Cocaine potentiates cathepsin B secretion and neuronal apoptosis from HIV-infected macrophages

Abstract

Substance abuse is a risk factor for HIV infection and progression to AIDS. Recent evidence establishes that cocaine use promotes brain perivascular macrophage infiltration and microglia activation. The lysosomal protease cathepsin B is increased in monocytes from patients with HIV dementia and its secretion induces 10-15% of neurotoxicity. Here we asked if cocaine potentiates cathepsin B secretion from HIV-infected monocyte-derived macrophages (MDM) and its effect in neuronal apoptosis. Samples of plasma, CSF, and post-mortem brain tissue from HIV positive patients that used cocaine were tested for cathepsin B and its inhibitors to determine the in vivo relevance of these findings. MDM were inoculated with HIV-1ADA, exposed to cocaine, and the levels of secreted and bioactive cathepsin B and its inhibitors were measured at different time-points. Cathepsin B expression (p < 0.001) and activity (p < 0.05) increased in supernatants from HIV-infected cocaine treated MDM compared with HIV-infected cocaine negative controls. Increased levels of cystatin B expression was also found in supernatants from HIV-cocaine treated MDM (p < 0.05). A significant increase in 30% of apoptotic neurons was obtained that decreased to 5% with the specific cathepsin B inhibitor (CA-074) or with cathepsin B antibody. Cathepsin B was significantly increased in the plasma and post-mortem brain tissue of HIV/cocaine users over non-drug users. Our results demonstrated that cocaine potentiates cathepsin B secretion in HIV-infected MDM and increase neuronal apoptosis. These findings provide new evidence that cocaine synergize with HIV-1 infection in increasing cathepsin B secretion and neurotoxicity.

Figure 1. Increased cathepsin B and cystatin B in plasma samples of HIV-infected cocaine-abuse patients

Cathepsin B levels and activity were measured in plasma and CSF of HIV-seropositive patients and HIV-seropositive patients that are cocaine abusers. There was a significant increase (p<0.05) of cathepsin B expression (A) and activity (B) in plasma of HIV-seropositive women cocaine-users compared with non-cocaine users. No significant differences in cathepsin B levels and activity were observed in the CSF of these women patients (Data not shown). Cystatin B and Cystatin C levels were measured in the plasma and CSF of HIV-seropositive women (n=6) and HIV-seropositive women cocaine-users (n=6) Increased levels of cystatin B were observed in plasma of HIV-seropositive women that are cocaine abusers (C) (p<0.01). No differences in cystatin C were observed neither in plasma (D) samples. No differences in cystatins B or C were found in CSF samples (Data not shown).

Figure 2. Increased cathepsin B and cystatin B in HIV-infected cocaine treated MDM

Supernatants of uninfected, uninfected cocaine-treated, HIV-infected and HIV-infected cocaine-treated were collected at 3, 6, and 12dpi to measure the levels of cathepsin B extracellular expression by ELISA. A significantly increased cathepsin B expression (p<0.001) (A) and activity (p<0.05) (B) was found in HIV-infected cocaine-treated supernatants compared with MDM supernatants from HIV positive, cocaine negative cultures. The levels of cathepsin B inhibitors; cystatin B and cystatin C in MDM supernatants at 3, 6, and 12dpi were determined by ELISA. Significantly increased levels of cystatin B were observed in supernatant of MDM HIV-infected cocaine treated compared with HIV-infected without the drug (p<0.05), HIV-uninfected cocaine-treated (p<0.01) or HIV-uninfected cultures (p<0.001) (C). Results are representative of three different experiments. No differences were found in cystatin C expression between the two groups (D).

Figure 3. Cocaine potentiates cathepsin B neurotoxicity in HIV-infected MDM

Neuronal apoptosis was measured using the terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay adding serum-free MDM supernatant at 6 and 12 days post-infection as shown by green fluorescence in neurons (apoptosis) and the nucleus stained with DAPI (blue). The results are representative of four experiments (Panel A). The SK-N-SH were exposed to MCM from uninfected (1and 3), uninfected cocaine-treated (6 and 8), HIV-infected (11 and 13) and HIV-infected cocaine-treated (16 and 18) with the cathepsin B inhibitor ( 2, 7, 12, and 17) and cathepsin B antibody at 1:500 dilution (5, 10, 15, and 20) do not show apoptosis. Controls are shown in panels 21-24. Neuronal apoptosis increased after the exposure of either HIV infected MCM or cocaine-treated MCM (panels 13 and 18 respectively) in later points of infection. Quantitative analysis of staining ratio between apoptotic (green)/non-apoptotic (blue) cells were calculated using Image-based Tool for Counting Nuclei (ITCN) from Image J software (NIH) (Panel B). Results revealed a significant increased percentage of apoptosis in neurons treated with HIV-infected supernatant compared with HIV-infected cocaine-treated at 6 and 12 days post-infection (p<0.01). A significant decrease in neuronal apoptosis was observed in neurons pre-treated with cathepsin B inhibitor and cathepsin B antibody at 6 and 12 dpi.(p<0.001) .

Figure 4. Expression of cathepsin B and cystatin B in Basal Ganglia of Post-Mortem brain tissues

Basal ganglia tissue from HIV-seronegative (A, and D), HIV seropositive cocaine negative (B and E), and HIV-seropositive cocaine positive patients were stained with mouse anti-human cathepsin B followed by Alexa 488 conjugate goat anti-mouse (green). For cystatin B staining, mouse anti-human cystatin B followed by Alexa 488 conjugate goat anti-mouse (green) was used. For microglia staining, rabbit anti-human Iba-1 followed by Alexa 546 goat anti-rabbit (red), and nuclear staining by DAPI (blue) was used. Secondary antibody was used as control of non-specific staining as shown in G. Magnification: 40X.

Figure 4. Expression of cathepsin B and cystatin B in Basal Ganglia of Post-Mortem brain tissues

Basal ganglia tissue from HIV-seronegative (A, and D), HIV seropositive cocaine negative (B and E), and HIV-seropositive cocaine positive patients were stained with mouse anti-human cathepsin B followed by Alexa 488 conjugate goat anti-mouse (green). For cystatin B staining, mouse anti-human cystatin B followed by Alexa 488 conjugate goat anti-mouse (green) was used. For microglia staining, rabbit anti-human Iba-1 followed by Alexa 546 goat anti-rabbit (red), and nuclear staining by DAPI (blue) was used. Secondary antibody was used as control of non-specific staining as shown in G. Magnification: 40X.