Molecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits

doi:10.1016/j.neuroscience.2005.02.049

Neuroscience

Volume 134, Issue 1, 2005, Pages 145-153

https://doi.org/10.1016/j.neuroscience.2005.02.049 Get rights and content

Abstract

We investigated the effect of type 1 human immunodeficiency virus (HIV-1) regulatory protein Tat on N-methyl-d-aspartate (NMDA) receptors expressed in Xenopus oocytes by voltage-clamp recording and its role in NMDA-mediated neurotoxicity using cultured rat hippocampal neurons. Tat (0.01–1μM) potentiated NMDA-induced currents of recombinant NMDA receptors. However, in the presence of Zn²⁺, the potentiating effect of Tat was much more pronounced, indicating an additional Zn²⁺-related effect on NMDA receptors. Consistently, Tat potentiated currents of the particularly Zn²⁺-sensitive NR1/NR2A NMDA receptor with a higher efficacy, whereas currents from a Zn²⁺-insensitive mutant were only marginally augmented. In addition, chemical-modified Tat, deficient for metal binding, did not reverse Zn²⁺-mediated inhibition of NMDA responses, demonstrating that Tat disinhibits NMDA receptors from Zn²⁺-mediated antagonism by complexing the cation. We therefore investigated the interplay of Tat and Zn²⁺ in NMDA-mediated neurotoxicity using cultures of rat hippocampal neurons. Zn²⁺ exhibited a prominent rescuing effect when added together with the excitotoxicant NMDA, which could be reverted by the Zn²⁺-chelator tricine. Similar to tricine, Tat enhanced NMDA-mediated neurotoxicity in the presence of neuroprotective Zn²⁺ concentrations. Double-staining with antibodies against Tat and the NR1 subunit of the NMDA receptor revealed partial colocalization of the immunoreactivities in membrane patches of hippocampal neurons, supporting the idea of a direct interplay between Tat and glutamatergic transmission. We therefore propose that release of Zn²⁺-mediated inhibition of NMDA receptors by HIV-1 Tat contributes to the neurotoxic effect of glutamate and may participate in the pathogenesis of AIDS-associated dementia.

Section snippets

Reagents

NMDA, MK-801, D-APV and Trypan Blue were obtained from RBI Sigma (Taufkirchen, Germany). The recombinant Tat of HIV-1 (1–86) and the modified Tat (Tat-toxoid) were gifts from Dr. R. El Habib (Aventis-Pasteur, Lyon, France).

cRNA synthesis, mutagenesis and oocyte expression

NotI linearized plasmid cDNAs of the rat NR1-1a (Moriyoshi et al., 1991), the mouse NR2A (Meguro et al., 1992) and NR2B (Kutsuwada et al., 1992) NMDA receptor subunits were used for the in vitro synthesis of cRNA (mCAP mRNA Capping Kit, Stratagene, La Jolla, CA, USA) using T7

Potentiation of NMDA receptors by HIV-1 Tat involves reversal of Zn²⁺ inhibition

To gain insight into the mechanisms of NMDA receptor modulation by Tat, we analyzed the effect of recombinant Tat on NMDA-induced currents of recombinant NR1/NR2B receptors expressed in Xenopus laevis oocytes by voltage-clamp recording in the absence and presence of Zn²⁺. In the absence of Zn²⁺, a single application of Tat (0.1–10μM) did not alter NMDA receptor-mediated currents generated at saturating concentrations of NMDA and glycine (data not shown). However, in the presence of 1μM Zn²⁺,

Discussion

HIV-Tat causes apoptotic neuronal death which can be antagonized by NMDA receptor blockers, indicating that Tat-induced neuronal injury interferes with glutamatergic transmission in the CNS. We demonstrate in this study that HIV-Tat impairs Zn²⁺ inhibition of NMDA receptors, and thereby potentiates the NMDA-mediated death of cultured hippocampal neurons. Thus, the data presented here constitute evidence that complex Zn²⁺-dependent potentiating effects of the Tat-protein on NMDA

Acknowledgments

We thank Prof. P. Chandra for his interest in our project and making Tat available for our research, and Prof. H. Betz for critically reading the manuscript. This study was supported by Dr. Robert Pfleger-Stiftung and Deutsche Forschungsgemeinschaft (La 1086/4–1).

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Attenuated neurotoxicity of the transactivation-defective HIV-1 Tat protein in hippocampal cell cultures
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This study reports that the cysteine 22 → glycine 22 substitution in the HIV-1 Tat 1–86 B significantly attenuates its neurotoxicity. Consistent with previous studies, direct interactions of rat hippocampal cells with Tat 1–86 B were shown to cause dose-dependent and time-dependent neurotoxicity associated with activation of caspases from the mitochondrial apoptotic pathway. Despite the similar binding/uptake properties, Cys22 Tat 1–86 B failed to induce significant neurotoxicity and activation of caspases 9 and 3/7 in hippocampal primary cultures. Results of the study underscore the important role of cysteine-rich domain in mechanism of Tat-mediated neurotoxicity.
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Citation Excerpt :
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¹: Present address: Wellcome Laboratory of Molecular Pharmacology, Department of Pharmacology, University College London, Gower Street, London WC1E 6BT, UK.

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Molecular neuroscienceMolecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits

Abstract

Section snippets

Reagents

cRNA synthesis, mutagenesis and oocyte expression

Potentiation of NMDA receptors by HIV-1 Tat involves reversal of Zn2+ inhibition

Discussion

Acknowledgments

Neurosci Lett

Biochem Biophys Res Com

Neurochem Int

Neurobiol Dis

Curr Opin Neurobiol

FEBS Lett

Lancet Neurol

Neuroscience

Neurosci Lett

Neuron

Prog Neurobiol

Brain Res

Neurosci

Trends Pharmacol Sci

Neurosci Lett

Neurosci Lett

Release of endogenous Zn2+ from brain tissue during activity

Nature

Optimized survival of hippocampal neurons in B27-supplemented Neurobasal™, a new serum-free medium combination

J Neurosci Res

p75 Neurotrophin receptor is required for constitutive and NGF-induced survival signalling in PC12 cells and rat hippocampal neurones

J Neurochem

Differential sensitivity of recombinant N-methyl-d-aspartate receptor subtypes to zinc inhibition

Mol Pharmacol

Pharmacology of glutamate neurotoxicity in cortical cell cultureattenuation by NMDA antagonists

J Neurosci

Three pairs of cysteine residues mediate both redox and Zn2+ modulation of the NMDA receptor

J Neurosci

Molecular neuroscience
Molecular interactions of the type 1 human immunodeficiency virus transregulatory protein Tat with N-methyl-d-aspartate receptor subunits

Potentiation of NMDA receptors by HIV-1 Tat involves reversal of Zn²⁺ inhibition

Release of endogenous Zn²⁺ from brain tissue during activity

Three pairs of cysteine residues mediate both redox and Zn²⁺ modulation of the NMDA receptor