Our study indicates a circadian regulation of the expression and secretion of retinoschisin in the chick retina. Protein expression and secretion of retinoschisin were higher during the subjective night and lower during the subjective day, and the highest mRNA expression was 4 hours more advanced than the peak of protein expression. The mRNA and protein rhythms of retinoschisin have the same patterns in constant darkness (DD) as in LD cycles (data not shown). In addition, we found that Ras, MAP kinase Erk, and CaMKII were part of the circadian output pathway regulating the rhythmicity of retinoschisin. This Ras-MAP kinase-CaMKII pathway also regulates the affinity rhythm of cGMP-gated ion channels to cGMP21,25
and the circadian expression of L-type VGCCs.24
Therefore, the Ras-MAP kinase-CaMKII pathway may serve as the “universal” output pathway in regulating photoreceptor physiology.
Interestingly, the rhythmicity of retinoschisin was concurrent with the circadian rhythms of L-type VGCC currents. Blockage of L-type VGCCs dampened the retinoschisin rhythm but did not completely block the secretion of retinoschisin. By contrast, the release of melatonin is an L-type VGCC-dependent process. Nitrendipine, an L-type VGCC blocker, inhibits the melatonin synthesis enzyme arylalkylamine N
-acetyltransferase, and it also blocks the release of melatonin in chick retina photoreceptors.16,17
Hence, L-type VGCCs played a role in the circadian regulation of retinoschisin content and secretion, but the molecular mechanism underlying retinoschisin secretion did not entirely depend on L-type VGCCs. Even though inhibition of L-type VGCCs did not completely inhibit the secretion of retinoschisin, we cannot rule out the possibility that the secretion of retinoschisin could be a calcium-dependent process.
Retinoschisin is known to serve as an anchor protein in maintaining the architecture of the retina synapses, especially around the photoreceptor synapses.1,10,13
Normally, upon secretion, retinoschisin interacts with proteins and phospholipids at the surfaces of photoreceptor membranes of the inner segments and the outer plexiform layer,34,35
and retinoschisin forms a stabilizing scaffold as a multimolecular complex for retinal synapses.36
The retinas of patients with human X-linked retinoschisis (XLRS) display macular atrophy.4,37
The electroretinogram (ERG) recordings from XLRS patients show that the synaptic currents between photoreceptors and bipolar cells (b-wave) are significantly altered, and the cone-driven ERG responses are more severely affected than rod-driven responses.4,37
In retinoschisin-deficient mice, the number of photoreceptors decreases with photoreceptor displacement,10
and the extracellular space increases in the region of photoreceptor ribbon synapses.10
Replacement of the RS1
gene in retinoschisin-knockout mice leads to an improvement in retinal structure and function.11,12
Hence, retinoschisin is believed to play an important role in maintaining the proper architecture of the retina during development.1,10
However, the protein expression of retinoschisin remains high throughout adulthood, and there is an especially heavy concentration of retinoschisin along the inner segments and synaptic regions of photoreceptors.13
Therefore, retinoschisin could have functions other than maintaining retinal architecture during development.
Photoreceptors undergo daily cycling changes in retinomotor movement of inner segments,29,38,39
outer segment disc shedding and membrane renewal,28,40,41
morphologic changes at synaptic ribbons,42,43
and functional properties of ion channels,21,24
among other photoreceptor activities in vertebrates. At photoreceptor synapses, the length and the shape of the photoreceptor synaptic ribbons change over 24-hour daily cycles in mice.42,43
The number of synaptic ribbons in photoreceptor terminals of fish retinas also changes on a circadian cycle.47
All of the evidence described above points to the circadian control of synaptic plasticity in the retina in vivo. The circadian expression of retinoschisin presented here supports the notion that retinoschisin plays an important role in daily photoreceptor synaptic plasticity and in maintaining photoreceptor stability during inner segment retinomotor movement and outer segment renewal during 24-hour cycles.
The ultrastructure and the length of synaptic ribbons are under circadian control but also respond to illumination.42,43,48
The ribbons form protrusions and release them into the cytoplasm within 30 to 60 minutes after lights on; the reverse occurs within 30 minutes after lights off.48
In a similar fashion, retinoschisin can respond to acute illumination changes, but this response depends on previous light exposure experience. We found that brief light exposure caused an increase of retinoschisin at night (ZT 16) under LD cycles and during the subjective day (at CT 4, in DD), whereas acute light exposure failed to elicit a transient increase of retinoschisin during the subjective night (at CT 16) and in embryos that were never exposed to the light before. Hence, retinoschisin plays an important role in the circadian regulation of photoreceptor physiology and function, and it may also participate in the circadian-dependent, and illumination-dependent, synaptic plasticity at ribbon synapses.