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Neurotoxicity of FIV and FIV envelope protein in feline cortical cultures
Brain Research 816 Ž1999. 431–437
Research report
Neurotoxicity of FIV and FIV envelope protein in feline cortical cultures D. Cristopher Bragg a , Rick B. Meeker a, ) , Barbara A. Duff a , Robert V. English b , Mary B. Tompkins b b
a Curriculum in Neurobiology and Department of Neurology, UniÕersity of North Carolina, Chapel Hill, NC 27599, USA Department of Microbiology, Parasitology and Pathology, College of Veterinary Medicine, North Carolina State UniÕersity, Raleigh, NC 27606, USA
Accepted 27 October 1998
Abstract The neurotoxic effects of the feline immunodeficiency virus ŽFIV. and FIV envelope proteins were measured in primary cultures of feline cortical neurons. Envelope protein from the FIV-PPR strain promoted neuronal swelling and death, whereas envelope protein from the FIV-34TF10 isolate produced intermediate or negligible toxicity. No effect was observed in control cultures treated with envelope protein from the Epstein–Barr virus. A concentration–effect curve showed that FIV-PPR protein produced maximal toxicity at 200 pM protein and decreased toxicity at higher concentrations, which is consistent with previous reports of the HIV-1 surface glycoprotein, gp120. These effects required the presence of low concentrations of glutamate. Using the natural host cells as targets, the effects of envelope protein and infectious virions were directly compared. All of the toxic activity could be attributed to non-infectious interactions between the viral envelope and target cells. Addition of 1 mM tetrodotoxin failed to block the effects of FIV-PPR in the presence of 20 mM glutamate. Toxicity would appear to involve two steps in which the envelope protein first sensitizes neurons through non-synaptic interactions ŽTTX insensitive. thereby setting the stage for enhanced synaptic activation via glutamate receptors ŽTTX sensitive.. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Feline immunodeficiency virus; gp120; Neurotoxicity; Glutamate
1. Introduction Previous studies have demonstrated that lentiviral infection within the central nervous system ŽCNS. frequently produces a significant loss of neurons in both cortical and subcortical regions w11,12,49x. However, the mechanisms underlying this neuronal loss remain unclear, and it has been particularly difficult to explain how these cells are depleted even in the absence of direct viral infection. Most studies indicate that the primary CNS targets of the human, simian, and feline immunodeficiency viruses are microglia and macrophages w1,7,9,26,50x. Although the interactions between lentiviruses and microgliarmacrophages are not fully understood, current evidence suggests that viral exposure induces these cells to release soluble factors that damage surrounding neurons w16,17,43x.
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Corresponding author. Department of Neurology, School of Medicine, University of North Carolina, CB a7025, Clinical Science Building, Chapel Hill, NC 27599, USA. Fax: q1-919-966-2922; E-mail: [email protected]
Considerable research has been recently devoted to identifying these soluble factors, as well as the mechanisms by which they are generated and the sites at which they act. Recent studies have demonstrated that AIDS-related neurotoxicity may be due to factors encoded by the host cell genome, such as proinflammatory cytokines w27,37x, as well as factors encoded by the viral genome, such as the regulatory protein, tat w35x, and the surface glycoprotein, gp120 w4x. The latter has been the subject of a large number of studies, and its potency as a neurotoxin appears well established by the observations that: cultures exposed to gp120 show a significant enhancement in glutamate-mediated excitotoxicity w29x, rats receiving intracerebro-ventricular injections of gp120 experience learning and memory deficits w24x, and transgenic mice expressing gp120 within the CNS develop severe neuropathology and neurologic disease w47x. That gp120 alone may produce these effects raises the additional possibility that non-infectious interactions between the viral envelope and neural cells may be sufficient to produce neuronal injury. Our laboratory has explored the mechanisms underlying this disease process using the feline immunodeficiency
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virus ŽFIV., a lymphotropic and neurotropic lentivirus that infects both wild and domestic cats worldwide w2x. FIV is structurally analogous to HIV-1 and SIV, shares similar cellular targets w13x, and like the primate lentiviruses produces a clinical disease that may include severe neurologic deficits w21,37,38x. In the current study we examine the neurotoxic effects of the FIV envelope protein. We have previously reported that cats experimentally infected with FIV develop a progressive loss of cortical neurons w32x and that neural cultures inoculated with FIV show a significant enhancement in glutamate-mediated excitotoxicity w31x. A primary goal of this study was to therefore determine whether these effects could be due, at least in part, to the viral envelope protein alone. The following report describes the neurotoxicity observed in primary cultures of feline cortex exposed to envelope proteins from two FIV isolates: FIV-PPR and FIV-34TF10. The cellular tropism and neurovirulence of these strains have been previously characterized: cats infected experimentally with FIV-PPR develop significant clinical disease that includes severe neurologic deficits w38x, whereas FIV-34TF10 replicates less efficiently in vivo and produces little disease w39,46x. Envelope protein from the Epstein–Barr virus ŽEBV., a lymphotropic virus that produces no neuropathology, was included as a control for non-specific effects of protein. In addition, we directly compared the toxicity produced by intact virions and purified envelope proteins. The results indicate that the FIVPPR envelope protein, like the HIV-1 surface glycoprotein, produces neurotoxicity and that non-infectious interactions between the virus and neural cells may be sufficient for neuronal loss to occur.
Dissociated cultures for long-term toxicity studies were prepared from the remaining cortex. Tissue was minced and incubated in 5–10 ml CMF-HBSS containing 2 Urml dispase ŽSigma. and 2.5 Urml DNase ŽLTI. for 10–15 min. Tissue was gently triturated using 5–6 passages through a 10-ml pipette, and cell clumps were allowed to settle for 2 min before suspended cells were transferred to a 50-ml culture tube containing 20 ml of complete medium. The remaining tissue was resuspended in 5-ml CMF-HBSS and the trituration procedure repeated until all cell clumps were completely dispersed. Suspended cells were collected by centrifugation, resuspended in complete medium at a concentration of approximately 10 6 cellsrml, and seeded at a density of approximately 50 000–100 000 cellsrcm2 . The resulting cultures contained a mixed population of neurons, glia and microglia. 2.2. Viral enÕelope proteins Partially purified preparations of envelope proteins from FIV-PPR, FIV-34TF10, and the Epstein–Barr virus ŽEBV. were generously provided by Dr. John Elder at the Scripps Research Institute. The expression system and purification scheme have been previously described w20x. Recombinant viral proteins were expressed in a baculovirus vector ŽInvitrogen. which was then used to infect TN5-B cells. Proteins were harvested from the cell supernatant, purified over a lentil lectin column Žmatrix from Sigma., and identified via SDS-PAGE and Western blot with appropriate antisera. Stock solutions were stored as aliquots to avoid multiple freeze-thaw cycles that could affect biologic activity. Protein samples used in toxicity studies
2. Materials and methods 2.1. Primary cultures of feline neurons Fetuses were obtained by cesarean section at approximately 25–40 days gestation. Brains were removed from the cranium, rinsed in calcium-, magnesium-free Hank’s Balanced Salt Solution ŽCMF-HBSS., and stripped of dura-arachnoid membranes. Punches were taken from frontal–parietal cortex using a 23-ga blunt-tip needle connected to a 1-ml syringe filled with sterile Dulbecco’s Modified Eagle Medium ŽDMEM. q 10% fetal bovine serum ŽFBS. q 20 mgrml gentamycin Žcomplete medium.. The punched pieces of tissue extracted with mild suction were then gently ejected onto poly-D-lysine Ž0.1 mgrml. treated glass coverslips. Cells were maintained in a 5% CO 2 incubator at 35–368C and fed every 2 days with complete medium. Neurons in the punch migrated out into the surrounding region providing discrete fields in which individual cells could be digitally imaged. Morphological features typical of cortical neurons were apparent by 6–8 days in vitro.
Fig. 1. Neuronal swelling in feline cortical cultures following acute exposure to viral envelope proteins. Cultures were treated with FIV-PPR envelope protein alone ŽPPR-env. or in the presence of 20 mM glutamate ŽPPRqGLU.. Control cultures were exposed to the Epstein–Barr virus envelope protein in the presence ŽEBVqGLU. or absence of added glutamate ŽEBV-env.. Cells were digitally traced at the given timepoints and increases were expressed as percent changes relative to baseline. The calculated increases reflect the mean of the population of neurons sampled in each condition Ž ns 25–40 cells.. Glutamate induced a significant increase Ž p- 0.05. in the average neuronal swelling only in cells pretreated with FIV-PPR envelope.
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Fig. 2. Example of the swelling response seen in individual cortical neurons treated with 20 mM glutamate following exposure to FIV-PPR envelope protein. Within 20 min of glutamate treatment, prominent swelling was seen in both the cell body Žlarge solid arrow. and dendrites Žsmall arrows. of individual neurons. After 60 min the swelling response was further increased. An adjacent astrocyte exhibited some shrinkage after 60 min Žopen arrow..
contained less than 0.1 unitsrml endotoxin activity as determined by a commercially available method ŽBiowhitaker.. The Epstein–Barr viral envelope protein, which typically produces no neuronal damage, served as a control for non-specific toxicity. 2.3. Acute neurotoxicity A Bioquant Image Analysis System ŽR & M Biometrics, Nashville, TN. was used to document morphologic changes in live cells following acute exposure to either FIV-PPR or EBV proteins in the presence and absence of glutamate. The culture medium was reduced to a volume of 600 ml and supplemented with 300 ml HBSS prior to stimulation with 100 ml of the test compound containing viral protein at a final concentration of 200 pM. Glutamate and glycine were added to the test compound Žat final concentrations of 20 mM and 2 mM, respectively. for cultures receiving concurrent glutamate challenge. Cells were imaged at a final magnification of 1584 = and the digitized images captured at 0, 5, 10, 20, 40, and 60 min following stimulation. Images were retrieved and analyzed with Bioquant Image Analysis software to quantify the area of individual neurons. Control cultures were treated with an equal volume of HBSS. 2.4. Long-term neurotoxicity Dissociated cultures were exposed to viral proteins ŽFIV-PPR, FIV-34TF10, or EBV; 200 pM in complete medium. in the presence and absence of glutamate for 24 h. Cell death was quantified by staining with the combined liverdead cell markers, calcein acetoxymethylester ŽAM.
and ethidium bromide homodimer ŽMolecular Probes, Eugene, OR.. Live cells convert the calcein AM to calcein, which fluoresces green, while the nuclei of dead cells fluoresce bright red after staining with ethidium. Fluorescent nuclei were automatically sized and counted by the Bioquant system in a defined field of 306 = . Dissociated cultures in which cells were seeded uniformly into a 24-well plate were chosen for analysis to guarantee equal numbers of cells in each condition. Five samples were systematically taken from each well and averaged to minimize sample bias. The mean number of dead cells per field in wells treated with FIV proteins was expressed relative
Fig. 3. Cell death in dissociated cortical cultures exposed to viral envelope proteins in the presence ŽqGLU. and absence ŽyGLU. of 20 mM glutamate. Cultures treated with the FIV-PPR protein in the presence of glutamate Ž ns 22 cultures. showed a significant Ž) p- 0.01. increase in cell death Ždead cellsrmm2 . relative to control cultures exposed to the EBV protein in the presence of glutamate Ž ns17. but not in the absence of glutamate Ž ns 7–16.. The FIV-34TF10 protein did not produce significant cell death in the presence Ž ns14. or absence Ž ns 7. of glutamate.
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3. Results
Fig. 4. A concentration–effect curve illustrating an increase in cell death with increasing concentrations of FIV-PPR envelope protein ŽPPR.. A maximal response was seen at 200 pM PPR, with an intermediate response from FIV-34TF10 envelope protein Ž ns12–14 wellsrtime point from each of three culture preparations.. The Epstein–Barr virus protein produced no effect Ž ns 4–6 wellsrtime point.. Concentration values are expressed as the log of the picomolar protein concentration. ) p- 0.05 relative to EBV control. )) p- 0.01 relative to EBV control.
to EBV controls. A dose–response curve was generated by inoculating cultures with different concentrations of viral proteins prepared by dilution of the original stock. The toxic effects of infectious virus and viral envelope were compared by measuring long-term neurotoxicity in dissociated cultures inoculated with either 10 3 tissue culture infectious doses ŽTCID50 . FIV-PPR or the envelope protein from the same strain Ž200 pM.. Control cultures were exposed to an ultrafiltrate prepared by passing the viral inoculum through a 100 000 M.W. cutoff filter. The contribution of intrinsic synaptic activity to the effects of envelope protein within the cultures was evaluated by measuring long-term neurotoxicity induced by infectious FIV-PPR and glutamate Ž20 mM with 2 mM glycine. in the presence and absence of tetrodotoxin ŽTTX, 1 mM..
Fig. 1 shows the timecourse for the average acute swelling response observed in neurons treated with viral envelope proteins. The FIV-PPR protein produced a significant Ž p - 0.05. increase in the mean neuronal cell area, whereas the EBV controls showed only negligible swelling with respect to baseline. The effect of FIV-PPR was dependent on concurrent challenge with glutamate and achieved a maximal response at 20 min post-stimulation. The mean increase measured in the total population of cells was due largely to a subset of neurons Ž32%. that showed dramatic increases that were well beyond the 95% confidence established by the EBV control cultures. An example of this swelling response in a large cortical neuron exposed to 200 pM FIV-PPRq 20 mM glutamate is illustrated in Fig. 2. By 20 min, prominent swelling of the cell body is visible Žlarge solid arrow.. Other neurons exhibit dendritic swelling Žsmall arrows.. By 60 min after treatment, extensive swelling of the cell body and dendrites can be seen. An adjacent astrocyte exhibits some shrinkage under the same conditions Žopen arrow.. Treatment with the FIV-PPR protein in the presence of exogenous glutamate resulted in a significant Ž p - 0.01. enhancement in cell death ŽFig. 3. in neuronal cultures as compared to the EBV protein. A concentration–effect curve indicated that maximal cell death was achieved at a protein concentration of approximately 200 pM ŽFig. 4.. Toxicity decreased slightly at a concentration of 2000 pM but was still significantly greater than controls. The FIV34TF10 protein produced enhanced toxicity only at the highest concentration tested Ž2000 pM.. Both infectious FIV-PPR and purified FIV-PPR protein produced a significant increase in cell death with respect to baseline Ž p - 0.0001. but there was no significant difference between the two treatments Ž p s 0.359. ŽFig. 5A..
Fig. 5. The cytotoxic activity of FIV envelope protein relative to infectious virus and the lack of dependence on synaptic activity. ŽA. Dissociated cultures were exposed to infectious virus ŽFIV-PPR, n s 40 cultures., viral envelope protein ŽPPR-ENV, n s 34 cultures. or an ultrafiltrate prepared from the viral inoculum ŽCONTROL, n s 27 cultures.. Both infectious virus and envelope protein produced significant increases in cell death Ždead cellsrmm2 . with respect to control cultures Ž) p’s - 0.0001., but there was no difference between the two treatments Ž p s 0.359.. ŽB. Failure of 1 mM tetrodotoxin to block the toxic effects of FIV. Feline cortical cultures were exposed to infectious FIV alone ŽFIV, n s 24 cultures. or FIV in the presence of 1 mM tetrodotoxin ŽFIV q TTX, n s 22 cultures. for 24 h. Control cultures were treated with an ultrafiltrate prepared from the viral inoculum Ž n s 12.. All cultures contained 20 mM glutamate and 2 mM glycine to provide a low level of postsynaptic receptor stimulation. FIV induced a significant increase in cell death Ždead cellsrmm2 . relative to controls Ž) p’s - 0.0006. but the effect of FIV was not blocked by TTX Ž p s 0.807..
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Furthermore, the increased toxicity produced by FIV-PPR in the presence of exogenous glutamate was not inhibited by addition of 1 mM TTX Ž p s 0.8074. ŽFig. 5B..
4. Discussion Previous studies have demonstrated that rats receiving intracerebroventricular injections of either the FIV envelope protein w42x or the HIV-1 surface glycoprotein, gp120 w36x, experience marked changes in sleep architecture that closely resemble the disturbances reported in both FIV-infected cats w41x and HIV-infected patients w5x. The current study provides further evidence that the neurotoxic effects of the FIV envelope protein may be measured in vitro and that this data is also consistent with previous descriptions of gp120. Neuronal swelling was observed in cultures exposed acutely to the FIV-PPR protein, whereas long-term exposure produced a significant increase in cell death. The initial increase in neuronal cell size most likely reflects the influx of sodium and chloride ions through non-NMDA receptor ion channels that may either precede or function independently of NMDA receptor activation. Gruol et al. w20x recently reported that the FIV-PPR protein facilitates the NMDA-dependent accumulation of intracellular calcium, and it is likely that a similar disruption in neuronal calcium homeostasis contributed to the cytotoxicity observed in the present study. We have previously shown that neural cultures inoculated with FIV experience an increased sensitivity to glutamate w31x. A similar mechanism appears to underlie the effects of the viral envelope, as the FIV-PPR protein produced significant neurotoxicity only in the presence of low concentrations of glutamate. Moreover, the current data suggests that a subset of cortical neurons may be particularly vulnerable to the effects of the viral protein, which correlates with the pattern of cortical cell loss previously documented in cats experimentally infected with FIV w32x. These findings are consistent with previous studies of the HIV-1 surface glycoprotein, gp120. Moreover, the dose–response curve generated by the FIV-PPR protein closely resembles the profile for gp120 previously described by Brenneman et al. w3x. Since that original report, a large number of investigators have independently demonstrated that gp120 may induce either excitotoxicity or apoptosis in neural cultures Žfor review, see Ref. w33x.. Most studies suggest that these neurotoxic effects require extracellular glutamate and NMDA receptor activation w10x. However, recent studies indicate that gp120 evokes a specific pattern of intracellular calcium accumulation w30x consisting of a sustained increase that is not blocked by NMDA receptor antagonists w34x. Tymianski et al. w48x observed the same pattern of calcium deregulation in neurons and provided convincing evidence that it is this slow, NMDA-independent accumulation of intracellular
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calcium that specifically leads to excitotoxic cell death. It thus appears that NMDA receptor activation may be required to initiate gp120-induced excitotoxicity and that the subsequent toxic accumulation of calcium may involve other channels andror intracellular stores that have not been fully identified. The importance of extracellular glutamate and postsynaptic NMDA receptor activation in this process has suggested that synaptic activity may play an important role in the neurotoxicity induced by lentiviral proteins. Lo et al. w30x and Diop et al. w8x both demonstrated recently that tetrodotoxin blocks toxicity in neural cultures exposed to gp120 alone. These studies clearly illustrate the importance of glutamate release from presynaptic terminals. However, it is still not clear how gp120 might sensitize neurons to the excitatory effects of glutamate. One hypothesis is that the viral protein may drive neurons to release not only glutamate but also other factors that alter the postsynaptic response. In the current study we addressed this issue by providing neural cultures with a low concentration of exogenous glutamate to control the level of postsynaptic receptor activation. Under these conditions, tetrodotoxin failed to block the neurotoxicity induced by FIV-PPR, suggesting that the increased sensitivity to glutamate does not require other factors released during synaptic transmission. The specific cellular targets of lentiviral envelope proteins remain unclear. The demonstration that neurons express chemokine receptors w22,25,44x raises the possibility that there may be toxic, non-infectious interactions between the viral envelope and the neuronal cell membrane. However, there is currently no data to either support or reject this hypothesis. An alternative explanation is that the neurotoxic effects of viral envelope proteins are mediated by non-neuronal cells, such as microglia andror astrocytes. The importance of microglia in AIDS-related neurotoxicity has been established by several lines of evidence, including the observations that: Ž1. the toxic effects of gp120 in vitro require the presence of microglia w28x, and Ž2. the severity of neurologic disease in vivo correlates closely with the density of microgliarmacrophages within the parenchyma w19x. There is now considerable evidence that lentiviruses may induce microglia to secrete a number of soluble factors, including tumor necrosis factor ŽTNFa . w14,45,51x, interleukins w45,51x, platelet activating factor ŽPAF. w15x, nitric oxide ŽNO. w6x, quinolinate w23x, and a new, partially characterized toxin, NTox w18x. The effects of viral envelope proteins may be mediated by any of these putative neurotoxins, although the specific mechanisms by which they bring about neuronal damage are not fully understood. In addition, there is increasing evidence that astrocytes may play an important role in AIDS-related neuropathogenesis. Yu et al. w52x recently reported that the infectious FIV-34TF10 clone induced neurotoxicity by decreasing the ability of astrocytes to remove glutamate from the synaptic
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cleft. However, in the current study the FIV-PPR protein was significantly more potent. It is possible that, under different conditions, both viral strains may produce neurotoxicity through separate, indirect pathways. That different viral strains may produce different effects within the CNS raises important questions about the mechanisms underlying AIDS-related neurotoxicity. Nath and Geiger w33x point out that previous studies of gp120 have examined envelope proteins only from lymphocytotropic strains of HIV-1. However, most studies indicate that virus within the brain is largely restricted to microglia and macrophages, and the extent to which these cells are efficiently infected by lymphocytotropic virus is currently unclear. Power et al. w40x have shown that specific HIV-1 sequences are detected within the brain, but to date there is no information about the specific contribution of each strain or the relative role of infection in neurotoxicity. In the current study, we directly compared the toxic effects of infectious virions and envelope proteins from the FIV-PPR isolate, which replicates efficiently in vivo and produces significant neurologic disease in cats w38x. The data showed no difference in the cytotoxicity produced by intact virus and purified protein, suggesting, at least for this viral isolate, that non-infectious interactions between the viral envelope and neural cells may be sufficient for neuronal injury to occur. In conclusion, the current study demonstrates that FIV induces neurotoxicity and that these effects may be due, at least in part, to the viral envelope protein. Furthermore, the data is consistent with previously described effects of the HIV-1 surface glycoprotein, gp120, suggesting that similar molecular mechanisms underlie the clinical syndromes produced by these lentiviruses. That these neurotoxic effects may be measured both in vitro and in vivo further establish FIV as an important tool for future investigation into AIDS-related neuropathogenesis.
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Research report
Neurotoxicity of FIV and FIV envelope protein in feline cortical cultures D. Cristopher Bragg a , Rick B. Meeker a, ) , Barbara A. Duff a , Robert V. English b , Mary B. Tompkins b b
a Curriculum in Neurobiology and Department of Neurology, UniÕersity of North Carolina, Chapel Hill, NC 27599, USA Department of Microbiology, Parasitology and Pathology, College of Veterinary Medicine, North Carolina State UniÕersity, Raleigh, NC 27606, USA
Accepted 27 October 1998
Abstract The neurotoxic effects of the feline immunodeficiency virus ŽFIV. and FIV envelope proteins were measured in primary cultures of feline cortical neurons. Envelope protein from the FIV-PPR strain promoted neuronal swelling and death, whereas envelope protein from the FIV-34TF10 isolate produced intermediate or negligible toxicity. No effect was observed in control cultures treated with envelope protein from the Epstein–Barr virus. A concentration–effect curve showed that FIV-PPR protein produced maximal toxicity at 200 pM protein and decreased toxicity at higher concentrations, which is consistent with previous reports of the HIV-1 surface glycoprotein, gp120. These effects required the presence of low concentrations of glutamate. Using the natural host cells as targets, the effects of envelope protein and infectious virions were directly compared. All of the toxic activity could be attributed to non-infectious interactions between the viral envelope and target cells. Addition of 1 mM tetrodotoxin failed to block the effects of FIV-PPR in the presence of 20 mM glutamate. Toxicity would appear to involve two steps in which the envelope protein first sensitizes neurons through non-synaptic interactions ŽTTX insensitive. thereby setting the stage for enhanced synaptic activation via glutamate receptors ŽTTX sensitive.. q 1999 Published by Elsevier Science B.V. All rights reserved. Keywords: Feline immunodeficiency virus; gp120; Neurotoxicity; Glutamate
1. Introduction Previous studies have demonstrated that lentiviral infection within the central nervous system ŽCNS. frequently produces a significant loss of neurons in both cortical and subcortical regions w11,12,49x. However, the mechanisms underlying this neuronal loss remain unclear, and it has been particularly difficult to explain how these cells are depleted even in the absence of direct viral infection. Most studies indicate that the primary CNS targets of the human, simian, and feline immunodeficiency viruses are microglia and macrophages w1,7,9,26,50x. Although the interactions between lentiviruses and microgliarmacrophages are not fully understood, current evidence suggests that viral exposure induces these cells to release soluble factors that damage surrounding neurons w16,17,43x.
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Corresponding author. Department of Neurology, School of Medicine, University of North Carolina, CB a7025, Clinical Science Building, Chapel Hill, NC 27599, USA. Fax: q1-919-966-2922; E-mail: [email protected]
Considerable research has been recently devoted to identifying these soluble factors, as well as the mechanisms by which they are generated and the sites at which they act. Recent studies have demonstrated that AIDS-related neurotoxicity may be due to factors encoded by the host cell genome, such as proinflammatory cytokines w27,37x, as well as factors encoded by the viral genome, such as the regulatory protein, tat w35x, and the surface glycoprotein, gp120 w4x. The latter has been the subject of a large number of studies, and its potency as a neurotoxin appears well established by the observations that: cultures exposed to gp120 show a significant enhancement in glutamate-mediated excitotoxicity w29x, rats receiving intracerebro-ventricular injections of gp120 experience learning and memory deficits w24x, and transgenic mice expressing gp120 within the CNS develop severe neuropathology and neurologic disease w47x. That gp120 alone may produce these effects raises the additional possibility that non-infectious interactions between the viral envelope and neural cells may be sufficient to produce neuronal injury. Our laboratory has explored the mechanisms underlying this disease process using the feline immunodeficiency
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virus ŽFIV., a lymphotropic and neurotropic lentivirus that infects both wild and domestic cats worldwide w2x. FIV is structurally analogous to HIV-1 and SIV, shares similar cellular targets w13x, and like the primate lentiviruses produces a clinical disease that may include severe neurologic deficits w21,37,38x. In the current study we examine the neurotoxic effects of the FIV envelope protein. We have previously reported that cats experimentally infected with FIV develop a progressive loss of cortical neurons w32x and that neural cultures inoculated with FIV show a significant enhancement in glutamate-mediated excitotoxicity w31x. A primary goal of this study was to therefore determine whether these effects could be due, at least in part, to the viral envelope protein alone. The following report describes the neurotoxicity observed in primary cultures of feline cortex exposed to envelope proteins from two FIV isolates: FIV-PPR and FIV-34TF10. The cellular tropism and neurovirulence of these strains have been previously characterized: cats infected experimentally with FIV-PPR develop significant clinical disease that includes severe neurologic deficits w38x, whereas FIV-34TF10 replicates less efficiently in vivo and produces little disease w39,46x. Envelope protein from the Epstein–Barr virus ŽEBV., a lymphotropic virus that produces no neuropathology, was included as a control for non-specific effects of protein. In addition, we directly compared the toxicity produced by intact virions and purified envelope proteins. The results indicate that the FIVPPR envelope protein, like the HIV-1 surface glycoprotein, produces neurotoxicity and that non-infectious interactions between the virus and neural cells may be sufficient for neuronal loss to occur.
Dissociated cultures for long-term toxicity studies were prepared from the remaining cortex. Tissue was minced and incubated in 5–10 ml CMF-HBSS containing 2 Urml dispase ŽSigma. and 2.5 Urml DNase ŽLTI. for 10–15 min. Tissue was gently triturated using 5–6 passages through a 10-ml pipette, and cell clumps were allowed to settle for 2 min before suspended cells were transferred to a 50-ml culture tube containing 20 ml of complete medium. The remaining tissue was resuspended in 5-ml CMF-HBSS and the trituration procedure repeated until all cell clumps were completely dispersed. Suspended cells were collected by centrifugation, resuspended in complete medium at a concentration of approximately 10 6 cellsrml, and seeded at a density of approximately 50 000–100 000 cellsrcm2 . The resulting cultures contained a mixed population of neurons, glia and microglia. 2.2. Viral enÕelope proteins Partially purified preparations of envelope proteins from FIV-PPR, FIV-34TF10, and the Epstein–Barr virus ŽEBV. were generously provided by Dr. John Elder at the Scripps Research Institute. The expression system and purification scheme have been previously described w20x. Recombinant viral proteins were expressed in a baculovirus vector ŽInvitrogen. which was then used to infect TN5-B cells. Proteins were harvested from the cell supernatant, purified over a lentil lectin column Žmatrix from Sigma., and identified via SDS-PAGE and Western blot with appropriate antisera. Stock solutions were stored as aliquots to avoid multiple freeze-thaw cycles that could affect biologic activity. Protein samples used in toxicity studies
2. Materials and methods 2.1. Primary cultures of feline neurons Fetuses were obtained by cesarean section at approximately 25–40 days gestation. Brains were removed from the cranium, rinsed in calcium-, magnesium-free Hank’s Balanced Salt Solution ŽCMF-HBSS., and stripped of dura-arachnoid membranes. Punches were taken from frontal–parietal cortex using a 23-ga blunt-tip needle connected to a 1-ml syringe filled with sterile Dulbecco’s Modified Eagle Medium ŽDMEM. q 10% fetal bovine serum ŽFBS. q 20 mgrml gentamycin Žcomplete medium.. The punched pieces of tissue extracted with mild suction were then gently ejected onto poly-D-lysine Ž0.1 mgrml. treated glass coverslips. Cells were maintained in a 5% CO 2 incubator at 35–368C and fed every 2 days with complete medium. Neurons in the punch migrated out into the surrounding region providing discrete fields in which individual cells could be digitally imaged. Morphological features typical of cortical neurons were apparent by 6–8 days in vitro.
Fig. 1. Neuronal swelling in feline cortical cultures following acute exposure to viral envelope proteins. Cultures were treated with FIV-PPR envelope protein alone ŽPPR-env. or in the presence of 20 mM glutamate ŽPPRqGLU.. Control cultures were exposed to the Epstein–Barr virus envelope protein in the presence ŽEBVqGLU. or absence of added glutamate ŽEBV-env.. Cells were digitally traced at the given timepoints and increases were expressed as percent changes relative to baseline. The calculated increases reflect the mean of the population of neurons sampled in each condition Ž ns 25–40 cells.. Glutamate induced a significant increase Ž p- 0.05. in the average neuronal swelling only in cells pretreated with FIV-PPR envelope.
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Fig. 2. Example of the swelling response seen in individual cortical neurons treated with 20 mM glutamate following exposure to FIV-PPR envelope protein. Within 20 min of glutamate treatment, prominent swelling was seen in both the cell body Žlarge solid arrow. and dendrites Žsmall arrows. of individual neurons. After 60 min the swelling response was further increased. An adjacent astrocyte exhibited some shrinkage after 60 min Žopen arrow..
contained less than 0.1 unitsrml endotoxin activity as determined by a commercially available method ŽBiowhitaker.. The Epstein–Barr viral envelope protein, which typically produces no neuronal damage, served as a control for non-specific toxicity. 2.3. Acute neurotoxicity A Bioquant Image Analysis System ŽR & M Biometrics, Nashville, TN. was used to document morphologic changes in live cells following acute exposure to either FIV-PPR or EBV proteins in the presence and absence of glutamate. The culture medium was reduced to a volume of 600 ml and supplemented with 300 ml HBSS prior to stimulation with 100 ml of the test compound containing viral protein at a final concentration of 200 pM. Glutamate and glycine were added to the test compound Žat final concentrations of 20 mM and 2 mM, respectively. for cultures receiving concurrent glutamate challenge. Cells were imaged at a final magnification of 1584 = and the digitized images captured at 0, 5, 10, 20, 40, and 60 min following stimulation. Images were retrieved and analyzed with Bioquant Image Analysis software to quantify the area of individual neurons. Control cultures were treated with an equal volume of HBSS. 2.4. Long-term neurotoxicity Dissociated cultures were exposed to viral proteins ŽFIV-PPR, FIV-34TF10, or EBV; 200 pM in complete medium. in the presence and absence of glutamate for 24 h. Cell death was quantified by staining with the combined liverdead cell markers, calcein acetoxymethylester ŽAM.
and ethidium bromide homodimer ŽMolecular Probes, Eugene, OR.. Live cells convert the calcein AM to calcein, which fluoresces green, while the nuclei of dead cells fluoresce bright red after staining with ethidium. Fluorescent nuclei were automatically sized and counted by the Bioquant system in a defined field of 306 = . Dissociated cultures in which cells were seeded uniformly into a 24-well plate were chosen for analysis to guarantee equal numbers of cells in each condition. Five samples were systematically taken from each well and averaged to minimize sample bias. The mean number of dead cells per field in wells treated with FIV proteins was expressed relative
Fig. 3. Cell death in dissociated cortical cultures exposed to viral envelope proteins in the presence ŽqGLU. and absence ŽyGLU. of 20 mM glutamate. Cultures treated with the FIV-PPR protein in the presence of glutamate Ž ns 22 cultures. showed a significant Ž) p- 0.01. increase in cell death Ždead cellsrmm2 . relative to control cultures exposed to the EBV protein in the presence of glutamate Ž ns17. but not in the absence of glutamate Ž ns 7–16.. The FIV-34TF10 protein did not produce significant cell death in the presence Ž ns14. or absence Ž ns 7. of glutamate.
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3. Results
Fig. 4. A concentration–effect curve illustrating an increase in cell death with increasing concentrations of FIV-PPR envelope protein ŽPPR.. A maximal response was seen at 200 pM PPR, with an intermediate response from FIV-34TF10 envelope protein Ž ns12–14 wellsrtime point from each of three culture preparations.. The Epstein–Barr virus protein produced no effect Ž ns 4–6 wellsrtime point.. Concentration values are expressed as the log of the picomolar protein concentration. ) p- 0.05 relative to EBV control. )) p- 0.01 relative to EBV control.
to EBV controls. A dose–response curve was generated by inoculating cultures with different concentrations of viral proteins prepared by dilution of the original stock. The toxic effects of infectious virus and viral envelope were compared by measuring long-term neurotoxicity in dissociated cultures inoculated with either 10 3 tissue culture infectious doses ŽTCID50 . FIV-PPR or the envelope protein from the same strain Ž200 pM.. Control cultures were exposed to an ultrafiltrate prepared by passing the viral inoculum through a 100 000 M.W. cutoff filter. The contribution of intrinsic synaptic activity to the effects of envelope protein within the cultures was evaluated by measuring long-term neurotoxicity induced by infectious FIV-PPR and glutamate Ž20 mM with 2 mM glycine. in the presence and absence of tetrodotoxin ŽTTX, 1 mM..
Fig. 1 shows the timecourse for the average acute swelling response observed in neurons treated with viral envelope proteins. The FIV-PPR protein produced a significant Ž p - 0.05. increase in the mean neuronal cell area, whereas the EBV controls showed only negligible swelling with respect to baseline. The effect of FIV-PPR was dependent on concurrent challenge with glutamate and achieved a maximal response at 20 min post-stimulation. The mean increase measured in the total population of cells was due largely to a subset of neurons Ž32%. that showed dramatic increases that were well beyond the 95% confidence established by the EBV control cultures. An example of this swelling response in a large cortical neuron exposed to 200 pM FIV-PPRq 20 mM glutamate is illustrated in Fig. 2. By 20 min, prominent swelling of the cell body is visible Žlarge solid arrow.. Other neurons exhibit dendritic swelling Žsmall arrows.. By 60 min after treatment, extensive swelling of the cell body and dendrites can be seen. An adjacent astrocyte exhibits some shrinkage under the same conditions Žopen arrow.. Treatment with the FIV-PPR protein in the presence of exogenous glutamate resulted in a significant Ž p - 0.01. enhancement in cell death ŽFig. 3. in neuronal cultures as compared to the EBV protein. A concentration–effect curve indicated that maximal cell death was achieved at a protein concentration of approximately 200 pM ŽFig. 4.. Toxicity decreased slightly at a concentration of 2000 pM but was still significantly greater than controls. The FIV34TF10 protein produced enhanced toxicity only at the highest concentration tested Ž2000 pM.. Both infectious FIV-PPR and purified FIV-PPR protein produced a significant increase in cell death with respect to baseline Ž p - 0.0001. but there was no significant difference between the two treatments Ž p s 0.359. ŽFig. 5A..
Fig. 5. The cytotoxic activity of FIV envelope protein relative to infectious virus and the lack of dependence on synaptic activity. ŽA. Dissociated cultures were exposed to infectious virus ŽFIV-PPR, n s 40 cultures., viral envelope protein ŽPPR-ENV, n s 34 cultures. or an ultrafiltrate prepared from the viral inoculum ŽCONTROL, n s 27 cultures.. Both infectious virus and envelope protein produced significant increases in cell death Ždead cellsrmm2 . with respect to control cultures Ž) p’s - 0.0001., but there was no difference between the two treatments Ž p s 0.359.. ŽB. Failure of 1 mM tetrodotoxin to block the toxic effects of FIV. Feline cortical cultures were exposed to infectious FIV alone ŽFIV, n s 24 cultures. or FIV in the presence of 1 mM tetrodotoxin ŽFIV q TTX, n s 22 cultures. for 24 h. Control cultures were treated with an ultrafiltrate prepared from the viral inoculum Ž n s 12.. All cultures contained 20 mM glutamate and 2 mM glycine to provide a low level of postsynaptic receptor stimulation. FIV induced a significant increase in cell death Ždead cellsrmm2 . relative to controls Ž) p’s - 0.0006. but the effect of FIV was not blocked by TTX Ž p s 0.807..
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Furthermore, the increased toxicity produced by FIV-PPR in the presence of exogenous glutamate was not inhibited by addition of 1 mM TTX Ž p s 0.8074. ŽFig. 5B..
4. Discussion Previous studies have demonstrated that rats receiving intracerebroventricular injections of either the FIV envelope protein w42x or the HIV-1 surface glycoprotein, gp120 w36x, experience marked changes in sleep architecture that closely resemble the disturbances reported in both FIV-infected cats w41x and HIV-infected patients w5x. The current study provides further evidence that the neurotoxic effects of the FIV envelope protein may be measured in vitro and that this data is also consistent with previous descriptions of gp120. Neuronal swelling was observed in cultures exposed acutely to the FIV-PPR protein, whereas long-term exposure produced a significant increase in cell death. The initial increase in neuronal cell size most likely reflects the influx of sodium and chloride ions through non-NMDA receptor ion channels that may either precede or function independently of NMDA receptor activation. Gruol et al. w20x recently reported that the FIV-PPR protein facilitates the NMDA-dependent accumulation of intracellular calcium, and it is likely that a similar disruption in neuronal calcium homeostasis contributed to the cytotoxicity observed in the present study. We have previously shown that neural cultures inoculated with FIV experience an increased sensitivity to glutamate w31x. A similar mechanism appears to underlie the effects of the viral envelope, as the FIV-PPR protein produced significant neurotoxicity only in the presence of low concentrations of glutamate. Moreover, the current data suggests that a subset of cortical neurons may be particularly vulnerable to the effects of the viral protein, which correlates with the pattern of cortical cell loss previously documented in cats experimentally infected with FIV w32x. These findings are consistent with previous studies of the HIV-1 surface glycoprotein, gp120. Moreover, the dose–response curve generated by the FIV-PPR protein closely resembles the profile for gp120 previously described by Brenneman et al. w3x. Since that original report, a large number of investigators have independently demonstrated that gp120 may induce either excitotoxicity or apoptosis in neural cultures Žfor review, see Ref. w33x.. Most studies suggest that these neurotoxic effects require extracellular glutamate and NMDA receptor activation w10x. However, recent studies indicate that gp120 evokes a specific pattern of intracellular calcium accumulation w30x consisting of a sustained increase that is not blocked by NMDA receptor antagonists w34x. Tymianski et al. w48x observed the same pattern of calcium deregulation in neurons and provided convincing evidence that it is this slow, NMDA-independent accumulation of intracellular
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calcium that specifically leads to excitotoxic cell death. It thus appears that NMDA receptor activation may be required to initiate gp120-induced excitotoxicity and that the subsequent toxic accumulation of calcium may involve other channels andror intracellular stores that have not been fully identified. The importance of extracellular glutamate and postsynaptic NMDA receptor activation in this process has suggested that synaptic activity may play an important role in the neurotoxicity induced by lentiviral proteins. Lo et al. w30x and Diop et al. w8x both demonstrated recently that tetrodotoxin blocks toxicity in neural cultures exposed to gp120 alone. These studies clearly illustrate the importance of glutamate release from presynaptic terminals. However, it is still not clear how gp120 might sensitize neurons to the excitatory effects of glutamate. One hypothesis is that the viral protein may drive neurons to release not only glutamate but also other factors that alter the postsynaptic response. In the current study we addressed this issue by providing neural cultures with a low concentration of exogenous glutamate to control the level of postsynaptic receptor activation. Under these conditions, tetrodotoxin failed to block the neurotoxicity induced by FIV-PPR, suggesting that the increased sensitivity to glutamate does not require other factors released during synaptic transmission. The specific cellular targets of lentiviral envelope proteins remain unclear. The demonstration that neurons express chemokine receptors w22,25,44x raises the possibility that there may be toxic, non-infectious interactions between the viral envelope and the neuronal cell membrane. However, there is currently no data to either support or reject this hypothesis. An alternative explanation is that the neurotoxic effects of viral envelope proteins are mediated by non-neuronal cells, such as microglia andror astrocytes. The importance of microglia in AIDS-related neurotoxicity has been established by several lines of evidence, including the observations that: Ž1. the toxic effects of gp120 in vitro require the presence of microglia w28x, and Ž2. the severity of neurologic disease in vivo correlates closely with the density of microgliarmacrophages within the parenchyma w19x. There is now considerable evidence that lentiviruses may induce microglia to secrete a number of soluble factors, including tumor necrosis factor ŽTNFa . w14,45,51x, interleukins w45,51x, platelet activating factor ŽPAF. w15x, nitric oxide ŽNO. w6x, quinolinate w23x, and a new, partially characterized toxin, NTox w18x. The effects of viral envelope proteins may be mediated by any of these putative neurotoxins, although the specific mechanisms by which they bring about neuronal damage are not fully understood. In addition, there is increasing evidence that astrocytes may play an important role in AIDS-related neuropathogenesis. Yu et al. w52x recently reported that the infectious FIV-34TF10 clone induced neurotoxicity by decreasing the ability of astrocytes to remove glutamate from the synaptic
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cleft. However, in the current study the FIV-PPR protein was significantly more potent. It is possible that, under different conditions, both viral strains may produce neurotoxicity through separate, indirect pathways. That different viral strains may produce different effects within the CNS raises important questions about the mechanisms underlying AIDS-related neurotoxicity. Nath and Geiger w33x point out that previous studies of gp120 have examined envelope proteins only from lymphocytotropic strains of HIV-1. However, most studies indicate that virus within the brain is largely restricted to microglia and macrophages, and the extent to which these cells are efficiently infected by lymphocytotropic virus is currently unclear. Power et al. w40x have shown that specific HIV-1 sequences are detected within the brain, but to date there is no information about the specific contribution of each strain or the relative role of infection in neurotoxicity. In the current study, we directly compared the toxic effects of infectious virions and envelope proteins from the FIV-PPR isolate, which replicates efficiently in vivo and produces significant neurologic disease in cats w38x. The data showed no difference in the cytotoxicity produced by intact virus and purified protein, suggesting, at least for this viral isolate, that non-infectious interactions between the viral envelope and neural cells may be sufficient for neuronal injury to occur. In conclusion, the current study demonstrates that FIV induces neurotoxicity and that these effects may be due, at least in part, to the viral envelope protein. Furthermore, the data is consistent with previously described effects of the HIV-1 surface glycoprotein, gp120, suggesting that similar molecular mechanisms underlie the clinical syndromes produced by these lentiviruses. That these neurotoxic effects may be measured both in vitro and in vivo further establish FIV as an important tool for future investigation into AIDS-related neuropathogenesis.
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