Interindividual differences in the levels of the glutamate transporters GLAST and GLT, but no clear correlation with Alzheimer's disease

Alzheimer's disease is a common progressive neurodegenerative disease of unknown etiology. Several different pathological processes have been identified in the brains of Alzheimer patients. To determine if reduced glutamate uptake is a contributing factor, we have measured the levels of the glutamate transporter proteins GLAST (EAAT1) and GLT (EAAT2) in human autopsy samples. The postmortem proteolysis of these proteins turned out to be fairly rapid. Brains from 10 Alzheimer and 10 control patients were therefore obtained with a relatively short postmortem delay (5 hr on average). GLT (N-terminal and central parts), GLAST (C-terminal), glial fibrillary acidic protein (GFAP) and inositol (1,4,5)-triphosphate (IP3)-receptor immunoreactivities were determined in the cingulate and inferior temporal gyri by immunoblotting. The Na+-dependent "binding" of D-[3H]aspartate and the glutamate uptake after solubilization and reconstitution in liposomes were determined for comparison. An individual variation in GLAST and GLT levels was found, but no significant correlation with Alzheimer's disease, except for a 14% lower ratio of N-terminal to central GLT immunoreactivity (P < 0.04). The levels of GLAST and GLT showed negative correlation in agreement with the idea that these proteins are differentially regulated. In conclusion, Alzheimer's disease brains can have both normal and reduced levels of GLAST and GLT.     

Demonstration of glutamate/aspartate uptake activity in nerve endings by use of antibodies recognizing exogenous D-aspartate

Nerve terminals as well as glial cells are thought to possess high-affinity Na(+)-dependent transport sites for excitatory amino acids. However, recent immunocytochemical results with antibodies against such a transporter isolated from rat brain showed a selective labelling of glial cells [Danbolt et al. (1992) Neuroscience 51, 295-310]. Critical evaluation of the literature indicates that previous evidence for nerve terminal uptake of acidic amino acids might possibly be attributed to glia. To find out whether there is indeed a glutamate transporter in nerve endings, we incubated hippocampal slices with D-aspartate (10 and 50 microM), a metabolically inert substrate for the high-affinity glutamate transport system. After fixation by glutaraldehyde/formaldehyde the slices were processed immunocytochemically with specific polyclonal antibodies raised against D-aspartate coupled to albumin by glutaraldehyde/formaldehyde. The electron-microscopic postembedding immunogold technique demonstrated a large accumulation of gold particles in nerve terminals making asymmetrical synapses, compared to their postsynaptic dendritic spines, as well as in glial cell processes. The labelled terminals include those of the glutamatergic Schaffer collaterals. Axosomatic boutons appeared unlabelled. Comparison with a test conjugate with known concentration of fixed D-aspartate (94 mM) suggests that the concentration attained in the terminals after incubation with 50 microM D-aspartate was in the lower millimolar range. The uptake was totally dependent on Na+, blocked by L-threo-3-hydroxyaspartate, and had a high affinity for D-aspartate (apparent Km about 20 microM). There was no labelling in slices incubated without D-aspartate. Compared to glia, the nerve terminals had a higher D-aspartate density and accounted for a much higher proportion of the total tissue uptake, but this relationship may be different in vivo. At the light-microscopic level the D-aspartate-like immunoreactivity showed a distinct laminar distribution, identical to that shown autoradiographically for D-[3H]aspartate and L-[3H]glutamate uptake sites [Taxt and Storm-Mathisen (1984) Neuroscience 11, 79-100], and corresponding to the terminal fields of the major excitatory fibre systems in the hippocampal formation. The novel approach described here establishes that glutamatergic nerve terminals as well as glia do sustain sodium-dependent high-affinity transport of excitatory amino acids, implying that more than one glutamate transporter must be present in the brain. Immunogold detection of D-aspartate gives a much higher anatomical resolution than electron microscopic autoradiography of D-[3H]aspartate or L-[3H]glutamate uptake, the only method that has been available previously for ultrastructural demonstration of uptake activity.(ABSTRACT TRUNCATED AT 400 WORDS)     

Localization and function of five glutamate transporters cloned from the salamander retina

Glutamate is the major excitatory neurotransmitter in the vertebrate retina. Native glutamate transporters have been well characterized in several retinal neurons, particularly from the salamander retina. We have cloned five distinct glutamate transporters from the salamander retina and examined their localization and functional properties: sEAAT1, sEEAAT2A, sEAAT2B, sEAAT5A and sEAAT5B. sEAAT1 is a homologue of the glutamate transporter EAAT1 (GLAST), sEAAT2A and sEAAT2B are homologues of EAAT2 (GLT-1) and sEAAT5A and sEAAT5B are homologues of the recently cloned human retinal glutamate transporter EAAT5. Localization was determined by immunocytochemical techniques using antibodies directed at portions of the highly divergent carboxy terminal. Glutamate transporters were found in glial, photoreceptor, bipolar, amacrine and ganglion cells. The pharmacology and ionic dependence were determined by two-electrode voltage clamp recordings from Xenopus laevis oocytes which had previously been injected with one of the glutamate transporter mRNAs. Each of the transporters behaved in a manner consistent with a glutamate transporter and there were some distinguishing characteristics which make it possible to link the function in native cells with the behavior of the cloned transporters in this study.     

Relevance of the antibody response against human immunodeficiency virus type 1 envelope to vaccine design

Understanding the antibody response in HIV-1 infection is important to vaccine design. We have studied the antibody response to HIV-1 envelope at the molecular level and determined the characteristics of neutralizing and non-neutralizing antibodies. These antibodies were isolated from phage display libraries prepared from long-term seropositive asymptomatic individuals. The HIV-1 envelope is presented to the immune system in several antigenically distinct configurations: unprocessed gp160, gp120 and gp41 subunits and native envelope, each of which may be important in eliciting an antibody response in HIV-1 infection. The antibodies tested characteristically had poor affinities for native envelope as expressed on the surface of virions or infected cells, but had high affinities against non-native forms of HIV-1 envelope (viral debris). An exceptionally potent neutralizing antibody in contrast, bound native envelope with equivalent or somewhat higher affinity than this. This indicates that the antibody response in HIV-1 infection is principally elicited by viral debris rather than virions, and that these antibodies bind and neutralize viruses sub-optimally. Potential vaccines should be designed to elicit responses against native envelope.     

Functional and molecular characterization of human monoclonal antibody reactive with the immunodominant region of HIV type 1 glycoprotein 41

The immunoreactivity, functional activity, and molecular features of a human monoclonal antibody (HMAb), F240, from an HIV-1-infected individual have been studied. Flow cytometric analysis demonstrated that F240 is reactive with cells infected with a broad range of laboratory isolates but not with uninfected cells. Reactivity of F240 is greatly enhanced by preincubation of infected cells with soluble CD4, and to a much lesser extent, with F105, an HMAb reactive with the CD4-binding site of gp120. This enhancement is temperature dependent, with maximum enhancement observed at 37 degrees C, and suggests that the F240 epitope may be more accessible after gp120 has bound to CD4 in vivo. Immunoblot analysis reveals antigen specificity of F240 for gp41 or its precursor gp160. F240 specificity is mapped to the immunodominant region of the gp41 ectodomain by Pepscan analysis. This epitope has been implicated in eliciting nonprotective antibodies that enhance infection in the presence of complement. Consistent with this, F240 failed to neutralize laboratory isolates and enhanced viral infection in a complement-dependent manner. The F240 VH demonstrates extensive somatic mutations compared with the product of its closest homologous germline gene VH3-3.11. Most amino acid substitutions occur in CDR2, characteristic of an antigen-driven response, and in FR3, a phenomenon observed in other anti-HIV-1 envelope HMAbs. Primary structure analysis of the F240 heavy chain revealed strong homology in the CDR domains to an HMAb (3D6) reactive with the same gp41 region, which suggests that these HMAbs could define a potential human antibody clonotype.     

HIV-1 Nef protein protects infected primary cells against killing by cytotoxic T lymphocytes

Cytotoxic T lymphocytes (CTLs) lyse virally infected cells that display viral peptide epitopes in association with major histocompatibility complex (MHC) class I molecules on the cell surface. However, despite a strong CTL response directed against viral epitopes, untreated people infected with the human immunodeficiency virus (HIV-1) develop AIDS. To resolve this enigma, we have examined the ability of CTLs to recognize and kill infected primary T lymphocytes. We found that CTLs inefficiently lysed primary cells infected with HIV-1 if the viral nef gene product was expressed. Resistance of infected cells to CTL killing correlated with nef-mediated downregulation of MHC class I and could be overcome by adding an excess of the relevant HIV-1 epitope as soluble peptide. Thus, Nef protected infected cells by reducing the epitope density on their surface. This effect of nef may allow evasion of CTL lysis by HIV-1-infected cells.     

Human immunodeficiency virus type 2 envelope glycoprotein binds to CD8 as well as to CD4 molecules on human T cells

We report here that human immunodeficiency virus type 2 (HIV-2) envelope glycoprotein (gp105), but not HIV-1 gp120, can bind to CD8 molecules as well as to CD4 molecules on human T cells. This phenomenon may lead to differences in the life cycles of HIV-1 and HIV-2, and it may be related to the differences in disease manifestations of HIV-1 and HIV-2 infection, including longer survival of HIV-2-infected patients.     

Long-term clinical angiographic follow-up undergoing PTCA more than 10 years

Previously, it has been shown that human immunodeficiency virus (HIV)-1 envelope proteins gp160 and gp41 bind to Candida albicans. Whether this interaction affects candidal virulence in vitro was investigated. HIV-1 gp160 or gp120 treatment of C. albicans significantly altered neither growth nor phospholipase activity of the fungus. However, treatment of C. albicans with gp160, but not with gp120, led to an elevation of free and cell-bound aspartate proteinase. In addition, culture supernatants obtained from C. albicans treated with gp160 or gp41, but not with gp120, showed a strong increase in proteinase activity. Finally, C. albicans viable yeast cells treated with gp160 or gp41 and serum were phagocytosed by polymorphonuclear leukocytes to a lesser extent than was C. albicans treated with gp120 and serum or serum alone. These findings suggest that the interaction between HIV-1 gp160 and C. albicans may promote the virulence of C. albicans in HIV-1-positive patients.     

Pseudotyping of murine leukemia virus with the envelope glycoproteins of HIV generates a retroviral vector with specificity of infection for CD4-expressing cells

CD4-expressing T cells in lymphoid organs are infected by the primary strains of HIV and represent one of the main sources of virus replication. Gene therapy strategies are being developed that allow the transfer of exogenous genes into CD4(+) T lymphocytes whose expression might prevent viral infection or replication. Insights into the mechanisms that govern virus entry into the target cells can be exploited for this purpose. Major determinants of the tropism of infection are the CD4 molecules on the surface of the target cells and the viral envelope glycoproteins at the viral surface. The best characterized and most widely used gene transfer vectors are derived from Moloney murine leukemia virus (MuLV). To generate MuLV-based retroviral gene transfer vector particles with specificity of infection for CD4-expressing cells, we attempted to produce viral pseudotypes, consisting of MuLV capsid particles and the surface (SU) and transmembrane (TM) envelope glycoproteins gp120-SU and gp41-TM of HIV type 1 (HIV-1). Full-length HIV-1 envelope glycoproteins were expressed in the MuLV env-negative packaging cell line TELCeB6. Formation of infectious pseudotype particles was not observed. However, using a truncated variant of the transmembrane protein, lacking sequences of the carboxyl-terminal cytoplasmic domain, pseudotyped retroviruses were generated. Removal of the carboxyl-terminal domain of the transmembrane envelope protein of HIV-1 was therefore absolutely required for the generation of the viral pseudotypes. The virus was shown to infect CD4-expressing cell lines, and infection was prevented by antisera specific for gp120-SU. This retroviral vector should prove useful for the study of HIV infection events mediated by HIV-1 envelope glycoproteins, and for the targeting of CD4(+) cells during gene therapy of AIDS.     

HIV-1 gene expression and replication in neuronal and glial cell lines with immature phenotype: effects of nerve growth factor

Encephalopathy and neurological disorders are a major manifestation of pediatric AIDS. Although HIV-1 can replicate in cells of neuronal and glial origin, it is yet unclear whether immature neural cells, which are present during nervous system development, can support HIV-1 replication and whether neurotrophic factors can modulate HIV-1 gene expression. In this study we show that a glial cell line with a phenotype closely resembling immature glial cells is more permissive to HIV-1 infection and replication than a neuroblastic cell line. After HIV-1 infection or after transfection of these cells with the HIV-1 LTR-CAT reporter gene alone or in the presence of Tat, both HIV-1 replication and viral gene expression progressively decrease in the neuronal cell line, while they increase in the glial cell line. In both cell types viral gene expression and replication are augmented by the addition to the cells of nerve growth factor (NGF) at concentrations which induce neuronal differentiation. However, these effects are again more evident with the glial cell type, suggesting that immature glial cells may represent one of the major targets and reservoirs of HIV-1 in the developing nervous system. As NGF and Tat act synergistically in inducing HIV-1 gene expression, these data also suggest that during development the presence of high levels of neural trophic factors may activate viral replication and render the CNS more susceptible to the deleterious effects of HIV-1 infection.     

Reduced cell surface expression of processed human immunodeficiency virus type 1 envelope glycoprotein in the presence of Nef

nef genes from two laboratory grown human immunodeficiency virus type 1 (HIV-1) strains and from two proviruses that had not been propagated in vitro were introduced into CD4+ lymphoblastoid CEM cells. The stable expression of all four Nef proteins was associated with an almost complete abrogation of CD4 cell surface localization. The consequences of the presence of Nef on gp160 cleavage, gp120 surface localization, and envelope-induced cytopathic effect were examined in CEM cells in which the HIV-1 env gene was expressed from a vaccinia virus vector. The presence of Nef did not modify the processing of gp160 into its subunits but resulted in a significant decrease of cell surface levels of gp120, associated with a dramatic reduction of the fusion-mediated cell death. Surface levels of mutant envelope glycoproteins unable to bind CD4 were not altered in Nef-expressing cells, suggesting that the phenomenon was CD4 dependent. The intracellular accumulation of fully processed envelope glycoproteins could significantly delay the cytopathic effect associated with envelope surface expression in HIV-infected cells and may be relevant to the selective advantage associated with Nef during the in vivo infectious process.     

A leucine zipper-like sequence from the cytoplasmic tail of the HIV-1 envelope glycoprotein binds and perturbs lipid bilayers

HIV-1 transmembrane envelope glycoprotein (gp41) has an unusually long cytoplasmic domain that has secondary associations with the inner leaflet of the membrane. Two highly amphiphatic alpha-helices in the cytoplasmic domain of gp41 have previously been shown to interact with lipid bilayers. We have detected a highly conserved leucine zipper-like sequence between the two alpha-helices. A peptide corresponding to this segment (residues 789-815, LLP-3) aggregates in aqueous solution, but spontaneously inserts into phospholipid membranes and dissociates into alpha-helical monomers. The peptide perturbs the bilayer structure resulting in the formation of micelles and other non-bilayer structures. Tryptophan fluorescence quenching experiments using brominated phospholipids revealed that the peptide penetrates deeply into the hydrophobic milieu of the membrane bilayer. The peptide interacts equally with zwitterionic and negatively-charged phospholipid membranes and is protected from proteolytic digestion in its membrane-bound state. Polarized attenuated total reflection Fourier transform infrared (ATR-FTIR) spectroscopy showed that the LLP-3 alpha-helix axis is about 70 degrees from the normal to the membrane plane. The ATR-FTIR CH2-stretching dichroic ratio increases when the peptide is incorporated into pure phospholipid membranes, further indicating that the peptide can deeply penetrate and perturb the bilayer structure. Integrating these data with what is already known about the membrane-associating features of adjacent segments, we propose a revised structural model in which a large portion of the cytoplasmic tail of the HIV-1 envelope glycoprotein is associated with the membrane.     

HIV-1 gp41 by a common immunological epitope induces increased levels of antibodies against human interferon-beta in HIV-1 positive individuals

Based on our finding that a similar epitope exists between human IFN-beta (aa128-134) and HIV-1 gp41 (aa586-595), we examined 20 sera from healthy and 20 from HIV-1 infected individuals for IFN-beta antibody levels by ELISA. The levels of anti-IFN-beta antibody in sera from HIV-infected individuals were increased by about 160% in comparison with HIV-negative. We affinity-purified anti-gp41 antibodies from sera of HIV-1-infected individuals using rsgp41-sepharose column. One of three antibodies could recognize human IFN-beta in comparison with antibodies from serum of a healthy individual. A mouse antiserum to human IFN-beta recognized rsgp41 (recombinant soluble gp41 Env amino acid 539-684), while the normal mouse serum (pre-immune serum) did not bind to rspg41. These results indicate that a common immunological epitope exists between human IFN-beta and HIV-1 gp41. The sequence-similarity suggests that this common immunological epitope may be located in the region aa128-134 of human IFN-beta and the immunosuppressive domain (aa583-599) of HIV-1 gp41. The increased levels of antibodies against interferon-beta in HIV-1 positive individuals may be explained by a common immunological epitope on human IFN-beta and HIV-1 gp41.     

Resistance to a drug blocking human immunodeficiency virus type 1 entry (RPR103611) is conferred by mutations in gp41

A triterpene derived from betulinic acid (RPR103611) blocks human immunodeficiency virus type 1 (HIV-1) infection and fusion of CD4+ cells with cells expressing HIV-1 envelope proteins (gp120 and gp41), suggesting an effect on virus entry. This compound did not block infection by a subtype D HIV-1 strain (NDK) or cell-cell fusion mediated by the NDK envelope proteins. The genetic basis of drug resistance was therefore addressed by testing envelope chimeras derived from NDK and a drug-sensitive HIV-1 strain (LAI, subtype B). A drug-resistant phenotype was observed for all chimeras bearing the ectodomain of NDK gp41, while the origins of gp120 and of the membrane anchor and cytoplasmic domains of gp41 had no apparent role. The envelope gene of a LAI variant, fully resistant to the antiviral effect of RPR103611, was cloned and sequenced. Its product differed from the parental sequence at two positions in gp41, with changes of arginine 22 to alanine (R22A) and isoleucine 84 to serine (I84S), the gp120 being identical. In the context of LAI gp41, the I84S substitution was sufficient for drug resistance. Therefore, in two different systems, differences in gp41 were associated with sensitivity or resistance to RPR103611. Modifications of gp41 can affect the quaternary structure of gp120 and gp41 and the accessibility of gp120 to antiviral agents such as neutralizing antibodies. However, a direct effect of RPR103611 on a gp41 target must also be envisioned, in agreement with the blocking of apparently late steps of HIV-1 entry. This compound could be a valuable tool for structure-function studies of gp41.     

Crystal structure of the simian immunodeficiency virus (SIV) gp41 core: conserved helical interactions underlie the broad inhibitory activity of gp41 peptides

The gp41 subunit of the envelope protein complex from human and simian immunodeficiency viruses (HIV and SIV) mediates membrane fusion during viral entry. The crystal structure of the HIV-1 gp41 ectodomain core in its proposed fusion-active state is a six-helix bundle. Here we have reconstituted the core of the SIV gp41 ectodomain with two synthetic peptides called SIV N36 and SIV C34, which form a highly helical trimer of heterodimers. The 2.2 A resolution crystal structure of this SIV N36/C34 complex is very similar to the analogous structure in HIV-1 gp41. In both structures, three N36 helices form a central trimeric coiled coil. Three C34 helices pack in an antiparallel orientation into highly conserved, hydrophobic grooves along the surface of this coiled coil. The conserved nature of the N36-C34 interface suggests that the HIV-1 and SIV peptides are functionally interchangeable. Indeed, a heterotypic complex between HIV-1 N36 and SIV C34 peptides is highly helical and stable. Moreover, as with HIV-1 C34, the SIV C34 peptide is a potent inhibitor of HIV-1 infection. These results identify conserved packing interactions between the N and C helices of gp41 and have implications for the development of C peptide analogs with broad inhibitory activity.     

Expression and purification of recombinant tick anticoagulant peptide (Y1W/D10R) double mutant secreted by Saccharomyces cerevisiae

In determining levels of expression of HIV-1 Nef protein within the central nervous system (CNS) we assessed antibody responses to the protein both peripherally and in CNS. Antibodies to Nef were not detected within the CNS despite detection of antibodies to both gp41 and Nef in peripheral blood and representative virus isolates derived from CNS and peripheral blood (PB) samples containing full length nef sequence and virus-infected cells expressing Nef protein. We conclude from this that expression of Nef within the CNS is such that little or no antibody production occurs and that these differences indicate that Nef protein may not be directly contributing to the AIDS dementia complex. Expression of Nef protein in PHA-activated peripheral blood mononuclear cells from CNS derived isolates was different to that of coincidental PB derived isolates in that partial surface expression was observed for the latter. The results suggest that antigenic presentation of Nef within the CNS is anomalous and that Nef protein expression, at least for the limited number of in vitro derived isolates tested, has a different localization pattern.     

Cultured blood dendritic cells retain HIV-1 antigen-presenting capacity for memory CTL during progressive HIV-1 infection

Dendritic cells (DC) are potent APC that may be involved in the pathogenesis of HIV-1 infection. We studied the APC function of DC from HIV-1-infected subjects that were derived from monocyte-depleted PBMC by culture in human IL-4 and human granulocyte-macrophage CSF. The cultured cells from the HIV-1-infected subjects had similar morphology and phenotype of mature DC (CD80 = 41 +/- 8%, CD86 = 77 +/- 5%, CD40 = 87 +/- 6%, CD1a = 1 +/- 1%) to DC cultured from seronegative subjects. The yield of these DC was lower than from HIV-1-seronegative subjects (4 +/- 0% vs 11 +/- 2%, p < 0.01), and the lower DC yields correlated with lower numbers of blood CD4+ T cells (r = 0.60, p < 0.01) and higher plasma viral load (r = -0.49, p < 0.01). DC from HIV-1-infected subjects were infected with recombinant vaccinia virus vectors expressing Gag, Pol, and Env and were able to stimulate equal or higher levels of MHC class I-restricted, anti-HIV-1 memory CTL (CTLm) than were similarly treated, autologous B lymphocyte cell lines. DC pulsed with peptides representing HIV-1 CTL epitopes stimulated higher levels of anti-HIV-1 CTLm responses than did DC infected with the vaccinia virus-HIV-1 constructs. Allogeneic, MHC class I-matched DC also stimulated anti-HIV-1 CTLm activity in cells from HIV-1-infected subjects. DC from early and late stages of HIV-1 infection had a similar ability to activate CTLm specific for targets expressing either HIV-1 genes via vaccinia virus vectors or HIV-1 immunodominant synthetic peptides. However, DC from either early or late stages of HIV-1 infection could not overcome the defect in anti-HIV-1 CTLm response in advanced infection.     

Fine specificity of anti-V3 antibodies induced in chimpanzees by HIV candidate vaccines

The fine specificity of the anti-V3 antibody responses induced in chimpanzees immunized by various human immunodeficiency type 1 (HIV-1) candidate vaccines and challenged by heterologous strains of HIV-1 was analyzed by enzyme-linked immunosorbent assay (ELISA) and Pepscan epitope mapping. Two chimpanzees immunized with the recombinant canarypox virus ALVAC-HIV (vCP125) expressing gp160MN and boosted with purified gp160MN/LAI alone, then with both immunogens in combination, were not protected against challenge with HIV-1 SF2. Their sera mainly recognized one epitope of the V3 loop, located in the NH2-terminal half. By contrast, immunization of two other chimpanzees with purified gp160MN/LAI and boosting with a synthetic V3MN peptide elicited a strong anti-V3 antibody response with a broader specificity directed against multiple epitopes all along the V3 loop. These chimpanzees were protected against infection by HIV-1 SF2. However, when these two chimpanzees were challenged later with a HIV-1 clade E strain virus, they became infected. We failed to detect any reactivity with the peptide of the ectodomain of gp41 of sera harvested after immunization with the various immunogens or after challenge with HIV-1 SF2 or HIV-1 90CR402. These results demonstrated that anti-V3 antibodies with a restricted fine specificity were induced in chimpanzees immunized with gp160 purified or expressed by recombinant canarypox confirming our previous results obtained in three different species (human, guinea pig and, macaque). In contrast, a boost with the V3 peptide broadened antibody responses, suggesting that the mode of presentation of the V3 loop to the immune system strongly influences the epitope specificity of the resulting antibody response.