Human immunodeficiency virus-1 protein Tat induces excitotoxic loss of presynaptic terminals in hippocampal cultures

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Abstract

Human immunodeficiency virus (HIV) infection of the CNS produces dendritic damage that correlates with cognitive decline in patients with HIV-associated neurocognitive disorders (HAND). HIV-induced neurotoxicity results in part from viral proteins shed from infected cells, including the HIV transactivator of transcription (Tat). We previously showed that Tat binds to the low density lipoprotein receptor-related protein (LRP), resulting in overactivation of NMDA receptors, activation of the ubiquitin–proteasome pathway, and subsequent loss of postsynaptic densities. Here, we show that Tat also induces a loss of presynaptic terminals. The number of presynaptic terminals was quantified using confocal imaging of synaptophysin fused to green fluorescent protein (Syn-GFP). Tat-induced loss of presynaptic terminals was secondary to excitatory postsynaptic mechanisms because treatment with an LRP antagonist or an NMDA receptor antagonist inhibited this loss. Treatment with nutlin-3, an E3 ligase inhibitor, prevented Tat-induced loss of presynaptic terminals. These data suggest that Tat-induced loss of presynaptic terminals is a consequence of excitotoxic postsynaptic activity. We previously found that ifenprodil, an NR2B subunit-selective NMDA receptor antagonist, induced recovery of postsynaptic densities. Here we show that Tat-induced loss of presynaptic terminals was reversed by ifenprodil treatment. Thus, Tat-induced loss of presynaptic terminals is reversible, and this recovery can be initiated by inhibiting a subset of postsynaptic NMDA receptors. Understanding the dynamics of synaptic changes in response to HIV infection of the CNS may lead to the design of improved pharmacotherapies for HAND patients.

Introduction

Human immunodeficiency virus (HIV) infection is a worldwide epidemic that affects approximately 30 million people (Kaul et al., 2001). Neurocognitive deficits are a significant consequence of HIV infection and affect approximately 30–50% of HIV-infected patients (Cysique et al., 2004, Tozzi et al., 2005). Neurological symptoms range in severity from mild cognitive impairment to severe HIV-associated dementia, and are collectively known as HIV-associated neurocognitive disorders (HAND) (Ellis et al., 2007). HAND is a major consequence of HIV infection; progression of these neurological symptoms often renders patients incapable of functioning without daily assistance (Hult et al., 2008, Kaul and Lipton, 2006, Minagar et al., 2008). Additionally, while the advent of combined anti-retroviral therapies has reduced the incidence of HIV-associated dementia, the increased lifespan of HIV-infected patients has increased the prevalence of HAND diagnoses, providing a compelling need to develop improved therapies to combat the rising incidence of HAND.

HIV induces neurotoxicity and subsequent neurocognitive deficits by an indirect mechanism. The virus infects macrophages and microglia, not neurons in the CNS, and these infected cells in turn secrete inflammatory cytokines and shed viral proteins that are toxic to neurons (Genis et al., 1992, Speth et al., 2001). One such toxic protein is the HIV transactivator of transcription (Tat), which is shed by infected cells. Tat mRNA and protein are found in the CNS of HAND patients (Del Valle et al., 2000, Hofman et al., 1994, Hudson et al., 2000, Wiley et al., 1996) and Tat protein induces HAND neuropathologies in vivo (Fitting et al., 2010, Kim et al., 2003). In vitro effects of Tat include dendritic pruning, decreased spine density, and synapse loss (Eugenin et al., 2007, Kim et al., 2008, Liu et al., 2000). Clinical studies have shown that the extent of cognitive decline in HAND patients correlates closely with dendritic damage and synapse loss, rather than overt neuronal death (Sa et al., 2004, Wiley et al., 1999).

Tat induces the loss of excitatory synapses via a mechanism that is distinct from that by which it elicits cell death (Kim et al., 2008). Tat binds to the low density lipoprotein receptor-related protein (LRP), and activates NMDA receptors. The subsequent postsynaptic calcium influx triggers two independent pathways. Loss of the postsynaptic density results from calcium-induced activation of an ubiquitin ligase. Cell death results from calcium-dependent activation of neuronal nitric oxide synthase (nNOS) (Kim et al., 2008). Interestingly, Tat-induced loss of postsynaptic densities is reversible. What is not known, however, is how loss and recovery of postsynaptic densities relates to the dynamics of presynaptic terminals during exposure to HIV-1 Tat.

Synaptophysin is an abundant membrane glycoprotein found on synaptic vesicles at the presynaptic terminal (Johnston and Sudhof, 1990, Rehm et al., 1986, Wiedenmann and Franke, 1985). Synaptophysin is a calcium-binding protein that is nonessential for vesicle release (McMahon et al., 1996), and is a major component of small synaptic vesicles. It is recruited to presynaptic active zones during synaptogenesis, albeit later than precursor proteins such as Bassoon or Piccolo (Fletcher et al., 1991, Friedman et al., 2000, Ziv and Garner, 2004), and is thus a good marker for presynaptic terminals.

In this study, we examined the effects of HIV-1 Tat on presynaptic terminals by tracking the expression of a synaptophysin-GFP fusion protein. HIV-1 Tat decreased the number of presynaptic terminals on hippocampal neurons in culture. Moreover, this loss was triggered by NMDA receptor activity, indicating that loss of presynaptic terminals was initiated by postsynaptic mechanisms. Tat-induced loss of presynaptic terminals was reversible, and this recovery was initiated by modulating NMDA receptor activity. These results suggest that Tat-induced synapse loss and recovery are driven by postsynaptic mechanisms.

Section snippets

Synaptophysin-GFP labels presynaptic terminals

The number of presynaptic terminals in rat hippocampal cultures was measured by confocal imaging of neurons transfected with an expression construct for the presynaptic protein synaptophysin fused to GFP (Syn-GFP). Synaptophysin is part of the neurotransmitter release machinery and is an established marker for presynaptic terminals. The neurons were co-transfected with an expression construct for DsRed2, a red fluorescent protein that filled the neuron and enabled visualization of cell

Discussion

Dendritic pruning and synapse loss are hallmarks of HAND pathology (Everall et al., 1999, Masliah et al., 1997). The HIV-1 protein Tat is shed by infected cells in the central nervous system and in vitro, it binds LRP to activate NMDA receptors, resulting in the loss of postsynaptic densities (Kim et al., 2008, Shin et al., 2012). Here, we examined the effects of Tat on presynaptic terminals. Tat induced loss of presynaptic terminals that mirrored loss of postsynaptic densities with respect to

Conclusions

In conclusion, our study has shown that HIV-1 Tat exposure can induce a reversible loss of presynaptic terminals in hippocampal neurons in culture. This loss and recovery mirrors the effects of Tat on postsynaptic densities and was inhibited by pharmacological agents that act on postsynaptic targets. Thus, the mechanisms initiating both processes are located postsynaptically, in contrast to the mechanisms of synaptogenesis. Elucidating the pathways by which Tat affects the loss and recovery of

Materials

Materials were obtained from the following sources: the Syn-GFP expression vector (pSynaptophysin-GFP-C1) was kindly provided by Jane Sullivan (University of Washington, Seattle, WA); the expression vector for DsRed2 (pDsRed2-N1) was from Clontech (Mountain View, CA); HIV-1 Tat (Clade B, full length, recombinant) was from Prospec Tany TechnoGene Ltd. (Rehovot, Israel) and through the NIH AIDS Research and Reference Reagent Program (HIV-1 Tat protein (full length, Clade B) from Dr. John Brady

Acknowledgments

The authors would like to thank the NIH AIDS Research and Reference Reagent Program for providing HIV-1 Tat protein, and Dr. Jane Sullivan (University of Washington, Seattle, WA) for providing the synaptophysin-GFP expression construct. The National Institute on Drug Abuse grant DA 07304 supported this work. A National Institute on Drug Abuse Training Grant (DA 07234) supported AS.

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