Fig. 6.

Meth and human immunodeficiency virus-1 (HIV) increase glutamate release and meth increases HIV-induced apoptosis. (a) Fold changes of extracellular glutamate were quantified in culture supernatant of astrocytes by ELISA. Control cultures have low amount of extracellular glutamate (lines with squares). HIV-infected or meth-treated cultures have increased extracellular accumulation of glutamate. The combination of HIV infection and meth treatment resulted in an additive effect and increased the release of glutamate 8 fold. In addition, of hemichannel blockers (mimetic peptides) and gap junction blockers (AGA) resulted in prevention of glutamate dysregulation. (*p < 0.05 as compared to control conditions, #p < 0.05 as compared to meth or HIV-treated conditions. n = 4). (b) Meth increased the bystander apoptosis induced by HIV infection of astrocytes. Apoptosis was evaluated by TUNEL staining. Basal apoptosis was minimal in neurons and astrocytes (black bars). HIV infection of astrocytes resulted in ~ 60% apoptosis in neurons and ~ 18% in uninfected astrocytes after 14 days post infection. HIV-infected astrocytes are protected from apoptosis (red bars) but most uninfected astrocytes undergo apoptosis. Meth treatment alone (1 and 10 μM) did not induce apoptosis in neurons and astrocytes (blue bars). The combination of HIV infection and meth treatment increased apoptosis of neurons to ~ 82% and astrocytes to ~ 33% (light blue bars). Addition of a gap junction (GJ) blocker, AGA, totally blocks spread of apoptosis from HIV-infected astrocytes into uninfected surrounding cells, neurons and astrocytes (pink bars). As we previously demonstrated, NMDAR activity in neurons is essential to trigger apoptosis in uninfected neurons and astrocytes induced by HIV infection of astrocytes. In agreement, blocking NMDAR activation using MK801 or AP-5, totally blocks apoptosis of neurons and astrocytes induced by HIV-infected astrocytes and meth (green and dark blue bars, respectively). Addition of flupenthixol, a pan dopamine receptor blocker, only partially reduced the apoptosis induced by HIV-infected astrocytes and meth treatment, suggesting a partial participation. Even when higher concentrations of this blocker were used (×100), no significant improvement in blocking apoptosis was observed (brown bars). Addition of blockers alone or in the presence of Meth did not altered apoptosis levels (data not shown). (*p < 0.05 as compared to control conditions, #p < 0.05 as compared to HIV infection alone, &p < 0.05 as compared to HIV-infected and meth-treated conditions. n = 4)