Significance: The function of progressive rod-cone degeneration (PRCD) protein has remained a mystery since its gene and associated mutation were discovered as one of the most common causes of retinal degeneration in dogs. Furthermore, numerous mutations in the protein have since been identified to cause retinal degeneration in human patients diagnosed with retinitis pigmentosa. Here, we generated a PRCD knockout mouse and found that PRCD functions at the site of photoreceptor disc morphogenesis where it is required to keep newly forming discs flat as they protrude from the plasma membrane. Without PRCD, the newly forming discs bulge and release extracellular vesicles, which accumulate in the extracellular space and ultimately lead to retinal pathology.

The light-sensitive outer segment of the vertebrate photoreceptor is a highly modified primary cilium filled with disc-shaped membranes that provide a vast surface for efficient photon capture. The formation of each disc is initiated by a ciliary membrane evagination driven by an unknown molecular mechanism reportedly requiring actin polymerization. Since a distinct F-actin network resides precisely at the site of disc morphogenesis, we employed a unique proteomic approach to identify components of this network potentially driving disc morphogenesis. The only identified actin nucleator was the Arp2/3 complex, which induces the polymerization of branched actin networks. To investigate the potential involvement of Arp2/3 in the formation of new discs, we generated a conditional knockout mouse lacking its essential ArpC3 subunit in rod photoreceptors. This knockout resulted in the complete loss of the F-actin network specifically at the site of disc morphogenesis, with the time course of ArpC3 depletion correlating with the time course of F-actin loss. Without the actin network at this site, the initiation of new disc formation is completely halted, forcing all newly synthesized membrane material to be delivered to the several nascent discs whose morphogenesis had already been in progress. As a result, these discs undergo uncontrolled expansion instead of normal enclosure, which leads to formation of unusual, large membrane whorls. These data suggest a model of photoreceptor disc morphogenesis in which Arp2/3 initiates disc formation in a “lamellipodium-like” mechanism.

Retinal photoreceptor cells contain the highest concentration of docosahexaenoic acid (DHA) in our bodies, and it has been long assumed that this is critical for supporting normal vision. Indeed, early studies using DHA dietary restriction documented reduced light sensitivity by DHA-deprived retinas. Recently, it has been demonstrated that a major route of DHA entry in the retina is the delivery across the blood–retina barrier by the sodium-dependent lipid transporter, Mfsd2a. This discovery opened a unique opportunity to analyze photoreceptor health and function in DHA-deprived retinas using theMfsd2aknock-out mouse as animal model. Our lipidome analyses ofMfsd2a^−/−retinas and outer segment membranes corroborated the previously reported decrease in the fraction of DHA-containing phospholipids and a compensatory increase in phospholipids containing arachidonic acid. We also revealed an increase in the retinal content of monounsaturated fatty acids and a reduction in very long chain fatty acids. These changes could be explained by a combination of reduced DHA supply to the retina and a concomitant upregulation of several fatty acid desaturases controlled by sterol regulatory element-binding transcription factors, which are upregulated inMfsd2a^−/−retinas.Mfsd2a^−/−retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructureMfsd2a^−/−photoreceptors and their ability to produce light responses were essentially normal. These data demonstrate that, whereas maintaining the lysophosphatidylcholine route of DHA supply to the retina is essential for long-term photoreceptor survival, it is not important for supporting normal phototransduction.SIGNIFICANCE STATEMENTPhospholipids containing docosahexaenoic acid (DHA) are greatly enriched in the nervous system, with the highest concentration found in the light-sensitive membranes of photoreceptor cells. In this study, we analyzed the consequences of impaired DHA transport across the blood–retina barrier. We have found that, in addition to a predictable reduction in the DHA level, the affected retinas undergo a complex, transcriptionally-driven rebuilding of their membrane lipidome in a pattern preserving the overall saturation/desaturation balance of retinal phospholipids. Remarkably, these changes do not affect the ability of photoreceptors to produce responses to light but are detrimental for the long-term survival of these cells.

KIF17, a kinesin-2 motor that functions in intraflagellar transport, can regulate the onset of photoreceptor outer segment development. However, the function of KIF17 in a mature photoreceptor remains unclear. Additionally, the ciliary localization of KIF17 is regulated by a C-terminal consensus sequence (KRKK) that is immediately adjacent to a conserved residue (mouse S1029/zebrafish S815) previously shown to be phosphorylated by CaMKII. Yet, whether this phosphorylation can regulate the localization, and thus function, of KIF17 in ciliary photoreceptors remains unknown. Using transgenic expression in zebrafish photoreceptors, we show that phospho-mimetic KIF17 has enhanced localization along the cone outer segment. Importantly, expression of phospho-mimetic KIF17 is associated with greatly enhanced turnover of the photoreceptor outer segment through disc shedding in a cell-autonomous manner, while genetic mutants of kif17 in zebrafish and mice have diminished disc shedding. Lastly, cone expression of constitutively active tCaMKII leads to a kif17-dependent increase in disc shedding. Taken together, our data support a model in which phosphorylation of KIF17 promotes its photoreceptor outer segment localization and disc shedding, a process essential for photoreceptor maintenance and homeostasis. While disc shedding has been predominantly studied in the context of the mechanisms underlying phagocytosis of outer segments by the retinal pigment epithelium, this work implicates photoreceptor-derived signaling in the underlying mechanisms of disc shedding.

Purpose: We examined outer retinal remodeling of the euthermic and torpid cone-dominant 13-lined ground squirrel (13-LGS) retina using optical coherence tomography (OCT) imaging and histology. Methods: Retinas and corneas of living 13-LGSs were imaged during euthermic and torpid physiological states using OCT. Retinal layer thickness was measured at the visual streak from registered and averaged vertical B-scans. Following OCT, some retinas were collected immediately for postmortem histologic comparison using light microscopy, immunofluorescence, or transmission electron microscopy. Results: Compared to OCT images from euthermic retinae, OCT images of torpid retinae revealed significantly thicker inner and outer nuclear layers, as well as increases in the distances between outer retinal reflectivity bands 1 and 2, and bands 3 and 4. A significant decrease in the distance between bands 2 and 3 also was seen, alongside significant thinning of the choriocapillaris and choroid. OCT image quality was reduced in torpid eyes, partly due to significant thickening of the corneal stroma during this state. Conclusions: The torpid retina of the hibernating 13-LGS undergoes structural changes that can be detected by OCT imaging. Comparisons between in vivo OCT and ex vivo histomorphometry may offer insight to the origin of hyperreflective OCT bands within the outer retina of the cone-dominant 13-LGS.