Splenic monocytes drive pathogenic subretinal inflammation in age-related macular degeneration.

Age-related macular degeneration (AMD) is invariably associated with the chronic accumulation of activated mononuclear phagocytes in the subretinal space. The mononuclear phagocytes are composed of microglial cells but also of monocyte-derived cells, which promote photoreceptor degeneration and choroidal neovascularization. Infiltrating blood monocytes can originate directly from bone marrow, but also from a splenic reservoir, where bone marrow monocytes develop into angiotensin II receptor (ATR1)+ splenic monocytes. The involvement of splenic monocytes in neurodegenerative diseases such as AMD is not well understood. Using acute inflammatory and well-phenotyped AMD models, we demonstrate that angiotensin II mobilizes ATR1+ splenic monocytes, which we show are defined by a transcriptional signature using single-cell RNA sequencing and differ functionally from bone marrow monocytes. Splenic monocytes participate in the chorio-retinal infiltration and their inhibition by ATR1 antagonist and splenectomy reduces the subretinal mononuclear phagocyte accumulation and pathological choroidal neovascularization formation. In aged AMD-risk ApoE2-expressing mice, a chronic AMD model, ATR1 antagonist and splenectomy also inhibit the chronic retinal inflammation and associated cone degeneration that characterizes these mice. Our observation of elevated levels of plasma angiotensin II in AMD patients, suggests that similar events take place in clinical disease and argue for the therapeutic potential of ATR1 antagonists to inhibit splenic monocytes for the treatment of blinding AMD.

Structural and functional distinctions of co-resident microglia and monocyte-derived macrophages after retinal degeneration.

BACKGROUND: Both resident microglia and invading peripheral immune cells can respond to injury and degeneration in the central nervous system. However, after dead and dying neurons have been cleared and homeostasis is re-established, it is unknown whether resident immune cells fully resume normal functions and to what degree the peripheral immune cells take up residence. METHODS: Using flow cytometry, in vivo retinal imaging, immunohistochemistry, and single-cell mRNA sequencing, we assess resident microglia and monocyte-derived macrophages in the retina during and after the loss of photoreceptors in the Arr1-/- mouse model of inducible degeneration. RESULTS: We find that photoreceptor loss results in a small, sustained increase in mononuclear phagocytes and, after degeneration wanes, these cells re-establish a spatial mosaic reminiscent of healthy retinas. Transcriptomic analysis revealed the population remained unusually heterogeneous, with several subpopulations expressing gene patterns consistent with mildly activated phenotypes. Roughly a third of "new resident" cells expressed markers traditionally associated with both microglial and monocytic lineages, making their etiology ambiguous. Using an inducible Cre-based fluorescent lineage tracing paradigm to confirm the origins of new resident immune cells, we found approximately equal numbers of microglia and monocyte-derived macrophages after degeneration had subsided. In vivo retinal imaging and immunohistochemical analysis showed that both subpopulations remained functionally responsive to sites of local damage, though on average the monocyte-derived cells had less morphological complexity, expressed higher levels of MHCII, and had less migratory activity than the native resident population. CONCLUSIONS: Monocytic cells that infiltrate the retina during degeneration differentiate into monocyte-derived macrophages that can remain in the retina long-term. These monocyte-derived macrophages adopt ramified morphologies and microglia-like gene expression. However, they remain distinguishable in morphology and gene expression from resident microglia and appear to differ functionally, showing less responsiveness to subsequent retinal injuries. These findings support the idea that persistent changes in the local immune population that occur in response to cell loss in aging and progressive retinal diseases may include the establishment of subpopulations of bone marrow-derived cells whose ability to respond to subsequent insults wanes over time.

In vivo imaging reveals transient microglia recruitment and functional recovery of photoreceptor signaling after injury.

Microglia respond to damage and microenvironmental changes within the central nervous system by morphologically transforming and migrating to the lesion, but the real-time behavior of populations of these resident immune cells and the neurons they support have seldom been observed simultaneously. Here, we have used in vivo high-resolution optical coherence tomography (OCT) and scanning laser ophthalmoscopy with and without adaptive optics to quantify the 3D distribution and dynamics of microglia in the living retina before and after local damage to photoreceptors. Following photoreceptor injury, microglia migrated both laterally and vertically through the retina over many hours, forming a tight cluster within the area of visible damage that resolved over 2 wk. In vivo OCT optophysiological assessment revealed that the photoreceptors occupying the damaged region lost all light-driven signaling during the period of microglia recruitment. Remarkably, photoreceptors recovered function to near-baseline levels after the microglia had departed the injury locus. These results demonstrate the spatiotemporal dynamics of microglia engagement and restoration of neuronal function during tissue remodeling and highlight the need for mechanistic studies that consider the temporal and structural dynamics of neuron-microglia interactions in vivo.

PRCD is essential for high-fidelity photoreceptor disc formation.

Progressive rod-cone degeneration (PRCD) is a small protein residing in the light-sensitive disc membranes of the photoreceptor outer segment. Until now, the function of PRCD has remained enigmatic despite multiple demonstrations that its mutations cause blindness in humans and dogs. Here, we generated a PRCD knockout mouse and observed a striking defect in disc morphogenesis, whereby newly forming discs do not properly flatten. This leads to the budding of disc-derived vesicles, specifically at the site of disc morphogenesis, which accumulate in the interphotoreceptor matrix. The defect in nascent disc flattening only minimally alters the photoreceptor outer segment architecture beyond the site of new disc formation and does not affect the abundance of outer segment proteins and the photoreceptor's ability to generate responses to light. Interestingly, the retinal pigment epithelium, responsible for normal phagocytosis of shed outer segment material, lacks the capacity to clear the disc-derived vesicles. This deficiency is partially compensated by a unique pattern of microglial migration to the site of disc formation where they actively phagocytize vesicles. However, the microglial response is insufficient to prevent vesicular accumulation and photoreceptors of PRCD knockout mice undergo slow, progressive degeneration. Taken together, these data show that the function of PRCD is to keep evaginating membranes of new discs tightly apposed to each other, which is essential for the high fidelity of photoreceptor disc morphogenesis and photoreceptor survival.

Biological Role of Arrestin-1 Oligomerization.

Members of the arrestin superfamily have great propensity of self-association, but the physiological significance of this phenomenon is unclear. To determine the biological role of visual arrestin-1 oligomerization in rod photoreceptors, we expressed mutant arrestin-1 with severely impaired self-association in mouse rods and analyzed mice of both sexes. We show that the oligomerization-deficient mutant is capable of quenching rhodopsin signaling normally, as judged by electroretinography and single-cell recording. Like wild type, mutant arrestin-1 is largely excluded from the outer segments in the dark, proving that the normal intracellular localization is not due the size exclusion of arrestin-1 oligomers. In contrast to wild type, supraphysiological expression of the mutant causes shortening of the outer segments and photoreceptor death. Thus, oligomerization reduces the cytotoxicity of arrestin-1 monomer, ensuring long-term photoreceptor survival.SIGNIFICANCE STATEMENT Visual arrestin-1 forms dimers and tetramers. The biological role of its oligomerization is unclear. To test the role of arrestin-1 self-association, we expressed oligomerization-deficient mutant in arrestin-1 knock-out mice. The mutant quenches light-induced rhodopsin signaling like wild type, demonstrating that in vivo monomeric arrestin-1 is necessary and sufficient for this function. In rods, arrestin-1 moves from the inner segments and cell bodies in the dark to the outer segments in the light. Nonoligomerizing mutant undergoes the same translocation, demonstrating that the size of the oligomers is not the reason for arrestin-1 exclusion from the outer segments in the dark. High expression of oligomerization-deficient arrestin-1 resulted in rod death. Thus, oligomerization reduces the cytotoxicity of high levels of arrestin-1 monomer.

RBX2 maintains final retinal cell position in a DAB1-dependent and -independent fashion.

The laminated structure of the retina is fundamental for the organization of the synaptic circuitry that translates light input into patterns of action potentials. However, the molecular mechanisms underlying cell migration and layering of the retina are poorly understood. Here, we show that RBX2, a core component of the E3 ubiquitin ligase CRL5, is essential for retinal layering and function. RBX2 regulates the final cell position of rod bipolar cells, cone photoreceptors and Muller glia. Our data indicate that sustained RELN/DAB1 signaling, triggered by depletion of RBX2 or SOCS7 - a CRL5 substrate adaptor known to recruit DAB1 - causes rod bipolar cell misposition. Moreover, whereas SOCS7 also controls Muller glia cell lamination, it is not responsible for cone photoreceptor positioning, suggesting that RBX2, most likely through CRL5 activity, controls other signaling pathways required for proper cone localization. Furthermore, RBX2 depletion reduces the number of ribbon synapses and disrupts cone photoreceptor function. Together, these results uncover RBX2 as a crucial molecular regulator of retina morphogenesis and cone photoreceptor function.

Tracking distinct microglia subpopulations with photoconvertible Dendra2 in vivo.

BACKGROUND: The ability to track individual immune cells within the central nervous system has revolutionized our understanding of the roles that microglia and monocytes play in synaptic maintenance, plasticity, and neurodegenerative diseases. However, distinguishing between similar subpopulations of mobile immune cells over time during episodes of neuronal death and tissue remodeling has proven to be challenging. METHODS: We recombineered a photoconvertible fluorescent protein (Dendra2; D2) downstream of the Cx3cr1 promoter commonly used to drive expression of fluorescent markers in microglia and monocytes. Like the popular Cx3cr1-GFP line (Cx3cr1+/GFP), naïve microglia in Cx3cr1-Dendra2 mice (Cx3cr1+/D2) fluoresce green and can be noninvasively imaged in vivo throughout the CNS. In addition, individual D2-expressing cells can be photoconverted, resulting in red fluorescence, and tracked unambiguously within a field of green non-photoconverted cells for several days in vivo. RESULTS: Dendra2-expressing retinal microglia were noninvasively photoconverted in both ex vivo and in vivo conditions. Local in vivo D2 photoconversion was sufficiently robust to quantify cell subpopulations by flow cytometry, and the protein was stable enough to survive tissue processing for immunohistochemistry. Simultaneous in vivo fluorescence imaging of Dendra2 and light scattering measurements (Optical Coherence Tomography, OCT) were used to assess responses of individual microglial cells to localized neuronal damage and to identify the infiltration of monocytes from the vasculature in response to large scale neurodegeneration. CONCLUSIONS: The ability to noninvasively and unambiguously track D2-expressing microglia and monocytes in vivo through space and time makes the Cx3cr1-Dendra2 mouse model a powerful new tool for disentangling the roles of distinct immune cell subpopulations in neuroinflammation.

Disrupted Blood-Retina Lysophosphatidylcholine Transport Impairs Photoreceptor Health But Not Visual Signal Transduction.

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 the Mfsd2a knock-out mouse as animal model. Our lipidome analyses of Mfsd2a-/- 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 in Mfsd2a-/- retinas. Mfsd2a-/- retinas undergo slow progressive degeneration, with ∼30% of photoreceptor cells lost by the age of 6 months. Despite this pathology, the ultrastructure Mfsd2a-/- 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 STATEMENT Phospholipids 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.

In vivo optophysiology reveals that G-protein activation triggers osmotic swelling and increased light scattering of rod photoreceptors.

The light responses of rod and cone photoreceptors have been studied electrophysiologically for decades, largely with ex vivo approaches that disrupt the photoreceptors' subretinal microenvironment. Here we report the use of optical coherence tomography (OCT) to measure light-driven signals of rod photoreceptors in vivo. Visible light stimulation over a 200-fold intensity range caused correlated rod outer segment (OS) elongation and increased light scattering in wild-type mice, but not in mice lacking the rod G-protein alpha subunit, transducin (Gαt), revealing these responses to be triggered by phototransduction. For stimuli that photoactivated one rhodopsin per Gαt the rod OS swelling response reached a saturated elongation of 10.0 ± 2.1%, at a maximum rate of 0.11% s-1 Analyzing swelling as osmotically driven water influx, we find the H2O membrane permeability of the rod OS to be (2.6 ± 0.4) × 10-5 cm⋅s-1, comparable to that of other cells lacking aquaporin expression. Application of Van't Hoff's law reveals that complete activation of phototransduction generates a potentially harmful 20% increase in OS osmotic pressure. The increased backscattering from the base of the OS is explained by a model combining cytoplasmic swelling, translocation of dissociated G-protein subunits from the disc membranes into the cytoplasm, and a relatively higher H2O permeability of nascent discs in the basal rod OS. Translocation of phototransduction components out of the OS may protect rods from osmotic stress, which could be especially harmful in disease conditions that affect rod OS structural integrity.

Monocyte infiltration rather than microglia proliferation dominates the early immune response to rapid photoreceptor degeneration.

BACKGROUND: Activation of resident microglia accompanies every known form of neurodegeneration, but the involvement of peripheral monocytes that extravasate and rapidly transform into microglia-like macrophages within the central nervous system during degeneration is far less clear. METHODS: Using a combination of in vivo ocular imaging, flow cytometry, and immunohistochemistry, we investigated the response of infiltrating cells in a light-inducible mouse model of photoreceptor degeneration. RESULTS: Within 24 h, resident microglia became activated and began migrating to the site of degeneration. Retinal expression of CCL2 increased just prior to a transient period of CCR2+ cell extravasation from the retinal vasculature. Proliferation of microglia and monocytes occurred concurrently; however, there was no indication of proliferation in either population until 72-96 h after neurodegeneration began. Eliminating CCL2-CCR2 signaling blocked monocyte recruitment, but did not alter the extent of retinal degeneration. CONCLUSIONS: These results demonstrate that the immune response to photoreceptor degeneration includes both resident microglia and monocytes, even at very early times. Surprisingly, preventing monocyte infiltration did not block neurodegeneration, suggesting that in this model, degeneration is limited by cell clearance from other phagocytes or by the timing of intrinsic cell death programs. These results show monocyte involvement is not limited to disease states that overwhelm or deplete the resident microglial population and that interventions focused on modulating the peripheral immune system are not universally beneficial for staving off degeneration.

Loss of cone function without degeneration in a novel Gnat2 knock-out mouse.

Rods and cones mediate visual perception over 9 log units of light intensities, with both photoreceptor types contributing to a middle 3-log unit range that comprises most night-time conditions. Rod function in this mesopic range has been difficult to isolate and study in vivo because of the paucity of mutants that abolish cone signaling without causing photoreceptor degeneration. Here we describe a novel Gnat2 knockout mouse line (Gnat2-/-) ideal for dissecting rod and cone function. In this line, loss of Gnat2 expression abolished cone phototransduction, yet there was no loss of cones, disruption of the photoreceptor mosaic, nor change in general retinal morphology up to at least 9 months of age. Retinal microglia and Müller glia, which are highly sensitive to neuronal pathophysiology, were distributed normally with morphologies indistinguishable between Gnat2-/- and wildtype adult mice. ERG recordings demonstrated complete loss of cone-driven a-waves in Gnat2-/- mice; comparison to WT controls revealed that rods of both strains continue to function at light intensities exceeding 104 photoisomerizations rod-1 s-1. We conclude that the Gnat2-/- mouse is a preferred model for functional studies of rod pathways in the retina when degeneration could be an experimental confound.

Rapid monocyte infiltration following retinal detachment is dependent on non-canonical IL6 signaling through gp130.

BACKGROUND: Retinal detachment (RD) can lead to proliferative vitreoretinopathy (PVR), a leading cause of intractable vision loss. PVR is associated with a cytokine storm involving common proinflammatory molecules like IL6, but little is known about the source and downstream signaling of IL6 and the consequences for the retina. Here, we investigated the early immune response and resultant cytokine signaling following RD in mice. METHODS: RD was induced in C57BL/6 J and IL6 knockout mice, and the resulting inflammatory response was examined using immunohistochemistry and flow cytometry. Cytokines and signaling proteins of vitreous and retinas were quantified by multiple cytokine arrays and Western blotting. To attempt to block IL6 signaling, a neutralizing antibody of IL6 receptor α (IL6Rα) or IL6 receptor ß (gp-130) was injected intravitreally immediately after RD. RESULTS: Within one day of RD, bone marrow-derived Cd11b + monocytes had extravasated from the vasculature and lined the vitreal surface of the retina, while the microglia, the resident macrophages of the retina, were relatively unperturbed. Cytokine arrays and Western blot analysis revealed that this sterile inflammation did not cause activation of IL6 signaling in the neurosensory retina, but rather only in the vitreous and aqueous humor. Monocyte infiltration was inhibited by blocking gp130, but not by IL6 knockout or IL6Rα blockade. CONCLUSIONS: Together, our results demonstrate that monocytes are the primary immune cell mediating the cytokine storm following RD, and that any resulting retinal damage is unlikely to be a direct result of retinal IL6 signaling, but rather gp130-mediated signaling in the monocytes themselves. These results suggest that RD should be treated immediately, and that gp130-directed therapies may prevent PVR and promote retinal healing.

New Developments in Murine Imaging for Assessing Photoreceptor Degeneration In Vivo.

Optical Coherence Tomography (OCT) is a powerful clinical tool that measures near infrared light backscattered from the eye and other tissues. OCT is used for assessing changes in retinal structure, including layer thicknesses, detachments and the presence of drusen in patient populations. Our custom-built OCT system for the mouse eye quantitatively images all layers of the neural retinal, the RPE, Bruchs' membrane and the choroid. Longitudinal assessment of the same retinal region reveals that the relative intensities of retinal layers are highly stable in healthy tissue, but show progressive increases in intensity in a model of retinal degeneration. The observed changes in OCT signal have been correlated with ultrastructural disruptions that were most dramatic in the inner segments and nuclei of the rods. These early changes in photoreceptor structure coincided with activation of retinal microglia, which migrated vertically from the inner to the outer retina to phagocytose photoreceptor cell bodies (Levine et al., Vis Res 102:71-79, 2014). We conclude that quantitative analysis of OCT light scattering signals may be a useful tool for early detection and subcellular localization of cell stress prior to cell death, and for assessing the progression of degenerative disease over time. Future efforts to develop sensitive approaches for monitoring microglial dynamics in vivo may likewise elucidate earlier signs of cellular stress during retinal degeneration.

Rhodopsin in the rod surface membrane regenerates more rapidly than bulk rhodopsin in the disc membranes in vivo.

Sustained vertebrate vision requires that opsin chromophores isomerized by light to the all-trans form be replaced with 11-cis retinal to regenerate the visual pigment. We have characterized the early receptor potential (ERP), a component of the electroretinogram arising from photoisomerization-induced charge displacements in plasma membrane visual pigment, and used it to measure pigment bleaching and regeneration in living mice. The mouse ERP was characterized by an outward 'R2' charge displacement with a time constant of 215 µs that discharged through a membrane with an apparent time constant of ∼0.6 ms. After complete bleaching of rhodopsin, the ERP recovered in two phases. The initial, faster phase had a time constant of ∼1 min, accounted for ∼20% of the total, and was not dependent on the level of expression of the retinal pigment epithelium isomerase, Rpe65. The slower, complementary phase had a time constant of 23 min in wild-type (WT) mice (C57Bl/6) and was substantially slowed in Rpe65(+/-) mice. Comparison of the ERPs of a mouse line expressing 150% of the normal level of cone M-opsin with those of WT mice revealed that M-opsin contributed 26% of the total WT ERP in these experiments, with the remaining 74% arising from rhodopsin. Thus, the fast regenerating fraction (20%) corresponds approximately to the fraction of the total ERP independently estimated to arise from M-opsin. Because both phases of the ERP recover substantially faster than previous measurements of bulk rhodopsin regeneration in living mice, we conclude that delivery of the highly hydrophobic 11-cis retinal to the interior of rod photoreceptors appears to be retarded by transit across the cytoplasmic gap between plasma and disc membranes.

Photoreceptor signaling: supporting vision across a wide range of light intensities.

For decades, photoreceptors have been an outstanding model system for elucidating basic principles in sensory transduction and biochemistry and for understanding many facets of neuronal cell biology. In recent years, new knowledge of the kinetics of signaling and the large-scale movements of proteins underlying signaling has led to a deeper appreciation of the photoreceptor's unique challenge in mediating the first steps in vision over a wide range of light intensities.

Spatiotemporal cGMP dynamics in living mouse rods.

Signaling of single photons in rod photoreceptors decreases the concentration of the second messenger, cyclic GMP (cGMP), causing closure of cGMP-sensitive channels located in the plasma membrane. Whether the spatiotemporal profiles of the fall in cGMP are narrow and deep, or broad and shallow, has important consequences for the amplification and the fidelity of signaling. The factors that determine the cGMP profiles include the diffusion coefficient for cGMP, the spontaneous rate of cGMP hydrolysis, and the rate of cGMP synthesis, which is powerfully regulated by calcium feedback mechanisms. Here, using suction electrodes to record light-dependent changes in cGMP-activated current in living mouse rods lacking calcium feedback, we have determined the rate constant of spontaneous cGMP hydrolysis and the longitudinal cGMP diffusion coefficient. These measurements result in a fully constrained spatiotemporal model of phototransduction, which we used to determine the effect of feedback to cGMP synthesis in spatially constricting the fall of cGMP during the single-photon response of normal rods. We find that the spatiotemporal cGMP profiles during the single-photon response are optimized for maximal amplification and preservation of signal linearity, effectively operating within an axial signaling domain of ~2 µm.

Membrane attachment is key to protecting transducin GTPase-activating complex from intracellular proteolysis in photoreceptors.

The members of the R7 regulator of G-protein signaling (RGS) protein subfamily are versatile regulators of G-protein signaling throughout the nervous system. Recent studies indicate that they are often found in complexes with membrane anchor proteins that serve as versatile modulators of their activity, intracellular targeting, and stability. One striking example is the interplay between the membrane anchor R9AP and the RGS9-1 · Gß5 GTPase-activating complex responsible for the rapid inactivation of the G-protein transducin in vertebrate photoreceptor cells during their recovery from light excitation. The amount of this complex in photoreceptors sets their temporal resolution and is precisely regulated by the expression level of R9AP, which serves to protect the RGS9-1 and Gß5 subunits from intracellular proteolysis. In this study, we investigated the mechanism by which R9AP performs its protective function in mouse rods and found that it is entirely confined to recruiting RGS9-1 · Gß5 to cellular membranes. Furthermore, membrane attachment of RGS9-1 · Gß5 is sufficient for its stable expression in rods even in the absence of R9AP. Our second finding is that RGS9-1 · Gß5 possesses targeting information that specifies its exclusion from the outer segment and that this information is neutralized by association with R9AP to allow outer segment targeting. Finally, we demonstrate that the ability of R9AP · RGS9-1 · Gß5 to accelerate GTP hydrolysis on transducin is independent of its means of membrane attachment, since replacing the transmembrane domain of R9AP with a site for lipid modification did not impair the catalytic activity of this complex.

Control of rhodopsin's active lifetime by arrestin-1 expression in mammalian rods.

In rod photoreceptors, deactivation of the light-activated G-protein-coupled receptor rhodopsin (R*) is initiated by phosphorylation and completed through subsequent binding of visual arrestin (Arr1). The in vivo kinetics of these individual interactions have proven difficult to determine with precision since R* lifetime is much shorter than the lifetimes of downstream G-protein and effector molecules. Here, we have used a transgenic mouse line with accelerated downstream deactivation kinetics to reveal the contribution of Arr1 binding to the overall time course of rhodopsin deactivation. Photoresponses revealed that the lifetime of R* is significantly increased in rods that express half of the normal amount of Arr1, in a manner consistent with a twofold decrease in the rate of Arr1 binding across a wide range of flash strengths. A basic model of photoresponse deactivation consistent with established photoreceptor biochemistry shows that R* phosphorylation and Arr1 binding occur with a time constant of approximately 40 ms in wild-type mouse rods, much faster than previous estimates.

Phosducin regulates transmission at the photoreceptor-to-ON-bipolar cell synapse.

The rate of synaptic transmission between photoreceptors and bipolar cells has been long known to depend on conditions of ambient illumination. However, the molecular mechanisms that mediate and regulate transmission at this ribbon synapse are poorly understood. We conducted electroretinographic recordings from dark- and light-adapted mice lacking the abundant photoreceptor-specific protein phosducin and found that the ON-bipolar cell responses in these animals have a reduced light sensitivity in the dark-adapted state. Additional desensitization of their responses, normally caused by steady background illumination, was also diminished compared with wild-type animals. This effect was observed in both rod- and cone-driven pathways, with the latter affected to a larger degree. The underlying mechanism is likely to be photoreceptor specific because phosducin is not expressed in other retina neurons and transgenic expression of phosducin in rods of phosducin knock-out mice rescued the rod-specific phenotype. The underlying mechanism functions downstream from the phototransduction cascade, as evident from the sensitivity of phototransduction in phosducin knock-out rods being affected to a much lesser degree than b-wave responses. These data indicate that a major regulatory component responsible for setting the sensitivity of signal transmission between photoreceptors and ON-bipolar cells is confined to photoreceptors and that phosducin participates in the underlying molecular mechanism.

Lessons from photoreceptors: turning off g-protein signaling in living cells.

Phototransduction in retinal rods is one of the most extensively studied G-protein signaling systems. In recent years, our understanding of the biochemical steps that regulate the deactivation of the rod's response to light has greatly improved. Here, we summarize recent advances and highlight some of the remaining puzzles in this model signaling system.