Brian S Mckay
- Professor, Ophthalmology - (Research Scholar Track)
- Professor, Ophthalmology - (Research Scholar Track) (Banner)
- Professor, Physiology
Chronology of Education:
University of Wisconsin, Milwaukee, WI 1982-1987 B.S.
Medical College of Wisconsin, Milwaukee, WI 1989-1995 Ph.D.
The Scripps Research Institute 1995-1997
Dissertation: “Development and Analysis of a Model System for Examining Epithelial Cell Organization”. Advisor: Janice M.Burke, Ph.D.
Major field(s) Cell Biology
Chronology of Employment :
Kohart Inc., Assistant Manager, 1981-1987
MedicalCollege of Wisconsin, Laboratory Technologist, 1987-1989
DukeUniversity, Research Assistant Professor, 1997-2002
University of Arizona, Assistant Professor, 2002-2008
University of Arizona, Associate Professor, 2008-Present
- University of Arizona, Tucson (2002 - Ongoing)
- Influential Health and Medical Leaders
- Tucson Media Association, Fall 2017
No activities entered.
Curr Rsrch Vis+NeurodegenIMB 696E (Fall 2020)
Honors Independent StudyNSCS 399H (Fall 2020)
Curr Rsrch Vis+NeurodegenPSIO 696E (Spring 2020)
Honors ThesisPSIO 498H (Spring 2020)
Curr Rsrch Vis+NeurodegenPSIO 696E (Fall 2019)
Honors ThesisPSIO 498H (Fall 2019)
Curr Rsrch Vis+NeurodegenPSIO 696E (Spring 2019)
Honors Independent StudyPSIO 399H (Spring 2019)
Curr Rsrch Opth+Vis SciPSIO 696E (Fall 2018)
Honors Independent StudyPSIO 399H (Fall 2018)
Directed ResearchBME 492 (Spring 2018)
Honors Independent StudyPSIO 399H (Spring 2018)
Honors Independent StudyPSIO 399H (Fall 2017)
- Mckay, B. S., Allingham, R. R., & Stamer, W. D. (2018). Is the a final common pathway affected in POAG. In Glaucoma Research and Clinical Advances 2018-2020(pp 97-108). Kugler Publications.
- Chung, A., Frye, J., Zbesko, J., Constantopoulos, E., Hayes, M., Figueroa, A., Day, A. W., Konhilas, J. P., Mckay, B. S., Nguyen, T. V., & Doyle, K. P. (2018). Liquefaction of the brain following stroke shares a similar molecular and morphological profile with atherosclerosis and mediates secondary neurodegeneration in an osteopontin dependent mechanism. eNeuro.
- McKay, B. S. (2019). Pigmentation and vision: Is GPR143 in control?. Journal of neuroscience research, 97(1), 77-87.More infoAlbinism, 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.
- Nguyen, T. V., Hayes, M., Zbesko, J. C., Frye, J. B., Congrove, N. R., Belichenko, N. P., McKay, B. S., Longo, F. M., & Doyle, K. P. (2018). Alzheimer's associated amyloid and tau deposition co-localizes with a homeostatic myelin repair pathway in two mouse models of post-stroke mixed dementia. Acta neuropathologica communications, 6(1), 100.More infoThe 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 β-amyloid (Aβ) 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 Aβ 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 Aβ 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 Aβ and tau deposits co-localized with increased levels of β-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, AβPP, resulting in greater Aβ seeding and pathogenesis.
- McKay, B. S., Lynch, R. M., & Stamer, W. D. (2017). Comment on "Identification of Novel G Protein-Coupled Receptor 143 Ligands as Pharmacologic Tools for Investigating X-Linked Ocular Albinism". Investigative ophthalmology & visual science, 58(11), 4733-4734.
- McKay, B. S., & Schwartz, S. G. (2017). Pigmentation and Macular Degeneration: Is There a Role for GPR143?. Journal of ocular pharmacology and therapeutics : the official journal of the Association for Ocular Pharmacology and Therapeutics, 32(1), 3-4.
- Brilliant, M. H., Vaziri, K., Connor, T. B., Schwartz, S. G., Carroll, J. J., McCarty, C. A., Schrodi, S. J., Hebbring, S. J., Kishor, K. S., Flynn, H. W., Moshfeghi, A. A., Moshfeghi, D. M., Fini, M. E., & McKay, B. S. (2016). Mining Retrospective Data for Virtual Prospective Drug Repurposing: L-DOPA and Age-related Macular Degeneration. The American journal of medicine, 129(3), 292-8.More infoAge-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.
- Locke, C. J., Congrove, N. R., Dismuke, W. M., Bowen, T. J., Stamer, W. D., & McKay, B. S. (2014). Controlled exosome release from the retinal pigment epithelium in situ. Experimental eye research, 129, 1-4.More infoRetinal 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.
- Summers, C. G., Connett, J. E., Holleschau, A. M., Anderson, J. L., De Becker, I., McKay, B. S., & Brilliant, M. H. (2014). Does levodopa improve vision in albinism? Results of a randomized, controlled clinical trial. Clinical & experimental ophthalmology, 42(8), 713-21.More infoDopamine 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.
- McKay, B. S., Congrove, N. R., Johnson, A. A., Dismuke, W. M., Bowen, T. J., & Stamer, W. D. (2013). A role for myocilin in receptor-mediated endocytosis. PloS one, 8(12), e82301.More infoMyocilin 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.
- Dismuke, W. M., McKay, B. S., & Stamer, W. D. (2012). Myocilin, a component of a membrane-associated protein complex driven by a homologous Q-SNARE domain. Biochemistry, 51(17), 3606-13.More infoMyocilin 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.
- Mckay, B., Falk, T., Congrove, N. R., Zhang, S., McCourt, A. D., Sherman, S. J., & Mckay, B. S. (2012). PEDF and VEGF-A output from human retinal pigment epithelial cells grown on novel microcarriers. Journal of biomedicine & biotechnology, 2012.More infoHuman 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.
- Mckay, B., Lopez, V. M., Decatur, C. L., Stamer, W. D., Lynch, R. M., & Mckay, B. S. (2008). L-DOPA is an endogenous ligand for OA1. PLoS biology, 6(9).More infoAlbinism 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.
- Bakall, B., Radu, R. A., Stanton, J. B., Burke, J. M., McKay, B. S., Wadelius, C., Mullins, R. F., Stone, E. M., Travis, G. H., & Marmorstein, A. D. (2007). Enhanced accumulation of A2E in individuals homozygous or heterozygous for mutations in BEST1 (VMD2). Experimental eye research, 85(1), 34-43.More infoBest vitelliform macular dystrophy (BMD) is an autosomal dominant inherited macular degenerative disease caused by mutations in the gene BEST1 (formerly VMD2). Prior reports indicate that BMD is characterized histopathologically by accumulation of lipofuscin in the retinal pigment epithelium (RPE). However, this accumulation has not been quantified and the chemical composition of lipofuscin in BMD has not been examined. In this study we characterize the histopathology of a donor eye from a rare individual homozygous for a mutation (W93C) in BEST1. We find that this individual's disease was not any more severe than has been described for heterozygotes. We then used this tissue to quantify lipofuscin accumulation by enriching intracellular granules from RPE cells on sucrose gradients and counting the granules in each density fraction. Granules from the homozygous donor eye as well as a donor eye from an individual heterozygous for the mutation T6R were compared with age-matched control eyes. Interestingly, the least dense fraction, representing classical lipofuscin granules was either not present or significantly diminished in the BMD donor eyes and the autoflourescence associated with lipofuscin had shifted to denser fractions. However, a substantial enrichment for granules in fractions of higher density was also noted in the BMD samples. Inspection of granules from the homozygous donor eye by electron microscopy revealed a complex abnormal multilobular structure. Analysis of granules by HPLC indicated a approximately 1.6- and approximately fourfold overall increase in A2E in the BMD eyes versus age-matched control eyes, with a shift of A2E to more dense granules in the BMD donor eyes. Despite the increase in A2E and total intracellular granules, the RPE in the homozygous donor eyes was relatively well preserved. Based on these data we conclude that the clinical and histopathologic consequences to the homozygous donor were not any more severe than has been reported previously for individuals who are established or presumptive heterozygotes. We find that A2E is a component of the lipofuscin accumulated in BMD and that it is more abundant than in control eyes suggesting that the etiology of BMD is similar to Stargardt's disease and Stargardt-like macular dystrophy. Finally, the changes we observe in the granules suggest that the histopathology and eventual vision loss associated with BMD may be due to defects in the ability of the RPE to fully degrade phagocytosed photoreceptor outer segments.
- Perkumas, K. M., Hoffman, E. A., McKay, B. S., Allingham, R. R., & Stamer, W. D. (2007). Myocilin-associated exosomes in human ocular samples. Experimental eye research, 84(1), 209-12.More infoMutations in myocilin result in ocular hypertension, likely due to decreased drainage of aqueous humor through the trabecular meshwork. Since less myocilin is found in the aqueous humor of those with disease-causing mutations, understanding myocilin's role in the aqueous humor is of clinical importance. Recently, myocilin was shown to exit cultured trabecular meshwork cells in association with shed vesicles called exosomes. To examine relevance of this finding in a physiological setting, the present study examined three different types of ocular samples for the presence of myocilin-associated exosomes. Using differential centrifugation steps, we found myocilin associated with exosomes isolated from effluent collected from human anterior segments in organ culture and aqueous humor obtained from human cadaveric eyes or from patients undergoing excisional surgery. Similar to results with cultured cells, myocilin associated predominately with exosomes in fresh samples, appeared mostly soluble at later times, and had biochemical properties (density of 1.13-1.19 g/ml in linear sucrose gradient) similar to those characteristics of exosomes. These data indicate that exosomes are present and may facilitate the transport of myocilin into the extracellular space of human ocular cells.
- Yu, L., Kelly, U., Ebright, J. N., Malek, G., Saloupis, P., Rickman, D. W., McKay, B. S., Arshavsky, V. Y., & Bowes Rickman, C. (2007). Oxidative stress-induced expression and modulation of Phosphatase of Regenerating Liver-1 (PRL-1) in mammalian retina. Biochimica et biophysica acta, 1773(9), 1473-82.More infoThe phosphatase of regenerating liver-1, PRL-1, gene was detected in a screen for foveal cone photoreceptor-associated genes. It encodes a small protein tyrosine phosphatase that was previously immunolocalized to the photoreceptors in primate retina. Here we report that in cones and cone-derived cultured cells both PRL-1 activity and PRL-1 gene expression are modulated under oxidative stress. Oxidation reversibly inhibited the phosphatase activity of PRL-1 due to the formation of an intramolecular disulfide bridge between Cys104 within the active site and another conserved Cys, Cys49. This modulation was observed in vitro, in cell culture and in isolated retinas exposed to hydrogen peroxide. The same treatment caused a rapid increase in PRL-1 expression levels in cultured cells which could be blocked by the protein translation inhibitor, cycloheximide. Increased PRL-1 expression was also observed in living rats subjected to constant light exposure inducing photooxidative stress. We further demonstrated that both oxidation and overexpression of PRL-1 upon oxidative stress are greatly enhanced by inhibition of the glutathione system responsible for cellular redox regulation. These findings suggest that PRL-1 is a molecular component of the photoreceptor's response to oxidative stress acting upstream of the glutathione system.
- Conley, S. M., McKay, B. S., Gandolfi, A. J., & Stamer, W. D. (2006). Alterations in human trabecular meshwork cell homeostasis by selenium. Experimental eye research, 82(4), 637-47.More infoEpidemiological evidence indicates that selenium supplementation may increase risk for glaucoma and ocular hypertension. The purpose of this study was to determine the effects of selenium on trabecular meshwork cells, a likely site of pathology for glaucoma. Human trabecular meshwork (HTM) cells and human umbilical vein endothelial cells (HUVECs) were treated with selenium (MSeA) at or near physiologically relevant concentrations. Selenium uptake by cells was monitored using mass spectrometry. Alterations in protein secretion, intracellular signaling, and cell morphology were monitored; and the role of integrin signaling in MSeA-induced morphological alterations was investigated using divalent cation treatments. Radiolabeling was used to assess protein synthesis and secretion, while luciferase and MTT assays monitored total cellular ATP and cell viability, respectively. Whereas detectible changes in intracellular selenium were observed after exposure to 1-10 microM MSeA for 24hr, the majority remained in the conditioned medium. Selenium-induced morphological changes (< or =3 hr) occurred before alterations in protein secretion and intracellular signaling (3-6 hr). Zinc treatment prevented selenium-mediated alterations in protein secretion and changes in cell-matrix adhesion. MSeA treatment (5 microM) led to a 60% decrease in protein synthesis after 3 hr and a 30% reduction in secretion, although significant alterations in cell viability and total ATP were not observed after MSeA treatment. Selenium altered several indicators of HTM cell homeostasis, but did not affect viability at physiologically relevant doses. Similar results with HUVECs have implications for understanding selenium's mechanisms of action as an anti-angiogenic agent.
- McKay, B. S., Goodman, B., Falk, T., & Sherman, S. J. (2006). Retinal pigment epithelial cell transplantation could provide trophic support in Parkinson's disease: results from an in vitro model system. Experimental neurology, 201(1), 234-43.More infoTransplantation of retinal pigment epithelial (RPE) cells in the basal ganglia could provide a novel cell-based therapy for Parkinson's disease by providing a constant source of dopamine replacement via the melanin synthetic pathway enzyme tyrosinase. We now demonstrate that human RPE cells also produce a neurotrophic effect on primary cultures of rat striatal (enkephalinergic) and mesencephalic (dopaminergic) neurons. Differentiation of RPE cells to a pigmented monolayer using a Ca(++)-switch protocol increased the potency of the neurotrophic effect on dopaminergic neurons. Conditioned medium derived from differentiated RPE cells increased neurite outgrowth in dopaminergic neurons by 125% compared to 25% for undifferentiated RPE cells. The neurotrophic effect was not due to tyrosinase activity. Differentiation of RPE cells doubled the production of pigment-derived epithelial factor (PEDF). However, PEDF accounted for only a portion of the neurotrophic effect as determined by depletion experiments and dose-response comparisons with purified PEDF, indicating that differentiation increased the production of other trophic factors as well. Conditioned medium from differentiated RPE cells also provided a neurotrophic effect on a subset of enkephalinergic striatal neurons increasing neurite outgrowth by 78%. Survival of enkephalinergic neurons in vitro was increased by RPE conditioned medium. In untreated cultures the number of enkephalinergic neurons declined 62% over a 2-week period compared to a 29% decline in RPE-treated cultures. These results indicate that transplantation RPE cells could potentially provide a dual benefit in Parkinson's disease producing both dopamine and neurotrophic support of the basal ganglia.
- Mckay, B., Rak, D. J., Hardy, K. M., Jaffe, G. J., & Mckay, B. S. (2006). Ca++-switch induction of RPE differentiation. Experimental eye research, 82(4).More infoCultured retinal pigment epithelial (RPE) cells are commonly used as a model of the tissue to study their involvement in visual diseases. Unfortunately, cultured RPE often lose their differentiated phenotype reducing their usefulness as a model of the RPE in vivo. In this study, we used a Ca++-switch protocol to initiate the patterned expression of several phenotypic and functional markers of RPE differentiation. Cultured RPE cells from adult donors were maintained through at least six serial passages prior to assay to minimize their differentiated properties. The cells were then subjected to the Ca++-switch protocol and maintained at confluence for up to 4 months. Paired control and Ca++-switch cells were examined for phenotype, pigmentation, and the expression of tyrosinase, CRABP, myocilin, and bestrophin by western blot analysis. The Ca++-switch protocol led to a rapid restriction of N-cadherin to lateral cell borders, and to expression of tyrosinase by day 4. After 8 weeks, the experimental RPE monolayers began to accumulate visible pigment, and after 12 weeks CRABP expression was observed. Myocilin was observed at 4 months after the Ca++-switch but bestrophin was not detected at any time point. Our results suggest this protocol may drive epithelial morphogenesis in RPE cells. We note two specific differences in cells plated in low Ca++, reduced spreading on the substrate and coordinated development of cadherin adhesion when the Ca++-concentration is returned to normal. Thus, we suggest that this method produces phenotypic changes through multiple cell signalling pathways.
- Stamer, W. D., Perkumas, K. M., Hoffman, E. A., Roberts, B. C., Epstein, D. L., & McKay, B. S. (2006). Coiled-coil targeting of myocilin to intracellular membranes. Experimental eye research, 83(6), 1386-95.More infoMutations in myocilin (MYOC) associate with glaucoma and ocular hypertension. Unfortunately, the specific role of MYOC, a widely expressed protein of unknown function, in ocular hypertension is unknown. Since MYOC localizes both to intracellular membranes and to the cytosol, we tested the hypothesis that MYOC is a cytosolic protein that associates with cellular membranes via its coiled-coil domain. Using green fluorescent protein (GFP) chimeras in expression and metabolic labeling studies, we observed that MYOC's putative signal peptide failed to traffic GFP into the secretory machinery and out of transfected cells. Next, we tested which of MYOC's three folding domains were responsible for targeting. In cell fractionation and immunofluorescence microscopy studies, the coiled-coil, but not the helix-turn-helix or olfactomedin domains, was necessary and sufficient to target GFP chimeras to cell membranes. Interestingly, a vesicular phenotype required sequential addition of the helix-turn-helix and olfactomedin domains to the coiled-coil. Taken together, these data indicate that the coiled-coil domain, not the putative signal sequence, is responsible for the targeting of MYOC to the secretory machinery.
- Hardy, K. M., Hoffman, E. A., Gonzalez, P., McKay, B. S., & Stamer, W. D. (2005). Extracellular trafficking of myocilin in human trabecular meshwork cells. The Journal of biological chemistry, 280(32), 28917-26.More infoMyocilin (MYOC) is a protein with a broad expression pattern, but unknown function. MYOC associates with intracellular structures that are consistent with secretory vesicles, however, in most cell types studied, MYOC is limited to the intracellular compartment. In the trabecular meshwork, MYOC associates with intracellular vesicles, but is also found in the extracellular space. The purpose of the present study was to better understand the mechanism of extracellular transport of MYOC in trabecular meshwork cells. Using a biochemical approach, we found that MYOC localizes intracellularly to both the cytosolic and particulate fractions. When intracellular membranes were separated over a linear sucrose gradient, MYOC equilibrated in a fraction less dense than traditional secretory vesicles and lysosomes. In pulse-labeling experiments that followed nascent MYOC over time, the characteristic doublet observed for MYOC by SDS-PAGE did not change, even in the presence of brefeldin A; indicating that MYOC is not glycosylated and is not released via a traditional secretory mechanism. When conditioned media from human trabecular meshwork cells were examined, both native and recombinant MYOC associated with an extracellular membrane population having biochemical characteristics of exosomes, and containing the major histocompatibility complex class II antigen, HLA-DR. The association of MYOC with exosome-like membranes appeared to be specific, on the extracellular face, and reversible. Taken together, data suggest that MYOC appears in the extracellular space of trabecular meshwork cells by an unconventional mechanism, likely associated with exosome-like vesicles.
- Mckay, B., Hoffman, E. A., Conley, S. M., Stamer, W. D., & Mckay, B. S. (2005). Barriers to productive transfection of trabecular meshwork cells. Molecular vision, 11.More infoA critical function of trabecular meshwork cells is to degrade cellular debris, including DNA. We hypothesize that low transfection efficiencies of primary human trabecular meshwork (HTM) cell cultures with plasmid DNA are a function of retained capacity to efficiently degrade exogenous DNA in vitro.
- Yang, P., McKay, B. S., Allen, J. B., & Jaffe, G. J. (2004). Effect of NF-kappa B inhibition on TNF-alpha-induced apoptosis in human RPE cells. Investigative ophthalmology & visual science, 45(7), 2438-46.More infoIn many cell types, tumor necrosis factor (TNF)-alpha-induced apoptosis is prevented by production of TNF-alpha-induced antiapoptotic protein, a process mediated by nuclear transcription factor (NF)-kappa B. TNF-alpha is widely expressed in proliferative vitreoretinopathy (PVR) membranes and is present in the vitreous of eyes with PVR. To understand mechanisms responsible for RPE cell survival and death in this disease, this study was conducted to determine whether specific NF-kappa B blockade by mutant inhibitory (I)-kappa B (I kappa B) affects TNF-alpha-induced cell death.
- Mckay, B., Stamer, W. D., Bok, D., Hu, J., Jaffe, G. J., & Mckay, B. S. (2003). Aquaporin-1 channels in human retinal pigment epithelium: role in transepithelial water movement. Investigative ophthalmology & visual science, 44(6).More infoAquaporin (AQP) is a hexahelical integral membrane protein that functions as a constitutive channel for water and regulated channel for cations in fluid transporting tissues, including many in the eye. Although AQP1 has been cloned from a cDNA library prepared from cultures of retinal pigment epithelial (RPE) cells isolated from human fetal tissue, three separate studies failed with various immunochemical techniques to detect AQP1 protein in adult human or rat RPE preparations. The purpose of this study was to examine specifically the expression and distribution of AQP1 in adult human RPE in situ by using alternative methodologies and model systems and to determine the contribution of AQP1 to water movement across cultured RPE cells isolated from human cadaveric and fetal eyes.
- Yang, P., McKay, B. S., Allen, J. B., Roberts, W. L., & Jaffe, G. J. (2003). Effect of mutant IkappaB on cytokine-induced activation of NF-kappaB in cultured human RPE cells. Investigative ophthalmology & visual science, 44(3), 1339-47.More infoThe nuclear transcription factor (NF)-kappaB is a central regulator of multiple inflammatory cytokines. The current study was conducted to determine whether infection of human retinal pigment epithelial (RPE) cells by adenovirus carrying a mutant inhibitory (I)-kappaB (IkappaB) transgene inhibits cytokine-induced activity of NF-kappaB and expression of NF-kappaB-dependent cytokines by preventing degradation of IkappaB. The persistence of recombinant protein expression and function after the viral infection was also examined.
- McKay, B. S., & Burke, J. M. (1997). Cell association increases RPE outgrowth from primary explant. Current eye research, 16(9), 891-9.More infoMost studies of cell growth of the RPE employ cultures which have been previously passaged, but here we investigated freshly-explanted RPE cells, which may have been growth-quiescent for years, within the eye to determine whether co-culture affects initial outgrowth in primary culture.
- McKay, B. S., Irving, P. E., Skumatz, C. M., & Burke, J. M. (1997). Cell-cell adhesion molecules and the development of an epithelial phenotype in cultured human retinal pigment epithelial cells. Experimental eye research, 65(5), 661-71.More infoFor most epithelial cells, the adherens junction protein E-cadherin is an epithelial morphogen, inducing the development of an epithelial phenotype in vitro after cell contact at confluency. Here retinal pigment epithelial cells (RPE), which lack E-cadherin but express a cadherin that is also found in many non-epithelial cells (N-cadherin), were examined for the ability to produce an epithelial phenotype in vitro. Subpopulations of grossly epithelioid or fusiform cells were selected for analysis from RPE cultures derived from adult human donors. After confluency, epithelioid RPE cells were observed to undergo time-dependent changes that were similar to those previously found in epithelial cells expressing E-cadherin: the cadherin gradually developed a zonular distribution of detergent-resistant protein that co-localized with forming circumferential actin bundles; Na/K ATPase accumulated at cell contact sites, then polarized to its tissue-specific domain (the apical membrane for RPE); the cells formed elevated domes on the impermeant culture substrate. In contrast to cells expressing E-cadherin, these events in RPE required weeks rater than days at confluency. Additional proteins were examined in epithelioid RPE cells revealing that cytokeratins reorganized after confluency producing a zonular array, and several other adhesion proteins (alpha5beta1 integrin, ICAM-1, PECAM-1, NCAM) became enriched at cell-cell contact sites, each developing a distinct pattern at a distinct postconfluency interval. In contrast to epithelioid RPE, in fusiform RPE the adhesion molecules did not develop discrete distribution patterns after confluency, although the same complement of adhesion proteins was expressed. In cells expressing E-cadherin, the absence of epithelial properties is often due to underexpression of the cadherin or of the catenins, adherens junction proteins that link the cadherin to actin. Fusiform RPE, however, were not deficient in these proteins, expressing amounts of N-cadherin, alpha-catenin, beta-catenin, plakoglobin, p120, alpha-actinin and vinculin that were equivalent to epithelioid cells. It appears, therefore, that a subset of epithelial cells that express N-cadherin can produce a highly-developed epithelial phenotype in vitro through a slow morphogenetic process. However, the expression alone of adhesion molecules, including those with a morphoregulatory function in other cells, is insufficient to produce an epithelial phenotype in all cells derived from the pigment epithelium.
- Burke, J. M., Skumatz, C. M., Irving, P. E., & McKay, B. S. (1996). Phenotypic heterogeneity of retinal pigment epithelial cells in vitro and in situ. Experimental eye research, 62(1), 63-73.More infoWe have previously developed methods based upon differential cell adhesion to select for cells of two different phenotypes, epithelioid and fusiform, from cultures of human RPE. Here we considered whether the differences in cell shape correlated with differences in protein tyrosine phosphorylation since it is known that elevated phosphorylation perturbs the stability of the adherens junction, a major determinant of epithelial phenotype. In cultures of both phenotypes we found low tyrosine phosphorylation levels in postconfluent cultures, and the same complement of tyrosine phosphoproteins after treatment with a phosphatase inhibitor. However, in contrast to epithelioid cells, fusiform RPE failed to localize the phosphoproteins to junctional sites on the cell periphery. We also re-evaluated primary and passaged RPE cultures for additional cell shape variants. Several discrete phenotypes were identified within the same cultures. They required several weeks at confluency to develop in primary as well as in passaged cultures, but after developing they remained stable for months. Since explanted RPE cells manifest several shape variants in an identical culture environment we examined bovine RPE cells in situ to determine whether the cells were heterogenous with regards to some cell surface and cytoskeletal proteins that might contribute to cell shape. Circumferential actin microfilament bundles and the occluding junction protein ZO-1 had fairly uniform distributions among cells in the monolayer, but the intermediate filament protein vimentin and the pericellular expression of phosphotyrosine varied among individual cells. Therefore, despite its grossly homogeneous appearance, the RPE monolayer in situ might be considered a mosaic of structurally heterogeneous cells which can give rise to phenotypically-distinct subpopulations when propagated in vitro.
- McKay, B. S., Annis, D. S., Honda, S., Christie, D., & Kunicki, T. J. (1996). Molecular requirements for assembly and function of a minimized human integrin alphaIIbbeta3. The Journal of biological chemistry, 271(48), 30544-7.More infoIntegrin subunit compatibility within and between species plays a major role in heterodimer assembly and ligand specificity. As an example, human alphaIIb pairs only with human beta3 and does not assemble a heterodimer with beta3 from other species. We use interspecies subunit chimeras to identify molecular requirements for subunit compatibility and show that species-restricted heterodimer assembly depends on a unique hexapeptide VGSDNH in an extended loop of the hypothetical human beta3 MIDAS domain. This allows us to express alphaIIb(1-233) and beta3(111-318) as a soluble, mini-integrin that retains RGD-dependent ligand recognition. Thus, in the case of one integrin, alphaIIbbeta3, the molecular requirements for integrin subunit compatibility and ligand recognition are intimately related.
- McKay, B. S., & Burke, J. M. (1994). Separation of phenotypically distinct subpopulations of cultured human retinal pigment epithelial cells. Experimental cell research, 213(1), 85-92.More infoLike most epithelial cells that are isolated from tissues and placed in culture, the phenotype of human retinal pigment epithelial cells (RPE) in vitro is more variable than for the same cells in situ. The phenotypic heterogeneity of the cultures has been attributed to varying stages of differentiation of the cells induced by the culture environment. In this study we show that within the same cultures RPE cells exist as phenotypic variants which are distinct and stable. Two phenotypically distinct subpopulations were identified, one epithelioid and one fusiform, that were present from the first spreading event in primary culture through multiple serial passages and when maintained for extended periods at confluency. Cell aggregation studies indicated differences in cellular adhesions, known determinants of cell shape, between the subpopulations. Methods to separate the subpopulations were developed which are based on the differential trypsin sensitivity of adhesions. The separated subpopulations had the same RPE cytokeratins by immunoblotting, but cytokeratin filaments (and actin filaments) had different organizations. The study indicates that RPE cell cultures contain at least two subpopulations of phenotypically distinct cells under identical culture conditions that can be separated and propagated independently. The phenotypic variants offer a model system for investigating determinants of epithelial cell shape in RPE. Further, the separation methods might be applied to test for phenotypic variants in other types of cultured epithelia.
- Mckay, B. S. (2019, May/ Spring). Parkinson drug may prevent and delay AMD. ARVO. Vancouver, CA: ARVO.
- Mckay, B. S., Congrove, N. R., Figueroa, A. G., & Sillik, S. A. (2018, Fall). GPR43 Signaling and Retinal Degeneration. XVIII International Symposium on Retinal Degeneration (2018). Killarney, Ireland: RD Society.
- Brennan, B. M., Timothy, F., Anna, F. G., Mckay, B. S., & Robert, S. W. (2019, May/ Spring). Short Term Effects of Carbidopa-Levodopa in Neovascular AMD. ARVO. Vancouver, CA: Association for Research in Vision and Ophthalmology.
- Figurueroa, A. G., Congrove, N. R., Liu, Y., & Mckay, B. S. (2019, May/ Spring). Dopaminergic control of constitutive exosome release from in situ RPE. ARVO. Vancouver, CA: ARVO.
- Morrison-Colvin, R., Sara, S., Nicole, C. R., Mckay, B. S., & Robert, S. W. (2019, May/ Spring). Circadian Patterns of Secretion of VEGF by RPE. ARVO 2019. Vancouver, CA: Association for Research in Vision and Ophthalmology.
- Chung, A., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M. I., Figueroa, A. G., Day, W. A., Konhilas, J. P., Mckay, B. S., Nguyen, T., & Doyle, K. (2018, June). Liquefaction of the brain following stroke shares a similar molecular and morphological profile with atherosclerosis and mediates secondary neurodegeneration in an osteopontin dependent mechanism. Keystone Symposia Conference: New Frontiers in Neuroinflammation: What Happens when CNS and Periphery Meet?. Keystone, Colorado: Keystone Symposia Conference.
- Chung, A., Frye, J. B., Zbesko, J. C., Constantopoulos, E., Hayes, M. I., Figueroa, A. G., Day, W. A., Konhilas, J. P., Mckay, B. S., Nguyen, T., & Doyle, K. (2018, November). Liquefaction of the brain following stroke shares characteristics with atherosclerosis. Society for Neuroscience Conference. San Diego, California: Society for Neuroscience.
- Figueroa, A., Congrove, N. R., Sillik, S. A., Sadideen, D. T., Falk, T., Rickman, C. B., & McKay, B. S. (2018, March). Exosome uptake is selective but not species or tissue-specific. INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE.
- Mckay, B. S., Sillik, S. A., Dismuke, W. M., Locke, C. J., Cojngrove, N. R., & Stamer, W. D. (2018, Fall). Endosomal Function and Glaucoma. XXIII Biennial Meeting of the International Society for Eye Research. Belfast, Northern Ireland: International Society for Eye Research.
- Piechowicz, M., Figueroa, A., Congrove, N. R., Sillik, S. A., & McKay, B. S. (2018, May). L-DOPA activation of GPR143 decreases lysosomal pH in RPE. INVESTIGATIVE OPHTHALMOLOGY & VISUAL SCIENCE. Honolulu, HI: ARVO.
- Figueroa, A., Falk, T., & Mckay, B. S. (2017, Fall). Myocilin-Associated Exosome Release in Glaucoma-Related Diseases.. Minority Access, 18th Annual Conference Abstracts.