Fluorogenic Substrates for Visualizing Acidic Organelle Enzyme Activities

• Published in: PloS one 2016-05, Vol.11 (5), p.e0156312-e0156312
• Main Authors: Harlan, Fiona Karen, Lusk, Jason Scott, Mohr, Breanna Michelle, Guzikowski, Anthony Peter, Batchelor, Robert Hardy, Jiang, Ying, Naleway, John Joseph
• Format: Article
• Language: English
• Subjects: Alzheimer's disease; Amines - metabolism; Analysis; Apoptosis; Autophagy; Biology and Life Sciences; Bone remodeling; Case-Control Studies; Cell lines; Cell Membrane - metabolism; Cells, Cultured; Chemical compounds; Chloroquine; Cholesterol; Disease; Drug development; Enzymatic activity; Enzyme activation; Enzyme activity; Enzymes; Esterase; Fibroblasts - enzymology; Fibroblasts - pathology; Fluorescence; Fluorescent dyes; Fluorescent Dyes - metabolism; Fluorescent indicators; Format; Gaucher Disease - enzymology; Gaucher Disease - pathology; Homeostasis; Humans; Huntington's disease; Leukocytes; Leukodystrophy; Leukodystrophy, Globoid Cell - enzymology; Leukodystrophy, Globoid Cell - pathology; Leukodystrophy, Metachromatic - enzymology; Leukodystrophy, Metachromatic - pathology; Lipid metabolism; Lipids; Localization; Lysosomal storage diseases; Lysosomes; Lysosomes - enzymology; Mammalian cells; Medicine and Health Sciences; Metabolism; Monitoring; Mutation; Nervous system diseases; Neurodegenerative diseases; Neurological diseases; Organelles; Parkinson's disease; Pharmacology; Proteins; R&D; Research & development; Research and Analysis Methods; Signaling; Skin - enzymology; Skin - pathology; Staining; Substrates
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0156312

Lysosomes are acidic cytoplasmic organelles that are present in all nucleated mammalian cells and are involved in a variety of cellular processes including repair of the plasma membrane, defense against pathogens, cholesterol homeostasis, bone remodeling, metabolism, apoptosis and cell signaling. Defects in lysosomal enzyme activity have been associated with a variety of neurological diseases including Parkinson's Disease, Lysosomal Storage Diseases, Alzheimer's disease and Huntington's disease. Fluorogenic lysosomal staining probes were synthesized for labeling lysosomes and other acidic organelles in a live-cell format and were shown to be capable of monitoring lysosomal metabolic activity. The new targeted substrates were prepared from fluorescent dyes having a low pKa value for optimum fluorescence at the lower physiological pH found in lysosomes. They were modified to contain targeting groups to direct their accumulation in lysosomes as well as enzyme-cleavable functions for monitoring specific enzyme activities using a live-cell staining format. Application to the staining of cells derived from blood and skin samples of patients with Metachromatic Leukodystrophy, Krabbe and Gaucher Diseases as well as healthy human fibroblast and leukocyte control cells exhibited localization to the lysosome when compared with known lysosomal stain LysoTracker® Red DND-99 as well as with anti-LAMP1 Antibody staining. When cell metabolism was inhibited with chloroquine, staining with an esterase substrate was reduced, demonstrating that the substrates can be used to measure cell metabolism. When applied to diseased cells, the intensity of staining was reflective of lysosomal enzyme levels found in diseased cells. Substrates specific to the enzyme deficiencies in Gaucher or Krabbe disease patient cell lines exhibited reduced staining compared to that in non-diseased cells. The new lysosome-targeted fluorogenic substrates should be useful for research, diagnostics and monitoring the effect of secondary therapeutic agents on lysosomal enzyme activity in drug development for the lysosomal storage disorders and allied diseases.