Differential effects of Best disease causing missense mutations on bestrophin-1 trafficking.

Mutations in bestrophin-1 (Best1) cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited retinal degenerative disease. Best1 is a homo-oligomeric anion channel localized to the basolateral surface of retinal pigment epithelial (RPE) cells. A number of Best1 mutants mislocalize in Madin-Darby canine kidney (MDCK) cells. However, many proteins traffic differently in MDCK and RPE cells, and MDCK cells do not express endogenous Best1. Thus, effects of Best1 mutations on localization in MDCK cells may not translate to RPE cells. To determine whether BVMD causing mutations affect Best1 localization, we compared localization and oligomerization of Best1 with Best1 mutants V9M, W93C, and R218C. In MDCK cells, Best1 and Best1(R218C) were basolaterally localized. Best1(W93C) and Best1(V9M) accumulated in cells. In cultured fetal human retinal pigment epithelium cells (fhRPE) expressing endogenous Best1, Best1(R218C) and Best1(W93C) were basolateral. Best1(V9M) was intracellular. All three mutants exhibited similar fluorescence resonance energy transfer (FRET) efficiencies to, and co-immunoprecipitated with Best1, indicating unimpaired oligomerization. When human Best1 was expressed in RPE in mouse eyes it was basolaterally localized. However, Best1(V9M) accumulated in intracellular compartments in mouse RPE. Co-expression of Best1 and Best1(W93C) in MDCK cells resulted in basolateral localization of both Best1 and Best1(W93C), but co-expression of Best1 with Best1(V9M) resulted in mislocalization of both proteins. We conclude that different mutations in Best1 cause differential effects on its localization and that this effect varies with the presence or absence of wild-type (WT) Best1. Furthermore, MDCK cells can substitute for RPE when examining the effects of BVMD causing mutations on Best1 localization if co-expressed with WT Best1.

Bestrophin-1 influences transepithelial electrical properties and Ca2+ signaling in human retinal pigment epithelium.

PURPOSE: Mutations in BEST1, encoding Bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD) and other inherited retinal degenerative diseases. Best1 is an integral membrane protein localized to the basolateral plasma membrane of the retinal pigment epithelium (RPE). Data from numerous in vitro and in vivo models have demonstrated that Best1 regulates intracellular Ca2+ levels. Although it is known from in vitro and crystal structure data that Best1 is also a calcium-activated anion channel, evidence for Best1 functioning as a channel in human RPE is lacking. To assess Best1-associated channel activity in the RPE, we examined the transepithelial electrical properties of fetal human RPE (fhRPE) cells, which express endogenous Best1. METHODS: Using adenovirus-mediated gene transfer, we overexpressed Best1 and the BVMD mutant Best1W93C in fhRPE cells and assessed resting transepithelial potential (TEP), transepithelial resistance, short circuit current (Isc), and intracellular Ca2+ levels. Cl- currents were directly measured in transfected HEK293 cells using whole-cell patch clamp. RESULTS: Best1W93C showed ablated Cl- currents and, when co-expressed, suppressed the channel activity of Best1 in HEK293 cells. In fhRPE, overexpression of Best1 increased TEP and Isc, while Best1W93C diminished TEP and Isc. Substitution of Cl- in the bath media resulted in a significant reduction of Isc in monolayers overexpressing Best1, but no significant Isc change in monolayers expressing Best1W93C. We removed Ca2+ as a limit on transepithelial electrical properties by treating cells with ionomycin, and found that changes in Isc and TEP for monolayers expressing Best1 were absent in monolayers expressing Best1W93C. Similarly, inhibition of calcium-activated anion channels with niflumic acid reduced both Isc and TEP of control and Best1 monolayers, but did not notably affect Best1W93C monolayers. Stimulation with extracellular ATP induced an increase in TEP in control monolayers that was greater than that observed in those expressing Best1(W93C). Examination of [Ca2+]i following ATP stimulation demonstrated that the expression of Best1W93C impaired intracellular Ca2+ signaling. CONCLUSIONS: These data indicate that Best1 activity strongly influences electrophysiology and Ca2+ signaling in RPE cells, and that a common BVMD mutation disrupts both of these parameters. Our findings support the hypothesis that Best1 functions as an anion channel in human RPE.

Suppression of Ca2+ signaling in a mouse model of Best disease.

Mutations in BEST1, encoding bestrophin-1 (Best1), cause Best vitelliform macular dystrophy (BVMD), a dominantly inherited macular degeneration characterized by a diminished electrooculogram light peak (LP), lipofuscin in retinal pigment epithelial cells (RPE), and fluid- and debris-filled retinal detachments. To understand the pathogenesis of BVMD we generated knock-in mice carrying the BVMD-causing mutation W93C in Best1. Both Best1(+/W93C)and Best1(W93C/W93C) mice had normal ERG a- and b-waves, but exhibited an altered LP luminance response reminiscent of that observed in BVMD patients. Morphological analysis identified fluid- and debris-filled retinal detachments in mice as young as 6 months of age. By 18-24 months of age Best1(+/W93C)and Best1(W93C/W93C) mice exhibited enhanced accumulation of lipofuscin in the RPE, and a significant deposition of debris composed of unphagocytosed photoreceptor outer segments and lipofuscin granules in the subretinal space. Although Best1 is thought to function as a Ca(2+)-activated Cl(-) channel, RPE cells from Best1(W93C) mice exhibited normal Cl(-) conductances. We have previously shown that Best1(-/-) mice exhibit increased [Ca(2+)](i) in response to ATP stimulation. However, ATP-stimulated changes in [Ca(2+)](i) in RPE cells from Best1(+/W93C) and Best1(W93C/W93C) mice were suppressed relative to Best1(+/+) littermates. Based on these data we conclude that mice carrying the Best1(W93C) mutation are a valid model for BVMD. Furthermore, these data suggest that BVMD is not because of Best1 deficiency, as the phenotypes of Best1(+/W93C) and Best1(W93C/W93C) mice are distinct from that of Best1(-/-) mice with regard to lipofuscin accumulation, and changes in the LP and ATP Ca(2+) responses.

Targeted disruption of fibulin-4 abolishes elastogenesis and causes perinatal lethality in mice.

Elastic fibers provide tissues with elasticity which is critical to the function of arteries, lungs, skin, and other dynamic organs. Loss of elasticity is a major contributing factor in aging and diseases. However, the mechanism of elastic fiber development and assembly is poorly understood. Here, we show that lack of fibulin-4, an extracellular matrix molecule, abolishes elastogenesis. fibulin-4-/- mice generated by gene targeting exhibited severe lung and vascular defects including emphysema, artery tortuosity, irregularity, aneurysm, rupture, and resulting hemorrhages. All the homozygous mice died perinatally. The earliest abnormality noted was a uniformly narrowing of the descending aorta in fibulin-4-/- embryos at embryonic day 12.5 (E12.5). Aorta tortuosity and irregularity became noticeable at E15.5. Histological analysis demonstrated that fibulin-4-/- mice do not develop intact elastic fibers but contain irregular elastin aggregates. Electron microscopy revealed that the elastin aggregates are highly unusual in that they contain evenly distributed rod-like filaments, in contrast to the amorphous appearance of normal elastic fibers. Desmosine analysis indicated that elastin cross-links in fibulin-4-/- tissues were largely diminished. However, expression of tropoelastin or lysyl oxidase mRNA was unaffected in fibulin-4-/- mice. In addition, fibulin-4 strongly interacts with tropoelastin and colocalizes with elastic fibers in culture. These results demonstrate that fibulin-4 plays an irreplaceable role in elastogenesis.

Bestrophin-2 is involved in the generation of intraocular pressure.

PURPOSE: The bestrophin family of proteins has been demonstrated to generate or regulate Ca2+-activated Cl(-) conductances. Mutations in bestrophin-1 (Best1) cause several blinding eye diseases, but little is known about other bestrophin family members. This study involved disruption of the Best2 gene in mice. METHODS: The mouse Best2 gene was disrupted by replacing exons 1, 2, and part of exon 3 with a Lac Z. The expression profile of Bestrophin-2 (Best2) was examined using RT-PCR, X-gal staining, and immunohistochemistry. Intraocular pressure (IOP) was measured by anterior chamber cannulation. RESULTS: RT-PCR of mouse tissues revealed Best2 mRNA in eye, colon, nasal epithelia, trachea, brain, lung, and kidney. X-gal staining, confirmed expression in colon epithelia and in the eye, in the nonpigmented epithelia (NPE). Best2 was not expressed in RPE cells. Best2 protein was observed only in NPE and colon epithelia. The absence of Best2 had no obvious deleterious effect on the mice. However, the Best2-/- mice were found to have significantly (P < 0.02) diminished IOP with respect to the Best2+/+ and Best2+/- littermates. The Best2-/- and Best2+/- mice responded better to the carbonic anhydrase inhibitor brinzolamide than did their Best2+/+ littermates, although the beta-blocker timolol brought IOP to the same level, regardless of genotype. CONCLUSIONS: Best2 plays a role in the generation of IOP by regulating formation of aqueous humor, and inhibition of Best2 function represents an attractive new avenue for regulating IOP in individuals with glaucoma.

Hydrodynamic properties of porcine bestrophin-1 in Triton X-100.

Bestrophin-1 (Best-1) is an integral membrane protein, defects in which cause Best vitelliform macular dystrophy. Best-1 is proposed to function as a Cl- channel and/or a regulator of Ca++ channels. A tetrameric (or pentameric) stoichiometry has been reported for recombinant best-1. Using a combination of gel exclusion chromatography and velocity sedimentation we examined the quaternary structure of native best-1 and found that it migrates as a single species with a Stokes radius of 7.3 nm, sedimentation coefficient (S20,w) of 4.9, and partial specific volume (nu) of 0.80 ml/g. The mass of the protein-detergent complex is calculated to be 206 kDa, with the protein component estimated to be approximately 138 kDa. Given a monomeric mass of 68 kDa, we conclude that native best-1 solubilized with Triton X-100 is a homodimer. The differences between this observation and a prior report were examined by comparing recombinant best-1 with tissue derived best-1 using gel exclusion chromatography. Much of the recombinant best-1 eluted in the column void (Vo) fraction, unlike that extracted from RPE cells. We conclude that the minimal functional unit of best-1 is dimeric. This stoichiometry differs from that previously measured for recombinant best-1, suggesting that further studies are necessary to determine the stoichiometry of functional best-1 in RPE membranes.

The light peak of the electroretinogram is dependent on voltage-gated calcium channels and antagonized by bestrophin (best-1).

Mutations in VMD2, encoding bestrophin (best-1), cause Best vitelliform macular dystrophy (BMD), adult-onset vitelliform macular dystrophy (AVMD), and autosomal dominant vitreoretinochoroidopathy (ADVIRC). BMD is distinguished from AVMD by a diminished electrooculogram light peak (LP) in the absence of changes in the flash electroretinogram. Although the LP is thought to be generated by best-1, we find enhanced LP luminance responsiveness with normal amplitude in Vmd2-/- mice and no differences in cellular Cl- currents in comparison to Vmd2+/+ littermates. The putative Ca2+ sensitivity of best-1, and our recent observation that best-1 alters the kinetics of voltage-dependent Ca2+ channels (VDCC), led us to examine the role of VDCCs in the LP. Nimodipine diminished the LP, leading us to survey VDCC beta-subunit mutant mice. Lethargic mice, which harbor a loss of function mutation in the beta4 subunit of VDCCs, exhibited a significant shift in LP luminance response, establishing a role for Ca2+ in LP generation. When stimulated with ATP, which increases [Ca++]I, retinal pigment epithelial cells derived from Vmd2-/- mice exhibited a fivefold greater response than Vmd2+/+ littermates, indicating that best-1 can suppress the rise in [Ca2+]I associated with the LP. We conclude that VDCCs regulated by a beta4 subunit are required to generate the LP and that best-1 antagonizes the LP luminance response potentially via its ability to modulate VDCC function. Furthermore, we suggest that the loss of vision associated with BMD is not caused by the same pathologic process as the diminished LP, but rather is caused by as yet unidentified effects of best-1 on other cellular processes.

Acute Ischemia Induced by High-Density Culture Increases Cytokine Expression and Diminishes the Function and Viability of Highly Purified Human Islets of Langerhans.

BACKGROUND: Encapsulation devices have the potential to enable cell-based insulin replacement therapies (such as human islet or stem cell-derived ß cell transplantation) without immunosuppression. However, reasonably sized encapsulation devices promote ischemia due to high ß cell densities creating prohibitively large diffusional distances for nutrients. It is hypothesized that even acute ischemic exposure will compromise the therapeutic potential of cell-based insulin replacement. In this study, the acute effects of high-density ischemia were investigated in human islets to develop a detailed profile of early ischemia induced changes and targets for intervention. METHODS: Human islets were exposed in a pairwise model simulating high-density encapsulation to normoxic or ischemic culture for 12 hours, after which viability and function were measured. RNA sequencing was conducted to assess transcriptome-wide changes in gene expression. RESULTS: Islet viability after acute ischemic exposure was reduced compared to normoxic culture conditions (P < 0.01). Insulin secretion was also diminished, with ischemic ß cells losing their insulin secretory response to stimulatory glucose levels (P < 0.01). RNA sequencing revealed 657 differentially expressed genes following ischemia, with many that are associated with increased inflammatory and hypoxia-response signaling and decreased nutrient transport and metabolism. CONCLUSIONS: In order for cell-based insulin replacement to be applied as a treatment for type 1 diabetes, oxygen and nutrient delivery to ß cells will need to be maintained. We demonstrate that even brief ischemic exposure such as would be experienced in encapsulation devices damages islet viability and ß cell function and leads to increased inflammatory signaling.

A model of best vitelliform macular dystrophy in rats.

PURPOSE: The VMD2 gene, mutated in Best macular dystrophy (BMD) encodes bestrophin, a 68-kDa basolateral plasma membrane protein expressed in retinal pigment epithelial (RPE) cells. BMD is characterized by a depressed light peak (LP) in the electro-oculogram. Bestrophin is thought to be the Cl channel that generates the LP. The goal was to generate an animal model of BMD and to determine the effects of bestrophin overexpression on the RPE-generated components of the ERG. METHODS: Bestrophin or bestrophin mutants (W93C or R218C) were overexpressed in the RPE of rats by injection of replication-defective adenovirus. Immunofluorescence microscopy and ERG recordings were used to study subsequent effects. RESULTS: Bestrophin was confined to the basolateral plasma membrane of the RPE. Neither wild-type (wt) nor mutant bestrophin affected the a- or b-waves of the ERG. Wt bestrophin, however, increased the c-wave and fast oscillation (FO), but not the LP. In contrast, both mutants had little or no effect on the c-wave and FO, but did reduce LP amplitude. LP amplitudes across a range of stimuli were not altered by wt bestrophin, though the luminance response function was desensitized. LP response functions were unaffected by bestrophin R218C but were significantly altered by bestrophin W93C. CONCLUSIONS: A model of BMD was developed in the present study. Because overexpression of wt bestrophin shifted luminance response but did not alter the range of LP response amplitudes, the authors conclude that the rate-limiting step for generating LP amplitude occurs before activation of bestrophin or that bestrophin does not directly generate the LP conductance.

Noninvasive recording and response characteristics of the rat dc-electroretinogram.

In response to light, the retinal pigment epithelium (RPE) generates a series of potentials that can be recorded using the dc-electroretinogram (dc-ERG). As these potentials can be related to specific cellular events, they provide information about RPE function and how that may be altered by disease or experimental manipulation. The purposes of the present study were to define a noninvasive means for recording the rat dc-ERG, to use this to define the stimulus-response properties of the major components, and to relate these results to measures of the rat electrooculogram (EOG). Parallel studies were conducted in two strains of rats (Long-Evans, LE; Sprague-Dawley, SD) that are commonly used in vision research. Rats were sedated with ketamine/xylazine and placed on a heating pad. Ag/AgCl wire electrodes were bridged with capillary tubes filled with Hanks balanced salt solution. The active electrode was placed in contact with the corneal surface and referenced to a second electrode placed within the orbit. The dc-ERG signal was amplified (dc-100 Hz), digitized, and stored offline. The duration of full-field flash stimuli was controlled using a mechanical shutter and flash luminance was controlled with neutral density filters. EOGs were recorded using subdermal platinum needle electrodes placed near the eye. In response to a 5-min light exposure, the dc-ERG of LE and SD rats included a distinct b-wave, after potential, c-wave, fast oscillation, and a slow potential of positive polarity the characteristics of which are consistent with a light peak. In LE rats, the final plateau of this slow positive potential was often lower than the prestimulus baseline; in SD rats, this potential achieved a level above the baseline. Analysis of EOGs recorded from these two strains yielded results consistent with the amplitude of the slow potential relative to the prestimulus baseline. Specifically, the amplitude of the EOG of SD rats increased when the eye was exposed to light. In LE rats, this increase did not occur, and in some cases light reduced the amplitude of the EOG. The two strains also differed with respect to c-wave implicit time, which was faster in SD rats. These results indicate that many of the major components of the dc-ERG are readily measured in the rat. Therefore, we believe that the rat may provide a useful animal model in which to conduct pharmacological analysis of nonneuronal responses and to develop animal models of human retinal disorders involving the RPE, such as Best Vitelliform Macular Dystrophy.

Bestrophin interacts physically and functionally with protein phosphatase 2A.

Bestrophin is a 68-kDa basolateral plasma membrane protein expressed in retinal pigment epithelial cells (RPE). It is encoded by the VMD2 gene, which is mutated in Best macular dystrophy, a disease characterized by a depressed light peak in the electrooculogram. Recently it was proposed that bestrophin is a chloride channel responsible for generating the light peak. To investigate its function further, we immunoaffinity purified a bestrophin complex from RPE lysates and identified bestrophin and the beta-catalytic subunit of protein phosphatase 2A (PP2A) as members of the complex by matrix-assisted laser desorption ionization time-of-flight mass spectrometry. Protein-protein interaction between bestrophin and PP2Ac and the structural subunit of PP2A, PR65, was confirmed by reciprocal immunoprecipitation. The C-terminal cytoplasmic domain of bestrophin was sufficient for the interaction with PP2A as demonstrated by a pulldown assay using a fusion of this domain with glutathione S-transferase. Bestrophin was phosphorylated when expressed in RPE-J cells and this phosphorylation was sensitive to okadaic acid. Purified PP2A effectively dephosphorylated bestrophin in vitro. These data suggest that bestrophin is in the signal transduction pathway that modulates the light peak of the electrooculogram, that it is regulated by phosphorylation, and that phosphorylation of bestrophin is in turn regulated by PP2A.