Mori Naoki, Ishiba Yasutsugu, Kubota Shinya, Kobayashi Akira, Higashide Tomomi, McLaren Margaret J, Inana George
Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida 33136, USA.
Invest Ophthalmol Vis Sci. 2006 Apr;47(4):1281-92. doi: 10.1167/iovs.05-0493.
To characterize the time course of apoptosis and degeneration in a transgenic mouse model of retinal degeneration based on truncated mutant HRG4; to investigate the nature of binding of the mutant HRG4 to its target, ADP-ribosylation factor-like (ARL)2; to study its effects on the downstream molecules Binder-of-ARL2 (BART) and adenine nucleotide transporter (ANT)-1 and on the induction of apoptosis.
Saturation binding, microscopic morphometric, Western blot, immunofluorescence, and TUNEL analyses were used.
Increased apoptosis did not occur until 20 months in the transgenic retina, consistent with the delayed-onset degeneration in this model. The truncated HRG4 protein exhibited approximately threefold greater affinity for ARL2 than the wild-type HRG4, likely resulting in nonfunctional sequestration of ARL2. A significant decrease in ARL2 was present by 20 months, accompanied by a 50% decrease in ANT-1 in the photoreceptor synaptic mitochondria, with evidence of mitochondrial dysfunction. Preapoptotic degeneration in the photoreceptor synapse was demonstrated with cytochrome c release and caspase 3 activation within the synapse-without evidence of TUNEL-positive apoptosis in the photoreceptor cell body-indicating an initial event in the synapse leading to apoptosis. Caspase 3 was activated in the accompanying secondary neuron, consistent with transsynaptic degeneration.
The results support a novel mechanism of retinal degeneration in which preapoptotic degeneration starts in the photoreceptor synapse because of a deficiency in ANT-1 and spreads to the secondary neuron transsynaptically, followed by apoptosis and degeneration in the cell body of the photoreceptor.
在基于截短突变体HRG4的视网膜变性转基因小鼠模型中，描述细胞凋亡和变性的时间进程；研究突变体HRG4与其靶标ADP核糖基化因子样（ARL）2的结合性质；研究其对下游分子ARL2结合蛋白（BART）和腺嘌呤核苷酸转运体（ANT）-1的影响以及对细胞凋亡诱导的影响。
采用饱和结合、显微镜形态计量、蛋白质印迹、免疫荧光和TUNEL分析。
转基因视网膜中直到20个月才出现凋亡增加，这与该模型中延迟发生的变性一致。截短的HRG4蛋白对ARL2的亲和力比野生型HRG4高约三倍，可能导致ARL2的无功能隔离。到20个月时，ARL2显著减少，同时光感受器突触线粒体中的ANT-1减少50%，有证据表明存在线粒体功能障碍。光感受器突触中的细胞色素c释放和半胱天冬酶3激活证明了光感受器突触中的凋亡前期变性，而光感受器细胞体中没有TUNEL阳性凋亡的证据，这表明突触中的初始事件导致细胞凋亡。半胱天冬酶3在伴随的次级神经元中被激活，这与跨突触变性一致。
结果支持一种视网膜变性的新机制，即由于ANT-1缺乏，凋亡前期变性始于光感受器突触，并通过跨突触扩散到次级神经元，随后光感受器细胞体发生凋亡和变性。
