Differences in NMDA receptor expression during human development determine the response of neurons to HIV-tat-mediated neurotoxicity

Abstract

HIV infection of the CNS can result in neurologic dysfunction in a significant number of infected individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV infection in neurons. Thus, neuronal damage and dropout are likely due to indirect effects of HIV infection of other CNS cells, through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivating protein tat. We and others demonstrated that tat induces apoptosis in differentiated mature human neurons. We now demonstrate that the high level of tat toxicity observed in human neurons involves specific developmental stages that correlate with N-methyl-D-aspartate receptor (NMDAR) expression, and that tat toxicity is also dependent upon the species being analyzed. Our results indicate that tat treatment of primary cultures of differentiated human neurons with significant amounts of NMDAR expression induces extensive apoptosis. In contrast, tat treatment induces only low levels of apoptosis in primary cultures of immature human neurons with low or minimal expression of NMDAR. In addition, tat treatment has minimal effect on rat hippocampal neurons in culture, despite their high expression of NMDAR. We propose that this difference may be due to low expression of the NR2A subunit. These findings are important for an understanding of the many differences among tissue culture systems and species used to study HIV-tat-mediated toxicity.

Fig. 1

Expression of NMDAR during the development of human neurons. Cultures of human neurons obtained from different stages of gestation, immature and mature human neurons characterized by RT-PCR (a) and Western blot (b). a Relative levels of NR1, NR2A, and actin mRNA were measured by semiquantitative RT-PCR as described in the “Materials and Methods”. Immature and mature neuronal cultures were evaluated for expression of those genes. Both cultures expressed the mRNA subunits. Lane 1 is a 100 bp DNA ladder as a molecular weight marker. Lane 2 is a negative control without enzyme. Lane 3 is the product with primers for the NR1 subunit mRNA. Lane 4 is the product with primers for the NR2A subunit mRNA. Lanes 5 and 6 are actin loading controls. b Levels of NR1 and NR2A were determined by Western blot analysis of homogenates of neuronal cultures (150 μg of protein per lane) prepared from immature human brain tissue or mature tissue and probed with NR1 and NR2A antibodies. (+) is rat brain lysate as a positive control. N is lysate of human neuronal cultures. Arrows indicate NR1 and NR2A bands (n = 5)

Fig. 2

Neurons isolated from different stages of development respond differently by calcium imaging to tat treatment. Neurons were preloaded with the calcium indicator dye, fluo-4, for 15–30 min at room temperature before time lapse imaging. Baseline mean fluorescence was determined for each individual neuron by averaging the mean fluorescence for the first four frames of the time lapse, before treatment with tat or vehicle. The response to tat treatment (100 ng/ml) varied depending on the cell culture utilized. Cultures of immature neurons (a–c), mature neurons (d–f), and rat hippocampal neurons were analyzed (g–h). a and b, d and e, and g and h, correspond to photographs before and after tat treatment. Arrows denote neurons that respond by increasing calcium after 30 min of tat treatment. (c, f, i). Bar: 25 μm. Quantification of the increase in calcium in response to tat treatment. In these graphs bars show the means ± SD. Filled circles, x’s, and open circles indicate high, low, and mean results (P <0.005, control vs. tat, n = 4)

Fig. 3

Tat induces apoptosis in neuronal cultures that depends on their maturity and is prevented by MK801, implicating NMDAR mediation. The percentage of total neurons that are apoptotic, Neuronal apoptosis (%), is plotted for control, after 24 h of tat treatment(100 ng/ml), and after 24 h tat treatment following pretreatment with MK801 (5 μM). For all experiments, control apoptosis was low, 2–5%. Tat treatment increased apoptosis to varying degrees and the effect of tat was largely blocked by MK801 pretreatment, indicating mediation by NMDARs. a Tat treatment induced a low rate of apoptosis in neuronal cultures obtained before 22 weeks (immature human neurons). b Tat treatment induced only slightly, but not significantly, more apoptosis in immature neuronal cultures that were cultured for 6–10 weeks. c Mature human neurons showed a much greater incidence of apoptosis. d, e Undifferentiated and differentiated human neural progenitor cells exhibited a small increase in apoptosis in response to tat treatment. f Apoptosis of rat hippocampal neurons, which exhibit strong expression of NMDARs, was only moderately increased in response to tat. * P <0.05, tat compared to control, and ^# P <0.05, MK801 pretreatment compared to tat alone (n = 6 for each panel)

Fig. 4

Tat induces the formation of an LRP–NMDAR complex that is not formed in immature neuronal cultures, which express little NMDAR protein. a NR2A was immunoprecipitated (IP) from neuronal lysates of control (C) cultures or cultures treated with tat (T) for 15 and 30 min, and analyzed by Western blot (W) with antibodies to LRP. Tat treatment markedly increased the amount of LRP that was coimmunoprecipitated with NR2A in mature human neuronal cultures, but not in immature neuronal cultures. b In cultures of rat hippocampal neurons tat treatment did not increase association of LRP with NR2A or NR2B. LRP was immunoprecipitated from neuronal lysates of control (C) cultures or cultures treated with tat (T) and analyzed by Western blot (W) with antibodies to NR2A or NR2B as indicated. Total human fetal cortex lysate was used as a positive control for NR2A and NR2B (+). Tat treatment did not increase LRP/ NR2A or LRP/NR2B complex formation (n = 3)

Fig. 5

Rat hippocampal neurons express LRP/NMDAR complexes, but the distribution of these proteins is not altered by tat treatment. Confocal microscopy indicates that tat treatment (100 ng/ml, 15 min) did not alter the colocalization of LRP and NR1 on the surface of rat hippocampal neurons. Double immunofluorescence labeling for LRP (Cy3 staining, red) and NR1 (FITC staining, green) shows LRP and NR1 in control (a–c) and tat (d–f) treated cultures. Specificity was confirmed by replacing the primary antibody with a non-specific myeloma protein of the same isotype or the appropriate polyclonal reagent (data not shown). Bar: 20 μm. (n = 3)