Human immunodeficiency virus type 1 alters brain-derived neurotrophic factor processing in neurons

Abstract

The molecular mechanisms leading to synaptic simplification and neuronal apoptosis in human immunodeficiency virus type 1 (HIV-1)-positive subjects are unknown. The HIV protein gp120 reduced the length of neuronal processes similarly to the proneurotrophin pro-brain-derived neurotrophic factor (proBDNF). Intriguingly, the effects of both proBDNF and gp120 were blocked by inhibitors of the p75 neurotrophin receptor, suggesting that proBDNF and gp120 share a similar mechanism of neurotoxicity. Therefore, we tested the hypothesis that gp120 affects the release of proBDNF. Using rat primary neurons, we observed that gp120 promotes a time-dependent intracellular and extracellular accumulation of proBDNF concomitantly with a decrease in mature BDNF. A similar imbalance in the ratio proBDNF/mature BDNF was confirmed in postmortem brains of HIV-positive subjects cognitively impaired and motor impaired. Therefore, it is conceivable to formulate the hypothesis that HIV neurotoxicity includes a gp120-mediated alteration of BDNF processing. To determine the cellular mechanism whereby gp120 produces an accumulation of proBDNF, we examined the levels of intracellular and extracellular enzymes that proteolytically cleave proBDNF furin and tissue plasminogen, respectively. In rat neurons exposed to gp120, intracellular furin levels decreased before cell death, whereas tissue plasminogen changed only during apoptosis. Our data suggest that HIV, through gp120, reduces proBDNF processing by affecting furin levels, and therefore causes an altered balance between antiapoptotic and proapoptotic neurotrophins. Our studies identify a new mechanism that may explain how HIV promotes neuronal injury.

Figure 1.

gp120 increases proBDNF. CGCs were exposed to gp120 for the indicated times, and levels of mBDNF and proBDNF were determined by ELISA and Western blot, respectively, in the medium and cell lysates. A, Representative Western blot analysis of CGC medium immunoprecipitated with an anti-BDNF antibody. The blot was analyzed with a BDNF antibody that recognizes either BDNF species (left) or proBDNF only (right). B, Semiquantification of the 34 kDa band by densitometric analysis. C, Levels of BDNF as determined by ELISA. Data expressed as mean ± SEM represent the average of three experiments (n = 6 each experiment). ^#p < 0.05, *p < 0.001 vs control (ANOVA and Sheffe's test).

Figure 2.

gp120 alters the release of mBDNF/proBDNF in cortical neurons. Cortical neurons, prepared as described previously (Avdoshina et al., 2010), were exposed to gp120 for the indicated times. The levels of mBDNF and proBDNF in the medium were determined by ELISA and Western blot, respectively, as described in Figure 1. Data are expressed as mean ± SEM (n = 6 each time point). ^#p < 0.05, *p < 0.001 vs control (ANOVA and Sheffe's test).

Figure 3.

gp120-induced neurite simplification is p75NTR dependent. A–F, Cortical neurons were exposed to boiled gp120 (A) or medium (D), gp120 (B), and proBDNF (50 ng/ml; E) alone or in combination with TAT-Pep5 (100 nm; C, F) for 6 h. Cells were then fixed and stained for class III β tubulin. G, H, Quantification of neurite processes was then done as described in Materials and Methods. Data are the mean ± SEM of 25 neurons. ^#p < 0.05, *p < 0.01 vs control (ANOVA and Sheffe's test). Scale bar, 50 μm.

Figure 4.

The toxic effect of gp120 is p75NTR mediated. A–D, Cortical neurons were exposed to gp120 (A, B) or proBDNF (50 ng/ml; C, D) alone or in the presence of K252a (100 nm), TAT-Pep5 (100 nm), or p75ba (10 μg/ml). In A and B, BDNF (50 ng/ml) was added 3 h before gp120. Neuronal survival was determined 18 h later by cell count (A, C) or MTT (B, D) as described previously (Bachis et al., 2003). Data are expressed as the mean ± SEM. n = 18. ^#p < 0.05 vs proBDNF or gp120; *p < 0.001 vs control (ANOVA and Sheffe's test).

Figure 5.

p75NTR-dependent reduction of neuronal survival. A, B, CGCs were exposed to gp120 (A) or proBDNF (50 ng/ml; B) alone or in the presence of K252a (100 nm), TAT-Pep5 (100 nm), or p75ba (10 μg/ml). In A, BDNF (50 ng/ml) was added 3 h before gp120. Neuronal survival was determined 18 h later as described in Figure 4. Data are expressed as the mean ± SEM. n = 18. p̂ < 0.05 vs proBDNF or gp120; *p < 0.001 vs control (ANOVA and Sheffe's test).

Figure 6.

BDNF levels are decreased in the brain of HAD subjects. A, B, BDNF (A) or NGF (B) levels were measured by ELISA in human CX, HP, and ST. Samples were from HIV-negative subjects, HIV-positive subjects with normal neurocognitive diagnosis (HIV but not HAD), HIV-positive subjects with HAD, and HIV-positive subjects with one or more opportunistic infections, such as encephalitis [HIV+OI (opportunistic infections)]. Data are the mean ± SEM of subjects described in Table 1. *p < 0.005; ^#p < 0.01 vs control (ANOVA and Sheffe's test). C, D, Representative Western blots of cortical lysates from HIV and HAD subjects analyzed with an anti-BDNF antibody or an anti-proBDNF antibody, respectively. E, Relative levels of proBDNF in the indicated areas as determined by densitometric analysis of the 34 kDa band. Data are expressed as the mean ± SEM (n = 7). *p < 0.05 vs HIV but not HAD (ANOVA and Sheffe's test).

Figure 7.

Neuronal localization of proBDNF in human brains. A, B, Sections from the cortex of HIV-positive subjects without (A) and with HAD (B) were processed as described in Materials and Methods. Sections were incubated with a rabbit anti-proBDNF antibody (Alomone Labs), rinsed in TBS, and incubated with a biotinylated anti-rabbit secondary antibody. Sections were then rinsed and incubated with ABC Elite solution. The reaction was visualized by using TBS with 0.05% 3,3′-diaminobenzidine and 0.01% H₂O₂. Sections were then rinsed, dehydrated, and coverslipped. Scale bar, 200 μm. Note the higher number of proBDNF-positive neurons in the HAD section.

Figure 8.

HIV and gp120 decrease furin levels. A, Intracellular and extracellular furin levels were measured by ELISA in cortical neurons exposed to gp120 for the indicated times. Data are the means ± SEM of six samples (*p < 0.001 vs control, ANOVA and Sheffe's test). B, Furin levels were measured in the indicated human brain areas by ELISA. Data are the means ± SEM of five samples. ^#p < 0.05; *p < 0.001 vs HIV negative (ANOVA and Sheffe's test). C, Example of a Western blot analysis of medium of neurons exposed to gp120 for the indicated times. D, Relative levels of tPA in the medium as determined by densitometric analysis of a 63 kDa band. Data expressed as mean ± SEM represent the average of three experiments (n = 4 in each experiment). *p < 0.05, **p < 0.001 vs control (ANOVA and Sheffe's test).