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Product# 1031 HIV-1 gp120 (YU2)

 

Abstract

Despite the recent advances in antiretroviral therapy, HIV-1 remains a global health threat. HIV-1 affects the central nervous system by releasing viral proteins that trigger neuronal death, neuroinflammation, and promotes alterations known as HIV-associated neurocognitive disorders (HAND). This disorder is not fully understood and no specific treatments are available. Recently, we demonstrated that the HIV-1 envelope protein gp120IIIB induces a functional upregulation of the α7-nicotinic acetylcholine receptor (α7) in neuronal cells. Furthermore, this upregulation promotes cell death that can be abrogated with receptor antagonists, suggesting that α7 may play an important role in the development of HAND. The partial duplication of the gene coding for the α7, known as CHRFAM7A, negatively regulates α7 expression but its role in HIV infection has not been studied. Hence, we studied both CHRNA7 and CHRFAM7A regulation pattern in various gp120IIIB in vitro conditions. In addition, we measured CHRNA7 and CHRFAM7A expression levels in postmortem brain samples from patients suffering from different stages of HAND. Our results demonstrate the induction of CHRNA7 expression accompanied by a significant down-regulation of CHRFAM7A in neuronal cells when exposed to pathophysiological concentrations of gp120IIIB. Our results suggest a dysregulation of CHRFAM7A and CHRNA7 expression in the basal ganglia from postmortem brain samples of HIV+ subjects and expand the current knowledge about the consequences of HIV infection in the brain.

Keywords: gp120, HIV, HAND, acetylcholine receptor, CHRNA7, CHRFAM7A

 

Introduction

The human immunodeficiency virus type 1 (HIV-1) is considered one of the principal pandemics of the twenty-first century with approximately 34 million of subjects infected globally (Joint United Nations Programme on HIV/AIDS (UNAIDS 2013). In addition to developing acquired immunodeficiency syndrome (AIDS), infected individuals may also develop neurological complications known as HIV-associated neurocognitive disorders (HAND). HAND includes asymptomatic neurocognitive impairment (ANI), mild neurocognitive disorder (MND), and HIV-associated dementia (HAD) (Antinori et al. 2007). HAD results in disabling cognitive impairment accompanied by motor dysfunction, speech problems, and overt behavioral changes (González-Scarano and Martín-García 2005; Clifford and Ances 2013). Although the incidence of HAD has decreased (Bhaskaran et al. 2008), the prevalence of HAND, mostly of the milder forms of neurocognitive impairment (ANI and MND), could be as high as 50% of patients (Sacktor et al. 2002; Cysique et al. 2004; Heaton et al. 2011). Moreover, the high prevalence of HAND occurs despite administration of combined antiretroviral therapy (cART) (Mothobi and Brew 2012). For instance, under cART, HAND persists despite systemic or brain viral load reduction or control (Cysique and Brew 2011; Koneru et al. 2014).

HIV is unable to infect neurons due to their lack of primary CD4 receptors, however, neuronal expression of both CCR5 and CXCR4 secondary receptors could allow viral interactions (Hesselgesser et al. 1997). Several hypotheses have emerged to explain the cause of HAND including the neurotoxic properties of viral proteins and the severe uncontrolled chronic neuroinflammation (Kong et al. 1996; Heaton et al. 2011). Particularly, the HIV-1 viral envelope protein gp120 has been reported to have various neurotoxic properties in vitro and in vivo including the inhibition of adult neural progenitor cells proliferation, neuronal damage and induction of apoptosis, and cell death of human neuronal cells (Toggas et al. 1994; Meucci and Miller 1996; Hesselgesser et al. 1998; Jana and Pahan 2004; Bardi et al. 2006; Okamoto et al. 2007; Ballester et al. 2012). Moreover, the severity of brain damage correlates with gp120 levels (Desai et al. 2013).

The alpha7 nicotinic acetylcholine receptor (α7) is one of the most common receptors expressed in the mammalian brain (Dani and Lester 2001). The α7 subunit is encoded by the CHRNA7 gene in chromosome 15, and is composed of 10 exons (Gault et al. 1998). Interestingly, the CHRNA7 has a counterpart gene named CHRFAM7A (Gault et al. 1998). The CHRFAM7A gene product, dupα7, exerts a regulatory/inhibitory role on the α7 ion channel activity and expression (de Lucas-Cerrillo et al. 2011; Araud et al. 2011), although a recent work has challenged these results showing that dupα7 and α7 can form functional heteropentamers with altered responses to choline and varenicline (Wang et al. 2014). This may be due to differences in the expression system used that could influence ion channel functionality and assembly –the first study used oocytes while the most recent used Neuro2a cells–, and the use of α7's chaperone RIC-3 in Neuro2a cells but not in oocytes. For a comprehensive review about dupα7 refer to (Costantini et al. 2014). Notwithstanding, although the α7 has been amply studied in CNS, very little is known about its role in the neuropathology of HIV infection. We recently demonstrated that gp120IIIB induces a functional α7 upregulation in neuronal cells and that the expression of gp120IIIB in the brain of a transgenic mouse model also induces the overexpression of α7 in the brain, particularly in the striatum, basal ganglia's primary input (Ballester et al. 2012). Moreover, we found that the activation of upregulated α7 in these neuronal cells induces cell death in a calcium-dependent manner (Ballester et al. 2012). In light of the possible role of α7 in the HIV neuropathogenesis, we evaluated the mRNA expression patterns of CHRNA7, CHRFAM7A and the expression ratio CHRNA7:CHRFAM7A upon gp120IIIB application in a human neuronal cell line and in post-mortem brain samples from HIV-infected patients expressing different severity stages of neurocognitive impairment.

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Materials and methods

Reagents

All reagents were purchased from Sigma Aldrich unless otherwise specified.

Cell culture and treatments

SH-SY5Y neuronal cell line was obtained from ATCC (Manassas, VA). Cells were incubated at 37 °C with 5% CO2 in DMEM/F-12 media supplemented with 10% fetal bovine serum, 1% penicillin/streptomycin and 1.2 g of NaHCO3. Cultures were performed in 12-well plates followed by treatments with gp120IIIB (Fitzgerald Industries International, Concord, MA) at 0.0015, 0.015, 0.15, 1.5 or 15 nM for the indicated time. For time-dependent experiments, the concentration of gp120IIIB was 0.15 nM. The CXCR4 antagonist, AMD3100 (EMD Chemicals, Inc., Gibbstown, NJ), was used at 1 μM and added 10 min prior to gp120IIIB application. The range of gp120 concentrations tested here were based on gp120 quantification studies using plasma, serum and tissues from HIV-infected subjects (Gilbert et al. 1991; Oh et al. 1992; Santosuosso et al. 2009; Rychert et al. 2010). To our knowledge there is no quantification studies to determine gp120 in the brain or cerebrospinal fluid (CSF). However, there is a robust body of evidence demonstrating that indeed gp120 is present in the central nervous system and CSF, even though no evidence of quantification is available in the literature (Buzy et al. 1989; Rolfs and Schumacher 1990; Ruta et al. 1998; Cashion et al. 1999; Jones et al. 2000; Ohagen et al. 2003; Pillai et al. 2006). Moreover, the existence of anti-gp120 antibodies in the CNS unequivocally attests to its presence (Lucey et al. 1993; Di Stefano et al. 1996; Trujillo et al. 1996).

RNA extraction and quantitative RT-PCR assay

In SH-SY5Y neuronal cells, total RNA samples were extracted using TriZol Reagent (Invitrogen Corporation, Carlsbad, CA). To eliminate possible genomic contamination, extracted RNA was treated with DNase using the Ambion DNA free kit (Ambion, Austin, TX). Quantification of total RNA was performed using a Nanodrop system (Thermo Scientific, Waltham, MA). The cDNA synthesis was carried out using 0.75 μg of total RNA with the iScript™ cDNA Synthesis Kit (Bio-Rad Laboratories, Inc., Hercules, CA) following the manufacturer's instructions. After optimization of the PCR conditions, real-time PCR experiments were performed using the iQ™ SYBR® Green Supermix (Bio-Rad Laboratories, Inc., Hercules, CA) in a Mastercycler® Ep Realplex Thermal Cycler (Eppendorf, NY). CHRNA7 and GAPDH primers where used at a final concentration of 400 nM, CHRFAM7A primers at 100 nM together with 100 ng of cDNA. Primers were designed using IDT Designer Software (Integrated DNA Technologies). The primers employed to amplify the genes of interest from cells and tissue samples were the following: CHRNA7 forward, 5′-GCTCCGGGACTCAACATG-3′; reverse, 5′-GGGATTGTAGTTCTTGACCAGC-3′; CHRFAM7A forward, 5′-CCGAAGTTACTGGCCTCTATC-3′ reverse, 5′-CTGAGTCGTGTAGATAAGCTCTC-3′, and for GAPDH: forward, 5′-GCTCTCTGCTCCTCCTGTTC-3′, reverse, 5′-GACTCCGACCTTCACCTTCC-3′. All primers were used with an annealing temperature of 55 °C.

Tissue processing and RNA extraction

Post-mortem brain tissues from HIV-infected patients were obtained from the Texas NeuroAIDS Research Center (IRB#: 98-402), California NeuroAIDS Tissue Network (IRBs#: 00000353, 00000354, 00000355, and 000002758), and UCLA National Neurological AIDS Bank (IRB#: 10000525) Tissue samples were pulverized in liquid nitrogen under RNAse free conditions. RNA extraction was performed using TriZol reagent (Invitrogen) following manufacturer's instructions. The RNA integrity was assayed in 1% electrophoresis agarose gel. Samples were processed for qRT-PCR as described above.

Statistical analyses

To evaluate the statistical significance of changes in expression levels of CHRNA7 and CHRFAM7A in neuronal cells we used One-way ANOVA followed by Holm-Sidak's multiple comparison test which allowed corrections for multiple comparisons with a fixed alpha value (0.05). Spearman correlation was used to identify correlations between CHRFAM7A and CHRNA7 expression levels in neuronal cells. The detected outliers were excluded from analysis. Statistical analysis was conducted using the GraphPad Prism 6 software (GraphPad Software, San Diego, CA, www.graphpad.com).

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Results

gp120IIIB promotes the downregulation of CHRFAM7A in neuronal cells

Neuronal cells were exposed to various concentrations of gp120IIIB including those within the pathophysiological range quantified in HIV-infected patients (0.0015, 0.015 and 0.15 nM) (Gilbert et al. 1991; Oh et al. 1992; Santosuosso et al. 2009; Rychert et al. 2010). Measurements of CHRNA7 and CHRFAM7A levels after addition of pathophysiological relevant gp120IIIB concentrations show that the CHRFAM7A was downregulated in a dose-dependent manner, and that the expression of CHRNA7 was induced (Fig. 1a). Noteworthy is that this effect in CHRFAM7A expression levels is sustained even when supraphysiological concentrations of gp120IIIB were used (15 nM). Further evaluation shows that CHRNA7:CHRFAM7A expression ratios increase with the gp120IIIB treatment (Fig. 1b).

Fig. 1

gp120IIIB induces the downregulation of CHRFAM7A in neuronal cells. a Neuronal cells were incubated with gp120IIIB 0.0015, 0.015, 0.15, 1.5 and 15.0 nM for 12 hours. A downregulation of CHRFAM7A was observed under pathophysiological (0.015 and 0.15 nM) and supraphysiological doses (15 nM). b CHRNA7:CHRFAM7A expression ratio shows a significant increase under pathophysiological and supraphysiological conditions as compared to control. In panel a and b, results were normalized to control. *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001. Statistical analysis: One-way ANOVA followed by Holm-Sidak's multiple comparison tests, error bars represents SEM. For all panels n = 4 independent experiments.

A pathophysiological dose of gp120IIIB time-dependently dysregulates CHRNA7 and CHRFAM7A expression in neuronal cells

CXCR4 is a coreceptor employed by HIV to infect immune cells and is expressed by neurons (Hesselgesser et al. 1997). Neuronal cells exposed to gp120IIIB (0.15 nM) at different time points showed that the α7 gene, CHRNA7, was upregulated after 12 hours post gp120IIIB exposure whereas CHRFAM7A downregulation initiated as early as 15 minutes post gp120IIIB application, and lasts 24 hours (Fig. 2a). Moreover, a ratio analysis demonstrates an early increase in the CHRNA7:CHRFAM7A expression (Fig 2b).

Fig. 2

Time-dependent responses of CHRNA7 and CHRFAM7A in neuronal cells exposed to HIV-1-gp120IIIB. a Neuronal cells were incubated with gp120IIIB (0.15 nM) at various time points. As compared to untreated control cells, downregulation of CHRFAM7A was observed at all-time points while CHRNA7 was upregulated after 12 hours of gp120IIIB application. b CHRNA7:CHRFAM7A expression ratio showed a significant increase after 15 min, 12h, and 24h post gp120IIIB application. Results were normalized and compared to control cells. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001. n = 4 independent experiments. Statistical analysis: One-way ANOVA followed by Holm-Sidak's multiple comparison test.

A CXCR4 antagonist abrogates the gp120IIIB-induced dysregulation of CHRNA7

To determine whether the CHRNA7 and CHRFAM7A dysregulation depends on CXCR4 stimulation, an antagonist (AMD3100) was applied prior to gp120IIIB addition. Our results show that CXCR4 blockade abrogates gp120IIIB-induced upregulation of CHRNA7 (Fig. 3). Unexpectedly, CHRFAM7A was downregulated by AMD3100 in the absence of gp120IIIB (Fig 3a).

Fig. 3

Dysregulation of the CHRFAM7A:CHRNA7 ratio by gp120IIIB is dependent on CXCR4-gp120 interaction. Neuronal cells were incubated with 0.15 nM gp120IIIB for 12 hours with and without a pre-incubation of AMD3100. The CHRNA7 upregulation induced by gp120IIIB was obliterated by AMD3100 treatment. AMD3100 treatment alone downregulates the CHRFAM7A. Results were normalized and compared to control cells. *P ≤ 0.05, **P ≤ 0.01. n = 3 independent experiments. Statistical analysis: One way ANOVA followed by a Holm-Sidak's multiple comparison tests.

The CHRNA7 and CHRFAM7A expression levels in the basal ganglia of HIV-infected subjects

It is known that the basal ganglia is an area of the brain that is severely affected in HIV-infected patients (Woods et al. 2009) and contain some of the brain's highest viral load (Kure et al. 1990). We recently found that the CHRNA7 gene product, α7, is upregulated in the striatum (a component of the basal ganglia) of mice expressing gp120IIIB in the brain (Ballester et al. 2012). Here we examined CHRNA7, CHRFAM7A, and CHRNA7:CHRFAM7A levels in the basal ganglia of HIV-infected post mortem basal ganglia samples representing different stages of neurological impairment. Table 1 summarizes the subject characteristics. Evaluation of CHRFAM7A and CHRNA7 genes in HIV+ patients shows that CHRNA7 is significantly expressed at higher levels than CHRFAM7A (Fig 4a), which is consistent with what we observed in the neuronal cells (Fig. 1a). The CHRNA7:CHRFAM7A ratio of the HIV+ group was increased in these patients (Fig. 4b). Examination of CHRNA7 levels in basal ganglia from HIV-infected subjects suffering from different stages of cognitive impairment showed no significant differences (Fig. 4c). In terms of CHRFAM7A, at first glance, patients with normal cognition are not different from HIV- (Fig. 4d) but detailed examination of the distribution of CHRFAM7A levels in normal cognition patients demonstrate two distinguishable groups identified as subgroups A and B (Fig. 4d). Evaluation of these groups revealed that subgroup A is upregulated while subgroup B is downregulated for CHRFAM7A expression (Fig. 4d). Furthermore, examination of CHRFAM7A levels in the MCMD group suggests that only HIV+ patients with low CHRFAM7A levels develop MCMD (Fig. 4e). Ratio analysis demonstrates no significant differences in the CHRNA7:CHRFAM7A expression ratio in the basal ganglia of these patients and a linear trend analysis showed a non-significant (P = 0.08) increment in CHRNA7:CHRFAM7A with increasing cognitive impairment severity (Fig. 4f).

Fig. 4

Dysregulation of the CHRFAM7A transcript in the basal ganglia of HIV+ individuals. a HIV-infected subjects exhibited significant higher levels of CHRNA7, HIV- (n = 5), HIV+ (n = 31). b CHRNA7:CHRFAM7A expression ratio appears higher in HIV-infected subjects HIV+ (n = 5), HIV- (n = 31). c Analysis of CHRNA7 mRNA levels in HIV-infected individuals. For control n = 5, normal cognition n = 13, MCMD n = 9, and HAD n = 11. d Two subgroups of HIV+ individuals with normal cognition exhibited different responses in CHRFAM7A regulation and are significantly different when compared to HIV- individuals. For control n = 5, normal cognition n = 16, subgroup A n = 7, and sub-group B n = 9. e No significant changes were detected in CHRFAM7A levels in MCMD or HAD suffering patients when compared to HIV- whereas normal cognition subgroups are significant different from HIV- subjects. For control n = 5, sub-group A n = 7, and sub-group B n = 9, MCMD n = 9, and HAD n = 13. f Analysis of variance followed by a linear trend test comparing all groups (P = 0.08). For control n = 5, normal cognition n = 10, MCMD n = 9, and HAD n = 12. For all panels, results were normalized to HIV- individuals. *P ≤ 0.05, **P ≤ 0.01, *** P ≤ 0.001.

Table 1

Subject characteristics

	HIV –	HIV +
General characteristics	(n = 5)	Normal Cognition (n = 16)	MCMD (n = 9)	HAD (n =13)
* Age (years)	51.0 (48.0,51.0)	53.0 (46.0, 59.0)	44.0 (40.5, 58.5)	51.0 (44.5, 54.5)
Gender	5 male	15 male/1 female	9 male	13 male
Treatment	N/A	RTV (1), 3TC (5), CBV (1), DRV (1), NVP (1), ABC (1), NA (6)	RTV (1), APV (1), CBV (2), 3TC (1), NA (4)	ZVD (1), FTC (1), RTV (1), APV (1), CBV (1), EFV (1), ABC (1), 3TC (1), NA (5)
Viral-immune profile
* CD4 cell count (cells/mm3)	NA	171.0 (18.0, 352.0)	26.5 (7.8, 159.8)	247.0 (25.0, 365.0)
* Plasma HIV RNA (log 10)	N/A	4.6 (2.6, 5.2)	5.1 (4.3, 5.8)	4.3 (2.6, 5.1)

*Values presented as median (25th, 75th percentiles)

N/A = not applicable

NA = information not available

RTV = ritonavir (Norvir), 3TC = lamivudine (Epivir), CBV = zidovudine + lamivudine (Combivir), DRV = TMC-114, darunavir (Prezista), NVP = nevirapine (Viramune), ABC = abacavir (Ziagen), APV = amprenavir (Agenerase), CBV = zidovudine + lamivudine (Combivir), ZDV = AZT, zidovudine (Retrovir), FTC = emtricitabine (Coviracil; Emtriva), EFV = efavirenz (Sustiva).

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Discussion

HIV-infected patients suffer from cognitive disorders associated with the infection. In a previous report we demonstrated that gp120IIIB is capable of inducing a functional upregulation of the α7 in neuronal cells and that this upregulation promotes cell death in a calcium-dependent manner (Ballester et al. 2012). In the current study, we expand these observations demonstrating that gp120IIIB induces the upregulation of the α7 gene CHRNA7 and the downregulation of its partial duplication, CHRFAM7A, in neuronal cells. The significant reduction in CHRFAM7A expression could imply that dupα7's dominant negative effect on α7's functionality may be concomitantly reduced, thus providing a modulatory/regulatory explanation for our previous observations (Ballester et al. 2012). Because of dupα7's dominant negative regulatory effect on α7, we evaluated the CHRNA7:CHRFAM7A ratio as indicative of the α7 functionality and found that gp120IIIB indeed does modify the ratio. In our study, we also used different concentrations of gp120IIIB to better understand its effects on CHRNA7 and CHRFAM7A expression. Our results show that the greatest observed reduction in CHRFAM7A expression together with a CHRNA7 induction occurs within the pathophysiological range of gp120IIIB documented for HIV-infected patients.

We also studied the kinetics of the gp120IIIB-induced CHRNA7 and CHRFAM7A dysregulation. Our results demonstrate that the gp120IIIB first induces a reduction in CHRFAM7A expression (15 min) followed by CHRNA7 induction (12 h), shedding light on the regulatory/modulatory mechanism behind the α7 upregulation which points to an early regulatory mechanism (before 15 min) by the CHRFAM7A gene. These results, together with our previous published observations demonstrating that the functional upregulation of α7 in neuronal cells promote cell death and that the α7 upregulation appears to be restricted to the basal ganglia (Ballester et al. 2012), are consistent with: (i) the neuronal apoptosis and cell death in the presence of gp120IIIB (×4), gp120 R5, and supernatants containing HIV-1 (Hesselgesser et al. 1998; Catani et al. 2000; Xu et al. 2004), (ii) the neuronal apoptosis identified in postmortem brain from adults and pediatric HIV-infected patients (Adle-Biassette et al. 1995; Gelbard et al. 1995), (iii) the basal ganglia neuronal density reduction in HIV-infected patients (Everall et al. 1995), (iv) autopsy studies of patients with HAD showing that the greatest burden of neuropathology is found in the basal ganglia (Brew et al. 1995), (v) the large accumulation of gp120 in humans' basal ganglia (Jones et al. 2000), and (vi) the neuronal dysfunction and cellular destruction identified in a transgenic mice expressing gp120 in the brain (Corboy et al. 1992; Toggas et al. 1994; Berrada et al. 1995).

Although neurons do not express CD4, they express functional CXCR4 and CCR5 coreceptors enabling gp120 to interact with them and activate signaling pathways leading to neuronal cell death (Kaul et al. 2005; Kaul et al. 2007). The role of CXCR4 in the gp120-mediated neurotoxicity can be direct, through the activation of neuronal receptors by gp120; or indirect through the stimulation of glial cells leading to release of neurotoxic factors (Ghafouri et al. 2006). The activation of CXCR4 by SDF-1α (CXCR4 endogenous agonist) or gp120 has been implicated in the mechanism for neuronal dysfunction during HAD (Hesselgesser et al. 1998; Zheng et al. 1999). Herein we report alterations in the gene expression of a cholinergic receptor and its partial duplication which are both amply distributed through the brain. Dysregulation of these genes under neuropathological settings is not new. For instance, the ratio of CHRNA7:CHRFAM7A mRNA levels is different in bipolar subjects when compared to unaffected controls (De Luca et al. 2006). Moreover, in vitro studies have demonstrated that two pro-inflammatory mediators characteristic of HIV-I infection, LPS and IL-1β, decrease CHRFAM7A expression leading to the suggestion that chronic pro-inflammatory responses might change the CHRFAM7A:CHRNA7 expression ratio (Benfante et al. 2011; van der Zanden et al. 2012). gp120IIIB is unable to promote CHRNA7:CHRFAM7A alterations in the presence of AMD3100, an antagonist of CXCR4, suggesting that the gp120IIIB-induced CHRNA7:CHRFAM7A dysregulation is CXCR4-dependent.

The cognitive impairments observed in HIV-infected subjects are the consequence of neurological alterations in the brain that compromise neural tracts resulting in significant damage and alterations of specific areas. The basal ganglia, which is one of the most severely affected areas in these patients (Berger and Nath 1997; Berger and Arendt 2000; Berger et al. 2000; von Giesen et al. 2001; Woods et al. 2009), contains cholinergic neurons and interneurons that express α7 (Azam et al. 2003; Bonsi et al. 2011). To better understand the clinical implications of our findings, analysis of CHRNA7 and CHRFAM7A genes were performed on post-mortem basal ganglia samples from HIV-infected individuals with different levels of neurological impairment severity. Our results demonstrate that regardless of the neurological impairment severity, the CHRNA7 was not significantly altered as compared to HIV- subjects. However, comparing the expression of the CHRNA7 and CHRFAM7A genes within HIV-infected patients reveals that the CHRNA7 expression is significantly increased in these patients (Fig. 4a). Interestingly, a closer look at the CHRFAM7A gene expression levels revealed two distinct populations within the normal cognition group: subgroup A and B. Of note, a significant increase was detected in the expression of the CHRFAM7A gene in subgroup A when compared to the HIV- group and subgroup B, and a significant reduction in the expression of CHRFAM7A in subgroup B was detected when compared to the HIV- group and subgroup A. In addition, comparing the CHRFAM7A expression in both subgroups reveals a statistically significant difference. A provocative hypothesis on the existence of these two discernible subgroups within the normal cognition group is that the patients exhibiting elevated levels of CHRFAM7A are less likely of suffering from HIV-associated cognition problems, and those with low levels of CHRFAM7A, within sub-group B, are more susceptible to develop neurological impairment as lower CHRFAM7A expression levels could imply a potentiation of the α7 receptor expression, increased calcium influx, and ultimate neuronal cell death (de Lucas-Cerrillo et al. 2011; Araud et al. 2011; Ballester et al. 2012). Because the tissues were collected before patients presenting ANI were distinguished from patients displaying normal cognition, subgroups A and B could comprise patients with either normal cognition or ANI. It is tempting to hypothesize that subgroup A comprise patients with normal cognition, and subgroup B comprise patients that presented ANI as patients presenting ANI are known to progress to more severe stages (Grant et al. 2014). Taking this into account, our results may imply that alterations in the expression of CHRNA7 and CHRFAM7A, or the CHRNA7:CHRFAM7A ratio might be detrimental to the cognitive performance of these patients.

In this study we tested the hypothesis that higher levels of neurological impairment could be associated with alterations in CHRNA7 or CHRFAM7A expression levels. Whether this dysregulation is responsible for the destruction of cholinergic neurons within the basal ganglia of HIV-infected patients remains to be determined. However, the available evidence points in that direction. For instance, (i) the basal ganglia of HIV-infected patients is compromised (Berger and Nath 1997; Berger et al. 2000; von Giesen et al. 2001; Woods et al. 2009), (ii) the α7 upregulation in the basal ganglia of transgenic mice expressing gp120 in the brain predispose this area to cell death events similar to what was detected in α7-upregulated neuronal cells (Ballester et al. 2012). Together, this evidence lead us to suggest that the alterations in the CHRNA7:CHRFAM7A expression might be implicated in the basal ganglia alterations observed in HIV-infected subjects with neurological impairments. This interpretation is supported by several lines of evidence showing that the motor dysfunction suffered by subjects, under pathological circumstances, involve compromised basal ganglia interneurons (Bonsi et al. 2011) reminiscent of MCMD-suffering patients.

In conclusion, we showed that gp120IIIB is capable of dysregulating the CHRNA7/CHRFAM7A expression in neuronal cells. Moreover, this dysregulation was detected in postmortem brain samples recovered from HIV-infected patients with different stages of HAND. The present study is limited in that the results from HIV+ patients basal ganglia may be hindered by the lack of statistical power to detect small changes in expression levels as statistically significant given the dispersion in the data; and that the normal cognition group may actually include HIV+ patients that presented asymptomatic neurocognitive impairment (ANI) because the tissues were collected before ANI was established as a classification category of HIV-induced neurocognitive disorders. Nevertheless, our results raises fundamental questions about the role of α7 and dupα7 in HIV-induced neurological disorders and warrants further statistically powered investigations using an increased number of brain samples from HIV-infected subjects under different stages of HAND. In addition, further studies aimed at exploring the CCR5 tropic gp120 (gp120JRFL) effects on α7 expression in neuronal cells are warranted. It would be interesting to determine whether CCR5 stimulation influences α7 expression as occurs with the CXCR4 tropic-specific gp120IIIB. In fact, it is known that activation of these G-protein coupled receptors produce similar signaling pathways (Davis et al. 1997; Lee et al. 2003) that, in the presence of gp120, could lead to death of neuronal cells (Catani et al. 2000), therefore, it would not be surprising that both gp120s produce similar responses.

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Acknowledgments

We are very grateful for Dr. Valerie Wojna's valuable suggestions and in depth review of this manuscript. We appreciate the National NeuroAIDS Tissue Consortium (NNTC) and the Data Coordinating Center (DCC) which is a project funded by the National Institute of Mental Health and the National Institute of Neurological Disorders and Stroke under the following grants: U24MH100928 (California NeuroAIDS Tissue Network), U24MH100930 (Texas NeuroAIDS Research Center), and U24MH100929 (UCLA National Neurological AIDS Bank). This research was supported by the National Institutes of Health-NINDS (2U54NS43011 to José A. Lasalde-Dominicci), the RISE Program from NIGMS (2R25GM061151 to Manuel Delgado-Vélez and Orestes Quesada) and MARC Program from NIGMS (5T34GM007821 to Orestes Quesada and Felix M. Ramos). Research reported in this publication was supported by the National Institute on Minority Health and Health Disparities of the National Institutes of Health under Award Number U54MD007587 (Carlos A. Báez-Pagán). The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

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Footnotes

Conflict of interest: The authors declare that they have no competing interests.

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