Paul S. Bernstein

Inactivity of human β,β-carotene-9′,10′-dioxygenase (BCO2) underlies retinal accumulation of the human macular carotenoid pigment

Significance Among mammals, only primates can accumulate high levels of xanthophyll carotenoids in their retinas. We discovered that, unlike most other mammals, the major xanthophyll cleavage enzyme β,β-carotene-9′,10′-dioxygenase (BCO2) is inactive in humans, explaining the unique accumulation of lutein, zeaxanthin, and meso-zeaxanthin in primate macula. We confirmed this discovery by demonstrating that BCO2 knockout mice accumulate zeaxanthin in their retinas. The macula of the primate retina uniquely concentrates high amounts of the xanthophyll carotenoids lutein, zeaxanthin, and meso-zeaxanthin, but the underlying biochemical mechanisms for this spatial- and species-specific localization have not been fully elucidated. For example, despite abundant retinal levels in mice and primates of a binding protein for zeaxanthin and meso-zeaxanthin, the pi isoform of glutathione S-transferase (GSTP1), only human and monkey retinas naturally contain detectable levels of these carotenoids. We therefore investigated whether or not differences in expression, localization, and activity between mouse and primate carotenoid metabolic enzymes could account for this species-specific difference in retinal accumulation. We focused on β,β-carotene-9′,10′-dioxygenase (BCO2, also known as BCDO2), the only known mammalian xanthophyll cleavage enzyme. RT-PCR, Western blot analysis, and immunohistochemistry (IHC) confirmed that BCO2 is expressed in both mouse and primate retinas. Cotransfection of expression plasmids of human or mouse BCO2 into Escherichia coli strains engineered to produce zeaxanthin demonstrated that only mouse BCO2 is an active zeaxanthin cleavage enzyme. Surface plasmon resonance (SPR) binding studies showed that the binding affinities between human BCO2 and lutein, zeaxanthin, and meso-zeaxanthin are 10- to 40-fold weaker than those for mouse BCO2, implying that ineffective capture of carotenoids by human BCO2 prevents cleavage of xanthophyll carotenoids. Moreover, BCO2 knockout mice, unlike WT mice, accumulate zeaxanthin in their retinas. Our results provide a novel explanation for how primates uniquely concentrate xanthophyll carotenoids at high levels in retinal tissue.

Macular pigment Raman detector for clinical applications

Clinical studies of carotenoid macular pigments (MP) have been limited by the lack of noninvasive, objective instruments. We introduce a novel noninvasive optical instrument, an MP Raman detector, for assessment of the carotenoid status of the human retina in vivo. The instrument uses resonant excitation of carotenoid molecules in the visible wavelength range, and quantitatively measures the highly specific Raman signals that originate from the single- and double-bond stretch vibrations of the pi-conjugated carotenoid molecules carbon backbone. The instrument is a robust, compact device and suitable for routine measurements of MP concentrations in a clinical setting. We characterized and tested the instrument in clinical studies of human subjects to validate its function and to begin to establish its role as a possible screening test for macular pathologies. We also show that the MP Raman spectroscopy technology has potential as a novel, highly specific method for rapid screening of carotenoid antioxidant levels in large populations at risk for vision loss from age-related macular degeneration, the leading cause of blindness of the elderly in the developed world.

Macular Pigment Imaging in AREDS2 Participants: An Ancillary Study of AREDS2 Subjects Enrolled at the Moran Eye Center

PURPOSE Age-Related Eye Disease Study 2 (AREDS2) is a randomized, placebo-controlled study designed to determine whether supplementation with 10 mg of lutein and 2 mg of zeaxanthin per day can slow the rate of progression of age-related macular degeneration (AMD). Although some biomarkers of response to carotenoid supplementation such as serum concentrations are part of the AREDS2 protocol, measurement of carotenoid concentrations in the eye and other tissues is not. In this approved ancillary study, macular pigment optical density (MPOD), macular pigment distributions, and skin carotenoid levels at enrollment and at each annual visit were measured to assess baseline carotenoid status and to monitor response to assigned interventions. METHODS All subjects enrolled at the Moran Eye Center had MPOD and macular pigment spatial distributions measured by dual-wavelength autofluorescence imaging and total skin carotenoids measured by resonance Raman spectroscopy. Results. Baseline MPOD in enrolled subjects was unusually high relative to an age-matched control group that did not consume carotenoid supplements regularly, consistent with the high rate of habitual lutein and zeaxanthin consumption in Utah AREDS2 subjects prior to enrollment. MPOD did not correlate with serum or skin carotenoid measurements. CONCLUSIONS Useful information is provided through this ancillary study on the ocular carotenoid status of AREDS2 participants in the target tissue of lutein and zeaxanthin supplementation: The macula. When treatment assignments are unmasked at the conclusion of the study, unique tissue-based insights will be provided on the progression of AMD in response to long-term, high-dose carotenoid supplementation versus diet alone. (ClinicalTrials.gov number, NCT00345176.).

Retinoid receptors trigger neuritogenesis in retinal degenerations

Anomalous neuritogenesis is a hallmark of neurodegenerative disorders, including retinal degenerations, epilepsy, and Alzheimers disease. The neuritogenesis processes result in a partial reinnervation, new circuitry, and functional changes within the deafferented retina and brain regions. Using the light‐induced retinal degeneration (LIRD) mouse model, which provides a unique platform for exploring the mechanisms underlying neuritogenesis, we found that retinoid X receptors (RXRs) control neuritogenesis. LIRD rapidly triggered retinal neuron neuritogenesis and up‐regulated several key elements of retinoic acid (RA) signaling, including retinoid X receptors (RXRs). Exogenous RA initiated neuritogenesis in normal adult retinas and primary retinal cultures and exacerbated it in LIRD retinas. However, LIRD‐induced neuritogenesis was partly attenuated in retinol dehydrogenase knockout (Rdh12−/−) mice and by aldehyde dehydrogenase inhibitors. We further found that LIRD rapidly increased the expression of glutamate receptor 2 and β Ca2+/ calmodulin‐dependent protein kinase II (pCaMKII). Pulldown assays demonstrated interaction between PCaMKII and RXRs, suggesting that CaMKII pathway regulates the activities of RXRs. RXR antagonists completely prevented and RXR agonists were more effective than RA in inducing neuritogenesis. Thus, RXRs are in the final common path and may be therapeutic targets to attenuate retinal remodeling and facilitate global intervention methods in blinding diseases and other neurodegenerative disorders.—Lin, Y., Jones, B. W., Liu, A., Tucker, J. F., Rapp, K., Luo, L., Baehr, W., Bernstein, P. S., Watt, C. B., Yang, J.‐H., Shaw, M. V., Marc, R. E. Retinoid receptors trigger neuritogenesis in retinal degenerations. FASEB J. 26, 81–92 (2012). www.fasebj.org

Resonance Raman based skin carotenoid measurements in newborns and infants.

We describe Resonance Raman based skin carotenoid measurements in newborns and infants. Skin- and serum carotenoid levels correlate with high statistical significance in healthy newborns and infants, and with reduced accuracy also in prematurely born infants, who in general feature very low carotenoid levels and thin transparent skin giving rise to large background absorption effects. Skin carotenoid levels can be easily compared among subjects and/or tracked in longitudinal studies with the highly molecule-specific Raman method. It therefore holds promise as a rapid, non-invasive, carotenoid antioxidant assessment method for newborns and infants in the field of pediatrics.

Correlations Between Macular, Skin, and Serum Carotenoids

Purpose Ocular and systemic measurement and imaging of the macular carotenoids lutein and zeaxanthin have been employed extensively as potential biomarkers of AMD risk. In this study, we systematically compare dual wavelength retinal autofluorescence imaging (AFI) of macular pigment with skin resonance Raman spectroscopy (RRS) and serum carotenoid levels in a clinic-based population. Methods Eighty-eight patients were recruited from retina and general ophthalmology practices from a tertiary referral center and excluded only if they did not have all three modalities tested, had a diagnosis of macular telangiectasia (MacTel) or Stargardt disease, or had poor AFI image quality. Skin, macular, and serum carotenoid levels were measured by RRS, AFI, and HPLC, respectively. Results Skin RRS measurements and serum zeaxanthin concentrations correlated most strongly with AFI macular pigment volume under the curve (MPVUC) measurements up to 9° eccentricity relative to MPVUC or rotationally averaged macular pigment optical density (MPOD) measurements at smaller eccentricities. These measurements were reproducible and not significantly affected by cataracts. We also found that these techniques could readily identify subjects taking oral carotenoid-containing supplements. Conclusions Larger macular pigment volume AFI and skin RRS measurements are noninvasive, objective, and reliable methods to assess ocular and systemic carotenoid levels. They are an attractive alternative to psychophysical and optical methods that measure MPOD at a limited number of eccentricities. Consequently, skin RRS and MPVUC at 9° are both reasonable biomarkers of macular carotenoid status that could be readily adapted to research and clinical settings.