Effects on photoreceptor cell death in Q344X fish.

<p>(A and B) Retina sections of eyes from rhodopsin Q344X transgenic at 5 dpf. Animals were reared in constant darkness (A) or in constant light (B). Light exposure reduces the survival of rod photoreceptor cells. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.) Light accelerated the rod cell death. (C) Graph of the number of rod photoreceptors in rhodopsin Q344X transgenic fish at 5 dpf. Darkness and light exposure are compared. (Bars mean SD, ** means p<0.01.) (D and E) Eye sections of eyes treated by anti-transducin morpholinos (E) and control MO (D) in Q344X at 5 dpf. Suppression of transducin α expression enhances the survival of rod photoreceptor cells. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.). (F) Graph of the number of rods in Q344X, control morpholino-treated and anti-transducin morpholinos. (Bars mean SD, * means p<0.05.) (G and H) Eye sections of eyes treated by anti-phosphodiesterase 6β morpholinos (H) and control MO (G) in Q344X at 5 dpf. Suppression of phosphodiesterase expression reduces the survival of rod photoreceptor cells. Rod photoreceptors are visualized with EGFP (Bar = 100 µm.). (I) Graph of the number of rods in Q344X, control morpholino-treated and anti-phosphodiesterase 6β morpholinos. (Bars mean SD, ** means p<0.01.) (J and K) Eye ections of Q344X transgenic fish bred in SQ22536-treated water (K) and normal control water (J) at 5 dpf. Rod photoreceptors are visualized by EGFP (Bar = 100 µm.) ADCY antagonist rescued rod photoreceptor cell death. (L) Graph of the number of rod photoreceptor cells in Q344X 5 dpf. Black dots indicate control and red dots indicate SQ22536-treated (10, 20 and 100 mM) water. (Bars mean SD, * means p<0.05.)</p

Inhibitor of ADCY suppresses photoreceptor cell death.

<p>(A–D) Sections of <i>ovl</i> mutants bred in SQ22536-treated water (B–D) and control water (A). (E) The number of surviving rod photoreceptors from <i>ovl</i> mutants in control water (black dots) and SQ22536-treated water. SQ22536 increased survival rod photoreceptors in concentrations of 1 and 10 mM. (Bars mean SD, * means p<0.05.)</p

Rod photoreceptor cell death in rhodopsin Q344X transgenic fish.

<p>(A) RT-PCR analysis of expression of ectopic rhodopsin Q344X transgene. (B) Sequence analysis of transgene in Q344X animal at 5 dpf. (C–H) Sections of normal rhodopsin fish at 3 dpf (C), 5 dpf (E), 7 dpf (G) and rhodopsin Q344X transgenic fish at 3 dpf (D), 5 dpf (F), 7 dpf (H). Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (Bar = 100 µm.) (I) Graph of the number of rod photoreceptor of normal rhodopsin and rhodopsin Q344X mutant at 3, 5 and 7 dpf. (Bars mean SD, * means p<0.05, ** means p<0.01.) Rod photoreceptors decreased by 5 dpf. (J and K) Immunohistochemistry sections of retina of wild-type (J) and Q344X (K) animal. F-actin is visualized with phalloidin (red), rod opsin with antibodies (green) and nuclei with Hoechst33342 (blue). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) Cell localization of rhodopsin is abnormal in Q344X. (L and M) TUNEL (green) assay of sections of normal rhodopsin (L) and rhodopsin Q344X transgenic (M) animals. F-actin is visualized with phalloidin (red), and nuclei with DAPI (blue). Arrows indicate TUNEL positive photoreceptor cells. TUNEL staining in ONL was observed only in Q344X. (N) Graph of the number of TUNEL assay positive cells, comparing normal rhodopsin (black dots) and rhodopsin Q344X (red dots) transgenic animals. (Bars mean SD, * means p<0.05.)</p

SQ22536 treatment of <i>rd10</i> mice.

<p>(A and B) HE (Hematoxilin-Eosin) stained sections from eyes of <i>rd10</i> mice at P28. Control PBS treated eye (A) and SQ22536 treated eye (B). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.). (C) Graph of the thickness of INL (outlined bar) and ONL (solid bar) in <i>rd10</i> mice at P28. Control group (black) and SQ treated group (red) are compared. (Bars mean SD, * means p<0.05.) (D) Graph of the ONL/INL ratio of SQ22536 treated control (black bar) and untreated retina (red bar) in <i>rd10</i> mice. (E) Schematic illustration of adenylyl cyclase and apoptosis in rod photoreceptors. OS: outer segment, CC: connecting cilium, IS: inner segment, R: rhodopsin, T: transducin, AC: adenylyl cyclase.</p

Treatment with cAMP analogue, cGMP analogue, and KT5720 in <i>ovl</i>.

<p>(A–E) Eye sections at 5 dpf <i>ovl</i> treated with different concentration of a cAMP analogue, 8-Bromo-cAMP. 8-Bromo-cAMP (B–E) or control water (A). Rod photoreceptors are visualized by EGFP (green) and F-actin by phalloidin (red). (Bar = 100 µm.) (F) Graph of survival rod photoreceptors of <i>ovl</i> mutants in control water (black dots) and cAMP analogue-treated water. cAMP analogue accelerated rod photoreceptor death. (Bars mean SD, * means p<0.05.) (G and H) Eye sections at 5 dpf <i>ovl</i> treated with an cGMP analogue, 8-Bromo-cGMP. (H) or control water (G). Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (I) Graph of survival of <i>ovl</i> mutant rod photoreceptors in control water (black dots) and cGMP analogue-treated water. cGMP does not accelerate rod photoreceptor death. (Bars mean SD.) (J and K) Eye sections at 5 dpf <i>ovl</i> treated with KT5720 (K) or control water (J). Rod photoreceptors are visualized by EGFP (green) and F-actin by phalloidin (red). (L) Graph of survival of <i>ovl</i> mutant rod photoreceptors in control water (black dots) and KT5720 analogue-treated water. KT5720 suppresses rod photoreceptor death. (Bars mean SD, * means p<0.05.)</p

Mislocalized ADCY in rod outer segments induces photoreceptor cell death.

<p>(A) Expression analysis of adenylyl cyclases in wild-type retina by RT-PCR. (B) RT-PCR analysis of recombinant adenylyl cyclase 2B from wild-type (lane1), ADCY RHO tail (−) (lane2) and ADCY RHO tail (+) (lane3). Lanes 4 to 6 are B-actin expression of each group. Ectopic expressions were confirmed. (C) Immunohistochemistry (IHC) section of retina of wild-type. F-actin is visualized with phalloidin (red), ADCY2 with antibodies (green) and nuclei with Hoechst33342 (blue). OS: outer segment, IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) ADCY did not expressed at OS. (D) Schematic diagrams of over-expression constructs. ADCY RHO tail (−) and (+) are downstream of zebrafish RH1 promoter between tol2 arms. (E and F) IHC sections of retina of ADCY RHO tail (−) fish (E) and ADCY RHO tail (+) fish (F) at 14 dpf. F-actin is visualized with phalloidin (red), ADCY2 with antibodies (green) and nuclei with Hoechst33342 (blue). Arrows indicate outer segments. IS: inner segment, ONL: outer nuclear layer (Bar = 10 µm.) ADCY is mis-localized at OS in only tail(+) animals. (G and H) Eye sections of ADCY RHO tail (−) and (+) animals at 14 dpf. Rod photoreceptors are visualized with EGFP (green) and F-actin with phalloidin (red). (Bar = 100 µm.) The number of rod photoreceptors was significantly decreased in tail (+) animals. (I) Graph of the number of rod photoreceptor of ADCY RHO tail (−) (black dots) and (+) (red dots). (Bars mean SD, ** means p<0.01.) (J and K) TUNEL (green) assay of sections in ADCY RHO tail (−) (J) and (+) (K) animals. F-actin is visualized with phalloidin (red), and nuclei with DAPI (blue). The signals of outer nuclear layer were observed only in tail (+) animals. (L) Magnification of the white square in (K). INL: inner uclear layer, ONL: outer nuclear layer. (M) Graph of the number of TUNEL assay positive cells, comparing ADCY RHO tail (−) (black dots) and (+) (red dots) animals. (Bars mean SD, ** means p<0.01.)</p

Dynamic Increase in Extracellular ATP Accelerates Photoreceptor Cell Apoptosis via Ligation of P2RX7 in Subretinal Hemorrhage

<div><p>Photoreceptor degeneration is the most critical cause of visual impairment in age-related macular degeneration (AMD). In neovascular form of AMD, severe photoreceptor loss develops with subretinal hemorrhage due to choroidal neovascularization (CNV), growth of abnormal blood vessels from choroidal circulation. However, the detailed mechanisms of this process remain elusive. Here we demonstrate that neovascular AMD with subretinal hemorrhage accompanies a significant increase in extracellular ATP, and that extracellular ATP initiates neurodegenerative processes through specific ligation of Purinergic receptor P2X, ligand-gated ion channel, 7 (P2RX7; P2X7 receptor). Increased extracellular ATP levels were found in the vitreous samples of AMD patients with subretinal hemorrhage compared to control vitreous samples. Extravascular blood induced a massive release of ATP and photoreceptor cell apoptosis in co-culture with primary retinal cells. Photoreceptor cell apoptosis accompanied mitochondrial apoptotic pathways, namely activation of caspase-9 and translocation of apoptosis-inducing factor (AIF) from mitochondria to nuclei, as well as TUNEL-detectable DNA fragmentation. These hallmarks of photoreceptor cell apoptosis were prevented by brilliant blue G (BBG), a selective P2RX7 antagonist, which is an approved adjuvant in ocular surgery. Finally, in a mouse model of subretinal hemorrhage, photoreceptor cells degenerated through BBG-inhibitable apoptosis, suggesting that ligation of P2RX7 by extracellular ATP may accelerate photoreceptor cell apoptosis in AMD with subretinal hemorrhage. Our results indicate a novel mechanism that could involve neuronal cell death not only in AMD but also in hemorrhagic disorders in the CNS and encourage the potential application of BBG as a neuroprotective therapy.</p> </div

Clinical characteristics of patients with MH, ERM, and AMD with VH.

<p>The patients underwent pars plana vitrectomy and the collected vitreous samples were subjected to ATP measurement by luciferase assay. There were no significant differences in age or sex ratio among the three groups.</p

Photoreceptor cell apoptosis in primary retinal cell cultures with blood clots.

<p>(A) Schematic image of the double chamber co-culture system of primary retinal cells and blood clots. (B) and (C) The viability of primary retinal cells in the lower chamber was accessed by calcein AM or MitoTracker CMTMRos after 24 h of culture with addition of a clot in the upper chamber (calcein in green, CMTMRos in red, recoverin in blue). The frequency of calcein⁺ or CMTMRos⁺ photoreceptors significantly decreased after incubation with clots. Apyrase treatment significantly rescued photoreceptors. (D<b>)</b> The ATP levels of culture medium in the lower chamber were significantly increased by clot exposure, and reversed by apyrase treatment. (E<b>)</b> The ATP levels in plasma and blood. <i>n = </i>10 per group; **<i>P<</i>0.01. Scale bar: 20 µm.</p

Photoreceptor cell apoptosis by subretinal injection of ATP.

<p>Exogenous ATP was injected into the subretinal space of Wt or P2rx7^−/− mice. TUNEL-positive apoptotic cells developed in the ONL 24 h after the subretinal injection of 1 mM ATP (TUNEL in green and Hoechst 33342 in blue). <i>n = </i>6 per group; **<i>P<</i>0.01. Scale bar: 20 µm.</p