Metallothionein-3 regulates lysosomal function in cultured astrocytes under both normal and oxidative conditions.
Cellular zinc plays a key role in lysosomal change and cell death in neurons and astrocytes under oxidative stress. Here, using astrocytes lacking metallothionein-3 (MT3), a potential source of labile zinc in the brain, we studied the role of MT3 in oxidative stress responses. H(2)O(2) induced a large increase in labile zinc in wild-type (WT) astrocytes, but stimulated only a modest rise in MT3-null astrocytes. In addition, H(2)O(2)-induced lysosomal membrane permeabilization (LMP) and cell death were comparably attenuated in MT3-null astrocytes. Expression and glycosylation of Lamp1 (lysosome-associated membrane protein 1) and Lamp2 were increased in MT3-null astrocytes, and the activities of several lysosomal enzymes were significantly reduced, indicating an effect of MT3 on lysosomal components. Consistent with lysosomal dysfunction in MT3-null cells, the level of LC3-II (microtubule-associated protein 1 light chain 3), a marker of early autophagy, was increased by oxidative stress in WT astrocytes, but not in MT3-null cells. Similar changes in Lamp1, LC3, and cathepsin-D were induced by the lysosomal inhibitors bafilomycin A1, chloroquine, and monensin, indicating that lysosomal dysfunction may lie upstream of changes observed in MT3-null astrocytes. Consistent with this idea, lysosomal accumulation of cholesterol and lipofuscin were augmented in MT3-null astrocytes. Similar to the results seen in MT3-null cells, MT3 knockdown by siRNA inhibited oxidative stress-induced increases in zinc and LMP. These results indicate that MT3 may play a key role in normal lysosomal function in cultured astrocytes.
Lee
Sook-Jeong
SJ
Neural Injury Research Lab, University of Ulsan College of Medicine, Seoul, Korea.
Park
Mi-Ha
MH
Kim
Hyun-Jae
HJ
Koh
Jae-Young
JY
D000818:Animals; D001253:Astrocytes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D002402:Cathepsin D / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000187:drug effects / Q000201:enzymology / Q000502:physiology; D002784:Cholesterol / Q000378:metabolism; D006031:Glycosylation / Q000187:drug effects; D006861:Hydrogen Peroxide / Q000633:toxicity; D008062:Lipofuscin / Q000378:metabolism; D052119:Lysosomal-Associated Membrane Protein 2 / Q000378:metabolism; D051907:Lysosomal Membrane Proteins / Q000378:metabolism; D008247:Lysosomes / Q000187:drug effects / Q000201:enzymology / Q000502:physiology*; D008297:Male; D008668:Metallothionein / Q000235:genetics / Q000378:metabolism; D000090023:Metallothionein 3; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology*; D052118:Lysosomal-Associated Membrane Protein 1
D002402:Cathepsin D; D002784:Cholesterol; D006861:Hydrogen Peroxide; D008062:Lipofuscin; D052119:Lysosomal-Associated Membrane Protein 2; D051907:Lysosomal Membrane Proteins; D008668:Metallothionein; D000090023:Metallothionein 3; D008869:Microtubule-Associated Proteins; D009419:Nerve Tissue Proteins; C052255:Lamp1 protein, mouse; D052118:Lysosomal-Associated Membrane Protein 1; C480860:Map1lc3b protein, mouse; C491054:Mt2 protein, mouse; C000715430:Mt3 protein, mouse; C525355:metallothionein-1, mouse; C533923:Ctsd protein, mouse