Molecular genetics of retinal degeneration: A Drosophila perspective

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP₂, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.     

Molecular genetics of retinal degeneration

Inherited retinal degeneration in Drosophila has been explored for insights into similar processes in humans. Based on the mechanisms, I divide these mutations in Drosophila into three classes. The first consists of genes that control the specialization of photoreceptor cells including the morphogenesis of visual organelles (rhabdomeres) that house the visual signaling proteins. The second class contains genes that regulate the activity or level of the major rhodopsin, Rh1, which is the light sensor and also provides a structural role for the maintenance of rhabdomeres. Some mutations in Rh1 (NinaE) are dominant due to constitutive activity or folding defects, like autosomal dominant retinitis pigmentosa (ADRP) in humans. The third class consists of genes that control the Ca²⁺ influx directly or indirectly by promoting the turnover of the second messenger and regeneration of PIP_2, or mediate the Ca²⁺-dependent regulation of the visual response. These gene products are critical for the increase in cytosolic Ca²⁺ following light stimulation to initiate negative regulatory events. Here I will focus on the signaling mechanisms underlying the degeneration in norpA, and in ADRP-type NinaE mutants that produce misfolded Rh1. Accumulation of misfolded Rh1 in the ER triggers the unfolded protein response (UPR), while endosomal accumulation of activated Rh1 may initiate autophagy in norpA. Both autophagy and the UPR are beneficial for relieving defective endosomal trafficking and the ER stress, respectively. However, when photoreceptors fail to cope with the persistence of these stresses, a cell death program is activated leading to retinal degeneration.     

Arrestin1 mediates light-dependent rhodopsin endocytosis and cell survival

BACKGROUND: Arrestins are pivotal, multifunctional organizers of cell responses to GPCR stimulation, including cell survival and cell death. In Drosophila norpA and rdgC mutants, endocytosis of abnormally stable complexes of rhodopsin (Rh1) and fly photoreceptor Arrestin2 (Arr2) triggers cell death, implicating Rh1/Arr2-bearing endosomes in pro-cell death signaling, potentially via arrestin-mediated GPCR activation of effector kinase pathways. In order to further investigate arrestin function in photoreceptor physiology and survival, we studied Arr2's partner photoreceptor arrestin, Arr1, in developing and adult Drosophila compound eyes. RESULTS: We report that Arr1, but not Arr2, is essential for normal, light-induced rhodopsin endocytosis. Also distinct from Arr2, Arr1 is essential for light-independent photoreceptor survival. Photoreceptor cell death caused by loss of Arr1 is strongly suppressed by coordinate loss of Arr2. We further find that Rh1 C-terminal phosphorylation is essential for light-induced endocytosis and also for translocation of Arr1, but not Arr2, from dark-adapted photoreceptor cytoplasm to photosensory membrane rhabdomeres. In contrast to a previous report, we do not find a requirement for photoreceptor myosin kinase NINAC in Arr1 or Arr2 translocation. CONCLUSIONS: The two Drosophila photoreceptor arrestins mediate distinct and essential cell pathways downstream of rhodopsin activation. We propose that Arr1 mediates an endocytotic cell-survival activity, scavenging phosphorylated rhodopsin and thereby countering toxic Arr2/Rh1 accumulation; elimination of toxic Arr2/Rh1 in double mutants could thus rescue arr1 mutant photoreceptor degeneration.     

Rhodopsin activation causes retinal degeneration in Drosophila rdgC mutant

Drosophila rdgC (retinal degeneration-C) mutants show normal retinal morphology and photoreceptor physiology at young ages. Dark-reared rdgC flies retain this wild-type phenotype, but light-reared mutants undergo retinal degeneration. rdgC photoreceptors with low levels of rhodopsin as a result of vitamin A deprivation or a mutant rhodopsin (ninaE) gene fail to show rdgC-induced degeneration even after prolonged light treatment, demonstrating that degeneration occurs as a result of light stimulation of rhodopsin. Analysis of norpA; rdgC flies shows that the norpA-encoded phospholipase C, the target enzyme of the G protein activated by rhodopsin, is not required for rdgC-induced degeneration. Thus the rdgC+ gene product is required to prevent retinal degeneration that results from a previously unrecognized consequence of rhodopsin stimulation.     

Role of asparagine-linked oligosaccharides in rhodopsin maturation and association with its molecular chaperone, NinaA

Many proteins require N-linked glycosylation for conformational maturation and interaction with their molecular chaperones. In Drosophila, rhodopsin (Rh1), the most abundant rhodopsin, is glycosylated in the endoplasmic reticulum (ER) and requires its molecular chaperone, NinaA, for exit from the ER and transport through the secretory pathway. Studies of vertebrate rhodopsins have generated several conflicting proposals regarding the role of glycosylation in rhodopsin maturation. We investigated the role of Rh1 glycosylation and Rh1/NinaA interactions under in vivo conditions by analyzing transgenic flies expressing Rh1 with isoleucine substitutions at each of the two consensus sites for N-linked glycosylation (N20I and N196I). We show that Asn(20) is the sole site for glycosylation. The Rh1(N20I) protein is retained within the secretory pathway, causing an accumulation of ER cisternae and dilation of the Golgi complex. NinaA associates with nonglycosylated Rh1(N20I); therefore, retention of nonglycosylated rhodopsin within the ER is not due to the lack of Rh1(N20I)/NinaA interaction. We further show that Rh1(N20I) interferes with wild type Rh1 maturation and triggers a dominant form of retinal degeneration. We conclude that during maturation Rh1 is present in protein complexes containing NinaA and that Rh1 glycosylation is required for transport of the complexes through the secretory pathway. Failure of this transport process leads to retinal degeneration.     

A Fluorescence-Based Genetic Screen to Study Retinal Degeneration in Drosophila

The Drosophila visual system has been proved to be a powerful genetic model to study eye disease such as retinal degeneration. Here, we describe a genetic method termed “Rh1::GFP ey-flp/hid” that is based on the fluorescence of GFP-tagged major rhodopsin Rh1 in the eyes of living flies and can be used to monitor the integrity of photoreceptor cells. Through combination of this method and ERG recording, we examined a collection of 667 mutants and identified 18 genes that are required for photoreceptor cell maintenance, photoresponse, and rhodopsin synthesis. Our findings demonstrate that this “Rh1::GFP ey-flp/hid” method enables high-throughput F1 genetic screens to rapidly and precisely identify mutations of retinal degeneration.     

Light activation, adaptation, and cell survival functions of the Na+/Ca2+ exchanger CalX

In sensory neurons, Ca(2+) entry is crucial for both activation and subsequent attenuation of signaling. Influx of Ca(2+) is counterbalanced by Ca(2+) extrusion, and Na(+)/Ca(2+) exchange is the primary mode for rapid Ca(2+) removal during and after sensory stimulation. However, the consequences on sensory signaling resulting from mutations in Na(+)/Ca(2+) exchangers have not been described. Here, we report that mutations in the Drosophila Na(+)/Ca(2+) exchanger calx have a profound effect on activity-dependent survival of photoreceptor cells. Loss of CalX activity resulted in a transient response to light, a dramatic decrease in signal amplification, and unusually rapid adaptation. Conversely, overexpression of CalX had reciprocal effects and greatly suppressed the retinal degeneration caused by constitutive activity of the TRP channel. These results illustrate the critical role of Ca(2+) for proper signaling and provide genetic evidence that Ca(2+) overload is responsible for a form of retinal degeneration resulting from defects in the TRP channel.     

Regulation of the rhodopsin protein phosphatase, RDGC, through interaction with calmodulin.

Hundreds of G protein-coupled receptors (GPCRs) and at least six GPCR kinases have been identified, but the only GPCR phosphatase that has been definitively demonstrated is the rhodopsin phosphatase encoded by the rdgC locus of Drosophila. Mutations in rdgC result in defects in termination of the light response and cause severe retinal degeneration. In the current work, we demonstrate that RDGC binds to calmodulin, and a mutation in an IQ motif that eliminates the calmodulin/RDGC interaction prevents dephosphorylation of rhodopsin in vivo and disrupts termination of the photoresponse. Our data indicate that RDGC is a novel calmodulin-dependent protein phosphatase and raise the possibility that regulation of other GPCRs through dephosphorylation may be controlled by calmodulin-dependent protein phosphatases related to RDGC.     

Drosophila crumbs is required to inhibit light-induced photoreceptor degeneration

Mutations in the human transmembrane protein CRB1 are associated with severe forms of retinal dystrophy, retinitis pigmentosa 12 (RP12), and Leber's congenital amaurosis (LCA). The Drosophila homolog, crumbs, is required for polarity and adhesion in embryonic epithelia and for correct formation of adherens junctions and proper morphogenesis of photoreceptor cells. Here, we show that mutations in Drosophila crumbs result in progressive, light-induced retinal degeneration. Degeneration is prevented by expression of p35, an inhibitor of apoptosis, or by reduction of rhodopsin levels through a vitamin A-deficient diet. In the dark, rhabdomeres survive but exhibit morphogenetic defects. We demonstrate that it is the extracellular portion of the Crumbs protein that is essential to suppress light-induced programmed cell death, while proper morphogenesis depends on the intracellular part. We conclude that human and Drosophila Crumbs proteins are functionally conserved to prevent light-dependent photoreceptor degeneration. This experimental system is now ideally suited to study the genetic and molecular basis of RP12- and LCA-related retinal degeneration.     

A diffusible factor from normal retinal cells promotes rod photoreceptor survival in an in vitro model of retinitis pigmentosa

Transgenic mice expressing a dominant mutation in the gene for the phototransduction molecule rhodopsin undergo retinal degeneration similar to that experienced by patients with the retinal degenerative disease, retinitis pigmentosa (RP). Although the mutation is thought to cause photoreceptor degeneration in a cell‐autonomous manner, the fact that rod photoreceptor degeneration is slowed in chimeric wild‐type/mutant mice suggests that cellular interactions are also important for maintaining photoreceptor survival. To more fully characterize the nature of the cellular interactions important for rod degeneration in the RP mutant mice, we have used an in vitro approach. We found that when the retinas of the transgenic mice were isolated from the pigmented epithelium and cultured as explants, the rod photoreceptors underwent selective degeneration with a similar time course to that observed in vivo. This selective rod degeneration also occurred when the cells were dissociated and cultured as monolayers. These data indicate that the mutant rod photoreceptors degenerate when removed from their normal cellular relationships and without contact with the pigmented epithelium, thus confirming the relative cell autonomy of the mutant phenotype. We next tested whether normal retinal cells could rescue the mutant photoreceptors in a coculture paradigm. Coculture of transgenic mouse with wild‐type mouse or rat retinal cells significantly enhanced transgenic rod photoreceptor survival; this survival‐promoting activity was diffusible through a filter, was heat labile, and not present in transgenic retinal cells. Several peptide growth factors known to be present in the retina were tested as the potential survival‐promoting molecule responsible for the effects of the conditioned medium; however, none of them promoted survival of the photoreceptors expressing the Pro23His mutant rhodopsin. Nevertheless, we were able to demonstrate that the mutant photoreceptors could be rescued by an antagonist to a retinoic acid receptor, suggesting that the endogeneous survival‐promoting activity may function through this pathway. These data thus confirm and extend the findings of previous work that local trophic interactions are important in regulating rod photoreceptor degeneration in retinitis pigmentosa. A diffusible factor found in normal but not transgenic retinal cells has a protective effect on the survival of rod photoreceptors from Pro23His mutant rhodopsin mice. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 475–490, 1999     

Crumbs regulates rhodopsin transport by interacting with and stabilizing myosin V

The evolutionarily conserved Crumbs (Crb) complex is crucial for photoreceptor morphogenesis and homeostasis. Loss of Crb results in light-dependent retinal degeneration, which is prevented by feeding mutant flies carotenoid-deficient medium. This suggests a defect in rhodopsin 1 (Rh1) processing, transport, and/or signaling, causing degeneration; however, the molecular mechanism of this remained elusive. In this paper, we show that myosin V (MyoV) coimmunoprecipitated with the Crb complex and that loss of crb led to severe reduction in MyoV levels, which could be rescued by proteasomal inhibition. Loss of MyoV in crb mutant photoreceptors was accompanied by defective transport of the MyoV cargo Rh1 to the light-sensing organelle, the rhabdomere. This resulted in an age-dependent accumulation of Rh1 in the photoreceptor cell (PRC) body, a well-documented trigger of degeneration. We conclude that Crb protects against degeneration by interacting with and stabilizing MyoV, thereby ensuring correct Rh1 trafficking. Our data provide, for the first time, a molecular mechanism for the light-dependent degeneration of PRCs observed in crb mutant retinas.     

Functional EF-Hands in Neuronal Calcium Sensor GCAP2 Determine Its Phosphorylation State and Subcellular Distribution In Vivo,and Are Essential for Photoreceptor Cell Integrity

The neuronal calcium sensor proteins GCAPs (guanylate cyclase activating proteins) switch between Ca²⁺-free and Ca²⁺-bound conformational states and confer calcium sensitivity to guanylate cyclase at retinal photoreceptor cells. They play a fundamental role in light adaptation by coupling the rate of cGMP synthesis to the intracellular concentration of calcium. Mutations in GCAPs lead to blindness. The importance of functional EF-hands in GCAP1 for photoreceptor cell integrity has been well established. Mutations in GCAP1 that diminish its Ca²⁺ binding affinity lead to cell damage by causing unabated cGMP synthesis and accumulation of toxic levels of free cGMP and Ca²⁺. We here investigate the relevance of GCAP2 functional EF-hands for photoreceptor cell integrity. By characterizing transgenic mice expressing a mutant form of GCAP2 with all EF-hands inactivated (EF⁻GCAP2), we show that GCAP2 locked in its Ca²⁺-free conformation leads to a rapid retinal degeneration that is not due to unabated cGMP synthesis. We unveil that when locked in its Ca²⁺-free conformation in vivo, GCAP2 is phosphorylated at Ser201 and results in phospho-dependent binding to the chaperone 14-3-3 and retention at the inner segment and proximal cell compartments. Accumulation of phosphorylated EF⁻GCAP2 at the inner segment results in severe toxicity. We show that in wildtype mice under physiological conditions, 50% of GCAP2 is phosphorylated correlating with the 50% of the protein being retained at the inner segment. Raising mice under constant light exposure, however, drastically increases the retention of GCAP2 in its Ca²⁺-free form at the inner segment. This study identifies a new mechanism governing GCAP2 subcellular distribution in vivo, closely related to disease. It also identifies a pathway by which a sustained reduction in intracellular free Ca²⁺ could result in photoreceptor damage, relevant for light damage and for those genetic disorders resulting in “equivalent-light” scenarios.     

Inhibiting autophagy reduces retinal degeneration caused by protein misfolding

Mutations in the genes necessary for the structure and function of vertebrate photoreceptor cells are associated with multiple forms of inherited retinal degeneration. Mutations in the gene encoding RHO (rhodopsin) are a common cause of autosomal dominant retinitis pigmentosa (adRP), with the Pro23His variant of RHO resulting in a misfolded protein that activates endoplasmic reticulum stress and the unfolded protein response. Stimulating macroautophagy/autophagy has been proposed as a strategy for clearing misfolded RHO and reducing photoreceptor death. We found that retinas from mice heterozygous for the gene encoding the RHO^P23H variant (hereafter called P23H) exhibited elevated levels of autophagy flux, and that pharmacological stimulation of autophagy accelerated retinal degeneration. In contrast, reducing autophagy flux pharmacologically or by rod-specific deletion of the autophagy-activating gene Atg5, improved photoreceptor structure and function. Furthermore, proteasome levels and activity were reduced in the P23H retina, and increased when Atg5 was deleted. Our findings suggest that autophagy contributes to photoreceptor cell death in P23H mice, and that decreasing autophagy shifts the degradation of misfolded RHO protein to the proteasome and is protective. These observations suggest that modulating the flux of misfolded proteins from autophagy to the proteasome may represent an important therapeutic strategy for reducing proteotoxicity in adRP and other diseases caused by protein folding defects.     

Ectopic expression of a microbial-type rhodopsin restores visual responses in mice with photoreceptor degeneration

The death of photoreceptor cells caused by retinal degenerative diseases often results in a complete loss of retinal responses to light. We explore the feasibility of converting inner retinal neurons to photosensitive cells as a possible strategy for imparting light sensitivity to retinas lacking rods and cones. Using delivery by an adeno-associated viral vector, here, we show that long-term expression of a microbial-type rhodopsin, channelrhodopsin-2 (ChR2), can be achieved in rodent inner retinal neurons in vivo. Furthermore, we demonstrate that expression of ChR2 in surviving inner retinal neurons of a mouse with photoreceptor degeneration can restore the ability of the retina to encode light signals and transmit the light signals to the visual cortex. Thus, expression of microbial-type channelrhodopsins, such as ChR2, in surviving inner retinal neurons is a potential strategy for the restoration of vision after rod and cone degeneration.     

视紫红质及RPE65蛋白在光致视网膜变性疾病中的作用机制

视紫红质是感光细胞中的一种视色素,在光线的接收和视觉电位的产生方面具有重要的生理作用,由视紫红质介导的过度光信号传导是光性视网膜变性的主要原因。近年的研究表明,视网膜色素上皮细胞中的RPE65蛋白作为影响视紫红质再生的关键因素,与视网膜光损伤的易感性密切相关。就视紫红质和RPE65蛋白在光致视网膜变性中的作用机理作一探讨。     

Accumulation of Rhodopsin in Late Endosomes Triggers Photoreceptor Cell Degeneration

Progressive retinal degeneration is the underlying feature of many human retinal dystrophies. Previous work using Drosophila as a model system and analysis of specific mutations in human rhodopsin have uncovered a connection between rhodopsin endocytosis and retinal degeneration. In these mutants, rhodopsin and its regulatory protein arrestin form stable complexes, and endocytosis of these complexes causes photoreceptor cell death. In this study we show that the internalized rhodopsin is not degraded in the lysosome but instead accumulates in the late endosomes. Using mutants that are defective in late endosome to lysosome trafficking, we were able to show that rhodopsin accumulates in endosomal compartments in these mutants and leads to light-dependent retinal degeneration. Moreover, we also show that in dying photoreceptors the internalized rhodopsin is not degraded but instead shows characteristics of insoluble proteins. Together these data implicate buildup of rhodopsin in the late endosomal system as a novel trigger of death of photoreceptor neurons.     

The Gos28 SNARE Protein Mediates Intra-Golgi Transport of Rhodopsin and Is Required for Photoreceptor Survival

SNARE proteins play indispensable roles in membrane fusion events in many cellular processes, including synaptic transmission and protein trafficking. Here, we characterize the Golgi SNARE protein, Gos28, and its role in rhodopsin (Rh1) transport through Drosophila photoreceptors. Mutations in gos28 lead to defective Rh1 trafficking and retinal degeneration. We have pinpointed a role for Gos28 in the intra-Golgi transport of Rh1, downstream from α-mannosidase-II in the medial- Golgi. We have confirmed the necessity of key residues in Gos28''s SNARE motif and demonstrate that its transmembrane domain is not required for vesicle fusion, consistent with Gos28 functioning as a t-SNARE for Rh1 transport. Finally, we show that human Gos28 rescues both the Rh1 trafficking defects and retinal degeneration in Drosophila gos28 mutants, demonstrating the functional conservation of these proteins. Our results identify Gos28 as an essential SNARE protein in Drosophila photoreceptors and provide mechanistic insights into the role of SNAREs in neurodegenerative disease.     

Effect of Purified Murine NGF on Isolated Photoreceptors of a Rodent Developing Retinitis Pigmentosa

A number of different studies have shown that neurotrophins, including nerve growth factor (NGF) support the survival of retinal ganglion neurons during a variety if insults. Recently, we have reported that that eye NGF administration can protect also photoreceptor degeneration in a mice and rat with inherited retinitis pigmentosa. However, the evidence that NGF acts directly on photoreceptors and that other retinal cells mediate the NGF effect could not be excluded. In the present study we have isolated retinal cells from rats with inherited retinitis pigmentosa (RP) during the post-natal stage of photoreceptor degenerative. In presence of NGF, these cells are characterized by enhanced expression of NGF-receptors and rhodopsin, the specific marker of photoreceptor and better cell survival, as well as neuritis outgrowth. Together these observations support the hypothesis that NGF that NGF acts directly on photoreceptors survival and prevents photoreceptor degeneration as previously suggested by in vivo studies.