Astrocyte Modulated Genes And Uses Thereof
The present invention relates to Astrocyte Modulated Genes (AMGs). AMGs are genes whose expression are modulated in human astrocytes grown in primary cell culture following the exposure of these cells to either the human immunodeficiency virus HIV-1 or to the HIV-1 protein gp120. AMGs comprise both Astrocyte Enhanced Genes (AEGs) and Astrocyte Suppressed Genes (ASGs). Thus, the present invention further relates to Astrocyte Enhanced Genes (AEGs), the expression of which are up-regulated in human astrocytes grown in primary cell culture that are exposed to either the human immunodeficiency virus HIV-1 or to the HIV-1 protein gp120, and to Astrocyte Suppressed Genes (ASGs), the expression of which are downregulated in human astrocytes grown in primary cell culture that are exposed to either the human immunodeficiency virus HIV-1 or to the HIV-1 protein gp120. Because they may play a role in HIV-associated dementia (“HAD”), AMGs may be used as markers in methods for screening for drugs that treat or prevent HAD.
The subject matter described herein was supported in part by National Institutes of Health Grant 5P01NS031492, so that the United States Government has certain rights herein.
1. INTRODUCTIONThe present invention relates to Astrocyte Modulated Genes (AMGs). AMGs are genes whose expressions are modulated in human astrocytes grown in primary cell culture and exposed to either the human immunodeficiency virus HIV-1 or to the HIV-1 protein gp120. AMGs comprise Astrocyte Enhanced Genes (AEGs) and Astrocyte Suppressed Genes (ASGs) among those astrocyte genes whose expression patterns are altered following exposure to HIV-1 or gp120. Because they may play a role in HIV-associated dementia (“HAD”), AMGs may be used as markers in methods for screening for drugs that treat or prevent HAD.
2. BACKGROUND OF THE INVENTIONGlial cells are the predominant cell type in the brain, constituting more than 50% of the total cell count and outnumbering neurons 10-fold (Schubert, 1984, Developmental Biology of Cultured Nerve, Muscle, and Glia. Wiley and Sons, New York; Rutka et al., 1997, J. Neurosurg. 87, 420-430). Two major subtypes of glia are distinguished by their distinct functions in the nervous system: oligodendrocytes, which form myelin sheaths around nerve cell axons, and astrocytes, which maintain brain homeostasis and respond to pathogens and brain injury (Benveniste, 1992, Am. J. Physiol. 263, C1-16; Verkhratsky et al., 1998, Physiol. Rev. 78, 100-130).
Historically, astrocytes were considered to provide mostly passive support to neurons and the overall function of the nervous system. Recent studies question this hypothesis and suggest that astrocytes also play a critical role in several aspects of signal transmission and that defects in these functions may lead to neurodegeneration (Choi, 1988; Neuron 1, 623-634; Rothstein et al., 1996, Neuron 16, 675-686; Verkhratsky et al., 1998, Physiol. Rev. 78, 100-130; Anderson & Swanson, 2000, Glia 32, 1-14). For example, the high-affinity excitatory amino acid transport systems acting through astrocyte-specific transporters EAAT1 and EAAT2 are thought to be primarily responsible for maintenance of low levels of free intrasynaptic L-glutamate, the major excitatory neurotransmitter in the brain (Choi, 1988; Neuron 1, 623-634; Benveniste, 1992, Am. J. Physiol. 263, C1-16; Anderson & Swanson, 2000, Glia 32, 1-14). Defects that specifically abrogate this function result in accumulation of extracellular glutamate in synaptic clefts and overexcitation and death of neurons, a phenomenon referred to as excitotoxicity (Choi, 1988; Neuron 1, 623-634; Gegelashvili, G. & Schousboe, A. (1997). Molec. Pharmacol., 52, 6-15; Tanaka et al., 1997, Science 276, 1699-1702).
At least one neurodegenerative disease, Amyotropic Lateral Sclerosis (ALS), has been linked to a significant decrease of high-affinity sodium-dependent glutamate transport in synaptic membranes (Rothstein et al., 1992, New Engl. J. Med. 326, 1464-1468) and a selective loss of the transporter EAAT2 (Bristol & Rothstein, 1996, Ann. Neurol. 39, 676-679). Similar defects are implicated in Alzheimer's disease, stroke/ischemia, epilepsy, and HIV-1-associated dementia (HAD) (Choi, 1988, Neuron 1, 623-634; Kaul et al., 2001, Nature 410, 988-994; Maragakis & Rothstein, 2001, Arch. Neurol., 58, 365-370).
In addition to their role in glutamate removal from synapses, astrocytes significantly increase the number of synapses and enhance synaptic efficacy by altering pre- and post-synaptic functions in vitro (Oliet et al., 2001, Science 292, 923-926; Ullian et al., 2001, Science 291, 657-660). Finally, astrocytes appear to share many properties with neurons, including expression of functional neuronal nicotinic acetylcholine receptors (nACHRs) and competence for Ca++-dependent glutamate release, thus permitting intercellular signaling between astrocytes and neurons and, possibly, modulation of neuronal signal transmission by astrocytes (Iino et al., 2001, Science 292, 926-929; Sharma & Vijayaraghavan, 2001, Proc Natl Acad Sci USA 98, 4148-4153; Ullian et al., 2001, Science 291, 657-660).
Overall, these studies suggest that astrocytes and neurons are functionally integrated and that pathogenic stimuli that adversely affect astrocytes will directly or indirectly impact on neuronal function and survival.
Studies have focused on investigation of HIV-1 infection in neural cells and the potential contribution of such infections to neurodegeneration and HAD. The pathogenic events triggered by HIV-1 in the brain, which ultimately result in neuronal loss and CNS dysfunction (Navia et al., 1986a, Ann. Neurol. 19, 525-535; Navia et al., 1986, Ann. Neurol. 19, 517-524; reviewed in Lipton & Gendelman, 1995, New Engl. J. Med. 233, 934-940), have not been fully resolved. Neurons are rarely infected in vivo (Wiley et al., 1986, Proc. Natl. Acad. Sci. USA 83, 7089-93) and it is unlikely that neuronal loss in HIV-1 dementia is caused by cytopathic infection of these cells.
Numerous neuropathology, immunocytochemistry, in situ hybridization, and virus isolation studies indicate that macrophages and microglial cells are the primary host cells for productive HIV-1 infection in the CNS (Koening et al., 1986, Science 233, 1089-1093; Brew et al., 1995, Ann. Neurol. 38, 563-570; reviewed in Lipton & Gendelman, 1995, New Engl. J. Med. 233, 934-940). It has been suggested that HIV-1 infection and subsequent activation of infected cells causes neuroinflammatory responses involving production of chemokines, cytokines, nitric oxide, and other factors, some of which were shown to be neurotoxic in vitro (reviewed in Lipton & Gendelman, 1995, New Engl. J. Med. 233, 934-940; Kaul et al., 2001, Nature 410, 988-994). Viral products secreted by infected cells, including gp120 and Tat, can also induce neurotoxicity in vitro and in animal models (Lipton & Gendelman, 1995, New Engl. J. Med. 233, 934-940; Kaul et al., 2001, Nature 410, 988-994).
Astrocytes also can be infected with HIV-1 in vitro and in vivo, although with lower efficiency than T cells and macrophages (Dewhurst et al., 1987, J. Virol. 61, 3774-3782, J. Virol. 61, 3774-3782; Tornatore et al., 1991, J. Virol. 65, 6094-6100; Saito et al., 1994, Neurology 44, 474-481; Tornatore et al., 1994, Neurology 44, 481-487; reviewed in Brack-Werner, 1999, AIDS 13, 1-22). The limited infection of astrocytes has been attributed to various mechanisms including intracellular restrictions to virus expression (Tornatore et al., 1994b, J. Virol. 68, 93-102; Gorry et al., 1999, J. Virol. 73, 352-61; Ludwig et al., 1999, J. Virol. 73, 8279-8289) or, as has been shown recently, inefficient virus entry (Bencheikh et al., 1999, J. Neurovirol. 5, 115-124; Canki et al., 2001, J. Virol. 75, 7925-7933). There is general agreement, however, that HIV-1 can persist in astrocytes for prolonged periods in a low productive, non-cytolytic state, from which it can be induced by physiologic stimuli such as tumor necrosis factor-α (TNF-α) (Tornatore et al., 1991, J. Virol. 65, 6094-6100; Shahabuddin et al., 1992, Pathobiology 60, 195-205).
Surveys of autopsy tissues using in situ PCR and sensitive immunocytochemistry techniques indicate that the frequency of HIV-1-positive astrocytes in selected tissue sections from brains of patients with dementia can achieve 1% (Saito et al., 1994, Neurology 44, 474-481; Tornatore et al., 1994a, Neurology 44, 481-487; Takahashi et al., 1996, Ann. Neurol. 39, 705-711). Considering that the number of astrocytes in the brain is between 1011 to 1012 cells (Verkhratsky et al., 1998, Physiol. Rev. 78, 100-130), these cells clearly constitute a major target for HIV-1 infection in the brain.
The consequences of this infection with respect to HAD pathogenesis are unknown but they may be significant. Persistent, non-cytolytic HIV-1 infection in culture alters gene expression in lymphocytes (Shahabuddin et al., 1994, AIDS Res. Hum. Retroviruses 10, 1525-1529; Geiss et al., 2000, Virology 266, 8-16) and astrocytes (Schneider-Schaulies et al., 1992, Virology 191, 765-772; He et al., 1997, Proc. Natl. Acad. Sci. USA 94, 3954-3959), indicating that such infections may affect cell function. Exposure of astrocytes to recombinant HIV-1 envelope glycoprotein gp120 alters cell physiology (Benos et al., 1994a, Adv. Neuroimmunol. 4, 175-179), including a potential effect on glutamate transport as indicated by increased D-aspartate efflux in astrocytes treated with gp120 (Benos et al., 1994b, Proc. Natl. Acad. Sci. USA 91, 494-498). Impairment of glutamate transport was also observed after incubation of human astrocytes with TNF-α (Fine et al., 1996, J. Biol. Chem. 271, 15303-15306) or co-cultivation with T cells infected with human T cell leukemia virus type I (HTLVI) (Szymocha et al., 2000, J. Virol. 74, 6433-6441), and similar defects were found in feline astrocytes after infection with feline immunodeficiency virus (FIV) (Yu et al., 1998, Proc. Natl. Acad. Sci. USA 95, 2624-2629).
More recent studies indicate that ligation of the HIV-1 coreceptor on astrocytes, CXCR4, by either stromal cell-derived factor 1 (SDF-1) or gp120 can stimulate a novel signaling pathway that involves Ca2+-dependent release of glutamate (Sharma & Vijayaraghavan, 2001, Proc Natl Acad Sci USA 98, 4148-4153) in a process including activation of the CXCR4 receptor, an autocrine/paracrine TNF-α-dependent signaling, and prostaglandin (Bezzi et al., 2001, Nat. Neurosci. 4, 702-710). These results suggest that HIV-1, gp120, and other neuropathogenic agents can alter specific signaling pathways in astrocytes in a way that may impair important physiological functions of these cells in neuronal signal transmission and response to brain injury.
3. SUMMARY OF THE INVENTIONThe present invention relates to Astrocyte Modulated Genes (AMGs). AMGs are genes whose expressions are modulated in human astrocytes grown in primary cell culture following the exposure of these cells to either the human immunodeficiency virus HIV-1 or to the HIV-1 protein gp120. AMGs comprise Astrocyte Enhanced Genes (AEGs) and Astrocyte Suppressed Genes (ASGs).
The invention is based, at least in part, on the results of a series of experiments in which a rapid subtraction hybridization (RaSH) method (Jiang et al., 2000, Proc. Natl. Acad. Sci. USA 97, 12684-12689) was used to globally identify human genes whose expression in astrocytes display temporal alterations following HIV-1 infection. Using this methodology, it was discovered that 15 AEGs, including 13 known and 2 novel genes, and 10 ASGs, including 9 known and 1 novel gene, displayed altered levels of expression in early passage human fetal astrocytes as a consequence of HIV-1 infection or treatment with gp120. These AMGs may be used as markers of HIV infection and/or pathology, and, as such, may be used as reporter genes in screening assays for identifying agents that inhibit or prevent such infection and/or pathology.
The invention is further based on the discovery that upregulation of one particular AMG, termed AEG-1, results in the downregulation of GLT-1, thereby increasing extracellular levels of the potentially excitotoxic neurotransmitter L-glutamate. In various embodiments of the invention, inhibition of AEG-1 may be used to facilitate glutamate transport and thereby prevent or decrease its toxic effects. As AEG-1 is constitutively present in astrocytes, inhibition of AEG-1 may be useful in preventing or decreasing glutamate toxicity in a variety of contexts, not limited to HIV infection, but including other pathologies as well, such as cerebral infarction and/or ischemia, Alzheimer's Disease, epilepsy, and amyotrophic lateral sclerosis. AEG-1 may be inhibited, for example, by antisense molecules or antagonists directed at the expressed protein. Such antagonists may be identified using screening assays which monitor AEG-1 levels, GLT-1 levels, and/or glutamate transport capability.
3.1. DefinitionsAs used herein, the term “nucleic acid molecule” includes both DNA and RNA and, unless otherwise specified, includes both double-stranded and single-stranded nucleic acids. Also included are molecules comprising both DNA and RNA, either DNA/RNA heteroduplexes, also known as DNA/RNA hybrids, or chimeric molecules containing both DNA and RNA in the same strand. Reference to a nucleic acid sequence can also include modified bases as long as the modification does not significantly interfere either with binding of a ligand such as a protein by the nucleic acid or Watson-Crick base pairing between two complementary nucleic acid molecules.
As used herein, the term “gene” refers to a DNA molecule that either directly or indirectly encodes a nucleic acid or protein product that has a defined biological activity.
As used herein, a specific, non-limiting example of stringent conditions for detecting hybridization of nucleic acid molecules are as set forth in “Current Protocols in Molecular Biology”, Volume I. Ausubel et al., eds. John Wiley: New York N.Y., pp. 2.10.1-2.10.16, first published in 1989 but with annual updating, wherein maximum hybridization specificity for DNA samples immobilized on nitrocellulose filters may be achieved through the use of repeated washings of a hybridized filter in a solution comprising 0.1-2×SSC (15-30 mM NaCl, 1.5-3 mM sodium citrate, pH 7.0) and 0.1% SDS (sodium dodecylsulfate) at temperatures of 65-68° C. or greater. For DNA samples immobilized on nylon filters, a stringent hybridization washing solution may be comprised of 40 mM NaPO4, pH 7.2, 1-2% SDS and 1 mM EDTA. Again, a washing temperature of at least 65-68° C. is recommended, but the optimal temperature required for a truly stringent wash will depend on the length of the nucleic acid probe, its GC content, the concentration of monovalent cations and the percentage of formamide, if any, that was contained in the hybridization solution. Stringent hybridizations are designed to identify molecules with 80% identity or preferably 90% identity or more preferably 95% identity over lengths of at least 15 nucleotides and preferably at least 50 nucleotides.
An Astrocyte Modulated Gene, or “AMG”, as the term is used herein, is a gene the expression of which is modified in astrocytes as a result of HIV-1 infection and/or exposure to HIV-1 gp120 protein. AMGs comprise Astrocyte Enhanced Genes (AEGs), whose expression levels are upregulated following exposure of cultured human fetal astrocytes to HIV-1 and/or to the HIV-1 gp120 protein, and Astrocyte Suppressed Genes (ASGs), whose expression levels are down-regulated following exposure of cultured human fetal astrocytes to HIV-1 and/or to the HIV-1 gp120 protein. AMGs also comprise genes whose pattern of expression, e.g. the timing of expression, is changed following exposure of cultured human fetal astrocytes to HIV-1 and/or to the HIV-1 gp120 protein. Increased expression is associated with higher levels of mRNA and/or protein, while reduced expression is associated with lower levels of mRNA and/or protein.
Examples of AEGs include: AEG-2 (G-binding protein), AEG-3 (GA17 protein) (Ryo et al., 2000, AIDS Res. Hum. Retroviruses 16, 995-1005), AEG-4 (unr/NRU) (Jeffers et al., 1990, Nucl. Acids. Res. 18, 4891-4899; Boussadia et al., 1993, Biochim. Biophys. Acta 1172, 64-72), AEG-5 (hGNT-IV-H) (Furukawa et al., 1999, J. Hum. Genet. 44, 397-401), AEG-6 (fibronectin), AEG-7 (human CTL2) (O'Regan et al., 2000, Proc. Natl. Acad. Sci. USA 97, 1835-1840), AEG-8 (acidic ribosomal phosphoprotein) (Rich & Stietz, 1987, Mol. Cell. Biol. 7, 4065-4074), AEG-9 (calnexin) (Honore et al., 1994, Electrophoresis 15, 482-490; Rubio & Wenthold, 1999, J. Neurochem. 73, 942-948; Shi et al., 2001, Cell 105, 331-342), AEG-10 (autotaxin) (Stracke et al., 1994, J. Biol. Chem. 267, 2524-2529; Kawagoe et al., 1997, Cancer Res. 57, 2516-25), AEG-12 (thymosin β-4) (Gondo et al., 1987, J. Immunol. 139, 3840-3848), AEG-13 (human non-muscle α-actinin) (Youssoufian et al., 1990, Am. J. Hum. Genet. 47, 62-72), AEG-14 (Schneider et al., 1988, Cell 54, 787-793; Gonos, 1998, Ann. N.Y. Acad. Sci. 851, 466-469; Prieto et al., 1999, Brain Res. 816, 646-661) and AEG-15 (PGK-1) (Michelson et al., 1983, Proc. Natl. Acad. Sci. USA 80, 472-476; Tsukada et al., 1991, J. Gerontol. 46, B213-B216), and the novel AEGs AEG-1 and AEG-11.
Examples of ASGs include: ASG-2 (human cDNA FLJ10705; GenBank Acc. No. AK001567, ASG-3 (Platelet-endothelial cell tetra-span antigen 3; CD151/PETA-3; Fitter et al., 1995, Blood 86, 1348-55; Yáñez-Mó et al., 1998, J. Cell Biol. 141, 791-804), ASG-4 (guanine nucleotide-releasing factor; C3G; Schweighoffer et al, 1993, Oncogene 8, 1477-85; Tanaka et al, 1994, Proc. Natl. Acad. Sci. USA 91, 3443-3447), ASG-5 (neuronatin; ASG-5; Dou & Joseph, 1996, Brain Res. 723, 8-22; Usui et al, 1997, J. Mol. Neurosci. 9, 55-60), ASG-6 (neuroendocrine differentiation factor; CGI149; Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86, 4504-11), ASG-7 (cysteine/glycine-rich protein 1; CSRP1; Liebhaber et al. 1990, Nucleic Acids Res 18, 3871-3879), ASG-8 (MLL5; Emerling et al, 2002, Oncogene 21, 4849-54), ASG-9 (human mitochondrion encoding RNA), ASG-10 (signal recognition particle 9 kD; SRP9KD; (Lütcke, 1995, Eur. J. Biochem. 228, 531-550), and the novel ASG ASG-1.
The present invention relates to compositions and/or methods which contain and/or utilize AMG nucleic acid molecules, proteins, or related reagents. The nucleic acid molecules of the invention may or may not comprise protein-encoding sequences. For clarity of presentation, and not by way of limitation, the detailed description is divided into the following subsections:
(i) AMG nucleic acids;
(ii) AMG proteins;
(iii) anti-AMG antibodies;
(iv) screening methods; and
(v) methods of diagnosis and treatment.
5.1. AMG Nucleic AcidsThe present invention provides for nucleic acid molecules corresponding to AMGs, which may comprise AEGs or ASGs. The nucleic acid molecules of the invention include but are not limited to “full-length” nucleic acid molecules which contain, in DNA or RNA form, a complete protein coding sequence, in sense or antisense orientation. The nucleic acid molecules of the invention also include molecules which are not “full-length” but which represent, in DNA or RNA form, a portion of an mRNA molecule, which may constitute protein coding and/or untranslated regions in sense or antisense orientation; such molecules may be useful as probes for detecting RNA levels, as PCR primers or as antisense inhibitors.
In particular non-limiting embodiments, the present invention provides for nucleic acid molecules of the following AEGs: AEG-2 (G-binding protein), AEG-3 (GA17 protein) (Ryo et al., 2000, AIDS Res. Hum. Retroviruses 16, 995-1005), AEG-4 (unr/NRU) (Jeffers et al., 1990, Nucl. Acids. Res. 18, 4891-4899; Boussadia et al., 1993, Biochim. Biophys. Acta 1172, 64-72), AEG-5 (hGNT-IV-H) (Furukawa et al., 1999, J. Hum. Genet. 44, 397-401), AEG-6 (fibronectin), AEG-7 (human CTL2) (O'Regan et al., 2000, Proc. Natl. Acad. Sci. USA 97, 1835-1840), AEG-8 (acidic ribosomal phosphoprotein) (Rich & Stietz, 1987, Mol. Cell. Biol. 7, 4065-4074), AEG-9 (calnexin) (Honore et al., 1994, Electrophoresis 15, 482-490; Rubio & Wenthold, 1999, J. Neurochem. 73, 942-948; Shi et al., 2001, Cell 105, 331-342), AEG-10 (autotaxin) (Stracke et al., 1994, J. Biol. Chem. 267, 2524-2529; Kawagoe et al., 1997, Cancer Res. 57, 2516-25), AEG-12 (thymosin β-4) (Gondo et al., 1987, J. Immunol. 139, 3840-3848), AEG-13 (human non-muscle α-actinin) (Youssoufian et al., 1990, Am. J. Hum. Genet. 47, 62-72), AEG-14 (Schneider et al., 1988, Cell 54, 787-793; Gonos, 1998, Ann. N. Y Acad. Sci. 851, 466-469; Prieto et al., 1999, Brain Res. 816, 646-661) and AEG-15 (PGK-1) (Michelson et al., 1983, Proc. Natl. Acad. Sci. USA 80, 472-476; Tsukada et al., 1991, J. Gerontol. 46, B213-B216), and the novel AEGs AEG-1 and AEG-11. The present invention further provides for nucleic acid molecules which hybridize to these nucleic acid molecules under stringent conditions, having lengths of at least 15 nucleotides and preferably at least 50 nucleotides.
In other non-limiting embodiments, the present invention provides for nucleic acid molecules encoding the novel AEGs AEG-1 and AEG-11. For example, the present invention provides for nucleic acid molecules having the sequence set forth as SEQ ID NO:1 or as SEQ ID NO:3, as depicted in
In particular non-limiting embodiments, the present invention also provides for nucleic acid molecules of the following ASGs: ASG-2 (human cDNA FLJ10705; GenBank Acc. No. AK001567, ASG-3 (Platelet-endothelial cell tetra-span antigen 3; CD151/PETA-3; Fitter et al., 1995, Blood 86, 1348-55; Yáñez-Mó et al., 1998, J. Cell Biol. 141, 791-804), ASG-4 (guanine nucleotide-releasing factor; C3G; Schweighoffer et al., 1993, Oncogene 8, 1.477-85; Tanaka et al., 1994, Proc. Natl. Acad. Sci. USA 91, 3443-3447), ASG-5 (neuronatin; ASG-5, Dou & Joseph, 1996, Brain Res. 723, 8-22; Usui et al., 1997, J. Mol. Neurosci. 9, 55-60), ASG-6 (neuroendocrine differentiation factor; CGI149; Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86, 4504-11), ASG-7 (cysteine/glycine-rich protein 1; CSRP1; Liebhaber et al. 1990, Nucleic Acids Res 18, 3871-3879), ASG-8 (MLL5; Emerling et al., 2002, Oncogene 21, 4849-54), ASG-9 (human mitochondrion encoding RNA), ASG-10 (signal recognition particle 9 kD; SRP9KD; (Lütcke, 1995, Eur. J. Biochem. 228, 531-550), and the novel ASG ASG-1. The present invention further provides for nucleic acid molecules which hybridize to these nucleic acid molecules under stringent conditions, having lengths of at least 15 nucleotides and preferably at least 50 nucleotides.
In other non-limiting embodiments, the present invention provides for nucleic acid molecules for the novel ASG ASG-1. For example, the present invention provides for nucleic acid molecules having the sequences set forth as SEQ ID NO:4, as depicted in
Any aforesaid nucleic acid molecule may be linked to a heterologous nucleic acid, as discussed, for example, below.
For some purposes, an AMG may be engineered such that it is in an “expressible form.” An “expressible form” is one in which an AMG is linked to one or more elements necessary or desirable for transcription and/or translation. For example, the AMG nucleic acid may be operatively linked to a suitable promoter element in an expression cassette which may further comprise a transcription initiation and termination site, nucleic acid encoding a nuclear localization sequence, a ribosome binding site, a polyadenylation site, and/or a mRNA stabilizing sequence, etc.
Examples of suitable promoter elements, include, but are not limited to, the cytomegalovirus immediate early promoter, the Rous sarcoma virus long terminal repeat promoter, the human elongation factor-1α promoter, the human ubiquitin c promoter, etc. It may be desirable, in certain embodiments of the invention, to use a regulatable promoter. Non-limiting examples of regulatable promoters include the murine mammary tumor virus promoter (inducible with dexamethasone), commercially-available steroid- or tetracycline-responsive promoters, or ecdysone-inducible promoters, etc. It may further be desirable, in certain embodiments of the invention, to use astrocyte-specific promoters. Non-limiting examples of astrocyte-specific promoters include the specific glial fibrillary acidic protein (GFAP) promoter, the murine cytomegalovirus immediate-early (MCMV-IE) gene promoter, and the alpha tubulin promoter. Other suitable constitutive, regulatable, or cell- or tissue-specific promoter systems are known to those of ordinary skill in the art.
A nucleic acid molecule of the invention, whether or not it is to be expressed as a protein, may be inserted into a suitable vector for duplication purposes. Suitable vectors include but are not limited to plasmids, cosmids, phages, phagemids, artificial chromosomes, replicons, and various virus-based vector systems known in the art.
Where an AMG protein or peptide is to be expressed, suitable expression vectors include virus-based vectors and non-virus based DNA or RNA delivery systems. Examples of appropriate virus-based gene transfer vectors include, but are not limited to, those derived from retroviruses, for example Moloney murine leukemia-virus based vectors such as LX, LNSX, LNCX or LXSN (Miller and Rosman, 1989, Biotechniques 7, 980-989); lentiviruses, for example human immunodeficiency virus (“HIV”), feline leukemia virus (“FIV”) or equine infectious anemia virus (“EIAV”)-based vectors (Case et al., 1999, Proc. Natl. Acad. Sci. USA 96, 2988-2993; Curran et al., 2000, Molecular Ther. 1, 31-38; Olsen, 1998, Gene Ther. 5, 1481-1487); adenoviruses (Zhang, 1999, Cancer Gene Ther. 6, 113-138; Connelly, 1999, Curr. Opin. Mol. Ther. 1, 565-572), for example Ad5/CMV-based E1-deleted vectors (Li et al., 1993, Human Gene Ther. 4, 403-409); adeno-associated viruses, for example pSub201-based AAV2-derived vectors (Walsh et al., 1992, Proc. Natl. Acad. Sci. U.S.A. 89, 7257-7261); herpes simplex viruses, for example vectors based on HSV-1 (Geller & Freese, 1990, Proc. Natl. Acad. Sci. U.S.A. 87, 1149-1153); baculoviruses, for example AcMNPV-based vectors (Boyce & Bucher, 1996, Proc. Natl. Acad. Sci. U.S.A. 93, 2348-2352); SV40, for example SVluc (Strayer & Milano, 1996, Gene Ther. 3, 581-587); Epstein-Barr viruses, for example EBV-based replicon vectors (Hambor et al., 1988, Proc. Natl. Acad. Sci. U.S.A. 85, 4010-4014); alphaviruses, for example Semliki Forest virus- or Sindbis virus-based vectors (Polo et al., 1999, Proc. Natl. Acad. Sci. U.S.A. 96, 4598-4603); vaccinia viruses, for example modified vaccinia virus (MVA)-based vectors (Sutter & Moss, 1992, Proc. Natl. Acad. Sci. U.S.A. 89, 10847-10851) or any other class of viruses that can efficiently transduce human tumor cells and that can accommodate the nucleic acid sequences required for therapeutic efficacy.
Non-limiting examples of non-virus-based delivery systems that may be used according to the invention include, but are not limited to, so-called naked nucleic acids (Wolff et al., 1990, Science 247, 1465-1468), nucleic acids encapsulated in liposomes (Nicolau et al., 1987, Methods in Enzymology 149, 157-176), nucleic acid/lipid complexes (Legendre & Szoka, 1992, Pharmaceutical Research 9, 1235-1242), and nucleic acid/protein complexes (Wu & Wu, 1991, Biother. 3, 87-95).
AMGs may also be produced using nucleic acid contained in plasmids, such as pCEP4 (Invitrogen, San Diego, Calif.), pMAMneo (Clontech, Palo Alto, Calif.; see below), pcDNA3.1 (Invitrogen, San Diego, Calif.), etc. Vectors useful in expressing AMG-1 in bacterial systems include but are not limited to the GST vector (Amersham) and the chitin binding domain vector (TYB-12) (New England Biolabs).
In a preferred embodiment, an AMG vector comprises the AEG-1 gene of SEQ ID NO:1 operatively linked to a heterologous regulatory element and a polyadenylation signal, both of which are active in mammalian cells. The resulting expression cassette may be contained within a plasmid or a virus-based vector. In preferred embodiments, the expression cassette is contained within an adenovirus vector derived from human adenovirus type 2 or 5, or within an adeno-associated virus (AAV) vector derived from human AAV2.
5.2. AMG ProteinsThe present invention provides for AMG proteins. These include, but are not limited to, the following AEG proteins: AEG-2 (G-binding protein), AEG-3 (GA17 protein) (Ryo et al., 2000, AIDS Res. Hum. Retroviruses 16, 995-1005), AEG-4 (unr/NRU) (Jeffers et al., 1990, Nucl. Acids. Res. 18, 4891-4899; Boussadia et al., 1993, Biochim. Biophys. Acta 1172, 64-72), AEG-5 (hGNT-IV-H) (Furukawa et al., 1999, J. Hum. Genet. 44, 397-401), AEG-6 (fibronectin), AEG-7 (human CTL2) (O'Regan et al., 2000, Proc. Natl. Acad. Sci. USA 97, 1835-1840), AEG-8 (acidic ribosomal phosphoprotein) (Rich & Stietz, 1987, Mol. Cell. Biol. 7, 4065-4074), AEG-9 (calnexin) (Honore et al., 1994, Electrophoresis 15, 482-490; Rubio & Wenthold, 1999, J. Neurochem. 73, 942-948; Shi et al., 2001, Cell 105, 331-342), AEG-10 (autotaxin) (Stracke et al., 1994, J. Biol. Chem. 267, 2524-2529; Kawagoe et al., 1997, Cancer Res. 57, 2516-25), AEG-12 (thymosin β-4) (Gondo et al., 1987, J. Immunol. 139, 3840-3848), AEG-13 (human non-muscle α-actinin) (Youssoufian et al., 1990, Am. J. Hum. Genet. 47, 62-72), AEG-14 (Schneider et al., 1988, Cell 54, 787-793; Gonos, 1998, Ann. N.Y. Acad. Sci. 851, 466-469; Prieto et al., 1999, Brain Res. 816, 646-661) and AEG-15 (PGK-1) (Michelson et al., 1983, Proc. Natl. Acad. Sci. USA 80, 472-476; Tsukada et al., 1991, J. Gerontol. 46, B213-B216). The invention also encompasses peptide fragments of these AEG proteins comprising at least 20 amino acids which cross-react with an immunoglobulin which specifically binds to the corresponding full-length protein.
In specific, non-limiting embodiments, the present invention provides for proteins encoded by nucleic acids AEG-1 and AEG-11. In one particular embodiment, the invention provides for a protein encoded by a nucleic acid sequence as set forth in SEQ ID NO:1 (depicted in
The present invention also provides for ASG proteins, including, but not limited to, the following: ASG-2 (human cDNA FLJ10705; GenBank Acc. No. AK001567, ASG-3 (Platelet-endothelial cell tetra-span antigen 3; CD151/PETA-3; Fitter et al., 1995, Blood 86, 1348-55; Yáñez-Mó et al., 1998, J. Cell Biol. 141, 791-804), ASG-4 (guanine nucleotide-releasing factor; C3G; Schweighoffer et al., 1993, Oncogene 8, 1477-85; Tanaka et al., 1994, Proc. Natl. Acad. Sci. USA 91, 3443-3447), ASG-5 (neuronatin; ASG-5; Dou & Joseph, 1996, Brain Res. 723, 8-22; Usui et al., 1997, J. Mol. Neurosci. 9, 55-60), ASG-6 (neuroendocrine differentiation factor; CG1149; Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86, 4504-11), ASG-7 (cysteine/glycine-rich protein 1; CSRP 1; Liebhaber et al. 1990, Nucleic Acids Res 18, 3871-3879), ASG-8 (MLL5; Emerling et al., 2002, Oncogene 21, 4849-54), ASG-9 (human mitochondrion encoding RNA), ASG-10 (signal recognition particle 9 kD; SRP9KD; (Lütcke, 1995, Eur. J. Biochem. 228, 531-550), and the novel ASG ASG-1. The invention also encompasses peptide fragments of these ASG proteins comprising at least 20 amino acids which cross-react with an immunoglobulin which specifically binds to the corresponding full-length protein.
In specific, non-limiting embodiments, the present invention provides for proteins encoded by ASG-1 nucleic acids. In one particular embodiment, the invention provides for a protein encoded by nucleic acid sequences as set forth in SEQ ID NO:4 (depicted in
The AMG proteins and peptides of the invention may be prepared by standard techniques, including recombinant DNA-related techniques and chemical synthesis, or by collection from natural sources. For recombinant DNA expression, a non-limiting list of suitable expression vectors is set forth in the preceding section.
Expression systems which may be used to produce AMG proteins include prokaryotic and eukaryotic expression systems, including eukaryotic cells, bacteria, fungi (e.g. yeast), insects, etc. Depending on the expression system used, nucleic acid may be introduced by any standard technique, including transfection, transduction, electroporation, bioballistics, microinjection, etc.
5.3. Anti-AMG AntibodiesThe present invention also provides for antibody molecules which react with AMG proteins and peptides. In specific, non-limiting examples, the invention provides for antibody molecules (as defined infra) which bind specifically to proteins having an amino acid sequence as set forth in SEQ ID NO:2 (AEG-1). According to the invention, an AMG protein or peptide, derivatives (e.g. a histidine-tagged protein), or analogs thereof, may be used as an immunogen to generate antibodies. Such antibodies include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments, and a Fab expression library.
Various procedures known in the art may be used for the production of polyclonal antibodies which specifically bind to an AMG protein or peptide. For the production of antibodies, various host animals can be immunized by injection with the protein or peptide, including but not limited to rabbits, mice, rats, goats, etc. Various adjuvants may be used to increase the immunological response, depending on the host species, and including but not limited to Freund's (complete or incomplete) adjuvant, mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacille Calmette-Guerin) and Corynebacterium parvum.
For preparation of monoclonal antibodies directed toward an AMG protein or polypeptide fragments thereof, any technique which provides for the production of antibody molecules by continuous cell lines in culture also may be used. Examples of such techniques include the hybridoma technique originally developed by Kohler & Milstein (1975, Nature 256, 495-497), as well as the trioma technique, the human B-cell hybridoma technique (Kozbor et al., Immunology Today 1983; 4, 72-79), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., 1985, pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing the technology disclosed in International Patent Application PCT/US89/02545. According to the invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al., 1983, Proc. Natl. Acad. Sci. U.S.A. 80, 2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al., in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, 1985, pp. 77-96). Further, according to the invention, techniques developed for the production of “chimeric antibodies” (Morrison et al., 1984, Proc. Nat. Acad. Sci. U.S.A. 81, 6851-6855; Neuberger et al., 1984, Nature 312, 604-608; Takeda et al., 1985, Nature 314, 452-454) by splicing the genes from a mouse antibody molecule specific for an AMG protein or peptide together with genes from a human antibody molecule of appropriate biological activity may be used; such antibodies are within the scope of this invention.
According to the invention, techniques described for the production of single chain antibodies (U.S. Pat. No. 4,946,778) may be adapted to produce AMG protein or peptide-specific single chain antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al., 1989, Science 246, 1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity.
Antibody fragments which contain the idiotype of the molecule can be generated by known techniques. For example, such fragments include but are not limited to: the F(ab′)2, fragment which can be produced by pepsin digestion of the antibody molecule; the Fab′ fragments which can be generated by reducing the disulfide bridges of the F(ab′)2, fragment, the Fab fragments which can be generated by treating the antibody molecule with papain and a reducing agent.
5.4. Screening MethodsThe present invention provides for methods of screening for agents that counteract the effect of HIV infection on astrocytes. Such methods comprise culturing early passage human fetal astrocytes, contacting the cultured astrocytes with HIV-1, further contacting the cultured astrocytes with an agent that is a candidate for counteracting the effects of HIV-1 infection on astrocytes, and monitoring the effect of the candidate agent on the expression of AEGs or ASGs, and comparing the level of expression of the AEGs, or ASGs in the presence and absence of the candidate agent, wherein an agent that counteracts the effects of HIV-1 expression in astrocytes is one that prevents the enhancement of expression of an AEG, or prevents the suppression of expression of an ASG.
According to such methods, the effect of a test agent on the expression of an AMG is monitored. Such methods are typically carried out in vitro using cultured astrocytes. An AMG is defined by a modulated level of expression, either upregulation or downregulation, or an altered temporal expression pattern, associated with HIV-1 infection and/or exposure to HIV-gp120; however, it may be found that other viruses or viral proteins may produce similar reductions in AMGs originally defined by an HIV-1/gp120 system, in which case such viruses or proteins may be used in the screening methods of the invention. For example, it may be preferable to use a crippled virus (e.g. incapable of proper packaging) rather than a wild-type virus to lessen the risks of infection.
According to the screening methods of the invention, a test agent may be evaluated for its ability to either prevent or decrease the enhancement of AEG expression associated with such viral infection or viral protein exposure, or to prevent or increase the reduction of ASG expression associated with such viral infection or viral protein exposure. The test agent may be introduced into an cell culture either prior to, concurrent with, or subsequent to introduction of virus or viral protein. The effect of the test agent on AMG expression may be monitored, for example, by measuring RNA and/or protein levels, using techniques known in the art. In specific, non-limiting embodiments of the invention, the AEGs to be monitored are AEG-1 through AEG-15, the ASGs to be monitored are ASG-1 through ASG-10. RNA levels of AEGs, or ASGs may be determined by Northern analysis or PCR. Protein levels may be determined by Western Blot Analysis using anti-AEG, or anti-ASG polyclonal or monoclonal antibodies. Similarly, other AMGs described herein may be monitored using similar methods.
Other screening methods of the invention are based on the discovery that enhanced AEG-1 expression decreases glutamate transport. Accordingly, the methods set forth above may be modified such that instead of measuring AEG levels directly, they may be measured indirectly, by determining the glutamate transport activity of the culture. For example, the amount of glutamate uptake of the astrocytes may be measured, for example, using detectably labeled glutamate. Intracellular levels of glutamate or the level of glutamate in the culture medium may be determined.
In related embodiments, the invention provides for screening methods for the identification of agents which increase glutamate transport. Such methods have the same steps as those intended to identify agents for treating or preventing HAD whereby either the expression level of AEG-1 or the glutamate transport activity is measured. Agents which increase glutamate transport may be useful in treating or preventing a variety of neurological conditions, including HAD, cerebral ischemia, amyotrophic lateral sclerosis, epilepsy and Alzheimer's disease.
In still other embodiments, the present invention provides for cell cultures and transgenic animals which overexpress an AEG or underexpress an ASG, thereby producing a model system for diseases associated with astrocyte pathology.
In the case of AEGs, the AEG may be placed under the control of a strong promoter. In particular non-limiting embodiments, the promoter may be inducible. Examples of suitable promoters are set forth in Section 5.1. The promoters are desirably active in astrocytes. In specific non-limiting embodiments of the invention, cells in culture or in a transgenic animal are engineered to contain AEG-1 under the control of a heterologous promoter, so that AEG-1 may be overexpressed and/or inducibly expressed. Such cells or animals may be used as model systems for study in HAD or other neurological conditions associated with AEG-1 overexpression, and may be used to identify agents which may treat or prevent such conditions.
In the case of ASGs, the expression of the ASG may be reduced in either the cell culture or transgenic animal model systems using an antisense construct or a ribozyme directed against the ASG mRNA sequence. Alternatively, triplex technology may also be used to reduce transcription of the ASG gene. ASG knock-out or knock-out/knock-in models may also be produced in which the ASG is disrupted by insertion of heterologous DNA.
Such cells or animals may be used as model systems for study in HAD or other neurological conditions associated with modulated expression of AMGs, and may be used to identify agents which may treat or prevent such conditions.
5.5. Methods of Diagnosis and TreatmentThe present invention further provides for methods of diagnosis and treatment of a disorder associated with a change in expression of one or more AMG. In one set of embodiments, the invention provides for methods of diagnosing HIV-1 infection and/or HAD in a subject comprising determining whether the level of an AEG is elevated in an astrocyte of the subject. The level of expression may be determined directly, for example using a brain biopsy, or indirectly, for example, by determining the level of an AEG protein or a metabolic product thereof in a body fluid of the subject, such as in serum or in cerebrospinal fluid. Metabolic imaging techniques analogous to PET scan may also be employed. An indirect metabolic product may also be used to detect increased AEG expression; for example, since AEG-1 decreases glutamate transport, an increase in cerebrospinal fluid levels of glutamate is consistent with enhanced AEG-1 expression.
In a second set of embodiments, the invention provides for methods of diagnosing HIV-1 infection and/or HAD in a subject comprising determining whether the level of an ASG gene is reduced in an astrocyte of the subject. The level of expression may be determined directly, for example using a brain biopsy, or indirectly, for example, by determining the level of an ASG protein or a metabolic product thereof in a body fluid of the subject, such as in serum or in cerebrospinal fluid. Metabolic imaging techniques analogous to PET scan may also be employed. An indirect metabolic product may also be used to detect decreased ASG expression.
Further, in view of the association between increased AEG-1 expression and decreased glutamate transport, the present invention provides for methods of decreasing extracellular glutamate levels, and hence treating or preventing conditions associated with neuronal glutamate toxicity, comprising antagonizing the effects of AEG-1. In one non-limiting embodiment, AEG-1 may be antagonized by inhibiting its expression using antisense RNA molecules complementary to all or a portion of AEG-1 nucleic acid, for instance having the sequence set forth as SEQ ID NO:1. The design of antisense molecules, including oligonucleotides, is known in the art. Conditions which may be treated or prevented in this manner include but are not limited to cerebral ischemia, amyotrophic lateral sclerosis, and Alzheimer's disease. The present invention further provides for the treatment or prevention of HAD by antagonizing the effects of AEG-1, for example using antisense molecules as set forth supra.
The present invention further provides for the treatment or prevention of HAD by overexpression of ASGs, for example by using vectors of various forms, both virus-based and non-virus-based, to introduce additional expressible copies of ASGs into astrocytes. Examples of such vectors for the expression of ASGs have been set forth supra.
6. EXAMPLE 6.1. Materials and MethodsHuman fetal astrocytes, other cells and cell culture conditions. Fetal astrocytes were isolated from second trimester (gestational age 16-19 wk) human fetal brains obtained from elective abortions in fill compliance with NIH guidelines, as previously described (Bencheikh et al., 1999, J. Neurovirol. 5, 115-124; Canki et al., 2001, J. Virol. 75, 7925-7933). Highly homogenous preparations of astrocytes were obtained using high-density culture conditions in the absence of growth factors in F12 Dulbecco's Modified Eagle Medium (GIBCO-BRL, Gaithersburg, Md.) containing 10% fetal bovine serum (FBS), penicillin, streptomycin, and gentamycin. Cells were maintained in this medium at 2-5×104 cells/cm2 and subcultured weekly up to six times. For each experiment a single batch of astrocytes of similar gestational age and passage was used. Cultures were regularly monitored for expression of the astrocytic marker glial fibrillary acidic protein (GFAP) and either HAM56 or CD68 to identify cells of monocyte/macrophage lineage. Only cultures that contained≧99% GFAP positive astrocytes and rare or no detectable HAM56- or CD68-positive cells were used in these experiments (Canki et al., 2001, J. Virol. 75, 7925-7933). Other cells used in this study were the human embryonal-kidney epithelial cell line 293T (Graham et al., 1977, J. Gen. Virol. 36, 59-72), used for HIV-1 propagation, and MAGI cells, a derivative of HeLa carrying the β-gal gene under the control of HIV-1 LTR and expressing HIV-1 receptors (Kimpton & Emerman, 1992, J. Virol. 66, 2232-2239), used as indicator cells for HIV-1 titration. Both cell lines were cultured in 90% DMEM/10% FBS supplemented with antibiotics and, for MAGI cells, 0.2 mg/ml G-418. Culture media and cells were tested for mycoplasma contamination using the Mycoplasma PCR ELISA kit (Roche Molecular Biochemicals, Indianapolis, Ind.) and found to be negative.
HIV-1 propagation. The HIV-1 strain used in this work was NL4-3, a prototypical X4-tropic laboratory clone of HIV-1 that expresses all known HIV-1 proteins (Adachi et al., 1986, J. Virol. 59, 284-91). Virus propagation was initiated by transfection of 15 μg of NL4-3 DNA into 1.5×106 293T cells as previously described (Bencheikh et al., 1999, J. Neurovirol. 5, 115-124). Culture supernatants were harvested 72 h after transfection, filtered through a 0.45 μm Millipore filters, and stored at −80° C. until use. Cell-free viral stock was tested for HIV-1 p24 core antigen content by ELISA using HIV-1 Ag kit according to the manufacturer's instructions (Coulter, Hialeah, Fla.) and for titers of infectious virus by multinuclear activation of a β-galactosidase indicator (MAGI) assay (Kimpton & Emerman, 1992, J. Virol. 66, 2232-2239). Culture supernatants contained 1-2 μg/ml of viral p24 protein and 1-2×106 infectious units (I.U.) per ml. In our experience, a MOI of one for CD4-positive T cells is approximately 1 pg of viral p24 per cell (Dewhurst et al., 1987, J. Virol. 61, 3774-3782; Canki et al., 2001, J. Virol. 75, 7925-7933)). Virus stocks were also tested for mycoplasma contamination as described above and for endotoxin using the E-TOXATE kit (Limulus Amebocyte Lysate, Sigma, St. Louis, Mo.), and found to be negative in both tests.
HIV-1 infection of astrocytes with HIV-1 or exposure of the cells to gp120, and preparation of samples for cellular RNA analysis. Confluent cultures of human fetal astrocytes in 225 cm2 culture flasks were exposed to HIV-1 in 10 ml of medium at 1 pg p24 per cell for 2 h at 37° C., washed 3 times in warm PBS, and cultured in astrocyte culture medium as described. Control astrocytes were treated as described above but without HIV-1. At 6 h, 12 h, 24 h, 3 d and 7 d after infection, culture supernatants were removed, and control and infected cells were washed 3 times in PBS and solubilized by addition of 10 ml of 4M guanidine isothiocyanate directly to culture flasks. Cell lysates were stored at −80° C. until further use. To insure preparation of sufficient amount of RNA for subsequent subtractive hybridization, astrocyte cultures and HIV-1 infections were scaled up to approximately 1×108 cells per time point (infected or control cells); the RNA yield was 5-10 μg per 106 cells. Infection of astrocytes with HIV-1 was verified by testing the levels of HIV-1 p24 antigen in culture supernatants by p24 ELISA as described previously (Bencheikh et al., 1999, J. Neurovirol. 5, 115-124; Canki et al., 2001, J. Virol. 75, 7925-7933). gp120 used for these experiments was a full-length, glycosylated protein from HIV-1MN produced from baculovirus vector and purified by ImmunoDiagnostics, and provided through the AIDS Research and Reference Reagent Program (Rockville, Md.). For gp120 treatment of astrocytes, large-scale cultures of cells prepared as described above were treated with gp120 at 1 nM in 10 ml medium for 2 h, and cells were washed, cultured, and extracted for RNA isolation as described for HIV-1 infection.
RNA isolation and Northern blot analysis. Uninfected, HIV-1 infected and exposed gp120 astrocytes were treated with 4M-guanidiniurn and total RNA was isolated by the guanidinium/phenol procedure and analyzed by Northern blotting as described previously (Jiang & Fisher, 1993, Mol. Cell. Different. 1, 285-299; Kang et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95, 13788-13793; Kang et al., 2001, Gene 267, 233-242). Northern blots were quantitated by densitometric analysis using a Molecular Dynamics densitometer (Sunnyvale, Calif.). Relative expression of the different AEGs or ASGs versus GAPDH expression was determined at different time points for HIV-1 infected (H-AEG/H-GAPDH or H-ASG/H-GAPDH), gp120-treated (G-AEG/G-GAPDH or G-ASG/G-GAPDH) and control uninfected cultures (C-AEG/C-GAPDH or C-ASG/C-GAPDH). Relative fold-change in expression of each AMG at 6 h, 12 h, 24 h, 3 d and 7 d was then determined for each condition by dividing H-AEG/H-GAPDH or H-ASG/H-GAPDH by C-ASG/C-GAPDH or C-ASG/C-GAPDH, respectively, to generate fold HIV modulation; or by dividing G-AEG/G-GAPDH or G-ASG/G-GAPDH by C-AEG/C-GAPDH or C-ASG/C-GAPDH to generate fold gp120 modulation. Poly(A) RNA was purified using Oligo(dT) cellulose columns (GIBCO BRL).
AEG Protein Analysis by Immunoblotting. Analysis of AEG protein products in astrocytes was determined by immunoblotting. Astrocytes were infected with HIV-1 at an M.O.I. of 1, washed, and cultured in parallel with uninfected controls as described above. At the designated times, cells were counted and lysed in a buffer containing 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, 5 mM iodoacetamide, 0.2 U/ml phenylmethylsulfonyl fluoride; cell lysates corresponding to equivalent number of cells were resolved by SDS-PAGE on 4-15% polyacrylamide ready gels (Bio-Rad, Hercules, Calif.) and transferred onto a 0.2 μm Trans-Blot nitrocellulose membrane (Bio-Rad). The membranes were incubated in 5% (w/v) skim milk in T-PBS (0.1% polyoxyethyline-sorbitan monolaurate in phosphate buffered saline) and then stained with the indicated primary antibodies followed by horseradish peroxidase-conjugated second antibody. Protein bands were visualized on x-ray film after luminescence reaction using an ECL kit (Amersham, Arlington, Ill.). Samples were standardized by their α-tubulin content prior to final evaluations. Antibodies used were: rabbit polyclonal anti-fibronectin antibody (Abcam, Cambridge, UK), monoclonal anti-α-actinin (Sigma), and monoclonal anti-β-tubulin (Sigma).
Primer design for RaSH procedure. The sequences of oligonucleotides that were used are as follows: XDPN-18 CTGATCACTCGAGAGATC (SEQ ID NO:5), XDPN-14 CTGATCACTCGAGA (SEQ ID NO:6), XDPN-12 GATCTCTCGAGT (SEQ ID NO:7). The adapters formed from the two sets of oligonucleotides contained an XhoI recognition site.
Preparation of PCR-based cDNA libraries used in RaSH procedure. To clone AEG cDNAs expressed at elevated levels in early passage human fetal astrocytes, 1 μg of poly(A) RNA from temporally spaced (6 h, 12 h, 24 h, and 3 d and 7 d) uninfected astrocytes (driver) or temporally spaced (6 h, 12 h, 24 h, and 3 d and 7 d) HIV-1 infected astrocytes (tester) prepared as described above was used for double-stranded cDNA synthesis using standard protocols (Gubler & Hoffman, 1983, Gene 25, 263-269). To clone ASG cDNAs expressed at reduced levels in early passage human fetal astrocytes, 1 μg of poly(A) RNA from temporally spaced (6 h, 12 h, 24 h, and 3 d and 7 d) HIV-1-infected astrocytes (driver) or temporally spaced (6 h, 12 h, 24 h, and 3 d and 7 d) mock-infected astrocytes (tester) prepared as described above was used for double-stranded cDNA synthesis using standard protocols (Gubler & Hoffman, 1983, Gene 25, 263-269).
The resulting cDNAs were digested with DpnII (New England Biolab, Beverly, Mass.) at 37° C. for 3 h followed by phenol/chloroform extraction and ethanol precipitation. The digested cDNAs were mixed with primers XDPN-14/XDPN-12 (final concentration 20 μM) in 30 μl of 1× ligation buffer (GIBCO BRL), heated at 55° C. for 1 min, and cooled down to 14° C. within 1 h. After adding 3 μl of T4 ligase (5 U/μl) (GIBCO BRL) to the mixtures individually, ligation was carried out at 14° C. overnight. The mixtures were diluted to 100 μl with TE buffer (pH 7.0), and at least 40 μl of the mixtures were used for PCR amplification. The PCR mixtures were set up as follows: 1 μl of the cDNA mixture, 10 μl 10×PCR buffer, 1 mM MgCl2, 0.4 mM dNTPs, 1 μM XDPN-18, and 1 U Taq polymerase (GIBCO BRL). The parameters for PCR were one cycle for 5 min at 72° C. followed by 25 cycles for 1 min at 94° C., 1 min at 55° C., 1 min at 72° C. preceded by one cycle for 3 min at 72° C. The PCR products were pooled and purified using Centricon columns (Amicon, Bedford, Mass.). Ten μg of the tester PCR products were digested with XhoI followed by phenol/chloroform extraction and ethanol precipitation.
Subtraction hybridization and generation of subtracted libraries. One hundred ng of the tester cDNA were mixed with 3 μg of the driver cDNA in 10 μl of a hybridization solution (0.5 M NaCl, 50 mM Tris pH 7.5, 0.2% SDS, 40% formamide), and after boiling for 5 min, incubated at 42° C. for 48 h. The hybridization mixture was phenol/chloroform extracted, ethanol precipitated, and dissolved in 20 μl of TE buffer. One μl of the mixture was ligated with 1 μg of XhoI-digested, CIP-treated pCRII plasmids, overnight at 14° C., and transformed into Shot-1 bacteria.
Colony screening. Bacterial colonies were randomly picked and PCR amplified. The PCR products were blotted onto filters and reverse Northern blotting was performed to identify cDNAs displaying differential expression in HIV-1 infected versus uninfected early passage human fetal astrocytes (Kang et al., 1998, Proc. Natl. Acad. Sci. U.S.A. 95, 13788-13793; Huang et al., 1999, Gene 236, 125-131; Jiang et al., 2000, Proc. Natl. Acad. Sci. USA. 97, 12684-12689). cDNAs displaying elevated expression in HIV-1 infected fetal astrocytes versus uninfected fetal astrocytes were designated AEG with a clone number of AEG-1 to AEG-15, while cDNAs displaying reduced expression in HIV-1 infected fetal astrocytes versus uninfected fetal astrocytes were designated ASG with a clone number of ASG-1 to ASG-10. Appropriate expression of the AMG clones identified by reverse Northern blotting was confirmed by Northern blotting. The sequences of these clones were determined using automated cycle sequencing at the DNA facility of Columbia University.
6.2. ResultsInfection of human astrocytes with HIV-1 and cloning of the AMGs using the RaSH approach. Human fetal astrocytes were cultured and infected with HIV-1 (NL4-3 clone) as previously described (Bencheikh et al., 1999, J. Neurovirol. 5, 115-124; Canki et al, 2001, J. Virol. 75, 7925-7933). Mock-infected cells were cultured and handled similarly in parallel as a control. HIV-1 infection of astrocytes alters gene expression and cell function (He et al., 1997, Proc. Natl. Acad. Sci. U.S.A. 94, 3954-3959; Kort, 1998, AIDS Res. Hum. Retroviruses 14, 1329-1339). To define the repertoire of genes modified as a consequence of infection of early passage fetal astrocytes with HIV-1, the efficient and rapid cloning approach RaSH was used (Jiang et al., 2000, Proc. Natl. Acad. Sci. USA. 97, 12684-12689; Kang et al., 2001, Gene 267, 233-242; Simm et al., 2001, Gene 269, 93-101). A schematic of this approach as applied to this fetal astrocyte model and HIV-1 infection is shown in
In general, the RaSH procedure followed that described for identification of cellular genes displaying elevated expression in astrocytes, as shown in
Selection of subtracted cDNAs for the identification of AEGs was achieved by matching the ends of the cDNA fragments to the ends of the plasmid vectors during ligation and construction of subtracted libraries (
Using this approach, 15 distinct AEGs displaying enhanced expression (
Sequence analysis of the RaSH-derived ESTs revealed nine previously identified ASGs and one unknown ASG, designated ASG-1 (novel), not reported in current databases (Table 1). Among the known ASG products are proteins involved in cell movement and cell differentiation (ASG-3, platelet-endothelial tetraspan antigen 3 (PETA-3); ASG-5, neuronatin; and ASG-6, a neuroendocrine differentiation factor), as well as intracellular regulators of signal transduction and gene expression (ASG-4, a guanine-nucleotide releasing protein C3 G; ASG-7, cysteine/glycine rich protein 1 (CSRP1); and ASG-10, signal recognition particle 9 (SRP9).
In contrast, the AEGs included a wider range of genes, including AEG-2 (G-binding protein), AEG-3 (GA 17 protein) (Ryo et al., 2000, AIDS Res. Hum. Retroviruses 16, 995-1005), AEG-4 (unr/NRU) (Jeffers et al., 1990, Nucleic Acids Res. 18, 4891-4899; Boussadia et al., 1993, Biochim. Biophys. Acta 1172, 64-72), AEG-5 (hGNT-IV-H) (Furukawa et al., 1999, J. Hum. Genet. 44, 397-401), AEG-6 (fibronectin), AEG-7 (human CTL2) (O'Regan et al., 2000, Proc. Natl. Acad. Sci. U.S.A. 97, 1835-1840), AEG-8 (acidic ribosomal phosphoprotein) (Rich & Stietz, 1987, Mol. Cell. Biol. 7, 4065-4074), AEG-9 (calnexin) (Honore et al., 1994, Electrophoresis 15, 482-490; Rubio & Wenthold, 1999, J. Neurochem. 73, 942-948; Shi et al., 2001, Cell 105, 331-342), AEG-10 (autotaxin) (Stracke et al., 1994, J. Biol. Chem. 267, 2524-2529; Kawagoe et al., 1997, Cancer Res. 57, 2516-2521), AEG-12 (thymosin β-4) (Gondo et al., 1987, J. Immunol. 139, 3840-3848), AEG-13 (human non-muscle α-actinin) (Youssoufian et al., 1990, Am. J. Hum. Genet. 47, 62-71), AEG-14 (Schneider et al., 1988, Cell 54, 787-793; Gonos, 1998, Ann. N.Y. Acad. Sci. 851, 466-469; Prieto et al., 1999, Brain Res. 816, 646-661) and AEG-15 (PGK-1) (Michelson et al., 1983, Proc. Natl. Acad. Sci. USA 80, 472-476; Tsukada et al., 1991, J. Gerontol. 46, B213-B216).
Expression analysis and characterization of AMGs. The differential expression of the RaSH-derived AMGs was confirmed by Northern blot analyses, shown in
Compared to their expression in control cells, an ˜1.5- to ˜4.5-fold temporal increase in expression of the various AEGs was apparent following HIV-1 infection and an ˜1.5- to ˜3.2-fold temporal increase was seen following gp120 treatment (Table 1; see below). The expression of specific ASGs was reduced at defined time-points up to ˜5.1-fold by HIV-1 infection and up to ˜4.2-fold following gp120 treatment (Table 2 and
In general, the temporal pattern and magnitude of enhanced AEG expression was similar in cells infected with HIV-1 or treated with gp120. However, expression of AEG-2 (G-binding protein), AEG-5 (hGNT-IV-H) (Furukawa et al., 1999), AEG-7 (human CTL2) (O'Regan et al., 2000), AEG-8 (acidic ribosomal phosphoprotein) (Rich & Stietz, 1987) and AEG-15 (PGK-1) (Michelson et al., 1983, Proc. Natl. Acad. Sci. USA 80, 472-476; Tsukada et al., 1991, J. Gerontol. 46, B213-B216) was elevated in fetal astrocyte cultures more rapidly following HIV-1 infection than gp120 treatment (
AEG-3 (GA17 protein), AEG-11 (novel) and AEG-13 (human non-muscle α-actinin) appear to be early response genes, which are elevated by 6 h after infection with HIV-1 or treatment with gp120 (
HIV-1 infection and gp120 treatment of early passage fetal astrocytes enhances fibronectin and α-actinin protein expression. To determine if elevated AEG mRNA correlated with enhanced protein expression, we obtained commercially available antibodies to fibronectin (AEG-6), calnexin (AEG-9) and human non-muscle α-actinin (AEG-13) and tested expression of these proteins in HIV-1 infected and control astrocytes by Western blotting. The results of analyses of fibronectin and α-actinin protein expression in two independently derived primary human fetal astrocyte cultures are shown in
Similar to differentially expressed gene products described above for AEGs, the ASGs also display distinct patterns of expression over time in response to HIV-1 infection of astrocytes, again stratifying into early and sustained versus late responders (Table 2,
Although the ASG library was constructed on the basis of transcripts from HIV-1 infected astrocytes (
Another implication of these results is that some modulatory effects of HIV-1 on cellular gene expression in human astrocytes, like the ones represented by changes in AEG and ASG, are independent of virus replication because they can be reproduced to a significant extent by treatment of cells with recombinant gp120 in the absence of other viral products (
The replication-independent modulation of cellular gene expression by HIV-1 is likely mediated by efficient viral interaction with surface receptors on astrocytes. Although these cells lack surface CD4, the canonical HIV-1 receptor on T cells and macrophages (Klatzman et al., 1984, Nature 312, 767-768; Cheng-Mayer et al., 1987, Proc. Natl. Acad. Sci. USA 84, 3526-3530), they do express a high-molecular weight protein of about 260 kDa which was shown to bind gp120 with high affinity (Ma et al., 1994, J. Virol. 68, 6824-6828) and which may be responsible for binding intact HIV-1 as well. Astrocytes also express the chemokine receptors CXCR4 and CCR5 (Andjelkovic et al., 1999, Glia 28, 225-235; Rezaie et al., 2002, Glia 37, 64-75) as well as galactocerebroside (Harouse et al., 1989, J. Virol. 63, 2527-2533), all of which can bind HIV-1 envelope. Further studies are needed to determine the identity of the membrane receptors on astrocytes that may mediate the HIV-1 effects on cellular gene expression observed here and in our other studies.
Although RNA transcripts of a single size is apparent in astrocytes after probing with ASG-3, -4, -5, -7 and -9, multiple hybridizing RNA species are apparent in Northern blots after probing astrocyte RNAs with ASG-1, -2, -6, -8 and -10 (
HIV-1-mediated dysregulation of cellular gene expression in human astrocytes and neuropathogenesis. The major implication of the findings presented here is that HIV-1 has profound, global effects on expression of a broad array of cellular genes in astrocytes, suggesting that this may be one route through which HIV-1 infected astrocytes contribute to HAD. Overall, 25 genes were identified as differentially expressed in astrocytes as a result of HIV-1 exposure, 15 of these were upregulated in their expression (AEGs) and 10 suppressed (ASGs). Based on the size of the RaSH-derived EST libraries utilized in these studies, the number of differentially expressed genes in this system may exceed 100. The magnitude of this HIV-1 effect on astrocyte biology is more remarkable as it occurs despite relatively inefficient viral expression in these cells (Tornatore et al., 1991, J. Virol. 65, 6094-6100; Canki et al., 2001, J. Virol. 75, 7925-7933; Su et al., 2002, Oncogene 21, 3592-3602) and it can be reproduced by treatment of astrocytes with isolated HIV-1 envelope glycoprotein (
The AEGs (Table 1) and ASGs (Table 2) identified thus far can provide leads for investigation of cellular pathways co-opted by HIV-1 in astrocytes. One important indicator is the time of gene activation or suppression relative to HIV-1 infection. Early modulation of cellular genes (6 h and 24 h after HIV-1 infection in our studies) may indicate direct cellular responses to HIV-1 mediated by signal transduction mechanisms activated by virus interaction with cell surface receptors or by disruption of the plasma membrane integrity during virus-cell fusion. Such responses were observed after HIV-1 exposure in T cells (Gupta & Vayuvegula, 1987, J Clin Immun 7, 486-489; Fermin & Garry, 1992, Virology 191, 941-946; Miller et al., 1993, Virology 196, 89-100), macrophages (Zheng et al., 1999, J Virol 73, 8256-8267; Choe et al., 2001, J Virol 75, 10738-10745), and neurons (Zheng et al., 1999, J Virol 73, 8256-8267). The early-responder genes are represented by ASG-4, -7, and -8, all of which show a decline in expression within 6-24 h after HIV-1 infection (
Similar to the AEG series of astrocyte genes, the majority of ASGs described here (6 out of 10) appear to be late-response genes, that is, their expression declined maximally only 3 d to 7 d after HIV-1 infection. These genes include ASG-1 (novel), ASG-3 (platelet-endothelial cell tetra-span antigen 3, or CD 151/PETA-3; Fitter et al., 1995, Blood 86, 1348-55; Yáñez-Mó et al., 1998, J Cell Biol. 141, 791-804), ASG-5 (neuronatin; Dou & Joseph, 1996, Brain Res. 723, 8-22; Usui et al., 1997, J. Mol. Neurosci. 9, 55-60), ASG-6 (neuroendocrine differentiation factor; CGI149; Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86, 4504-11), ASG-9 (human mitochondrion genomic DNA, this fragment is homologous to the 952 to 1232-bp region of genomic DNA which encodes 16s rRNA (from the 650 to 1603-bp region of genomic DNA), and ASG-10 (signal recognition particle, SRP9; Lütcke, 1995, Eur. J. Biochem. 228, 531-550). PETA-3 (ASG-3) is a glycoprotein of 253 amino acids that belongs to the tetraspanin family of surface proteins (Fitter et al., 1995, Blood 86, 1348-55; Testa et al., 1999, Cancer Res 59, 3812-3820). PETA-3 RNA is downmodulated almost 4-fold in infected versus uninfected astrocytes (Table 2). Although PETA-3 was originally identified as a platelet surface protein, recent data indicate that it functions as a component of integrin signaling complexes on endothelial cells and it may be involved in regulation of cell motility (Yáñez-Mó et al., 1998, J. Cell Biol. 141, 791-804; Testa et al, 1999, Cancer Res 59, 3812-3820). Dowmnodulation of PETA-3 in astrocytes by HIV-1 may affect astrocyte function in maintaining the integrity of the blood-brain-barrier (BBB) by reducing both the flexibility and adhesion strength of the astrocytic underlayer of the BBB (Morgello et al., 1995, Glia 14, 43-54). Neuronatin (ASG-5) is a brain-specific human protein that is selectively expressed during development (Dou & Joseph, 1996, Genomics 33, 292-297; Dou & Joseph, 1996, Brain Res. 723, 8-22) and therefore it is unlikely to play a role in the adult disease such as HAD. The five-fold downmodulation of neuronatin observed here is of interest because of the proposed function of the protein as a regulator of anion channels (Dou & Joseph, 1996, Brain Res. 723, 8-22), an activity that may be functionally related to the observed downregulation of glutamate transport in these cells (Wang et al., 2002, J. Virol., in press).
The neuroendocrine differentiation factor, NEDF (ASG-6), has been recently identified by a yeast two-hybrid screening as a novel intracellular protein that interacts with the IGF-binding protein-related protein-1 (IGFBP-rP1) and proposed to act together with IGFBP-rP1 in inducing neuroendocrine cell differentiation in response to IGF (Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86, 4504-11). ASG-6 is of interest in the context of HIV-1 infection because IGF-like growth factors appear to be protective during CNS injury (Bondy & Lee, 1993, Ann. N. Y. Acad. Sci. 692, 33-43) and HIV-1 disease correlates with defects in the insulin-like growth factor system (Frost et al., 1996, Clin. Endocrinol. 44, 501-514; Jain et al., 1998, Endocr. Rev. 5, 625-646). Also of note, it has been suggested that the NEDF (ASG-6)/IGFBP-rP1 complex acts through the Ras/MAPK signaling pathway (Wilson et al., 2001, J. Clin. Endocrinol. Metab. 86, 4504-11), an alternative to the Crk/C3G/Rap1 pathway discussed earlier in the context of observed downmodulation of C3G (ASG-4). Thus two genes in astrocytes whose expression is reduced by HIV-1, one early (ASG-4) and one late (ASG-6) after infection, encode products that transduce signals from RTKs, indicating that this signal transduction pathway is a major target for HIV-1 mediated dysregulation of astrocyte physiology. Experiments are now under way to investigate this possibility.
ASG-10 encodes a signal recognition particle SRP9, a component of Alu RNA binding heterodimer SRP9/14 (Lütcke, 1995, Eur. J. Biochem. 228, 531-550). SRP targets secretory and membrane proteins to rough endoplasmic reticulum in a complex, co-translational process that includes a temporary arrest of elongation (Lütcke, 1995, Eur. J. Biochem. 228, 531-550). In the CNS, the heterodimer SRP9/14 was found to be an integral part of the brain-specific BC200 RNA, a small non-messenger RNA that is a constituent of a ribonucleoprotein complex in neurons and that is believed to regulate protein biosynthesis in dendrites (Kremerskothen et al., 1998, Neurosci. Lett. 245, 123-126). Downmodulation of SRP9 expression by HIV-1 may impair the formation and function of the SRP9/14 heterodimer and, consequently, affect synthesis of secretory and membrane proteins by astrocytes. The virus could adopt this mechanism as a means for reducing exposure of infected astrocytes to immune recognition, similar in an outcome to the Nef-mediated downmodulation of HLA-Class I in T lymphocytes (Collins et al., 1998, Nature 391, 397-401).
Various publications are cited herein, the contents of which are hereby incorporated by reference in their entireties.
Claims
1-18. (canceled)
19. A protein encoded by a nucleic acid molecule, wherein the sequence of the nucleic acid molecule comprises nucleotides 220-1968 of SEQ ID NO:1.
20. (canceled)
21. (canceled)
22. A protein encoded by a nucleic acid molecule, wherein the nucleic acid molecule comprises residues which are at least 90 percent identical to nucleotides 220-1968 of SEQ ID NO:1.
23. (canceled)
24. (canceled)
25. A protein having an amino acid sequence as set forth in SEQ ID NO:2.
26-31. (canceled)
32. A peptide which is a fragment of the protein of claim 19, and which binds to an antibody which binds to a protein having an amino acid sequence as set forth in SEQ ID NO:2.
33. (canceled)
34. (canceled)
35. A peptide which is a fragment of the protein of claim 22, and which binds to an antibody which binds to a protein having an amino acid sequence as set forth in SEQ ID NO:2.
36. (canceled)
37. (canceled)
38. A peptide which is a fragment of the protein of claim 25, and which binds to an antibody which binds to a protein having an amino acid sequence as set forth in SEQ ID NO:2.
39-63. (canceled)
Type: Application
Filed: Apr 13, 2009
Publication Date: Aug 13, 2009
Inventors: Paul B. Fisher (Scarsdale, NY), Zhao-zhong Su (New York, NY), Dong-chul Kang (Dongan-gu)
Application Number: 12/422,882
International Classification: C07K 14/16 (20060101);
