar, 100 nm. (<b>c</b>) A molecular model of the OS disk membrane (from a segment of a single disk, <i>white box in b</i>). The density of rhodopsin, approximately 90% of the protein in the OS disk membranes <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030101#pone.0030101-Papermaster1" target="_blank">[32]</a> is illustrated to scale in the molecular homology model based upon the high-resolution bovine rhodopsin structure <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030101#pone.0030101-Okada1" target="_blank">[33]</a>. The rhodopsin-phospholipid molar ratio is presented to scale. (<b>e, f</b>) Representative images of sections of live <i>Xenopus</i> retina showing rod cells expressing either Rho-EGFP (e) or Rho^P23H-EGFP (f) The DIC image of a small piece of retina (<i>left</i>) and the corresponding three dimensional rendering of a confocal laser scanning z-stack using EGFP detection (<i>middle</i>) are shown. The outer and inner segments are labeled. To illustrate the range of transgene expression, a concentration heat map (red, maximum intensity) is shown (<i>right</i>). Scale bar, 5 µm.</p

Quantification of transgene expression from dual rhodopsin cassettes in <i>Xenopus</i> rods.

<p>(<b>a, b</b>) Measurement of the EGFP and mCherry fluorescence distributions in cells expressing Rho-mCherry/Rho-EGFP (a) and Rho-mCherry-Rho^P23H-EGFP (b). Control rods with no fluorescent protein had only background fluorescence which was set to zero. Images show the mCherry (<i>red</i>), EGFP (<i>green</i>) and merged distributions for representative rods. The merged image (a, <i>right</i>) shows synchronized changes in the expression level of both transgenes (Rho- EGFP and Rho-mCherry). The concentration of both proteins is comparable along the rod axis. (b) The Rho^P23H-EGFP expression level is significantly lower (b, <i>middle</i>) than the co-expressed wild type Rho-mCherry (b, <i>left</i>). The inset in panel (b, <i>middle</i>) shows the intensified EGFP channel of the original figure to clarify its profile. (<b>c, d</b>) Distributions were measured along the central axis of the cell (<i>white line</i>), converted to concentration using the calibration in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030101#pone.0030101.s003" target="_blank">Fig. S3</a> and plotted as a function of distance from the OS base. The dual wild type cassettes are shown in (c) at the same scale for both green and red channels, while for the dual cassette containing Rho^P23HEGFP (d), the EGFP values (<i>green</i>) are given on right Y-axis (<i>green</i>) and the mCherry values (<i>red</i>) on left Y-axis. The distance is measured from the base of the rod OS. The arrowhead indicates fluorescent foci in the Rho^P23H-EGFP rod. Scale bar, 5 µm.</p

Quantification of rhodopsin-EGFP transgenes in Xenopus rods.

<p>*<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030101#pone.0030101-Besharse1" target="_blank">[17]</a>.</p>§<p>Estimated from serial section western blot data <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030101#pone.0030101-Sokolov1" target="_blank">[34]</a>.</p

Aberrant expression and aggregation of Rho^P23H-EGFP in rod photoreceptors.

<p>Confocal images of representative live cells expressing rhodopsin-EGFP fusion proteins show distributions of Rho-EGFP (<b>a</b>) and mutant opsin, Rho^P23H-EGFP (<b>b</b>). DIC (<i>top</i>) and fluorescence (<i>middle</i>) images were used to calculate the fluorescence distribution, displayed as a heat map (<i>bottom</i>): red for most intense, green for mid-level and blue for least intense. The border between the IS and OS is indicated by the arrow. (<b>c</b>) The fluorescence profile distribution was computed along the z-spline path through the center of the cell (<i>white line</i>), with the origin arbitrarily set in the nuclear region for each cell. The fluorescence intensity was normalized to the maximum value along the spline. (<b>d</b>) High resolution images of a representative live rod expressing fluorescent protein from two rhodopsin cassettes: Rho-mCherry (<i>red</i>, encoding wild type opsin) and Rho^P23H-EGFP (<i>green</i>). The fluorescence intensity profile of two transgenes was determined simultaneously, and the individual distributions (mCherry, <i>top</i> and EGFP, <i>middle</i> are shown together with the distributions merged onto a DIC image (<i>bottom</i>). (<b>e</b>) Quantification of the fluorescent foci in two OS axial locations in live rods expressing Rho^P23H-EGFP. The OS was divided into two sections (I and II), comprising ∼60% of the length. The more distal region of the OS was not included to avoid regions prone to swelling or other <i>in vitro</i> damage. The number of isolated fluorescent foci in the outer segment regions was counted in a total of 27 cells from 16 transgenic tadpoles expressing Rho^P23H-EGFP. (<b>f</b>) Representative cross-sectional view of an OS from a live rod expressing two rhodopsin cassettes: Rho-mCherry and Rho^P23H-EGFP. The <i>z</i>-section for the CSLM was parallel to the rod axis, and the fluorescence from each channel is shown separately and merged. Scale bars, 5 µm.</p

Schematic model to explain OS defect formation in disks that express mutant opsin.

<p>Wild type rhodopsin (<i>red</i>) distributes randomly throughout the disk membrane (except incisures which are not shown). In rods expressing Rho^P23H (<i>green</i>), there is an initial random distribution of mutant with wild type protein in the disk, although the concentration of mutant protein is much lower than wild type protein. According to this model, over time, the Rho^P23H mutant begins to self-associate and form aggregates in the membrane, excluding wild type protein. The resulting mutant protein concentrates in a localized area that causes deformation or defects in the membrane structure, leading to vesiculation and disk breakdown. This could lead to structural instability in the OS, initiating a breakdown and potentially rod death.</p

Defects in OS disk membranes in rods expressing Rho^P23H-EGFP.

<p>(<b>a–e</b>) Images from a rod expressing fluorescent protein from two rhodopsin cassettes: Rho^P23H-EGFP and Rho-mCherry (<b>b</b> and <b>c</b>, respectively). Both the heat map representation of Rho^P23H-EGFP distribution (b, <i>arrow</i>) and reconstruction images of the cell fluorescence show Rho^P23H-EGFP fluorescent foci (<b>e</b>, <i>arrow and dotted lines</i>) near the base of the OS that correspond to inhomogeneous OS disturbances of the DIC image (d, <i>arrows</i>). Scale bars (a–e), 5 µm. (<b>f–i</b>) Electron micrographs of rod OS from transgenic retina expressing either Rho-EGFP (f, g) or Rho^P23H-EGFP (h, i). Membranes from Rho^P23H-EGFP transgenic animals exhibited vesiculotubular structures (h, i, <i>arrowheads</i>) of similar size to the fluorescent foci found in CSLM imaging. These structures were not observed in micrographs from Rho-EGFP transgenic animals (f). Mechanical disruption of retina prior to fixation (g) showed OS breaks and disk membrane separations but no vesiculotubular structures. Micrographs from four animals were examined for each group. (<b>j–m</b>) Electron micrographs of retina from wild-type (j) or Rho^P23H (GHL) transgenic mice (l). Membranes from Rho^P23H transgenic animals exhibited vesiculotubular structures (m, <i>arrowheads</i>). Vesiculotubular structures were not observed in micrographs from wild type animals (j, k). Scale bars, 100 nm (g, i, <i>k, m</i>), 200 nm (<i>f, h</i>) and 2 µm (j, l).</p

Ablation of the Proapoptotic Genes Chop or Ask1 Does Not Prevent or Delay Loss of Visual Function in a P23H Transgenic Mouse Model of Retinitis Pigmentosa

<div><p>The P23H mutation in rhodopsin (Rho^P23H) is a prevalent cause of autosomal dominant retinitis pigmentosa. We examined the role of the ER stress proteins, Chop and Ask1, in regulating the death of rod photoreceptors in a mouse line harboring the Rho^P23H rhodopsin transgene (<i>GHL⁺</i>). We used knockout mice models to determine whether Chop and Ask1 regulate rod survival or retinal degeneration. Electrophysiological recordings showed similar retinal responses and sensitivities for <i>GHL⁺</i>, <i>GHL⁺/Chop^−/−</i> and <i>GHL⁺/Ask1^−/−</i> animals between 4–28 weeks, by which time all three mouse lines exhibited severe loss of retinal function. Histologically, ablation of <i>Chop</i> and <i>Ask1</i> did not rescue photoreceptor loss in young animals. However, in older mice, a regional protective effect was observed in the central retina of <i>GHL⁺/Chop^−/−</i> and <i>GHL⁺/Ask1^−/−</i>, a region that was severely degenerated in <i>GHL⁺</i> mice. Our results show that in the presence of the Rho^P23H transgene, the rate of decline in retinal sensitivity is similar in <i>Chop</i> or <i>Ask1</i> ablated and wild-type retinas, suggesting that these proteins do not play a major role during the acute phase of photoreceptor loss in <i>GHL⁺</i> mice. Instead they may be involved in regulating secondary pathological responses such as inflammation that are upregulated during later stages of disease progression.</p></div

G3U inducible system in mammalian cell culture.

<p>(A) Diagram of G3U system using luciferase (pCMV:G3 and pUAS:Luciferase) or mCherry reporters (top, pCMV:G3 and pUAS:mCherry). A CMV promoter drives transcription of a chimeric transcription factor, G3, which encodes contains a GAL4 DNA binding domain, the VP16 transcription activation domain, a rat glucocorticoid receptor binding domain (GR) and eGFP. Synthesized G3 protein localizes to the cytosol. Dex treatment triggers the dimerization of G3, which translocates into the nucleus. Nuclear G3 activates a second construct containing five tandem repeats of the UAS sequence upstream of the hsp70 minimal promoter. The reporter gene (luciferase or mCherry) is under the control of this system. (B) Luciferase assay of G3U inducible system in cell culture. HEK293T cells transfected with pCMV:G3 and pUAS:Luciferase were lysed and luciferase activity measured at different concentrations (0-80 µM) and treatment durations (2-24 hr) of Dex. Relative luciferase activity is plotted as a function of duration and Dex concentration. (C) Live cell imaging of G3 translocation after induction. HEK293 cells were transfected with pCMV:G3 and pUAS:mCherry and induced with 10 μM Dex at 27°C and 37°C. Confocal images were taken before and 20 min after induction; G3 (eGFP), nucleus (Hoechst). Scale bar is 10 μm. (D) Nuclear translocation rate of G3 in HEK293T cells at different temperatures. Fluorescence intensity was measure in nuclear and cytoplasm of live 293T cells at 27°C and 37 °C. Dex (10 μM) was added and mixed into medium. Error bars represent standard deviation (n = 17 at 37 °C and n = 15 at 27 °C). (E) mCherry reporter expression after induction. HEK293T cells transfected with pCMV:G3-GFP and pUAS:mCherry were induced with 10 μM Dex and fixed at different times after induction. G3 (eGFP) and mCherry images show the movement of G3 and expression of mCherry. Scale bar is 10 μm.</p

ERG changes as a function of age in <i>GHL⁺</i> and <i>GHL⁺/Chop^−/−</i> mice.

<p>Maximal a- and b-wave amplitudes as a function of age in C57BL/6 (blue trace), <i>Chop^−/−</i> (orange trace) <i>GHL⁺</i> (black trace) and <i>GHL⁺/Chop^−/−</i> (red trace) mice. Colored lines are polynomial fits.</p

Disk displacement measured from the spatial distribution of induction response peak.

<p>(A) Diagram of the Dex treatment paradigm. (B) Correlation of distance of the peak response to the IS/OS junction and time of Dex treatment (Error bar is standard deviation, dash line is the linear regression line. (C) Histogram of peak-to-peak distances in following repetitive inductions with the eight day paradigm. The distance distribution was fit to a Gaussian curve with an R² = 0.96. The mean of peak-peak distance was 8.0 μm (SD = 2.4, n = 72).</p