gp120 as an etiologic agent for NeuroAIDS: Neurotoxicity and model systems

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Advances in Neuroimmunology Vol. 4, pp. 15%165, 1994

Pergamon

1994 Elsevier ScienceLtd Printed in Great Britain. 0960-5428/94 $26.00 0960-5428(94)00016-6

gp120 as an etiologic agent for NeuroAIDS" Neurotoxicity and model systems Douglas E. B r e n n e m a n , *, 1 Susan K. M c C u n e , 1,s R o n a l d F. Mervis 2 and Joanna M. Hill 1 1Section on Developmental and Molecular Pharmacology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD 20892, USA 2NeurometrixResearch, Inc., 749 Northwest Blvd, Columbus, OH 43212, USA 3Department of Neonatology, Children's National Medical Center, 111 Michigan Ave, N.W. Washington, D.C. 20010, USA

Summary The search for an agent that can mediate the symptoms of N e u r o A I D S has been directed at gp120, the major envelope protein from HIV. The toxicity associated with gp120 was examined as a model and predictor of the neuropathological and neuropsychiatric manifestations of AIDS. Studies of the neurotoxic effects of purified gp120 on neurons from the rodent CNS cell cultures indicated the following: potent and selective killing of subpopulations of hippocampal neurons; varying potency of gp120s obtained from various H I V isolates; complete and potent protection from gpl20 killing action after treatment with peptides related to vasoactive intestinal peptide; and obligatory presence of glia for gpl20-related toxicity. Investigations of gpl20 treatment of rodents revealed: cortical neurodystrophy with reduced arborizations and swollen processes; delays in developmental behaviors involving motor skills; peptide T prevention or attenuation of the morphological *Correspondence address: Dr Douglas E. Brenneman, SDMP/LDN/NICHD, Building 49, Rm 5A38, National Institutes of Health, Bethesda, MD 20892.

and behavioral deficits/delays produced by administration of gp120; and impairment of learning in the Morris swim maze. In addition, studies of subcutaneously administered, radiolabeled gp120 in neonatal animals demonstrated the presence of toxic fragments of gp120 in the developing brain. With the use of model test systems of nonhuman derived cell cultures and neonatal rats, we have captured and predicted a number of the morphological and behavioral deficits associated with AIDS. These multidisciplinary studies of the actions of gpl20 and associated fragments in rodents and rodent cells predict that the loss of cognitive and neurological function in patients with AIDS are attributed in part to interference of critical brain functions by the envelope protein, gpl20.

Introduction Cognitive dysfunction and neurological impairment are commonly observed features in patients with acquired immune deficiency syndrome (AIDS) (see Brenneman et al., 1990a). The human immunodeficiency virus 157

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(HIV) is the recognized etiological agent for this disease that damages both the immune and nervous systems. A major dilemma in our understanding of AIDS lies in the observation that very few HIV-infected cells are detected in either the immune or nervous system (Schnittman et al., 1989; Shaw et al., 1985), yet catastrophic functional impairment eventually occurs. Such pervasive impairment cannot be explained by direct damage through viral infection of individual cells and has led to the search for indirect mechanisms of HIV-related toxicity. A leading candidate for a cytotoxic product of HIV is the major external envelope protein, gp120, which has demonstrated toxicity in cultured cells from both CNS (Brenneman et a l . , 1988b) and immune systems (Weinhold et al., 1989). HIV-infected cells shed gp120 (Schneider et al., 1986) and gp120-1ike toxicity has been reported in the cerebrospinal fluid of AIDS patients (Buzy et al., 1992). Gp160/120 has been identified as circulating antigen in patients with AIDS (Oh et al., 1992). The envelope protein discovered in the serum appeared to be in the form of immune complexes. In the present review, the models employed to study neurotoxicity involve neonatal rats and cells from embryonic mice, species that do not exhibit productive infection with HIV. With this caveat apparent, why examine the effect of viral products in these species? The answer lies in the possible separation of the mechanism(s) relating to infectivity versus those pertaining in neurotoxicity. Whereas it is clear that HIV enters and replicates in cells only derived from humans or selected primates, studies in rodents suggest that AIDS-related symptoms/damage can be elicited by the envelope protein alone. This further implies that issues relating to neurotoxicity are a result of interference of brain functions that are not unique to the human and which do not involve interaction with the CD4 receptor, the recognized mediator of viral entry

(Klatzmann et al., 1984). The value of these or any other models lies in their predictive potential and relevance to clinical disease. It is the goal of this review to examine the neurotoxic consequences of gp120 in the rodent models and compare these findings with parallel observations made in patients with AIDS.

gpl20 neurotoxicity in CNS cultures The neurotoxic action of HIV envelope protein was first discovered in hippocampal cultures derived from fetal mice (Brenneman et al., 1988b). gpl20-induced toxicity was prevented by co-treatment with either vasoactive intestinal peptide (VIP) or antibodies against L3T4, the mouse homolog of the CD4 receptor, gp120 from different isolates all exhibited potent cell killing action, although the maximum effective dose ranged from 0.01 to 1 pm. These observations predicted that neuronal cell death would be a feature of HIV disease. Neuronal loss now has been reported in specific cortical regions of patients with HIV infection, in the absence of opportunistic infections or neoplasms (Ketzler et al., 1990; Everall et al., 1991). Testing for gp120 neurotoxicity under various culture conditions has given insight into contributing mechanistic factors. Serum in the nutrient medium did not influence the toxic response in this model system, as cultures maintained in serum-free medium showed similar responses to those containing 5% horse serum. However, in hippocampal cultures composed of >90% neurons, no neuronal cell death was observed over a wide range of concentrations of gpl20 treatment (Fig. 1). Thus, the toxicity of gp120 was contingent on the presence of glial cells. These data suggest an indirect mechanism for gp120 neurotoxicity, involving more than one cell type and entailing multiple biochemical intermediaries. The nature and complexity of these indirect effects and the

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Fig. 1. To test for potential differences in the vulnerability to neuronal cell killing action, hippocampal cultures composed of a mixture of cell types (neurons and glia) maintained in either 5% horse serum/MEM (filled circles) or in serum-free medium (open triangles) were compared to a hippocampal cultures that were neuron-enriched and gila-poor (open squares). Cultures above were compared for the relative abundance of neurons (neuron specific enolase immunoreactivity) vs astroglia (glial fibrillary acidic protein immunoreactivity). The mixed cultures were comprised of 26% neurons whereas the percentage of cells that were neurons in the neuron-enriched cultures was 91%. Significant decreases in neuronal cell counts were observed in mixed cultures (both those in the presence or absence of serum in the medium) treated with gp120 at concentrations -> 0.01 pM (P < 0.001). In the gila-poor cultures, no significant changes in neuronal cell counts were observed with any concentration gpl20 examined. Control counts between experiments ranged from 600-1500 neurons/100 fields. Values are the mean of 8-12 determinations from three separate dissections. The error bar is the standard error. identity of biochemical mechanisms is only beginning to be explored. The neurotoxic effects of gp120 were confirmed in rat retinal ganglion cells and extended with the observation that gpl20 produced increases in calcium which could be attenuated by N M D A antagonists or calcium channel blockers (Dreyer et al., 1990; Lipton et al., 1991). Excitatory amino acids and the generation of nitric oxide apparently have significant roles in mediating the toxic responses (Dawson et al., 1993). M a n y have hypothesized a role for cytotoxic cytokines in mediating the neurological imp a i r m e n t associated with A I D S (Merrill et al., 1992). Indeed, increased expression of

several cytokines, including interleukin-1 (IL-1) and t u m o r necrosis factor alpha, has b e e n observed in the brains of HIV-infected patients (Tyor et al., 1992). Infusion of gpl20 into rat brain induces an elevation in IL-1 and IL-l-like effects (Sundar et al., 1991). In regard to the cytotoxic action of IL-1 in rodent cultures, we have observed a potent neuronal cell killing action of I L - I alpha ( B r e n n e m a n et al., 1993) that occurred over a very narrow range of concentrations (1-10 pm). F u r t h e r m o r e , this toxic response was dependent on the age of the test culture. IL-1 alpha exhibited a trophic role in developing cultures ( B r e n n e m a n et al., 1992) and a killing action in 1 month-old cultures

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(Brenneman et al., 1993). The mechanism for this stage-dependent effect of IL-1 alpha is unknown. The above examples serve to point out the complexity of gpl20-induced toxic mechanisms, which involve several substances and multiple pathways.

gpl20 neurotoxicity: In vivo models The morphological and behavioral responses of developing rodents to gpl20 treatment have also been examined as models for " N e u r o A I D S " associated symptoms/damage. As shown in Fig. 2, neonatal animals injected subcutaneously with purified gpl20 exhibited dystrophic changes in all cortical areas examined (Hill et al., 1993). These abnormalities included thickened and coarse basilar and oblique dendritic branches, decreased branching and a significant reduction in the length of dendrites. Most dendritic spines were clumped and fused and characterized by thickened spine necks with no expanded heads. These changes are similar to those described for cortical pyramidal neurons from the brains of patients with H I V encephalopathy (Wiley et al., 1991; Masliah et al., 1992). In the neonatal rodent model described above, co-treatment with peptide T (an octapeptide from gpl20 with sequence homology to VIP, see section on cytoprotection) p r e v e n t e d gpl20-induced n e u r o d y s t r o p h y (Fig. 2d). Compared to controls, no changes in gross brain morphology or brain weight was apparent after treatment with gp120 or peptide T. These studies indicate that neuronal damage occurred in neonatal animals exposed to peripheral administration of gp120 and that this damage could be prevented with peptide T (Hill et al., 1993). To investigate the effects of gp120 on neurobehavioral development, a battery of tests was conducted on rat neonates treated daily from birth to 2 weeks of age with subcutaneous injections of gpl20 (Hill et al., 1993). As shown in Table 1, developmental

Table 1. Developmental behaviors and milestones of gpl20-treated rat neonates compared with control neonates Delayed behaviors Time to exhibit behavior

Fold delay Surface righting Negative geotaxis

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Day of onset Day delay Forelimb placing Hindlimb placing Forelimb grasp Air righting Ear twitch

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Undelayed behaviors Auditory startle Eyelid reflex Gait Eye opening Crossed extensor

From birth to day 20, rat pups received a subcutaneous injection of 5 ng gpl20, 5 ng gpl20 + 0.5 ixg peptide T, 0.5 txg peptide T or saline and daily observations were made of neonatal behaviors and developmental signs. Either the time to perform the behavior or the day of first appearance of the behavior was recorded. Compared with saline, gpl20 treatment resulted in the retardation of seven behaviors. Co-treatment with peptide T either prevented or attenuated the gpl20 effects. delays occurred in several behaviors involving complex motor responses. Whereas most tests showed delays in onset and decreased levels of performance, several assessments of sensory development, e.g. eye opening and auditory startle, showed no apparent delay. In additional studies, rats treated with gpl20 exhibited impaired performance in the Morris swim maze, a measure of learning and memory (Glowa et al., 1992). Delays in the achievement of developmental

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Fig. 2. Photomicrographs of layer V cortical pyramidal neurons stained by the rapid Golgi method from the brains of rats which had received daily subcutaneous injections from birth to day 28 of either: (a) sterile saline; (b) 5.0 ng gpl20; (c) 0.5 ~xg peptide T; (d) 5.0 ng gpl20 + 0.5 Ixg peptide T. Adjacent to each neuron is a higher magnification illustrating the spine morphology on its basilar dendritic branches. Saline, gpl20 + peptide T and peptide T-treated brains exhibit the normal morphological characteristics of soma and dendritic branching of pyramidal neurons. Brains from gp120-treated animals exhibited neuronal dystrophy, including reduced branching and thickened branches. Neuronal magnification was ×350 and x950 for the dendritic branch insert. (Reprinted, with permission, from Hill et al., 1993.)

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milestones are c o m m o n features in pediatric A I D S and previous studies have shown that a b o u t 50% of H I V - i n f e c t e d children with neurological dysfunction have manifestations of m o t o r i n v o l v e m e n t ( B e l m a n et

al., 1988). T o g e t h e r , these studies suggest that g p l 2 0 administration can result in brain dysfunction, the qualitative nature of which has similarities to those o b s e r v e d in patients with H I V disease.

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gp120 as an etiologic agent for NeuroAIDS

Toxic fragments of gpl20 Both the cortical neurodystrophy and delays in developmental milestones occurred after the systemic injection of purified gpl20 to neonatal rats. A priori, the probability appeared very low that a large glycoprotein could reach the CNS intact. To test for the possibility that a toxic fragment of gpl20 might mediate the observed deleterious effects, radiolabeled gpl20 was injected into newborn rats (Fig. 3). The brain supernatant from animals injected with gp120 contained several low molecular weight fragments that were shown to be toxic when tested on cerebral cortical cultures obtained from newborn rats (Hill et al., 1993). The cell death associated with fraction 54, the smallest fragment of gpl20 exhibiting toxicity, was prevented by co-treatment with peptide T in the in vitro toxicity assays. Importantly, no toxicity was found in the brain supernatant of control mice. These data indicate that a degradative product of gpl20 can be generated in vivo and our studies suggest that this substance enters the brain and produces dysfunction and neuronal damage/death. The structural nature of this fragment remains unidentified.

Cytoprotective mechanisms

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1990) and activity-dependent neurotrophic factor (Brenneman and Gozes, 1992). The latter substance is a novel protein that can also prevent neuronal cell killing in gp120-treatment cerebral cortical cultures (Brenneman and Gozes, 1993). These data suggest a pathway where gp120 toxicity is blocked by the release of trophic substances that have a cytoprotective action. Alternatively, the small sequence homology between VIP, peptide T and gpl20 has suggested that these substances act through a common receptor. The focus of this hypothesis relies on the observation that peptide T sequences were identified in all strains of gp120 and that these sequences have some homology to VIP (Ruff et al., 1987a,b; Smith et al., 1988). The active site in peptide T belongs to a family of gp120associated peptides that are more accurately classified as pharmacologically homologous rather than amino acid sequence homologous, as considerable divergence exists: e.g. TTNYT, TTSYS, TTSYT and STNYT. All these peptides can prevent gp120-associated cell death in dissociated hippocampal cultures (Brenneman et al., 1988a). TDNYT is the homologous sequence in VIP, a peptide that has been shown to be cytoprotective, albeit at 100-fold less potency than VIP (Brenneman et al., 1988a). The protective action of these peptides may be attributed to their ability to block the attachment of gpl20 or a neurotoxic fragment of gp120. Support for this hypothesis has been revealed in binding studies, HIV infectivity assays and monocyte chemotaxis measurements (Pert et al., 1986; Ruff et al., 1987a,b; Sacerdote et al., 1987; Smith et al., 1988).

The molecular mechanism for the cytoprotective effect of vasoactive intestinal peptide (VIP) and peptide T has not been resolved. Two hypotheses, that may not be mutually exclusive, have been considered: (1) a neurotrophic hypothesis in which a growth factor released by VIP/peptide T prevents or attenuates the deleterious actions of gp120; or (2) a ligand competition hypothesis based on the sequence homology shared between VIP, peptide T and gp120. In regard to the References neurotrophic hypothesis, VIP can act as an Belman, A. L., Diamond, G., Dickson, D., astroglia secretagogue for substances that Horoupian, D., Llena, J., Lantos, G. and increase the survival of neurons (Brenneman Rubinstein, A. (1988). Pediatric acquired et al., 1990b). Among the trophic substances immunodeficiency syndrome: Neurologic synreleased are protease nexin I (Festoff et al., , dromes. A m . J. Dis. Child. 142:29-35.

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