The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases
The cellular and compartmental profile of mouse retinal glycolysis, tricarboxylic acid cycle, oxidative phosphorylation, and ~P transferring kinases
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发表时间：
2016-07
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影响因子：
2.2
通讯作者：
Elda M. Rueda;Jerry E. Johnson;A. Giddabasappa;A. Swaroop;M. Brooks;Irena Sigel;S. Chaney;D. Fox
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作者：
Elda M. Rueda;Jerry E. Johnson;A. Giddabasappa;A. Swaroop;M. Brooks;Irena Sigel;S. Chaney;D. Fox
目的 细胞 ATP 的稳态调节是通过 ATP 利用、合成和缓冲的协调活动来实现的。葡萄糖是通过糖酵解和氧化磷酸化 (OXPHOS) 合成 ATP 的主要底物，而通过三羧酸 (TCA) 循环的中间代谢利用非葡萄糖衍生的单羧酸、氨基酸和 α 酮酸来支持线粒体 ATP 和 GTP 合成。细胞 ATP 由专门的平衡驱动的高能磷酸 (~P) 转移激酶缓冲。我们的目标有两个：1）表征整个小鼠视网膜、视网膜区室和/或细胞中能量代谢关键合成和调节酶的基因表达、蛋白质表达和活性；2）提供与功能相关的结果的综合分析。方法从我们的全视网膜Affymetrix芯片数据中提取能量相关基因的mRNA表达数据。使用成年 C57BL/6N 小鼠的固定冷冻视网膜进行免疫组织化学、激光扫描共聚焦显微镜和酶组织化学。使用我们建立的半定量共聚焦和成像技术获得了所有主要视网膜细胞及其区室的充分表征的抗体的免疫反应水平。通过组织化学测定定量细胞色素氧化酶（COX）和乳酸脱氢酶（LDH）活性。结果 Affymetrix 数据揭示了肌肉、肝脏和大脑中发现的 ATP 合成和调节酶的不同基因表达模式。共聚焦研究表明，参与葡萄糖、谷氨酸、谷氨酰胺、乳酸和肌酸代谢的同工酶存在差异细胞和区室分布。抗体以及 COX 和 LDH 活性的模式和强度显示光感受器有氧糖酵解和 OXPHOS 的高能力。丙酮酸竞争分析表明，LDH-5 位于光感受器内节。综合结果表明，糖酵解受到光感受器中己糖激酶 2、丙酮酸激酶 M1 和丙酮酸激酶 M2 的区室表达的调节，而内部视网膜神经元表现出较低的糖酵解和有氧糖酵解能力。核苷二磷酸激酶、线粒体相关腺苷酸激酶和几种线粒体相关肌酸激酶同工酶的表达在外视网膜中最高，而胞质腺苷酸激酶和脑肌酸激酶在视锥细胞、水平细胞和无长突细胞中表达较高，表明视网膜中 ATP 缓冲策略的多样性 神经元。根据抗体强度以及 COX 和 LDH 活性，Müller 胶质细胞 (MGC) 的糖酵解、有氧糖酵解和 OXPHOS 能力最低。然而，它们表现出谷氨酸脱氢酶、α-酮戊二酸脱氢酶、琥珀酸硫激酶、GABA转氨酶和~P转移激酶的高表达。这表明 MGC 利用 TCA 循环回补和回补来生成 GTP 和 ~P 转移激酶，以产生支持 MGC 能量需求的 ATP。结论 我们全面综合的结果表明，成年小鼠视网膜表达多种 ATP 合成、调节和缓冲基因亚型；表达糖酵解、OXPHOS、TCA 循环和 ~P 转移激酶蛋白的差异细胞和区室水平；并表现出逐层差异的 LDH 和 COX 活性。讨论了对细胞特异性和区室 ATP 和 GTP 产生、利用和缓冲策略及其与已知视网膜和细胞功能的关系的新见解。制定神经保护治疗策略以及以细胞特异性方式治疗视网膜缺陷和变性将需要这些知识。这项工作为未来的研究提供了一个平台，旨在识别调节这些过程的分子靶标和蛋白质。
Purpose The homeostatic regulation of cellular ATP is achieved by the coordinated activity of ATP utilization, synthesis, and buffering. Glucose is the major substrate for ATP synthesis through glycolysis and oxidative phosphorylation (OXPHOS), whereas intermediary metabolism through the tricarboxylic acid (TCA) cycle utilizes non-glucose-derived monocarboxylates, amino acids, and alpha ketoacids to support mitochondrial ATP and GTP synthesis. Cellular ATP is buffered by specialized equilibrium-driven high-energy phosphate (~P) transferring kinases. Our goals were twofold: 1) to characterize the gene expression, protein expression, and activity of key synthesizing and regulating enzymes of energy metabolism in the whole mouse retina, retinal compartments, and/or cells and 2) to provide an integrative analysis of the results related to function. Methods mRNA expression data of energy-related genes were extracted from our whole retinal Affymetrix microarray data. Fixed-frozen retinas from adult C57BL/6N mice were used for immunohistochemistry, laser scanning confocal microscopy, and enzymatic histochemistry. The immunoreactivity levels of well-characterized antibodies, for all major retinal cells and their compartments, were obtained using our established semiquantitative confocal and imaging techniques. Quantitative cytochrome oxidase (COX) and lactate dehydrogenase (LDH) activity was determined histochemically. Results The Affymetrix data revealed varied gene expression patterns of the ATP synthesizing and regulating enzymes found in the muscle, liver, and brain. Confocal studies showed differential cellular and compartmental distribution of isozymes involved in glucose, glutamate, glutamine, lactate, and creatine metabolism. The pattern and intensity of the antibodies and of the COX and LDH activity showed the high capacity of photoreceptors for aerobic glycolysis and OXPHOS. Competition assays with pyruvate revealed that LDH-5 was localized in the photoreceptor inner segments. The combined results indicate that glycolysis is regulated by the compartmental expression of hexokinase 2, pyruvate kinase M1, and pyruvate kinase M2 in photoreceptors, whereas the inner retinal neurons exhibit a lower capacity for glycolysis and aerobic glycolysis. Expression of nucleoside diphosphate kinase, mitochondria-associated adenylate kinase, and several mitochondria-associated creatine kinase isozymes was highest in the outer retina, whereas expression of cytosolic adenylate kinase and brain creatine kinase was higher in the cones, horizontal cells, and amacrine cells indicating the diversity of ATP-buffering strategies among retinal neurons. Based on the antibody intensities and the COX and LDH activity, Müller glial cells (MGCs) had the lowest capacity for glycolysis, aerobic glycolysis, and OXPHOS. However, they showed high expression of glutamate dehydrogenase, alpha-ketoglutarate dehydrogenase, succinate thiokinase, GABA transaminase, and ~P transferring kinases. This suggests that MGCs utilize TCA cycle anaplerosis and cataplerosis to generate GTP and ~P transferring kinases to produce ATP that supports MGC energy requirements. Conclusions Our comprehensive and integrated results reveal that the adult mouse retina expresses numerous isoforms of ATP synthesizing, regulating, and buffering genes; expresses differential cellular and compartmental levels of glycolytic, OXPHOS, TCA cycle, and ~P transferring kinase proteins; and exhibits differential layer-by-layer LDH and COX activity. New insights into cell-specific and compartmental ATP and GTP production, as well as utilization and buffering strategies and their relationship with known retinal and cellular functions, are discussed. Developing therapeutic strategies for neuroprotection and treating retinal deficits and degeneration in a cell-specific manner will require such knowledge. This work provides a platform for future research directed at identifying the molecular targets and proteins that regulate these processes.