Mechanisms of HIV-tat-induced phosphorylation of N-methyl-D-aspartate receptor subunit 2A in human primary neurons: implications for neuroAIDS pathogenesis

Abstract

HIV infection of the central nervous system results in neurological dysfunction in a large number of individuals. NeuroAIDS is characterized by neuronal injury and loss, yet there is no evidence of HIV-infected neurons. Neuronal damage and dropout must therefore be due to indirect effects of HIV infection of other central nervous system cells through elaboration of inflammatory factors and neurotoxic viral proteins, including the viral transactivator, tat. We previously demonstrated that HIV-tat-induced apoptosis in human primary neurons is dependent on N-methyl-D-aspartate receptor (NMDAR) activity. NMDAR activity is regulated by various mechanisms including NMDAR phosphorylation, which may lead to neuronal dysfunction and apoptosis in pathological conditions. We now demonstrate that tat treatment of human neurons results in tyrosine (Y) phosphorylation of the NMDAR subunit 2A (NR2A) in a src kinase-dependent manner. In vitro kinase assays and in vivo data indicated that NR2A Y1184, Y1325, and Y1425 are phosphorylated. Tat treatment of neuronal cultures enhanced phosphorylation of NR2A Y1325, indicating that this site is tat sensitive. Human brain tissue sections from HIV-infected individuals with encephalitis showed an increased phosphorylation of NR2A Y1325 in neurons as compared with uninfected and HIV-infected individuals without encephalitis. These findings suggest new avenues of treatment for HIV-associated cognitive impairment.

Figure 1

The human NR2A subunit is tyrosine phosphorylated after tat treatment. A: Western blots from two separate, representative experiments demonstrating tat-induced tyrosine phosphorylation of NR2A (IP:NR2A WB:P-Tyr). Human neuronal cultures were treated with HIV-tat protein (10 or 100 ng/ml) for 5–60 minutes; 5–15 minutes shown. After cell lysis, NR2A was immunoprecipitated and electrophoresed. Membranes were probed with a pan phosphotyrosine antibody. The time of maximal phosphorylation varied among experiments, but always occurred between 5 and 15 minutes in patterns similar to those represented. A representative control (IP:NR2A WB:NR2A) demonstrates that changes in tyrosine phosphorylation were not due to changes in the total amount of NR2A immunoprecipitated. B: Densitometric analyses of the maximal time point for each individual experiment, normalized to the amount of NR2A immunoprecipitated, indicated a significant increase in the tyrosine phosphorylation of NR2A after tat treatment. (**P < 0.01, n = 5, 10, or 100 ng/ml of tat).

Figure 2

Src is associated with the NMDAR after tat treatment. A: Western blots demonstrating association of active (IP:NR2A WB:Active src) and total src (IP:NR2A WB:Total src) association with NR2A by co-immunoprecipitation and representative control Western blot showing approximately equal amounts of total NR2A immunoprecipitated (IP:NR2A WB:NR2A). Neuronal cultures were treated with tat (10 ng/ml) for 5, 10, and 15 minutes. NR2A was immunoprecipitated from the lysates, and membranes were probed for active src, total src, and NR2A. Changes in association were not due to nonspecific immunoprecipitation of src or NR2A proteins (IP: IgG1 WB: NR2A and WB: src). B and C: Densitometric analyses of active src (*P < 0.05, n = 3) (B) and of total src (*P < 0.05, n = 3) (C) indicated that maximal association with the NMDAR occurred five minutes after tat treatment.

Figure 3

Src activity is necessary for tat-induced tyrosine phosphorylation of NR2A. A: Western blots demonstrating reduced phosphorylation after tat treatment when src activity is inhibited. Neurons were pretreated with 5 μmol/L src inhibitor, SrcI, or vehicle for 10 minutes before tat treatment (10 or 100 ng/ml). NR2A was immunoprecipitated, and Western blotting was performed for phosphotyrosine (IP:NR2A WB:P-Tyr) or NR2A (IP:NR2A WB:NR2A). Tat induced tyrosine phosphorylation of NR2A, which was blocked by pretreatment with src inhibitor. B: Densitometric analyses indicated that tat induced tyrosine phosphorylation of NR2A (*P < 0.05, Control versus Tat), and pretreatment with the src inhibitor prevented tat induced phosphorylation (**P < 0.01, Tat versus SrcI + Tat). Although the src inhibitor sometimes induced minimal phosphorylation, it was not significant as compared with control levels (Control versus Src Inhibitor, not significant) (n = 3).

Figure 4

Identification of phosphorylation sites on human NR2A. A: Schematic depicting the tyrosine residues in the cytoplasmic tail of NR2A that were predicted to have a high likelihood of being phosphorylated according to the computer analysis program NetPhos 2.0. The tyrosines marked with a circled P were phosphorylated in our in vitro kinase assay, and Y1325, marked in red, was also found to be phosphorylated after tat treatment in our culture system. B: In vitro kinase assay. Pure recombinant src was preincubated in reaction buffer with ATP for 5 minutes, allowing for src to trans-autophosphorylate and become maximally active before adding target peptides. CON, src target consensus sequence, our positive control, and Y# indicates the tyrosine residue of the NR2A cytoplasmic tail. Three of seven residues tested were capable of being phosphorylated in vitro, Y1184, Y1325, and Y1423 (n = 3). C: Y1325 is phosphorylated in our neuronal culture system after tat treatment. Immune sera were generated against the phosphorylated forms of Y1184, Y1325 and Y1423, preabsorbed with the corresponding unphosphorylated peptide, and used for Western blotting after immunoprecipitation for NR2A. Western blotting indicated that Y1325 was a likely target of tat induced phosphorylation. Phosphorylation of Y1184 and Y1423 was present basally but did not change in response to tat treatment (100 ng/ml).

Figure 5

Monoclonal antibodies to phosphorylated Y1325 were specific, and NR2A was phosphorylated on Y1325 after tat treatment. A: Monoclonal antibodies from clone 11.1 were specific for phosphorylated Y1325. Hybridoma supernatant collected from clone 11.1 was diluted up to 1:20,000 in 1% bovine serum albumin in PBS, and enzyme-linked immunosorbent assays were performed. The supernatant titered to 1:20,000 against phosphorylated Y1325 (pY1325, black square), but did not recognize the unphosphorylated form of the peptide (Y1325, black circle), other phosphorylated peptides (pY1423, gray inverted triangle), or bovine serum albumin (gray triangle), used in the blocking solution. B: Y1325 is phosphorylated after tat treatment. Neuronal cultures were treated with tat (100 ng/ml) for 5–30 minutes, and NR2A was immunoprecipitated from the lysates. Western blotting was performed with the hybridoma supernatant, diluted 1:1000. Phosphorylation occurred between 5 and 15 minutes after tat treatment (IP:NR2A WB:pY1325). C: Densitometric analyses demonstrated that NR2A was phosphorylated at Y1325 after tat treatment (*P < 0.01, n = 4, 100 or 300 ng/ml tat).

Figure 6

Immunofluorescent staining indicates that tat treatment induces increased phosphorylation of Y1325 and that this is blocked by treatment with src inhibitor. A–L: Neuronal cultures were pretreated with SrcI or vehicle for ten minutes before being treated with tat (300 ng/ml) or vehicle for 5, 10, and 15 minutes. The 5-minute treatment is shown. Cover slips were subsequently fixed and stained for MAP2 (red), a neuronal marker, and pY1325 (green). Overlay of pY1325 and MAP2 is shown in the right-hand column (merge). Tat treatment (D–F) induced an increase in phosphorylation over controls (A–C). Pretreatment for ten minutes with src inhibitor prevented tat induced phosphorylation (G–I, M). SrcI treatment alone did not affect phosphorylation at the time point examined (J–L). M: The number of pixels per frame of each color was counted, and the ratio of green pixels to red pixels was calculated. Only those images with at least one spot of pY1325 staining were counted. Tat induced enhanced phosphorylation (*P < 0.05). Src inhibitor blocked tat induced phosphorylation (**P < 0.01, compared with tat treatment). There was no difference between control and SrcI + tat treated cultures (n = 3, with at least three pictures for each cover slip and two cover slips per experiment).

Figure 7

Phosphorylation of Y1325 of NR2A is increased in HIV encephalitic tissue. Tissue sections from HIV encephalitic brains (HIVE, n = 4), HIV-infected without encephalitis (HIV) or from control brains (Control) with a similar interval to autopsy and fixation (n = 4) were immunostained for MAP-2 (red, A, D, and G), a neuronal marker and phosphorylated Y1325, labeled pY1325 (green, B, E, and H). Micrographs labeled C, F, and I are the merged images. A–C: Control tissue demonstrated robust MAP-2 staining and minimal staining for phosphorylated Y1325. D–F: HIV tissue sections without encephalitis (HIV) show a similar minimal staining pattern to control conditions. The merged images show that all Y1325 staining colocalizes with MAP-2 and thus is found only in the neurons (C and F). HIV encephalitic tissue had greatly increased staining for pY1325 relative to control levels. There was also diminished MAP-2 staining relative to controls cells, perhaps indicative of neuronal damage, as there were also fewer neurons. The MAP-2 staining is so intense for all panels, such that in the merged images it is difficult to see the colocalization of green and red (orange). However, the reactivity with pY1325 is very apparent in H, as compared with B (Control) and E (HIV). The quantification of the fluorescence intensity for Y1325 and MAP-2 staining are described in Results.