title: Laser capture microdissection-directed profiling of glycolytic and mTOR pathways in areas of selectively ablated Müller cells in the murine retina.
abstract: OBJECTIVE
  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 of Müller 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 pathway-associated 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.
authors: Macular Research Group, Department of Clinical Ophthalmology and Eye Health, Save Sight Institute, the University of Sydney, Sydney, Australia.
authors: Sook Hyun
authors: SH
authors: Chung
authors: Weiyong
authors: W
authors: Shen
authors: Mark C
authors: MC
authors: Gillies
mesh_terms: D000818:Animals; D015415:Biomarkers / Q000378:metabolism; D004195:Disease Models, Animal; D063928:Ependymoglial Cells / Q000378:metabolism*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation; D006019:Glycolysis / Q000502:physiology*; D007150:Immunohistochemistry; D061346:Laser Capture Microdissection; D051379:Mice; D008822:Mice, Transgenic; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D012160:Retina / Q000166:cytology / Q000378:metabolism*; D012162:Retinal Degeneration / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D058570:TOR Serine-Threonine Kinases / Q000378:metabolism
chemical_list: D015415:Biomarkers; C546842:MTOR protein, human; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases
keywords: Müller cells; glycolysis; laser capture microdissection; mTOR pathway; photoreceptors