Colocalization of organelle-specific proteins to autofluorescent astrocyte granules by laser scanning confocal microscopy.
Astrocytes in aging periventricular brain regions and in cysteamine-treated neonatal brain cell cultures contain cytoplasmic inclusions that exhibit an affinity for Gomori stains, orange-red autofluorescence, and non-enzymatic peroxidase activity. In order to delineate the cellular constituents participating in the biogenesis of these astrocytic inclusions, colocalization of FITC-immunolabeled organelles to the red autofluorescent granules was analyzed using a laser scanning confocal imaging system. Areas of true colocalization exhibited yellow fluorescence which persisted in Z-axis image reconstructions. We observed no colocalization of catalase- and PMP70-positive peroxisomes or the Golgi apparatus to the autofluorescent inclusions. The rough endoplasmic reticulum exhibited infrequent dot-like regions of colocalization consistent with previous electron microscopic observations. A minority of early endosomes colocalized to the autofluorescent inclusions in perinuclear cytoplasm. There was extensive colocalization of lgp120-labeled lysosomes to larger autofluorescent granules in close proximity to the nucleus whereas smaller autofluorescent granules often remained unlabeled. A macroautophagic process involving lysosomes and to lesser extent, early endosomes and the rough endoplasmic reticulum, may participate in the biogenesis of autofluorescent astrocytic inclusions in cysteamine-treated glial cultures and in the aging periventricular brain.
Bloomfield Centre for Studies in Aging, Lady Davis Institute for Medical Research, Sir Mortimer B. Davis-Jewish General Hospital, Montreal, Quebec, Canada.
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Schipper
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Walton
D000375:Aging; D000818:Animals; D000831:Animals, Newborn; D001253:Astrocytes / Q000187:drug effects / Q000648:ultrastructure*; D001343:Autophagy / Q000187:drug effects; D001921:Brain / Q000648:ultrastructure; D002478:Cells, Cultured / Q000187:drug effects / Q000648:ultrastructure; D003543:Cysteamine / Q000494:pharmacology; D004721:Endoplasmic Reticulum / Q000648:ultrastructure; D016650:Fluorescein-5-isothiocyanate; D002479:Inclusion Bodies / Q000737:chemistry / Q000187:drug effects / Q000648:ultrastructure*; D008247:Lysosomes / Q000378:metabolism; D008928:Mitochondria / Q000648:ultrastructure; D009195:Peroxidase / Q000032:analysis; D051381:Rats
D003543:Cysteamine; D009195:Peroxidase; D016650:Fluorescein-5-isothiocyanate