Laser Capture Microdissection-Directed Profiling of Glycolytic and mTOR Pathways in Areas of Selectively Ablated Müller Cells in the Murine Retina

Abstract

Citation: Chung SH, Shen W, Gillies MC. Laser capture microdissectiondirected profiling of glycolytic and mTOR pathways in areas of selectively ablated müller cells in the murine retina. Invest Ophthalmol Vis Sci. 2013;54:6578-6584. DOI:10.1167/ iovs.13-12311 PURPOSE. We have reported previously down-regulation of key metabolic pathways, the glycolytic and mTOR pathways, from a global retinal microarray analysis after selective Müller cell ablation in a novel transgenic model. The purpose of the present study was to examine changes in expression of key molecules of glycolytic and mTOR pathways specifically in patches of Müller cell loss.METHODS. Eyes were enucleated 1 and 3 months after induced Müller cell ablation, directly embedded in optimal cutting temperature medium, and snap frozen in liquid nitrogen. Laser capture microdissection (LCM) was conducted to dissect patches of Müller cell loss for quantitative RT-PCR (qRT-PCR) analysis of key genes of the glycolytic (glyceraldehyde-3-phosphate dehydrogenase, enolase 1 and 2, lactate dehydrogenase A and B) and mTOR pathways (insulin-like growth factor receptor 1, phosphatidylinositide-3-kinase, Akt1, and regulatory-associated protein of mTOR). Protein validations were performed by immunohistochemistry. RESULTS. The LCM-directed qRT-PCR analysis ofMüller cell ablated specimens demonstrated reduced transcription of genes involved in the glycolytic and mTOR metabolic pathways. Of the proteins we chose to study, only enolase 1 was expressed by Müller cells. Other glycolytic and mTOR pathway proteins were expressed by photoreceptor inner and outer segments, which were lost in patches of Müller cell ablation.CONCLUSIONS. We found suppression of genes encoding various glycolytic and mTOR pathwayassociated enzymes in areas of Müller cell loss. This appeared mainly to be due to loss of photoreceptor inner and outer segments. The consequences of metabolic derangement caused by Müller cell ablation warrant further investigation.Keywords: Müller cells, glycolysis, mTOR pathway, photoreceptors, laser capture microdissection M¨u ller cells are the principal glial cells of the mammalian retina. They provide an anatomic and functional link between retinal neurons and their surrounding compartments, since they span the entire thickness of the retina from the microvilli projecting from the outer limiting membrane, formed by the apical processes of Müller cells, to the Müller cell end feet, which form the inner limiting membrane. They have critical roles in maintaining retinal homeostasis, such as producing neuroactive substances through a neurotransmitter recycling system, 1 maintaining the blood-retinal barrier (BRB), 2 and regulating ion, water content, and pH. 3,4 Müller cells also provide metabolic support and nutrients to neurones via metabolic pathways, such as the glycolytic pathway. 5 There is strong evidence that the Müller cell is one of the major cells responsible for metabolizing glucose into pyruvate and lactate to fuel neurons in the retina. [6][7][8][9][10][11...

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