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Stargardt disease (STGD1), associated with biallelic variants in the ABCA4 gene, is the most common heritable macular dystrophy and is currently untreatable. To identify potential treatment targets, we characterized surviving STGD1 photoreceptors. We used clinical data to identify macular regions with surviving STGD1 photoreceptors. We compared the hyperreflective bands in the optical coherence tomographic (OCT) images that correspond to structures in the STGD1 photoreceptor inner segments to those in controls. We used adaptive optics scanning light ophthalmoscopy (AO-SLO) to study the distribution of cones and AO-OCT to evaluate the interface of photoreceptors and retinal pigment epithelium (RPE). We found that the profile of the hyperreflective bands differed dramatically between patients with STGD1 and controls. AO-SLOs showed patches in which cone densities were similar to those in healthy retinas and others in which the cone population was sparse. In regions replete with cones, there was no debris at the photoreceptor-RPE interface. In regions with sparse cones, there was abundant debris. Our results raise the possibility that pharmaceutical means may protect surviving photoreceptors and so mitigate vision loss in patients with STGD1. [ABSTRACT FROM AUTHOR]

N-retinylidene-phosphatidylethanolamine (N-Ret-PE), the Schiff-base conjugate formed through the reversible reaction of retinal (Vitamin A-aldehyde) and phosphatidylethanolamine, plays a crucial role in the visual cycle and visual pigment photoregeneration. However, N-Ret-PE can react with another molecule of retinal to form toxic di-retinoids if not removed from photoreceptors through its transport across photoreceptor membranes by the ATP-binding-cassette transporter ABCA4. Loss-of-function mutations in ABCA4 are known to cause Stargardt disease (STGD1), an inherited retinal degenerative disease associated with the accumulation of fluorescent di-retinoids and severe loss in vision. A larger assessment of retinal-phospholipid Schiff-base conjugates in photoreceptors is needed, along with further investigation of ABCA4 residues important for N-Ret-PE binding. In this study we show that N-Ret-PE formation is dependent on pH and phospholipid content. When retinal is added to liposomes or photoreceptor membranes, 40 to 60% is converted to N-Ret-PE at physiological pH. Phosphatidylserine and taurine also react with retinal to form N-retinylidene-phosphatidylserine and N-retinylidenetaurine, respectively, but at significantly lower levels. N-retinylidene-phosphatidylserine is not a substrate for ABCA4 and reacts poorly with retinal to form di-retinoids. Additionally, amino acid residues within the binding pocket of ABCA4 that contribute to its interaction with N-Ret-PE were identified and characterized using site-directed mutagenesis together with functional and binding assays. Substitution of arginine residues and hydrophobic residues with alanine or residues implicated in STGD1 significantly reduced or eliminated substrateactivated ATPase activity and substrate binding. Collectively, this study provides important insight into conditions which affect retinal-phospholipid Schiff-base formation and mechanisms underlying the pathogenesis of STGD1. [ABSTRACT FROM AUTHOR]

G protein-coupled receptor (GPCR) signaling is ubiquitous. As an archetype of this signaling motif, rod phototransduction has provided many fundamental, quantitative details, including a dogma that one active GPCR molecule activates a substantial number of downstream G protein/enzyme effector complexes. However, rod phototransduction is light-activated, whereas GPCR pathways are predominantly ligand-activated. Here, we report a detailed study of the ligand-triggered GPCR pathway in mammalian olfactory transduction, finding that an odorant-receptor molecule when (one-time) complexed with its most effective odorants produces on average much less than one downstream effector. Further experiments gave a nominal success probability of tentatively ~10-4 (more conservatively, ~10-2 to ~10-5). This picture is potentially more generally representative of GPCR signaling than is rod phototransduction, constituting a paradigm shift. [ABSTRACT FROM AUTHOR]

Author(s): Haining Zhong [1]; Laurie L. Molday [2]; Robert S. Molday (corresponding author) [2]; King-Wai Yau (corresponding author) [1, 3, 4] Cyclic nucleotide-gated (CNG) channels are crucial for visual and [...]

Deleterious mutations in RS1 encoding retinoschisin are associated with X-linked juvenile retinoschisis (RS), a common form of macular degeneration in males. The disorder is characterized by a negative electroretinogram pattern and by a splitting of the inner retina. To gain further insight into the function of the retinoschisin protein and its role in the cellular pathology of RS, we have generated knockout mice deficient in Rs1h, the murine ortholog of the human RS1 gene. We show that pathologic changes in hemizygous Rs1[h.sup.-/Y] male mice are evenly distributed across the retina, apparently contrasting with the macula-dominated features in human. Similar functional anomalies in human and Rs1[h.sup.-/Y] mice, however, suggest that both conditions are a disease of the entire retina affecting the organization of the retinal cell layers as well as structural properties of the retinal synapse.

Biochemical studies are conducted to identify proteins in retinal cell extracts that interact with RP2 in order to define the role of RP2 in the retina and X-linked retinitis pigmentosa (XLRP). The studies have shown that RP2, in addition to serving as a regulator of ADP ribosylation factor-like 3 (Arl3), has interacted with N-ethylmaleimide sensitive factor (NSF) and the complex has played a vital role in membrane protein trafficking in photoreceptors and other cells of the retina.

The interaction of wild-type and mutant retinoschisin (RS1) with various carbohydrates coupled to agarose supports were analyzed to gain insight into the role of RS1 in retinal cell biology and X-linked retinoschisis. The cell expression and protein purification method developed based on effective displacement of RS1 bound to galactose-agarose by incubation with isopropyl [beta]-D-1-thiogalactopyranoside (IPTG) could be used to isolate RS1 for detailed structure-function studies.

In mammalian olfactory transduction, odorants activate a cAMPmediated signaling pathway that leads to the opening of cyclic nucleotide-gated (CNG), nonselective cation channels and depolarization. The Ca2+ influx through open CNG channels triggers an inward current through Ca2+-activated Cl channels (ANO2), which is expected to produce signal amplification. However, a study on an Ano2-/- mouse line reported no elevation in the behavioral threshold of odorant detection compared with wild type (WT). Subsequent studies by others on the same Ano2-/- line, nonetheless, found subtle defects in olfactory behavior and some abnormal axonal projections from the olfactory receptor neurons (ORNs) to the olfactory bulb. As such, the question regarding signal amplification by the Cl current in WT mouse remains unsettled. Recently, with suction-pipette recording, we have successfully separated in frog ORNs the CNG and Cl currents during olfactory transduction and found the Cl current to predominate in the response down to the threshold of action-potential signaling to the brain. For better comparison with the mouse data by others, we have now carried out similar current-separation experiments on mouse ORNs. We found that the Cl current clearly also predominated in the mouse olfactory response at signaling threshold, accounting for ~80% of the response. In the absence of the Cl current, we expect the threshold stimulus to increase by approximately sevenfold. [ABSTRACT FROM AUTHOR]