Elsevier

Neurobiology of Disease

Volume 49, January 2013, Pages 128-136
Neurobiology of Disease

Astrocytic expression of HIV-1 Nef impairs spatial and recognition memory

https://doi.org/10.1016/j.nbd.2012.08.007Get rights and content

Abstract

Despite the widespread use of antiretroviral therapy that effectively limits viral replication, memory impairment remains a dilemma for HIV infected people. In the CNS, HIV infection of astrocytes leads to the production of the HIV-1 Nef protein without viral replication. Post mortem studies have found Nef expression in hippocampal astrocytes of people with HIV associated dementia suggesting that astrocytic Nef may contribute to HIV associated cognitive impairment even when viral replication is suppressed. To test whether astrocytic expression of Nef is sufficient to induce cognitive deficits, we examined the effect of implanting primary rat astrocytes expressing Nef into the hippocampus on spatial and recognition memory. Rats implanted unilaterally with astrocytes expressing Nef showed impaired novel location and novel object recognition in comparison with controls implanted with astrocytes expressing green fluorescent protein (GFP). This impairment was correlated with an increase in chemokine ligand 2 (CCL2) expression and the infiltration of peripheral macrophages into the hippocampus at the site of injection. Furthermore, the Nef exposed rats exhibited a bilateral loss of CA3 neurons. These results suggest that Nef protein expressed by the implanted astrocytes activates the immune system leading to neuronal damage and spatial and recognition memory deficits. Therefore, the continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HIV associated cognitive impairment.

Highlights

► Astrocyte HIV-1 Nef production in hippocampus produces learning impairment in rats. ► Nef expression in astrocytes stimulates CCL2 production. ► CCL2 induced by Nef stimulates peripheral monocyte infiltration into hippocampus. ► Neuronal loss is associated with Nef expression, CCL2 induction and monocyte infiltration.

Introduction

HIV/AIDS remains a global epidemic affecting over 33 million people, including 2.6 million new infections and 1.8 million deaths in 2009 (UNAIDS/WHO 2009 Global Summary). HIV infection is associated with a spectrum of neurological disorders and opportunistic infections in the CNS. The virus quickly invades the brain and establishes a foothold predominantly in microglia and infiltrating macrophages that support viral replication, and in astrocytes that produce a limited set of viral neurotoxins (Messam and Major, 2000, Tornatore et al., 1991, Wiley et al., 1986). In the beginning of the epidemic, 10  20% of the patients with HIV showed severe manifestations of neuropathology known as the AIDS dementia complex, characterized by low CD4, high viral loads, cognitive and motor impairments and behavioral changes (McArthur et al., 1993, Price et al., 1988). Since the advent of combination antiretroviral therapy (cART), the incidence of dementia has declined significantly; however, milder forms of neuropathology still affect as many as 50% of HIV positive persons (Heaton et al., 2011, McArthur et al., 1999, McArthur et al., 2003, Robertson et al., 2007). Even though viral replication is well controlled by cART, viral proteins persist in viral reservoir areas (Popovic et al., 2005) suggesting that neurotoxic viral proteins may contribute to the continued cognitive decline. Patients with HIV associated neurocognitive disorders (HAND) show deficits in attention, behavior changes and memory impairment (McArthur et al., 1999, McArthur et al., 2003, Sacktor et al., 2002). While it is known that HAND is a result of HIV infection in the brain, the roles of the various infected cell types remain an area of active investigation.

Replication in permissive cells in the brain such as microglia and perivascular macrophages induces the production of new virus and viral proteins, immune system activation and inflammation that result in neurotoxicity and cell death (Anderson et al., 2002). HIV crosses into the brain predominantly through infected mononuclear cells (Haase, 1986, Peluso et al., 1985) which release viral particles that infect permissive cells and produce latent infection of astrocytes (Conant et al., 1994, Thompson et al., 2001, Tornatore et al., 1994). Infection of astrocytes is widespread and the magnitude of astrocyte infection, particularly near perivascular macrophages, correlates with the severity of neuropathogenesis (Churchill et al., 2009). Astrocytes support viral persistence in the brain during suppressive cART (Dayton, 2008), since viral activity in astrocytes is not a target of antiretroviral treatment directed at replicating virus. Recent work showed TCF-4/β-catenin-mediated transcriptional repression of HIV-1 is crucial to restricted repression in astrocytes (Henderson et al., 2012). However, HIV-infected astrocytes do produce early viral proteins (Haughey and Mattson, 2002, Messam and Major, 2000, Van Marle et al., 2004), including Nef, which may constitute an important source of neurotoxins throughout the neurocognitive decline observed in cART-treated populations.

HIV Nef was found to be expressed in astrocytes of post mortem sub-cortical tissue from people with HIV associated dementia (Fiala et al., 2008, Ranki et al., 1995), suggesting that astrocytic expression of Nef may contribute to HIV associated cognitive deficits. Similarly, Nef mRNA was found post mortem in hippocampal tissue of patients who had suffered from HIV associated dementia (Torres-Munoz et al., 2001). In a non-human primate model of AIDS, rhesus macaques infected with a neurovirulent strain of the simian immunodeficiency virus (SIV) developed encephalitis. Post mortem analysis of this study found Nef expression in astrocytes of these monkeys (Overholser et al., 2003). These findings provide compelling evidence that Nef is present in astrocytes and in the hippocampus during HIV infection. However, it is unknown whether the Nef expressed by the astrocytes contributes to memory loss. We hypothesized that astrocytic Nef expression in the hippocampus could produce memory deficits. To test this hypothesis, we examined the effect of implanting astrocytes expressing Nef into the hippocampus on spatial and novel object recognition memory in rats.

Section snippets

Animals

Thirty-day-old, male Sprague Dawley rats were tested in the novel location recognition and novel object recognition tasks. We used three groups: rats implanted with astrocytes expressing Nef (N = 14), green fluorescent protein (GFP, N = 12), and a naive group (N = 8). After surgery, rats were placed in an air purified biobubble and housed in pairs or three to a cage to avoid isolation stress. For pain and discomfort after surgery, a topical antibiotic with pain reliever (3.5 mg neomycin, 400 units of

Nef expression in primary astrocytes is maintained for seven days

We developed a model system to provide expression of the HIV-1 Nef protein in astrocytes to assess if endogenous production of Nef protein by astrocytes is sufficient to cause learning deficits. We extracted primary astrocytes from Sprague Dawley rats, cultured them in vitro and verified the predominance of astrocytes in the cultures by western blot and immunofluorescent analysis of GFAP reactivity. Fig. 1A shows three independent isolations of primary rat astrocytes (lanes 1–3) as well as an

Discussion

While several properties of Nef including direct neurotoxicity (Trillo-Pazos et al., 2000) are well characterized in vitro, the functional importance of HIV-1 Nef protein in the development and progression of neuropathology, especially in the era of cART, is less clear. To begin to address this issue, we created an animal model that mimics the astrocytic expression of Nef found in patients with HIV-associated dementia. We found that the implantation of primary astrocytes expressing HIV-1 Nef

Conclusion

We have demonstrated that Nef expression from astrocytes in the hippocampus is sufficient to produce spatial memory deficits from a short exposure of seven days. Based on our findings and reports from the literature, we propose the following model (Fig. 6). Astrocytes expressing Nef in the hippocampus secrete CCL2 and promote the transmigration of the CD163-positive mononuclear cells into the brain. Inflammatory proteins are known to be released from Nef-expressing astrocytes (Lehmann et al.,

Conflict of interest

None.

Acknowledgments

This research was supported by funds from NIH grants DA026722 and GM008239 to RJN, RCMI Behavioral Facility and RCMI Molecular Biology Core Laboratory (RR003050, MD007579), and MBRS RISE Program (R25GM082406). Special thanks to Janet Colon (western blots and immunofluorescence); Maria Colon, Samary Mendez, Anitza Hernandez, Ana Lopez, Michael Manoharan, and Eliezer Ruiz (surgery and behavioral experiments); and Myrella Cruz, Alcira Benitez, Angelica Perez-Burgos, and Tirtsa Porrata-Doria

References (60)

  • G. Trillo-Pazos et al.

    Recombinant nef HIV-IIIB protein is toxic to human neurons in culture

    Brain Res.

    (2000)
  • G. Van Marle et al.

    Human immunodeficiency virus type 1 Nef protein mediates neural cell death: a neurotoxic role for IP-10

    Virology

    (2004)
  • Z. Wang et al.

    Reduced expression of glutamate transporter EAAT2 and impaired glutamate transport in human primary astrocytes exposed to HIV-1 or gp120

    Virology

    (2003)
  • E. Anderson et al.

    HIV-1-associated dementia: a metabolic encephalopathy perpetrated by virus-infected and immune-competent mononuclear phagocytes

    J. Acquir. Immune Defic. Syndr.

    (2002)
  • M.J. Churchill et al.

    Extensive astrocyte infection is prominent in human immunodeficiency virus‐associated dementia

    Ann. Neurol.

    (2009)
  • A.I. Dayton

    Hitting HIV where it hides

    Retrovirology

    (2008)
  • C. Depboylu et al.

    Lentiviral infection of Rhesus macaques causes long-term injury to cortical and hippocampal projections of prostaglandin-expressing cholinergic basal forebrain neurons

    J. Neuropathol. Exp. Neurol.

    (2012)
  • M. Fiala et al.

    HIV-1 antigens in neurons of cocaine-abusing patients

    Open Virol. J.

    (2008)
  • T. Fischer-Smith et al.

    Monocyte/macrophage trafficking in acquired immunodeficiency syndrome encephalitis: lessons from human and nonhuman primate studies

    J. Neurovirol.

    (2008)
  • T. Fischer-Smith et al.

    CD163/CD16 coexpression by circulating monocytes/macrophages in HIV: potential biomarkers for HIV infection and AIDS progression

    AIDS Res. Hum. Retroviruses

    (2008)
  • B. Gelman et al.

    Prefrontal dopaminergic and enkephalinergic synaptic accommodation in HIV-associated neurocognitive disorders and encephalitis

    J. NeuroImmune Pharmacol.

    (2012)
  • G.J. Guillemin et al.

    Kynurenine pathway metabolism in human astrocytes: a paradox for neuronal protection

    J. Neurochem.

    (2001)
  • A.T. Haase

    Pathogenesis of lentivirus infections

    Nature

    (1986)
  • N.J. Haughey et al.

    Calcium dysregulation and neuronal apoptosis by the HIV-1 proteins Tat and gp120

    J. Acquir. Immune Defic. Syndr.

    (2002)
  • R. Heaton et al.

    HIV-associated neurocognitive disorders before and during the era of combination antiretroviral therapy: differences in rates, nature, and predictors

    J. Neurovirol.

    (2011)
  • L.J. Henderson et al.

    Identification of novel TCF-4 binding sites on the HIV LTR which associate with TCF-4, β-catenin and SMAR1 to repress HIV transcription

    J. Virol.

    (2012)
  • M.B. Huang et al.

    Characterization of Nef-CXCR4 interactions important for apoptosis induction

    J. Virol.

    (2004)
  • Y. Huang et al.

    Glutaminase dysregulation in HIV-1-infected human microglia mediates neurotoxicity: relevant to HIV-1-associated neurocognitive disorders

    J. Neurosci.

    (2011)
  • W. Kelder et al.

    Beta-chemokines MCP-1 and RANTES are selectively increased in cerebrospinal fluid of patients with human immunodeficiency virus-associated dementia

    Ann. Neurol.

    (1998)
  • U. Koedel et al.

    HIV type 1 Nef protein is a viral factor for leukocyte recruitment into the central nervous system

    J. Immunol.

    (1999)
  • Cited by (55)

    • HIV-1 and drug abuse comorbidity: Lessons learned from the animal models of NeuroHIV

      2021, Neuroscience Letters
      Citation Excerpt :

      These rats also showed bilateral loss of CA3 neurons. This study thus showed that continued expression of Nef by astrocytes in the absence of viral replication has the potential to contribute to HAND [128]. Interestingly, cell culture studies have demonstrated that Nef overexpressing astrocytes (via adenovirus vectors) can release Nef in the extracellular vesicles, which, in turn, can be up taken by the neurons, leading to neuronal dysfunction in HAND [129].

    • Pananx notoginseng saponins attenuate CCL2-induced cognitive deficits in rats via anti-inflammation and anti-apoptosis effects that involve suppressing over-activation of NMDA receptors

      2020, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      CCL2 is elevated in the CNS of HIV-1 infected people with HAND, which is critical for neuroAIDS. Further, HIV-1 Nef protein may contribute to HIV-induced cognitive impairment, as cognitive impairment in patients with HIV correlates to increased CCL2 expression [11]. Buprenorphine, which can reduce CCL2-mediated neuroinflammation, may be used for HAND treatment [8,12].

    • Astrocytes autophagy in aging and neurodegenerative disorders

      2020, Biomedicine and Pharmacotherapy
      Citation Excerpt :

      Among HIV-1 viral proteins, Nef is a small HIV-1 protein and abundant expression of HIV Nef was observed in HIV-1-infected astrocytes [150]. Furthermore, recent studies indicated that Nef could induce neurocognitive impairments in rats [19,116]. Overexpression of Nef in human astrocytes caused an increase in ATG8/LC3 and p62 [150].

    View all citing articles on Scopus
    View full text