Apoptotic mechanisms in targeted neuronal cell death by chromophore-activated photolysis.
Apoptosis influences early development and later refinement in adult tissues. Experiments in which embryonic neurons or multipotent neural precursor cells are transplanted into regions of neuronal degeneration following targeted photolytic cell death show similar regulation of neuronal migration and differentiation. In those experiments, transplanted cells sought to restore normal cytoarchitecture by preferential migration into neuron deficient regions, assumption of pyramidal morphology, and early process elongation. Control transplants into intact and kainic acid lesioned cortex failed to elicit similar responses. We investigated the possibility that mechanisms of neuronal death common to apoptosis and targeted photolysis could explain the similar developmental influences. We assessed the pathways of cellular injury and eventual cell death in neuroblastoma and PC12 cell cultures labeled with nanospheres carrying the chromophore NH4-chlorin e6 and subjected to photoactivation (1) pharmacologically by scavengers of singlet oxygen and inhibitors of lysosomal proteases, (2) histologically by electron, fluorescence, and light microscopy, and (3) biochemically with binding of cellular DNA by propridium iodide, 3'-OH DNA end terminal labeling, and gel electrophoresis. We found that nanospheres were incorporated into lysosomes, and exposure to light energy led to singlet oxygen (1O2) production and cell death within both neuroblastoma and PC-12 cell lines. Scavengers of 1O2 prevented cell toxicity, while inactivation of lysosomal proteases reduced cell death. Morphologically, degenerating cells revealed release of proteases from lysosomes and disruption of cytoskeletal proteins. Apoptotic characteristics including early loss of cell adhesion, plasma membrane blebbing, and nuclear condensation and convolution were observed. Biochemically, DNA fragmentation was present in cells stained with propridium iodide and observed by 3'-OH end terminal labeling and gel electrophoresis. Thus, cells targeted by photolytically generated 1O2 undergo a form of cell autolysis whose final common pathway is apoptotic. The slow, nonnecrotic process of targeted neuronal cell death in vivo may activate many of the same physiological cues activated by programmed cell death during normal development and during organizational refinement in the adult vertebrate nervous system. This may potentially explain the migration and differentiation of neocortical neurons and neural precursors transplanted into these regions of neuronal degeneration.
Sheen
V L
VL
Department of Neurology, Harvard Medical School, Boston, Massachusetts.
Macklis
J D
JD
D000818:Animals; D017209:Apoptosis; D016923:Cell Death / Q000187:drug effects; D002735:Chlorophyllides; D003594:Cytoplasmic Granules / Q000378:metabolism; D016166:Free Radical Scavengers / Q000494:pharmacology; D008247:Lysosomes / Q000378:metabolism; D051379:Mice; D009474:Neurons / Q000502:physiology*; D010100:Oxygen / Q000737:chemistry / Q000378:metabolism; D016716:PC12 Cells; D010782:Photolysis; D011166:Porphyrins / Q000494:pharmacology*; D011480:Protease Inhibitors / Q000494:pharmacology; D000644:Quaternary Ammonium Compounds / Q000494:pharmacology; D051381:Rats; D014407:Tumor Cells, Cultured
D002735:Chlorophyllides; D016166:Free Radical Scavengers; D011166:Porphyrins; D011480:Protease Inhibitors; D000644:Quaternary Ammonium Compounds; C062985:phytochlorin; D010100:Oxygen