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- Materials and methods
Recent studies have demonstrated that inhibition of the proteasome, an enzyme responsible for the majority of intracellular proteolysis, may contribute to the toxicity associated with oxidative stress. In the present study we demonstrate that exposure to oxidative injury (paraquat, H2O2, FeSO4) 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 vulnerability 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 proteasomal 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.
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 identification of which biochemical alterations are disrupted, and those responsible for mediating oxidative stress toxicity, has not been fully elucidated.
Cells of the CNS can rapidly increase the intracellular levels of heat shock protein (Hsp) family members in response to a wide variety of environmental stimuli, including oxidative stress (Omar and Pappolla 1993; Ohtsuka and Hata 2000; Ohtsuka and Suzuki 2000). Recent studies have demonstrated that increased expression of Hsp's may suppress the toxicity of oxidative stress (Liu et al. 1997; Beucamp et al. 1998), while decreased Hsp expression results in increased levels of oxidative stress-induced toxicity (Nakano et al. 1997; Yu et al. 1999). However, the exact mechanism by which Hsps confer resistance to oxidative injury has not been determined. In the present study we examined the possible neuroprotective effects of the molecular chaperone HDJ-1, which is a member of the Hsp40 family.
In the present study, we sought to determine the role of proteasome inhibition in oxidative stress toxicity, and determine if the neuroprotective effect of Hsp expression is mediated in part by attenuating the toxicity associated with proteasome inhibition. Together, these data demonstrate a possible role for proteasome inhibition in oxidative stress toxicity, and suggest that Hsp-conferred neuroprotection is associated with a preservation of proteasome function, and resistance to the toxicity associated with proteasome inhibition.
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- Materials and methods
The data presented in this study indicate a possible role for proteasome inhibition in oxidative stress-induced neurotoxicity. A causal role for proteasome inhibition as a mediator of neurotoxicity is supported by the fact that: (1) proteasome inhibition occurred rapidly following application of oxidative stressors, preceding cell death; (2) application of proteasome inhibitors alone was sufficient to induce cell death; (3) application of proteasome inhibitors increased the toxicity of oxidative stressors. Previous studies have suggested that the accumulation of oxidized, damaged and aggregated proteins following oxidative injury may be the result of proteasome inhibition (Grune et al. 1995; Grune and Davies 1997; Okada et al. 1999). These previous reports suggested that, while proteasome activity is harnessed towards the degradation of proteins modified during oxidative stress, the activity of the proteasome becomes less efficient, or possibly inhibited, resulting in the accumulation of oxidized, aggregated, or damaged proteins. It is possible that proteasome activity may become inhibited as the result of the oxidized or aggregated protein substrates themselves. For example, exposure to excessively oxidized or aggregated proteins, potently inhibits proteasome activity in vitro and in vivo (Friguet and Szweda 1997; Reinheckel et al. 1998; Okada et al. 1999). Consistent with a role for oxidative stress-modified proteins modulating the activity of the proteasome, in the present study we demonstrated that cells expressing increased levels of the Hsp (HDJ-1), had preserved proteasome activity following application of oxidative stressors. The ability of increased levels of HDJ-1 to attenuate proteasome inhibition did not appear to be due to a decrease in ROS levels, or altered levels of proteasome subunits. However, these data do not rule out the possibility that increased levels of HDJ-1 suppress the levels of non-DCF detectable ROS. Previous studies have demonstrated that Hsp play important roles in protein trafficking, protein folding and inhibiting or reversing protein aggregation (Omar and Pappolla 1993; Ohtsuka and Hata 2000; Ohtsuka and Suzuki 2000). It is therefore possible that increased Hsp levels preserve proteasome activity following oxidative injury, by delaying or reversing the deleterious effects of oxidative stress on intracellular proteins.
Data from the present study suggest that the beneficial effects of elevated Hsp expression to neural survival may be mediated, at least in part, via their amelioration of proteasome inhibition-associated toxicity. Previous studies have suggested that Hsps may play important roles in regulating proteasome activity via their roles in recruitment of substrates to the proteasome, or through beneficial Hsp–proteasome interactions (Ciechanover et al. 2000). For example, studies have demonstrated co-immunopreciptiation or co-localization of the proteasome with Hsps (Cummings et al. 1998; Luders et al. 2000), and cellular reconstitution experiments have demonstrated a requirement for some Hsps for proper proteasome activity (Conconi et al. 1996, 1998; Luders et al. 2000). Our data suggest that HDJ-1 may perform important chaperone functions that aid considerably in maintaining proteasome activity, particularly during oxidative injury. Although no previous work has identified a role for HDJ-1 in the suppression of oxidative stress-induced toxicity, previous studies have demonstrated that increased expression of HDJ proteins confers neuroprotection to the neurotoxicity associated with polyglutamine expansion (Chai et al. 1999; Jana et al. 2000; Kobayashi et al. 2000). It is interesting to note that the beneficial effects of increased HDJ expression in previous studies has been suggested to mediated, in part, by the attenuation of protein aggregation (Chai et al. 1999; Jana et al. 2000; Muchowski et al. 2000). Other evidence supporting an essential role for HDJ-1 in mediating neuron survival comes from antisense studies, in which antisense mediated down regulation of HDJ-1 in neural SH-SY5Y cells resulted in cell death (data not shown).
In the present study, proteasome inhibitors induced cell death in neural SH-SY5Y cells, and increased the toxicity of oxidative stressors. Although the mechanism by which proteasome inhibitors induced cell death, and increased vulnerability to oxidative stress is likely multifactoral, it is interesting to note the effects of proteasome inhibition and oxidative stress on mitochondrial membrane potential. At present it is unclear as to how proteasome inhibition may cause the loss of mitochondrial membrane potential. Based on the ability of increased HDJ-1 expression to attenuate proteasome inhibition-induced loss of mitochondrial membrane potential, it is likely that protein aggregation or deleterious protein–protein interactions contribute to the loss of mitochondrial membrane potential. Disruption of mitochondrial function is believed to play a contributing role to neuron death in a number of neurodegenerative conditions associated with oxidative stress (Chan 1996; Facchinetti et al. 1998; Keller et al. 1998). Data from the present study suggest that proteasome inhibition may therefore possibly contribute to the disruption of mitochondrial membrane potential in conditions associated with oxidative stress.
Although the data presented in the current study are in agreement with numerous reports (Boutillier et al. 1999; Canu et al. 2000; Keller and Markesbery 2000; Pasquini et al. 2000; Qui et al. 2000), indicating a role for proteasome inhibition in the neurodegenerative process, it is important to point out that proteasome inhibitor application has been demonstrated to attenuate neuronal death in some experimental paradigms (Sadoul et al. 1996; Favit et al. 2000). Although it is unclear how proteasome inhibitors mediate their neuroprotective effect, it is possible that proteasome inhibitors induce a beneficial form of heat shock in these experimental paradigms. For example, application of proteasome inhibitors has been demonstrated to be a potent inducer of Hsp family members in some cell types (Goldberg et al. 1997; Lee and Goldberg 1998). Alternatively, cells resistant to proteasome inhibitor toxicity may have higher levels of lysosomal proteases, and are therefore more efficient at shunting intracellular proteins to these proteases following proteasome inhibitor application, and thus prevent or delay the deleterious accumulation of toxic proteins. Recent studies also suggest that the proteasome may play a role in the regulation of some forms of NFkB activation (Goldberg et al. 1997; Tanaka 1998), which can have anti- or pro-apoptotic effects, depending on cell type (Mattson et al. 2000).
At present, the processes responsible for regulating the activity of the proteasome are poorly defined. Data in the present study provide in vitro evidence that non-proteasomal proteins, in particular Hsps, may play important roles in regulating proteasome activity. Because proteasome activity appears important to maintaining neuronal homeostasis and neuronal survival, data in the present study suggest that upregulation of Hsp family members may be of therapeutic benefit, via their amelioration of proteasome inhibition and proteasome inhibition-associated toxicity.