Long Interspersed Nuclear Element-1 Analytes in Extracellular Vesicles as Tools for Molecular Diagnostics of Non-Small Cell Lung Cancer.

Aberrant expression of the oncogenic retrotransposon LINE-1 is a hallmark of various cancer types, including non-small cell lung cancers (NSCLCs). Here, we present proof-of-principle evidence that LINE-1 analytes in extracellular vesicles (EVs) serve as tools for molecular diagnostics of NSCLC, with LINE-1 status in tumor cells and tissues mirroring the LINE-1 mRNA and ORF1p cargos of EVs from lung cancer cell culture conditioned media or human plasma. The levels of LINE-1 analytes in plasma EVs from ostensibly healthy individuals were higher in females than males. While the profiles of LINE-1 mRNA and ORF1p in African Americans compared to Hispanics were not significantly different, African Americans showed slightly higher ORF1p content, and 2-3 times greater ranges of LINE-1 values compared to Hispanics. Whole plasma ORF1p levels correlated with EV ORF1p levels, indicating that most of the circulating LINE-1 protein is contained within EVs. EV LINE-1 mRNA levels were elevated in patients with advanced cancer stages and in select patients with squamous cell carcinoma and metastatic tumors compared to adenocarcinomas. The observed EV LINE-1 mRNA profiles paralleled the patterns of ORF1p expression in NSCLC tissue sections suggesting that LINE-1 analytes in plasma EVs may serve to monitor the activity of LINE-1 retroelements in lung cancer.

Decoding Race and Age-Related Macular Degeneration: GPR 143 Activity Is the Key.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the developed world. Caucasians are eightfold more likely to develop AMD than any other race, indicating a racial bias in AMD incidence which is unexplained. We hypothesize that pigmentation of the retinal pigment epithelium (RPE) and choroid protects from AMD and underlies this peculiar racial bias. We investigated GPR143, a receptor in the pigmentation pathway, which is activated by a melanin synthesis by-product, l-dopa. In this model, greater pigmentation leads to greater l-dopa production and, in turn, greater GPR143 signaling. GPR143 activity upregulates PEDF and downregulates both VEGF and exosomes; all of which reduce the angiogenic potential in the retina. Moreover, we demonstrate that GPR143 signaling enhances the digestion of shed photoreceptor outer segments. Together, our data suggests a central role for GPR143 signaling in RPE-photoreceptor interaction which is critical to healthy vision.

Levodopa Is Associated with Reduced Development of Neovascular Age-Related Macular Degeneration.

OBJECTIVE: To determine whether levodopa (L-DOPA) is associated with a reduced likelihood of developing neovascular age-related macular degeneration (AMD). DESIGN: Three studies were performed: retrospective analyses in the Vestrum Health Retina Database (#1-2) and case-control analysis in the Merative MarketScan Research Databases (#3). PARTICIPANTS: Eyes with neovascular AMD and 2 years of follow-up (#1). Eyes with non-neovascular AMD and 1 to 5 years of follow-up (#2). Patients aged ≥ 55 years with newly diagnosed neovascular AMD matched to controls without neovascular AMD (#3). METHODS: Eyes were divided into 2 groups (#1-2): exposed to L-DOPA before or on the date of neovascular (#1) or nonneovascular (#2) AMD diagnosis, and eyes not exposed to L-DOPA. We extracted AMD risk factors, number of intravitreal injections (#1), and conversion rate to neovascular AMD (#2). We calculated the percentage of newly diagnosed neovascular AMD cases and matched controls exposed to any L-DOPA and determined the cumulative 2-year dose in grams by tertiles (< 100 mg, approximately 100-300 mg, and approximately > 300 mg per day, #3). MAIN OUTCOME MEASURES: Number of intravitreal injections (#1) and detection of new-onset neovascular AMD (#2-3) after adjusting for AMD risk factors. RESULTS: In the Vestrum database, eyes with neovascular AMD that were exposed to L-DOPA underwent 1 fewer intravitreal injection over 2 years (N = 84 088 control vs. 530 L-DOPA eyes, P = 0.006). In eyes with nonneovascular AMD (N = 42 081-203 155 control vs. 314-1525 L-DOPA eyes), L-DOPA exposure was associated with a reduced risk of conversion to neovascular AMD by 21% at year 2 (P = 0.029), 35% at years 3 to 4 (P < 0.001), and 28% at year 5 (P = 0.024). In the MarketScan databases (N = 86 900 per group), cumulative 2-year doses of L-DOPA between approximately 100 to 300 mg per day and approximately > 300 mg were associated with decreased odds of developing neovascular AMD by 15% (odds ratio [OR], 0.85; 95% confidence interval [CI], 0.75-0.97) and 23% (OR, 0.77; 95% CI, 0.67-0.87), respectively. CONCLUSIONS: Levodopa use was associated with reduced detection of new-onset neovascular AMD. A prospective, randomized clinical trial should be considered to investigate whether low-dose L-DOPA reduces neovascular AMD conversion. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Zinc Supplementation Induced Transcriptional Changes in Primary Human Retinal Pigment Epithelium: A Single-Cell RNA Sequencing Study to Understand Age-Related Macular Degeneration.

Zinc supplementation has been shown to be beneficial to slow the progression of age-related macular degeneration (AMD). However, the molecular mechanism underpinning this benefit is not well understood. This study used single-cell RNA sequencing to identify transcriptomic changes induced by zinc supplementation. Human primary retinal pigment epithelial (RPE) cells could mature for up to 19 weeks. After 1 or 18 weeks in culture, we supplemented the culture medium with 125 µM added zinc for one week. RPE cells developed high transepithelial electrical resistance, extensive, but variable pigmentation, and deposited sub-RPE material similar to the hallmark lesions of AMD. Unsupervised cluster analysis of the combined transcriptome of the cells isolated after 2, 9, and 19 weeks in culture showed considerable heterogeneity. Clustering based on 234 pre-selected RPE-specific genes divided the cells into two distinct clusters, we defined as more and less differentiated cells. The proportion of more differentiated cells increased with time in culture, but appreciable numbers of cells remained less differentiated even at 19 weeks. Pseudotemporal ordering identified 537 genes that could be implicated in the dynamics of RPE cell differentiation (FDR < 0.05). Zinc treatment resulted in the differential expression of 281 of these genes (FDR < 0.05). These genes were associated with several biological pathways with modulation of ID1/ID3 transcriptional regulation. Overall, zinc had a multitude of effects on the RPE transcriptome, including several genes involved in pigmentation, complement regulation, mineralization, and cholesterol metabolism processes associated with AMD.

LINE-1 Cargo and Reverse Transcriptase Activity Profiles in Extracellular Vesicles from Lung Cancer Cells and Human Plasma.

Long Interspersed Element-1 (LINE-1) is an oncogenic human retrotransposon that 'copies and pastes' DNA into new locations via reverse transcription. Given that enzymatically active LINE-1 can be exported in extracellular vesicles (EVs), and that LINE-1 mRNA and its two encoded proteins, ORF1p and ORF2p, are required for retrotransposition, the present study examined LINE-1 EV loading patterns relative to reverse transcriptase (RT) activity in vivo and in vitro. Density gradient ultracentrifugation identified conserved patterns of LINE-1 mRNA and protein distribution in EVs, with RT activity readily detected in EV fractions containing both LINE-1 mRNA and protein. Unlike whole cell and tissue lysates, the ORF1p in EVs was detected as a dimer. EVs from ostensibly healthy plasma donors showed variable but consistent ORF1p profiles, with residual levels of LINE-1 mRNA measured in some but not all samples. EVs from cancer cell lines had elevated mean LINE-1 levels and 5-85 times greater RT activity than EVs from normal cells or healthy plasma. EV RT activity was associated with EV LINE-1 mRNA content and was highest in cell lines that also expressed an elevated expression of ORF1p and ORF2p. Given that LINE-1 activation is a hallmark of many cancer types, our findings suggest that an EV LINE-1 'liquid biopsy' may be developed to monitor LINE-1 activity during the course of malignant progression.

Levodopa Positively Affects Neovascular Age-Related Macular Degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a common cause of blindness worldwide. Neovascular AMD (nAMD) is an advanced form of the disease, in which excess vascular endothelial growth factor (VEGF) induces growth of new blood vessels that leak fluid, accounting for 90% of vision loss in AMD. Dysfunction of the retinal pigment epithelium likely initiates AMD. Retinal pigment epithelial cells express a G protein-coupled receptor, GPR143, which downregulates VEGF in response to levodopa. Anti-VEGF therapy effectively treats nAMD, suggesting that excessive VEGF activity drives the pathology. METHODS: In an open-label pilot study, in patients with newly diagnosed nAMD and naïve to anti-VEGF injections (Cohort-1), the effects of carbidopa-levodopa on vision and anatomic outcomes were evaluated for 4 weeks. Then patients were followed 5 months further with ascending levodopa doses. Patients previously treated with anti-VEGF injection therapy (Cohort-2) were also treated with ascending levodopa doses and evaluated for 6 months. RESULTS: Levodopa was safe, well tolerated, and delayed anti-VEGF injection therapy while improving visual outcomes. In the first month, retinal fluid decreased by 29% (P = .02, n = 12) without anti-VEGF treatment. Through 6 months the decrease in retinal fluid was sustained, with a mean frequency of 0.38 injections/month. At month 6, mean visual acuity improved by 4.7 letters in Cohort-1 (P = .004, n = 15) and by 4.8 letters in Cohort-2 (P = .02, n = 11). Additionally, there was a 52% reduction in the need for anti-VEGF injections in Cohort-2 (P = .002). CONCLUSIONS: Our findings suggest efficacy and support the pharmacological targeting of GPR143 with levodopa for the treatment of nAMD in future studies.

A G-Protein Coupled Receptor and Macular Degeneration.

Age-related macular degeneration (AMD) is a leading cause of irreversible blindness in the world. The risk of AMD increases with age and is most common among the white population. Here, we discuss the convergence of factors related to race, pigmentation, and susceptibility to AMD, where the primary defect occurs in retinal support cells, the retinal pigment epithelium (RPE). We explore whether the observed racial bias in AMD incidence is related to innate differences in the basal level of pigmentation between races, and whether the pigmentation pathway activity in the RPE might protect from retinal degeneration. More specifically, we explore whether the downstream signaling activity of GPR143, a G-protein coupled receptor in the pigmentation pathway, might underly the racial bias of AMD and be a target to prevent the disease. Lastly, we summarize the past findings of a large retrospective study that investigated the relationship between the stimulation of GPR143 with L-DOPA, the pigmentation pathway, and AMD, to potentially help develop new ways to prevent or treat AMD. The reader of this review will come to understand the racial bias of AMD, which is related to the function of the RPE.

GPR143 Signaling and Retinal Degeneration.

Age-related macular degeneration (AMD) is the most common cause of irreversible blindness. We do not know the cause of the disease and have inadequate prevention and treatment strategies for those at risk or affected. The greatest risk factors include age and race, with the white population at the highest risk for the disease. We developed the hypothesis that pigmentation in the retinal pigment epithelium (RPE) protects darkly pigmented individuals from AMD. We have tested this hypothesis in multiple ways including dissecting the pigmentation pathway in RPE using albinism-related tools, identification of a G protein-coupled receptor in the pigmentation pathway that drives expression of trophic factors, and using a very large retrospective chart analysis to test whether the ligand for the receptor prevents AMD. In total, our results indicate that pigmentation of the RPE is a cornerstone of RPE-retinal interaction and support and that the receptor in the pigmentation pathway most likely underlies the racial bias of the disease. The ligand for that receptor is an ideal candidate as a preventative and treatment for AMD. Here we summarize these results, discussing the research in its entirety with one overall goal, treatment or prevention of AMD.

Pigmentation and vision: Is GPR143 in control?

Albinism, typically characterized by decreased melanin synthesis, is associated with significant visual deficits owing to developmental changes during neurosensory retina development. All albinism is caused by genetic mutations in a group of diverse genes including enzymes, transporters, G-protein coupled receptor. Interestingly, these genes are not expressed in the neurosensory retina. Further, regardless of cause of albinism, all forms of albinism have the same retinal pathology, the extent of which is variable. In this review, we explore the possibility that this similarity in retinal phenotype is because all forms of albinism funnel through the same final common pathway. There are currently seven known genes linked to the seven forms of ocular cutaneous albinism. These types of albinism are the most common, and result in changes to all pigmented tissues (hair, skin, eyes). We will discuss the incidence and mechanism, where known, to develop a picture as to how the mutations cause albinism. Next, we will examine the one form of albinism which causes tissue-specific pathology, ocular albinism, where the eye exhibits the retinal albinism phenotype despite near normal melanin synthesis. We will discuss a potential way to treat the disease and restore normal retinal development. Finally, we will briefly discuss the possibility that this same pathway may intersect with the most common cause of permanent vision loss in the elderly.

Liquefaction of the Brain following Stroke Shares a Similar Molecular and Morphological Profile with Atherosclerosis and Mediates Secondary Neurodegeneration in an Osteopontin-Dependent Mechanism.

Here we used mouse models of heart and brain ischemia to compare the inflammatory response to ischemia in the heart, a protein rich organ, to the inflammatory response to ischemia in the brain, a lipid rich organ. We report that ischemia-induced inflammation resolves between one and four weeks in the heart compared to between eight and 24 weeks in the brain. Importantly, we discovered that a second burst of inflammation occurs in the brain between four and eight weeks following ischemia, which coincided with the appearance of cholesterol crystals within the infarct. This second wave shares a similar cellular and molecular profile with atherosclerosis and is characterized by high levels of osteopontin (OPN) and matrix metalloproteinases (MMPs). In order to test the role of OPN in areas of liquefactive necrosis, OPN-/- mice were subjected to brain ischemia. We found that at seven weeks following stroke, the expression of pro-inflammatory proteins and MMPs was profoundly reduced in the infarct of the OPN-/- mice, although the number of cholesterol crystals was increased. OPN-/- mice exhibited faster recovery of motor function and a higher number of neuronal nuclei (NeuN) positive cells in the peri-infarct area at seven weeks following stroke. Based on these findings we propose that the brain liquefies after stroke because phagocytic cells in the infarct are unable to efficiently clear cholesterol rich myelin debris, and that this leads to the perpetuation of an OPN-dependent inflammatory response characterized by high levels of degradative enzymes.

Alzheimer's associated amyloid and tau deposition co-localizes with a homeostatic myelin repair pathway in two mouse models of post-stroke mixed dementia.

The goal of this study was to determine the chronic impact of stroke on the manifestation of Alzheimer's disease (AD) related pathology and behavioral impairments in mice. To accomplish this goal, we used two distinct models. First, we experimentally induced ischemic stroke in aged wildtype (wt) C57BL/6 mice to determine if stroke leads to the manifestation of AD-associated pathological ß-amyloid (Aß) and tau in aged versus young adult wt mice. Second, we utilized a transgenic (Tg) mouse model of AD (hAPP-SL) to determine if stroke leads to the worsening of pre-existing AD pathology, as well as the development of pathology in brain regions not typically expressed in AD Tg mice. In the wt mice, there was delayed motor recovery and an accelerated development of cognitive deficits in aged mice compared to young adult mice following stroke. This corresponded with increased brain atrophy, increased cholinergic degeneration, and a focal increase of Aß in areas of axonal degeneration in the ipsilateral hemisphere of the aged animals. By contrast, in the hAPP-SL mice, we found that ischemia induced aggravated behavioral deficits in conjunction with a global increase in Aß, tau, and cholinergic pathology compared to hAPP-SL mice that underwent a sham stroke procedure. With regard to a potential mechanism, in both models, we found that the stroke-induced Aß and tau deposits co-localized with increased levels of ß-secretase 1 (BACE1), along with its substrate, neuregulin 1 (NGR1) type III, both of which are proteins integral for myelin repair. Based on these findings, we propose that the chronic sequelae of stroke may be ratcheting-up a myelin repair pathway, and that the consequent increase in BACE1 could be causing an inadvertent cleavage of its alternative substrate, AßPP, resulting in greater Aß seeding and pathogenesis.

Mining Retrospective Data for Virtual Prospective Drug Repurposing: L-DOPA and Age-related Macular Degeneration.

BACKGROUND: Age-related macular degeneration (AMD) is a leading cause of visual loss among the elderly. A key cell type involved in AMD, the retinal pigment epithelium, expresses a G protein-coupled receptor that, in response to its ligand, L-DOPA, up-regulates pigment epithelia-derived factor, while down-regulating vascular endothelial growth factor. In this study we investigated the potential relationship between L-DOPA and AMD. METHODS: We used retrospective analysis to compare the incidence of AMD between patients taking vs not taking L-DOPA. We analyzed 2 separate cohorts of patients with extensive medical records from the Marshfield Clinic (approximately 17,000 and approximately 20,000) and the Truven MarketScan outpatient and databases (approximately 87 million) patients. We used International Classification of Diseases, 9th Revision codes to identify AMD diagnoses and L-DOPA prescriptions to determine the relative risk of developing AMD and age of onset with or without an L-DOPA prescription. RESULTS: In the retrospective analysis of patients without an L-DOPA prescription, AMD age of onset was 71.2, 71.3, and 71.3 in 3 independent retrospective cohorts. Age-related macular degeneration occurred significantly later in patients with an L-DOPA prescription, 79.4 in all cohorts. The odds ratio of developing AMD was also significantly negatively correlated by L-DOPA (odds ratio 0.78; confidence interval, 0.76-0.80; P <.001). Similar results were observed for neovascular AMD (P <.001). CONCLUSIONS: Exogenous L-DOPA was protective against AMD. L-DOPA is normally produced in pigmented tissues, such as the retinal pigment epithelium, as a byproduct of melanin synthesis by tyrosinase. GPR143 is the only known L-DOPA receptor; it is therefore plausible that GPR143 may be a fruitful target to combat this devastating disease.

Controlled exosome release from the retinal pigment epithelium in situ.

Retinal Pigment Epithelial cells (RPE) express both GPR143 and myocilin, which interact in a signal transduction-dependent manner. In heterologous systems, activation of GPR143 with ligand causes transient recruitment of myocilin to internalized receptors, which appears to be the entry point of myocilin to the endocytic pathway. In some but not all cells, myocilin also traffics through the multivesicular body (MVB) and is released on the surface of exosomes in a signal transduction-dependent fashion. Little is known regarding the role of exosomes in RPE, but they likely serve as a mode of communication between the RPE and the outer retina. In this study, we used posterior poles with retina removed from fresh human donor eyes as a model to test the relationship between GPR143, myocilin, and exosomes in an endogenous system. We isolated exosomes released by RPE using differential centrifugation of media conditioned by the RPE for 25 min, and then characterized the exosomes using nanoparticle tracking to determine the number and size of the exosomes. Next, we tested whether ligand stimulation of GPR143 using l-DOPA altered RPE exosome release. Finally, we investigated whether myocilin was present on the exosomes released by RPE and whether l-DOPA stimulation of GPR143 caused recruitment of myocilin to the endocytic pathway, as we have previously observed using cultured cells. Activation of GPR143 halted RPE exosome release, while simultaneously recruiting myocilin to the endocytic compartment. Together, our results indicate that GPR143 and myocilin function in a signal transduction system that can control exosome release from RPE.

Does levodopa improve vision in albinism? Results of a randomized, controlled clinical trial.

BACKGROUND: Dopamine is an intermediate product in the biosynthesis of melanin pigment, which is absent or reduced in albinism. Animal research has shown that supplying a precursor to dopamine, levodopa, may improve visual acuity in albinism by enhancing neural networks. This study examines the safety and effectiveness of levodopa on best-corrected visual acuity in human subjects with albinism. DESIGN: Prospective, randomized, placebo-controlled, double-masked clinical trial conducted at the University of Minnesota. PARTICIPANTS: Forty-five subjects with albinism. METHODS: Subjects with albinism were randomly assigned to one of three treatment arms: levodopa 0.76 mg/kg with 25% carbidopa, levodopa 0.51 mg/kg with 25% carbidopa, or placebo and followed for 20 weeks, with best-corrected visual acuity measured at enrollment, and at weeks 5, 10, 15, and 20 after enrollment. Side-effects were recorded with a symptom survey. Blood was drawn for genotyping. MAIN OUTCOME MEASURES: Side-effects and best-corrected visual acuity 20 weeks after enrolment. RESULTS: All subjects had at least one mutation found in a gene known to cause albinism. Mean age was 14.5 years (range: 3.5 to 57.8 years). Follow up was 100% and compliance was good. Minor side-effects were reported; there were no serious adverse events. There was no statistically significant improvement in best-corrected visual acuity after 20 weeks with either dose of levodopa. CONCLUSIONS: Levodopa, in the doses used in this trial and for the time course of administration, did not improve visual acuity in subjects with albinism.

A role for myocilin in receptor-mediated endocytosis.

Myocilin is a broadly expressed protein that when mutated uniquely causes glaucoma. While no function has been ascribed to explain focal disease, some properties of myocilin are known. Myocilin is a cytoplasmic protein that also localizes to vesicles specifically as part of a large membrane-associated complex with properties similar to the SNARE machinery that function in vesicle fusion. Its role in vesicle dynamics has not been detailed, however myocilin intersects with the endocytic compartment at the level of the multivesicular body. Since internalized GPCRs are sorted in the multivesicular body, we investigated whether myocilin functions in ligand-dependent GPR143 endocytosis. Using recombinant systems we found that the kinetics of myocilin recruitment to biotinylated membrane proteins was similar to that of arrestin-3. We also co-localized myocilin with GPR143 and Arrestin-2 by confocal microscopy. However, wild-type myocilin differed significantly in its association kinetics and co-localization with internalized proteins from mutant myocilin (P370L or T377M). Moreover, we found that myocilin bound to the cytoplasmic tail of GPR143, an interaction mediated by its amino terminal helix-turn-helix domain. Hydrodynamic analyses show that the myocilin-GPR143 protein complex is >158 kD and stable in 500 mM KCl, but not 0.1% SDS. Collectively, data indicate that myocilin is recruited to the membrane compartment, interacting with GPCR proteins during ligand-mediated endocytosis and that GPCR signaling underlies pathology in myocilin glaucoma.

PEDF and VEGF-A output from human retinal pigment epithelial cells grown on novel microcarriers.

Human retinal pigment epithelial (hRPE) cells have been tested as a cell-based therapy for Parkinson's disease but will require additional study before further clinical trials can be planned. We now show that the long-term survival and neurotrophic potential of hRPE cells can be enhanced by the use of FDA-approved plastic-based microcarriers compared to a gelatin-based microcarrier as used in failed clinical trials. The hRPE cells grown on these plastic-based microcarriers display several important characteristics of hRPE found in vivo: (1) characteristic morphological features, (2) accumulation of melanin pigment, and (3) high levels of production of the neurotrophic factors pigment epithelium-derived factor (PEDF) and vascular endothelial growth factor-A (VEGF-A). Growth of hRPE cells on plastic-based microcarriers led to sustained levels (>1 ng/ml) of PEDF and VEGF-A in conditioned media for two months. We also show that the expression of VEGF-A and PEDF is reciprocally regulated by activation of the GPR143 pathway. GPR143 is activated by L-DOPA (1 µM) which decreased VEGF-A secretion as opposed to the previously reported increase in PEDF secretion. The hRPE microcarriers are therefore novel candidate delivery systems for achieving long-term delivery of the neuroprotective factors PEDF and VEGF-A, which could have a value in neurodegenerative conditions such as Parkinson's disease.

Myocilin, a component of a membrane-associated protein complex driven by a homologous Q-SNARE domain.

Myocilin is a widely expressed protein with no known function; however, mutations in myocilin appear to manifest uniquely as ocular hypertension and the blinding disease of glaucoma. Using the protein homology/analogy recognition engine (Phyre), we find that the olfactomedin domain of myocilin is similar in sequence motif and structure to a six-blade, kelch repeat motif based on the known crystal structures of such proteins. Additionally, using sequence analysis, we identify a coiled-coil segment of myocilin with homology to human Q-SNARE proteins (inset). Using COS-7 cells expressing full-length human myocilin and a version lacking the C-terminal olfactomedin domain, we identified a membrane-associated protein complex containing myocilin by hydrodynamic analysis. The myocilin construct that included the coiled-coil but lacked the olfactomedin domain formed complexes similar to the full-length protein, indicating that the coiled-coil domain of myocilin is sufficient for myocilin binding to the large detergent-resistant complex. In human retina and retinal pigment epithelium, which express myocilin, we detected the protein in a large, sodium dodecyl sulfate-resistant, membrane-associated complex. We characterized myocilin in human tissues as either a 15 S complex with an M(r) of 405000-440000 yielding a slightly elongated globular shape similar to that of known SNARE complexes or a 6.4 S dimer with an M(r) of 108000. By identifying the Q-SNARE homology within the second coil of myocilin and documenting its participation in a SNARE-like complex, we provide evidence of a SNARE domain-containing protein associated with a human disease.

L-DOPA is an endogenous ligand for OA1.

Albinism is a genetic defect characterized by a loss of pigmentation. The neurosensory retina, which is not pigmented, exhibits pathologic changes secondary to the loss of pigmentation in the retina pigment epithelium (RPE). How the loss of pigmentation in the RPE causes developmental defects in the adjacent neurosensory retina has not been determined, but offers a unique opportunity to investigate the interactions between these two important tissues. One of the genes that causes albinism encodes for an orphan GPCR (OA1) expressed only in pigmented cells, including the RPE. We investigated the function and signaling of OA1 in RPE and transfected cell lines. Our results indicate that OA1 is a selective L-DOPA receptor, with no measurable second messenger activity from two closely related compounds, tyrosine and dopamine. Radiolabeled ligand binding confirmed that OA1 exhibited a single, saturable binding site for L-DOPA. Dopamine competed with L-DOPA for the single OA1 binding site, suggesting it could function as an OA1 antagonist. OA1 response to L-DOPA was defined by several common measures of G-protein coupled receptor (GPCR) activation, including influx of intracellular calcium and recruitment of beta-arrestin. Further, inhibition of tyrosinase, the enzyme that makes L-DOPA, resulted in decreased PEDF secretion by RPE. Further, stimulation of OA1 in RPE with L-DOPA resulted in increased PEDF secretion. Taken together, our results illustrate an autocrine loop between OA1 and tyrosinase linked through L-DOPA, and this loop includes the secretion of at least one very potent retinal neurotrophic factor. OA1 is a selective L-DOPA receptor whose downstream effects govern spatial patterning of the developing retina. Our results suggest that the retinal consequences of albinism caused by changes in melanin synthetic machinery may be treated by L-DOPA supplementation.