Abstract

Human immunodeficiency virus-1 (HIV-1)-encoded transactivator of transcription (Tat) potentiated the depolarization-evoked exocytosis of [3H]D-aspartate ([3H]D-ASP) from human neocortical terminals. The metabotropic glutamate (mGlu) 1 receptor antagonist 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) prevented this effect, whereas the mGlu5 receptor antagonist 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP) was ineffective. Western blot analysis showed that human neocortex synaptosomes possess mGlu1 and mGlu5 receptors. Tat potentiated the K+-evoked release of [3H]D-ASP or of endogenous glutamate from mouse neocortical synaptosomes in a CPCCOEt-sensitive and MPEP-insensitive manner. Deletion of mGlu1 receptors (crv4/crv4 mice) or mGlu5 receptors (mGlu5−/−mouse) silenced Tat effects. Tat enhanced inositol 1,4,5-trisphosphate production in human and mouse neocortical synaptosomes, consistent with the involvement of group I mGlu receptors. Tat inhibited the K+-evoked release of [3H]γ-aminobutyric acid ([3H]GABA) from human synaptosomes and that of endogenous GABA or [3H]GABA from mouse nerve terminals; the inhibition was insensitive to CPCCOEt or MPEP. Tat-induced effects were retained by Tat37–72 but not by Tat48–85. In mouse neocortical slices, Tat facilitated the K+- and the veratridine-induced release of [3H]D-ASP in a CPCCOEt-sensitive manner and was ineffective in crv4/crv4 mouse slices. These observations are relevant to the comprehension of the pathophysiological effects of Tat in central nervous system and may suggest new potential therapeutic approaches to the cure of HIV-1–associated dementia.

Introduction

Central nervous system (CNS) disorders often accompany the acquired immunodeficiency syndrome (AIDS) and are typified by neuropsychiatric symptoms, such as cognitive and motor impairments (Lanska 1999; McArthur et al. 2004; Lipton and Gendelman 2005; Tattevin et al. 2006), sometimes paralleled by neuropathological hallmarks. Collectively, these events are referred to as human immunodeficiency virus-1 (HIV-1)–associated dementia (HAD). Before the advent of the highly active antiretroviral therapy (HAART), about 20% of adult patients but as many as 40% of children/adolescent infected subjects developed HAD. Nowadays, in the era of HAART, the prevalence of HAD has decreased, but a more subtle form of disorder, referred to as minor cognitive–motor disorder (MCMD), has emerged in about 20% of symptomatic HIV-1–seropositive patients, including those receiving HAART (McArthur et al. 2004; Selnes 2005; Nath and Sacktor 2006).

The mechanisms underlying the AIDS-related neuropsychiatric symptoms are poorly understood, but the possibility exists that, besides evident neurotoxic damages, subtle modulations of neurotransmission are caused by proteins shed by the virus or actively released by HIV-1–infected cells in the CNS (Ensoli et al. 1993; Kaul et al. 2005), as well as by endogenous factors whose production is triggered by the viral infection (Nath and Geiger 1998; Pocernich et al. 2005). Among the many candidates, the viral protein transactivator of transcription (Tat) has attracted much interest. In the CNS, this protein is secreted by infected cells, including microglia, astrocytes, and macrophages (King et al. 2006). Tat mRNA levels are elevated in the serum and in the brain of patients with HIV-1 dementia (Wessenligh et al. 1993; Westendorp et al. 1995) or HIV encephalitis (Wiley et al. 1996). Furthermore, when introduced into the CNS, exogenous Tat was reported to produce pathological effects similar to those observed in HAD patients (Kim et al. 2003; Maragos et al. 2003).

Tat can excite neurons (Sabatier et al. 1991; Nath and Geiger 1998) and mobilize Ca2+ from intracellular stores (Haughey et al. 2001; Feligioni et al. 2003), compatible with a potential role of neurotransmitter release modulator. Accordingly, the protein was shown to enhance the basal release of acetylcholine from human and rodent neocortical cholinergic terminals (Feligioni et al. 2003). This Tat-mediated release appeared restricted to the cholinergic terminals because the basal release of other neurotransmitters, including glutamate and γ-aminobutyric acid (GABA), was not modified by the protein (Feligioni et al. 2003). We have now reconsidered this topic by investigating whether Tat could affect the exocytosis of glutamate and GABA evoked by depolarizing stimuli from human and mouse neocortical nerve endings. Tat facilitated glutamate exocytosis by acting as an extremely potent agonist at metabotropic glutamate (mGlu) 1 autoreceptors located on human and mouse neocortical glutamatergic nerve endings. Although mGlu5 autoreceptors seem not directly involved, their presence seems to be required for the expression of functional mGlu1 autoreceptors. Surprisingly, Tat potently inhibited GABA exocytosis by acting at hitherto unknown binding sites on GABAergic nerve endings. The unbalance between glutamate and GABA transmission may play a role in the neuropsychiatric disorders characteristic of HAD and MCMD.

Materials and Methods

Human Brain Tissue Samples

Samples of human cerebral cortex were obtained from informed and consenting HIV-1–negative patients undergoing neurosurgery to reach deeply seated tumors. The samples represented parts of frontal, parietal, and temporal lobes obtained from 10 women and 21 men (ages 24–70 years). Patients had been treated with levetiracetam (1 g every 12 h) starting from 24 h before neurosurgery. After a premedication with midazolam (2 mg/kg), anesthesia was induced with propofol (2 mg/kg), cisatracurium (10–14 mg), and fentanyl (0.1 mg) and maintained with propofol (5 μg/kg/h) and remifentanyl (0.005–0.008 μg/min) during neurosurgery. Immediately after removal, the tissue was placed in a physiological salt solution at 2–4 °C, and purified synaptosomes were prepared within 30–40 min. The experimental procedure was approved by the Ethical Committee of the University of Genova.

Animals

Adult male mice (Swiss; 20–25 g) were used in all the experiments except those carried out with mGlu1 receptor lacking cervelet-4 (crv4) mice and mGlu5−/− receptor knockout mice. The crv4 mutation is a spontaneous recessive mutation occurred in the BALB/c/Pas inbred strain. It consists in an LTR intronic insertion that disrupts splicing of mGlu1 receptor gene and causes absence of the protein (Conti et al. 2006). Crv4 homozygous mice present mainly with an ataxic phenotype. Affected (crv4/crv4) and control (+/+) mice are maintained on the same genetic background by intercrossing +/crv4 mice at the animal facility of the National Institute of Cancer Research (Genova, Italy). DNA was extracted from wild-type and crv4 mice tails and was amplified via polymerase chain reaction (PCR) using primers (5′-GAGTGTTCACTAGTTCACCCAAGA-3′ and 5′-TCAGGCAACAATAAGGCAAG-3′) that flank the insertion; the PCR products amplified with these primers were 688 bp for the crv4 mutant and 498 bp for the wild type (see also Musante, Neri, et al. 2008). Heterozygous mGlu5 receptor knockout mice (129-Gprc1etmt1rod) were obtained from The Jackson Laboratory (Bar Harbor, ME). Mice heterozygous for the targeted mutation were intercrossed to homozygosity at the “Istituto Neurologico Mediterraneo Neuromed” (Pozzilli, IS, Italy). Homozygous females and males were fertile but poor breeders. Thus, all mice were generated by heterozygous breeding. Primers for the genotyping of knockout mice were designed at the Jackson Laboratory. Mice were identified by PCR analysis of tail samples after birth. Transgenic mice were then sent to the animal facility of our laboratory (Department of Experimental Medicine, Pharmacology and Toxicology Section, University of Genoa). Mice were kept under environmentally controlled conditions (ambient temperature = 22 ± 1 °C, humidity = 50%) on a 12-h light/dark cycle with food and water ad libitum.

The animals were killed by decapitation, brain cortices were rapidly removed, and purified synaptosomes or slices were prepared within minutes. The experimental procedures were approved by the Department Ethical Committee, in accordance with the European legislation (European Communities Council directive of November 24, 1986, 86/609/EEC). Experiments were performed following the Guide for the Care and the Use of laboratory animals of the National Institutes of Health (NIH publication No. 86-23, revised 1987). Adequate measure was taken to minimize pain or discomfort.

Preparation of Synaptosomes

Purified synaptosomes were prepared essentially according to Dunkley et al. (1986), with minor modifications. The tissue was homogenized in 10 volumes of 0.32 M sucrose, buffered to pH 7.4 with Tris (final concentration 0.01 M) using a glass Teflon tissue grinder (clearance 0.25 mm). The homogenate was centrifuged at 1000 × g for 5 min to remove nuclei and debris, and the supernatant was gently stratified on a discontinuous Percoll gradient (6%, 10%, and 20% v/v in Tris-buffered sucrose) and centrifuged at 33 500 × g for 5 min. The layer between 10% and 20% Percoll (synaptosomal fraction) was collected and washed by centrifugation. The synaptosomal pellets were always resuspended in a physiological medium having the following composition (mM): NaCl, 125; KCl, 3; MgSO4, 1.2; CaCl2, 1.2; NaH2PO4, 1; NaHCO3, 22; glucose, 10 (aeration with 95% O2 and 5% CO2); pH 7.2–7.4. In GABA release experiments, the medium contained aminooxyacetic acid, 50 μM, to avoid GABA metabolism. Synaptosomal protein contents were determined according to Bradford (1976).

Preparation of Slices

Slices (0.4 mm thick) were prepared using a Mcllwain tissue chopper (Mickle Laboratory Engineering Co. Ltd, Surrey, UK) and then placed in a physiological salt solution (see above) at 2–4 °C where rinsed 3 times by changing the physiological solution every 20 min.

Experiments of Release from Superfused Synaptosomes

Synaptosomes were labeled in the absence (experiments of release of endogenous glutamate or GABA) or in the presence of [3H]D-aspartate ([3H]D-ASP; final concentration 30–60 nM) or [3H]γ-aminobutyric acid ([3H]GABA; final concentration 50 nM) at 37 °C, for 15 min, in a rotary water bath and in an atmosphere of 95% O2 and 5% CO2. After the labeling period, identical portions of the synaptosomal suspensions were layered on microporous filters at the bottom of parallel superfusion chambers (Ugo Basile, Comerio, Varese, Italy; Raiteri and Raiteri 2000) thermostated at 37 °C and superfused at 0.5 ml/min with standard physiological solution aerated with 95% O2 and 5% CO2. The amount of human neocortical synaptosomal protein in each superfusion chamber amounted to 0.110 ± 0.023 mg protein/chamber. In the experiments aimed at studying [3H] neurotransmitter release from mouse neocortical synaptosomes, the synaptosomal proteins amounted to 0.070 ± 0.014 mg protein/chamber (release of tritiated neurotransmitter); in the experiments aimed at investigating the release of endogenous neurotransmitter, the synaptosomal protein content amounted to 0.23 ± 0.074 mg protein/chamber (release of endogenous neurotransmitter).

When studying the effects of Tat- and of mGlu1/5-selective ligands on the basal release of neurotransmitters, synaptosomes were first equilibrated during 36 min of superfusion and then 4 consecutive 3-min fractions (termed b1–b4) were collected. Compounds were introduced at the end of the first fraction collected (b1; t = 39 min) and maintained until the end of the superfusion. When studying the effect of Tat and of (RS)-3,5-dihydroxyphenylglycine (3,5-DHPG) on neurotransmitter overflows evoked by high K+, synaptosomes were transiently (90 s) exposed at t = 39 min to a solution containing 12 mM KCl (NaCl substituting for an equimolar concentration of KCl). Tat or 3,5-DHPG was added concomitantly with the depolarizing stimulus. Antagonists were added 8 min before agonists. In these experiments, fractions were collected according to the following scheme: two 3-min fractions (basal release), one before (t = 36–39 min, b1) and one after (t = 45–48 min, b3), and a 6-min sample (t = 39–45 min; evoked release, b2) containing the transmitter released by the depolarizing stimulus. Superfusion was always performed with medium enriched with dialyzed 0.1% of Polypep to avoid protein sticking to glass walls and tubings. Collected fractions and superfused synaptosomes were counted for radioactivity or analyzed for the endogenous neurotransmitter content.

Experiments of Release from Superfused Slices

Slices were labeled with [3H]D-ASP (final concentration 90 nM) or with [3H]GABA (final concentration 60 nM) at 37 °C for 15 min in a rotary water bath and in an atmosphere of 95% O2 and 5% CO2. Slices were transferred to parallel superfusion chambers (one slice per chamber) and superfused (1 ml/min at 37 °C) with standard medium. After 60 min of superfusion to equilibrate the system, six 5-min samples were collected. Slices were exposed to high K+ or veratridine (concentrations as indicated) for 3 min starting at t = 71 min to t = 74 min of superfusion and then replaced with superfusion medium. Tat was applied contemporary to the depolarizing stimulus, 7-(hydroxyimino)cyclopropa[b]chromen-1a-carboxylate ethyl ester (CPCCOEt) 30 min before Tat. Superfusion was performed with medium enriched with dialyzed 0.1% of Polypep to avoid sticking to glass walls and tubings. Samples collected and solubilized slices (Soluene; Canberra Packard, Milan, Italy) were counted for radioactivity.

Endogenous Amino Acid Determination

Endogenous glutamate and GABA were measured by HPLC analysis after precolumn derivatization with o-phthalaldehyde and separation on a C18 reverse-phase chromatographic column (10 × 4.6 mm, 3 μm; at 30 °C; Chrompack, Middleburg, The Netherlands) coupled with fluorimetric detection (excitation wavelength, 350 nm; emission wavelength, 450 nm). Buffers and gradient program are described elsewhere (see Luccini et al. 2007). Homoserine was used as internal standard.

Endogenous Inositol 1,4,5-Trisphosphate Determination

Human (0.165 ± 0.021 mg protein/sample) or mouse (0.095 ± 0.023 mg protein/sample) neocortical synaptosomes were first preincubated in physiological medium for 5 min at 37 °C to equilibrate the system. Synaptosomes were then incubated for 6 min in the absence of depolarizing stimulus (control) as well as in the presence of K+ (final concentration 12 mM), Tat (final concentration 1 nM), 3,5DHPG (final concentration 50 μM), and CPCCOEt (final concentration 5 μM) as indicated. Synaptosomes were finally lysed with iced cold water, the suspension was centrifuged to remove membranes, and the endogenous inositol 1,4,5-trisphosphate (IP3) content in the supernatant was determined by a commercially available radioimmunoassay kit (Amersham Radiochemical Center, Buckinghamshire, UK). Synaptosomal protein contents were determined according to Bradford (1976).

Isolation of Detergent-Soluble Fractions from Synaptosomes

Purified synaptosomes were prepared as previously described (Musante, Longordo, et al. 2008) and collected by centrifugation at 14 000 × g for 15 min at 4 °C. The pellet was diluted and lysed in 1 ml of ice-cold 20 mM Tris/HCl (pH 7.4, containing 10 mM NaCl and protease inhibitor cocktail). In order to isolate the detergent-soluble membrane fraction (DS-Syn), after 15 min at 4 °C, 450 μg of synaptosome lysate (Tot-Syn) were centrifuged at 200 000 × g, 4 °C for 15 min, and the pellet was solubilized in 800 μl of 20 mM Tris/HCl (pH 7.4, containing 140 mM NaCl, 0.2% Triton X-100, and protease inhibitor cocktail). After 15 min at 4 °C, samples were centrifuged at 200 000 × g, 4 °C for 15 min, and the supernatant, the DS-Syn, was collected. Proteins were quantified using the Pierce BCA Protein Assay Kit (Pierce Biochemical, Rockford, IL).

Immunoprecipitation

DS-Syn (100 μg of proteins) was diluted in 0.5 ml of Triton X buffer having the following composition: 20 mM Tris/HCl, 140 mM NaCl, protease inhibitors, 0.5% (v/v) Triton X-100, pH 7.4, and precleaned by incubating the suspension with 20 μl of protein A–agarose beads (Santa Cruz Biotechnology, Santa Cruz, CA) at 4 °C for 1 h. Bead suspension was centrifuged and the supernatant incubated with 6 μg of the specific antibody (anti-mGlu1 or mGlu5 receptor antibody) at 4 °C overnight. The supernatant was added with 30 μl of protein A–agarose beads, incubated for 1 h at 4 °C, and the bead suspension was centrifuged. The pellet was subjected to 5 × 0.2 ml washes with the Triton X buffer, followed by 5 × 0.2 ml washes with the same buffer from where Triton X-100 was removed. The adsorbed proteins were resuspended in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loading sample buffer and eluted by heating the suspension for 5 min at 95 °C.

Immunoblotting

Proteins (25 μg/lane) were separated by SDS-PAGE 7.5% polyacrylamide and then transferred onto polyvinylidene difluoride membranes. Membranes were blocked in 20 mM sodium phosphate buffer (PBS, pH 7.4, containing 140 mM NaCl, 5% nonfat dry milk, 0.1% Tween-20) and probed with one of the following primary antibodies (60 min at 20 °C): rabbit anti-mGlu1 receptor (1:500), rabbit anti-mGlu5 receptor (1:1000), mouse anti-syntaxin-1A (anti Stx-1A, 1:5000), and mouse anti-glial fibrillary acidic protein (anti-GFAP, 1:1000) as appropriate. After extensive washes, membranes were incubated for 1 h at 20 °C with the appropriate horseradish peroxidase–linked secondary antibody (1:4000), and immunoblots were visualized with an enhanced chemiluminescence western blotting detection system.

Calculations

The amount of endogenous amino acid from neocortical synaptosomes in each superfusate fractions was expressed as picomoles per milligram per protein (pmol/mg/protein). The effects of Tat on the basal release of endogenous transmitters were evaluated by calculating the ratio between the amino acid content in the fraction where the maximal effect was reached (the third fraction collected) and the amount in the first fraction and comparing this ratio with the corresponding ratio obtained under control conditions (no drug added). The amount of radioactivity released into each superfusate fraction was expressed as % of the total synaptosomal tritium content at the start of the fraction collected (fractional efflux). Drug-induced changes to the spontaneous release of tritiated neurotransmitters from neocortical synaptosomes were expressed as % increase over basal release and were evaluated as the ratio between the percentage of tritium released into the third fraction and that in the first fraction collected. This ratio was compared with the corresponding ratio obtained under control conditions (no drug added).

The K+-induced overflow of endogenous neurotransmitters from neocortical synaptosomes was expressed as pmol/mg protein, whereas the induced tritium overflow was expressed as % of total radioactivity. In both cases, the induced overflow was estimated by subtracting the neurotransmitter content into the first and the third fractions collected (basal release, b1 and b3) from that in the 6-min fraction collected during and after the depolarization pulse (evoked release, b2). When the time course of drug-induced modification to K+-evoked endogenous neurotransmitter release is described, endogenous neurotransmitter released in each superfusate fractions was expressed as pmol/mg protein. When the time course of drug-induced modification to K+-evoked tritiated neurotransmitter release is described, the amount of radioactivity in each superfusate fraction collected is expressed as % of the total synaptosomal tritium content at the start of the fraction collected (fractional efflux).

The tritium overflow evoked by the depolarizing stimuli from slices was expressed as induced overflow and was calculated by subtracting the neurotransmitter content into the second and the fifth fractions collected (basal release) from that in the third and fourth fractions collected (evoked release). Endogenous IP3 content in synaptosomal supernatant was expressed as pmol/mg protein. Analysis of variance was performed followed by Newman Keuls or Dunnett's multiple comparison test, as appropriate; direct comparisons were performed by applying Student's t-test. Data were considered significant for P < 0.05. Appropriate controls with antagonists were always run in parallel.

Drugs

1-[7,8 3H]D-aspartate ([3H]D-ASP specific activity 16.3 Ci/mmol) and 4-amino-n-[2,3-3H]butyric acid ([3H]GABA, specific activity 87 Ci/mmol) were purchased from Amersham Radiochemical Center. Veratridine, o-(carboximethyl)hydroxylamine hemihydrochloride, anti-GFAP monoclonal mouse purified immunoglobulin G (IgG) and Protease Inhibitor Cocktail were purchased from Sigma Aldrich Inc. (St Louis, MO). (RS)-3,5-d3,5-DHPG, 2-methyl-6-(phenylethynyl) pyridine hydrochloride (MPEP), and CPCCOEt were obtained from Tocris Cookson (Bristol, UK). Anti-mGlu5 receptor polyclonal rabbit immunoaffinity-purified IgG and anti-mGlu1 receptor polyclonal rabbit immunoaffinity IgG were purchased from Upstate Biotechnology (Lake Placid, NY). Anti-syntaxin-1A monoclonal mouse IgG was obtained from Synaptic System (Germany). Horseradish peroxidase–conjugated anti-mouse and anti-rabbit secondary antibodies and the ECL Plus western blotting detection system were purchased from GE Healthcare. Recombinant Tat corresponded to the full-length sequence of the protein (aa1-86); Tat was kindly gifted by Centre for AIDS Reagents (supported by EU Programme EVA/MRC, contract QLK2-CT-1999-00609) and by the UK Medical Research Council. Tat peptide 37–72 and Tat peptide 48–85 were kindly donated by “AIDS Reagent Project” (ARP 7004.2 and 7005) and by European program EVA (779.1-779-8) (NIBSC, Hertfordshire, UK). The amino acid sequence of the Tat peptides perfectly overlaps the corresponding sequences of the full-length protein.

Results

Effects of Tat on the Release of [3H]D-ASP and [3H]GABA Evoked by KCl from Human Neocortical Synaptosomes

Purified human neocortical synaptosomes were prelabeled with [3H]D-ASP (an unmetabolizable glutamate analogue routinely used in release studies as a marker of the endogenous excitatory amino acid transmitter) or with [3H]GABA, and the release of the 2 neurotransmitters was monitored during superfusion. Exposure to 12 mM K+ is known to elicit external Ca2+-dependent, exocytotic release of [3H]D-ASP and [3H]GABA (reviewed by Raiteri 2006).

Addition of Tat concomitantly with the depolarizing stimulus potentiated the K+-evoked overflow of [3H]D-ASP (Fig. 1A) but inhibited the overflow of [3H]GABA (Fig. 1B). Tat failed to affect the poststimulation neurotransmitter baseline (Fig. 1C,D). The protein was maximally effective when added at 1–3 nM. The basal release of [3H]D-ASP or [3H]GABA had been shown to be unaffected by nanomolar concentrations of the viral protein (Feligioni et al. 2003). The neurotoxic fragment Tat37–72 (1 nM) mimicked the entire protein in facilitating glutamate exocytosis and in inhibiting K+-evoked GABA release; on the contrary, Tat48–85 (1 nM), a fragment peptide devoid of neurotoxicity (Sabatier et al. 1991; Nath et al. 1996), failed to affect glutamate and GABA exocytosis (Table 1).

Table 1

Effects of Tat37–72 and Tat48–85 on [3H]D-ASP and [3H]GABA exocytosis from human and mouse neocortical synaptosomes

 Human
 
Mouse
 
 [3H]D-ASP release—% induced overflow [3H]GABA release—% induced overflow [3H]D-ASP release—% induced overflow [3H]GABA release—% induced overflow 
Control 1.23 ± 0.09 2.57 ± 0.22 1.20 ± 0.13 6.96 ± 0.46 
1 nM Tat37–72 1.73 ± .16* 1.56 ± 0.18* 2.16 ± 0.23* 4.24 ± 0.31* 
1 nM Tat48–86 1.31 ± 0.12 2.85 ± 0.18 1.29 ± 0.10 7.11 ± 0.56 
 Human
 
Mouse
 
 [3H]D-ASP release—% induced overflow [3H]GABA release—% induced overflow [3H]D-ASP release—% induced overflow [3H]GABA release—% induced overflow 
Control 1.23 ± 0.09 2.57 ± 0.22 1.20 ± 0.13 6.96 ± 0.46 
1 nM Tat37–72 1.73 ± .16* 1.56 ± 0.18* 2.16 ± 0.23* 4.24 ± 0.31* 
1 nM Tat48–86 1.31 ± 0.12 2.85 ± 0.18 1.29 ± 0.10 7.11 ± 0.56 

Note: Effects of Tat peptides on the 12 mM K+-induced modulation of [3H]D-ASP and [3H]GABA exocytosis from human and mouse neocortical synaptosomes. Results are expressed as induced overflow. Data are mean ± standard error of the mean of 3 experiments run in triplicate.

*P < 0.05 at least versus respective control.

Figure 1.

Effects of Tat on [3H]D-ASP (A) and [3H]GABA (B) exocytosis from human neocortical synaptosomes. Gray bars: 12 mM K+-evoked neurotransmitter overflow in the absence of Tat; crosshatched gray bars: 12 mM K+-evoked neurotransmitter overflow in the presence of Tat (concentration as indicated). Results are expressed as induced overflow. The neurotransmitter released in the first 3-min fraction (b1) collected (see Material and Methods) amounted to: [3H]D-ASP = 0.65 ± 0.05%; [3H]GABA = 3.15 ± 0.28%. The induced neurotransmitter overflow in the absence of Tat and K+ amounted to: [3H]D-ASP release, 0.08 ± 0.05% induced overflow; [3H]GABA release, −0.013 ± 0.21% induced overflow. Time course relationship of the 12 mM K+-evoked release of [3H]D-ASP (C) and [3H]GABA (D) in the absence (gray circles) and in the presence (black circles) of 1 nM Tat. Results are expressed as % of total tritium content. When not shown, error bars are within graph symbols. Data are means ± standard error of the mean of 5 (A, C) and 4 (B, D) experiments run in triplicate (three superfusion chambers for each experimental condition). *P < 0.05 at least versus 12 mM K+-evoked tritiated neurotransmitter overflow; #P < 0.05 at least versus respective 12 mM K+-evoked neurotransmitter release (expressed as % of total tritium content).

Figure 1.

Effects of Tat on [3H]D-ASP (A) and [3H]GABA (B) exocytosis from human neocortical synaptosomes. Gray bars: 12 mM K+-evoked neurotransmitter overflow in the absence of Tat; crosshatched gray bars: 12 mM K+-evoked neurotransmitter overflow in the presence of Tat (concentration as indicated). Results are expressed as induced overflow. The neurotransmitter released in the first 3-min fraction (b1) collected (see Material and Methods) amounted to: [3H]D-ASP = 0.65 ± 0.05%; [3H]GABA = 3.15 ± 0.28%. The induced neurotransmitter overflow in the absence of Tat and K+ amounted to: [3H]D-ASP release, 0.08 ± 0.05% induced overflow; [3H]GABA release, −0.013 ± 0.21% induced overflow. Time course relationship of the 12 mM K+-evoked release of [3H]D-ASP (C) and [3H]GABA (D) in the absence (gray circles) and in the presence (black circles) of 1 nM Tat. Results are expressed as % of total tritium content. When not shown, error bars are within graph symbols. Data are means ± standard error of the mean of 5 (A, C) and 4 (B, D) experiments run in triplicate (three superfusion chambers for each experimental condition). *P < 0.05 at least versus 12 mM K+-evoked tritiated neurotransmitter overflow; #P < 0.05 at least versus respective 12 mM K+-evoked neurotransmitter release (expressed as % of total tritium content).

The Tat-induced effects can be attributed to direct action of the protein on glutamatergic and GABAergic nerve terminals because the characteristics of the technique used to monitor release (up–down superfusion of monolayers of purified synaptosomes) prevent or minimize indirect effects (see Raiteri and Raiteri 2000).

The Tat Facilitation of [3H]D-ASP Release from Human Neocortex Nerve Endings Is Sensitive to an mGlu1 Receptor Antagonist

Previous results had shown that group I mGlu receptors mediated the effects of Tat on the basal release of acetylcholine and noradrenaline in human brain (Feligioni et al. 2003; Longordo et al. 2006). We therefore investigated whether group I mGlu receptors could also mediate the Tat effects here observed. CPCCOEt, a selective mGlu1 receptor antagonist, and MPEP, a selective mGlu5 receptor antagonist, were tested against the effects of Tat on the release of glutamate and GABA evoked by 12 mM K+ from human neocortical synaptosomes. CPCCOEt and MPEP were added at concentrations (5 and 1 μM, respectively) previously shown to prevent Tat effects under our experimental conditions (Feligioni et al. 2003; Longordo et al. 2006). CPCCOEt totally prevented the potentiation of the K+-evoked [3H]D-ASP overflow caused by 1 nM Tat (Fig. 2A). In contrast, the effect of Tat was insensitive to MPEP. The K+-evoked release of [3H]D-ASP was not significantly modified when 5 μM CPCCOEt or 1 μM MPEP was added alone. The antagonists failed to affect on their own the spontaneous (Table 2) or the 12 mM K+-evoked (Fig. 2A) release of [3H]D-ASP.

Table 2

Effects of the group I mGlu receptor agonist 3,5DHPG and of the antagonists CPCCOEt and MPEP on the spontaneous release of [3H]D-ASP and of [3H]GABA from human and mouse neocortical synaptosomes

 Human
 
Mouse
 
 [3H]D-ASP release—% increase [3H]GABA release—% increase [3H]D-ASP release—% increase [3H]GABA release—% increase 
50 μM 3,5DHPG 0.69 ± 0.86 6.52 ± 4.13 3.55 ± 2.67 4.96 ± 3.55 
5 μM CPCCOEt 0.91 ± .36 0.54 ± 4.47 4.98 ± 6.64 1.09 ± 0.85 
1 μM MPEP 1.31 ± 0.95 8.55 ± 4.66 5.31 ± 2.78 3.45 ± 1.17 
 Human
 
Mouse
 
 [3H]D-ASP release—% increase [3H]GABA release—% increase [3H]D-ASP release—% increase [3H]GABA release—% increase 
50 μM 3,5DHPG 0.69 ± 0.86 6.52 ± 4.13 3.55 ± 2.67 4.96 ± 3.55 
5 μM CPCCOEt 0.91 ± .36 0.54 ± 4.47 4.98 ± 6.64 1.09 ± 0.85 
1 μM MPEP 1.31 ± 0.95 8.55 ± 4.66 5.31 ± 2.78 3.45 ± 1.17 

Note: Synaptosomes were superfused with standard medium (control) and then exposed to 3,5DHPG, CPCCOEt, or MPEP until the end of the superfusion medium. Results are expressed as the percentage of increase over basal release. Data are mean ± standard error of the mean of 3 experiments run in triplicate.

Figure 2.

Effects of group I mGlu receptor antagonists on the Tat-induced modulation of [3H]D-ASP and [3H]GABA exocytosis from human neocortical synaptosomes. (A) Effects of CPCCOEt and MPEP on the release of [3H]D-ASP evoked by high K+ in absence or in presence of 1 nM Tat . (B) Effects of CPCCOEt and MPEP on the release of [3H]GABA evoked by K+ in absence or in presence of 1 nM Tat. Results are expressed as induced overflow. Data are means ± standard error of the mean of 4 (A) and 3 (B) experiments run in triplicate. *P < 0.05 at least versus respective control.

Figure 2.

Effects of group I mGlu receptor antagonists on the Tat-induced modulation of [3H]D-ASP and [3H]GABA exocytosis from human neocortical synaptosomes. (A) Effects of CPCCOEt and MPEP on the release of [3H]D-ASP evoked by high K+ in absence or in presence of 1 nM Tat . (B) Effects of CPCCOEt and MPEP on the release of [3H]GABA evoked by K+ in absence or in presence of 1 nM Tat. Results are expressed as induced overflow. Data are means ± standard error of the mean of 4 (A) and 3 (B) experiments run in triplicate. *P < 0.05 at least versus respective control.

This result was expected considering that, in our superfusion system, the compounds released are immediately removed by the up–down superfusing solution before they can feedback on presynaptic targets (see Raiteri and Raiteri 2000). CPCCOEt (5 μM) and MPEP (1 μM) did not modify the inhibition of the K+-evoked [3H]GABA release caused by 1 nM Tat (Fig. 2B). The two antagonists, added alone, had not effect on the K+-evoked release of [3H]GABA (Fig. 2B), nor they modified the spontaneous release of [3H]GABA (Table 2).

Glutamatergic but not GABAergic Human Neocortical Nerve Endings Are Endowed with Functional mGlu1 Receptors

The results so far described were suggestive of the existence of mGlu1 presynaptic autoreceptors located on human neocortical glutamatergic terminals. To verify this hypothesis, we investigated the effects of 3,5-DHPG, a wide spectrum agonist at group I mGlu receptors, on the release of [3H]D-ASP from human neocortical terminals. 3,5DHPG did not affect the spontaneous release of tritium (Table 2) but, when added at 50 μM, significantly potentiated the [3H]D-ASP overflow (Fig. 3B,D). The mGlu1/5 receptor agonist left unmodified the poststimulation neurotransmitter baseline (Fig. 3C). The pharmacological characterization of the mGlu receptors involved was then carried out using CPCCOEt and MPEP. The effect of 3,5-DHPG was halved by 1 μM MPEP but totally prevented by CPCCOEt (Fig. 3A). The antagonists failed to affect on their own the spontaneous (Table 2) or the 12 mM K+-evoked (Fig. 3A) release of [3H]D-ASP.

Figure 3.

Effects of 3,5-DHPG on [3H]D-ASP and [3H]GABA-evoked exocytosis from human neocortical synaptosomes: antagonism by CPCCOEt or MPEP and western blot analysis of mGlu1/5 receptor proteins. (A) K+ (12 mM)-evoked release of [3H]D-ASP. (B) K+ (12 mM)-evoked release of [3H]GABA. Results are expressed as induced overflow. Time course relationship of the 12 mM K+-evoked release of [3H]D-ASP (C) and [3H]GABA (D) in the absence (gray circles) and in the presence (black circles) of 50 μM 3,5DHPG. Results are expressed as % of total tritium content. When not shown, error bars are within graph symbols. Data are means ± standard error of the mean of 4 (A, C) and 3 (B, D) experiments run in triplicate. *P < 0.05 at least versus 12 mM K+; §P < 0.05 at least versus 12 mM K+/1 μM MPEP; #P < 0.05 at least versus respective 12 mM K+-evoked neurotransmitter release (expressed as % of total tritium content). (E) Western blot analysis of mGlu1 and mGlu5 receptor proteins in DS-Syn fractions isolated from human neocortical synaptosomes. The figure represents a western blot comparing the mGlu1 and mGlu5 receptor immunoreactivities in a fraction enriched in human neocortical synaptosomal membranes. Anti-Stx-1A was used as a selective neuronal marker; 25-μg protein/lane were applied to the SDS-PAGE gel. Protein weights are in kDa. The blot in the figure is representative of 4 blots from synaptosomal preparations obtained from different patients, in different days.

Figure 3.

Effects of 3,5-DHPG on [3H]D-ASP and [3H]GABA-evoked exocytosis from human neocortical synaptosomes: antagonism by CPCCOEt or MPEP and western blot analysis of mGlu1/5 receptor proteins. (A) K+ (12 mM)-evoked release of [3H]D-ASP. (B) K+ (12 mM)-evoked release of [3H]GABA. Results are expressed as induced overflow. Time course relationship of the 12 mM K+-evoked release of [3H]D-ASP (C) and [3H]GABA (D) in the absence (gray circles) and in the presence (black circles) of 50 μM 3,5DHPG. Results are expressed as % of total tritium content. When not shown, error bars are within graph symbols. Data are means ± standard error of the mean of 4 (A, C) and 3 (B, D) experiments run in triplicate. *P < 0.05 at least versus 12 mM K+; §P < 0.05 at least versus 12 mM K+/1 μM MPEP; #P < 0.05 at least versus respective 12 mM K+-evoked neurotransmitter release (expressed as % of total tritium content). (E) Western blot analysis of mGlu1 and mGlu5 receptor proteins in DS-Syn fractions isolated from human neocortical synaptosomes. The figure represents a western blot comparing the mGlu1 and mGlu5 receptor immunoreactivities in a fraction enriched in human neocortical synaptosomal membranes. Anti-Stx-1A was used as a selective neuronal marker; 25-μg protein/lane were applied to the SDS-PAGE gel. Protein weights are in kDa. The blot in the figure is representative of 4 blots from synaptosomal preparations obtained from different patients, in different days.

The group I mGlu agonist 3,5DHPG failed to modify the spontaneous (Table 2) or the 12 mM K+-evoked release of [3H]GABA (Fig. 3B,D).

In order to ascertain the existence of mGlu1 and/or mGlu5 receptor proteins in human neocortical terminals, DS-Syn isolated from purified synaptosomes were probed with anti-mGlu1 and anti-mGlu5 receptor antibodies. To validate the purity of the synaptosomal membrane preparations, we also investigated the presence of the selective neuronal marker Stx-1A. The presence of the GFAP, a specific astrocyte marker, was also evaluated: GFAP immunoreactivity was barely detectable, suggesting that, in our synaptosomal preparations, contamination by gliosomes is very low (not shown, but see Musante, Longordo, et al. 2008). Selective antibodies recognized immunoreactive protein components with an apparent mass corresponding to those of mGlu1 and 5 receptors, respectively (Fig. 3C), suggesting the existence of both receptors in human neocortex. By combining the results from release experiments with those from western blot and considering that glutamatergic nerve terminals are extremely abundant in the neocortex (∼80% according to Millán et al. 2003), it seems reasonable to assume that presynaptic mGlu1 and mGlu5 autoreceptors exist on human glutamatergic nerve endings. Coexistence on the same terminal remains to be established.

Effects of Tat on Glutamate and GABA Release Evoked by KCl from Mouse Neocortical Synaptosomes

It was important to verify if the effects of Tat observed in human brain could be reproduced in a laboratory animal that could become an appropriate model to further study the effects of Tat in the CNS. In mouse neocortical synaptosomes, as in human neocortical nerve endings (Feligioni et al. 2003), Tat failed to affect significantly the spontaneous release of [3H]D-ASP (control = 1.20 ± 0.85%; +1 nM Tat = 0.75 ± 1.02%, n = 3, results expressed as % of total synaptosomal tritium content, nonsignificant [NS]), but the protein facilitated the release induced by 12 mM K+ (Fig. 4A). Similarly, the basal release of endogenous glutamate from mouse neocortical synaptosomes was not modified by Tat (control = 85.3 ± 9.6; 1 nM Tat = 97.5 ± 10.2, n = 3, results expressed as pmol/mg protein, NS), whereas the 12 mM K+-evoked glutamate exocytosis was increased (Fig. 4B). Analysis of the time course relationship of Tat-induced facilitation of glutamate and [3H]D-ASP exocytosis showed that the viral protein did not cause modifications to poststimulation neurotransmitter baseline (Fig. 4C,D).

Figure 4.

Effects of Tat on the K+-evoked exocytosis of glutamate from mouse neocortical synaptosomes. (A) Effects of Tat on the K+ (12 mM)-evoked release of [3H]D-ASP from mouse neocortical synaptosomes. (B) Effects of Tat on the K+ (12 mM)-evoked release of endogenous glutamate from mouse neocortical synaptosomes. Gray bars: 12 mM K+-evoked neurotransmitter overflow in the absence of Tat; cross-hatched gray bars: 12 mM K+-evoked neurotransmitter overflow in the presence of Tat (concentration as indicated). Results are expressed as induced overflow. The neurotransmitter released in the first 3-min fraction (b1) collected amounted to: endogenous glutamate = 138 ± 28 pmol/mg protein; [3H]D-ASP = 0.83 ± 0.08%. The induced neurotransmitter overflow in the absence of Tat and K+ amounted to: [3H]D-ASP release, 0.06 ± 0.05% induced overflow; endogenous glutamate, 25 ± 3.5 pmol/mg protein. Time course relationship of the 12 mM K+-evoked release of [3H]D-ASP (C) and endogenous glutamate (D) in the absence (gray circles) and in the presence (black circles) of 1 nM Tat. Results are expressed as percentage of respective total tritium content (C) as well as the amount (pmol/mg protein) of endogenous glutamate in each fraction collected (D). When not shown, error bars are within graph symbols. Data are means ± standard error of the mean of 5 (A, C) and 4 (B, D) experiments run in triplicate. *P < 0.05 at least versus respective control; #P < 0.05 at least versus respective 12 mM K+-evoked neurotransmitter release (expressed as % of total tritium content or as pmol/mg protein).

Figure 4.

Effects of Tat on the K+-evoked exocytosis of glutamate from mouse neocortical synaptosomes. (A) Effects of Tat on the K+ (12 mM)-evoked release of [3H]D-ASP from mouse neocortical synaptosomes. (B) Effects of Tat on the K+ (12 mM)-evoked release of endogenous glutamate from mouse neocortical synaptosomes. Gray bars: 12 mM K+-evoked neurotransmitter overflow in the absence of Tat; cross-hatched gray bars: 12 mM K+-evoked neurotransmitter overflow in the presence of Tat (concentration as indicated). Results are expressed as induced overflow. The neurotransmitter released in the first 3-min fraction (b1) collected amounted to: endogenous glutamate = 138 ± 28 pmol/mg protein; [3H]D-ASP = 0.83 ± 0.08%. The induced neurotransmitter overflow in the absence of Tat and K+ amounted to: [3H]D-ASP release, 0.06 ± 0.05% induced overflow; endogenous glutamate, 25 ± 3.5 pmol/mg protein. Time course relationship of the 12 mM K+-evoked release of [3H]D-ASP (C) and endogenous glutamate (D) in the absence (gray circles) and in the presence (black circles) of 1 nM Tat. Results are expressed as percentage of respective total tritium content (C) as well as the amount (pmol/mg protein) of endogenous glutamate in each fraction collected (D). When not shown, error bars are within graph symbols. Data are means ± standard error of the mean of 5 (A, C) and 4 (B, D) experiments run in triplicate. *P < 0.05 at least versus respective control; #P < 0.05 at least versus respective 12 mM K+-evoked neurotransmitter release (expressed as % of total tritium content or as pmol/mg protein).

Tat also failed to affect the spontaneous release of [3H]GABA (control = 2.76 ± 0.25%; 1 nM Tat = 3.01 ± 0.32%, n = 4, results expressed as % of total synaptosomal content, NS), but it inhibited the 12 mM K+-evoked exocytosis of the tritiated neurotransmitter from mouse neocortical synaptosomes (Fig. 5A). The basal release of endogenous GABA was insensitive to the viral protein (control = 55 ± 5.7; 1 nM Tat = 43.5 ± 8.2 pmol/mg protein (n = 3), but the 12 mM K+-evoked exocytosis of GABA was significantly reduced by Tat (Fig. 5B). Again, the analysis of the time course relationship of Tat-induced facilitation of GABA and [3H]GABA exocytosis demonstrated that changes did not occur to poststimulation neurotransmitter baseline (Fig. 5C,D). Also in this case, the neurotoxic fragment Tat37–72 (1 nM) mimicked the entire protein in facilitating glutamate exocytosis and in inhibiting K+-evoked GABA release from mouse neocortical synaptosomes, whereas Tat48–85 (1 nM) was inactive (Table 1).

Figure 5.

Effects of Tat on the K+-evoked exocytosis of GABA from mouse neocortical synaptosomes. (A) Effects of Tat on the K+ (12 mM)-evoked release of [3H]GABA from mouse neocortical synaptosomes. (B) Effects of Tat on the K+ (12 mM)-evoked release of endogenous GABA from mouse neocortical synaptosomes Gray bars: 12 mM K+-evoked neurotransmitter overflow in the absence of Tat; cross-hatched gray bars: 12 mM K+-evoked neurotransmitter overflow in the presence of Tat (concentration as indicated). Results are expressed as induced overflow. The neurotransmitter released in the first 3-min fraction (b1) collected amounted to: endogenous GABA = 163 ± 32 pmol/mg protein; [3H]GABA = 2.78 ± 0.65% induced overflow. The induced neurotransmitter overflow in the absence of Tat and K+ (baseline) amounted to: [3H]GABA release, 0.015 ± 0.21 5 % induced overflow; endogenous glutamate, 18 ± 4.3 pmol/mg protein. Time course relationship of the 12 mM K+-evoked release of [3H]GABA (C) and endogenous GABA (D) in the absence (gray circles) and in the presence (black circles) of 1 nM Tat. Results are expressed as % of respective total tritium content (C) as well as the amount (pmol/mg protein) of endogenous GABA in each fraction collected (D). When not shown, error bars are within graph symbols. Data are means ± standard error of the mean of 5 (A, C) and 4 (B, D) experiments run in triplicate. *P < 0.05 at least versus respective control; #P < 0.05 at least versus respective 12 mM K+-evoked neurotransmitter release (expressed as % of total tritium content or as pmol/mg protein).

Figure 5.

Effects of Tat on the K+-evoked exocytosis of GABA from mouse neocortical synaptosomes. (A) Effects of Tat on the K+ (12 mM)-evoked release of [3H]GABA from mouse neocortical synaptosomes. (B) Effects of Tat on the K+ (12 mM)-evoked release of endogenous GABA from mouse neocortical synaptosomes Gray bars: 12 mM K+-evoked neurotransmitter overflow in the absence of Tat; cross-hatched gray bars: 12 mM K+-evoked neurotransmitter overflow in the presence of Tat (concentration as indicated). Results are expressed as induced overflow. The neurotransmitter released in the first 3-min fraction (b1) collected amounted to: endogenous GABA = 163 ± 32 pmol/mg protein; [3H]GABA = 2.78 ± 0.65% induced overflow. The induced neurotransmitter overflow in the absence of Tat and K+ (baseline) amounted to: [3H]GABA release, 0.015 ± 0.21 5 % induced overflow; endogenous glutamate, 18 ± 4.3 pmol/mg protein. Time course relationship of the 12 mM K+-evoked release of [3H]GABA (C) and endogenous GABA (D) in the absence (gray circles) and in the presence (black circles) of 1 nM Tat. Results are expressed as % of respective total tritium content (C) as well as the amount (pmol/mg protein) of endogenous GABA in each fraction collected (D). When not shown, error bars are within graph symbols. Data are means ± standard error of the mean of 5 (A, C) and 4 (B, D) experiments run in triplicate. *P < 0.05 at least versus respective control; #P < 0.05 at least versus respective 12 mM K+-evoked neurotransmitter release (expressed as % of total tritium content or as pmol/mg protein).

Group I mGlu Receptors of the mGlu1 Subtype Mediate the Facilitation of Glutamate Release from Mouse Neocortical Nerve Endings

CPCCOEt (5 μM) failed to affect the K+-evoked release of [3H]D-ASP, whereas MPEP (1 μM) decreased it (−23.02 ± 1.8%, P < 0.05). The inhibition by MPEP was not significantly affected by the contemporary addition of CPCCOEt (−21.6 ± 4.3%, P < 0.05; Fig. 6A). Tat could not facilitate the overflow of [3H]D-ASP evoked by 12 mM K+ in the presence of CPCCOEt but significantly potentiated the K+-evoked overflow in the presence of MPEP (Fig. 6A). CPCCOEt did not affect the K+-evoked release of endogenous glutamate but blocked the release-enhancing effect of 1 nM Tat (Fig. 6B). On the contrary, the effect of Tat was not prevented by 1 μM MPEP (Fig. 6B). As in the case of [3H]D-ASP release, the K+-evoked release of endogenous glutamate was significantly inhibited by 1 μM MPEP added alone (Fig. 6B). The antagonists failed to affect on their own the spontaneous release of [3H]D-ASP and glutamate (Table 2).

Figure 6.

Effects of CPCCOEt or MPEP on the Tat-induced modulation of glutamate and GABA exocytosis from mouse neocortical synaptosomes. (A) Effects of CPCCOEt and MPEP on the Tat facilitation of the K+ (12 mM)-evoked release [3H]D-ASP. (B) Effects of CPCCOEt or MPEP on the Tat facilitation by Tat of the K+(12 mM)-evoked release of endogenous glutamate. (C) Effects of CPCCOEt or MPEP on the inhibition by Tat of the K+(12 mM)-evoked release of [3H]GABA. (D) Effects of CPCCOEt or MPEP on the inhibition by Tat of the K+(12 mM)-evoked release of endogenous GABA. Results are expressed as induced overflow. Data are means ± standard error of the mean of 5 (A), 4 (B), 5 (C), and 4 (D) experiments run in triplicate. *P < 0.05 at least versus 12 mM K+; #P < 0.05 at least versus 12 mM K+/1 μM MPEP.

Figure 6.

Effects of CPCCOEt or MPEP on the Tat-induced modulation of glutamate and GABA exocytosis from mouse neocortical synaptosomes. (A) Effects of CPCCOEt and MPEP on the Tat facilitation of the K+ (12 mM)-evoked release [3H]D-ASP. (B) Effects of CPCCOEt or MPEP on the Tat facilitation by Tat of the K+(12 mM)-evoked release of endogenous glutamate. (C) Effects of CPCCOEt or MPEP on the inhibition by Tat of the K+(12 mM)-evoked release of [3H]GABA. (D) Effects of CPCCOEt or MPEP on the inhibition by Tat of the K+(12 mM)-evoked release of endogenous GABA. Results are expressed as induced overflow. Data are means ± standard error of the mean of 5 (A), 4 (B), 5 (C), and 4 (D) experiments run in triplicate. *P < 0.05 at least versus 12 mM K+; #P < 0.05 at least versus 12 mM K+/1 μM MPEP.

CPCCOEt and MPEP had no effect, on their own, on the spontaneous (Table 2) or the K+-evoked release of [3H]GABA and endogenous GABA from mouse cortical synaptosomes (Table 2 and Fig. 6C and D). Neither of the 2 antagonists was able to prevent the inhibitory effect of 1 nM Tat on the K+-evoked release of [3H]GABA (Fig. 6C) or of endogenous GABA (Fig. 6D). Furthermore, 3,5-DHPG failed to modify the 12 mM K+-evoked release of GABA from mouse neocortical synaptosomes (control: 6.54 ± 0.71; 0.50 μM 3,5-DHPG: 5.69 ± 0.45, NS). For further investigations on the receptor(s) potentially involved in Tat-induced inhibition of GABA release, please see Supplementary results.

Deletion of mGlu1 or mGlu5 Receptors Prevents the Facilitation of Glutamate Exocytosis but not the Inhibition of GABA Exocytosis Caused by Tat in Mouse Cortical Synaptosomes

To better understand the role of mGlu1 and mGlu5 autoreceptors in the Tat-induced facilitation of glutamate release, the effects of Tat were investigated in synaptosomes isolated from the cerebrocortex of mouse lacking mGlu1 (crv4/crv4 mice, see Fig. 7C) or mGlu5 (mGlu5−/− mice) receptors.

Figure 7.

Effects of Tat on the release of [3H]D-ASP (A) or [3H]GABA (B) evoked by 12 mM KCl in synaptosomes isolated from the cortex of mice lacking mGlu1 (crv4/crv4) or mGlu5−/− receptors. The neurotransmitter released in the first 3-min fraction (b1) collected from crv4/crv4 mouse neocortical synaptosomes amounted to: [3H]D-ASP = 1.13 ± 0.08% induced overflow t; [3H]GABA = 2.56 ± 0.35% induced overflow. The neurotransmitter released in the first 3-min fraction (b1) collected from mGlu5−/− mouse neocortical synaptosomes amounted to: [3H]D-ASP = 0.92 ± 0.12% induced overflow t; [3H]GABA = 2.32 ± 0.28% induced overflow. Data are means ± standard error of the mean of 5 (A, B) experiments run in triplicate. *P < 0.05 at least versus respective control. (C) Western blot of mGlu1 protein expression in DS-Syn isolated from wt (a) and crv4/crv4 (b) mouse cortical synaptosomal membranes. The figure is representative of 3 blots from synaptosomal preparations obtained from different animals. (D) Western blot analysis of mGlu1 and mGlu5 receptor proteins in DS-Syn isolated from mouse neocortical synaptosomes. mGlu1 (c) and mGlu5 (d) receptor proteins were immunoprecipitated and aliquots of the pellets were subjected to western blotting with anti-mGlu5 and anti-mGlu1 receptor antibodies. Protein weights are in kDa. The figure is representative of 4 blots from synaptosomal preparations obtained from different animals.

Figure 7.

Effects of Tat on the release of [3H]D-ASP (A) or [3H]GABA (B) evoked by 12 mM KCl in synaptosomes isolated from the cortex of mice lacking mGlu1 (crv4/crv4) or mGlu5−/− receptors. The neurotransmitter released in the first 3-min fraction (b1) collected from crv4/crv4 mouse neocortical synaptosomes amounted to: [3H]D-ASP = 1.13 ± 0.08% induced overflow t; [3H]GABA = 2.56 ± 0.35% induced overflow. The neurotransmitter released in the first 3-min fraction (b1) collected from mGlu5−/− mouse neocortical synaptosomes amounted to: [3H]D-ASP = 0.92 ± 0.12% induced overflow t; [3H]GABA = 2.32 ± 0.28% induced overflow. Data are means ± standard error of the mean of 5 (A, B) experiments run in triplicate. *P < 0.05 at least versus respective control. (C) Western blot of mGlu1 protein expression in DS-Syn isolated from wt (a) and crv4/crv4 (b) mouse cortical synaptosomal membranes. The figure is representative of 3 blots from synaptosomal preparations obtained from different animals. (D) Western blot analysis of mGlu1 and mGlu5 receptor proteins in DS-Syn isolated from mouse neocortical synaptosomes. mGlu1 (c) and mGlu5 (d) receptor proteins were immunoprecipitated and aliquots of the pellets were subjected to western blotting with anti-mGlu5 and anti-mGlu1 receptor antibodies. Protein weights are in kDa. The figure is representative of 4 blots from synaptosomal preparations obtained from different animals.

As already reported (Musante, Neri, et al. 2008), the 12 mM K+-induced [3H]D-ASP overflow from crv4/crv4 neocortical synaptosomes was significantly greater (+50, 76 ± 7.3%, P < 0.05) than that released from control synaptosomes. In contrast, deletion of the mGlu5 receptor subtype did not affect the tritium released by the depolarizing stimulus (Fig. 6A, Musante, Neri, et al. 2008). Lack of either mGlu1 or mGlu5 receptors prevented the enhancing effects of Tat on glutamate exocytosis (Fig. 6A). Indeed, the viral protein, added at 1 nM, failed to facilitate the 12 mM K+-evoked release of [3H]D-ASP not only from crv4/crv4 but also from mGlu5−/− neocortical synaptosomes (Fig. 6A). These results are compatible with the idea that mGlu1 and mGlu5 receptor interact with each other, perhaps, because they are coexpressed on the same glutamatergic nerve endings as previously hypothesized (Musante, Neri, et al. 2008). To further test this hypothesis (or further support these findings), we immunoprecipitated mGlu1 and mGlu5 receptor proteins and, subsequently, analyzed the immunoprecipitates with mGlu5 and mGlu1 receptor antibodies. Immunoprecipitation of neocortical synaptosomes with the anti-mGlu1 receptor antibody produced a marked signal for the mGlu5 receptor protein and vice versa (Fig. 7D). Considering that glutamatergic terminals represent by far the most abundant of the cortical synaptosomal populations (Millán et al. 2003), it seems reasonable to assume that mGlu1 and mGlu5 autoreceptors coexist on glutamatergic nerve endings. Deletion of mGlu1 or mGlu5 receptors failed to affect the amount of [3H]GABA released by 12 mM KCl. Furthermore, the inhibition of the evoked [3H]GABA exocytosis by 1 nM Tat was not modified in synaptosomes prepared from crv4/crv4 or mGlu5−/− mice (Fig. 7B).

Tat Stimulates IP3 Production in Human and Mouse Neocortical Synaptosomes

Human and mouse neocortical synaptosomes were analyzed for their endogenous IP3 content. Synaptosomes were first analyzed for their IP3 content in control (in the absence of depolarizing stimulus) and in depolarizing (i.e., exposure to 12 mM K+) conditions; the results indicate that depolarization increases, although not significantly, the endogenous synaptosomal amount of the second messenger. We then investigated the impact of Tat on the 12 mM K+-evoked IP3 production. Table 3 shows that the addition of 1 nM Tat almost doubled the endogenous IP3 content in human neocortical synaptosomes exposed to 12 mM KCl. Concomitant addition of 5 μM CPCCOEt significantly reduced the endogenous content of the second messenger in human neocortical synaptosomes exposed to 12 mM KCl/1 nM Tat. Similarly, Tat significantly increased the endogenous IP3 content in mouse neocortical synaptosomes depolarized with 12 mM K+. Again, the 12 mM KCl/1 nM Tat-induced IP3 production was abolished by 5 μM CPCCOEt (Table 3). Similar results were obtained when analyzing the effect of 3,5-DHPG on the 12 mM K+-evoked production of IP3. Table 3 shows that 50 μM 3,5DHPG significantly increased the endogenous IP3 content in both human and mouse neocortical synaptosomes. In both cases, the 3,5DHPG-induced IP3 production was largely prevented by 5 μM CPCCOEt. CPCCOEt did not modify, on its own, the IP3 content in human and mouse neocortex synaptosomes exposed to 12 mM K+ (data not shown).

Table 3

Effects of Tat on the IP3 content in human and mouse synaptosomes

 Endogenous IP3 content (pmol/mg protein)
 
 Human Mouse 
Control 70.97 ± 9.32 176.18 ± 15.03 
12 mM K+ 102.86 ± 10.01 225.88 ± 29.03 
12 mM K+/1 nM Tat 182.92 ± 12.43* 322.91 ± 14.65* 
12 mM K+/1 nM Tat/5 μM CPCCOEt 117,75 ± 16.75# 238.73 ± 18.91# 
12 mM K+/50 μM 3,5DHPG 162.97 ± 10.21* 319.15 ± 13.53* 
12 mM K+/50 μM 3,5DHPG/5 μM CPCCOEt 120.56 ± 9.34# 253.39 ± 13.47# 
 Endogenous IP3 content (pmol/mg protein)
 
 Human Mouse 
Control 70.97 ± 9.32 176.18 ± 15.03 
12 mM K+ 102.86 ± 10.01 225.88 ± 29.03 
12 mM K+/1 nM Tat 182.92 ± 12.43* 322.91 ± 14.65* 
12 mM K+/1 nM Tat/5 μM CPCCOEt 117,75 ± 16.75# 238.73 ± 18.91# 
12 mM K+/50 μM 3,5DHPG 162.97 ± 10.21* 319.15 ± 13.53* 
12 mM K+/50 μM 3,5DHPG/5 μM CPCCOEt 120.56 ± 9.34# 253.39 ± 13.47# 

Note: Synaptosomes were superfused with standard medium (control) or were exposed to the depolarizing stimulus (12 mM K+) in the absence or in the presence of drugs, as indicated. Results are expressed as pmol/mg protein; data are mean ± standard error of the mean of 3–6 experiments run in triplicate.

*P < 0.05 versus 12 mM K+; #P < 0.05 versus 12 mM K+/1 nM Tat; §P < 0.05 versus 12 mM K+/50 μM 3,5DHPG.

Effects of Tat on the Release of [3H]D-ASP and of [3H]GABA from Mouse Neocortical Slices: Role of mGlu1 Receptors

It was important to investigate if the Tat effects observed in synaptosomes, a relatively simplified preparation that often facilitates interpretation of results, could be reproduced in brain slices. Mouse neocortical slices preloaded with [3H]D-ASP or with [3H]GABA were first exposed in superfusion to 1 nM Tat. Under these conditions, the protein was unable to modify the basal release of radioactivity (data not shown).

We then studied the effects of Tat on the release of [3H]D-ASP or [3H]GABA induced by different depolarizing stimuli. Figure 8A shows that the exposure of slices to 30 mM K+ or to 10 μM veratridine caused overflows that were significantly potentiated by 1 nM Tat. CPCCOEt, added alone, did not affect the veratridine-induced [3H]D-ASP overflow but totally prevented the facilitation of release caused by Tat (10 μM veratridine/5 μM CPCCOEt = 2.74 ± 0.52; 10 μM veratridine/5 μM CPCCOEt/1 nM Tat = 2.68 ± 0.39, expressed as % induced [3H]D-ASP overflow). Veratridine also evoked the release of [3H]GABA from mouse neocortical slices. A concentration of the alkaloid of 3 μM was chosen because the overflow elicited by 3 μM veratridine was quantitatively comparable to that of [3H]D-ASP elicited by 10 μM veratridine. Tat was unable to modulate [3H]GABA overflow elicited by veratridine from slices (3 μM veratridine =2.4 ± 0.26; 3 μM veratridine/1 nM Tat = 2.45 ± 0.27, expressed as % induced [3H]GABA overflow).

Figure 8.

Effects of Tat on the release of [3H]D-ASP evoked by different depolarizing stimuli from neocortical slices: effects of wild-type and mGlu1 receptor lacking mice. (A) Effects of Tat on the release of [3H]D-ASP evoked by 30 mM K+ or 10 μM veratridine from neocortical slices prepared from control mice. (B) Effects of Tat on the release of [3H]D-ASP evoked by 10 μM veratridine from neocortical slices prepared from crv4/crv4 mice. Results are expressed as induced overflow. The amount of neurotransmitter released in the first 3-min fraction (b3) collected (see Material and Methods) during superfusion of mouse neocortical slices amounted to: [3H]D-ASP = 0.18 ± 0.04%. *P < 0.05 at least versus respective control; #P < 0.05 at least versus respective control.

Figure 8.

Effects of Tat on the release of [3H]D-ASP evoked by different depolarizing stimuli from neocortical slices: effects of wild-type and mGlu1 receptor lacking mice. (A) Effects of Tat on the release of [3H]D-ASP evoked by 30 mM K+ or 10 μM veratridine from neocortical slices prepared from control mice. (B) Effects of Tat on the release of [3H]D-ASP evoked by 10 μM veratridine from neocortical slices prepared from crv4/crv4 mice. Results are expressed as induced overflow. The amount of neurotransmitter released in the first 3-min fraction (b3) collected (see Material and Methods) during superfusion of mouse neocortical slices amounted to: [3H]D-ASP = 0.18 ± 0.04%. *P < 0.05 at least versus respective control; #P < 0.05 at least versus respective control.

To further support the involvement of mGlu1 receptors in the Tat-induced glutamate release, neocortical slices were prepared from crv4/crv4 mice and exposed to 10 μM veratridine alone or in the presence of 1 nM Tat. The veratridine-induced [3H]D-ASP release in crv4/crv4 neocortical slices did not differ significantly from that in control slices. The lack of mGlu1 receptors totally impeded the effect of Tat on the veratridine-induced overflow (Fig. 8B).

Discussion

The principal finding of the present investigation is that low nanomolar concentrations of the HIV-1 viral protein Tat can exert opposite effects on the depolarization-evoked release of the two major neurotransmitters of the mammalian CNS. Tat facilitated the exocytosis of the excitatory neurotransmitter glutamate but decreased that of the inhibitory neurotransmitter GABA from human neocortical nerve endings. The effects of Tat may therefore cause unbalances between excitation and inhibition in neuronal circuitries involved in HAD and MCMD. Tat appears to activate directly release-enhancing glutamate autoreceptors belonging to the mGlu1 subtype. The targets that mediate the inhibition of GABA release remain to be identified. The data obtained with mouse synaptosomes paralleled those with human brain, indicating that this animal is a suitable model to study the effect of Tat in CNS.

The possibility that Tat modulates glutamatergic neurotransmission in the CNS had been already considered on the basis of its excitotoxic effects (Nath et al. 1996; Cheng et al 1998). Tat was found to regulate extracellular glutamate levels in human cortical cultured cells (Eugenin et al. 2003), to enhance glutamate transmission in the mouse suprachiasmatic nucleus (Clark et al. 2005) and to increase the frequency of miniature excitatory postsynaptic currents in rat cortical neurons (Brailoiu et al. 2008). This latter observation was considered predictive of a presynaptic effect, which is confirmed by the present results showing that Tat could facilitate glutamate exocytosis from adult human and mouse neocortical nerve terminals.

The facilitation of glutamate release is due to activation by Tat of group I mGlu presynaptic autoreceptors. In particular, based on the results with selective group I mGlu receptor antagonists, mGlu1 receptors seem to play the major role because Tat facilitation of glutamate exocytosis from human and mouse neocortical nerve endings was antagonized by CPCCOEt but scarcely inhibited by MPEP. Group I mGlu receptors are known to trigger phospholipase C–dependent IP3 production and consequent Ca2+mobilization from the endoplasmic reticulum (Ferraguti et al. 2008). In line with the proposed involvement of IP3-linked group I mGlu receptors, a significant production of IP3 was induced by Tat both in human and mouse neocortical synaptosomes.

The existence of release-enhancing mGlu autoreceptors of group I on glutamate nerve endings of rodent cerebrocortex, first described by Herrero et al. (1992), has been the object of several studies (see, for a recent review, Raiteri 2008). Most authors classified these autoreceptors as mGlu5 subtype (Rodriguez-Moreno et al. 1998; Sistiaga et al. 1998; Fazal et al. 2003; Rodrigues et al. 2005), although evidence has been provided that mGlu1 receptors may also be present on glutamatergic terminals of rodent brain (Reid et al. 1999, Musante, Neri, et al. 2008). The apparent discrepancies seem to have been clarified by Musante, Neri, et al. (2008) who found that, in mouse nerve endings, the group I mGlu receptor agonist 3,5-DHPG enhanced the evoked release of [3H]D-ASP through mGlu5 autoreceptors when added at low micromolar concentrations (<1 μM) but also through mGlu1 autoreceptors when added at concentrations ≥30 μM. Here we show that group I mGlu autoreceptors of the mGlu1 subtype also exist on human neocortical glutamatergic terminals, based on the findings that 1) 3,5-DHPG, added at 50 μM, potentiated the K+-evoked release of glutamate; 2) this potentiation was totally antagonized by CPCCOEt but only in part by MPEP, as expected if 3,5-DHPG preferentially activated mGlu1 autoreceptors; and 3) based on western blot data, both mGlu1 and mGlu5 receptor exist on human synaptosomal membranes.

Consistent with a prevalent role of mGlu1 receptors in the releasing effect of Tat, synaptosomes from animals lacking mGlu1 (crv4/crv4 mice) exhibited no functional responses to Tat. Curiously, however, Tat was also devoid of efficacy in synaptosomes prepared from the cortex of mGlu5−/− mice, a finding in apparent contrast with the poor ability of MPEP in antagonizing the Tat-induced facilitation of glutamate release in wild-type mice. Previous results, however, had shown that mGlu1 receptor-mediated functions, including the presynaptic control of glutamate release, were altered in mGlu5−/− mice (Volk et al. 2006; Musante, Neri, et al. 2008). These observations could be predictive of a physical connection between mGlu1 and mGlu5 receptor proteins; on the other hand, they also suggest that there may be alterations in mGlu1 receptor localization and function consequent to mGlu5 deletion. The functional observations in knockout mice, together with the results from western blot analysis showing coimmunoprecipitation of mGlu1 and mGlu5 receptor proteins, support the idea that mGlu5 autoreceptors exert a permissive role on the function of co-existing mGlu1 autoreceptors. If this were the case, the lack of efficacy of Tat in mGlu5−/− synaptosomes would represent the consequence of the altered function of mGlu1 autoreceptors due to mGlu5 deletion, instead of suggesting a direct binding of Tat at mGlu5 autoreceptors.

It was proposed that Tat can act directly on G protein (Haughey et al. 2001). In fact, the protein was found able to enter cells by process of internalization (Frankel and Pabo 1988; Ferrari et al. 2003). This probably does not occur under our conditions because the lack of effect of Tat in neocortical synaptosomes from crv4/crv4 or mGlu5−/− mice indicates that Tat directly binds at the mGlu receptor proteins (Longordo et al. 2006).

The Tat-induced facilitation of glutamate exocytosis observed in isolated nerve endings could also be seen using much more intact preparations, that is, the mouse neocortical slices. Receptors of the mGlu1 subtype appear to play a major role also in neocortical slices because 1) CPCCOEt prevented the effect of the viral protein and 2) the facilitation by Tat of glutamate exocytosis could not be observed in slices from crv4/crv4 mice.

The low nanomolar concentrations of Tat (1–3 nM) that facilitated glutamate exocytosis inhibited the exocytosis of GABA from human and mouse neocortical nerve terminals. The characteristics of the superfusion technique used allow us to say that Tat acts directly on GABAergic nerve endings; however, the nature of the binding sites is at present unknown. Receptors of the mGlu1 or mGlu5 subtype appear not to be involved because selective antagonists could not prevent the inhibitory effect of Tat and 3,5-DHPG did not mimic Tat in inhibiting GABA exocytosis. Moreover, the Tat inhibition persisted in mice lacking mGlu1 or mGlu5 receptors.

The present results may have pathological implications. Considering that the concentrations of Tat here used are lower than those causing overt neurotoxicity (Magnuson et al. 1995; New et al. 1997; Cheng et al. 1998; Haughey et al. 2001), the Tat-induced effects here observed may not necessarily represent steps on the path toward neurotoxicity. Importantly, the concentrations of Tat found to be active on the release are those measured in the CNS of AIDS patients (∼2.5 nM, Westendorp et al. 1995); therefore, the impairments of neurotransmission consequent to enhancement of glutamate exocytosis and inhibition of GABA exocytosis may reasonably occur in the brain of HIV-1–infected patients. Glutamate receptors of the mGlu1 subtype may represent targets through which Tat can act as an extremely potent “pathological agonist” to produce impairments in glutamatergic (present results), cholinergic (Feligioni et al. 2003), and noradrenergic (Longordo et al. 2006) neurotransmissions. It is surprising that mGlu1 autoreceptors require high micromolar concentrations of classic agonist to be activated but fully respond to 1–3 nM Tat. Because Tat may not be easily removed from the CNS of HIV-1–infected patients, mGlu1 receptors could be activated by Tat even in the presence of physiological levels of glutamate. On the other hand, the Tat-induced reduction of GABA release should weaken the inhibition of glutamate exocytosis exerted by GABA spilling over onto presynaptic GABAB heteroreceptors presynaptically located on glutamatergic terminals (Raiteri 2006), thus worsening the Tat-mediated exacerbation of glutamate transmission. These neurotransmitter unbalances may play roles in the neuropsychiatric manifestations, including loss of memory and seizures, often observed in HIV-1–infected patients (Bartolomei et al. 1999; Lawrence and Major 2002; Modi et al. 2002) and in part exhibited by animals administered Tat (Sabatier et al. 1991; Zink et al. 2002; Fitting et al. 2006) or expressing Tat in their CNS (Kim et al. 2003).

To conclude, the available information (Feligioni et al. 2003; Longordo et al. 2006) together with the present results suggest that mGlu1/5 receptor negative modulators/antagonists, in association with HAART, could represent novel therapeutic agents able to relieve the neuropsychiatric symptoms characteristic of HAD and MCMD.

Supplementary Material

Supplementary material can be found at: http://www.cercor.oxfordjournals.org/.

Funding

Italian Ministero dell'Istruzione, dell'Università e della Ricerca Scientifica (Project n. 200728AA57_002 to A.P. and 2007YYL5J9_004 to M.R.); University of Genoa “Progetto Ricerca Ateneo” (to A.P. and M.R.); Istituto Superiore di Sanità (Programma Nazionale di Ricerca sull'AIDS: Progetto “Patologia, Clinica e Terapia dell'AIDS to M.R.).

We thank FIT Biotech Oyj Plc (Tampere, Finland), the Centre for AIDS Reagents, the EU Programme EVA/MRC (contract QLKZ-CT-1999-00609), and the UK Medical Research Council for kindly providing us with the HIV-1 viral protein Tat. We wish to thank Dr Marco Pedrazzi for helpful technical and scientific support in western blot analysis, and Maura Agate and Silvia E. Smith (University of Utah) for editorial assistance and for reviewing the manuscript, respectively. Conflict of Interest: None declared

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