Synaptic pathology in retinoschisis knockout (Rs1-/y) mouse retina and modification by rAAV-Rs1 gene delivery.
Synaptic pathology in retinoschisis knockout (Rs1-/y) mouse retina and modification by rAAV-Rs1 gene delivery.
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DOI：
10.1167/iovs.07-1071
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发表时间：
2008-08
影响因子：
4.4
通讯作者：
Sieving PA
中科院分区：
文献类型：
--
作者：
Takada Y;Vijayasarathy C;Zeng Y;Kjellstrom S;Bush RA;Sieving PA
在早期，视网膜裂素敲除（Rs1-KO）小鼠视网膜发生进行性光感受器变性，并伴有外丛状层（OPL）的严重破坏，随着年龄的增长，这种破坏会减少。视网膜电图（ERG）发生平行变化。来自双极细胞的b波振幅在年轻时不成比例地减少到光感受器的a波，但在老年时却没有。研究不同年龄Rs1-KO小鼠OPL蛋白表达及形态学变化，探讨突触层在这些ERG变化中的作用。采用光镜、电镜和生物化学方法对野生型（Wt）和Rs1-KO小鼠出生后7 ~ 12个月的视网膜进行了观察。通过免疫荧光和免疫电镜对PSD95（突触后密度蛋白）、mGluR6（代谢性谷氨酸受体亚型6）、视黄质裂素（Rs1）、<s:1> ller细胞蛋白胶质纤维酸性蛋白（GFAP）和谷氨酰胺合成酶（GS）、双极细胞标记蛋白激酶Cα (PKCα)和水平细胞标记calbindin进行定位。定量Western blot检测PSD95和mGluR6水平。在P14时单眼玻璃内注射rAAV(2/2)-CMV-Rs1治疗的Rs1-KO小鼠，在8个月时通过视野反射性ERG反应和视网膜免疫组织化学进行评估。Rs1与野生型（Wt）视网膜突触膜外表面相关。PSD95和mGluR6在Rs1-KO视网膜的OPL中与P14并列，这表明突触结构已经形成。在P14时，Wt和Rs1-KO的视网膜光镜形态相似，但在P21时，Rs1-KO的OPL被破坏，一些PSD95和mGluR6在外核层（ONL）错位。GFAP表达跨越所有视网膜层。EM显示突触结构靠近感光核。Western blot检测1个月时PSD95和mGluR6水平正常，4个月时分别下降到Wt的59% （P < 0.001）和55% （P < 0.05）。此后12个月的水平没有进一步下降。注射AAV-Rs1的眼睛在8个月时通过免疫组化研究，与未注射的眼睛相比，PSD95和mGluR6的表达更高，GFAP的表达更低。在Rs1-KO小鼠中，视网膜层的形成和OPL中突触蛋白的表达在P14之前是正常的，这意味着突触连接的正常发育。P21对突触蛋白的异常定位表明，在光敏受体数量和突触蛋白水平正常的年轻时期，发育和/或成熟突触的移位导致b波减少。Rs1-KO视网膜1- 12个月间PSD95和mGluR6的下降反映了b波变化的过程，为ERG和OPL变化之间的因果关系提供了证据。这些发现以及Rs1基因转移治疗后OPL结构完整性和b波振幅的改善为解释该模型中视网膜信号的变化提供了细胞和分子基础。（Invest Ophthalmol Vis Sci. 2008; 49:3677-3686） DOI:10.1167/iovs.07-1071
At an early age, the retinoschisin knockout (Rs1-KO) mouse retina has progressive photoreceptor degeneration with severe disruption of the outer plexiform layer (OPL) that decreases at older ages. The electroretinogram (ERG) undergoes parallel changes. The b-wave amplitude from bipolar cells is reduced disproportionately to the photoreceptor a-wave at young but not at older ages. The protein expression and morphology of the OPL in Rs1-KO mice was investigated at different ages, to explore the role of the synaptic layer in these ERG changes. Retinas of wild-type (Wt) and Rs1-KO mice from postnatal day (P)7 to 12 months were evaluated by light and electron microscopy (EM) and biochemistry. PSD95 (postsynaptic density protein), mGluR6 (metabotropic glutamate receptor subtype 6), retinoschisin (Rs1), the Müller cell proteins glial fibrillary acidic protein (GFAP) and glutamine synthetase (GS), the bipolar cell marker protein kinase C alpha (PKCα), and the horizontal cell marker calbindin were localized by immunofluorescence and immuno-EM. Levels of PSD95 and mGluR6 were determined by quantitative Western blot. Rs1-KO mice treated by intravitreous injection of rAAV(2/2)-CMV-Rs1 in one eye at P14 were evaluated at 8 months by full-field scotopic ERG responses and retinal immunohistochemistry. Rs1 was associated with the outer surface of synaptic membranes in wild-type (Wt) retinas. PSD95 and mGluR6 were juxtaposed in the OPL of the Rs1-KO retinas by P14, implying that synaptic structures are formed. Light microscopic retinal morphology was similar in Wt and Rs1-KO at P14, but by P21, the OPL was disrupted in Rs1-KO, and some PSD95 and mGluR6 was mislocalized in the outer nuclear layer (ONL). GFAP expression spanned all retinal layers. EM showed synaptic structures adjacent to photoreceptor nuclei. PSD95 and mGluR6 levels were normal at 1 month on Western blot but declined to 59% (P < 0.001) and 55% (P < 0.05) of Wt, respectively, by 4 months. Levels thereafter showed no further reduction out to 12 months. Eyes injected with AAV-Rs1 were studied at 8 months by immunohistochemistry and had higher expression of PSD95 and mGluR6 and less GFAP expression compared with fellow untreated eyes. In the Rs1-KO mouse, retinal layer formation and synaptic protein expression in the OPL is normal up to P14, implying normal development of synaptic connections. Aberrant localization of synaptic proteins by P21 indicates that displacement of developing and/or mature synapses contributes to the b-wave reduction at young ages, when photoreceptor numbers and synaptic protein levels are normal. The subsequent decline in PSD95 and mGluR6 between 1 and 12 months in Rs1-KO retina mirrors the course of b-wave change and provides evidence of causal relationship between the ERG and OPL changes. These findings and the improved structural integrity of the OPL and b-wave amplitude after Rs1 gene transfer therapy provide a cellular and molecular basis for interpreting the changes in retinal signaling in this model. (Invest Ophthalmol Vis Sci. 2008; 49:3677-3686) DOI:10.1167/iovs.07-1071
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