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Abstract
We previously demonstrated that the lentivirus lytic peptide 1 (LLP-1) corresponding to the carboxyl terminus of HIV-1 gp41 induced cell death in human neuronal cells. Present study was conducted to further elucidate the pathogenic mechanisms involved in HIV-1 gp41-induced neurodegeneration in AIDS patients with cognitive deficits. The effect of LLP-1 on activation of calpain-1, a calcium-activated cysteine protease, which has been implicated in neuronal degeneration and death, was monitored by the proteolysis of spectrin in rat organotypic hippocampal slice cultures. Protease specific spectrin breakdown products revealed that LLP-1 generated ∼150/145-kDa fragments characteristic of calpain-1 activation in hippocampus undergoing cell death as evidenced by LDH release. This spectrin cleavage pattern was further confirmed by in vitro calpain-1 proteolysis. Futhermore, calpectin and MDL28170, inhibitors of calpain activity, blocked calpain-1-mediated spectrin cleavage. Spectrin cleavage likely occurred in the absence of overt synaptic loss, as suggested by the preserved levels of synaptophysin. Among pharmacological agents tested, apocynin, NADPH oxidase inhibitor, ameliorated the LLP-1-induced spectrin. Given the role of spectrin essential for synapse stabilization, LLP-1-induced spectrin cleavage as occurs with the activation of calpain-1 may be an important effector in LLP-1-mediated cell injury in hippocampus, which is primarily linked to cognitive dysfunction.
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Figure 1.
LLP-1 increases LDH release in cultured media of organotypic hippocampal slice cultures. Dose response of LDH release by LLP-1 was examined by incubation with increasing concentrations of peptides as indicated for 30 h. LDH activity in cultured media from hippocampal slices was assayed after exposing rat hippocampal slice cultures to gp41 derived LLP-1 or LLP-1 analog 2 (Ana2). Values are expressed as a percentage of the vehicle treated controls, considering the values obtained in the hippocampal slices as 100%.
Figure 2.
LLP-1-induced cell death was accompanied by calpain-1 activation as analyzed by spectrin cleavage. Western blot analysis of spectrin and synaptophysin content in whole cell extracts prepared from hippocampal slices as described in Fig. 1 was performed as detailed in Materials and Methods section. LLP-1 treatment for 30 h clearly generated ∼150/145-kDa spectrin breakdown products (SBP) characteristic of calpain-1 activation. Representative blots from two to three experiments with similar results are shown. Uniformity of gel loading was confirmed with β-actin as the standard.
Figure 3.
In vitro calpain-1 proteolysis confirms the spectrin cleavage pattern induced by LLP-1. Representative immunoblots of in vitro spectrin proteolysis by calpain-1. Spectrin or synaptophysin present in total lysate of hippocampal slices treated with vehicle only for 30 h are digested with increasing concentrations of calpain-1 for 1 h as indicated. Western blot analysis of spectrin and synaptophysin contents was performed as described in Fig. 2. Representative blots from two to three experiments with similar results are shown.
Figure 4.
Calpain-1 inhibitors blocked the spectrin cleavage pattern. Effects of specific calpain-1 inhibitors, calpectin and MDL-28170 on spectrin proteolysis in vitro by calpain-1 were analyzed. Whole cell extracts prepared from hippocampal slices treated with vehicle only were digested with calpain-1 in the presence of either calpectin or MDL28170 as indicated for 1 h. Western blot analysis of spectrin and synaptophysin contents was performed as described in Fig. 2. Representative blots from two to three experiments with similar results are shown.
Figure 5.
Apocynin reduced the LLP-1-induced spectrin cleavage. Effects of apocynin, NADPH inhibitor, on spectrin proteolysis triggered by LLP-1 in hippocampal slices were analyzed by 1 h pretreatment with apocynin before exposure of hippocampal slices to LLP-1 for 30 h. Western blot analysis of spectrin content in whole cell extracts prepared from hippocampal slices was conducted as described in Fig. 1 and Fig. 2.
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