Delineating HIV-Associated Neurocognitive Disorders ... - Springer Link

J Neuroimmune Pharmacol (2012) 7:319–331 DOI 10.1007/s11481-011-9310-7

INVITED REVIEW

Delineating HIV-Associated Neurocognitive Disorders Using Transgenic Models: The Neuropathogenic Actions of Vpr Christopher Power & Elizabeth Hui & Pornpun Vivithanaporn & Shaona Acharjee & Maria Polyak

Received: 24 June 2011 / Accepted: 17 August 2011 / Published online: 15 September 2011 # Springer Science+Business Media, LLC 2011

Abstract HIV-associated neurocognitive disorders (HAND) represent a constellation of neurological disabilities defined by neuropsychological impairments, neurobehavioral abnormalities and motor deficits. To gain insights into the mechanisms underlying the development of these disabilities, several transgenic models have been developed over the past two decades, which have provided important information regarding the cellular and molecular factors contributing to the neuropathogenesis of HAND. Herein, we concentrate on the neuropathogenic effects of HIV-1 Vpr expressed under the control of c-fms, resulting transgene expression in myeloid cells in both the central and peripheral nervous systems. Vpr’s actions, possibly through its impact on cell cycle machinery, in brain culminate in neuronal and astrocyte injury and death through apoptosis involving activation of caspases-3, -6 and −9 depending on the individual target cell type. Indeed, these outcomes are also induced by soluble Vpr implying Vpr’s effects stem from direct interaction with target cells. Remarkably, in vivo transgenic Vpr expression induces a C. Power : E. Hui : P. Vivithanaporn : S. Acharjee : M. Polyak Department of Medicine (Neurology), University of Alberta, Heritage Medical Research Center, Edmonton, AB T6G 2S2, Canada P. Vivithanaporn Department of Pharmacology, Faculty of Science, Mahidol University, Rama VI Road, Ratchathewi, Bangkok 10400, Thailand C. Power (*) Division of Neurology, 6–11 Heritage Medical Research Centre, University of Alberta, Edmonton, AB T6G 2S2, Canada e-mail: [email protected] URL: www.BrainPowerLab.ualberta.ca

neurodegenerative phenotype defined by neurobehavioral deficits and neuronal loss in the absence of frank inflammation. Implantation of another viral protein, hepatitis C virus (HCV) core, into Vpr transgenic animals’ brains stimulated neuroinflammation and amplified the neurodegenerative disease phenotype, thereby recapitulating HCV’s putative neuropathogenic actions. The availability of different transgenic models to study HIV neuropathogenesis represents exciting and innovative approaches to understanding disease mechanisms and perhaps developing new therapeutic strategies in the future. Keywords HIV-1 . Neuropathogenesis . Transgenic mouse . Vpr . Dementia

Background Over 33 million individuals worldwide are currently infected with human immunodeficiency virus-1 (HIV-1). Neurological disorders caused by HIV-1 infection involve the central (CNS) and peripheral (PNS) nervous systems at any point during the course of infection, affecting between 40% and 70% of infected individuals in the absence of treatment (reviewed in Power et al. 2009). Despite the availability of combination antiretroviral therapy (cART), HIV-related neurological disorders contribute to substantial personal, economic, and societal burdens although most is known about HIV-1 B clade virus-related neurological disease (Pandya et al. 2005; Yeung et al. 2006). Neurodegenerative syndromes caused directly by the HIV-1 with concurrent immunosuppression have emerged as the most common nervous system disorders in industrialized countries’ clinics today (McArthur et al. 2005). These

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disorders represent the virus’ immediate deleterious effects on neural cells, causing damage to the brain, spinal cord, and peripheral nerves (Jones and Power 2006). The most significant of these primary HIV-associated brain disorders include HIV-associated dementia (also termed AIDS Dementia Complex or HIV encephalopathy) and its frequently antecedent disorder, Minor Neurocognitive Disorder (MND). In addition, asymptomatic neurocognitive impairment in HIV infection has been proposed to be part of the spectrum of HIV associated neurocognitive disorders in HIV infection but its phenotype and impact remains incompletely defined (Antinori et al. 2007). Collectively, these syndromes are termed HIV-associated Neurocognitive Disorders (HAND), but HAD and MND affect up to 10– 20% of HIV-infected patients receiving cART. HAND is characterized by a complex constellation of signs and symptoms including neurocognitive impairments (impaired memory and executive tasks), neurobehavioral disturbances (anxiety, apathy, mania), and motor dysfunction (psychomotor slowing, ataxia), indicative of injury to multiple brain areas (cerebral cortex and subcortical structures including white matter, basal ganglia and hypothalamus) (Boisse et al. 2008). Opportunistic infections of the nervous system arise as consequences of HIV-induced immunosuppression and are infrequently seen in patients in active care and receiving cART. The incidence of severe cases of HIV-associated dementia has declined among HIV-infected persons since the introduction of cART in 1996 (Sacktor et al. 2001). However, the incidence of less severe HAND (mild HAD and MND) has remained unchanged, if not increased, in recent years, perhaps due to the inability of some antiretroviral drugs to cross the blood–brain barrier or the chronic (and systemic) immune activation that persists despite cART(Letendre et al. 2008).

Pathogenic mechanisms of NeuroAIDS HIV-1 neuroinvasion and neurotropism HIV-1 infects leukocytes in the blood and lymphoid organs through engagement of the CD4 molecule and a co-receptor, typically using the chemokine receptors, CXCR4 or CCR5 (Gras and Kaul 2010). These events result in chronic (innate) immune activation and eventual immune exhaustion resulting in immunosuppression. Infected and/or activated leukocytes pass through the blood–brain brain, transporting virus to permissive neural cells including microglia, perivascular macrophages and astrocytes (Fig. 1), which express the requisite receptors for infection (but not neurons or oligodendrocytes) (Gonzalez-Scarano and Martin-Garcia 2005; Yadav and Collman 2009). HIV-1 strains entering the CNS are macrophage-tropic/CCR5dependent although viruses in the peripheral circulation are

J Neuroimmune Pharmacol (2012) 7:319–331

often T cell tropic/CXCR4-dependent (Gorry et al. 2001; Ohagen et al. 1999; Power et al. 1998). The different viral genotypes residing in diverse areas of brain combined with their differential tropism suggest multiple neurotropic variants exist, which might influence the neurological manifestations during HIV infection (Smit et al. 2001). Host factors modulate susceptibility to infection and disease progression including specific polymorphisms, individual host genes (APOBEC3G, tetherin. TRIM5-alpha, etc.) as well as host defense strategies including anti-viral cellular microRNAs (Strebel et al. 2009). HIV-1 neurovirulence HIV infection’s neuropathological hallmarks include neuroinflammation defined by multinucleated giant cells, perivascular cuffs, and diffuse white matter pallor with ensuing neurodegeneration, represented by neuronal or synapto-dendritic damage and loss, all of which are often present in HAND and are correlated with MRI and MRS findings (Jones and Power 2006). The development of HIV-induced neurological disease is defined by neural cell injury and loss resulting from (a) chronic neuroinflammation with excess production of host innate immune molecules (cytokines, chemokines, free radicals, proteases) in the brain, which damage cells, together with (b) molecular diversity and variable expression of HIV-1 proteins (Vpr, gp120, Tat and Nef) by infected macrophages/microglia, which cause neurodegeneration through their cytotoxic actions including autophagy suppression, apoptosis and process (pre- and post-synaptic) retraction in neurons. Concurrent immunosuppression is usually a requisite feature of HIV neurovirulence, likely leading to dysregulation of innate immunity within the brain predicated on the activation of glial cells (microglia and astrocytes) complemented by a distinctive neuroimmune profile in HIV-infected brains (induction of TNF-α, Il-1β, MMP-2, CXCL12, CXCL10, iNOS) (Noorbakhsh et al. 2009; Vergote et al. 2006; Zhang et al. 2003). This neuroimmune milieu has been associated with the hallmarks of the unfolded protein response and endoplasmic reticulum stress, in keeping with other studies (Antony et al. 2007; Noorbakhsh et al. 2006) suggesting a link between innate inflammation and the unfolded protein response (Zhang and Kaufman 2008). Whether this phenomenon is driven by host or viral genes remains unresolved to date. While HIV-1 genome and protein can be detected in the brain, viral abundance in nervous system tissue in terms of viral RNA, proviral DNA or protein is not correlated consistently with the diagnosis or severity of HAND (McArthur et al. 2005). Autopsy studies also show that specific viral genotype polymorphisms distinguish patients with and without HAND, underscoring the conceptualization of HAND as a virus-driven dementia (Power et al.


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Fig. 1 Pathogenesis of HIVassociated neurocognitive disorders (HAND): HIV-1 infects circulating leukocytes (macrophages and lymphocytes), which cross the blood–brain barrier and infect (and activate) perivascular macrophages, (PVMϕ), microglia and astrocytes (nonproductive for astrocytes). 1 Infected/activated cells express (Nef) and secrete (Vpr, gp120) viral proteins. Nef induces the release of neurotoxic molecules including cleaved CXCL12, MCP-1, TNF-α and proteases which damage neurons and astrocytes. 2 In addition, secreted viral proteins (e.g. Vpr) are neurotoxic to neurons and astrocytes. Adapted from Jones & Power, Neurobiol Dis 2006 and van Marle & Power, JNeuroVirol 2005

1994; Power et al. 1998). While apoptosis in neurons has garnered substantial attention in the literature, it is becoming apparent that other types of programmed cell death (autophagy) likely contribute to reduced neural cell viability (Gonzalez-Scarano and Martin-Garcia 2005); for example, we showed that neuronal autophagy was suppressed in HAD brains through a mechanism by which MMP-2 cleaved CXCL12 and the resulting cleaved chemokine switched its receptor-engagement preference from CXCR4 to CXCR3 with ensuing suppression of neuronal autophagy and eventual cell death (Zhu et al. 2009).

Transgenic animals in neuroAIDS pathogenesis Since the initial reports of genetically modified animal models, many transgenic animals have been generated, which has facilitated a broader understanding of disease

mechanisms. Early in the HIV epidemic transgenic mice were developed by several groups including the first model designed by the group of Clements and colleagues in which the activity of the HIV-1 LTR in brain was assessed. These studies showed that depending on the individual LTR sequence, its activity in brain was enhanced and in particular, a LTR derived from a neurotropic HIV-1 strain was most active although a disease phenotype was not assessed in this model (Corboy et al. 1992). Mucke and colleagues reported a transgenic mouse in which the HIV-1 gp120 was expressed under the control of the glial fibrillary acidic protein (GFAP) promoter; this animal model exhibited neuropathological features of HIV infection including gliosis and neuronal injury (Toggas et al. 1994). Subsequent studies showed that these animals had neurobehavioral deficits with clear expression of the transgene. Recently this same animal model was shown to have features of distal sensory polyneuropathy after exposure to

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neurotoxic nucleoside reverse transcriptase inhibitors (Keswani et al. 2006). The group of Jolicoeur has also generated several valuable transgenic animal models including the initial report of the partial expression of full length HIV-1 NL4-3 strain under the control of the neurofilament-light chain promoter (Thomas et al. 1994); these animals exhibited a neurobehavioral phenotype with concurrent evidence of both PNS and CNS disease. Soon thereafter, this same group reported a unique model of HIV-induced (vacuolar) myelopathy in a transgenic animal in which the NL4-3 provirus was partially expressed under the myelin basic protein promoter. These animals clearly showed convergent neurobehavioral and neuropathological phenotypes (Goudreau et al. 1996). Surprisingly, this same group produced a HIV-1 nef transgenic animal model with systemic immune abnormalities when expressed under the control of the CD4 promoter but without apparent neurological phenotype(s) (Hanna et al. 1998). Browning Paul et al. reported an interesting transgenic mouse in which the HIV-1 JR-CSF provirus was expressed and produced infectious virus particles (Browning Paul et al. 2000); more recently this same model was found to have a neurological phenotype (Sun et al. 2008). Another group reported a transgenic rat model in which the full length HIV-1 NL4-3 provirus expressed, albeit without gag-pol encoded proteins; this model also shows neurological phenotype (Reid et al. 2001). The He group described a unique model in which the HIV-1 tat was expressed under the control of a doxycycline-dependent GFAP promoter (Kim et al. 2003); this model has been widely used by multiple groups showing a diversity of neuropathogenic mecha-

nisms coupled with a neurological phenotype. More recently, we reported a transgenic animal in which the Vpr derived from HIV-1 NL4-3 was expressed under the control of the c-fms promoter, which ensured expression in myeloid cells. These animals show systemic abnormalities (Dickie et al. 2004) but relevant to this review, this model exhibited a robust neurological phenotype defined by neurobehavioral abnormalities and neuronal apoptosis (Jones et al. 2007) and associated synaptic injury caused by Vpr’s toxic effects on mitochondria (Fig. 2). Subsequent studies using this model showed that Vpr also mediated astrocyte injury through a mechanism involving activation of caspase-6 (Noorbakhsh et al. 2010). When this same model was crossed with RAG1 null mice, the transgenic homozygote knockout progeny manifested a neurobehavioral phenotype indicative of a distal sensory polyneuropathy (DSP), which was supported by neuropathological and molecular studies (Acharjee et al. 2010). These latter studies prompted us to look more closely into the contribution of Vpr to HIV neuropathogenesis as reviewed below.

HIV-1 Vpr transgenic expression Vpr structure and function The HIV-1 Vpr gene expresses a 14 KDa, 96 amino acid protein (Fig. 2) and is the only accessory gene of HIV-1 that is incorporated efficiently into virions (Muller et al. 2000). It is expressed late during the viral cycle but is believed to function early in HIV-1 replication. Vpr is conserved among primate lentiviruses TATFIIB

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MEQAPEDQGPQREPYNEWTLELLEELKSEAVRHFPRIWLHNLGQHIYETY α-helix α helix1

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NL Cell cycle arrest / Apoptosis Fig. 2 Vpr structure: Amino acid sequence of Vpr (based on HIV-1 NL4-3 strain). The positions of the α-helices (in boxes), the acidic amino-terminus and the basic carboxyl terminus are indicated. Functions associated with Vpr sequences are indicated below. aa = amino acid


HIV-1 and −2, and simian immunodeficiency viruses (SIV) (Tristem et al. 1998). Based on NMR analysis, the proposed protein structure of Vpr consists of three amphipathic αhelices, from amino acids 17–33, 38–50 and 55–77, respectively (Morellet et al. 2003; Schuler et al. 1999; Wecker and Roques 1999). The structure is characterized by a flexible, acidic N-terminus and a flexible, basic Cterminus. The α-helices are joined by flexible loops and fold around a hydrophobic core in which residues are positioned to allow interactions with various cellular and viral partners in a manner which reflect many of its biological functions. Vpr has been associated with various functions in host cells; the best established functions include nuclear import of the pre-integration complex (PIC) (Heinzinger et al. 1994; Yao et al. 1995), induction of cell cycle arrest (He et al. 1995; Jowett et al. 1995), induction of apoptosis (Stewart et al. 1997), modulation of reverse transcription (Mansky et al. 2000), virion packaging (Kondo et al. 1995; Lu et al. 1993) and activation of LTRmediated transcription (Felzien et al. 1998; Hogan et al. 2003). These will be discussed briefly below (for additional reviews see Romani and Engelbrecht 2009; Zhao et al. 2011). The ability of Vpr to promote the nuclear import of the PIC is supported by its known ability to move between the cytoplasm and the nucleus (Heinzinger et al. 1994). In HIVinfected PBMCs, Vpr was localized predominantly in the nucleus and in the nuclear membrane (Lu et al. 1993) but Vpr is also required for efficient replication in nonreplicating macrophages (Connor et al. 1995; Heinzinger et al. 1994). Since Vpr has not known to contain a nuclear localization signal (NLS), it is generally assumed that nuclear localization of Vpr is mediated by a distinct pathway that does not require an NLS, but might use a modification of the classical NLS-cargo machinery or is facilitated by various cellular factors, such HSP70 (Zhao et al. 2011). Vpr is able to inhibit cell growth by blocking infected cells in the G2/M phase of the cell cycle (He et al. 1995; Jowett et al. 1995; Re et al. 1995). It is believed that the cell cycle G2 arrest provides a replication advantage for the virus by allowing maximal virus production. An important step in the progression of cells in the G2 phase into the M involves the cyclin-dependent serine/threonine kinase CDC2. Dephosphorylation of this kinase, specifically at Thr14 and Tyr15, results in activation of CDC2 kinase and subsequent phosphorylation events leads to mitosis (Solomon et al. 1990). DNA damage can arrest cells in G2 phase by activation of pathways that result in hyperphosphorylation of CDC2 kinase (Murray 1992). Jowett et al. (1995) showed that Vpr is necessary and sufficient to induce cell cycle arrest and that this induction was correlated with an increase in the hyperphosphorylation of

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the CDC2 kinase. More recently, Vpr was found to recruit Cul4A-DDB1 (VPRBP), which is an E3 ubiquitin ligase involved in induction of G2 arrest (Belzile et al. 2010; Belzile et al. 2007). These authors found that Vpr via its C-terminal domain could target chromatin for ubiquitination through direct interaction with VPRBP, presumably leading to induction of DNA stress and G2 arrest. This association implies that Vpr is involved in protein ubiquitination and proteasome-mediated proteolysis. Vpr can also cause apoptotic cell death. The significance of Vpr-induced apoptosis during HIV-1 infection is unclear, although it may contribute to the depletion of CD4+ T cells during an infection. Further, the proposed underlying molecular mechanisms are complex. For example, Vpr was found to induce apoptosis subsequent to G2 arrest but continued G2 arrest was not required to maintain Vprinduced apoptosis (Stewart et al. 1997). Studies on Vprinduced apoptosis suggest that mitochondrial intermembrane proteins, including adenine nucleotide translocator (ANT) and cytochrome c are released during apoptosis. It has been demonstrated that Vpr can permeabilize mitochondrial membrane by binding to the permeability transition pore complex (PTPC); this event results in the release of proteins from the inner mitochondrial space, leading to apoptosis (Jacotot et al. 2000). Mitochondrial membrane permeability is believed to responsible for the strong binding between Vpr and ANT and that this interaction forms conductance channels, with PTPC, in the mitochondrial membranes, resulting in the release of mitochondrial proteins (Jacotot et al. 2001). The released cytochrome c molecules will bind to apoptotic peptidaseactivating factor 1 (Apaf-1) and form complexes that will become activated by caspase-9 (Kim et al. 2005). During an HIV infection, the virus enters the cytoplasm and reverse transcription occurs to convert viral RNA to proviral DNA. The HIV-1 reverse transcriptase is an error prone enzyme, misincorporating one nucleotide approximately in every 2000–5000 bases (Basavapathruni and Anderson 2007). Vpr appears to have a role in reverse transcription by its interaction with the nuclear form of uracil-N-glycosylase (UNG2). UNG2 is a DNA-repair enzyme that specifically removes uracil from nuclear DNA (Parikh et al. 2000). Without Vpr, a four-fold increase in mutations has been found in each round of HIV-1 replication (Chen et al. 2002). It has been shown that viral incorporation of Vpr can lower the rate of base pair mutation during replication (Mansky 1996). Further, UNG2 can be recruited into HIV virus particles and that this recruitment requires Vpr incorporation (Mansky et al. 2000). It is possible that Vpr could function to potentiate the activity of UNG2 and promote reverse transcription fidelity. As mentioned above, incorporation of Vpr into virions in essential to the operation of various Vpr functions.

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Conserved residues (L-X-S-L-F-G) in the C terminus of the p6 domain of the Gag precursor protein Pr55gag are required for Vpr incorporation, presumably acting as an anchor to the assembling capsid (Kondo and Gottlinger 1996). Oligomerization of Vpr and specific residues on the hydrophobic and hydrophilic faces of the N-terminal αhelix that mediate protein-protein interactions are also important for Vpr virion incorporation (Venkatachari et al. 2010; Yao et al. 1995). Upon HIV-1 proviral DNA integration into host chromosomes, Vpr promotes viral gene transcription by direct interaction with the long terminal repeat (LTR). Vprmediated gene transcription occurs by Vpr’s association with various transcriptional factors or co-factors on the LTR promoter. For example, Vpr-LTR binding includes transcriptional binding sites of: the CCAAT enhancer binding protein (C/EBP) and adjacent sequences, including a NFκB site, which may mediate promoter activation (Hogan et al. 2003); p300/CBP (a CREB-binding protein) to form a stable complex with the ligand-bound GR (glucocorticoid receptor) via the glucocorticoid response element (GRE) in which Vpr serves as an adapter for LTR gene transcription (Felzien et al. 1998); and transcriptional factor IIB (TFIIB), interacting with Vpr, may activate transcription by promoting conformational changes of TFIIB (Agostini et al. 1996). Vpr-mediated effects in the CNS HIV-1 Vpr is expressed in perivascular macrophages and microglia in brains of HIVinfected individuals (Jones et al. 2007; Wheeler et al. 2006) and is detectable in cerebrospinal fluid (CSF) at similar levels to serum in HIV-infected individuals with neurological diseases (Levy et al. 1994). In vitro studies demonstrated that extracellular application of soluble HIV-1 Vpr protein is cytotoxic to primary rat and human neurons and at high concentrations astrocytes (Huang et al. 2000; Jones et al. 2007; Noorbakhsh et al. 2010). HIV-1 Vpr forms ion channels, alters neuronal membrane responses and induces neuronal apoptosis (Jones et al. 2007; Patel et al. 2000; Piller et al. 1999). In addition, mitochondrial dysfunction caused by soluble HIV-1 Vpr protein resulted in inhibition of axonal outgrowth (Kitayama et al. 2008). Our recent studies indicated that soluble Vpr protein induced intracellular calcium flux and activated caspase-6 pathway in astrocytes, leading to astrocyte apoptosis, albeit at higher concentrations, than in neurons (Noorbakhsh et al. 2010) (Fig. 3a). In microglia, expression of Vpr contributes to induction of RANTES/CCL5 expression in HIV-infected cells (Si et al. 2002). In Vpr transgenic mice with a FVB/N background and the Vpr gene under the control of the c-fms promoter, HIV-1 Vpr transcript was detectable in cortex, basal ganglia and hindbrain. Vpr immunoreactivity was present in monocytoid cells. All three regions of brains also expressed


reduced IL-6 mRNA transcripts compared to their wildtype littermate while transcript levels of other proinflammatory cytokines (i.e. IL-1β and TNFα) and activated monocytoid cells were similar between both groups (Jones et al. 2007). Nissl staining of cerebral cortex revealed fewer numbers and lower density of neurons in Vpr transgenic animals. Immunohistochemistry using anti-glial fibrillary acidic protein (GFAP) and anti-ionized calcium binding adaptor molecule 1 (Iba-1) antibodies as markers for astrocytes and macrophage/microglia, respectively, showed reduced GFAP-positive cells without macrophage/microglia activation in Vpr transgenic animals (Fig. 3b). These neuropathological analyses were consistent with in vitro neurotoxicity assays and reduced immunoreactivity of synaptophysin and GFAP detected by Western blotting in protein extracts from animal forebrains (Jones et al. 2007; Noorbakhsh et al. 2010). Similarly, intrastriatal implantation of full-length Vpr or a Vpr peptide (amino acids 70– 96) with an arginine at amino acid residue 77 reduced neuronal numbers and astrocyte immunoreactivity (Na et al. 2011). The lower expression of GAD65 and VAChT in Vpr transgenic animal brains indicated that expression of Vpr in vivo damaged both GABAergic and cholinergic neurons (Jones et al. 2007). The up-regulation of cleaved caspase-3 and caspase-6 in Vpr transgenic mouse brains highlighted that in vivo Vpr expression induced apoptosis in neurons and astrocytes (Jones et al. 2007; Noorbakhsh et al. 2010). These latter studies support earlier studies showing that implantation of viral vectors expressing HIV-1 Vpr into the cortex or ventricles of neonatal C57Bl/6j mice caused neuronal and astrocyte apoptosis in the cortex despite Vpr expression not being detectable after 1 week post implantation (Cheng et al. 2007). The reduction of the GFAP transcript in our Vpr transgenic model was accompanied by lower levels of glutamate transporters, EAAT1 and EAAT2, together with suppression of the neurotrophic factors, IGF-1 and BDNF. Glutamate levels in cortex and basal ganglia of Vpr transgenic mice were higher than in wild-type littermate, implying that in vivo Vpr expression leads to disruption of astrocyte function (Noorbakhsh et al. 2010). The similar pathogenic effects of HIV-1 Vpr in both FVB/N and C57Bl/6j mouse strains suggest that Vpr-induced neuropathogenesis might be mouse strain-independent. The same HIV-1 Vpr transgenic animals also exhibit neurobehavioral abnormalities. The deficits in motor function as measured by inverted screen and horizontal bar tests were apparent in 4-month old mice (Jones et al. 2007). The reduced locomotor activity was observed in 1year old animals (Noorbakhsh et al. 2010). To extend analyses of the effects of the Vpr transgene on neurobehavioral performance, we measured spatial memory using T-maze with food reward alteration protocol.


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Fig. 3 HIV-1 Vpr causes neuronal and astrocyte injury. a Exposure of soluble HIV-1 Vpr protein to primary human fetal neurons and astrocytes reduce cell viability in a dose-dependent manner. At the same concentration, HIV-1 Vpr is more toxic to neurons than astrocytes. Data are presented as the mean ± SEM. (**, p