Age-Related Decline in Rod Phototransduction Sensitivity in Rhesus Monkeys Fed an n-3 Fatty Acid-Deficient Diet

PURPOSE. Docosahexaenoic acid (DHA), an n-3 fatty acid, is the major polyunsaturate in rod outer segments. The effect of long-term n-3 fatty acid deficiency on rod and cone phototransduction was investigated in the rhesus monkey. METHODS. From birth to Х9 years rhesus monkeys were fed an n-3-deficient diet (n ϭ 9) known to reduce retinal DHA by 80%. Monkeys in the control group (n ϭ 12) received either 8% ␣-linolenic acid (ALA) or 0.6% DHA, both of which support normal retinal DHA levels. None of the diets contained carotenoids. Photoactivation kinetics were assessed from the rate of increase and a P3 model fit of the ERG a-wave. Maximal cone amplitude and sensitivity were measured from the cone a-wave at 4 ms. The rod photoresponse and rod recovery were derived by using a paired flash method. RESULTS. Rod sensitivity was reduced by 40% in the n-3-deficient monkeys at 9 but not 4.5 years. The onset of the rising phase of the photoresponse was significantly delayed (P Ͻ 0.004) at 9 years. Rod recovery was delayed by 20% in n-3-deficient monkeys at both ages, but only for bright saturating flashes. Cone phototransduction was not altered by n-3 deficiency. CONCLUSIONS. Long-term dietary n-3 deficiency in the rhesus monkey was associated with two changes in retinal function. First, there was a delay in rod recovery that has remained relatively constant throughout life. Second, there was an agedependent loss in rod phototransduction sensitivity; the lack of dietary carotenoids may have contributed to this decline. (Invest Ophthalmol Vis Sci. 2009;50:4360 -4367) DOI:10.1167/ iovs.09-3640 D ocosahexaenoic acid (DHA or 22:6n-3), an n-3 fatty acid with six double bonds, is the major polyunsaturated fatty acid in rod outer segments (ROS). The highest concentration of DHA is found in the retina, particularly within the ROS where DHA accounts for up to 60% of phosphatidylethanolamine (PE) acyl chains. 1 PE is the major phospholipid class of the outer bi-lipid layer of ROS discs. The cone-dominated retinas of the goldfish and chick are also highly enriched in DHA. 1,2 The effects of dietary n-3 deficiency on retinal lipid composition have been well described. The most consistent change in retinal function in n-3-deficient rodents, as measured with the ERG, is a reduction in ERG amplitude. 7 described a nonlinear function that related the reduction in rod sensitivity to retinal DHA levels, but only at 16 weeks of age, by which time guinea pigs are adults. In contrast, there were no significant losses of rod sensitivity from n-3-deficient guinea pigs at 6 or 11 weeks of age, despite 35% to 40% reductions in retinal DHA at these time points. At 16 weeks, similar reductions in retinal DHA levels were associated with significantly reduced rod sensitivity. The results indicate lower retinal DHA in combination with aging alters rod photoreceptor function in the guinea pig. In our earlier studies of the rhesus monkey, the most consistent change in retinal function of n-3-deficient animals was a delay in rod recovery as measured from either ERG a-or b-waves, a change seen starting in infancy. 5,8 However, we found no alteration in rod phototransduction sensitivity in n-3-deficient rhesus monkeys at 4.5 years of age when these animals were juveniles or subadults. 8 Given the interaction between age and n-3 deficiency in guinea pigs, the purpose of the present study was to investigate the effect of long-term n-3 deficiency on phototransduction mechanisms in our rhesus monkeys, now mature adults aged 9.2 Ϯ 1.2 years. The monkeys examined in the present study are of particular interest, as their diets were also free of carotenoids, including the xanthophylls lutein and zeaxanthin, which form the macular pigment. In a previous study of monkeys fed the same diets as we used, no lutein or zeaxanthin was present in the serum, and there was no detectable macular pigment. 10 Although the effects of varying dietary carotenoid levels were not examined in the monkeys described herein, the effect of long-term deficiency of both carotenoids and n-3 fatty acids on retinal function and aging is of particular interest. These two nutrients have been identified as risk factors for age-related macular degeneration (AMD) and are being evaluated in a large-scale trial of AMD progression 11 (www.areds2.org/ Age-Related Eye Disease Study)

Elevated Fundus Autofluorescence in Monkeys Deficient in Lutein, Zeaxanthin, and Omega-3 Fatty Acids

PURPOSE. We quantified fundus autofluorescence (FAF) in the nonhuman primate retina as a function of age and diets lacking lutein and zeaxanthin (L/Z) and omega-3 fatty acids. METHODS. Quantitative FAF was measured in a cross-sectional study of rhesus macaques fed a standard diet across the lifespan, and in aged rhesus macaques fed lifelong diets lacking L/Z and providing either adequate or deficient levels of omega-3 fatty acids. Macular FAF images were segmented into multiple regions of interest, and mean gray values for each region were calculated using ImageJ. The resulting FAF values were compared across ages within the standard diet animals, and among diet groups and regions. RESULTS. Fundus autofluorescence increased with age in the standard diet animals, and was highest in the perifovea. Monkeys fed L/Z-free diets with either adequate or deficient omega-3 fatty acids had significantly higher FAF overall than age-matched standard diet monkeys. Examined by region, those with adequate omega-3 fatty acids had higher FAF in the fovea and superior regions, while monkeys fed the diet lacking L/Z and omega-3 fatty acids had higher FAF in all regions. CONCLUSIONS. Diets devoid of L/Z resulted in increased retinal autofluorescence, with the highest values in animals also lacking omega-3 fatty acids. The increase was equivalent to a 12-to 20-year acceleration in lipofuscin accumulation compared to animals fed a standard diet. Together these data add support for the role of these nutrients as important factors in lipofuscin accumulation, retinal aging, and progression of macular disease

Brain xanthophyll content and exploratory gene expression analysis: subspecies differences in rhesus macaque

BACKGROUND: The dietary xanthophylls, lutein and zeaxanthin, accumulate in primate retina and brain, and emerging evidence indicates neural lutein content may be beneficial for cognition. Neural xanthophyll content in primates varies greatly among individuals, and genetic factors are likely to be significant contributors. Subspecies of rhesus macaques originating from different geographic locations are known to differ genetically, but the effect of origin on gene expression and carotenoid status has not been determined. The study objective was to determine whether xanthophyll status and expression of carotenoid-related genes, as well as genes with known variants between subspecies, differ between the brains of adult rhesus monkeys of Indian and Chinese origin. METHODS: Samples of prefrontal cortex, cerebellum, and striatum were collected from adult monkeys (n = 9) fed a standard stock diet containing carotenoids. Serum and brain carotenoids were determined using reverse-phase high-performance liquid chromatography. For each brain region, RNA sequencing and real-time quantitative polymerase chain reaction were used to determine differentially expressed genes between the subspecies. RESULTS: Indian-origin monkeys had higher xanthophyll levels in brain tissue compared to Chinese-origin monkeys despite consuming similar amounts of dietary carotenoids. In a region-specific manner, four genes related to carotenoid and fatty acid metabolism (BCO2, RPE65, ELOVL4, FADS2) and four genes involved in the immune response (CD4, CD74, CXCL12 LTBR) were differentially expressed between Indian- and Chinese-origin monkeys. Expression of all four genes involved in carotenoid and fatty acid metabolism were correlated with brain xanthophyll concentration in a region-specific manner. CONCLUSIONS: These results indicate that origin is related to differences in both gene expression and xanthophyll content in the brain. Findings from this study may have important implications regarding genetic diversity, lutein status, and cognition in primates

Utilizing cell-free DNA to validate targeted disruption of MYO7A in rhesus macaque pre-implantation embryos

Direct injection of CRISPR/Cas9 into zygotes enables the production of genetically modified nonhuman primates (NHPs) essential for modeling specific human diseases, such as Usher syndrome, and for developing novel therapeutic strategies. Usher syndrome is a rare genetic disease that causes loss of hearing, retinal degeneration, and problems with balance, and is attributed to a mutation in MYO7A, a gene that encodes an uncommon myosin motor protein expressed in the inner ear and retinal photoreceptors. To produce an Usher syndrome type 1B (USH1B) rhesus macaque model, we disrupted the MYO7A gene in developing zygotes. Identification of appropriately edited MYO7A embryos for knockout embryo transfer requires sequence analysis of material recovered from a trophectoderm (TE) cell biopsy. However, the TE biopsy procedure is labor intensive and could adversely impact embryo development. Recent studies have reported using cell-free DNA (cfDNA) from embryo culture media to detect aneuploid embryos in human in vitro fertilization (IVF) clinics. The cfDNA is released from the embryo during cell division or cell death, suggesting that cfDNA may be a viable resource for sequence analysis. Moreover, cfDNA collection is not invasive to the embryo and does not require special tools or expertise. We hypothesized that selection of appropriate edited embryos could be performed by analyzing cfDNA for MYO7A editing in embryo culture medium, and that this method would be advantageous for the subsequent generation of genetically modified NHPs. The purpose of this experiment is to determine whether cfDNA can be used to identify the target gene mutation of CRISPR/Cas9 injected embryos. In this study, we were able to obtain and utilize cfDNA to confirm the mutagenesis of MYO7A, but the method will require further optimization to obtain better accuracy before it can replace the TE biopsy approach

Additional file 1: Table S1. of Brain xanthophyll content and exploratory gene expression analysis: subspecies differences in rhesus macaque

Mean number of reads (± SD) and quality score (PHRED format ± SD) for high and low lutein content in each brain region. Table S2. Primer sequences utilized in RT-PCR analysis. (DOCX 12 kb