Materials.

Materials were obtained from the following sources: the PSD95-GFP expression vector (pGW1-CMV-PSD95-EGFP) was kindly provided by Donald B. Arnold; the alternative reading frame polypeptide (ARF) expression vector (pcDNA3-myc-ARF) was kindly provided by Yanping Zhang; the expression vector for DsRed2 (pDsRed2-N1) was from Clontech (Mountain View, CA); HIV-1 gp120 IIIB was from Protein Sciences Corporation (Meriden, CT); Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum, and horse serum were from Invitrogen (Carlsbad, CA); IL-1-ra was from R&D Systems (Minneapolis, MN); dizocilpine (MK-801), [(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt] (Win55,212-2), 6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-methoxyphenyl)methanone (AM630), 1,1′-[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride (AMD3100), threonine-lysine-proline (TKP), and all other reagents were from Sigma-Aldrich (St. Louis, MO). PSD95-GFP lacking the PEST sequence (PSD95ΔPEST-GFP) was produced by QuikChange Site-Directed mutagenesis (Stratagene, La Jolla, CA); primers were designed to delete amino acids 10 to 25 of rat PSD-95 as described previously (Kim et al., 2008a).

Cell Culture.

Rat hippocampal neurons were grown in primary culture as described previously (Kim et al., 2008a) with minor modifications. Fetuses were removed on embryonic day 17 from maternal rats, anesthetized with CO₂, and sacrificed by decapitation. Hippocampi were dissected and placed in Ca²⁺and Mg²⁺-free HEPES-buffered Hanks’ salt solution, pH 7.45. HEPES-buffered Hanks’ salt solution was composed of the following: 20 mM HEPES, 137 mM NaCl, 1.3 mM CaCl₂, 0.4 mM MgSO₄, 0.5 mM MgCl₂, 5.0 mM KCl, 0.4 mM KH₂PO₄, 0.6 mM Na₂HPO₄, 3.0 mM NaHCO₃, and 5.6 mM glucose. Cells were dissociated by trituration through a 5-ml pipette and a flame-narrowed Pasteur pipette, pelleted, and resuspended in DMEM without glutamine, supplemented with 10% fetal bovine serum and penicillin/streptomycin (100 U/ml and 100 μg/ml, respectively). Dissociated cells were then plated at a density of 100,000 to 200,000 cells/dish onto a 25-mm round coverglass (#1) glued to cover a 19-mm diameter opening drilled through the bottom of a 35-mm Petri dish. The coverglass was precoated with Matrigel (200 μl, 0.2 mg/ml). Neurons were grown in a humidified atmosphere of 10% CO₂ and 90% air, pH 7.4, at 37°C, and fed on days 1 and 6 by exchange of 75% of the media with DMEM, supplemented with 10% horse serum and penicillin/streptomycin. Cells used in these experiments were cultured without mitotic inhibitors for at least 12 days.

Immunocytochemistry.

Neuronal cultures were prepared as described above and fixed between day 13 and 14 in vitro. Cells were stained with 1 μM Hoescht 33342 (Sigma-Aldrich) for 15 min at 37°C, washed with PBS, and fixed with ice-cold methanol for 10 min at −20°C. Cells were washed with PBS three times and blocked for 1 h at room temperature in 10% bovine serum albumin (BSA; Sigma-Aldrich) in PBS. After blocking, cells were incubated with affinity-purified antibodies in PBS containing 3% BSA for 16 h at 4°C. The following antibodies were used: mouse anti-MAP2 (Sigma-Aldrich, 1:400 titer), mouse anti-glial fibrillary acidic protein (Sigma-Aldrich, 1:400 titer), or mouse anti-OX42 (AbCam, Cambridge, UK; 1:200 titer) to identify neurons, astrocytes, or microglia, respectively. Immunolabeled cells were visualized by incubating with fluorescein isothiocyanate-conjugated anti-mouse antibody (Dako Denmark A/S, Glostrup, Denmark; 1:300 titer) in PBS containing 3% BSA for 1 h at room temperature. Coverslips were mounted in Fluoromount (Southern Biotech, Birmingham, AL). Images of immunolabeled cells were captured on an Olympus IX70 inverted microscope (Olympus America, Melville, NY) equipped with a 20× objective (0.4 numerical aperture), a charge-coupled device camera (DVC Co., Austin, TX), and DVC View software (version 2.2.8). Fluorescein isothiocyanate was excited at 455 to 495 nm, and emission was collected at 510 nm. Hoechst 33342 was excited at 330 to 385 nm and emission was collected at 420 nm. In each region, the total number of Hoechst-stained cells was counted (100%) and compared with the number of cells labeled with primary antibody to calculate the percentage of each cell type. At least six separate images were counted for each cell type over two different cultures.

Transfection.

Rat hippocampal neurons were transfected between 10 and 13 days in vitro using a modification of a protocol described previously (Kim et al., 2008a). In brief, hippocampal cultures were incubated for at least 20 min in DMEM supplemented with 1 mM kynurenic acid, 10 mM MgCl₂, and 5 mM HEPES to reduce neurotoxicity. A DNA/calcium phosphate precipitate containing 1 μg of plasmid DNA per well was prepared, allowed to form for 30 min at room temperature, and added to the culture. After a 90-min incubation, cells were washed once with DMEM supplemented with MgCl₂and HEPES and then returned to conditioned media, saved at the beginning of the procedure. The transfection efficiency ranged from 2 to 11% based on the percentage of neurons that expressed DsRed2.

Confocal Imaging.

Transfected neurons were transferred to the stage of a laser-scanning confocal microscope (Olympus FluoView 300) and viewed through a 60× oil-immersion objective (numerical aperture, 1.40). Because of the low transfection efficiency, only one cell in the field expressed fluorescent proteins. For experiments in which the same neuron was imaged before and after a 24-h interval, the location of the cell was recorded using micrometers attached to the stage of the microscope. Nine optical sections spanning 8 μm in the z-dimension were collected (1-μm steps) and combined through the z-axis into a compressed z stack. GFP was excited at 488 nm with an argon ion laser, and emission was collected at 530 nm (10 nm bandpass). The excitation (HeNe laser) and emission wavelengths for DsRed2 were 543 nm and >605 nm, respectively.

Image Processing.

To count and label PSD95-GFP puncta, an automated algorithm was created using MetaMorph 6.2 image processing software (Molecular Devices, Sunnyvale, CA) described previously (Waataja et al., 2008). In brief, maximum z-projection images were created from the DsRed2 and GFP image stacks. Next, a threshold set 1 S.D. above the image mean was applied to the DsRed2 image. This created a one-bit image that was used as a mask via a logical AND function with the GFP maximum z-projection. A top-hat filter (80 pixels) was applied to the masked PSD95-GFP image. A threshold set 1.5 S.D. above the mean intensity inside the mask was then applied to the contrast-enhanced image. Structures between 8 and 80 pixels (approximately 0.37–3.12 μm in diameter) were counted as PSDs. The structures were then dilated and superimposed on the DsRed2 maximum z-projection for visualization. PSD counts were presented as mean ± S.E.M., where n is the number of cells, each from a separate coverglass over multiple cultures. We used Student’s t test for single or ANOVA with Bonferroni post test for multiple statistical comparisons.

Toxicity.

Cell death was quantified using propidium iodide (PI) fluorescence as described previously (Kim et al., 2008a). Cell culture was performed as described above except that 100,000 cells/well were plated in 96-well plates and grown for 12 to 14 days in vitro. The experiment was started by replacing 100 μl (approximately two-thirds volume) of the cell culture medium with fresh DMEM containing 10% horse serum, penicillin/streptomycin, 70 μM PI, and either neurotoxin (1 mM glutamate or gp120 at various concentrations) or vehicle (control). The plate was placed in a FluoStar Galaxy multiwell fluorescent plate scanner (BMG Technologies GmbH, Offenburg, Germany) and maintained at 37°C. PI fluorescence intensity measurements (excitation 544 nm ± 15, emission 620 nm ± 15) were taken at times 0, 24, and 48 h. Between measurements, cells were returned to the incubator and kept at 37°C in 10% CO₂. Drugs, when present, were applied 15 min before application of the neurotoxin and included in the media exchange. Each treatment was performed in triplicate; thus, a set of three wells from a single plating of cells was defined as an individual experiment (n = 1).

ELISA.

IL-1β protein levels were determined using a commercially available rat IL-1 ELISA kit (R&D Systems). The assays were performed according to the manufacturer’s instructions. Absorbance was read at 450 nm using a FluoStar Galaxy multiwell fluorescent plate scanner (BMG Technologies GmbH). The concentration of secreted IL-1β is expressed as picograms per milliliter.

Quantitative Real-Time Reverse Transcription-PCR.

RNA was extracted from cultures using an RNA isolation kit (Zymo Research, Irvine, CA). For real-time PCR, RNA was amplified using a SYBR Green Brilliant II qRT-PCR kit (Stratagene) following the manufacturer’s recommendations. In brief, 12.5 μl of SYBR Green qRT-PCR master mix was combined with 100 ng of isolated RNA, 100 nM sense and antisense primers, and 1 μl of RT/RNase block enzyme mix. Reverse transcription was performed by incubating samples at 50°C for 30 min. Samples were then transferred into an MX3005P cycler. Samples were monitored using MxPro-Mx3005P version 4.01 (Stratagene) software during the following thermocycling protocol: initial denaturation, 95°C for 10 min, followed by 40 cycles of 95°C for 30 s, and 60°C for 1 min. IL-1β was amplified using primers 5′-GGAAGGCAGTGTCACTCATTGTGG-3′ and 5′-CAGCTCACATGGGTCAGACAGCAC-3′ that were designed as shown previously (Nam et al., 2008). As an internal reference control, the glyceraldehyde-3-phosphate dehydrogenase gene was PCR-amplified using QuantiTect primers (QIAGEN, Valencia, CA). For each sample, two IL-1β reactions and two glyceraldehyde-3-phosphate dehydrogenase reactions were run in parallel and averaged (n = 1). Quantitative analysis was performed using the 2^−ΔΔC_t method.

gp120 Induces Synapse Loss.

We have described previously a quantitative assay to track changes in the number of postsynaptic sites visualized by confocal imaging of hippocampal neurons expressing PSD95-GFP and DsRed2 (Waataja et al., 2008). Figure 1A shows representative images of neurons 48 h after transfection with expression plasmids for PSD95-GFP and DsRed2. PSD95-GFP expressed as discrete puncta that contrasted well from diffuse green fluorescence found throughout the cell. DsRed2 expression filled the soma and dendrites and was used to track morphological changes, as a mask for image processing and to determine cell viability based on cytoplasmic retention of the fluorescent protein. Image processing identified and counted puncta by locating intensity peaks of the appropriate size (mean diameter = 0.52 μm) in contact with the DsRed2 mask. In a series of previously published control experiments, we demonstrated that PSD95-GFP puncta represent functional postsynaptic sites as indicated by colocalization with neurotransmitter release sites, NMDA-induced Ca²⁺ increases, and NMDA receptor immunoreactivity (Waataja et al., 2008).

Fig. 1.

HIV-1 gp120 IIIB induced PSD loss and cell death in a time and concentration-dependent manner. A, confocal fluorescent images display maximum z-projections of neurons expressing PSD95-GFP and DsRed2 before and 24 h after treatment with 600 pM gp120. Processing …

We investigated the effects of gp120 on the number of synaptic connections between hippocampal neurons in culture. Treatment with 600 pM gp120 for 24 h induced a 37 ± 4% (n = 44) loss of PSD95-GFP puncta (Fig. 1A). The time course of gp120-induced changes in the number of fluorescent puncta is shown in Fig. 1B. A 24-h exposure to gp120 significantly decreased the number of synaptic sites. gp120-induced synapse loss was concentration-dependent with an EC₅₀ of 153 ± 50 pM (Fig. 1C). Treatment with gp120 for 24 h did not significantly affect cell survival relative to control as defined by retention of dsRed (Fig. 1A) and uptake of propidium iodide (Fig. 1D). However, by 48 h, gp120 elicited significant cell death (EC₅₀ = 85 ± 45 pM). These findings suggest that synapse loss preceded neuronal death.

gp120-Induced Synapse Loss Requires Sequential Activation of CXCR4, the IL-1 Receptor, and the NMDA Receptor.

gp120 exerts neurotoxic effects by both direct actions on neurons (Pattarini et al., 1998) and indirect actions via the evoked release of cytokines from glia (Kaul and Lipton, 1999). Immunocytochemistry experiments revealed that the hippocampal cultures used for these studies were composed of 18 ± 2% neurons, 70 ± 3% astrocytes, and 9 ± 3% microglia. To determine whether gp120 was acting indirectly on microglia present in the mixed cultures studied here, we pretreated the cultures with the tuftsin-derived tripeptide TKP that inhibits microglial activation (Auriault et al., 1983) (Fig. 2A). TKP (50 μM) blocked gp120-induced synapse loss, implicating microglia as the primary target for gp120. The initial number of PSDs was 67 ± 8 and increased to 75 ± 16 after treatment with TKP. The IIIB strain of gp120 used in the present study binds to the chemokine receptor CXCR4 (Hesselgesser et al., 1998). Thus, we tested the CXCR4 receptor antagonist AMD3100, which prevents HIV-1 entry, to investigate the role of CXCR4 in gp120-induced synapse loss (Donzella et al., 1998). Pretreatment with AMD3100 (1 μM) significantly reduced gp120-induced synapse loss, indicating that CXCR4 was required. Both direct and indirect neurotoxicity elicited by gp120 requires the activation of NMDA receptors (Dawson et al., 1993; Pattarini et al., 1998; Kaul and Lipton, 1999). Similar to the mechanism of gp120-induced cell death, pretreatment with dizocilpine (MK801; 10 μM) blocked gp120-induced synapse loss. If gp120 acts indirectly, as suggested by the TKP experiment, then microglia-derived cytokines might act on neurons to activate NMDA receptors. Indeed, application of the endogenously produced IL-1 receptor antagonist, IL-1-ra, completely prevented gp120-induced synapse loss (Fig. 2A), suggesting the activation of IL-1 receptors expressed by neurons (Viviani et al., 2006).

Fig. 2.

gp120-induced synapse loss required sequential activation of CXCR4, IL-1β, and NMDA receptors. A, inhibition of CXCR4, IL-1β, or NMDA receptors prevents gp120-induced synapse loss. Bar graph summarizes changes in PSD95-GFP puncta (PSDs) …

If the synapse loss induced by gp120 is mediated by IL-1β receptors, then IL-1β should mimic the effects of gp120. Exogenous application of IL-1β (3 ng/ml) induced a loss of PSD95-GFP puncta, which was abolished by 1 μg/ml IL-1-ra (Fig. 2B). In contrast to the actions of gp120, IL-1β-induced synapse loss was not affected by pretreatment with 1 μM AMD3100, consistent with the idea that CXCR4 is upstream from the IL-1β receptor. Activation of IL-1β receptors stimulates the tyrosine kinase Src (Viviani et al., 2003), which phosphorylates NMDA receptors and sensitizes them to glutamate (Viviani et al., 2006; Salter et al., 2009). Here, we show that pretreatment with 10 μM MK801 or 10 μM pyrazolopyrimidine 2, a specific inhibitor of Src family kinases, prevented IL-1β-induced synapse loss (Fig. 2B). These results suggest that gp120-induced release of IL-1β from microglia might lead to the loss of synaptic connections as a result of overstimulation of NMDA receptors.

This hypothesis predicts that gp120 will elevate IL-1β levels in hippocampal cultures. Treating hippocampal cultures with 600 pM gp120 led to a 25-fold increase in IL-1β as detected by ELISA (Fig. 3A). This result is consistent with previous studies showing that infection with HIV-1 IIIB or intracerebroventricular microinfusion of gp120 induced glial release of IL-1β (Merrill et al., 1992; Ilyin and Plata-Salamán, 1997). IL-1β measured in the media collected from gp120-treated hippocampal cultures was near the limit of detection with commercially available ELISAs (Fig. 3A). Because the evoked release of IL-1β is accompanied by increased transcription of IL-1β message, we used a qRT-PCR assay to measure IL-1β production. Treating hippocampal cultures with 600 pM gp120 evoked a time-dependent increase in the expression of IL-1β mRNA that peaked 12 h after gp120 treatment (Fig. 3B). Thus, subsequent assays were performed at 12 h. Increases in IL-1β mRNA were blocked by pretreatment with either TKP or AMD3100 (Fig. 3C), suggesting that gp120 acts via CXCR4 on microglia to evoke IL-1β production and subsequent synapse loss.

Fig. 3.

gp120 induced IL-1β production in hippocampal cultures. A, incubation with 600 pM gp120 for 24 h induced the release of IL-1β in rat hippocampal cultures. IL-1β expression was measured by ELISA as described underMaterials and …

The gp120-Induced Loss of PSD95-GFP Puncta Is Mediated by the Ubiquitin-Proteasome Pathway.

In a previous study, gp120 was shown to reduce spine density and corresponding PSD95 levels (Viviani et al., 2006). We have found that activation of NMDA receptors results in the loss of postsynaptic sites by activation of the ubiquitin-proteasome pathway (Kim et al., 2008a; Waataja et al., 2008). Thus, here we determined whether gp120 might induce the loss of PSD95-GFP puncta by activating a ubiquitin ligase. Murine double minute 2 (Zhang et al., 1998) is an E3 ligase present in dendritic spines and known to ubiquitinate PSD95, targeting it for proteasomal degradation (Colledge et al., 2003). Nutlin-3 inhibits ubiquitin ligase activity (Kim et al., 2008a) and when applied at a concentration of 100 nM significantly reduced gp120-induced loss of PSDs (Fig. 4, A and B). p14 ARF binds to and inhibits murine double minute 2 (Colledge et al., 2003). Cultured hippocampal neurons were transfected with expression vectors for ARF (pcDNA3-myc-ARF) (Zhang et al., 1998), PSD95-GFP, and DsRed2 as described previously (Kim et al., 2008a). Hippocampal neurons expressing ARF were protected from gp120-induced synapse loss (Fig. 4B). These data suggest that gp120 induces the loss of synaptic connections through activation of an E3 ligase. Furthermore, gp120 did not significantly reduce the number of puncta in cells expressing PSD95ΔPEST-GFP. This construct lacks the PEST sequence at the N terminus of PSD95 that is required for ubiquitination (Colledge et al., 2003). Thus, our results suggest that gp120-induced synapse loss results from NMDA receptor-mediated activation of the ubiquitin-proteasome pathway as described for other excitotoxic stimuli (Kim et al., 2008a,b; Waataja et al., 2008). Neuronal nitric-oxide synthase (nNOS) mediates neuronal death triggered by gp120 (Dawson et al., 1993). However, treatment with N^G-nitro-l-arginine methyl ester hydrochloride (l-NAME), an inhibitor of nNOS, 15 min before and during 24-h exposure to gp120 did not significantly affect PSD loss (Fig. 4B). This result suggests that separate pathways downstream of the NMDA receptor mediate synapse loss and cell death.

Fig. 4.

gp120 induced PSD loss via the ubiquitin-proteasome pathway and induced cell death by activating NOS. A, processed images display labeled PSDs superimposed on DsRed2 fluorescence acquired before and after 24-h treatment with 600 pM gp120 in control cultures …

We tested this hypothesis using cell survival assays to determine whether gp120-induced neuronal death and synapse loss would show distinct pharmacological profiles (Fig. 4C). Cell death induced by various concentrations of gp120 was quantified by uptake of propidium iodide measured after 48-h exposure. l-NAME (100 μM) blocked gp120-induced neurotoxicity, consistent with previous reports (Dawson et al., 1993). In contrast, treatment with nutlin-3 (100 nM) did not significantly affect the gp120 concentration-response curve (EC₅₀ = 11 ± 2 pM) relative to control (EC₅₀ = 11 ± 9 pM). This concentration of nutlin-3 blocked gp120-induced PSD loss (Fig. 4B). These findings suggest that a path different from that leading to neuronal death mediates the synapse loss induced by gp120.

Cannabinoid Receptor Agonists Inhibit gp120-Induced Synapse Loss.

Cannabinoids modulate neurotoxic and inflammatory processes (Stella, 2009). Thus, we examined the effects of cannabinoids on HIV protein-induced synapse loss. The cannabinoid receptor full agonist Win55212-2 inhibited synapse loss induced by gp120 (Fig. 5, A and B) but not that evoked by the HIV-1 protein Tat. We examined the effects of cannabinoid receptor antagonists to determine whether the protection of synapses afforded by Win55212-2 was mediated via type 1 or 2 cannabinoid receptors (CB1 or CB2). The selective CB1 antagonist rimonabant did not affect the action of Win55212-2 on gp120-induced synapse loss. In contrast, the CB2 antagonist AM630 (100 nM) blocked the protection from gp120-induced synapse loss afforded by Win55212-2, indicating that the effects of Win55212-2 are mediated by CB2. Because CB2 receptors are expressed by activated microglia, but not hippocampal neurons, our results are consistent with an initial involvement of microglia in the toxic affect of gp120 (Stella, 2010).

Fig. 5.

Cannabinoid receptor agonist, Win55,212-2, prevented PSD loss induced by gp120, but not that induced by Tat. A, confocal images show PSD95-GFP puncta before and 24-h after exposure to 600 pM gp120 in the absence (untreated) or presence of Win55,212-2 …

Cannabinoids Inhibit gp120-Induced IL-1β Production in Microglia.

Cannabinoids act on glia and neurons to inhibit the release of proinflammatory molecules (Sheng et al., 2009; Stella, 2009). Thus, we examined the effects of cannabinoids on the production and release of IL-1β. gp120 evoked the expression of IL-1β mRNA by 12 h (Fig. 3B). This increase was blocked by Win55212-2 (Fig. 5C). AM630, but not rimonabant, antagonized the protective effects of Win55212-2 on IL-1β mRNA expression induced by gp120 (Fig. 5C), consistent with CB2 receptors mediating the effects of Win55212-2 on gp120-induced synapse loss.

HAD

HIV-associated dementia

HAND

HIV-associated neurocognitive disorders

CB1

cannabinoid type 1 receptor

CB2

cannabinoid type 2 receptor

PSD

postsynaptic density protein

PSD95-GFP

postsynaptic density protein 95 fused to green fluorescent protein

ARF

alternative reading frame polypeptide

DMEM

Dulbecco’s modified Eagle’s medium

MK801

dizocilpine

Win55212-2

[(R)-(+)-[2,3-dihydro-5-methyl-3[(4-morpholinyl)methyl]pyrrolo[1,2,3-de]-1,4-benzoxazinyl]-(1-naphthalenyl) methanone mesylate salt]

AM630

6-iodo-2-methyl-1-[2-(4-morpholinyl)ethyl]-1H-indol-3-yl(4-methoxyphenyl)methanone

AMD3100

1,1′-[1,4-phenylenebis(methylene)]bis-1,4,8,11-tetraazacyclotetradecane octahydrochloride

TKP

threonine-lysine-proline

HHSS

HEPES-buffered Hanks’ salt solution

qRT-PCR

quantitative real-time polymerase chain reaction

nNOS

neuronal nitric-oxide synthase

PCR

polymerase chain reaction

IL-1β

interleukin-1β

l-NAME

N^G-nitro-l-arginine methyl ester hydrochloride

nutlin-3

(±)-4-[4,5-bis(4-chlorophenyl)-2-(2-isopropoxy-4-methoxy-phenyl)-4,5-dihydro-imidazole-1-carbonyl]-piperazin-2-one

ELISA

enzyme-linked immunosorbent assay

NMDA

N-methyl-d-aspartate

PBS

phosphate-buffered saline

BSA

bovine serum albumin

GFP

green fluorescent protein

ANOVA

analysis of variance

PI

propidium iodide

gp120

glycoprotein 120.