Neuronal excitatory properties of human immunodeficiency virus type 1 tat protein

doi:10.1016/S0306-4522(97)00174-7

Neuroscience

Volume 82, Issue 1, 25 September 1997, Pages 97-106

https://doi.org/10.1016/S0306-4522(97)00174-7 Get rights and content

Abstract

Neuronal dysfunction and cell death in patients with human immunodeficiency virus type-1 (HIV-1) infection may be mediated by HIV-1 proteins and products released from infected cells. Two HIV-1 proteins, the envelope glycoprotein gp120 and nonstructural protein Tat, are neurotoxic. We have determined the neuroexcitatory properties of HIV-1 tat protein using patch-clamp recording techniques. When fmoles of Tat were applied extracellularly, it elicited dose-dependent depolarizations of human fetal neurons in culture and rat CA1 neurons in slices, both in the absence and presence of tetrodotoxin. These responses were voltage-dependent, reversed at ∼0 mV, and were significantly increased by repetitive applications with no evidence of desensitization. That these responses to Tat were due to direct actions on neurons was supported by observations that Tat dose-dependently depolarized outside-out patches excised from cultured human neurons. Removal of extracellular Ca²⁺ decreased the responses both in neurons and membrane patches.

This is the first demonstration that an HIV-1 protein can, in the absence of accessory cells, directly excite neurons and leads us to speculate that Tat may be a causative agent in HIV-1 neurotoxicity.

Section snippets

Culture and immunofluorescence assay of human fetal neurons

Brain specimens were obtained from fetuses of 14 to 15 weeks gestational age with consent from women undergoing elective termination of pregnancy and approval by the University of Manitoba Committee for the Protection of Human Subjects. Cultures of human fetal neurons were established as described previously.[23]Briefly, the cells were mechanically dissociated, suspended in OptiMEM with 1% heat-inactivated fetal bovine serum, 0.2% N2 supplement (GIBCO), and 1% antibiotic solution (penicillin G

Characterization of human fetal neurons

Cultured human fetal brain cells were >70% neurons as determined by immunostaining for neuron-specific microtubule-associated protein-2 (Fig. 1A) and only those cells exhibiting morphological characteristics of neurons were used in electrophysiological studies. In comparison to rat hippocampal CA1 neurons, human fetal neurons had significantly (P<0.01) lower resting membrane potentials (more negative), longer membrane time constants, and higher thresholds for action potential generation (Table 1

Discussion

The principle finding reported here is that the HIV-1 tat protein directly excites neurons. We found that Tat produces dose-dependent depolarizations of neurons in culture and slice preparations, even in the presence of TTX, suggesting that Tat actions are independent of synaptic interactions. Tat was also found to induce dose-dependent depolarizations in outside-out membrane patches excised from cultured human neurons. When administered intracellularly, however, Tat did not alter neuronal

Conclusions

Sustained activation of neurons by Tat released from HIV-1-infected cells may result in neurotoxicity in patients infected with HIV-1. Although Tat concentrations in situ remain to be determined, it is conceivable that levels sufficient to increase neuronal excitation and neurotoxicity through direct actions on neuronal membranes and to induce Ca²⁺-sensitive, non-desensitizing activation of neurons may well be achieved near HIV-1-infected glia cells.

Acknowledgements

We thank Carolyn Gibbs, Mark Bernier, and Carol Martin for excellent technical assistance. The tat genome of HIV-1BRU and the HL3T1 cells were obtained as gifts from the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. This project was supported in part by the National Health and Research Development Program (NHRDP) and the Medical Research Council (MRC) of Canada. J. D. Geiger is a MRC Scientist, A. Nath is a NHRDP AIDS Scholar, and M. Ma is a recipient of a Manitoba

References (40)

D.J Benos et al.
Cytokines and HIV envelope glycoprotein gp120 stimulate Na⁺/H⁺ exchange in astrocytes
J. biol. Chem.
(1994)
D.W Choi
Calcium-mediated neurotoxicity: relationship to specific channel types and role in ischemic damage
Trends Neurosci.
(1988)
A.G Diop et al.
Tetrodotoxin blocks HIV coat protein (gp120) toxicity in primary neuronal cultures
Neurosci. Lett.
(1994)
M Hayman et al.
Neurotoxicity of peptide analogues of the transactivating protein tat from Maedi-Visna virus and human immunodeficiency virus
Neuroscience
(1993)
V Philippon et al.
The basic domain of the lentiviral Tat protein is responsible for damages in mouse brain: involvement of cytokines
Virology
(1994)
R.E Twyman et al.
Glutamate receptor antibodies activate a subset of receptors and reveal an agonist binding site
Neuron
(1995)
P Ascher et al.
Quisqualate- and kainate-activated channels in mouse central neurones in culture
J. Physiol., Lond.
(1988)
J.R Brorson et al.
Ca²⁺ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells
J. Neurosci.
(1994)
J.R Brorson et al.
AMPA receptor desensitization predicts the selective vulnerability of cerebellar Purkinje cells to excitotoxicity
J. Neurosci.
(1995)
D.W Choi
Excitotoxic cell death
J. Neurobiol.
(1992)

D.W Choi

Calcium and excitotoxic neuronal injury

Ann. N. Y. Acad. Sci.

(1994)

B Ensoli et al.

Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation

J. Virol.

(1993)

L.G Epstein et al.

Human immunodeficiency virus type 1 infection of the nervous system: pathogenic mechanisms

Ann. Neurol.

(1993)

M.B Feinberg et al.

The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation

Proc. natn. Acad. Sci. U.S.A.

(1991)

G Garthwaite et al.

AMPA neurotoxicity in rat cerebellar and hippocampal slices: histological evidence for three mechanisms

Eur. J. Neurosci.

(1991)

F Gray et al.

Prominent cortical atrophy with neuronal loss as correlate of human immunodeficiency virus encephalopathy

Acta. neuropath., Berlin

(1991)

D.M Hartley et al.

Glutamate receptor-induced 45Ca²⁺ accumulation in cortical cell culture correlates with subsequent neuronal degeneration

J. Neurosci.

(1993)

M Hollmann et al.

Ca²⁺ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition

Science

(1991)

M Iino et al.

Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurones

J. Physiol., Lond.

(1990)

P Jonas et al.

Glutamate receptor channels in isolated patches from CA1 and CA3 pyramidal cells of rat hippocampal slices

J. Physiol., Lond.

(1992)

Cited by (133)

Astrocytes: Role in pathogenesis and effect of commonly misused drugs in the HIV infected brain
2023, Current Research in Neurobiology
The roles of astrocytes as reservoirs and producers of a subset of viral proteins in the HIV infected brain have been studied extensively as a key to understanding HIV-associated neurocognitive disorders (HAND). However, their comprehensive role in the context of intersecting substance use and neurocircuitry of the reward pathway and HAND has yet to be fully explained. Use of methamphetamines, cocaine, or opioids in the context of HIV infection have been shown to lead to a faster progression of HAND. Glutamatergic, dopaminergic, and GABAergic systems are implicated in the development of HAND-induced cognitive impairments. A thorough review of scientific literature exploring the variety of mechanisms in which these drugs exert their effects on the HIV brain and astrocytes has revealed marked areas of convergence in overexcitation leading to increased drug-seeking behavior, inflammation, apoptosis, and irreversible neurotoxicity. The present review investigates astrocytes, the neural pathways, and mechanisms of drug disruption that ultimately play a larger holistic role in terms of HIV progression and drug use. There are opportunities for future research, therapeutic intervention, and preventive strategies to diminish HAND in the subset population of patients with HIV and substance use disorder.
Citrate shuttling in astrocytes is required for processing cocaine-induced neuron-derived excess peroxidated fatty acids
2022, iScience
Citation Excerpt :
Likewise, little to no additive effects were observed with cocaine and Tat together. Of note, the choice of Tat from among the HIV components was based upon its well-established stimulation of inflammatory cytokine and ROS production, induction of cytotoxicity, and presumed presence in the brain in people with HIV with undetectable plasma virus (Sabatier et al., 1991; Cheng et al., 1998; Haughey et al., 2001; Li et al., 2009). This evidence makes it all the more surprising that cocaine and Tat failed to exert additive or synergistic effects on either neurons or astrocytes.
Disturbances in lipid metabolism in the CNS contribute to neurodegeneration and cognitive impairments. Through tight metabolic coupling, astrocytes provide energy to neurons by delivering lactate and cholesterol and by taking up and processing neuron-derived peroxidated fatty acids (pFA). Disruption of CNS lipid homeostasis is observed in people who use cocaine and in several neurodegenerative disorders, including HIV. The brain’s main source of energy is aerobic glycolysis, but numerous studies report a switch to β-oxidation of FAs in response to cocaine. Unlike astrocytes, in response to cocaine, neurons cannot efficiently consume excess pFAs for energy. Accumulation of pFA in neurons induces autophagy and release of pFA. Astrocytes endocytose the pFA for oxidation as an energy source. Our data show that blocking mitochondrial/cytosolic citrate transport reduces the neurotrophic capacity of astrocytes, leading to decreased neuronal fitness.
HIV-1 Tat protein promotes neuronal dysregulation by inhibiting E2F transcription factor 3 (E2F3)
2019, Journal of Biological Chemistry
Individuals who are infected with HIV-1 accumulate damage to cells and tissues (e.g. neurons) that are not directly infected by the virus. These include changes known as HIV-associated neurodegenerative disorder (HAND), leading to the loss of neuronal functions, including synaptic long-term potentiation (LTP). Several mechanisms have been proposed for HAND, including direct effects of viral proteins such as the Tat protein. Searching for the mechanisms involved, we found here that HIV-1 Tat inhibits E2F transcription factor 3 (E2F3), CAMP-responsive element–binding protein (CREB), and brain-derived neurotropic factor (BDNF) by up-regulating the microRNA miR-34a. These changes rendered murine neurons dysfunctional by promoting neurite retraction, and we also demonstrate that E2F3 is a specific target of miR-34a. Interestingly, bioinformatics analysis revealed the presence of an E2F3-binding site within the CREB promoter, which we validated with ChIP and transient transfection assays. Of note, luciferase reporter assays revealed that E2F3 up-regulates CREB expression and that Tat interferes with this up-regulation. Further, we show that miR-34a inhibition or E2F3 overexpression neutralizes Tat's effects and restores normal distribution of the synaptic protein synaptophysin, confirming that Tat alters these factors, leading to neurite retraction inhibition. Our results suggest that E2F3 is a key player in neuronal functions and may represent a good target for preventing the development of HAND.
Neuroprotective effects of fatty acid amide hydrolase catabolic enzyme inhibition in a HIV-1 Tat model of neuroAIDS
2018, Neuropharmacology
The HIV-1 transactivator of transcription (Tat) is a neurotoxin involved in the pathogenesis of HIV-1 associated neurocognitive disorders (HAND). The neurotoxic effects of Tat are mediated directly via AMPA/NMDA receptor activity and indirectly through neuroinflammatory signaling in glia. Emerging strategies in the development of neuroprotective agents involve the modulation of the endocannabinoid system. A major endocannabinoid, anandamide (N-arachidonoylethanolamine, AEA), is metabolized by fatty acid amide hydrolase (FAAH). Here we demonstrate using a murine prefrontal cortex primary culture model that the inhibition of FAAH, using PF3845, attenuates Tat-mediated increases in intracellular calcium, neuronal death, and dendritic degeneration via cannabinoid receptors (CB₁R and CB₂R). Live cell imaging was used to assess Tat-mediated increases in [Ca²⁺]_i, which was significantly reduced by PF3845. A time-lapse assay revealed that Tat potentiates cell death while PF3845 blocks this effect. Additionally PF3845 blocked the Tat-mediated increase in activated caspase-3 (apoptotic marker) positive neurons. Dendritic degeneration was characterized by analyzing stained dendritic processes using Imaris and Tat was found to significantly decrease the size of processes while PF3845 inhibited this effect. Incubation with CB₁R and CB₂R antagonists (SR141716A and AM630) revealed that PF3845-mediated calcium effects were dependent on CB₁R, while reduced neuronal death and degeneration was CB₂R-mediated. PF3845 application led to increased levels of AEA, suggesting the observed effects are likely a result of increased endocannabinoid signaling at CB₁R/CB₂R. Our findings suggest that modulation of the endogenous cannabinoid system through inhibition of FAAH may be beneficial in treatment of HAND.
Dose-dependent neurocognitive deficits following postnatal day 10 HIV-1 viral protein exposure: Relationship to hippocampal anatomy parameters
2018, International Journal of Developmental Neuroscience
Citation Excerpt :
Gp120, a structural viral gene product, is vital for viral entry into the CNS (Hao and Lyman, 1999) and may cause neurotoxicity by binding to cell surface receptors, ultimately leading to neuronal death (Lipton, 1991; Corasaniti et al., 2001a; Haughey and Mattson, 2002). Preclinical assessments, including both in vitro and in vivo studies, have extensively examined both Tat and gp120 (Brenneman et al., 1988; Lipton et al., 1995; Nath et al., 1996; Cheng et al., 1998; Aksenov et al., 2001; Corasaniti et al., 2001b; Bruce-Keller et al., 2003; Aksenov et al., 2006; Aksenova et al., 2006; Fitting et al., 2006a; Aksenov et al., 2008; Adams et al., 2010; Zhu et al., 2011; Bertrand et al., 2013; Fitting et al., 2013; Hahn et al., 2013; Bertrand et al., 2014, 2015; Marks et al., 2016), providing strong evidence for the role of HIV-1 viral proteins in neurocognitive and neuroanatomical alterations in adulthood. Although preclinical and clinical studies have characterized HIV-1 associated neurocognitive disorders (HAND) in adults (e.g., review, Woods et al., 2009; Heaton et al., 2010), neurocognitive deficits in HIV-infected children are poorly understood (Crowell et al., 2014).
Despite the availability of antiretroviral prophylactic treatment, pediatric human immunodeficiency virus type 1 (HIV-1) continues to be a significant risk factor in the post-cART era. The time of infection (i.e., during pregnancy, delivery or breastfeeding) may play a role in the development of neurocognitive deficits in pediatric HIV-1. HIV-1 viral protein exposure on postnatal day (P)1, preceding the postnatal brain growth spurt in rats, had deleterious effects on neurocognitive development and anatomical parameters of the hippocampus (Fitting et al., 2008a,b). In the present study, rats were stereotaxically injected with HIV-1 viral proteins, including Tat_1–86 and gp120, on P10 to further examine the role of timing on neurocognitive development and anatomical parameters of the hippocampus (Fitting et al., 2010). The dose-dependent virotoxin effects observed across development following P10 Tat_1–86 exposure were specific to spatial learning and absent from prepulse inhibition and locomotor activity. A relationship between alterations in spatial learning and/or memory and hippocampal anatomical parameters was noted. Specifically, the estimated number of neurons and astrocytes in the hilus of the dentate gyrus explained 70% of the variance of search behavior in Morris water maze acquisition training for adolescents and 65% of the variance for adults; a brain-behavior relationship consistent with observations following P1 viral protein exposure. Collectively, late viral protein exposure (P10) results in selective alterations in neurocognitive development without modifying measures of somatic growth, preattentive processing, or locomotor activity, as characterized by early viral protein exposure (P1). Thus, timing may be a critical factor in disease progression, with children infected with HIV earlier in life being more vulnerable to CNS disease.
Neonatal intrahippocampal HIV-1 protein Tat<inf>1-86</inf> injection: Neurobehavioral alterations in the absence of increased inflammatory cytokine activation
2014, International Journal of Developmental Neuroscience
Pediatric AIDS caused by human immunodeficiency virus type 1 (HIV-1) remains one of the leading worldwide causes of childhood morbidity and mortality. HIV-1 proteins, such as Tat and gp120, are believed to play a crucial role in the neurotoxicity of pediatric HIV-1 infection. Detrimental effects on development, behavior, and neuroanatomy follow neonatal exposure to the HIV-1 viral toxins Tat_1–72 and gp120. The present study investigated the neurobehavioral effects induced by the HIV-1 neurotoxic protein Tat_1–86, which encodes the first and second exons of the Tat protein. In addition, the potential effects of HIV-1 toxic proteins Tat_1–86 and gp120 on inflammatory pathways were examined in neonatal brains. Vehicle, 25 μg Tat_1–86 or 100 ng gp120 was injected into the hippocampus of male Sprague–Dawley pups on postnatal day 1 (PD1). Tat_1–86 induced developmental neurotoxic effects, as witnessed by delays in eye opening, delays in early reflex development and alterations in prepulse inhibition (PPI) and between-session habituation of locomotor activity. Overall, the neurotoxic profile of Tat_1–86 appeared more profound in the developing nervous system in vivo relative to that seen with the first exon encoded Tat_1–72 (Fitting et al., 2008b), as noted on measures of eye opening, righting reflex, and PPI. Neither the direct PD1 CNS injection of the viral HIV-1 protein variant Tat_1–86, nor the HIV-1 envelope protein gp120, at doses sufficient to induce neurotoxicity, necessarily induced significant expression of the inflammatory cytokine IL-1β or inflammatory factors NF-κβ and I-κβ. The findings agree well with clinical observations that indicate delays in developmental milestones of pediatric HIV-1 patients, and suggest that activation of inflammatory pathways is not an obligatory response to viral protein-induced neurotoxicity that is detectable with behavioral assessments. Moreover, the amino acids encoded by the second tat exon may have unique actions on the developing hippocampus.

View all citing articles on Scopus

View full text

Neuronal excitatory properties of human immunodeficiency virus type 1 tat protein

Abstract

Section snippets

Culture and immunofluorescence assay of human fetal neurons

Characterization of human fetal neurons

Discussion

Conclusions

Acknowledgements

J. biol. Chem.

Trends Neurosci.

Neurosci. Lett.

Neuroscience

Virology

Neuron

Quisqualate- and kainate-activated channels in mouse central neurones in culture

J. Physiol., Lond.

Ca2+ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells

J. Neurosci.

AMPA receptor desensitization predicts the selective vulnerability of cerebellar Purkinje cells to excitotoxicity

J. Neurosci.

Excitotoxic cell death

J. Neurobiol.

Calcium and excitotoxic neuronal injury

Ann. N. Y. Acad. Sci.

Release, uptake, and effects of extracellular human immunodeficiency virus type 1 Tat protein on cell growth and viral transactivation

J. Virol.

Human immunodeficiency virus type 1 infection of the nervous system: pathogenic mechanisms

Ann. Neurol.

The role of Tat in the human immunodeficiency virus life cycle indicates a primary effect on transcriptional elongation

Proc. natn. Acad. Sci. U.S.A.

AMPA neurotoxicity in rat cerebellar and hippocampal slices: histological evidence for three mechanisms

Eur. J. Neurosci.

Prominent cortical atrophy with neuronal loss as correlate of human immunodeficiency virus encephalopathy

Acta. neuropath., Berlin

Glutamate receptor-induced 45Ca2+ accumulation in cortical cell culture correlates with subsequent neuronal degeneration

J. Neurosci.

Ca2+ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition

Science

Permeation of calcium through excitatory amino acid receptor channels in cultured rat hippocampal neurones

J. Physiol., Lond.

Glutamate receptor channels in isolated patches from CA1 and CA3 pyramidal cells of rat hippocampal slices

J. Physiol., Lond.

Ca²⁺ entry via AMPA/KA receptors and excitotoxicity in cultured cerebellar Purkinje cells

Glutamate receptor-induced 45Ca²⁺ accumulation in cortical cell culture correlates with subsequent neuronal degeneration

Ca²⁺ permeability of KA-AMPA-gated glutamate receptor channels depends on subunit composition