ABSTRACT
Synapse loss and neuronal death are key features of Alzheimer's disease pathology. Disrupted axonal transport of mitochondria is a potential mechanism that could contribute to both. As the major producer of ATP in the cell, transport of mitochondria to the synapse is required for synapse maintenance. However, mitochondria also play an important role in the regulation of apoptosis. Investigation of aluminum (Al) maltolate induced apoptosis in human NT2 cells led us to explore the relationship between apoptosis related changes and the disruption of mitochondrial transport. Similar to that observed with tau over expression, NT2 cells exhibit peri-nuclear clustering of mitochondria following treatment with Al maltolate. Neuritic processes largely lacked mitochondria, except in axonal swellings. Similar, but more rapid results were observed following staurosporine administration, indicating that the clustering effect was not specific to Al maltolate. Organelle clustering and transport disruption preceded apoptosis. Incubation with the caspase inhibitor zVAD-FMK effectively blocked apoptosis, however failed to prevent organelle clustering. Thus, transport disruption is associated with the initiation, but not necessarily the completion of apoptosis. These results, together with observed transport defects and apoptosis related changes in Alzheimer disease brain suggest that mitochondrial transport disruption may play a significant role in synapse loss and thus the pathogenesis or Alzheimer's disease.
Subject(s)
Apoptosis/drug effects , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Mitochondria/drug effects , Mitochondria/ultrastructure , Organometallic Compounds/toxicity , Pyrones/toxicity , Alzheimer Disease/pathology , Amino Acid Chloromethyl Ketones/toxicity , Animals , Antineoplastic Agents/toxicity , Cell Line , Cytochromes c/metabolism , Enzyme Inhibitors/toxicity , Humans , Hydrogen Peroxide/toxicity , Immunohistochemistry , In Situ Nick-End Labeling , Microtubules/drug effects , Neurites/ultrastructure , Neuroprotective Agents/toxicity , Nocodazole/toxicity , Organelles/drug effects , Organelles/ultrastructure , Rabbits , Staurosporine/toxicityABSTRACT
Aluminum (Al) compounds are neurotoxic and have been shown to induce experimental neurodegeneration although the mechanism of this effect is unclear. In order to study this neurotoxic effect of Al, we have developed an in vitro model system using Al maltolate and human NT2 cells. Al maltolate at 500 microM caused significant cell death with a 24-h incubation and this toxicity was even more evident after 48 h. Lower doses of Al maltolate were also effective, but required a longer incubation for cell death. Nuclear fragmentation suggestive of apoptosis was observed as early as three hours and increased substantially through 24 h. Chromatin condensation and nuclear fragmentation were confirmed by electron microscopy. In addition, TUNEL positive nuclei were also observed. The release of cytochrome c was demonstrated with Western blot analysis. This in vitro model using human cells adds to our understanding of Al neurotoxicity and could provide insight into the neurodegenerative processes in human disease.
Subject(s)
Apoptosis/drug effects , Cytochromes c/metabolism , Neurons/drug effects , Organometallic Compounds/toxicity , Pyrones/toxicity , Aluminum/toxicity , Blotting, Western , Cell Line, Tumor , Cell Nucleus/drug effects , Cell Nucleus/ultrastructure , Cell Survival/drug effects , Humans , In Situ Nick-End Labeling , L-Lactate Dehydrogenase/metabolism , Microscopy, Electron , Mitochondria/drug effects , Mitochondria/ultrastructure , Neurons/enzymologyABSTRACT
Neurodegenerative diseases including Alzheimer's disease are characterized by a progressive and selective neuronal loss via an apoptosis mechanism, and there is a growing body of evidence which supports a central role of mitochondria in this apoptotic cell death. Release of cytochrome c from the mitochondria to the cytosol is considered a critical step in apoptosis. Here we report that aluminum maltolate induces cytochrome c translocation into the cytosol as early as 3 hours in aged but not in young rabbit hippocampus. Pretreatment with cyclosporin A, an inhibitor of the mitochondria permeability transition pore (MTP), blocks cytochrome crelease. Therefore, it appears that aluminum maltolate-induced cytochrome c release results from opening of the MTP. This effect implicates aging as a prerequisite factor, since the MTP does not open in young animals. Mitochondrial injury thus may represent a primary initiator of neurodegeneration.
