Nestor Mas Gomez

The Role of Bestrophin-1 in Intracellular Ca 2+ Signaling

Mutations in the BEST1 gene lead to a variety of retinal degenerations, among them Bests vitelliforme macular degeneration. To clarify the mechanism of the disease, the understanding of the function of BEST1 gene product, bestrophin-1, is mandatory. In overexpression studies bestrophin-1 appeared to function as a Ca(2+)-dependent Cl channel. On the other hand, bestrophin-1 is able to participate in intracellular Ca(2+) signaling. Endogenously expressed bestrophin-1 largely localized to the cytosolic compartment close to the basolateral membrane of the retinal pigment epithelium (RPE) as it can be shown using differential centrifugation, immunohistochemistry, and transmission electron microscopy. To elucidate a cytosolic function of bestrophin-1, we explored the store-operated Ca(2+) entry in short-time cultured porcine RPE cells. Depletion of cytosolic Ca(2+)stores by SERCA inhibition led to activation of Orai-1 Ca(2+) channels. This resulted in an influx of extracellular Ca(2+) into the cell which was reduced when bestrophin-1 expression was knocked down using siRNA techniques. Quantification of Ca(2+) which can be released from cytosolic Ca(2+) stores revealed that after reduction of bestrophin-1 expression less Ca(2+) is stored in ER Ca(2+) stores. Thus, bestrophin-1 functions as an intracellular Cl channel which helps to accumulate and to release Ca(2+) from stores by conducting the counterion for Ca(2+).

Bestrophinopathy: An RPE-photoreceptor interface disease

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.

Contribution of Ion Channels in Calcium Signaling Regulating Phagocytosis: MaxiK, Cav1.3 and Bestrophin-1

Mutations in the BEST1 gene lead to a variety of retinal degenerations including Bests vitelliforme macular degeneration. The BEST1 gene product, bestrophin-1, is expressed in the retinal pigment epithelium (RPE). It is likely that mutant bestrophin-1 impairs functions of the RPE which support photoreceptor function and will thus lead to retinal degeneration. However, the RPE function which is influenced by bestrophin-1 is so far not identified. Previously we showed that bestrophin-1 interacts with L-type Ca²⁺ channels of the CaV1.3 subtype and that the endogenously expressed bestrophin-1 is required for intracellular Ca²⁺ regulation. A hallmark of Bests disease is the fast lipofuscin accumulation occurring already at young ages. Therefore, we addressed the hypothesis that bestrophin-1 might influence phagocytosis of photoreceptor outer segments (POS) by the RPE. Here, siRNA knock-down of bestrophin-1 expression as well as inhibition of L-type Ca²⁺ channel activity modulated the POS phagocytosis in vitro. In vivo CaV1.3 expression appeared to be diurnal regulated with a higher expression rate in the afternoon. Compared to wild-type littermates, Ca V 1.3 (-/-) mice showed a shift in the circadian POS phagocytosis with an increased activity in the afternoon. Thus we suggest that mutant bestrophin-1 leads to an impaired regulation of the POS phagocytosis by the RPE which would explain the fast lipofuscin accumulation in Best patients.

Compromised phagosome maturation underlies RPE pathology in cell culture and whole animal models of Smith-Lemli-Opitz Syndrome

ABSTRACT Treatment of rats with the cholesterol pathway inhibitor AY9944 produces an animal model of Smith-Lemli-Opitz syndrome (SLOS), an autosomal recessive disease caused by defective cholesterol synthesis. This SLOS rat model undergoes progressive and irreversible degeneration of the neural retina, with associated pathological features of the retinal pigmented epithelium (RPE). Here, we provide further insights into the mechanism involved in the RPE pathology. In the SLOS rat model, markedly increased RPE apical autofluorescence is observed, compared to untreated animals, which correlates with increased levels of A2E and other bisretinoids. Utilizing cultured human induced pluripotent stem cell (iPSC)- derived SLOS RPE cells, we found significantly elevated steady-state levels of 7-dehydrocholesterol (7DHC) and decreased cholesterol levels (key biochemical hallmarks of SLOS). Western blot analysis revealed altered levels of the macroautophagy/autophagy markers MAP1LC3B-II and SQSTM1/p62, and build-up of ubiquitinated proteins. Accumulation of immature autophagosomes was accompanied by inefficient degradation of phagocytized, exogenously supplied retinal rod outer segments (as evidenced by persistence of the C-terminal 1D4 epitope of RHO [rhodopsin]) in SLOS RPE compared to iPSC-derived normal human control. SLOS RPE cells exhibited lysosomal pH levels and CTSD activity within normal physiological limits, thus discounting the involvement of perturbed lysosomal function. Furthermore, 1D4-positive phagosomes that accumulated in the RPE in both pharmacological and genetic rodent models of SLOS failed to fuse with lysosomes. Taken together, these observations suggest that defective phagosome maturation underlies the observed RPE pathology. The potential relevance of these findings to SLOS and the requirement of cholesterol for phagosome maturation are discussed.

Stimulation of TLR3 triggers release of lysosomal ATP in astrocytes and epithelial cells that requires TRPML1 channels

Cross-reactions between innate immunity, lysosomal function, and purinergic pathways may link signaling systems in cellular pathologies. We found activation of toll-like receptor 3 (TLR3) triggers lysosomal ATP release from both astrocytes and retinal pigmented epithelial (RPE) cells. ATP efflux was accompanied by lysosomal acid phosphatase and beta hexosaminidase release. Poly(I:C) alkalinized lysosomes, and lysosomal alkalization with bafilomycin or chloroquine triggered ATP release. Lysosomal rupture with glycyl-L-phenylalanine-2-naphthylamide (GPN) eliminated both ATP and acid phosphatase release. Secretory lysosome marker LAMP3 colocalized with VNUT, while MANT-ATP colocalized with LysoTracker. Unmodified membrane-impermeant 21-nt and “non-targeting” scrambled 21-nt siRNA triggered ATP and acid phosphatase release, while smaller 16-nt RNA was ineffective. Poly(I:C)-dependent ATP release was reduced by TBK-1 block and in TRPML1−/− cells, while TRPML activation with ML-SA1 was sufficient to release both ATP and acid phosphatase. The ability of poly(I:C) to raise cytoplasmic Ca2+ was abolished by removing extracellular ATP with apyrase, suggesting ATP release by poly(I:C) increased cellular signaling. Starvation but not rapamycin prevented lysosomal ATP release. In summary, stimulation of TLR3 triggers lysosomal alkalization and release of lysosomal ATP through activation of TRPML1; this links innate immunity to purinergic signaling via lysosomal physiology, and suggests even scrambled siRNA can influence these pathways.

Membrane localization of the Repeats-in-Toxin (RTX) Leukotoxin (LtxA) produced by Aggregatibacter actinomycetemcomitans

The oral bacterium, Aggregatibacter actinomycetemcomitans, which is associated with localized aggressive periodontitis, as well as systemic infections including endocarditis, produces numerous virulence factors, including a repeats-in-toxin (RTX) protein called leukotoxin (LtxA), which kills human immune cells. The strains of A. actinomycetemcomitans most closely associated with disease have been shown to produce the most LtxA, suggesting that LtxA plays a significant role in the virulence of this organism. LtxA, like many of the RTX toxins, can be divided into four functional domains: an N-terminal hydrophobic domain, which contains a significant fraction of hydrophobic residues and has been proposed to play a role in the membrane interaction of the toxin; the central domain, which contains two lysine residues that are the sites of post-translational acylation; the repeat domain that is characteristic of the RTX toxins, and a C-terminal domain thought to be involved in secretion. In its initial interaction with the host cell, LtxA must bind to both cholesterol and an integrin receptor, lymphocyte function-associated antigen-1 (LFA-1). While both interactions are essential for toxicity, the domains of LtxA involved remain unknown. We therefore undertook a series of experiments, including tryptophan quenching and trypsin digestion, to characterize the structure of LtxA upon interaction with membranes of various lipid compositions. Our results demonstrate that LtxA adopts a U-shaped conformation in the membrane, with the N- and C-terminal domains residing outside of the membrane.

The P2Y12 Receptor Antagonist Ticagrelor Reduces Lysosomal pH and Autofluorescence in Retinal Pigmented Epithelial Cells From the ABCA4-/- Mouse Model of Retinal Degeneration

The accumulation of partially degraded lipid waste in lysosomal-related organelles may contribute to pathology in many aging diseases. The presence of these lipofuscin granules is particularly evident in the autofluorescent lysosome-associated organelles of the retinal pigmented epithelial (RPE) cells, and may be related to early stages of age-related macular degeneration. While lysosomal enzymes degrade material optimally at acidic pH levels, lysosomal pH is elevated in RPE cells from the ABCA4-/- mouse model of Stargardt’s disease, an early onset retinal degeneration. Lowering lysosomal pH through cAMP-dependent pathways decreases accumulation of autofluorescent material in RPE cells in vitro, but identification of an appropriate receptor is crucial for manipulating this pathway in vivo. As the P2Y12 receptor for ADP is coupled to the inhibitory Gi protein, we asked whether blocking the P2Y12 receptor with ticagrelor could restore lysosomal acidity and reduce autofluorescence in compromised RPE cells from ABCA4-/- mice. Oral delivery of ticagrelor giving rise to clinically relevant exposure lowered lysosomal pH in these RPE cells. Ticagrelor also partially reduced autofluorescence in the RPE cells of ABCA4-/- mice. In vitro studies in ARPE-19 cells using more specific antagonists AR-C69931 and AR-C66096 confirmed the importance of the P2Y12 receptor for lowering lysosomal pH and reducing autofluorescence. These observations identify P2Y12 receptor blockade as a potential target to lower lysosomal pH and clear lysosomal waste in RPE cells.