HIV-tat induces formation of an LRP-PSD-95- NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes

Abstract

HIV infection of the central nervous system can result in neurologic dysfunction with devastating consequences in AIDS patients. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence that HIV can infect neurons. Here we show that the HIV-encoded protein tat triggers formation of a macromolecular complex involving the low-density lipoprotein receptor-related protein (LRP), postsynaptic density protein-95 (PSD-95), N-methyl-d-aspartic acid (NMDA) receptors, and neuronal nitric oxide synthase (nNOS) at the neuronal plasma membrane, and that this complex leads to apoptosis in neurons negative as well as positive for NMDA receptors and also in astrocytes. Blockade of LRP-mediated tat uptake, NMDA receptor activation, or neuronal nitric oxide synthase significantly reduces ensuing neuronal apoptosis, suggesting that formation of this complex is an early step in tat toxicity. We also show that the inflammatory chemokine, CCL2, protects against tat toxicity and inhibits formation of the complex. These findings implicate the complex in HIV-induced neuronal apoptosis and suggest therapeutic targets for intervention in the pathogenesis of NeuroAIDS.

Fig. 1.

LRP is necessary for tat-induced apoptosis. (A) Schematic illustrating cocultures, with neurons growing on top of astrocytes; neurons were imaged in a more superficial optical section. (B–D) Double staining for TUNEL (apoptosis) and MAP-2 (neurons). (E–G) Staining for TUNEL and GFAP (astrocytes). There was minimal apoptosis in untreated cultures (B and E), increased apoptosis after 24 h of tat treatment (C and F), and reduced apoptosis when the LRP inhibitor, RAP, was applied 15 min before and 6 h after tat (D and G). (H and I) Percentage of TUNEL-positive cells after different durations of treatment with tat alone (●) or with tat plus RAP. Control cells without treatment did not show increased apoptosis (data not shown). Addition of RAP once (15 min before tat treatment, RAP, ■), twice (a second application 6 h after tat treatment, RAPX2, ▴), or three times (at 6 and 12 h after tat treatment, RAPX3, ▾) reduced tat-induced apoptosis in neurons and astrocytes (P < 0.05 for all treatments vs. control, n = 7, no significant difference between RAP treatments).

Fig. 2.

NR2A and LRP form a complex after tat treatment, and PSD-95 may mediate complex formation. (A) LRP or NR2A antibodies were used for immunoprecipitation (IP) from lysates of control (time 0) cultures or cultures treated with tat or tat plus CCL2 for 5, 10, 15, 30, 45, 60, and 180 min. Samples were analyzed by Western blotting (WB) with the antibody indicated; input samples were used as loading controls. Human cortex lysate (Cortex) was used as a positive control for NR2A and LRP. (B and C) Densitometric analysis of the CoIP protein compared with the amount in the input (n = 5). Tat treatment increased association of NR2A and LRP maximally at 10–45 min (∗, P < 0.05 vs. control). The association was largely blocked by CCL2 (#, P < 0.05 vs. tat alone). (D) PSD-95 was immunoprecipitated from lysates of control cultures (time 0) or cultures treated with tat or tat plus CCL2 and analyzed by WB with antibodies to NR2A and to LRP. (E and F) Quantification of these data (n = 5). Tat treatment increased association of PSD-95 with LRP and with NR2A (∗, P < 0.05 vs. control). These effects were blocked by CCL2 (#, P < 0.05 vs. tat treatment). (G) The total input protein was used as a loading control for each IP (Input and data not shown).

Fig. 3.

Tat treatment enhances interaction between NR2A and nNOS. (A) NR2A was immunoprecipitated (IP) from lysates of control, untreated cultures (time 0), or cultures treated with tat or tat plus CCL2 for 5, 10, 15, 30, 45, 60, and 180 min, and precipitates were analyzed by Western blotting (WB) for nNOS interaction. Total protein lysate was used as a loading control (Input). (B) Densitometric analysis (n = 3) of nNOS abundance in the CoIP relative to input. No or low interaction between NR2A and nNOS was found in control cells. Tat treatment resulted in nNOS association with NR2A that was maximal between 30 and 60 min after tat treatment (∗, P < 0.05 vs. control; #, P < 0.05 vs. 10–30 min tat treated). CCL2 cotreatment significantly inhibited tat-induced NR2A/nNOS complex formation (&, P < 0.05 vs. tat alone).

Fig. 4.

Tat induces NO production, primarily through nNOS activation, resulting in neuron and astrocyte apoptosis. (A) Cultures were treated with tat (●) or tat plus NPA (▴), CCL2 (▶), MK801 (◀), or L-NAME (▾). At different time points, medium was collected and NO production was measured by the Griess reaction. Tat treatment induced significant NO production over the untreated condition (■; P < 0.001). MK801 or L-NAME abolished tat-induced production of NO (P < 0.001 vs. tat alone). NPA or CCL2 reduced tat-induced production of NO (P < 0.001) but not to basal levels, suggesting a source of NO in addition to nNOS. The addition of MK801, L-NAME, NPA, or CCL2 alone did not change basal NO production (data not shown). ∗, P < 0.001 vs. control; #, P < 0.001 for a treatment compared with tat alone (n = 4). (B and C) Summary data of apoptosis in neurons and astrocytes after 24-h treatment. Tat induced a high percentage of apoptosis compared with control (first two bars on the left). The NMDA blockers, MK801 and AP5, provided substantial protection (third and fourth bars). The general NOS antagonist, L-NAME, blocked apoptosis in both cell types. The nNOS-specific blocker, NPA, greatly inhibited neuronal apoptosis with little effect on astrocyte apoptosis (sixth bar). The NO donor, SIN, alone (seventh bar) induced almost as much apoptosis as tat [∗, P < 0.001 vs. control conditions and #, P < 0.001 compared with tat treatment (n ≥ 6)]. (D) Proposed mechanism of tat-induced apoptosis. Tat induces formation of a macromolecular complex of LRP, PSD-95 [protein with three PDZ domains, an SH3 and guanylyl kinase (GK)-like domain], NMDAR, and nNOS. The formation of this complex generates proapoptotic signals, such as NO, that can be transmitted to cells lacking NMDARs, resulting in extensive apoptosis.