The hippocampus of Alzheimers disease brain has been shown to be highly oxidized compared to age-matched controls. One of the most sensitive targets of oxidation is anionic sulfur which can be found within the active site of members of the cysteine-protease family. Thus, while members of the cysteine-protease family such as the calpains and caspases have been found to be in an active conformation in vulnerable brain regions in AD it is possible that their proteolytic activity is hampered due to the robust oxidative stress found at these locations. To address this issue, the amount of casemolytic activity present in the hippocampus from post-mortem brain samples of AD and age-matched controls was determined. No difference in casemolytic activity in the absence of exogenous reducing agent was observed between AD and control. However, after addition of the thiol-specific reducing agent, dithiothreitol (DTT), the amount of caseinolytic activity was significantly increased in AD compared to the DTT-rriediated increase in control. This suggests that the cysteine proteases are more oxidized in AD brain and that latent proteolytic activity in AD brain can be released by antioxidants. Further testing revealed that the calcium-dependent casemolytic activity was significantly lower in AD brain compared to controls. This is despite the fact that the major calcium-dependent thiol protease, calpain, is threefold more activated in AD brain based oil autolytic activation measured by Western blotting. This calcium-dependent protease difference between AD and control brains was negated by addition of DTT. These data suggest that cysteine protease activity in AD brain is inactivated by oxidants, which is evident by the ability of thiol-specific reducing agents such as DTT to rescue and recover activity. (c) 2005 Elsevier Inc. All rights reserved.