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. Author manuscript; available in PMC: 2011 Aug 1.
Published in final edited form as: Eur J Neurosci. 2010 Jul 28;32(4):570–578. doi: 10.1111/j.1460-9568.2010.07325.x

M-tropic HIV envelope protein gp120 exhibits a different neuropathological profile than T-tropic gp120 in rat striatum

Alessia Bachis 1, Maria I Cruz 1, Italo Mocchetti 1
PMCID: PMC2924467  NIHMSID: NIHMS210979  PMID: 20670282

Abstract

Most early human immunodeficiency virus type 1 (HIV-1) strains are macrophage (M)-tropic HIV variants and use the chemokine receptor CCR5 for infection. Neuronal loss and dementia are less severe among individuals infected with M-tropic strains. However, after several years, the T cell (T)-tropic HIV strain, which uses CXCR4 variant, can emerge in conjunction with brain abnormalities, suggesting strain-specific differences in neuropathogenicity. The molecular and cellular mechanisms of such diversity remain under investigation. We have previously demonstrated that HIV envelope protein gp120IIIB, which binds to CXCR4, causes neuronal apoptosis in rodents. Thus, we have used a similar experimental model to examine the neurotoxic effects of M-tropic gp120BaL. Gp120BaL was microinjected in the rat striatum and neuronal apoptosis was examined in the striatum, as well as in anatomically connected areas, such as the somatosensory cortex and the substantia nigra. Gp120BaL promoted neuronal apoptosis and tissue loss that were confined to the striatum. Apoptosis was associated with microglial activation and increased levels of interleukin-1β. Intriguingly, gp120BaL increased brain-derived neurotrophic factor in the striatum. Overall, our data show that gp120BaL demonstrates a different neuropathological profile than gp120IIIB. A better understanding of the pathogenic mechanisms mediating HIV neurotoxicity is vital for developing effective neuroprotective therapies against Acquired Immune Deficiency Syndrome-associated Dementia Complex.

Keywords: BDNF, gp120BaL, CXCR4, CCR5, microglia

Introduction

The chemokine receptors CXCR4 and CCR5 are necessary for the fusion of human immunodeficiency virus type 1 (HIV) strains with target cell membranes and consequently, for viral entry (Berger et al., 1999). Macrophage-tropic HIV primarily uses CCR5 as a co-receptor, whereas T-cell line-tropic (T-tropic) viruses use CXCR4 (Pierson et al., 2004). Both CCR5- and CXCR4-using viruses are cytotoxic, although their pathogenic effects might be distinct (Grivel & Margolis, 1999; Harouse et al., 1999). HIV also enters the central nervous system during the early phase of infection and infects primarily macrophages and microglia (He et al., 1997; Bissel & Wiley, 2004). Nevertheless, it has been established that HIV, in the late phase of infection, promotes loss of neurons (Adle-Biassette et al., 1995; Gelbard & Epstein, 1995; Power et al., 1998; Petito et al., 2001) and other brain abnormalities defined by the term HIV-associated dementia (HAD), a neurological disease characterized by cognitive and motor impairments (Price et al., 1988; Masliah et al., 1996; Price, 1996).

Complex mechanisms have been suggested to explain why HIV causes neurological diseases such as HAD. Two prominent hypotheses have been proposed, namely that HIV causes neuronal apoptosis and white matter atrophy by either promoting neuroinflammatory events, such as the release of pro-apoptotic cytokines (Mastroianni et al., 1992; Glass et al., 1993; Persidsky et al., 1997), excitatory amino acids (Heyes et al., 1991; Bezzi et al., 2001), or nitric oxide (Adamson et al., 1996; Bagetta et al., 1996); or by directly affecting neuronal viability via releasing viral proteins such as the envelope protein gp120. Gp120 has been shown to produce neuronal apoptosis by acting on CXCR4 (Hesselgesser et al., 1998; Meucci et al., 1998; Zheng et al., 1999; Kaul et al., 2001) or CCR5 (Kaul & Lipton, 1999; Bachis & Mocchetti, 2005). Both chemokine receptors are expressed in neurons as well as in non–neuronal cells (Albright et al., 1999; van der Meer et al., 2000; Westmoreland et al., 2002). Thus, one may predict that gp120 from M- or T- tropic strains could be equally neurotoxic. However, the relative potency of CCR5-selective gp120 versus CXCR4-specific gp120 has not yet been fully defined. In vitro studies have shown that activation of CCR5 by two natural ligands, CCL5 (or RANTES), or macrophage inflammatory proteins, protects neurons from gp120-induced apoptosis (Meucci et al., 1998; Kaul & Lipton, 1999; Bachis & Mocchetti, 2005). Neuroprotection by CCL5 has also been confirmed by studies showing that cortical cultures from CCR5 knock-out mice are more sensitive to the neurotoxic effect of T-tropic gp120 (Kaul et al., 2007). On the other hand, stromal cell-derived factor 1 alpha or CXCL12, the natural ligand of CXCR4, promotes neuronal apoptosis (Hesselgesser et al., 1998; Kaul & Lipton, 1999; Zheng et al., 1999; Bezzi et al., 2001). In addition, it has been shown that HIV-infected individuals with higher cerebrospinal fluid concentrations of macrophage inflammatory proteins and CCL5 perform better on neuropsychological measures than those with low or undetectable levels (Sozzani et al., 1997; Letendre et al., 1999). Moreover, higher levels of CCL5 correlate with lower plasma HIV (Zanussi et al., 1996; Saha et al., 1998; Paxton et al., 2001). These findings suggest that CCL5 might slow down the progression of HAD.

We have previously reported that gp120IIIB, a strain that binds selectively to CXCR4, causes a time-dependent neuronal apoptosis in the rodent brain both at the site of injection as well as in distal areas (Bachis et al., 2006; Nosheny et al., 2007). In the current study, we have used a similar experimental design to examine the neurotoxic property of gp120BaL, a CCR5 specific strain (McDyer et al., 1999), as well as to dissect the role of the chemokine receptor CCR5 in M-tropic gp120-induced neurotoxicity. We report that gp120BaL exhibits a different profile than gp120IIIB.

Materials and Methods

Animal treatment

All surgical procedures on rats were performed in strict accordance with the Laboratory Animal Welfare Act, with National Institutes of Health Guide for the Care and Use of Laboratory Animals, and after approval from the Georgetown University Animal Care and Use Committee. Coordinates for injecting gp120 strain BaL (NIH AIDS Research Reference Reagent Program) in 0.1% bovine serum albumin or vehicle control (VEH, boiled gp120 in 0.1% bovine serum albumin) into the rat striatum (anteroposterior +0.7 mm; mediolateral ±3.0 mm; dorsoventral −6.0 mm from bregma), were according to Paxinos & Watson (Paxinos & Watson, 1998). Details of injection procedures have been described previously (Nosheny et al., 2004; Bachis et al., 2006). In brief, VEH or gp120BaL (400 ng) were delivered by a microperfusion pump (0.2 μl/min for 10 min) in anesthetized (ketamine/xylazine, 80/10 mg/kg i.p.) adult male Sprague Dawley rats (Taconic Farm, Hudson, NY). After completion of each injection, the needle was left in place for an additional 4 min to accomplish quantitative diffusion of the volume delivered. Animals were then returned to their cages. At the appropriate survival times, animals were deeply anesthetized with ketamine/xylazine (80 and 10 mg/kg, i.p.), followed by intracardiac perfusion of 4% paraformaldehyde in 0.1 M phosphate buffer, pH 7.4, for immunohistochemical analyses, or euthanized by decapitation for the biochemical determinations.

Immunohistochemistry

Brains were removed and post-fixed in 4% paraformaldehyde, then transferred into buffered graded sucrose (10%, 20% and 30%), and serial cross sections (16 μm) were prepared. For apoptosis, sections were analyzed for in situ terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling (TUNEL) according to the manufacturer instructions (ApopTag® Fluorescein In Situ Apoptosis Detection Kit, Cat. # S7110, Millipore, Bedford, MA) and activated caspase-3 as previously described (Nosheny et al., 2004; Bachis et al., 2006). In brief, for caspase-3 and cellular markers, after incubation with 5% normal goat serum, sections were incubated with a cleaved caspase-3 antibody (1:150 dilution; Cell Signaling, Beverly, MA; cat. # 9661) for 48 hr at 4°C, and NeuN (1:500, Millipore; cat. #MAB377) to visualize neurons. CD68 (1:500; Serotec, Raleigh, NC; cat. # MCA341R), or glia fibrillary acidic protein (GFAP, 1:200; Millipore; cat. # MAB360) were used to visualize microglia or astrocytes, respectively. After three washes, sections were incubated with Alexa-Fluor® 488 and Alexa-Fluor® 594 secondary antibodies (1:1000, Millipore; cat. # A11034 and A11032) to visualize caspase-3 and cellular markers, respectively. Control sections were incubated either with primary or secondary antibody only. A positive control for the TUNEL staining was done by incubating sections with DNase I (0.5 μg/ml, Sigma) in 30 mM Trizma base, pH 7.2, 4 mM MgCl2, 0.1 mM DTT at room temperature for 10 min. For negative control, terminal deoxynucleotidyl transferase was omitted.

For tyrosine hydroxylase (TH) and caspase-3 immunoreactivity, sections were incubated with an anti-TH antibody (1:200; Millipore; cat. # MAB5280) together with the caspase-3 antibody described above for 48 hr at 4°C. Sections were then incubated with Alexa-Fluor® 594 and Alexa-Fluor® 488 (1:1000, Millipore) secondary antibodies to visualize TH and caspase-3, respectively.

Histological analysis

Sections were analyzed with a Zeiss fluorescence microscope Axioplan2 supported with AxioVision software (Carl Zeiss Microimaging, Inc., Thornwood, NY) as previously described (Bachis et al., 2006; Nosheny et al., 2007). In brief, positive cells were counted using a 20× objective and MetaMorph® Imaging software (Universal Imaging Corp, Downingtown, PA). The number of TUNEL and/or caspase-3 and CD68 positive cells in the striatum was assessed in an area of at least 0.25 mm2 per section after excluding the sections with the needle tract (~40 μm). A total of at least 10 sections (1 section every 100 μm) rostral and caudal to the injection site per animal were used (N=5 rats per group). The entire striatum was analyzed. For the substantia nigra (SN), caspase-3 positive cells were counted along its entire length. Caspase-3 positive neurons in the somatosensory cortex were counted in a total of 15 sections (1 section every 100 μm). To ensure equal overall staining intensity between sections, total intensity of fluorescent signal in the corpus callosum was compared between sections.

For TH immunoreactivity in the SN, total intensity of fluorescent signal was measured using MetaMorph® software in a series of 20X digitized microscope images (6 sections/animal; n=5 animals/group) from the following regions of interest: −5.00 to −5.30 mm from bregma (medial SN), −5.80 to −6.30 mm from bregma (lateral SN) (Paxinos & Watson, 1998). TH values for each group were then averaged and expressed as percentage of control levels. Size and shape of the lateral and 3rd ventricles were examined to ensure that sections from identical coronal planes were used for each animal.

Enzyme-Linked ImmunoSorbent Assay (ELISA) for cytokines

Interleukin-1β (IL-1β) levels were measured in the striatum by ELISA. Tissues were homogenized using RIPA buffer (Millipore; cat. # 20-188) containing 1 μg/mL aprotinin, 1 μg/mL leupeptin, and 1 μg/mL pepstatin (all from Sigma). Samples were centrifuged for 10 min at 15,000 g and protein content was measured in the supernatant by the Bradford Comassie Blue colorimetric assay (Bio-Rad). The ELISA assay was performed according to the manufacturer instructions (Rat IL-1 beta/IL-1F2 DuoSet, cat. # DY501; R&D, Minneapolis, MN) using a 96 well plate covered with a monoclonal anti IL-1β antibody overnight at room temperature. A seven point standard curve using 2-fold serial dilutions was also prepared. The highest standard had a concentration of 2000 pg of IL-1β per 100 μl. Each sample was done in triplicate. Wells were then washed and incubated with anti-rat IL-1β polyclonal antibody for 2 hr at room temperature. After washing, 100 μl of Streptavidin-HRP (1:200 dilution, R&D) were added to each well and incubated for 20 minutes at room temperature, followed by three washes and a 20-minute incubation with a solution containing 50% H2O2 and 50% Tetramethylbenzidine (Substrate solution, R&D). The color was developed by adding 50 μl of Stop Solution (2N H2SO4, R&D). The optical density was immediately determined using a microplate reader set to 450 nm. Total IL-1β proteins for each sample were calculated by plotting sample absorbance values against the standard curve.

Cerebellar granule cells

Cerebellar granule cells were prepared from 7-day-old Sprague Dawley rat pups as described previously (Bachis et al., 2003). In brief, neurons were plated onto poly-L-lysine precoated 6 well plates at a density of 1×106 cells/ml and grown in Basal Medium Eagle (Invitrogen Corp., Grand Island, NY) containing fetal calf serum(10%), L-glutamine (0.5 mM), KCl (25 mM), gentamycin (50 μg/ml), and penicillin-streptomycin(10,000U/ml). Cells were maintained at 37°C in 5% CO2-95% air for 7 days before adding gp120s.

ELISA for neurotrophic factors

Brain-derived neurotrophic factor (BDNF), glial-cell derived neurotrophic factor (GDNF) and nerve growth factor (NGF) levels were determined using the Emax immunoassay systems from Promega, Madison, WI (Cat. # G7610, G7620 and G7630, respectively), as previously described (Marini et al., 1998; Nosheny et al., 2004). When neurotrophic factor levels were measured in brain tissue we used 100 μg of tissue homogenate prepared as described above. For the measurement of BDNF in cerebellar granule cells, we used 100 μl of the medium. Each sample was done in triplicate. Total levels of neurotrophic factors for each sample were calculated by plotting sample absorbance values against a standard curve.

Statistical analysis

Data were evaluated by ANOVA with post hoc Holm-Sidak’ test (SigmaStat(R) software, Systat Software, Inc, Point Richard, CA).

Results

Gp120BaL causes cell death at the site of injection

We have previously shown that gpBaL causes caspase-3-dependent neuronal cell death in vitro (Bachis & Mocchetti, 2005). To determine whether gp120BaL is neurotoxic in vivo, adult male rats received an acute injection of gp120BaL (400 ng) or vehicle control (VEH) in the striatum. Animals were sacrificed 1, 7 and 15 days after the injection. Brain sections where then analyzed for activated caspase-3 and TUNEL (Fig. 1), both markers of apoptosis, along with NeuN, a neuronal marker. The striata of VEH-injected rats exhibited few caspase-3 positive cells at all times examined (Figs. 1A and C). In gp120-injected rats there was a time dependent increase in apoptotic cells. In fact, by 7 days and up to 15 days, several sections from the striatum exhibited caspase-3 (arrows in Fig. 1B, Fig. 1C) and TUNEL positive cells (Fig. 1D) which were also NeuN positive, suggesting neurons.

Figure 1. gp120BaL promotes neuronal apoptosis.

Figure 1

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VEH or gp120BaL was microinjected into the rat striatum. Animals were sacrificed at the indicated times after the injection. Caspase-3, TUNEL and NeuN immunoreactivity were examined by immunohistochemistry in serial sections from the striatum. Representative sections from VEH (A) and gp120BaL (B) injected rats taken 50 μm from the injection site showing caspase-3 immunoreactivity (green) in NeuN (red) positive cells 7 days after injection. Arrows indicate examples of apoptotic neurons. Bar = 200 μm. C and D, The average number of caspase-3 and TUNEL positive neurons per section was determined by MetaMorph®. Data are expressed as the mean ± SEM of four animals per group (at least 10 sections per animal). *p<0.001 versus VEH control (1C: F = 73.663 for 7 days, F = 50.855 for 15 days; 1D: F = 89.832 for 7 days, F = 80.074).

The extent of tissue damage was then determined by calculating the length and area of TUNEL and caspase-3 immunoreactivity, rostral and caudal to the injection site, as well as the area of tissue loss, as described previously (Bachis et al., 2006). By 7 days, gp120BaL caused an area of tissue damage extending several μm rostral and caudal to the injection site with multiple TUNEL- and caspase-3-positive cells (Table 1). Nevertheless, when compared to the effect of gp120IIIB previously described (Nosheny et al., 2004), the M-tropic gp120 did not cause neuronal injury as extensive as that caused by gp120IIIB.

Table 1.

Extent of tissue damage after acute parenchymal gp120BaL injection

Groups Length (μm) Lesion area (μm2) Penumbra area (μm2)
VEH ND 3.2±1.4 ND
Gp120BaL 860 ± 26.5 11.8 ± 8.3 220 ± 35.2
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Striatal sections from VEH and gp120-treated rats (400 ng, 7 days after the injection) were processed for TUNEL and caspase-3 immunoreactivity. Tissue damage was determined rostrally and caudally from the injection site, by calculating the length of an area showing TUNEL and caspase-3 immunoreactivity, a lesion area (showing no tissue), and a penumbra area (area with caspase-3 positive cells surrounding the lesion area). ND= not detected. Data are the mean ± SEM of at least 15 sections from 3 rats per group. In a previous study (Nosheny et al., 2004), we have found that gp120IIIB-mediated tissue damage was: length 1800 ± 20μm, lesion area 33.8 ± 9.3 μm2 and penumbra area 366.9 ± 45.2 μm2. Thus, gp120BaL evokes a tissue damage that is significantly less than that obtained with gp120IIIB.

Gp120BaL does not affect neurons’ viability in distal areas

The data presented so far indicate that the pathological profile of M-tropic gp120 is different from that of T-tropic gp120. We have recently shown that intrastriatal gp120IIIB causes neuronal damage in the substantia nigra (SN) (Bachis et al., 2006; Nosheny et al., 2007). Thus, to further analyze the neurotoxic effect of gp120BaL, we determined neuronal loss in areas that send axons to the striatum, such as the SN and the somatosensory cortex (CX). Sections were double stained with an antibody against cleaved caspase-3 and an antibody against NeuN. The SN and CX of control rats exhibited an average number of caspase-3 positive neurons per section that was 0.9 ± 1 and 4±1, respectively. This number did not change through the investigation (Fig. 2). Gp120BaL failed to induce more neuronal apoptosis in these brain areas at either 1, 7 or 15 days (Fig. 2). In addition, we did not observed an effect of gp120BaL on TH immunoreactivity in the SN. Indeed, there was no difference between VEH and gp120-treated in TH immunoreactivity at any time point (Fig. 3). These data indicate that M-tropic gp120 does not cause retrograde degeneration.

Figure 2. gp120BaL does not cause apoptosis in distal areas.

Figure 2

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Rats were microinjected with VEH or gp120BaL into the striatum and were sacrificed at the indicated days after the injection. Sections from the SN and somatosensory cortex (CX) were processed for caspase-3 and NeuN. Data are the mean ± SEM of the number of caspase-3 positive neurons determined in at least 15 sections per area from five rats per group.

Figure 3. Analysis of TH immunoreactivity.

Figure 3

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TH immunoreactivity was analyzed in the SN of rats microinjected with VEH or gp120BaL into the striatum. TH immunoreactivity was quantified using MetaMorph® software in a series of sections from the SN pars compacta (pc) and SN pars reticulata (pr) as previously described (Nosheny et al., 2006). Data, expressed as percentage of controls, are the mean ± SEM of at least 15 sections per rat, n=5 rats per time point.

Gp120BaL promotes activation of microglia

We have previously shown that gp120IIIB causes neuronal apoptosis in vivo without promoting microglial activation (Bachis et al., 2006). To determine whether gp120BaL behaves in a similar fashion, sections from the rat striatum were stained with CD68, a marker for microglia. In control (VEH) rats we observed CD68+ cells only in an area immediately surrounding the injection site along the needle tract (Fig. 4A). In gp120-treated rats, the area containing CD68 positive cells in each section was wider than VEH (Fig. 4B). Time-course studies revealed that gp120BaL causes a time-dependent increase in CD68 immunoreactivity in several sections that did not include the injection site (Fig. 4C). Moreover, we did not find any difference in GFAP staining between VEH and gp120-treated rats (data not shown).

Figure 4. gp120BaL causes microglia activation at the site of injection.

Figure 4

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Animals were microinjected with VEH or gp120BaL into the striatum and were sacrificed at various days after the injection. Activated microglia was detected using an antibody against CD68. A and B. Representative striatal sections of VEH and gp120-treated rats, respectively, showing CD68 immunoreactivity. Sections were taken 50 μm from the injection site. Bar= 100 μm. C. CD68 positive cells were counted in the striatum at the indicated times after gp120 injection. Data, expressed as the mean ± SEM of five animals per group (10 sections per animal), are the average of the number of CD68 positive cells in an area of 0.25 mm2 per section. *p< 0.001 vs VEH (F = 37.496 for 1 day; F = 34.535 for 7 days; F = 54.983 for 15 days).

To test the hypothesis that gp120BaL promotes microglia activation and perhaps an inflammatory response we examined levels of a pro-inflammatory cytokine, IL-1β, in rat striatum 1, 7 and 15 days after injection. Gp120BaL elicited a significant increase in IL-1 β levels by 24 hr and up to 15 days (Fig. 5). Overall, these data suggest that inflammatory responses may help evoke and maintain gp120BaL-mediated neurotoxicity and that gp120BaL toxicity may also involve an indirect mechanism.

Figure 5. IL-1β is increased after gp120BaL.

Figure 5

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Striatal levels of IL-1βwere measured by ELISA 1, 7 and 15 days after the striatal injection of VEH or gp120BaL. Data expressed as % of VEH-treated animals are the mean ± SEM of five animals per group. *p<0.001 vs VEH (F = 166.967 for 1 day; F = 80.438 for 7 days; F = 27.695 for 15 days). The amount of IL-1βin ipsilateral VEH was 41 ± 8.2 pg/100 μg proteins.

Gp120BaL increases BDNF

Gp120IIIB has been shown to reduce BDNF and GDNF levels in the striatum and SN, respectively (Nosheny et al., 2004; Nosheny et al., 2006), and to increase nerve growth factor (NGF) in the hippocampus (Bagetta et al., 1996). Alterations of neurotrophic support has been suggested to contribute to neuronal loss in chronic neurodegenerative diseases (Mocchetti & Brown, 2008). Thus, gp120BaL toxicity may involve a reduction of neurotrophic factors. To test this hypothesis, rats received gp120BaL or VEH control in the striatum, and were sacrificed 1, 7 and 15 days later. The levels of BDNF, GDNF and NGF were measured in the ipsilateral and contralateral striatum, SN and cortex by ELISA. No difference was observed in the levels of GDNF (Fig. 6B) and NGF (Fig. 6C) between VEH and gp120-injected animals in all tissues examined at all time points. Interestingly, gp120BaL increased the levels of BDNF in the ipsilateral striatum by one day, and up to 15 days (Fig. 6A). No changes in BDNF were observed in the contralateral striatum, SN or cortex (Fig. 6A).

Figure 6. gp120BaL increases BDNF.

Figure 6

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The levels of BDNF, GDNF and NGF were determined in the indicated brain areas 1, 7 and 15 days after gp120BaL injection into the striatum (IP ST=ipsilateral striatum, CL ST=contralateral striatum). Data are the mean ± SEM of five animals per group. *p< 0.05 vs VEH (F = 20.361); #p< 0.001 (F = 45.475 for 7 days; F = 38.738 for 15 days). BDNF, GDNF and NGF levels in the IP striatum of VEH-treated rats were (in pg/100 μg proteins): 400 ± 21; 22.5 ± 4.2; 18.5 ± 2.0, respectively.

Striatal BDNF is mostly produced by cortical neurons (Altar et al., 1997). Thus, the apparent discrepancy between the increase in striatal BDNF and the lack of effect of gp120BaL on cortical BDNF may be attributed to the fact that gp120Bal affects only the release of BDNF. To examine this mechanism, we used cerebellar granule cells in culture which express both CXCR4 and CCR5 receptors (Bachis et al., 2003) and release BDNF upon appropriate stimuli (Marini et al., 1998). Neurons were exposed to gp120s (5 nM, each) or VEH and the medium was collected at different time points to determine released BDNF. We found that BDNF accumulation in the medium varies depending upon the strain of gp120 used. In fact, gp120BaL evoked a time-dependent increase in BDNF levels (Fig. 7). The opposite was obtained by gp120IIIB (Fig. 7). These data help support the notion that gp120BaL exhibits a different cellular mechanism than gp120IIIB.

Figure 7. BDNF release is differentially regulated by gp120s.

Figure 7

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Cerebellar granule cells were exposed to gp120BaL or gp120IIIB (5 nM, each) and medium was collected at the indicated time points. BDNF was determined in 100 μl fraction of the medium by ELISA. BDNF levels in the medium of control cells were 75 ± 10 pg/ml. Data are the mean ± SEM of three separate samples each group. #p<0.05 vs VEH (F = 9.914); *p<0.001 vs VEH (gp120BaL: F = 28.743 for 6 hr; F = 42.202 for 12 hr. gp120IIIB: F = 87.432 for 6 hr; F = 174.988 for 12 hr).

Discussion

Several studies have shown that gp120 activation of CXCR4 is directly involved in HIV-associated neuronal damage (Hesselgesser et al., 1998; Meucci et al., 1998; Kaul & Lipton, 1999). Activation of CCR5 paradoxically can result in neuronal injury or protection depending upon the ligand used. Indeed, gp120 binding to CCR5 causes neuronal apoptosis whereas the natural CCR5 ligands RANTES (Kaul & Lipton, 1999; Bachis & Mocchetti, 2005) or MIP-1b (Meucci et al., 1998) confer neuroprotection against gp120. In this work, we have used gp120BaL, a M-tropic envelop protein, in an attempt to provide information as to whether this strain is as toxic as gp120IIIB. We have found similarities as well as differences. Gp120BaL, similar to gp20IIIB, induced caspase-3 dependent neuronal apoptosis in the rat striatum. However, the extent of neuronal damage after the M-strain differed from that of gp120IIIB both anatomically and quantitatively. Indeed, gp120BaL-mediated apoptosis was seen only in the striatum and was restricted to an area smaller than 250 μm2 per section. Gp120IIIB-mediated apoptosis, instead, has been seen in distal areas from the injection site, such as the SN, and covered an area greater than 250 μm2 per section (Nosheny et al., 2004; Bachis et al., 2006). Thus, it appears that gp120BaL, unlike gp120IIIB, does not cause retrograde neuronal cell death. Furthermore, the apoptotic effect of M-tropic gp120 appears also to involve activated microglia, suggesting that gp120BaL may use cellular mechanisms that are different from that utilized by gp120IIIB.

The role of CXCR4 receptor in the neurotoxic effect of T-tropic strain of gp120 has been established (Hesselgesser et al., 1998; Meucci et al., 1998; Zheng et al., 1999). Consequently, it is not surprising that an intrastriatal injection of gp120IIIB evokes apoptosis only in CXCR4 positive neurons (Bachis et al., 2006; Nosheny et al., 2006). We have previously shown that gp120BaL or HIV-1BaL reduces the survival of neuronal cultures in a CCR5 dependent manner (Bachis & Mocchetti, 2005; Bachis et al., 2009). Thus, we were expecting a similar result in vivo. Instead, the toxic effect of gp120BaL was quantitatively weaker than that of gp120IIIB previously observed (Bachis et al., 2006; Nosheny et al., 2007). Both CXCR4 and CCR5 are expressed in the adult rat striatum (Banisadr et al., 2002; Bachis et al., 2006; Ahmed et al., 2008; Trecki et al., 2009). However, previous data have shown that CXCR4 expression in various brain areas is wider than that of CCR5 (van der Meer et al., 2000). This difference could account for the widespread neurotoxicity of gp120IIIB but, it is unlikely that the abundance of CCR5 in the striatum can solely account for gp120BaL weaker effect. On the other hand, CCR5 is also expressed by astrocytes and microglia (Rottman et al., 1997; van der Meer et al., 2000). The latter, when activated, can cause an inflammatory response that ultimately leads to neuronal injury. Such mechanism could account for gp120BaL neurotoxicity. We have observed that gp120BaL, unlike gp120IIIB (Bachis et al., 2006), promoted proliferation of microglial cells. Activated microglia is known to induce the release of pro-inflammatory molecules such as cytokines (Floyd, 1999; Raivich et al., 1999), and nitric oxide (Perry et al., 1993; Ogura et al., 1994), potent stimuli that trigger neuronal cell death. We have seen an increase in IL-1β in gp120BaL-treated rats. Levels of IL-1 β are elevated in the cerebrospinal fluid of AIDS patients (Gallo et al., 1989) and in postmortem brains (Tyor et al., 1992). IL-1 β and other cytokines can act on a variety of neuronal populations independently from the presence of chemokine receptors. Collectively, these results suggest that gp120BaL may activate CCR5 on non-neuronal cells that may have been recruited to inflammatory sites, which, in turn, induce the synthesis of pro-inflammatory cytokines. Overall, while we cannot underscore enough the role of CCR5 in gp120BaL-mediated neuronal apoptosis, our data point at a multi-factorial effect of gp120BaL which may include the ability of this strain to increase microglia activation/proliferation.

Intrastriatal gp120IIIB has been shown to cause a decrease in the levels of BDNF in the striatum (Nosheny et al., 2004) and GDNF in the SN (Nosheny et al., 2006). Other studies have shown that gp120IIIB increases NGF in the hippocampus (Bagetta et al., 1996). While NGF has little or no trophic activity on central dopamine neurons (Hagg, 1998), BDNF and GDNF are two of the most potent neurotrophic factors for the survival of dopaminergic neurons of the nigro-striatal system (Hyman et al., 1991; Altar et al., 1992; Beck et al., 1995; Choi-Lundberg et al., 1997). Indeed, dysregulation of BDNF and GDNF synthesis has been proposed to promote neurodegenerative diseases such as Parkinson’s disease (Nagatsu et al., 2000). Moreover, BDNF heterozygous mice are more sensitive to the neurotoxic action of gp120IIIB (Nosheny et al., 2004). A number of HIV infected individuals exhibits Parkinsonian-like motor abnormalities (Berger & Arendt, 2000; Koutsilieri et al., 2002; Nath & Berger, 2004) which are consistent with a nigro-striatal dysfunction. Thus, the dopaminergic loss observed in HAD may be caused by a lack of neurotrophic support. We have determined the overall neurotrophic factor environment after gp120BaL in an attempt to characterize its similarities with gp120IIIB. Our data show that gp120BaL increases BDNF and does not change GDNF levels in vivo whereas gp120IIIB decreases both (Nosheny et al., 2004; Nosheny et al., 2006). This diverse profile was also observed in vitro. In fact, gp120BaL increased whereas gp120IIIB decreased BDNF release. This and other evidence suggest that gp120BaL and gp120IIIB exhibit a different neuropathological profile.

An attractive albeit speculative suggestion that emerges from our studies is that gp120BaL may promote, in addition to a neurotoxic insult, a neuroprotective response via the increase in BDNF. The mechanism(s) whereby BDNF participates in a neuroprotective response evoked by gp120BaL is still a matter of speculation. BDNF is a strong neuroprotective agent against various toxins that are believed to be released by gp120. These include nitric oxide (Adamson et al., 1996; Bagetta et al., 1996; Kume et al., 1997) and glutamate (Lindholm et al., 1993; Marini et al., 1998; Bezzi et al., 2001). In addition, HIV/gp120-mediated apoptosis has been linked to the expression and trafficking of N-methyl-D-aspartate receptors (Eugenin et al., 2003; Viviani et al., 2006). BDNF has also been shown to down-regulate this receptor (Brandoli et al., 1998). Thus, BDNF may modulate negatively toxic responses that are activated by gp120s. Because these mechanisms may work synergistically, modulating one may lead to reduced toxicity. More studies are needed to characterize which of these mechanisms are most likely crucial for the limited (when compared to gp120IIIB) toxic effect of gp120BaL.

Collectively, our data suggest a speculative hypothesis that M-tropic gp120 has a weaker toxic property than T-tropic. M-tropic and CCR5-preferring HIV has been shown to be present in the early phase of infection whereas CXCR4-preferring strains occur in the late stage when a subset of individuals develops HAD. Moreover, dual-tropic viruses can use both co-receptors and appear to be specialized in replicating within the CNS (Gray et al., 2009). Our results might help explain the suggestion that M-tropic strains of HIV that infect the CNS are not sufficient to cause dementia or encephalitis (Power et al., 1994). Additional experiments with other M-tropic gp120s and/or M-tropic HIV are needed to fully support this hypothesis.

Acknowledgments

Special thanks to NIH AIDS Research Reference Reagent Program for gp120BaL and Diane M Leader for critical reading of the manuscript. This work was supported by NIH grants NS040670, NS059323 and DA026174.

Abbreviations

BDNF

Brain-derived neurotrophic factor

CX

somatosensory cortex

ELISA

enzyme-linked immunosorbent assay

GDNF

glial-cell derived neurotrophic factor

GFAP

glia fibrillary acidic protein

HAD

HIV-associated dementia

HIV

human immunodeficiency virus type 1

IL-1β

interleukin-1β

NGF

nerve growth factor

SN

substantia nigra

TH

tyrosine hydroxylase

TUNEL

in situ terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling

VEH

vehicle control

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