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Quantifying ABCA1/apoA-1 Signaling Pathways with AFM Imaging and Lipidomic Analysis.
Cruz-Wegener, Carolina; Kutell, Jonah; Aldaher, Louay; deHaseth, Noa; Bhattacharya, Sanjoy K; Ziebarth, Noel.
Afiliación
  • Cruz-Wegener C; Bascom Palmer Eye Institute, Miami Integrative Metabolomics Research Center, University of Miami, Miami, FL, USA.
  • Kutell J; Bascom Palmer Eye Institute, Miami Integrative Metabolomics Research Center, University of Miami, Miami, FL, USA.
  • Aldaher L; Bascom Palmer Eye Institute, Miami Integrative Metabolomics Research Center, University of Miami, Miami, FL, USA.
  • deHaseth N; Bascom Palmer Eye Institute, Miami Integrative Metabolomics Research Center, University of Miami, Miami, FL, USA.
  • Bhattacharya SK; Department of Biomedical Engineering, University of Miami, Coral Gables, USA.
  • Ziebarth N; Bascom Palmer Eye Institute, Miller School of Medicine at University of Miami, Miami, FL, USA.
Methods Mol Biol ; 2816: 205-222, 2024.
Article en En | MEDLINE | ID: mdl-38977601
ABSTRACT
The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3 days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transducción de Señal / Apolipoproteína A-I / Microscopía de Fuerza Atómica / Transportador 1 de Casete de Unión a ATP / Lipidómica Límite: Animals Idioma: En Revista: Methods Mol Biol Asunto de la revista: BIOLOGIA MOLECULAR Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos
Texto completo
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Texto completo: 1 Colección: 01-internacional Base de datos: MEDLINE Asunto principal: Transducción de Señal / Apolipoproteína A-I / Microscopía de Fuerza Atómica / Transportador 1 de Casete de Unión a ATP / Lipidómica Límite: Animals Idioma: En Revista: Methods Mol Biol Asunto de la revista: BIOLOGIA MOLECULAR Año: 2024 Tipo del documento: Article País de afiliación: Estados Unidos Pais de publicación: Estados Unidos