PrPC, the Cellular Isoform of the Human Prion Protein, Is a Novel Biomarker of HIV-Associated Neurocognitive Impairment and Mediates Neuroinflammation
2010; Elsevier BV; Volume: 177; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2010.091006
ISSN1525-2191
AutoresToni K. Roberts, Eliseo A. Eugenín, Susan Morgello, Janice E. Clements, M. Christine Zink, Joan W. Berman,
Tópico(s)Neurological diseases and metabolism
ResumoOf the 33 million people infected with the human immunodeficiency virus (HIV) worldwide, 40–60% of individuals will eventually develop neurocognitive sequelae that can be attributed to the presence of HIV-1 in the central nervous system (CNS) and its associated neuroinflammation despite antiretroviral therapy. PrPC (protease resistant protein, cellular isoform) is the nonpathological cellular isoform of the human prion protein that participates in many physiological processes that are disrupted during HIV-1 infection. However, its role in HIV-1 CNS disease is unknown. We demonstrate that PrPC is significantly increased in both the CNS of HIV-1–infected individuals with neurocognitive impairment and in SIV-infected macaques with encephalitis. PrPC is released into the cerebrospinal fluid, and its levels correlate with CNS compromise, suggesting it is a biomarker of HIV-associated neurocognitive impairment. We show that the chemokine (c-c Motif) Ligand-2 (CCL2) increases PrPC release from CNS cells, while HIV-1 infection alters PrPC release from peripheral blood mononuclear cells. Soluble PrPC mediates neuroinflammation by inducing astrocyte production of both CCL2 and interleukin 6. This report presents the first evidence that PrPC dysregulation occurs in cognitively impaired HIV-1–infected individuals and that PrPC participates in the pathogenesis of HIV-1–associated CNS disease. Of the 33 million people infected with the human immunodeficiency virus (HIV) worldwide, 40–60% of individuals will eventually develop neurocognitive sequelae that can be attributed to the presence of HIV-1 in the central nervous system (CNS) and its associated neuroinflammation despite antiretroviral therapy. PrPC (protease resistant protein, cellular isoform) is the nonpathological cellular isoform of the human prion protein that participates in many physiological processes that are disrupted during HIV-1 infection. However, its role in HIV-1 CNS disease is unknown. We demonstrate that PrPC is significantly increased in both the CNS of HIV-1–infected individuals with neurocognitive impairment and in SIV-infected macaques with encephalitis. PrPC is released into the cerebrospinal fluid, and its levels correlate with CNS compromise, suggesting it is a biomarker of HIV-associated neurocognitive impairment. We show that the chemokine (c-c Motif) Ligand-2 (CCL2) increases PrPC release from CNS cells, while HIV-1 infection alters PrPC release from peripheral blood mononuclear cells. Soluble PrPC mediates neuroinflammation by inducing astrocyte production of both CCL2 and interleukin 6. This report presents the first evidence that PrPC dysregulation occurs in cognitively impaired HIV-1–infected individuals and that PrPC participates in the pathogenesis of HIV-1–associated CNS disease. Approximately 33 million people are infected with the human immunodeficiency virus (HIV) worldwide.1UNAIDS Report on the global AIDS epidemic 2008. 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We compared CNS PrPC expression in HIV-1–infected individuals with and without cognitive impairment to uninfected individuals and evaluated its soluble form as a potential biomarker of CNS dysfunction. We used the pigtail macaque model of neuroAIDS to investigate alterations in PrPC during defined stages of an accelerated disease process. We found PrPC is increased significantly in the brain of HIV-1–infected individuals with neurocognitive impairment, relative to infected and uninfected individuals who are unimpaired, and in SIV-infected macaques with encephalitis, as compared to infected and uninfected animals without encephalitis. We found that elevated soluble PrPC (sPrPC) cerebrospinal fluid (CSF) levels predict neurocognitive impairment in HIV-1–infected individuals, suggesting that CSF sPrPC is a biomarker of HAND. We also showed that sPrPC in the CSF reflects the extent of encephalitis in SIV-infected macaques. We demonstrated that CCL2, a chemokine that is elevated in the CNS of individuals with HAND and is associated with neuropathology,27Cinque P Vago L Mengozzi M Torri V Ceresa D Vicenzi E Transidico P Vagani A Sozzani S Mantovani A Lazzarin A Poli G Elevated cerebrospinal fluid levels of monocyte chemotactic protein-1 correlate with HIV-1 encephalitis and local viral replication.AIDS. 1998; 12: 1327-1332Crossref PubMed Scopus (211) Google Scholar, 28Conant K Garzino-Demo A Nath A McArthur JC Halliday W Power C Gallo RC Major EO Induction of monocyte chemoattractant protein-1 in HIV-1 Tat-stimulated astrocytes and elevation in AIDS dementia.Proc Natl Acad Sci USA. 1998; 95: 3117-3121Crossref PubMed Scopus (502) Google Scholar significantly increases PrPC release from CNS cells and that HIV-1 infection alters the release of PrPC from peripheral blood mononuclear cells in vitro. We also showed that sPrPC contributes to neuroinflammation by increasing the production of CCL2 and interleukin (IL)-6 by astrocytes. This is the first demonstration of PrPC dysregulation in neurocognitively impaired individuals infected with HIV-1. Tissue was collected as part of the IRB-approved protocols of the Manhattan HIV Brain Bank. Research subjects undergo a rigorous battery of neuropsychological, neuromedical, and psychiatric assessments, as described previously,29Ryan EL Morgello S Isaacs K Naseer M Gerits P Neuropsychiatric impact of hepatitis C on advanced HIV.Neurology. 2004; 62: 957-962Crossref PubMed Scopus (141) Google Scholar and blood is evaluated for viral load and CD4 T-cell counts. Cognitive diagnoses are rendered by two systems. We operationalized American Academy of Neurology criteria for diagnoses of HIV-associated dementia and minor cognitive motor disorder.30Clinical confirmation of the American Academy of Neurology algorithm for HIV-1-associated cognitive/motor disorder. The Dana Consortium on Therapy for HIV Dementia and Related Cognitive Disorders.Neurology. 1996; 47: 1247-1253Crossref PubMed Scopus (131) Google Scholar Cerebral cortex was evaluated from four seronegative individuals without cognitive impairment, two HIV-1–infected individuals without cognitive impairment, four HIV-1–infected individuals with minor motor cognitive disorder (MCMD), two individuals with HIV-associated dementia (HAD), and two individuals with HIV encephalitis (HIVE). We also examined tissue from underlying white matter obtained from the same individuals described above with similar results (data not shown). CSF samples were evaluated from five uninfected individuals who are unimpaired, five HIV-1–infected individuals with MCMD, and five HIV-1–infected individuals with HAD. Sera samples were evaluated from three uninfected individuals without cognitive impairment, three uninfected individuals with neurocognitive impairment, five HIV-1–infected individuals without cognitive impairment, five HIV-1 seropositive individuals with MCMD, and five HIV-1–infected individuals with HAD (see details in Table 1).Table 1Elevated Expression of CNS PrPC Is Independent of Age, Gender, Race, Peripheral Viral Load, and CD4 T-Cell CountAge (years)Sex (male/female)RacePeripheral viral load (copies/ml)Peripheral CD4 count (cells/ml)HIV (−)57FWNANAHIV (−)58FHNANAHIV (−)48MBNANAHIV (−)50MHNANAHIV (+)46MWUndetectable233normalHIV (+)49MWUndetectable103normalHIV (+)58MBUndetectable204MCMDHIV (+)58FB576,00098MCMDHIV (+)28MA20975MCMDHIV (+)50MW26,62120MCMDHIV (+)49MB1521215HADHIV (+)51MH65136HADHIV (+)44MW389,1207HIVEHIV (+)47MB367,6202HIVEDemographics of individuals from which cerebral cortex tissue sections were evaluated for PrPC expression by immunohistochemistry. MCMD indicates minor motor cognitive disorder; HAD, HIV-associated dementia; HIVE, HIV encephalitis; F, female; M, male; W, white/Caucasian; H, Hispanic; B, black/African American; A, Asian; NA, not applicable and not assessed. Open table in a new tab Demographics of individuals from which cerebral cortex tissue sections were evaluated for PrPC expression by immunohistochemistry. MCMD indicates minor motor cognitive disorder; HAD, HIV-associated dementia; HIVE, HIV encephalitis; F, female; M, male; W, white/Caucasian; H, Hispanic; B, black/African American; A, Asian; NA, not applicable and not assessed. Pig-tailed macaques (Macaca nemestrina) were dual-inoculated intravenously with a virus swarm, SIV/DeltaB670, and with the molecular clone SIV/17E-Fr.31Zink MC Amedee AM Mankowski JL Craig L Didier P Carter DL Munoz A Murphey-Corb M Clements JE Pathogenesis of SIV encephalitis: selection and replication of neurovirulent SIV.Am J Pathol. 1997; 151: 793-803PubMed Google Scholar, 32Zink MC Suryanarayana K Mankowski JL Shen A Piatak Jr, M Spelman JP Carter DL Adams RJ Lifson JD Clements JE High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis.J Virol. 1999; 73: 10480-10488Crossref PubMed Google Scholar Three negative control animals were mock-inoculated. After inoculation, blood and CSF were collected weekly for the first six weeks, then biweekly for quantification of viral RNA and inflammatory proteins including CCL2. Animals with mild encephalitis had low RNA viral copy numbers and low CCL2, while those with severe encephalitis had high viremia and high CCL2.33Mankowski JL Queen SE Clements JE Zink MC Cerebrospinal fluid markers that predict SIV CNS disease.J Neuroimmunol. 2004; 157: 66-70Abstract Full Text Full Text PDF PubMed Scopus (71) Google Scholar, 34Witwer KW Gama L Li M Bartizal CM Queen SE Varrone JJ Brice AK Graham DR Tarwater PM Mankowski JL Zink MC Clements JE Coordinated regulation of SIV replication and immune responses in the CNS.PLoS One. 2009; 4: e8129Crossref PubMed Scopus (81) Google Scholar, 35Zink MC Clements JE A novel simian immunodeficiency virus model that provides insight into mechanisms of human immunodeficiency virus central nervous system disease.J Neurovirol. 2002; 8: 42-48Crossref PubMed Scopus (69) Google Scholar The animals were euthanized at 3 months postinoculation and complete necropsies were performed. All CNS tissues were microscopically examined by two pathologists and scored independently for the severity of the observed lesions.31Zink MC Amedee AM Mankowski JL Craig L Didier P Carter DL Munoz A Murphey-Corb M Clements JE Pathogenesis of SIV encephalitis: selection and replication of neurovirulent SIV.Am J Pathol. 1997; 151: 793-803PubMed Google Scholar, 32Zink MC Suryanarayana K Mankowski JL Shen A Piatak Jr, M Spelman JP Carter DL Adams RJ Lifson JD Clements JE High viral load in the cerebrospinal fluid and brain correlates with severity of simian immunodeficiency virus encephalitis.J Virol. 1999; 73: 10480-10488Crossref PubMed Google Scholar CNS tissue was evaluated from two uninfected animals, one SIV-infected animal without encephalitis, three SIV-infected animals with mild encephalitis, and three SIV-infected animals with severe encephalitis. CSF obtained from six uninfected animals without encephalitis, five SIV-infected animals with mild encephalitis, and five SIV-infected animals with severe encephalitis were evaluated. Plasma from three uninfected animals without encephalitis, three SIV-infected individuals without encephalitis, five SIV-infected animals with mild encephalitis, and five SIV-infected animals with severe encephalitis were also evaluated. Sections of cerebral cortex and underlying white matter were obtained at autopsy/necropsy, processed, embedded in paraffin, and affixed onto slides. The postmortem intervals with the exception of one HAD case were from 5 to 24 hours, with no significant differences between groups. Human samples were processed at the Manhattan HIV Brain Bank and simian tissue was processed at Johns Hopkins University School of Medicine as previously described.31Zink MC Amedee AM Mankowski JL Craig L Didier P Carter DL Munoz A Murphey-Corb M Clements JE Pathogenesis of SIV encephalitis: selection and replication of neurovirulent SIV.Am J Pathol. 1997; 151: 793-803PubMed Google Scholar Before immunostaining, tissue sections were deparaffinized with ethanol and xylene and washed in Tris-buffered saline. Triton antigen retrieval was then performed. Tissue was incubated in blocking solution (5 mmol/L EDTA, 1% fish gelatin, 1% essentially Ig-free BSA, and 2% horse serum) overnight at room temperature. Proteins were labeled with the following primary antibodies overnight at 4°C: MAP-2 (Sigma, St. Louis, MO), CD68 (Santa Cruz, CA), glial fibrillary acidic protein (GFAP; Sigma, St. Louis, MO), and PrPC (Clone SAF-32, Cayman Chemical, Ann Arbor, MI). Tissue was also incubated with an equal concentration of the following species or isotype-matched antibodies as a control: rabbit IgG, goat IgG, and mouse IgG2b (all from Santa Cruz, Santa Cruz, CA). After washing in phosphate-buffered saline (PBS), tissue was incubated in the following fluorescently labeled secondary antibodies for 1 hour at room temperature: sheep anti-mouse IgG-FITC, rabbit anti-goat IgG-Cy3, goat anti-rabbit IgG- Cy3, (all from Sigma, St. Louis, MO), or donkey anti-mouse IgG-Cy5 (Invitrogen, Carlsbad, CA). Tissue was again washed in PBS for 1 hour at room temperature, followed by mounting with Prolong Gold antifade reagent with DAPI (Molecular Probes, Junction City, OR). Tissue sections were imaged at ×40 magnification using a Leica AOBS laser scanning confocal microscope and its accompanying software. Antibody specificity was verified and nonspecific background immunofluorescence was detected using isotype- or species-matched control antibodies and was minimal in all experiments. Quantification of the PrPC staining in cortex as well as underlying white matter tissue sections of humans was performed using NIS Elements Advanced Research software (Nikon, Japan) to determine the total and the mean intensity of fluorescence in the PrPC channel. The background obtained with the respective irrelevant antibodies was subtracted before the analyses. Primary human neurons and astrocytes were obtained as part of an ongoing research protocol at the Albert Einstein College of Medicine and were cultured as previously described.36Eugenin EA Berman JW Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood-brain barrier.Methods. 2003; 29: 351-361Crossref PubMed Scopus (113) Google Scholar, 37Eugenin EA King JE Nath A Calderon TM Zukin RS Bennett MV Berman JW HIV-tat induces formation of an LRP-PSD-95- NMDAR-nNOS complex that promotes apoptosis in neurons and astrocytes.Proc Natl Acad Sci USA. 2007; 104: 3438-3443Crossref PubMed Scopus (174) Google Scholar For neuron enrichment, mixed cultures of neurons and astrocytes were plated at a density of 9 × 107 in DMEM supplemented with 1% penicillin/streptomycin and 1% nonessential amino acids (all from Gibco, Grand Island, NY), 10% heat-inactivated fetal bovine serum (Lonza, Walkersville, MD), and 2% HEPES (USB Corporation, Cleveland, OH). To enrich for astrocytes, mixed cultures of neurons and astrocytes were plated at a density of 4.5 × 107 in DMEM supplemented with 10% heat-inactivated fetal bovine serum, 1% penicillin/streptomycin, 1% nonessential amino acids (all from Gibco, Grand Island, NY), and 2% HEPES (USB Corporation, Cleveland, OH). After one week in culture, cells were dissociated and replated. To propagate pure neuron cultures, cells from the neuron-enriched flasks were plated onto 100-mm culture dishes coated with 0.005% poly-D-lysine (Sigma, St. Louis, MO) in neurobasal media supplemented with 1% penicillin/streptomycin and 1 bottle of N-2 supplement (all from Gibco, Grand Island, NY). Media was changed every 2–3 days until pure neuron cultures were obtained. Pure astrocyte cultures were propagated by plating directly onto plastic 100-mm culture dishes in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 1% penicillin/streptomycin (all from Gibco, Grand Island, NY). Neuron/astrocyte cocultures were propagated by plating directly onto plastic 100-mm culture dishes and growing in neurobasal media supplemented with 1% penicillin/streptomycin, 1 bottle of N-2 supplement (all from Gibco, Grand Island, NY), and 0.5% heat-inactivated fetal bovine serum (Lonza, Walkersville, MD). Experiments with pure neuron cultures and neuron/astrocyte cocultures were conducted 6–8 days after being plated onto 100-mm culture dishes. Pure astrocyte cultures were passaged once before conducting experiments. Primary human brain microvascular endothelial cells were obtained from Applied Cell Biology Research Institute (Kirkland, WA) and cultured as previously described.36Eugenin EA Berman JW Chemokine-dependent mechanisms of leukocyte trafficking across a model of the blood-brain barrier.Methods. 2003; 29: 351-361Crossref PubMed Scopus (113) Google Scholar Cells were grown to confluence on 0.2% gelatin (Fisher Scientific, Pittsburgh, PA)-coated 100 mm tissue culture dishes in M199 media (Gibco, Grand Island, NY) supplemented with 20% heat-inactivated newborn calf serum (Gibco, Grand Island, NY), 5% heat-inactivated human serum type AB (Lonza, Walkersville, MD), 1% penicillin/streptomycin (Gibco, Grand Island, NY), 0.8% l-glutamine (Gibco, Grand Island, NY), 0.1% heparin (Sigma, St. Louis, MO), 0.1% ascorbic acid (Sigma, St. Louis, MO), 0.25% endothelial cell growth supplement (Sigma, St. Louis, MO), and 0.06% bovine brain extract (Clonetics, Walkersville, MD). Human blood from healthy volunteers was obtained from the New York Blood Center and peripheral blood mononuclear cells (PBMCs) were isolated using Ficoll-Paque (GE Health care, Uppsala, Sweden). PBMCs were grown in suspension at a density of 2 × 106 cells/ml in 5 ml of 1640 RPMI media (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum (Lonza, Walkersville, MD), 1% HEPES (USB Corporation, Cleveland, OH), and 1% penicillin/streptomycin (Gibco, Grand Island, NY). Cells were activated for 2 days before experimentation by adding 5 μg/ml PHA (Sigma, St. Louis, MO) and 10 ng/ml IL-2 (PeproTech, Rocky Hill, NJ). Monocytes were isolated from PBMCs using CD14 magnetic beads and columns according to manufacturer's instructions (Miltenyi Biotec, Auburn, CA). Monocytes were grown in suspension at a density of 2 × 106 cells/ml in 5 ml of 1640 RPMI media (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated human serum type AB and 5% heat-inactivated fetal bovine serum (Lonza, Walkersville, MD), 1% HEPES (USB Corporation, Cleveland, OH), 1% penicillin/streptomycin (Gibco, Grand Island, NY), and 10 ng/ml M-CSF (Peprotech, Rocky Hill, NJ). Monocytes were grown in polypropylene tubes (BD Falcon, Bedford, MA) to avoid adherence to plastic and differentiation into macrophages. For studies with macrophages, CD14-isolated cells were plated at a density of 1 × 106 cells/ml in 4 ml 1640 RPMI media (Gibco, Grand Island, NY) supplemented with 10% heat-inactivated fetal bovine serum and 5% heat-inactivated human serum type AB (Lonza, Walkersville, MD), 1% HEPES (USB Corporation, Cleveland, OH), 1% penicillin/streptomycin (Gibco, Grand Island, NY), and 10 ng/ml M-CSF (Peprotech, Rocky Hill, NJ) onto 60 mm tissue culture dishes to facilitate adherence. Media was changed every 3 days. Twenty-four hours before treatment of neurons, astrocytes, neurons/astrocytes, and human brain microvascular endothelial cells (HBMVECs), cell media was replaced with 3 ml of fresh media. For CCL2 treatment of cell cultures, human recombinant CCL2/MCP-1 (R&D Systems, Minneapolis, MN) was added directly to cell culture media to a final concentration of 200 ng/ml. Control cultures were treated with an equal volume of 0.1% BSA in PBS, the CCL2 diluent. Twenty-four hours before media transfer, astrocyte media was changed from supplemented DMEM to 3 ml of supplemented neurobasal. Six hours in advance of media transfer, CCL2, or an equal volume of 0.1% BSA in PBS, was added to astrocyte cultures at a final volume 200 ng/ml. At time of transfer, media from astrocyte cultures was removed and saved for analysis of sPrPC levels by ELISA. Media from astrocytes was then transferred to the neuron cultures, with media from a repl
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