Brain Xanthophyll Content and Exploratory Gene Expression Analysis: Subspecies Differences in Rhesus Macaque. Macaca mulatta

Objective: The dietary xanthophylls, lutein and zeaxanthin, accumulate in primate brain and may be beneficial for cognition. Brain xanthophyll content varies greatly among individuals and genetic factors are likely to be significant contributors. Subspecies of rhesus macaques originating from different geographic locations 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: Next generation RNA sequencing was used to determine differentially expressed carotenoid-related genes and genes with known variants among rhesus monkey subspecies in the prefrontal cortex, cerebellum, and striatum of Indian-origin monkeys (n=3) versus Chinese-origin monkeys (n=3). Serum and brain xanthophylls were determined using HPLC. FastQC was performed on raw sequenced reads to determine the quality of each read. Reads were mapped to the Rhesus Macaque reference genome and differences in gene expression (FPKM) were determined using TopHat and Cuffdiff, respectively. Findings from RNAseq were validated using RT-PCR. 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, 4 genes related to carotenoid and fatty acid metabolism (BCO2, RPE65, ELOVL4, FADS2) and 4 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. Overall design: Sequenced reads were generated for rhesus monkeys of Indian origin (n=3) and Chinese origin (n=3) for 3 different brain regions (prefrontal cortex, cerebellum, and striatum) using single-end 50 base pair sequencing on the HiSeq Illumina 2500

研究目标：膳食叶黄素类（dietary xanthophylls），即叶黄素（lutein）与玉米黄质（zeaxanthin），可在灵长类（primate）大脑中蓄积，或对认知功能具有益处。不同个体间大脑叶黄素类含量差异显著，遗传因素（genetic factors）可能是主要贡献因素。不同地理起源的恒河猴（rhesus macaques）亚种存在遗传差异，但起源对基因表达（gene expression）及类胡萝卜素状态（carotenoid status）的影响尚未明确。本研究旨在探究印度起源与中国起源的成年恒河猴大脑中，叶黄素类状态、类胡萝卜素相关基因以及亚种间已知变异基因的表达是否存在差异。
研究方法：采用下一代RNA测序（Next generation RNA sequencing），对印度起源（n=3）与中国起源（n=3）恒河猴的前额叶皮层（prefrontal cortex）、小脑（cerebellum）及纹状体（striatum）中差异表达的类胡萝卜素相关基因，以及亚种间存在已知变异的基因进行分析。采用高效液相色谱（HPLC）检测血清与大脑组织中的叶黄素类含量。对原始测序读段执行FastQC质控，以评估每条读段的质量；将读段比对至恒河猴参考基因组（Rhesus Macaque reference genome），分别采用TopHat与Cuffdiff分析基于FPKM的基因表达差异；通过RT-PCR对RNA测序结果进行验证。
研究结果：尽管膳食类胡萝卜素摄入量相似，印度起源恒河猴的大脑组织叶黄素类水平显著高于中国起源个体。在脑区特异性模式下，4个与类胡萝卜素及脂肪酸代谢相关的基因（BCO2、RPE65、ELOVL4、FADS2）与4个参与免疫应答的基因（CD4、CD74、CXCL12、LTBR）在印度与中国起源恒河猴间存在差异表达。所有4个类胡萝卜素及脂肪酸代谢相关基因的表达均与脑区特异性的大脑叶黄素类浓度呈显著相关。
研究结论：上述结果表明，起源与恒河猴大脑的基因表达及叶黄素类含量差异均相关。本研究结果或对灵长类的遗传多样性、叶黄素状态及认知功能研究具有重要意义。
总体设计：采用Illumina HiSeq 2500平台进行单端50碱基对测序（single-end 50 base pair sequencing），对印度起源（n=3）与中国起源（n=3）恒河猴的3个不同脑区（前额叶皮层、小脑、纹状体）进行测序，生成测序读段。