Kirsten G. Locke

The Very Large G-Protein-Coupled Receptor VLGR1: A Component of the Ankle Link Complex Required for the Normal Development of Auditory Hair Bundles

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles.

Thickness of Receptor and Post-receptor Retinal Layers in Patients with Retinitis Pigmentosa Measured with Frequency-Domain Optical Coherence Tomography

PURPOSE To better understand the effects of retinitis pigmentosa (RP) on post-receptor anatomy, the thicknesses of the receptor, inner nuclear, retinal ganglion cell (RGC), and retinal nerve fiber layers (RNFL) were measured with frequency-domain optical coherence tomography (fdOCT). METHODS FdOCT scans were obtained from the horizontal midline in 30 patients with RP and 23 control subjects of comparable age. Raw images were exported and the thicknesses of photoreceptor/RPE, inner nuclear, RGC plus inner plexiform, and nerve fiber layers were measured with a manual segmentation procedure aided by a computer program. The RNFL thickness was also measured in 20 controls and 25 patients using circular peripapillary fdOCT scans. RESULTS Results from controls were consistent with known anatomy. In patients with RP, the pattern of photoreceptor loss with eccentricity was consistent with the field constriction characteristic of RP. INL and RGC layer measures were comparable to normal subjects, although some patients showed slightly thicker RGC layers. However, RNFL layer thickness was significantly greater than normal; a majority of patients showed a thicker RFNL on both horizontal midline scans and peripapillary scans. CONCLUSIONS To make optimal use of OCT RNFL thickness as a measure of the integrity of RGCs in patients with RP, a better understanding of the causes of the thickening seen in the majority of the patients is needed. As the RGC layer thickness can be measured with fdOCT, RGC layer thickness may turn out to be a more direct and valid indicator of the presence of RGCs in patients with RP.

A Comparison of Visual Field Sensitivity to Photoreceptor Thickness in Retinitis Pigmentosa

PURPOSE To explore the relationship between visual field sensitivity and photoreceptor layer thickness in patients with retinitis pigmentosa (RP). METHODS Static automated perimetry (central 30-2 threshold program with spot size III; Humphrey Field Analyzer; Carl Zeiss Meditec, Inc., Dublin, CA) and frequency domain optical coherence tomography (Fd-OCT) scans (Spectralis HRA+OCT; Heidelberg Engineering, Vista, CA) were obtained from 10 age-matched normal control subjects and 20 patients with RP who had retained good central vision (better than 20/32). The outer segment (OS+) thickness (the distance between retinal pigment epithelium [RPE])/Bruchs membrane [BM] to the photoreceptor inner-outer segment junction), outer nuclear layer (ONL), and total retinal thickness were measured at locations corresponding to visual field test loci up to 21 degrees eccentricity. RESULTS The average OS+ thickness in the control eyes was 63.1 +/- 5.2 microm, varying from approximately 69 microm in the foveal center to 56 microm at 21 degrees eccentricity. In patients with RP, OS+ thickness was below normal limits outside the fovea, and thickness decreased with loss in local field sensitivity, reaching an asymptotic value of 21.5 microm at approximately -10 dB. The ONL thickness also decreased with local field sensitivity loss. Although relative OS thickness was linearly related to visual field loss at all locations examined, a slightly better correlation was found between the product of OS and ONL thickness and visual field loss. CONCLUSIONS In patients with RP with good foveal sensitivity, the OS thickness and the product of OS thickness and ONL thickness (assumed to represent the number of photoreceptors) decreases linearly with loss of local field sensitivity. In general, in regions where perimetric sensitivity loss is -10 dB or worse, the OS+ thickness approaches the thickness of the RPE/BM complex.

The Transition Zone between Healthy and Diseased Retina in Patients with Retinitis Pigmentosa

PURPOSE To describe the structural changes in the transition zone from relatively healthy retinal regions to severely affected regions in patients with retinitis pigmentosa (RP) using frequency domain optical coherence tomography (fdOCT). METHODS FdOCT line scans of the horizontal meridian were obtained from one eye of 13 patients with RP and 30 control subjects. The patients had normal or near normal foveal sensitivities and visual field diameters ≥10°. Using a computer-aided manual segmentation procedure, the locations at which the outer segment (OS) and outer nuclear layer plus outer plexiform layer (ONL+) thicknesses fell below the 95% confidence interval of the controls were measured, as were the locations at which the OS layer disappeared and the locations at which the ONL+ was reduced to an asymptotically small thickness. RESULTS The progression from healthy to severely affected regions followed a common pattern in most patients. Region A, the central region including the foveal center, had normal OS and ONL+ thickness. Region B had abnormal OS but normal ONL+ thickness. Region C had abnormal but measurable OS and ONL+ thicknesses. In Region D, the OS layer disappeared, as did the IS/OS line, and the ONL+ thickness decreased further. In Region E, the ONL+ reached an asymptotic thickness. CONCLUSIONS The structural changes in the transition zone followed an orderly progression from a thinning of the OS layer, to a thinning of the ONL+, to a loss of the OS layer, to an ONL+ reduced to an asymptotically small level.

Spectral-domain optical coherence tomography measures of outer segment layer progression in patients with X-linked retinitis pigmentosa.

IMPORTANCE Determining the annual rate of change in the width of the inner segment ellipsoid zone (EZ; ie, inner/outer segment border) in the context of short-term variability should allow us to better understand the value of this measure for future treatment trials in X-linked retinitis pigmentosa (XLRP). OBJECTIVES To identify the width of the central region showing an EZ and to determine the short-term repeat variability and the annual rate of change in the width of the EZ from spectral-domain optical coherence tomography (SD-OCT) measures in RP. DESIGN Patients with recessive or simplex RP (age range, 8-65 years; mean age, 40.5 years) underwent scanning twice on the same day to evaluate test-retest variability. Patients with XLRP (age range, 8-27 years; mean age, 15.2 years) from a larger group participating in an ongoing double-blind treatment trial (docosahexaenoic acid vs placebo; clinicaltrials.gov NCT00100230) underwent spectral-domain optical coherence tomography line scanning across the horizontal meridian at 3 yearly intervals. SETTING Research center specializing in medical retina. PARTICIPANTS Forty-eight patients with RP, including 20 with recessive or simplex RP and 28 with XLRP, and 23 healthy control subjects. MAIN OUTCOME AND MEASURE Widths of the EZ calculated and compared among the 3 annual visits. RESULTS Test-retest differences were normally distributed, and the magnitude of the difference was independent of mean EZ width. The mean (SD) for test-retest differences in EZ width was 0.08° (0.22°) (range, -0.30° to 0.60°). Thus, 95% of all test-retest differences fall within ± 0.43° (124 μm). Of the 28 patients with XLRP, 27 showed a significant decrease in EZ width after 2 years. Patients with XLRP showed a mean annual decrease in EZ width of 0.86° (248 μm, or 7%). CONCLUSIONS AND RELEVANCE The mean rate of decline in EZ width (7%) translates into a mean rate of change of 13% for the equivalent area of functioning retina. This rate of change is consistent with that reported for visual fields and full-field electroretinograms. Unlike visual fields and electroretinograms, however, the repeat variability is less than the annual rate of change. These results support the validity of EZ width as an outcome measure in prospective clinical trials in RP.

Variability of full-field electroretinogram responses in subjects without diffuse photoreceptor cell disease

PURPOSE To evaluate test-retest variability in electroretinogram (ERG) responses in subjects without evidence of diffuse photoreceptor cell disease. DESIGN Cohort study. PARTICIPANTS Forty subjects without diffuse photoreceptor cell disease. METHODS Serial ERGs were performed on 40 subjects (mean age at the time of first ERG: 54 years; range: 38-75 years) over a period of 2 to 6 years. These subjects participated in a study by a pharmaceutical company investigating the effects of certain drugs, used for gastrointestinal disorders, on retinal function. None of the subjects showed any evidence of progressive change in retinal function related to the medications. The ERG responses that were evaluated included amplitudes and implicit times for the dark-adapted rod-isolated and rod-dominant responses, light-adapted single flash response, and both light- and dark-adapted 31-Hz flicker responses. MAIN OUTCOME MEASURES The data were analyzed by using analysis of variance methods, and a threshold criteria for significant change with 95% confidence was calculated for implicit times and an increase or decrease in ERG amplitudes. RESULTS The threshold for significant change varied depending on the ERG stimulus. For the dark-adapted stimuli, a significant decrease in amplitude varied from 35% to 42% as compared with a variation of 53% to 73% for a significant increase. For the light-adapted stimuli, a significant decrease in amplitude varied by 52% as compared with a variation of 109% to 110% for a significant increase. The threshold for significant change for implicit times varied from 3.0 milliseconds to 8.7 milliseconds. CONCLUSIONS The measured test-retest variability in ERG amplitudes and implicit times in subjects without diffuse photoreceptor cell disease underscores the importance of conducting similar comprehensive studies of variability in patients with acquired and hereditary retinal diseases. These data are also of value for monitoring disease progression and in future therapeutic trials.

Rates of decline in regions of the visual field defined by frequency-domain optical coherence tomography in patients with RPGR-mediated X-linked retinitis pigmentosa.

PURPOSE To determine whether annual decline in visual field sensitivity is greater in the transition zone at the edge of the frequency-domain optical coherence tomography (fdOCT) inner segment ellipsoid zone (EZ) than at other locations in the visual field. DESIGN Prospective, longitudinal, observational study. PARTICIPANTS Forty-four patients with X-linked retinitis pigmentosa (XLRP) resulting from a mutation in the RPGR gene. METHODS Static perimetric fields (Humphrey 30-2; Carl Zeiss Meditec, Dublin, CA) were obtained annually for 4 years. Beginning with year 2, fdOCT scans were obtained annually with a Heidelberg Spectralis HRA + OCT (Heidelberg Engineering, Heidelberg, Germany). MAIN OUTCOME MEASURES The rate of visual field decline at locations near the edge of the EZ compared with the rates for the macula and in the mid periphery. RESULTS Sensitivity just inside and outside the edge of the EZ declined at rates of 0.84 and 0.92 dB/year, respectively. By comparison, average sensitivity in the macula and mid periphery declined by 0.38 and 0.61 dB/year, respectively. CONCLUSIONS The edge of the EZ in each patient with XLRP indicates a transition zone between relatively healthy and relatively degenerate retina. The annual loss of sensitivity in the transition zone is more rapid than it is elsewhere in the retina.

A Comparison of Progressive Loss of the Ellipsoid Zone (EZ) Band in Autosomal Dominant and X-Linked Retinitis Pigmentosa

PURPOSE In patients with retinitis pigmentosa (RP), the inner segment ellipsoid zone (EZ; also known as the inner segment/outer segment [IS/OS] border) is a marker of the usable visual field at a given point in time and of the progression of the disease over time. Here we compare the change in the width per year of the EZ band in patients with autosomal dominant (ad) and x-linked (xl) RP. METHODS Using optical coherence tomography (OCT), 9-mm horizontal and vertical line scans through the fovea were obtained for one eye of 26 xlRP patients and 33 adRP patients. Scans were repeated on average 2.0 years later (range, 0.6-4.8 years). Using a manual segmentation procedure, the EZ band was delineated and its horizontal width (HW) and vertical width (VW) were determined. RESULTS The adRP and xlRP patients had similar initial EZ HW (xlRP: 11.8 ± 5.4°, adRP: 12.4 ± 6.3°, P = 0.69) and VW (xlRP: 8.5 ± 4.9°, adRP: 11.4 ± 7.1°, P = 0.09). However, between visits the absolute loss and percent loss of the EZ width per year was significantly greater for xlRP than adRP for both HW (xlRP: 1.0 ± 0.6°/y, 9.6 ± 5.6%/y; adRP: 0.4 ± 0.5°/y, 3.4 ± 5.4%/y; P < 0.001) and VW (xlRP: 0.8 ± 0.8°/y, 9.2 ± 8.9%/y; adRP: 0.3 ± 0.5°/y, 4.2 ± 6.4%/y; P < 0.01). There was a weak correlation between the loss of EZ width per year and the initial width for xlRP (r(2) = 0.17, P = 0.036), but no correlation for adRP (r(2) = 0.004, P = 0.73). The test-retest difference of EZ HW was 0.2 ± 0.5°. CONCLUSIONS The OCT data here support a faster rate of loss per year in the case of xlRP. (ClinicalTrials.gov number, NCT00100230.).

Macular atrophy in birdshot retinochoroidopathy: an optical coherence tomography and multifocal electroretinography analysis.

Purpose:The purpose of this study was to evaluate macular atrophy by frequency-domain optical coherence tomography (OCT) in patients with birdshot retinochoroidopathy and to compare the resulting thickness measures with visual acuity and multifocal electroretinography (mfERG). Methods:Measures were obtained from 14 eyes of 7 patients with birdshot retinochoroidopathy and 23 normal eyes. Optical coherence tomography-3 measures of macular thinning were related to visual acuity, mfERG response density, and time since diagnosis. Horizontal midline frequency-domain OCT scans identified which layers of the retina were primarily responsible for macular thinning. Results:All eyes with a history of birdshot retinochoroidopathy for >10 years had abnormal mfERG response densities. Compared with those without anatomic thinning (n = 8), eyes with anatomic thinning (n = 6) had significantly lower visual acuity (P = 0.0006), foveal response density (P = 0.006), and overall mfERG response density (P = 0.009). Segmentation of retinal layers on frequency-domain OCT scans showed that anatomic thinning was as a result of reduction in the receptor 1 layer (REC+), the thickness of the segment extending from the proximal border of the outer plexiform layer to the Bruch membrane-choroid interface. Conclusion:Macular atrophy, as reflected in OCT evidence of macular thinning and mfERG evidence of macular function, occurs in patients with long-standing birdshot retinochoroidopathy. Measures of retinal layer thicknesses by frequency-domain OCT suggest that the atrophy occurs primarily in the outer retina.

ApoER2 Function in the Establishment and Maintenance of Retinal Synaptic Connectivity

The cellular and molecular mechanisms responsible for the development of inner retinal circuitry are poorly understood. Reelin and apolipoprotein E (apoE), ligands of apoE receptor 2 (ApoER2), are involved in retinal development and degeneration, respectively. Here we describe the function of ApoER2 in the developing and adult retina. ApoER2 expression was highest during postnatal inner retinal synaptic development and was considerably lower in the mature retina. Both during development and in the adult, ApoER2 was expressed by A-II amacrine cells. ApoER2 knock-out (KO) mice had rod bipolar morphogenic defects, altered A-II amacrine dendritic development, and impaired rod-driven retinal responses. The presence of an intact ApoER2 NPxY motif, necessary for binding Disabled-1 and transducing the Reelin signal, was also necessary for development of the rod bipolar pathway, while the alternatively spliced exon 19 was not. Mice deficient in another Reelin receptor, very low-density lipoprotein receptor (VLDLR), had normal rod bipolar morphology but altered A-II amacrine dendritic development. VLDLR KO mice also had reductions in oscillatory potentials and delayed synaptic response intervals. Interestingly, age-related reductions in rod and cone function were observed in both ApoER2 and VLDLR KOs. These results support a pivotal role for ApoER2 in the establishment and maintenance of normal retinal synaptic connectivity.