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.