Attenuated neurotoxicity of the transactivation-defective HIV-1 Tat protein in hippocampal cell cultures

Abstract

This study reports that the cysteine 22-->glycine 22 substitution in the HIV-1 Tat 1-86 B significantly attenuates its neurotoxicity. Consistent with previous studies, direct interactions of rat hippocampal cells with Tat 1-86 B were shown to cause dose-dependent and time-dependent neurotoxicity associated with activation of caspases from the mitochondrial apoptotic pathway. Despite the similar binding/uptake properties, Cys22 Tat 1-86 B failed to induce significant neurotoxicity and activation of caspases 9 and 3/7 in hippocampal primary cultures. Results of the study underscore the important role of cysteine-rich domain in mechanism of Tat-mediated neurotoxicity.

Figure 1. Neuronal cell viability changes in hippocampal cell cultures induced by the original and cysteine 22-substituted Tat 1–86 clade B

(A) The dose-response of decreased neuronal cell viability in primary rat fetal hippocampal cell cultures exposed to Tat 1–86 or Cys 22 Tat 1–86. The graph shows the decrease in Live/Dead ratios produced by different doses of recombinant Tat 1–86 after 48 hours of treatment. Data presented as mean values, n of sister cultures analyzed = 8–15 per each Tat 1–86 concentration.*- marks significant (P<0.05) differences in Live/Dead ratios between cultures treated with Cys22 Tat 1–86 and vehicle-treated controls. **- marks significant (P<0.05) differences in Live/Dead ratios between cultures treated with Tat 1–86 and cultures exposed to the same dose of Cys22 Tat 1–86. (B) The time course of the changes in neuronal cell viability in primary rat fetal hippocampal cell cultures exposed to Tat 1–86 or Cys 22 Tat 1–86. The graph represents relative (compared to non-treated controls) changes in Live/Dead ratios following the addition of 50 nM Tat 1–86 or 50 nM Cys 22 Tat 1–86. Individual measurements were carried out in 4–8 sister cultures (wells of the 96-well plate) per each time point and the experiment was repeated three times to ensure the reproducibility of the results. Data presented as mean values. *- marks incubation time points when significant (P<0.05) differences in Live/Dead ratios between cultures treated with Cys22 Tat 1–86 and vehicle-treated controls have been observed. **-marks incubation time points when significant (P<0.05) differences in Live/Dead ratios between cultures treated with Tat 1–86 and Cys22 Tat 1–86 have been observed.

Figure 2. Direct interactions of Tat 1–86 and Cys22 Tat 1–86 with hippocampal cells and following changes in cell viability and caspase activation

Binding/uptake of Tat 1–86 or Cys22 Tat 1–86 by cultured rat fetal hippocampal neurons. (A) Images show the specific Tat (or Cys22 Tat) immunoreactivity in rat fetal hippocampal cell cultures exposed to 50 nM Tat 1–86 or 50 nM Cys 22 Tat 1–86 for 2 hours. (B) Western blots show the specific Tat immunoreactivity in cell lysates 2 hours after the addition of either 50 nM Tat or 50 nM of Cys22 Tat 1–86 to the cell culture medium. (C) The graph shows amounts of Tat 1–86 or Cys22 Tat 1–86 per well specifically absorbed by hippocampal cells during the first 2 hours of treatment. Data presented as mean values ± SEM (n=3 per each time point). Neurotoxic effects of the transient exposure of primary rat fetal hippocampal cell cultures to Tat 1–86 or Cys 22 Tat 1–86. (D) Cell cultures were exposed to a 50 nM dose of Tat 1–86 or Cys22 Tat 1–86 for different time periods ranging from 1 min to 2 hours. After exposure, the cell culture medium was replaced with a fresh portion of medium without Tat or Cys22 Tat. The graph shows Live/Dead ratios determined in hippocampal cell cultures 48 hours after different time periods of transient exposure to original and Cys22-substituted Tat 1–86. Data presented as mean values, n of sister cultures analyzed = 7–12 per each time point. *- marks time periods of transient exposure to Tat 1–86, which were sufficient to induce a significant (P<0.05) decrease in Live/Dead ratios compared to vehicle-treated controls. The caspase 9 and caspase 3/7 activities in primary rat fetal hippocampal cell cultures after the exposure to Tat 1–86 or Cys 22 Tat 1–86. (E) Representative images show results of the detection of the SR-LEHD (red) fluorescent signal attributed to activated caspase 9 in neurons treated for 2 hours and results of the detection of the FAM- DEVD (green) fluorescent signal attributed to activated caspase 3/7 in neurons treated for 24 hours (B) with either 50 nM Tat 1–86 or 50 nM Cys 22 Tat 1–86 . Cultures were co-stained with Hoechst (blue fluorescence).