A highly conserved zebrafish IMPDH retinal isoform produces the majority of guanine and forms dynamic protein filaments in photoreceptor cells.

Inosine monophosphate dehydrogenase (IMPDH) is a key regulatory enzyme in the de novo synthesis of the purine base guanine. Dominant mutations in human IMPDH1 cause photoreceptor degeneration for reasons that are unknown. Here, we sought to provide some foundational information on Impdh1a in the zebrafish retina. We found that in zebrafish, gene subfunctionalization due to ancestral duplication resulted in a predominant retinal variant expressed exclusively in rod and cone photoreceptors. This variant is structurally and functionally similar to the human IMPDH1 retinal variant and shares a reduced sensitivity to GTP-mediated inhibition. We also demonstrated that Impdh1a forms prominent protein filaments in vitro and in vivo in both rod and cone photoreceptor cell bodies, synapses, and to a lesser degree, in outer segments. These filaments changed length and cellular distribution throughout the day consistent with diurnal changes in both mRNA and protein levels. The loss of Impdh1a resulted in a substantial reduction of guanine levels, although cellular morphology and cGMP levels remained normal. Our findings demonstrate a significant role for IMPDH1 in photoreceptor guanine production and provide fundamental new information on the details of this protein in the zebrafish retina.

Mitochondrial glutathione peroxidase 4 is indispensable for photoreceptor development and survival in mice.

Glutathione peroxidase 4 (GPx4) is known for its unique function in the direct detoxification of lipid peroxides in the cell membrane and as a key regulator of ferroptosis, a form of lipid peroxidation-induced nonapoptotic cell death. However, the cytosolic isoform of GPx4 is considered to play a major role in inhibiting ferroptosis in somatic cells, whereas the roles of the mitochondrial isoform of GPx4 (mGPx4) in cell survival are not yet clear. In the present study, we found that mGPx4 KO mice exhibit a cone-rod dystrophy-like phenotype in which loss of cone photoreceptors precedes loss of rod photoreceptors. Specifically, in mGPx4 KO mice, cone photoreceptors disappeared prior to their maturation, whereas rod photoreceptors persisted through maturation but gradually degenerated afterward. Mechanistically, we demonstrated that vitamin E supplementation significantly ameliorated photoreceptor loss in these mice. Furthermore, LC-MS showed a significant increase in peroxidized phosphatidylethanolamine esterified with docosahexaenoic acid in the retina of mGPx4 KO mice. We also observed shrunken and uniformly condensed nuclei as well as caspase-3 activation in mGPx4 KO photoreceptors, suggesting that apoptosis was prevalent. Taken together, our findings indicate that mGPx4 is essential for the maturation of cone photoreceptors but not for the maturation of rod photoreceptors, although it is still critical for the survival of rod photoreceptors after maturation. In conclusion, we reveal novel functions of mGPx4 in supporting development and survival of photoreceptors in vivo.

Photoreceptor phosphodiesterase (PDE6): activation and inactivation mechanisms during visual transduction in rods and cones.

Rod and cone photoreceptors of the vertebrate retina utilize cGMP as the primary intracellular messenger for the visual signaling pathway that converts a light stimulus into an electrical response. cGMP metabolism in the signal-transducing photoreceptor outer segment reflects the balance of cGMP synthesis (catalyzed by guanylyl cyclase) and degradation (catalyzed by the photoreceptor phosphodiesterase, PDE6). Upon light stimulation, rapid activation of PDE6 by the heterotrimeric G-protein (transducin) triggers a dramatic drop in cGMP levels that lead to cell hyperpolarization. Following cessation of the light stimulus, the lifetime of activated PDE6 is also precisely regulated by additional processes. This review summarizes recent advances in the structural characterization of the rod and cone PDE6 catalytic and regulatory subunits in the context of previous biochemical studies of the enzymological properties and allosteric regulation of PDE6. Emphasis is given to recent advances in understanding the structural and conformational changes underlying the mechanism by which the activated transducin α-subunit binds to-and relieves inhibition of-PDE6 catalysis that is controlled by its intrinsically disordered, inhibitory γ-subunit. The role of the regulator of G-protein signaling 9-1 (RGS9-1) in regulating the lifetime of the transducin-PDE6 is also briefly covered. The therapeutic potential of pharmacological compounds acting as inhibitors or activators targeting PDE6 is discussed in the context of inherited retinal diseases resulting from mutations in rod and cone PDE6 genes as well as other inherited defects that arise from excessive cGMP accumulation in retinal photoreceptor cells.

The N termini of the inhibitory γ-subunits of phosphodiesterase-6 (PDE6) from rod and cone photoreceptors differentially regulate transducin-mediated PDE6 activation.

Phosphodiesterase-6 (PDE6) plays a central role in both rod and cone phototransduction pathways. In the dark, PDE6 activity is suppressed by its inhibitory γ-subunit (Pγ). Rhodopsin-catalyzed activation of the G protein transducin relieves this inhibition and enhances PDE6 catalysis. We hypothesized that amino acid sequence differences between rod- and cone-specific Pγs underlie transducin's ability to more effectively activate cone-specific PDE6 than rod PDE6. To test this, we analyzed rod and cone Pγ sequences from all major vertebrate and cyclostome lineages and found that rod Pγ loci are far more conserved than cone Pγ sequences and that most of the sequence differences are located in the N-terminal region. Next we reconstituted rod PDE6 catalytic dimer (Pαß) with various rod or cone Pγ variants and analyzed PDE6 activation upon addition of the activated transducin α-subunit (Gtα*-GTPγS). This analysis revealed a rod-specific Pγ motif (amino acids 9-18) that reduces the ability of Gtα*-GTPγS to activate the reconstituted PDE6. In cone Pγ, Asn-13 and Gln-14 significantly enhanced Gtα*-GTPγS activation of cone Pγ truncation variants. Moreover, we observed that the first four amino acids of either rod or cone Pγ contribute to Gtα*-GTPγS-mediated activation of PDE6. We conclude that physiological differences between rod and cone photoreceptor light responsiveness can be partially ascribed to ancient, highly conserved amino acid differences in the N-terminal regions of Pγ isoforms, demonstrating for the first time a functional role for this region of Pγ in the differential activation of rod and cone PDE6 by transducin.

The small GTPase RAB28 is required for phagocytosis of cone outer segments by the murine retinal pigmented epithelium.

RAB28, a member of the RAS oncogene family, is a ubiquitous, farnesylated, small GTPase of unknown function present in photoreceptors and the retinal pigmented epithelium (RPE). Nonsense mutations of the human RAB28 gene cause recessive cone-rod dystrophy 18 (CRD18), characterized by macular hyperpigmentation, progressive loss of visual acuity, RPE atrophy, and severely attenuated cone and rod electroretinography (ERG) responses. In an attempt to elucidate the disease-causing mechanism, we generated Rab28-/- mice by deleting exon 3 and truncating RAB28 after exon 2. We found that Rab28-/- mice recapitulate features of the human dystrophy (i.e. they exhibited reduced cone and rod ERG responses and progressive retina degeneration). Cones of Rab28-/- mice extended their outer segments (OSs) to the RPE apical processes and formed enlarged, balloon-like distal tips before undergoing degeneration. The visual pigment content of WT and Rab28-/- cones was comparable before the onset of degeneration. Cone phagosomes were almost absent in Rab28-/- mice, whereas rod phagosomes displayed normal levels. A protein-protein interaction screen identified several RAB28-interacting proteins, including the prenyl-binding protein phosphodiesterase 6 δ-subunit (PDE6D) and voltage-gated potassium channel subfamily J member 13 (KCNJ13) present in the RPE apical processes. Of note, the loss of PDE6D prevented delivery of RAB28 to OSs. Taken together, these findings reveal that RAB28 is required for shedding and phagocytosis of cone OS discs.

Sex-related differences in the progressive retinal degeneration of the rd10 mouse.

The retinal degeneration 10 (rd10) mouse is a model of autosomal recessive retinitis pigmentosa (RP), a disease that causes blindness through the progressive loss of photoreceptors. This study shows evidence of sex-related differences in RP onset and progression in rd10 retinas. The disease onset was considerably earlier in the female rd10 mice than in the male rd10 mice, as evidenced by a loss of PDE6ß proteins and rod-dominated electroretinogram (ERG) responses at an early age. Single photopic flash and flicker ERG responses and immunolabeling of opsin molecules were analyzed in both genders to assess the sex differences in the degeneration of cones in the RP retinas. The averaged amplitudes of cone-mediated ERG responses obtained from the females were significantly smaller than the amplitudes of the responses from the age-matched males in the late stages of the RP, suggesting that cones might degenerate faster in the female retinas as the disease progressed. The rapid degeneration of cones caused a more substantial decrease in the ERG responses derived from the On-pathway than the Off-pathway in the females. In addition, the male rd10 mice had heavier body weights than their female counterparts aged between postnatal (P)18 and P50 days. In summary, female rd10 mice were more susceptible to retinal degeneration, suggesting that the female sex might be a risk factor for RP. The results have important implications for future studies exploring potential sex-related differences in RP development and progression in the clinic.

CO2/bicarbonate modulates cone photoreceptor ROS-GC1 and restores its CORD6-linked catalytic activity.

This study with recombinant reconstituted system mimicking the cellular conditions of the native cones documents that photoreceptor ROS-GC1 is modulated by gaseous CO2. Mechanistically, CO2 is sensed by carbonic anhydrase (CAII), generates bicarbonate that, in turn, directly targets the core catalytic domain of ROS-GC1, and activates it to increased synthesis of cyclic GMP. This, then, functions as a second messenger for the cone phototransduction. The study demonstrates that, in contrast to the Ca2+-modulated phototransduction, the CO2 pathway is Ca2+-independent, yet is linked with it and synergizes it. It, through R787C mutation in the third heptad of the signal helix domain of ROS-GC1, affects cone-rod dystrophy, CORD6. CORD6 is caused firstly by lowered basal and GCAP1-dependent ROS-GC1 activity and secondly, by a shift in Ca2+ sensitivity of the ROS-GC1/GCAP1 complex that remains active in darkness. Remarkably, the first but not the second defect disappears with bicarbonate thus explaining the basis for CORD6 pathological severity. Because cones, but not rods, express CAII, the excessive synthesis of cyclic GMP would be most acute in cones.

Success of Gene Therapy in Late-Stage Treatment.

Retinal gene therapy has yet to achieve sustained rescue after disease onset - perhaps because transduction efficiency is insufficient ("too little") and/or the disease is too advanced ("too late") in humans. To test the latter hypothesis, we used a mouse model for retinitis pigmentosa (RP) that allowed us to restore the mutant gene in all diseased rod photoreceptor cells, thereby generating optimally treated retinas. We then treated mice at an advanced disease stage and analyzed the rescue. We showed stable, sustained rescue of photoreceptor structure and function for at least 1 year, demonstrating gene therapy efficacy after onset of degeneration. The results suggest that RP patients are treatable, even when the therapy is administered at late disease stages.

Overexpression of Type 3 Iodothyronine Deiodinase Reduces Cone Death in the Leber Congenital Amaurosis Model Mice.

Leber congenital amaurosis (LCA) is a devastating pediatric retinal degenerative disease, accounting for 20% of blindness in children attending schools for the blind. Mutations in the RPE65 gene, which encodes the retinal pigment epithelium-specific isomerohydrolase RPE65, account for 16% of all LCA cases. Recent findings have linked cone photoreceptor viability to thyroid hormone (TH) signaling. TH signaling regulates cell proliferation, differentiation, and metabolism. At the cellular level, TH action is regulated by the two iodothyronine deiodinases, DIO2 and DIO3. DIO2 converts the prohormone thyroxine (T4) to the bioactive hormone triiodothyronine (T3), and DIO3 inactivates T3 and T4. The present work investigates the effects of overexpression of DIO3 to suppress TH signaling and thereby modulate cone death/survival. Subretinal delivery of AAV5-IRBP/GNAT2-hDIO3 induced robust expression of DIO3 in the mouse retina and significantly reduced the number of TUNEL-positive cells in the cone-dominant LCA model Rpe65 -/- /Nrl -/- mice. Our work shows that suppressing TH signaling by overexpression of DIO3 preserves cones, supporting that suppressing TH signaling locally in the retina may represent a treatment strategy for LCA management.

PKG-Dependent Cell Death in 661W Cone Photoreceptor-like Cell Cultures (Experimental Study).

In humans cone photoreceptors are responsible for high-resolution colour vision. A variety of retinal diseases can compromise cone viability, and, at present, no satisfactory treatment options are available. Here, we present data towards establishing a reliable, high-throughput assay system that will facilitate the search for cone neuroprotective compounds using the murine-photoreceptor cell line 661 W. To further characterize 661 W cells, a retinal marker study was performed, followed by the induction of cell death using paradigms over-activating cGMP-dependent protein kinase G (PKG). We found that 661 W cells may be used to mimic specific aspects of cone degeneration and may thus be valuable for future compound screening studies.

Loss of the cone-enriched caspase-7 does not affect secondary cone death in retinitis pigmentosa.

Purpose: The apoptotic mechanisms responsible for secondary cone death in retinitis pigmentosa (RP) remain largely unknown. The cone-enriched apoptotic protease caspase-7 (Casp7) is thought to be triggered by endoplasmic reticulum (ER) stress and plays a pivotal role in mice deficient in the cone cyclic nucleotide-gated channels, a deficiency that causes achromatopsia in humans and in mice with autosomal dominant rhodopsin mutations, in particular the T17M mutation. Thus, we tested in two mouse models of RP whether the cone-enriched Casp7 plays a role during secondary cone death. Methods: Casp7 knockout mice were crossed to two different RP mouse models with significantly different rod and cone death kinetics: the rd1 mouse model, which carries a mutation in the Pde6b gene, and the rhodopsin knockout mouse model (Rho-KO or Rho-/- ). In both models, cone survival was assessed on retinal flat mounts by quantifying the percentage of cone arrestin staining over the retinal surface area. The analyses were performed at two different time points for each model. Results: Loss of Casp7 did not alter cone survival in either of the two mouse models tested regardless of the time point analyzed. Rod survival was also not affected in either model nor did loss of Casp7 affect rod or cone function in a wild-type background as assessed with electroretinogram analyses. Conclusions: Secondary cone death in retinitis pigmentosa is unlikely to be triggered by ER stress and is likely independent of Casp7 activity.

RDH13L, an enzyme responsible for the aldehyde-alcohol redox coupling reaction (AL-OL coupling reaction) to supply 11-cis retinal in the carp cone retinoid cycle.

Cone photoreceptors require effective pigment regeneration mechanisms to maintain their sensitivity in the light. Our previous studies in carp cones suggested the presence of an unconventional and very effective mechanism to produce 11-cis retinal, the necessary component in pigment regeneration. In this reaction (aldehyde-alcohol redox coupling reaction, AL-OL coupling reaction), formation of 11-cis retinal, i.e. oxidation of 11-cis retinol is coupled to reduction of an aldehyde at a 1:1 molar ratio without exogenous NADP(H) which is usually required in this kind of reaction. Here, we identified carp retinol dehydrogenase 13-like (RDH13L) as an enzyme catalyzing the AL-OL coupling reaction. RDH13L was partially purified from purified carp cones, identified as a candidate protein, and its AL-OL coupling activity was confirmed using recombinant RDH13L. We further examined the substrate specificity, subcellular localization, and expression level of RDH13L. Based on these results, we concluded that RDH13L contributes to a significant part, but not all, of the AL-OL coupling activity in carp cones. RDH13L contained tightly bound NADP(+) which presumably functions as a cofactor in the reaction. Mouse RDH14, a mouse homolog of carp RDH13L, also showed the AL-OL coupling activity. Interestingly, although carp cone membranes, carp RDH13L and mouse RDH14 all showed the coupling activity at 15-37 °C, they also showed a conventional NADP(+)-dependent 11-cis retinol oxidation activity above 25 °C without addition of aldehydes. This dual mechanism of 11-cis retinal synthesis attained by carp RDH13L and mouse RDH14 probably contribute to effective pigment regeneration in cones that function in the light.

AIPL1, A protein linked to blindness, is essential for the stability of enzymes mediating cGMP metabolism in cone photoreceptor cells.

Defects in the photoreceptor-specific gene encoding aryl hydrocarbon receptor interacting protein like-1 (AIPL1) are linked to blinding diseases, including Leber congenital amaurosis (LCA) and cone dystrophy. While it is apparent that AIPL1 is needed for rod and cone function, the role of AIPL1 in cones is not clear. In this study, using an all-cone animal model lacking Aipl1, we show a light-independent degeneration of M- and S-opsin containing cones that proceeds in a ventral-to-dorsal gradient. Aipl1 is needed for stability, assembly and membrane association of cone PDE6, an enzyme crucial for photoreceptor function and survival. Furthermore, RetGC1, a protein linked to LCA that is needed for cGMP synthesis, was dramatically reduced in cones lacking Aipl1. A defect in RetGC1 is supported by our finding that cones lacking Aipl1 exhibited reduced levels of cGMP. These findings are in contrast to the role of Aipl1 in rods, where destabilization of rod PDE6 results in an increase in cGMP levels, which drives rapid rod degeneration. Our results illustrate mechanistic differences behind the death of rods and cones in retinal degenerative disease caused by deficiencies in AIPL1.

Neuroprotective effect of activated 5'-adenosine monophosphate-activated protein kinase on cone system function during retinal inflammation.

BACKGROUND: Retinal inflammation can cause retinal neural disorders. In particular, functional disorder in the cone photoreceptor system influences visual acuity. However, the underlying mechanism is not yet fully understood. In this study, we evaluated cone system function and the role of 5'-adenosine monophosphate-activated protein kinase (AMPK) during retinal inflammation. RESULTS: Six to eight-week-old male C57BL/6 mice received an intraperitoneal injection of lipopolysaccharide (LPS) to induce retinal inflammation, and were treated with an AMPK activator, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR; 250 mg/kg body weight) or phosphate-buffered saline as vehicle 3 h before the LPS injection. The b-wave of the photopic electroretinogram, which represents cone system function, was decreased 24 h after LPS injection and this reduction was suppressed by AICAR treatment. At this time point, there was no remarkable morphological change in the cone photoreceptor cells. At 1.5 h after LPS injection, the retina mRNA levels of an inflammatory cytokine, Tnf-α, were increased, and those of a regulator of mitochondrial biogenesis, Pgc1-α, were decreased. However, AICAR treatment suppressed these changes in mRNA expression. Immunohistochemistry showed that induction of glial fibrillary acidic protein expression was also suppressed by AICAR 24 h after LPS injection. Furthermore, the mouse cone photoreceptor-derived cell line 661W was treated with AICAR to increase the level of phosphorylated and activated AMPK. After 3 h of AICAR incubation, 661W cells showed decreased Tnf-α mRNA levels and increased Pgc1-α mRNA levels. CONCLUSION: AMPK activation has a neuroprotective effect on cone system function during inflammation, and the effect may, at least in part, involve the regulation of inflammatory cytokines and mitochondrial condition.

Quantitative aspects of cGMP phosphodiesterase activation in carp rods and cones.

Cones are less light-sensitive than rods. We showed previously in carp that more light (>100-fold) is required in cones than in rods to activate 50% of cGMP phosphodiesterase (PDE). The lower effectiveness of PDE activation in carp cones is due partly to the fact that the activation rate of transducin (Tr) by light-activated visual pigment (R*) is 5-fold lower in carp cones than in rods. In this study, we tried to explain the remaining difference. First, we examined the efficiency of activation of PDE by activated Tr (Tr*). By activating PDE with known concentrations of the active (guanosine 5'-Ο-(γ-thio)triphosphate (GTPγS)-bound) form of Tr*, we found that Tr* activated PDE at a similar efficiency in rods and cones. Next, we examined the contribution of R* and Tr* lifetimes. In a comparison of PDE activation in the presence (with GTP) and absence (with GTPγS) of Tr* inactivation, PDE activation required more light (and was therefore less effective) when Tr* was inactivated in both rod and cone membranes. This is probably because inactivation of Tr* shortened its lifetime, thereby reducing the number of activated PDE molecules. The effect of Tr* inactivation was larger in cones, probably because the lifetime of Tr* is shorter in cones than in rods. The shorter lifetimes of Tr* and R* in cones seem to explain the remaining difference in the effectiveness of PDE activation between rods and cones.

Receptor interacting protein kinase mediates necrotic cone but not rod cell death in a mouse model of inherited degeneration.

Retinitis pigmentosa comprises a group of inherited retinal photoreceptor degenerations that lead to progressive loss of vision. Although in most cases rods, but not cones, harbor the deleterious gene mutations, cones do die in this disease, usually after the main phase of rod cell loss. Rod photoreceptor death is characterized by apoptotic features. In contrast, the mechanisms and features of subsequent nonautonomous cone cell death remain largely unknown. In this study, we show that receptor-interacting protein (RIP) kinase mediates necrotic cone cell death in rd10 mice, a mouse model of retinitis pigmentosa caused by a mutation in a rod-specific gene. The expression of RIP3, a key regulator of programmed necrosis, was elevated in rd10 mouse retinas in the phase of cone but not rod degeneration. Although rd10 mice lacking Rip3 developed comparable rod degeneration to control rd10 mice, they displayed a significant preservation of cone cells. Ultrastructural analysis of rd10 mouse retinas revealed that a substantial fraction of dying cones exhibited necrotic morphology, which was rescued by Rip3 deficiency. Additionally, pharmacologic treatment with a RIP kinase inhibitor attenuated histological and functional deficits of cones in rd10 mice. Thus, necrotic mechanisms involving RIP kinase are crucial in cone cell death in inherited retinal degeneration, suggesting the RIP kinase pathway as a potential target to protect cone-mediated central and peripheral vision loss in patients with retinitis pigementosa.

Exome sequencing and analysis of induced pluripotent stem cells identify the cilia-related gene male germ cell-associated kinase (MAK) as a cause of retinitis pigmentosa.

Retinitis pigmentosa (RP) is a genetically heterogeneous heritable disease characterized by apoptotic death of photoreceptor cells. We used exome sequencing to identify a homozygous Alu insertion in exon 9 of male germ cell-associated kinase (MAK) as the cause of disease in an isolated individual with RP. Screening of 1,798 unrelated RP patients identified 20 additional probands homozygous for this insertion (1.2%). All 21 affected probands are of Jewish ancestry. MAK encodes a kinase involved in the regulation of photoreceptor-connecting cilium length. Immunohistochemistry of human donor tissue revealed that MAK is expressed in the inner segments, cell bodies, and axons of rod and cone photoreceptors. Several isoforms of MAK that result from alternative splicing were identified. Induced pluripotent stem cells were derived from the skin of the proband and a patient with non-MAK-associated RP (RP control). In the RP control individual, we found that a transcript lacking exon 9 was predominant in undifferentiated cells, whereas a transcript bearing exon 9 and a previously unrecognized exon 12 predominated in cells that were differentiated into retinal precursors. However, in the proband with the Alu insertion, the developmental switch to the MAK transcript bearing exons 9 and 12 did not occur. In addition to showing the use of induced pluripotent stem cells to efficiently evaluate the pathogenicity of specific mutations in relatively inaccessible tissues like retina, this study reveals algorithmic and molecular obstacles to the discovery of pathogenic insertions and suggests specific changes in strategy that can be implemented to more fully harness the power of sequencing technologies.

Glutathione peroxidase 4 is required for maturation of photoreceptor cells.

Oxidative stress is implicated in the pathologies of photoreceptor cells, and the protective role of antioxidant enzymes for photoreceptor cells have been well understood. However, their essentiality has remained unknown. In this study we generated photoreceptor-specific conditional knock-out (CKO) mice of glutathione peroxidase 4 (GPx4) and showed the critical role of GPx4 for photoreceptor cells. In the wild-type retina the dominant GPx4 expression was in the mitochondria, indicating the mitochondrial variant was the major GPx4 in the retina. In the GPx4-CKO mice, although photoreceptor cells developed and differentiated into rod and cone cells by P12, they rapidly underwent drastic degeneration and completely disappeared by P21. The photoreceptor cell death in the GPx4-CKO mice was associated with the nuclear translocation of apoptosis-inducing factor (AIF) and TUNEL-positive cells. Photoreceptor cells before undergoing apoptosis (P11) exhibited decreased mitochondrial biomass, decreased number of connecting cilia, as well as disorganized structure of outer segments. These findings indicate that GPx4 is a critical antioxidant enzyme for the maturation and survival of photoreceptor cells.

Flow of energy in the outer retina in darkness and in light.

Structural features of neurons create challenges for effective production and distribution of essential metabolic energy. We investigated how metabolic energy is distributed between cellular compartments in photoreceptors. In avascular retinas, aerobic production of energy occurs only in mitochondria that are located centrally within the photoreceptor. Our findings indicate that metabolic energy flows from these central mitochondria as phosphocreatine toward the photoreceptor's synaptic terminal in darkness. In light, it flows in the opposite direction as ATP toward the outer segment. Consistent with this model, inhibition of creatine kinase in avascular retinas blocks synaptic transmission without influencing outer segment activity. Our findings also reveal how vascularization of neuronal tissue can influence the strategies neurons use for energy management. In vascularized retinas, mitochondria in the synaptic terminals of photoreceptors make neurotransmission less dependent on creatine kinase. Thus, vasculature of the tissue and the intracellular distribution of mitochondria can play key roles in setting the strategy for energy distribution in neurons.

Rod phosphodiesterase-6 (PDE6) catalytic subunits restore cone function in a mouse model lacking cone PDE6 catalytic subunit.

Rod and cone photoreceptor neurons utilize discrete PDE6 enzymes that are crucial for phototransduction. Rod PDE6 is composed of heterodimeric catalytic subunits (αß), while the catalytic core of cone PDE6 (α') is a homodimer. It is not known if variations between PDE6 subunits preclude rod PDE6 catalytic subunits from coupling to the cone phototransduction pathway. To study this issue, we generated a cone-dominated mouse model lacking cone PDE6 (Nrl(-/-) cpfl1). In this animal model, using several independent experimental approaches, we demonstrated the expression of rod PDE6 (αß) and the absence of cone PDE6 (α') catalytic subunits. The rod PDE6 enzyme expressed in cone cells is active and contributes to the hydrolysis of cGMP in response to light. In addition, rod PDE6 expressed in cone cells couples to the light signaling pathway to produce S-cone responses. However, S-cone responses and light-dependent cGMP hydrolysis were eliminated when the ß-subunit of rod PDE6 was removed (Nrl(-/-) cpfl1 rd). We conclude that either rod or cone PDE6 can effectively couple to the cone phototransduction pathway to mediate visual signaling. Interestingly, we also found that functional PDE6 is required for trafficking of M-opsin to cone outer segments.