Kinetics Of Dark Adaptation Research Articles

Interim safety and efficacy of gene therapy for RLBP1-associated retinal dystrophy: a phase 1/2 trial

Gene therapy holds promise for treatment of inherited retinal dystrophies, a group of rare genetic disorders characterized by severe loss of vision. Here, we report up to 3-year pre-specified interim safety and efficacy results of an open-label first-in-human dose-escalation phase 1/2 gene therapy clinical trial in 12 patients with retinal dystrophy caused by biallelic mutations in the retinaldehyde-binding protein 1 (RLBP1) gene of the visual cycle. The primary endpoints were systemic and ocular safety and recovery of dark adaptation. Secondary endpoints included microperimetry, visual field sensitivity, dominant eye test and patient-reported outcomes. Subretinal delivery of an adeno-associated viral vector (AAV8-RLBP1) was well tolerated with dose-dependent intraocular inflammation which responded to corticosteroid treatment, and focal atrophy of the retinal pigment epithelium as the dose limiting toxicity. Dark adaptation kinetics, the primary efficacy endpoint, improved significantly in all dose-cohorts. Treatment with AAV8-RLBP1 resulted in the resolution of disease-related retinal deposits, suggestive of successful restoration of the visual cycle. In conclusion, to date, AAV8-RLBP1 has shown preliminary safety and efficacy in patients with RLBP1-associated retinal dystrophy. Trial number: NCT03374657.

Photoreceptor Function and Structure in Autosomal Dominant Vitelliform Macular Dystrophy Caused by BEST1 Mutations.

The purpose of this study was to evaluate rod and cone function and outer retinal structure within macular lesions, and surrounding extralesional areas of patients with autosomal dominant Best vitelliform macular dystrophy caused by BEST1 mutations. Seventeen patients from seven families were examined with dark- and light-adapted chromatic perimetry and optical coherence tomography. Subsets of patients had long-term follow-up (14-22 years, n = 6) and dark-adaptation kinetics measured (n = 5). Within central lesions with large serous retinal detachments, rod sensitivity was severely reduced but visual acuity and cone sensitivity were relatively retained. In surrounding extralesional areas, there was a mild but detectable widening of the subretinal space in some patients and some retinal areas. Available evidence was consistent with subretinal widening causing slower dark-adaptation kinetics. Over long-term follow-up, some eyes showed formation of de novo satellite lesions at retinal locations that years previously demonstrated subretinal widening. A subclinical abnormality consisting of a retina-wide mild thickening of the outer nuclear layer was evident in many patients and thickening increased in the subset of patients with long-term follow-up. Outcome measures for future clinical trials should include evaluations of rod sensitivity within central lesions and quantitative measures of outer retinal structure in normal-appearing regions surrounding the lesions.

Intrinsic signal optoretinography of dark adaptation kinetics

Delayed dark adaptation due to impaired rod photoreceptor homeostasis has been reported as the earliest symptom of eye diseases such as age-related macular degeneration, diabetic retinopathy, and retinitis pigmentosa. Objective measurement of dark adaptation can facilitate early diagnosis to enable prompt intervention to prevent vision loss. However, there is a lack of noninvasive methods capable of spatiotemporal monitoring of photoreceptor changes during dark adaptation. Here we demonstrate functional optical coherence tomography (OCT) for in vivo intrinsic signal optoretinography (ORG) of dark adaptation kinetics in the C57BL/6J mouse retina. Functional OCT revealed a shortening of the outer retina, a rearrangement of the cone and rod photoreceptor interdigitation zone, and a reduction in intrinsic signal amplitude at the photoreceptor inner segment ellipsoid (ISe). A strong positive correlation between the outer retinal shortening and ISe intensity reduction was also confirmed. Functional OCT of dark adaptation kinetics promises an objective method for rapid ORG assessment of physiological integrity of retinal photoreceptors.

Influence of Lipid Compositions in the Events of Retinal Schiff Base of Bacteriorhodopsin Embedded in Covalently Circularized Nanodiscs: Thermal Isomerization, Photoisomerization, and Deprotonation.

Covalently circularized nanodiscs using circular membrane scaffold protein (MSP) serve as a suitable membrane mimetic for transmembrane proteins by providing stability and tunability in lipid compositions, providing controllable biological environments for targeted proteins. In this work, monomeric bacteriorhodopsin (mbR) was embedded in lipid nanodiscs of different lipid compositions using negatively charged lipid dioleoyl phosphatidylglycerol (DOPG) and the zwitterion lipid dioleoyl phosphatidylcholine (DOPC), and the events associated with the retinal Schiff base, including the thermal isomerization during the dark adaptation, photoisomerization, and deprotonation, were investigated. The retinal thermal isomerization from all-trans, 15-anti to the 13-cis, 15-syn configuration during the dark adaptation was accelerated in the DOPG bilayer, whereas the processes in the DOPC bilayer and in Triton X-100 micelles were similar. This observation indicated that the negatively charged lipid reduced the barrier for retinal thermal isomerization at C13═C14-C15═N in the ground electronic state. Furthermore, the broader absorption contour of mbR in the DOPC nanodisc probably indicated various retinal isomers in the light-adapted state, consistent with the observed nontwo-state dark adaptation kinetics. Moreover, the kinetics of the photoisomerization of the retinal was slightly decelerated upon increasing the content of DOPC. However, the cascading deprotonation of the protonated Schiff base is not dependent on the types of the surrounding lipids in the nanodiscs. In summary, our research deepens the understanding of the coupling between lipid membrane and the photochemistry of bR retinal Schiff base. Combined with the results of our previous works (Lee, T.-Y.; Yeh, V.; Chuang, J.; Chan, J. C. C.; Chu, L.-K.; Yu, T.-Y. Biophys. J. 2015, 109, 1899-1906; Kao, Y.-M.; Cheng, C.-H.; Syue, M.-L.; Huang, H.-Y.; Chen, I-C.; Yu, T.-Y.; Chu, L.-K. J. Phys. Chem. B 2019, 123, 2032-2039), these outcomes extend our understanding of the control of photochemistry and biophysical events for other photosynthetic proteins via altering the lipid environments.

Rhodopsin kinase and arrestin binding control the decay of photoactivated rhodopsin and dark adaptation of mouse rods.

Photoactivation of vertebrate rhodopsin converts it to the physiologically active Meta II (R*) state, which triggers the rod light response. Meta II is rapidly inactivated by the phosphorylation of C-terminal serine and threonine residues by G-protein receptor kinase (Grk1) and subsequent binding of arrestin 1 (Arr1). Meta II exists in equilibrium with the more stable inactive form of rhodopsin, Meta III. Dark adaptation of rods requires the complete thermal decay of Meta II/Meta III into opsin and all-trans retinal and the subsequent regeneration of rhodopsin with 11-cis retinal chromophore. In this study, we examine the regulation of Meta III decay by Grk1 and Arr1 in intact mouse rods and their effect on rod dark adaptation. We measure the rates of Meta III decay in isolated retinas of wild-type (WT), Grk1-deficient (Grk1(-/-)), Arr1-deficient (Arr1(-/-)), and Arr1-overexpressing (Arr1(ox)) mice. We find that in WT mouse rods, Meta III peaks ∼6 min after rhodopsin activation and decays with a time constant (τ) of 17 min. Meta III decay slows in Arr1(-/-) rods (τ of ∼27 min), whereas it accelerates in Arr1(ox) rods (τ of ∼8 min) and Grk1(-/-) rods (τ of ∼13 min). In all cases, regeneration of rhodopsin with exogenous 11-cis retinal is rate limited by the decay of Meta III. Notably, the kinetics of rod dark adaptation in vivo is also modulated by the levels of Arr1 and Grk1. We conclude that, in addition to their well-established roles in Meta II inactivation, Grk1 and Arr1 can modulate the kinetics of Meta III decay and rod dark adaptation in vivo.

Retinal characteristics during 1year of insulin pump therapy in type 1 diabetes: a prospective, controlled, observational study.

To investigate changes in retinal metabolism, function, structure and morphology in relation to initiation of insulin pump therapy (continuous subcutaneous insulin infusion, CSII). Visual acuity, retinopathy level, dark adaptation kinetics, retinal and subfoveal choroidal thickness, macular perfusion velocities, retinal vessel diameters and blood oxygen saturations were measured at baseline and after 1, 4, 16, 32 and 52weeks in 31 patients with type 1 diabetes who startedCSII and 20 patients who continued multiple daily insulin injections (MDI). One year of CSII reduced haemoglobin A1c (HbA1c ) by 1.6% (17.8mmol/mol) compared with 0.3% (3.1mmol/mol) in the MDI group (p<0.0001). Central retinal thickness increased by 1.5% in the CSII group (within-group p=0.0098; between-group p=0.063) without producing macular oedema. No detectable change was found in any other primary outcome measure. The proportion of patients with retinopathy worsening did not differ between groups. At baseline, longer disease duration was associated with higher retinal artery oxygen saturation (p=0.014) and lower macular venous perfusion velocity (p=0.045). One year of CSII led to an HbA1c reduction relative to continued MDI and a small increase in retinal thickness but not to early retinopathy worsening or to changes in retinal vascular, structural or functional characteristics. Longer duration of type 1 diabetes appears to be associated with lower macular venous perfusion velocity and higher retinal artery oxygen saturation. The latter could potentially reflect cumulative glycaemia.

Macular Pigment and Visual Performance in Low-Light Conditions.

By reducing rod intrusion and improving efficiency of neural signaling throughout the visual system, macular pigment (MP) could improve many aspects of visual performance in low-light level conditions. Our study examined this possibility for a variety of visual performance parameters, including spatial resolution, dark adaptation kinetics, and color detection. Twenty-seven subjects participated in the study. Spatial profiles of MP optical density (MPOD) were determined by using heterochromatic flicker photometry. Mesopic- and scotopic-adaptation level experiments were conducted in Maxwellian view. Subjects with higher MPOD required significantly lower contrast to detect the mesopic-level resolution targets; this effect became stronger with increasing spatial frequency. Dark adaptation recovery times were significantly faster as a function of MPOD (by nearly 2 minutes for the lowest mesopic-level task [high versus low MPOD]; P < 0.001). Absolute scotopic thresholds were also significantly associated with MPOD (P < 0.001). Macular pigment optical density was inversely associated with detection of yellow (P < 0.001), and, paradoxically, approached a significant positive correlation with the detection of blue (P = 0.06). Macular pigment appears to enhance visual function in low-light conditions. Based on the results of this study, it can be said that MP extends the range of foveal vision into lower light. Additionally, MP appears to enhance dark adaptation kinetics, which suggests that increased MPOD leads to more efficient photopigment regeneration. The findings of the color detection portion of the study are suggestive of an active compensatory mechanism that offsets absorption by MP in order to maintain normal color perception.

Endophenotypes for Age-Related Macular Degeneration: Extending Our Reach into the Preclinical Stages of Disease.

The key to reducing the individual and societal burden of age-related macular degeneration (AMD)-related vision loss, is to be able to initiate therapies that slow or halt the progression at a point that will yield the maximum benefit while minimizing personal risk and cost. There is a critical need to find clinical markers that, when combined with the specificity of genetic testing, will identify individuals at the earliest stages of AMD who would benefit from preventive therapies. These clinical markers are endophenotypes for AMD, present in those who are likely to develop AMD, as well as in those who have clinical evidence of AMD. Clinical characteristics associated with AMD may also be possible endophenotypes if they can be detected before or at the earliest stages of the condition, but we and others have shown that this may not always be valid. Several studies have suggested that dynamic changes in rhodopsin regeneration (dark adaptation kinetics and/or critical flicker fusion frequencies) may be more subtle indicators of AMD-associated early retinal dysfunction. One can test for the relevance of these measures using genetic risk profiles based on known genetic risk variants. These functional measures may improve the sensitivity and specificity of predictive models for AMD and may also serve to delineate clinical subtypes of AMD that may differ with respect to prognosis and treatment.

Measuring dark adaptation in the elderly: a predictor of who may develop macular degeneration?

and patient fatigue). The authors show that, after adjustment for age, 22% of subjects had impaired, baseline dark-adaptation kinetics despite absence of AMD, and that these abnormalities correlated with elevated C-reactive protein, a serological marker of inflammation, and with high blood pressure, and exhibited a J-shaped correlation with alcohol intake. This study is of special interest to AMD research in that it provides potential evidence for a functional biomarker that could precede the onset of detectable AMD. It will now be interesting to see if the abnormal dark-adaptation kinetics found at baseline will translate in greater risk of developing AMD and if they will correlate with any nutritional factor and/or genetic marker of AMD. If so, studies of dark adaptometry in the elderly may prove invaluable in predicting who is at greatest risk of AMD, leading to earlier and better treatment, and providing an important outcome measure for trials of AMD.

PH dependence of Anabaena sensory rhodopsin: retinal isomer composition, rate of dark adaptation, and photochemistry.

Microbial rhodopsins are photoactive proteins, and their binding site can accommodate either all-trans or 13-cis retinal chromophore. The pH dependence of isomeric composition, dark-adaptation rate, and primary events of Anabaena sensory rhodopsin (ASR), a microbial rhodopsin discovered a decade ago, are presented. The main findings are: (a) Two pKa values of 6.5 and 4.0 assigned to two different protein residues are observed using spectroscopic titration experiments for both ground-state retinal isomers: all-trans, 15-anti (AT) and 13-cis, 15-syn (13C). The protonation states of these protein residues affect the absorption spectrum of the pigment and most probably the isomerization process of the retinal chromophore. An additional pKa value of 8.5 is observed only for 13C-ASR. (b) The isomeric composition of ASR is determined over a wide pH range and found to be almost pH-independent in the dark (>96% AT isomer) but highly pH-dependent in the light-adapted form. (c) The kinetics of dark adaptation is recorded over a wide pH range, showing that the thermal isomerization from 13C to AT retinal occurs much faster at high pH rather than under acidic conditions. (d) Primary photochemical events of ASR at pH 5 are recorded using VIS hyperspectral pump-probe spectroscopy with <100 fs resolution and compared with the previously recorded results at pH 7.5. For AT-ASR, these are shown to be almost pH-independent. However, photochemistry of 13C-ASR is pH-dependent and slowed down in acidic environments.

A1120, a Nonretinoid RBP4 Antagonist, Inhibits Formation of Cytotoxic Bisretinoids in the Animal Model of Enhanced Retinal Lipofuscinogenesis

Excessive accumulation of lipofuscin is associated with pathogenesis of atrophic age-related macular degeneration (AMD) and Stargardt disease. Pharmacologic inhibition of the retinol-induced interaction of retinol-binding protein 4 (RBP4) with transthyretin (TTR) in the serum may decrease the uptake of serum retinol to the retina and reduce formation of lipofuscin bisretinoids. We evaluated in vitro and in vivo properties of the new nonretinoid RBP4 antagonist, A1120. RBP4 binding potency, ability to antagonize RBP4-TTR interaction, and compound specificity were analyzed for A1120 and for the prototypic RBP4 antagonist fenretinide. A1120 ability to inhibit RPE65-mediated isomerohydrolase activity was assessed in the RPE microsomes. The in vivo effect of A1120 administration on serum RBP4, visual cycle retinoids, lipofuscin bisretinoids, and retinal visual function was evaluated using a combination of biochemical and electrophysiologic techniques. In comparison to fenretinide, A1120 did not act as a RARα agonist, while exhibiting superior in vitro potency in RBP4 binding and RBP4-TTR interaction assays. A1120 did not inhibit isomerohydrolase activity in the RPE microsomes. A1120 dosing in mice induced 75% reduction in serum RBP4, which correlated with reduction in visual cycle retinoids and ocular levels of lipofuscin fluorophores. A1120 dosing did not induce changes in kinetics of dark adaptation. A1120 significantly reduces accumulation of lipofuscin bisretinoids in the Abca4(-/-) animal model. This activity correlates with reduction in serum RBP4 and visual cycle retinoids confirming the mechanism of action for A1120. In contrast to fenretinide, A1120 does not act as a RARα agonist indicating a more favorable safety profile for this nonretinoid compound.

The Mammalian Cone Visual Cycle Promotes Rapid M/L-Cone Pigment Regeneration Independently of the Interphotoreceptor Retinoid-Binding Protein

Rapid regeneration of the visual pigment following its photoactivation is critical for the function of cone photoreceptors throughout the day. Though the reactions of the visual cycle in the retinal pigment epithelium (RPE) that recycle chromophore for rod pigment regeneration are well characterized, the corresponding mechanisms that enable rapid regeneration of cone pigment are poorly understood. A key remaining question is the relative contribution of the recently discovered cone-specific retina visual cycle and the classic RPE-dependent visual cycle to mammalian cone pigment regeneration. In addition, it is not clear what role, if any, the abundant interphotoreceptor retinoid-binding protein (IRBP) presumed to facilitate the traffic of chromophore, plays in accelerating mammalian cone pigment regeneration. To address these issues, we used transretinal recordings to evaluate M/L-cone pigment regeneration in isolated retinas and eyecups from control and IRBP-deficient mice. Remarkably, the mouse retina promoted M/L-cone dark adaptation eightfold faster than the RPE. However, complete cone recovery required both visual cycles. We conclude that the retina visual cycle is critical for the initial rapid regeneration of mouse M/L-cone pigment during dark adaptation, whereas the slower RPE visual cycle is required to complete the process. While the deletion of IRBP reduced the amplitude and slowed the kinetics of mouse M/L-cone photoresponses, cone adaptation in bright, steady light and the kinetics of cone dark adaptation were not affected in isolated retina or in intact eyecup. Thus, IRBP does not accelerate cone pigment regeneration and is not critical for the function of mouse M/L-cones in bright light.

Macular and retinal dysfunction of unknown origin in adults with normal fundi: Evidence for an autoimmune pathophysiology

Here we report the discovery of and phenotypic characterization of a retinal disorder of unknown origin in adults using clinical, electrophysiological and psychophysical techniques, and to seek the presence of circulating retinal autoantibodies in the sera of these patients. Sixteen patients were identified with progressive bilateral visual loss over a period of months. Ten of the patients were male, and the average age was 55.3 years (range from 43 to 76 years). Known causes such as carcinoma-associated retinopathy, acute zonal occult outer retinopathy and hereditary cone dystrophy appeared unlikely. Investigations included electrophysiology, fundus autofluorescence imaging and psychophysical tests. The sera of these patients were analyzed with indirect immunocytochemistry and Western immunoblot analysis on murine (BALB/c) retinal tissue for the presence of retinal autoantibodies. Bilateral visual loss and photophobia progressed over a period of months to years (average 28.7 months, range 3–67) and subsequently stabilized. No abnormality was observed by biomicroscopy, angiography or autofluorescence imaging. Electrophysiology indicated predominant cone-system dysfunction, either macular or generalized, and post-phototransduction involvement in 9 patients (56%). Photopic and scotopic visual fields and dark adaptation kinetics showed both cone and rod system involvement in all cases. Heterogeneous immunohistochemical staining patterns were seen with the sera of these patients as compared with controls. A majority of the affected patients (9/15) stained with an antinuclear pattern. The retinal autoantibodies from the sera of most patients reacted with the retinal proteins of molecular weight between 34 and 40 kDa. The aetiology of this distinctive retinal disorder therefore appears to be mediated through an autoimmune mechanism.

Phototransduction, Dark Adaptation, and Rhodopsin Regeneration The Proctor Lecture

This article is divided into two sections. The first outlines the molecular events that underlie the onset of phototransduction, the photoreceptor’s response to light, and provides a quantitative model of its kinetics and amplification. Light responses measured both with single-cell techniques and with the electroretinogram (ERG) are compared with the predictions of the model, and an account is given of the differences in gain between amphibian and mammalian rods. The second section turns to recovery of the overall visual system after exposure of the eye to very intense “bleaching” exposures, and describes the cellular and molecular mechanisms involved in the regeneration of rhodopsin. A model is presented for the delivery of 11-cis retinal to opsin in the bleached rods, which accounts for the kinetics of psychophysical dark adaptation and rhodopsin regeneration, both in normal subjects and in a range of disease states. The slide presentation of the 2006 Proctor Lecture is available online as a PowerPoint file at http://www.iovs.org/cgi/ content/full/47/12/5138/DC1.

Hyperoxia, hyperglycemia, and photoreceptor sensitivity in normal and diabetic subjects

The aim of this study was to investigate the effects of an increase in the saturation of blood oxygen (SaO2) and/or serum glucose on photoreceptor sensitivity in normal subjects and in patients with diabetes mellitus. We monitored cone and rod sensitivity by recording dark-adaptation curves to both green and red test stimuli while inhaling either air (20% O2 + 80% N2) or 100% oxygen in 12 normal subjects and 12 diabetic patients with no (10) or mild (2) retinopathy. We also repeated the experiment in 10 of the normal subjects under hyperglycemia (mean serum glucose: 161 mg/dl). Results show that in normal subjects the dark-adapted cone sensitivity is improved by an increase in SaO2 or by hyperglycemia. Final rod sensitivity is unchanged during hyperoxia and during hyperglycemia when measured with a green test spot. However the kinetics of dark adaptation are altered during hyperglycemia, and an increase in final sensitivity is observed when measured with the red test spot. Inhalation of oxygen during hyperglycemia in normal subjects reduces cone sensitivity compared to that found during hyperglycemia alone (Pasteur effect). In diabetic subjects the dark-adapted cone threshold is comparable to that found in normal subjects, and sensitivity also increases with an increase in SaO2. The final rod threshold, however, is impaired compared to that of the control group, and rod sensitivity is improved by increasing the SaO2. The results suggest that the metabolism of rods and cones may differ in normal subjects: in cones, the rate of metabolism can be augmented by increasing the available oxygen or glucose, whereas rods appear more insensitive to increased blood oxygen saturation and hyperglycemia. In diabetic subjects, both cone and rod metabolism can be increased by supplemental oxygen, indicative of an early rod deficit. The study lends weight to the hypothesis that dark-adapted rods in diabetics are hypoxic before the onset of retinopathy.

Quantification of non-QB-reducing centers in leaves using a far-red pre-illumination.

An alternative approach to quantification of the contribution of non-QB-reducing centers to Chl a fluorescence induction curve is proposed. The experimental protocol consists of a far-red pre-illumination followed by a strong red pulse to determine the fluorescence rise kinetics. The far-red pre-illumination induces an increase in the initial fluorescence level (F(25 micros)) that saturates at low light intensities indicating that no light intensity-dependent accumulation of QA - occurs. Far-red light-dose response curves for the F(25 micros)-increase give no indication of superimposed period-4 oscillations. F(25 micros)-dark-adaptation kinetics following a far-red pre-pulse, reveal two components: a faster one with a half-time of a few seconds and a slower component with a half-time of around 100 s. The faster phase is due to the non-QB-reducing centers that re-open by recombination between QA - and the S-states on the donor side. The slower phase is due to the recombination between QB - and the donor side in active PS II reaction centers. The pre-illumination-induced increase of the F(25 micros)-level represents about 4-5% of the variable fluorescence for pea leaves ( approximately 2.5% equilibrium effect and 1.8-3.0% non-QB-reducing centers). For the other plant species tested these values were very similar. The implications of these values will be discussed.

Functional Characterization of Mouse RDH11 as a Retinol Dehydrogenase Involved in Dark Adaptation in Vivo

We previously cloned mouse RDH11 (mRDH11) as a gene regulated by the transcription factor sterol regulatory element-binding proteins and showed that it is a retinol dehydrogenase expressed in non-ocular tissues such as the liver and testis and in the retina (Kasus-Jacobi, A., Ou, J., Bashmakov, Y. K., Shelton, J. M., Richardson, J. A., Goldstein, J. L., and Brown, M. S. (2003) J. Biol. Chem. 278, 32380-32389). It was proposed to function in the recycling of the visual chromophore 11-cis-retinal after photoisomerization by a bleaching light, a pathway referred to as the visual cycle. In this work, we describe our studies on the ocular function of mRDH11. We created a knockout mouse by replacing the mrdh11 coding sequence with the lacZ reporter gene for expression profiling. 5-Bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining demonstrated active transcription of this gene in photoreceptor cells. We show by immunoblot analysis that mRDH11 is associated with retinal membranes purified from a non-outer segment fraction of the retina. No obvious retinal defect was found during development and aging of RDH11-deficient mice. The functional consequences of mRDH11 disruption were investigated by electroretinography. Dark adaptation was delayed by a factor of 2.5-3 compared with wild-type mice. However, the kinetics of 11-cis-retinal recycling during dark adaptation was not affected, suggesting that mRDH11 is not involved in the visual cycle. We propose that mRDH11 disruption affects retinoid metabolism in photoreceptor inner segments and delays the kinetics of dark adaptation through modulation of calcium homeostasis.

Symptomatic abnormalities of dark adaptation in patients with EFEMP1 retinal dystrophy (Malattia Leventinese/Doyne honeycomb retinal dystrophy).

To investigate the nature of symptomatic visual disturbance in patients with EFEMP1 retinal dystrophy in the absence of geographic atrophy or choroidal neovascularization. Patients presenting to a tertiary referral centre underwent clinical evaluation, fluorescein angiography, colour contrast sensitivity, focal, pattern, and standard electroretinography, electrooculography, scotopic threshold perimetry and dark adaptometry. Clinical features included reduced central vision, difficulty passing from light to dark, and diffuse submacular and peripapillary deposits, which were hyperfluorescent by fluorescein angiography. Colour contrast thresholds were abnormal in all six patients studied and both pattern and focal electroretinograms were abnormal in five of six patients. The scotopic and mixed rod-cone single flash ERG was normal but two patients demonstrated reduced oscillatory potentials and one had borderline delayed 30 Hz responses. Scotopic thresholds were elevated and rod-mediated dark adaptation kinetics were markedly prolonged in all six patients when measured over the central visible confluent deposits. In patients with EFEMP1 retinal dystrophy with confluent macular deposits, scotopic sensitivity is reduced and dark adaptation kinetics are prolonged over the macular deposits but are normal elsewhere. These results emphasize the localised nature of functional deficits in some patients with EFEMP1 retinal dystrophy and correlate well with the patient's visual symptoms. Symptomatic visual dysfunction may precede the development of clinically evident geographic atrophy or choroidal neovascularization in this disorder.

Contribution of extracellular Glu residues to the structure and function of bacteriorhodopsin. Presence of specific cation-binding sites.

Single and multiple mutants of extracellular Glu side chains of bacteriorhodopsin were analyzed by acid and calcium titration, differential scanning calorimetry, and thermal difference spectrophotometry. Acid titration spectra show that the second group protonating with Asp(85) is revealed in E204Q in the absence of Cl(-) but is not observed in the triple mutant E9Q/E194Q/E204Q or in the quadruple mutant E9Q/E74Q/E194Q/E204Q. The results point to Glu(9) as the second group protonating cooperatively with Asp(85). Comparison of the apparent pK(a) of Asp(85) protonation in water and in the deionized forms and results of calcium titration suggest that cation-binding sites are of low affinity in the multiple Glu mutants. Like for deionized wild type bacteriorhodopsin, differential scanning calorimetry reveals a lack of the pretransition in the multiple mutants, whereas in E9Q it appears at lower temperature and with lower cooperativity. Additionally, at neutral pH the band at 630 nm arising from cation release upon temperature increase is absent for the multiple mutants. Based on these results, we propose the presence of two cation-binding sites in the extracellular region of bacteriorhodopsin having as ligands Glu(9), Glu(194), Glu(204), and water molecules.

Multiple Phosphorylation of Rhodopsin and the In Vivo Chemistry Underlying Rod Photoreceptor Dark Adaptation

Dark adaptation requires timely deactivation of phototransduction and efficient regeneration of visual pigment. No previous study has directly compared the kinetics of dark adaptation with rates of the various chemical reactions that influence it. To accomplish this, we developed a novel rapid-quench/mass spectrometry-based method to establish the initial kinetics and site specificity of light-stimulated rhodopsin phosphorylation in mouse retinas. We also measured phosphorylation and dephosphorylation, regeneration of rhodopsin, and reduction of all- trans retinal all under identical in vivo conditions. Dark adaptation was monitored by electroretinography. We found that rhodopsin is multiply phosphorylated and then dephosphorylated in an ordered fashion following exposure to light. Initially during dark adaptation, transduction activity wanes as multiple phosphates accumulate. Thereafter, full recovery of photosensitivity coincides with regeneration and dephosphorylation of rhodopsin.