Mansergh Fiona, Orton Noelle C, Vessey John P, Lalonde Melanie R, Stell William K, Tremblay Francois, Barnes Steven, Rancourt Derrick E, Bech-Hansen N Torben
Department of Oncology, University of Calgary, Canada.
Hum Mol Genet. 2005 Oct 15;14(20):3035-46. doi: 10.1093/hmg/ddi336. Epub 2005 Sep 9.
Retinal neural transmission represents a key function of the eye. Identifying the molecular components of this vital process is helped by studies of selected human genetic eye disorders. For example, mutations in the calcium channel subunit gene CACNA1F cause incomplete X-linked congenital stationary night blindness (CSNB2 or iCSNB), a human retinal disorder with abnormal electrophysiological response and visual impairments consistent with a retinal neurotransmission defect. To understand the subcellular basis of this retinal disorder, we generated a mouse with a loss-of-function mutation by inserting a self-excising Cre-lox-neo cassette into exon 7 of the murine orthologue, Cacna1f. Electroretinography of the mutant mouse revealed a scotopic a-wave of marginally reduced amplitude compared with the wild-type mouse and absence of the post-receptoral b-wave and oscillatory potentials. Cone ERG responses together with visual evoked potentials and multi-unit activity in the superior colliculus were also absent. Calcium imaging in Fluo-4 loaded retinal slices depolarized with KCl showed 90% less peak signal in the photoreceptor synapses of the Cacna1f mutant than in wild-type mice. The absence of post-receptoral ERG responses and the diminished photoreceptor calcium signals are consistent with a loss of Ca((2+)) channel function in photoreceptors. Immunocytochemistry showed no detectable Ca(v)1.4 protein in the outer plexiform layer of Cacna1f-mutant mice, profound loss of photoreceptor synapses, and abnormal dendritic sprouting of second-order neurons in the photoreceptor layer. Together, these findings in the Cacna1f-mutant mouse reveal that the Ca(v)1.4 calcium channel is vital for the functional assembly and/or maintenance and synaptic functions of photoreceptor ribbon synapses. Moreover, the outcome of this study provides critical clues to the pathophysiology of the human retinal channelopathy of X-linked incomplete CSNB.
视网膜神经传递是眼睛的一项关键功能。对特定人类遗传性眼病的研究有助于确定这一重要过程的分子成分。例如，钙通道亚基基因CACNA1F的突变会导致不完全X连锁先天性静止性夜盲（CSNB2或iCSNB），这是一种人类视网膜疾病，其电生理反应异常，视觉障碍与视网膜神经传递缺陷一致。为了了解这种视网膜疾病的亚细胞基础，我们通过将一个自我切除的Cre-lox-neo盒插入小鼠同源基因Cacna1f的第7外显子中，产生了一个功能丧失突变的小鼠。对突变小鼠进行视网膜电图检查发现，与野生型小鼠相比，暗视a波的幅度略有降低，且没有感受器后b波和振荡电位。锥体视网膜电图反应以及上丘中的视觉诱发电位和多单位活动也不存在。在用氯化钾去极化的加载了Fluo-4的视网膜切片中进行钙成像显示，Cacna1f突变体的光感受器突触中的峰值信号比野生型小鼠少90%。感受器后视网膜电图反应的缺失和光感受器钙信号的减弱与光感受器中Ca(2+)通道功能的丧失一致。免疫细胞化学显示，在Cacna1f突变小鼠的外网状层中未检测到Ca(v)1.4蛋白，光感受器突触严重丧失，光感受器层中的二级神经元出现异常树突发芽。总之，在Cacna1f突变小鼠中的这些发现表明，Ca(v)1.4钙通道对于光感受器带状突触的功能组装和/或维持以及突触功能至关重要。此外，这项研究的结果为X连锁不完全CSNB的人类视网膜通道病的病理生理学提供了关键线索。
