ON bipolar neurons in retina detect the glutamate released by rods and cones via metabotropic glutamate receptor 6 (mGluR6), whose cascade is unknown. The trimeric G-protein G o might mediate this cascade because it colocalizes with mGluR6. To test this, we studied the retina in mice negative for the ␣ subunit of G o (G␣ o Ϫ/Ϫ). Retinal layering, key cell types, synaptic structure, and mGluR6 expression were all normal, as was the a-wave of the electroretinogram, which represents the rod and cone photocurrents. However, the b-wave of the electroretinogram, both rod-and cone-driven components, was entirely missing. Because the b-wave represents the massed response of ON bipolar cells, its loss in the G␣ o null mouse establishes that the light response of the ON bipolar cell requires G o . This represents the first function to be defined in vivo for the ␣ subunit of the most abundant G-protein of the brain .

The cellular mechanisms underlying axogenesis and dendritogenesis are not completely understood. The axons and dendrites of retinal bipolar cells, which contact their synaptic partners within specific laminae in the inner and outer retina, provide a good system for exploring these issues. Using transgenic mice expressing enhanced green fluorescent protein (GFP) in a subset of bipolar cells, we determined that axonal and dendritic arbors of these interneurons develop directly from apical and basal processes attached to the outer and inner limiting membranes, respectively. Selective stabilization of processes contributed to stratification of axonal and dendritic arbors within the appropriate synaptic layer. This unusual mode of axogenesis and dendritogenesis from neuroepithelial-like processes may act to preserve neighbor-neighbor relationships in synaptic wiring between the outer and inner retina.

Daily, the retinal pigment epithelium (RPE) ingests a bolus of lipid and protein in the form of phagocytized photoreceptor outer segments (OS). The RPE, like the liver, expresses enzymes required for fatty acid oxidation and ketogenesis. This suggests that these pathways play a role in the disposal of lipids from ingested OS, as well as providing a mechanism for recycling metabolic intermediates back to the outer retina. In this study, we examined whether OS phagocytosis was linked to ketogenesis. We found increased levels of β-hydroxybutyrate (β-HB) in the apical medium following ingestion of OS by human fetal RPE and ARPE19 cells cultured on Transwell inserts. No increase in ketogenesis was observed following ingestion of oxidized OS or latex beads. Our studies further defined the connection between OS phagocytosis and ketogenesis in wild-type mice and mice with defects in phagosome maturation using a mouse RPE explant model. In explant studies, the levels of β-HB released were temporally correlated with OS phagocytic burst after light onset. In the mouse where phagosome maturation is delayed, there was a temporal shift in the release of β-HB. An even more pronounced shift in maximal β-HB production was observed in the RPE, in which loss of the ATP-binding cassette A4 transporter results in defective phagosome processing and accumulation of lipid debris. These studies suggest that FAO and ketogenesis are key to supporting the metabolism of the RPE and preventing the accumulation of lipids that lead to oxidative stress and mitochondrial dysfunction.

Glutamate released onto retinal ON bipolar neurons binds to a metabotropic receptor to activate a heterotrimeric G-protein (G(o)) that ultimately closes a nonspecific cation channel. Signaling requires the alpha subunit (Galpha(o)), but its effector is unknown. Because Galpha(o) is transcribed into two splice variants (alpha(o1) and alpha(o2)) that differ in the key GTPase domain, the next step in elucidating this pathway was to determine which splice variant carries the signal. Here we show by reverse transcription-PCR and Western blots that retina expresses both splice variants. Furthermore, in situ hybridization and immunostaining on mouse retina deficient in one splice variant or the other show that both alpha(o1) and alpha(o2) are expressed by ON bipolar cells but that alpha(o1) is much more abundant. Finally, electroretinography performed on mice deficient for one splice variant or the other shows that the positive b-wave (response of ON bipolar cells to rod and cone input) requires alpha(o1) but not alpha(o2). Thus, the light response of the ON bipolar cell is probably carried by its strongly expressed splice variant, Galpha(o1).

Like other neurons, retinal cells utilize autophagic pathways to maintain cell homeostasis. The mammalian retina relies on heterophagy and selective autophagy to efficiently degrade and metabolize ingested lipids with disruption in autophagy associated degradation contributing to age related retinal disorders. The retinal pigment epithelium (RPE) supports photoreceptor cell renewal by daily phagocytosis of shed photoreceptor outer segments (OS). The daily ingestion of these lipid-rich OS imposes a constant degradative burden on these terminally differentiated cells. These cells rely on Microtubule-Associated Protein 1 Light Chain 3 (LC3) family of proteins for phagocytic clearance of the ingested OS. The LC3 family comprises of three highly homologous members, MAP1LC3A (LC3A), MAP1LC3B (LC3B), and MAP1LC3C (LC3C). The purpose of this study was to determine whether the LC3B isoform plays a specific role in maintaining RPE lipid homeostasis. We examined the RPE and retina of the LC3B-/- mouse as a function of age using in vivo ocular imaging and electroretinography coupled with ex vivo, lipidomic analyses of lipid mediators, assessment of bisretinoids as well as imaging of lipid aggregates. Deletion of LC3B resulted in defects within the RPE including increased phagosome accumulation, decreased fatty acid oxidation and a subsequent increase in RPE and sub-RPE lipid deposits. Age-dependent RPE changes included elevated levels of oxidized cholesterol, deposition of 4-HNE lipid peroxidation products, bisretinoid lipofuscin accumulation, and subretinal migration of microglia, collectively likely contributing to loss of retinal function. These observations are consistent with a critical role for LC3B-dependent processes in the maintenance of normal lipid homeostasis in the aging RPE, and suggest that LC3 isoform specific disruption in autophagic processes contribute to AMD-like pathogenesis.

Retinal ON bipolar cells comprise about 70% of all bipolar cells. Glutamate hyperpolarizes these cells by binding to the metabotropic glutamate receptor mGluR6, activating the G-protein G o1 , and closing an unidentified cation channel. To facilitate investigation of ON bipolar cells, we here report on the production of a transgenic mouse (Grm6-GFP) in which EGFP, under control of mGluR6 promoter, was expressed in all and only ON bipolar cells. We used the mouse to determine density of ON bipolar cells, which in central retina was 29,600 cells/mm 2 . We further sorted the fluorescent cells and created a pure ON bipolar cDNA library that was negative for photoreceptor unique genes. Using this library, we determined expression of 27 genes of interest. We obtained positive transcripts for: G o interactors: regulators of G-protein signaling (RGS), Ret-RGS1 (a variant of RGS20), RGS16, RGS7, purkinje cell protein 2 (PCP2, also called L7 or GPSM4), synembryn (RIC-8), LGN (GPSM2), RAP1GAP, Gβ5; cGMP modulators: guanylyl cyclase (GC) 1α1, GC1β1, phosphodiesterase (PDE) 1C, PDE9A; and channels: inwardly rectifying potassium channel Kir2.4, transient receptor potential TRPC2, sperm-specific cation channels CatSper 2-4. The following transcripts were not found in our library: AGS3 (GPSM1), RGS10, RGS19 (GAIP), calbindin, GC1α2, GC1β2, PDE5, PDE2A, amiloride-sensitive sodium channel ACCN4, and CatSper1. We then localized Kir2.4 to several cell types, and showed that in ON bipolar cells, the channel concentrates in their dendritic tips. The channels and modulators found in ON bipolar cells likely shape their light response. Additional uses of the Grm6-GFP mouse are also discussed.

Bestrophinopathies, one of the most common forms of inherited macular degenerations, are caused by mutations in the BEST1 gene expressed in the retinal pigment epithelium (RPE). Both human and canine BEST1-linked maculopathies are characterized by abnormal accumulation of autofluorescent material within RPE cells and bilateral macular or multifocal lesions; however, the specific mechanism leading to the formation of these lesions remains unclear. We now provide an overview of the current state of knowledge on the molecular pathology of bestrophinopathies, and explore factors promoting formation of RPE-neuroretinal separations, using the first spontaneous animal model of BEST1-associated retinopathies, canine Best (cBest). Here, we characterize the nature of the autofluorescent RPE cell inclusions and report matching spectral signatures of RPE-associated fluorophores between human and canine retinae, indicating an analogous composition of endogenous RPE deposits in Best Vitelliform Macular Dystrophy (BVMD) patients and its canine disease model. This study also exposes a range of biochemical and structural abnormalities at the RPE-photoreceptor interface related to the impaired cone-associated microvillar ensheathment and compromised insoluble interphotoreceptor matrix (IPM), the major pathological culprits responsible for weakening of the RPE-neuroretina interactions, and consequently, formation of vitelliform lesions. These salient alterations detected at the RPE apical domain in cBest as well as in BVMD- and ARB-hiPSC-RPE model systems provide novel insights into the pathological mechanism of BEST1-linked disorders that will allow for development of critical outcome measures guiding therapeutic strategies for bestrophinopathies.

Heterotrimeric G-proteins, comprising Gα and Gβγ subunits, couple metabotropic receptors to various downstream effectors and contribute to assembling and trafficking receptor based signaling complexes. A G-protein β-subunit, Gβ3 plays a critical role in several physiological processes as a polymorphism in its gene is associated with a risk factor for several disorders. Retinal ON bipolar cells express Gβ3, and they provide an excellent system to study its role. In the ON bipolar cells, mGluR6 inverts the photoreceptor’s signal via a cascade in which glutamate released from photoreceptors closes the TRPM1 channel. This cascade is essential for vision since deficiencies in its proteins lead to complete congenital stationary night blindness. Here we report that Gβ3 participates in the G-protein heterotrimer that couples mGluR6 to TRPM1. Gβ3 deletion in mouse greatly reduces the light response under both scotopic and photopic conditions, but it does not eliminate it. In addition, Gβ3 deletion causes mislocalization and downregulation of most cascade elements and modulators. Furthermore, Gβ3 may play a role in synaptic maintenance since in its absence, the number of invaginating rod bipolar dendrites is greatly reduced, a deficit that was not observed at 3 weeks, the end of the developmental period.