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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. H2O2 induced a large increase in labile zinc in wild‐type (WT) astrocytes, but stimulated only a modest rise in MT3‐null astrocytes. In addition, H2O2‐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. © 2010 Wiley‐Liss, Inc.