Journal of Neurochemistry, 2001, 77, 1010±1017 Proteasome inhibition in oxidative stress neurotoxicity: implications for heat shock proteins Qunxing Ding* and Jeffrey N. Keller* , ² *Department of Anatomy and Neurobiology, University of Kentucky, Lexington, Kentucky, USA ²Sanders-Brown Centeron Aging and Department of Gerontology, University of Kentucky, Lexington, Kentucky, USA Abstract Recent studies have demonstrated that inhibition of the proteasome, an enzyme responsible for the majority of intra- cellular proteolysis, may contribute to the toxicity associated with oxidative stress. In the present study we demonstrate that exposure to oxidative injury (paraquat, H 2 O 2 , FeSO 4 ) induces a rapid increase in reactive oxygen species (ROS), loss of mitochondrial membrane potential, inhibition of proteasome activity, and induction of cell death in neural SH-SY5Y cells. Application of proteasome inhibitors (MG115, epoxomycin) mimicked the effects of oxidative stressors on mitochondrial membrane potential and cell viability, and increased vulner- ability to oxidative injury. Neural SH-SY5Y cells stably transfected with human HDJ-1, a member of the heat shock protein family, were more resistant to the cytotoxicity associated with oxidative stressors. Cells expressing increased levels of HDJ-1 displayed similar degrees of ROS formation following oxidative stressors, but demonstrated a greater preservation of mitochondrial function and protea- somal activity following oxidative injury. Cells transfected with HDJ-1 were also more resistant to the toxicity associated with proteasome inhibitor application. These data support a possible role for proteasome inhibition in the toxicity of oxidative stress, and suggest heat shock proteins may confer resistance to oxidative stress, by preserving proteasome function and attenuating the toxicity of proteasome inhibition. Keywords: heat shock protein, neuron, oxidative stress, proteasome, SH-SY5Y. J. Neurochem. (2001) 77, 1010±1017. Cells within the CNS are continually exposed to reactive oxygen species (ROS), and must therefore systematically respond to ROS mediated damage, in order to prevent oxidative injury. Elevated levels of oxidative damage are evident in numerous neurodegenerative disorders, including Alzheimer's disease (AD) (Markesbery 1997) and ischemia- reperfusion injury (IRI) (Chan 1996), possibly contributing to the neuronal degeneration observed in those conditions (Facchinetti et al. 1998). Although it is likely that the mechanism whereby oxidative stress induces cell death is likely multifactoral, the identi®cation of which biochemical alterations are disrupted, and those responsible for mediat- ing oxidative stress toxicity, has not been fully elucidated. The proteasome is a large intracellular protease that is responsible for mediating the majority of intracellular proteolysis (Rock et al. 1994; Goldberg et al. 1997; Tanaka 1998), including the degradation of most oxidized proteins (Grune and Davies 1997; Grune et al. 1997). Recent studies indicate that proteasome activity is inhibited following exposure to oxidative stress (Reinheckel et al. 1998; Okada et al. 1999), and is inhibited in both AD (Keller et al. 2000a; Lopez Salon et al. 2000) and experimental models of IRI (Keller et al. 2000c). Pharmacological inhibition of the proteasome is suf®cient to cause cell death in primary neuron cultures (Boutillier et al. 1999; Canu et al. 2000; Keller and Markesbery 2000; Pasquini et al. 2000; Qui et al. 2000) and neuronal cell lines (Lopes et al. 1997; Keller et al. 2000b), suggesting that proteasome inhibition may play a role in the neurotoxicity associated with oxidative stress. Although these data indicate a possible role for proteasome inhibition in neuronal oxidative stress, direct evidence for oxidative injury inducing proteasome inhibition in neuronal 1010 q 2001 International Society for Neurochemistry, Journal of Neurochemistry, 77, 1010±1017 Received January 4, 2000; revised manuscript received February 8, 2000; accepted February 9, 2000. Address correspondence and reprint request to J. N. Keller, 101 Sanders-Brown, University of Kentucky, Lexington KY 40536±0230, USA. E-mail: Jnkell0@pop.uky.edu Abbreviations used: AD, Alzheimer's disease; BSA, bovine serum albumin; CNS, central nervous system; DCF, 2,7-dichloro¯uorescin; HSP, heat shock protein; IRI, ischemia-reperfusion injury; JC-1, 5,5 0 ,6,6 0 - tetrachloro-1,1 0 ,3,3 0 -tetraethyl-benzimidazolycarbocyanine iodide; ROS, reactive oxygen species.