α₂δ-4 Is Required for the Molecular and Structural Organization of Rod and Cone Photoreceptor Synapses

Abstract

αδ-4 is an auxiliary subunit of voltage-gated Ca1.4 L-type channels that regulate the development and mature exocytotic function of the photoreceptor ribbon synapse. In humans, mutations in the gene encoding αδ-4 cause heterogeneous forms of vision impairment in humans, the underlying pathogenic mechanisms of which remain unclear. To investigate the retinal function of αδ-4, we used genome editing to generate an αδ-4 knock-out (αδ-4 KO) mouse. In male and female αδ-4 KO mice, rod spherules lack ribbons and other synaptic hallmarks early in development. Although the molecular organization of cone synapses is less affected than rod synapses, horizontal and cone bipolar processes extend abnormally in the outer nuclear layer in αδ-4 KO retina. In reconstructions of αδ-4 KO cone pedicles by serial block face scanning electron microscopy, ribbons appear normal, except that less than one-third show the expected triadic organization of processes at ribbon sites. The severity of the synaptic defects in αδ-4 KO mice correlates with a progressive loss of Ca1.4 channels, first in terminals of rods and later cones. Despite the absence of b-waves in electroretinograms, visually guided behavior is evident in αδ-4 KO mice and better under photopic than scotopic conditions. We conclude that αδ-4 plays an essential role in maintaining the structural and functional integrity of rod and cone synapses, the disruption of which may contribute to visual impairment in humans with mutations. In the retina, visual information is first communicated by the synapse formed between photoreceptors and second-order neurons. The mechanisms that regulate the structural integrity of this synapse are poorly understood. Here we demonstrate a role for αδ-4, a subunit of voltage-gated Ca channels, in organizing the structure and function of photoreceptor synapses. We find that presynaptic Ca channels are progressively lost and that rod and cone synapses are disrupted in mice that lack αδ-4. Our results suggest that alterations in presynaptic Ca signaling and photoreceptor synapse structure may contribute to vision impairment in humans with mutations in the gene encoding αδ-4.

A New Zebrafish Model for CACNA2D4-Dysfunction

“…In this study we identified two zebrafish orthologs of human CACNA2D4, harboring the canonical protein domain compo-sition typical of all a 2 d family members, indicative of functional conservation. We found that Cacna2d4b is essential for synaptic expression of Cacna1fa in photoreceptors of larval zebrafish, comparable to mouse Cacna2d4 23,24 and other a 2 d subunits. [51][52][53] In zebrafish, the two paralogs show partial subfunctionalization: cacna2d4a-KO did not affect Cacna1fa expression, which is consistent with its more prominent expression in the INL.…”

“…Interestingly, even though we found the cone ERG responses to be significantly impaired, the amplitude of the b-wave was only slightly reduced, which is in accordance with the rather mild ERG defect in patients, 7,22,46 but in contrast to the more severe phenotype in cacna2d4-KO mice. 6,23,24 Furthermore, we were surprised to find that loss of Cacna2d4a and b together with the substantial reduction in Cacna1fa levels did not elicit a stronger effect on ERG response as well as on ribbon synapse structure and general retinal morphology in double-KO. This is particularly surprising in light of the strong defects in cacna1fa-KO zebrafish and the fact that not only Cacna1fa was proven important for ribbon synapse development and maintenance in zebrafish, 20 but also Cacna2d4 was shown to have a similar function in murine rod photoreceptors.…”

“…Of particular interest was the outer plexiform layer (OPL), the most affected retinal layer in cacna1fa-KO zebrafish 20 and cacna2d4 mutant mice. 6,23,24 Loss of any of the a 2 d 4 subunits leads to decreased eye size at 5 dpf (Fig. 5B).…”

“…Therefore, cacna2d4-KO zebrafish is a suitable model to study the pathophysiological mechanisms underlying CACNA2D4 dysfunction in human.Keywords: calcium channel, cone photoreceptor, zebrafish, disease model Recent studies on two different Cacna2d4-KO mice suggest a role for Cacna2d4 in structural organization and function of rod 23,24 and cone synapses. 24 Interestingly, in all three studies, Cacna2d4 mutation affects rods more severely than cones, and…”

“…20 In addition, mice with synaptopathies, including Cacna2d4-KO mice, show outgrowth of horizontal cell and bipolar cell synapses into the ONL, where they form ectopic synapses with photoreceptors. 4,17,23,24,[48][49][50] Therefore, the ectopic punctate Cacna1fa and Ribeyeb expression might reflect the presynaptic portion of such ectopically formed synapses. On the postsynaptic side, however, we did not detect changes in mGluR6b localization (Fig.…”