Alpha7 nicotinic acetylcholine receptor is required for amyloid pathology in brain endothelial cells induced by Glycoprotein 120, methamphetamine and nicotine

One of the most challenging issues in HIV-associated neurocognitive disorders (HAND) caused by HIV-1 virotoxins and drug abuse is the lack of understanding the underlying mechanisms that are commonly associated with disorders of the blood-brain barrier (BBB), which mainly consists of brain microvascular endothelial cells (BMEC). Here, we hypothesized that Glycoprotein 120 (gp120), methamphetamine (METH) and nicotine (NT) can enhance amyloid-beta (Aβ) accumulation in BMEC through Alpha7 nicotinic acetylcholine receptor (α7 nAChR). Both in vitro (human BMEC) (HBMEC) and in vivo (mice) models of BBB were used to dissect the role of α7 nAChR in up-regulation of Aβ induced by gp120, METH and NT. Aβ release from and transport across HBMEC were significantly increased by these factors. Methyllycaconitine (MLA), an antagonist of α7 nAChR, could efficiently block these pathogenic effects. Furthermore, our animal data showed that these factors could significantly increase the levels of Aβ, Tau and Ubiquitin C-Terminal Hydrolase L1 (UCHL1) in mouse cerebrospinal fluid (CSF) and Aβ in the mouse brains. These pathogenicities were significantly reduced by MLA, suggesting that α7 nAChR may play an important role in neuropathology caused by gp120, METH and NT, which are the major pathogenic factors contributing to the pathogenesis of HAND.

have been shown to be neurotoxic since they could mediate inflammation and oxidative stress 8 . An earlier study demonstrated that APP was accumulated in HIV encephalitis and that widespread axonal injury as well as an increased prevalence of amyloid plaques was found in brains of patients with AIDS compared with the age matched, non-HIV-infected controls 7 . This suggested that amyloid plaque formation could be facilitated by HIV-1-induced inflammatory response in the central nervous system (CNS) 6 . Since the firstly reported in 1997 7 , more studies have shown that amyloid deposition in the brains of HIV-1-infected patients was increased and correlated with the patient age 9 . There are cellular pathology similarities between Alzheimer's disease and HAND 6,9 . Both animal and human studies demonstrated that Aβ metabolism could be altered by HIV-1 infection 6,9 . It has been suggested that the BBB may play a critical role in Aβ metabolism and homeostasis 9 .
One of the most challenging issues in HAND caused by HIV-1 virotoxins [e.g., Glycoprotein 120(gp120)] and related comorbid factors [e.g., methamphetamine (METH) and nicotine (NT)] is the lack of understanding the underlying mechanisms of the shared comorbidities that are commonly associated with disorders of the BBB, which mainly consists of brain microvascular endothelial cells (BMEC). Our recent study suggests that alpha7 nicotinic acetylcholine receptor (α 7 nAChR) is an essential regulator of inflammation, which contributes to neuroinflammation and BBB disorders caused by microbial (e.g., HIV-1 Glycoprotein 41(gp41)/gp120, Cryptococcus neoforman, E. coli) and non-microbial (e.g., METH and nicotine) factors [10][11][12] . METH, NT and gp120 were able to significantly increase the blood levels of both molecular [Ubiquitin C-Terminal Hydrolase L1(UCHL1), S100B] and cellular (circulating BMEC, cBMEC) biomarkers for BBB injury. The enhancement of these biomarkers, CNS inflammation and BBB permeability was significantly reduced in α 7 nAChR knockout mice, suggesting the involvement of this important inflammatory regulator in CNS disorders and BBB injury caused by microbial and non-microbial factors. Alpha7 nAChR also contributes to the pathogenesis of neurodegenerative disorders, including Alzeimer's disease 13 . As mentioned above, Alzheimer's-like brain pathology has been observed in patients with HIV-1 infection. HIV virotoxins could promote the secretion of Aβ  in primary rat fetal hippocampal cell cultures. However, the underlying mechanisms remain obscure and it is unclear whether the α 7 nAChR cholinergic pathway is essential for the role of BBB disorders in the pathogenesis of HAND and Alzheimer's-like brain pathology caused by multiple pathogenic factors. We hypothesized that gp120, METH and NT can enhance Aβ accumulation in BMEC through α 7 nAChR-mediated signaling cascade, which is the common pathway.

Results
HIV-1 gp120, METH and NT exposure increases Aβ levels of the culture supernatants in human BMECs (HBMECs). The blood-brain barrier (BBB) plays a critical role in regulating Aβ levels in the brain 14 .
Therefore, we hypothesized that exposure to HIV-1 gp120, METH and NT might predispose brain endothelial cells to alterations of Aβ levels and thus contribute to brain amyloid pathology. To confirm the effects of HIV-1 gp120, NT and METH on cellular Aβ levels, HBMECs were treated with gp120 (50 ng/ml), NT (10 μ M), and METH (50 nM) either alone or in combination of them [METH (50 nM) + gp120 (50 ng/ml) or NT (10 μ M) + gp120 (50 ng/ml)] for 24 h. ELISA assays with the antibody specific to the 1-42 fragment of Aβ revealed the Aβ protein levels in HBMECs. As illustrated in Fig. 1, a 24 h exposure to HIV-1 gp120, NT or METH markedly increased Aβ expression in all experimental treatments as compared to the control cultures. In addition, HBMECs were exposed to different doses of gp120 (0-200 ng/ml) for 24 h or 50 ng/ml gp120, 50 nM METH, 10 μ M NT at Gp120-, METH-and NT-induced accumulation of Aβ in vivo/in vitro and gp120-induced Aβ transport is inhibited by MLA. Aβ is a potential cause of Alzheimer's disease, in which it accumulates in the brain and increases monocyte migration across the BBB. To confirm the effects of HIV-1 gp120, METH and NT on cellular Aβ levels in vivo and vitro. After an exposure to HIV-1 gp120, METH, and NT, HBMECs and mouse brain tissues were used for immunoblotting analysis. Immunoblot with the antibody specific to the 17-24 fragment of Aβ (SIG-39220 from Covance Research Products) revealed several Aβ -specific bands of molecular weights of ~40, 60, and 100 kDa in the brain tissues of C57BL/6 J mice (Fig. 3b). This antibody preferably reacts with the precursor and aggregated forms of Aβ proteins. These multiple Aβ immunoreactive bands may be caused by the formation of various Aβ oligomers as described earlier 15 , and/or by the binding of Aβ to other cellular proteins. Exposure to HIV-1 gp120, METH and NT significantly increased the Aβ levels in HBMECs and the brains of C57BL/6 J mice as compared to the controls (Fig. 3a,b). We also determined the effects of MLA on gp120-mediated elevation of Aβ transport. A 24 h exposure to gp120 results in a marked increase of Aβ transport. However, pretreatment with MLA appeared to decrease gp120-mediated increase in Aβ transport (Fig. 3c), suggesting that α 7 nAChR plays an important role in Aβ homeostasis.

Gp120-and METH-induced expression of biomarkers and receptors for BBB injury.
To further examine gp120-and METH-induced expression of BBB biomarkers and receptors, HBMECs stimulated with gp120 (50 ng/ml) or METH (50 nM) at different time points were subjected to immunoblotting analysis of α 7 nAChR and biomarkers [S100B, Tau, and receptor for advanced glycation end-products(RAGE)], which play an important role in Alzheimer's-like brain pathology 13,16 . Our studies indicated that gp120 (Fig. 4a) and METH (Fig. 4b) were able to significantly upregulate expression of α 7 nAChR, S100B, Tau, and RAGE in a time (0-24 h)-dependent manner (Fig. 4). These studies suggest that α 7 nAChR, S100B, Tau, and RAGE are upregulated by gp120 and METH.
HIV-1 gp120-, METH-and NT-induced enhancement of α7 nAChR and S100B levels in vivo. To determine whether the brain levels of α 7 nAChR and S100B, which play an important role in Alzheimer's-like brain pathology, were increased upon stimulation with HIV-1 gp120 (50 ng/mouse, 2d), METH (2, 4, 6, 8, 10, 10, 10, 10, 10, 10 mg/kg/d), NT (1.5 mg/kg/d, 3d), the expression of α 7 nAChR and S100B in the hippocampal dentate gyrus (DG) in gp120-, METH-and NT-treated mice was examined by immunohistochemical staining. As shown in Figs 5 and 6, gp120, METH or NT treatment significantly increased α 7 nAChR and S100B expression in the DG of the hippocampus. These findings were consistent with the in vitro data. It also concurred with previous studies that α 7 nAChR and its agonist nicotine play an important role in the pathogenesis of CNS inflammation 10,11 . These results showed that gp120, METH and NT might contribute to Alzheimer's-like brain pathology by increasing expression of α 7 nAChR and S100B, the pan marker of astrocytes which are an important component of the BBB 11,16 . These findings suggest that α 7 nAChR and S100B contribute to HIV-1 gp120-, METH-and NT-increased Aβ levels in the brain and may be involved in the increased BBB permeability by upregulating protein levels of BBB biomarkers and the cholinergic pathway.

Figure 4. HIV-1 gp120-and METH-induced biomarkers accumulation in HBMECs.
HBMECs were incubated with gp120 (50 ng/ml) (a) or METH (50 nM) (b) for 2, 6, and 24 h, respectively. Expression of α 7 nAChR (AchR7), S100B, Tau, and RAGE, the biomarkers of Alzheimer's-like brain pathology, was determined by Western blot using the antibodies as described in Materials and methods. 0 h: the control HBMECs without METH or gp120 stimulation. β -actin in both fractions was detected as internal loading controls.
However, neither the control group nor MLA treatment cells showed these morphological changes, and most of the cells maintained the normal morphology following the MLA treatment (Fig. 8a,c,e,g,i,k). Furthermor, the numbers of SA β -gal stained cells were significantly lower in both the control group with treatment and MLA-treated cells exposed to HIV-1 gp120, METH and NT (Fig. 8B). These results suggest that the α7 antagonist MLA suppresses HIV-1 gp120-, METH-and NT-induced senescence in HBMECs. Cerebrospinal fluid (CSF) levels of biomarkers for Alzheimer's-like brain pathology and BBB injury are reduced by chemical blockage of α7 nAChR (MLA) upon exposure to HIV-1 gp120, METH and NT. To further determine the role of α 7 nAChR in the CNS inflammation and Alzheimer's-like brain pathology, the levels of biomarkers in CSF, including Tau, Aβ (Alzheimer's-like brain pathology) and UCHL1 (BBB injury), were analyzed (Figs 9 and 10). The data showed that treatment with these factors alone or their combination (gp120, METH, NT, METH + gp120 and NT + gp120) could significantly increase the levels of UCHL1 (Fig. 9a)/Tau (Fig. 9b) in CSF and the concentrations of Aβ in both blood (Fig. 10a) and CSF (Fig. 10b), while the treatment with MLA resulted in a significant decrease of these biomarkers. METH/NT had a synergistic effect in increasing the Tau protein in CSF. Gp120-, METH-and NT-enhanced expression of these biomakers was significantly blocked in the animals treated with the α 7 antagonist MLA. These data suggested that α 7 nAChR could upregulate biomarkers of neurodegeneration, Alzheimer's-like brain pathology and BBB injury upon exposure to HIV-1 gp120, METH and NT.

Disscussion
Up to now, the mechanisms responsible for the pathogenesis of the CNS disorders caused by HIV-1 and drug abuse remain largely unknown. An important connection between the nervous system and the inflammatory response to diseases has been uncovered through the identification of α 7 nAChR as an essential regulator of inflammation [20][21][22] . In this report, we present the evidence that HIV-1 gp120, METH and NT significantly increased Aβ accumulation in HBMECS in an α 7 nAChR dependent manner. Our results also illustrated the effects of α 7 nAChR on the inflammatory response and BBB integrity after treatment with HIV-1 gp120, METH   and NT through upregulation of the cholinergic α 7 nAChR pathway. Hence, α 7 nAChR plays an essential role in Aβ homeostasis and inflammatory response in the brain, may be a shared molecular event of the neuronal response to injury incited by virotoxins that are thought to contribute to the development of cognitive deficits associated with chronic HIV-1 infection in vivo.
Aβ has been considered the main pathogenetic factor of Alzheimer's disease 23 . Non-aggregated Aβ monomers have a role in the support of neuronal repair, but their conversion into oligomers with anti-parallel β -sheet structure leads to neurotoxicity 24,25 .
HAND encompasses the entire spectrum of neurological disorders associated with HIV-1 infection. It is proved that Nef exosomes from patients with HAND have the ability to interact with the neuroblastoma cells, subsequently increased the expression and secretion of Aβ , and finally aggravated the cognitive impairment 26 . Interestingly, patients infected with the subtype C of HIV-1 virus have a decreased incidence of cognitive deficits,  confirming that Aβ peptides is linked to the HIV protein mediated neurotoxicity mechanisms 27 . The current study also demonstrated that HIV-1 gp120, METH and NT significantly increased the Aβ 1-42 levels in HBMECs, and also enhanced the Aβ accumulation in the mouse brain tissues, while MLA incubation remarkably inhibited gp120-mediated stimulation of Aβ transport. The results suggest that α 7 nAChR plays an important role in Aβ homeostasis and that misregulation of the Aβ production might worsen neuronal homeostasis 28 .
Alterations of BMEC are commonly associated with HIV-1 infection. It is well-known that the integrality of the BBB plays a pivotal role in preventing microorganism invasion, and also be involved in the regulation of Aβ levels in the brain 29,30 . However, the mechanism of the shedding and senescence of BMECs is still unclear up to now. Previously, we have illustrated that cBMECs were used as a cellular index of the BBB damage caused by microbial (E. coli K1 and gp120) and non-microbial (nicotine and METH) pathogenic insults 11 . Leakage of peripheral proteins into the CNS has been used to evaluate BBB permeability 31,32 , such as fibrinogen or albumin. There are a number of peripheral blood biomarkers for detection of BBB injury, including UCHL1, RAGE and its ligand S100B. UCHL1, which is a component of the ubiquitin proteosome system has a more specific tissue distribution than S100B and is found more exclusively in neurons [33][34][35] . Our previous data have demonstrated that elevated serum levels of UCHL1 increased in the animals treated with gp120 and nicotine, suggesting that this protein could be used as a new molecular biomarker for BBB injury caused by microbial and nonmicrobial factors 11 . Meanwhile, RAGE and its downstream effectors, and nuclear factor-k-gene binding(NF-κ B) have been shown to be involved in neuronal death and astroglial conversion to the pro-inflammatory neurodegenerative phenotype 16,36 . As a result, we found that the expression of Aβ , Tau and UCHL1 in CSF increased greatly after treatment with gp120, METH and NT, while had little effects on the levels of these biomarkers in the animals treated with the α 7 antagonist MLA. Additionally, our data showed that gp120 or METH enhanced the proteins of α 7 nAChR, S100B, Tau, and RAGE, and meanwhile efficiently induced the premature senescence of HBMECs. RAGE activation promotes vascular dysfunction by impairing endothelial nitric oxide bioavailability 37 , increasing the release of proinflammatory cytokines, thus sustaining a harmful vicious cycle enhancing vascular inflammation and BBB impairment. In this report, we have established that α 7 nAChR plays an essential role in regulation of the BBB function in vitro. The pathogenic insult-induced HBMECs senescence, which is correlated with increased BBB permeability, is significantly reduced in the cells treated with the α 7 antagonist MLA, suggesting that up-regulation of α 7 nAChR is detrimental to the brain endothelial functions, and might increase Aβ transport across to the brain.
Currently, the precise mechanism responsible for the pathogenic insult mediated increase in the BBB permeability during HAND still remains elusive. Recent studies have revealed that α 7 nAChR is a critical link between inflammation and neurodegeneration, which is closely associated with Alzheimer's disease, might be a potential therapeutic target for the pathogenicities-caused by HIV-1 and drugs of abuse 38 . It has been shown that gp120 could up-regulate α 7 nAChR, which is a previously unrecognized contributor to the neurotoxicity associated with HAND 38 . It has been shown that enhancing peptides (EPs) derived from the co-receptor binding region of gp120 could promptly accelerated the formation of amyloid fibrils through the EP-derived nanofibers and significantly enhance HIV-1 infection 39,40 . Recently, Zhang et al demonstrated that α 7 nAChR plays a detrimental role in the host defense against CNS inflammation caused by microbial (e.g., meningitic pathogens and gp41) and non-microbial factors (e.g., METH) via the NF-κ B signaling pathway 12 , which may be involved in regulation of the molecular marker (S100B) during various CNS disorders 41 . Similarly, the current study also showed that nicotine could upregulateα 7 nAChR through activation of RAGE and S100B in the dentate gyrus (DG) of the hippocampus upon exposure to gp120, METH or NT in vivo, meanwhile the decrease of α 7 nAChR and RAGE alleviated leukocyte (HL60 cell) transmigration across the HBMEC monolayers. These findings suggest that α 7 nAChR is required for the modulation of inflammatory response through the cholinergic pathway, which may be involved in the pathogenesis of CNS comorbidities caused by HIV-1 virotoxins (e.g., gp120) and related factors (e.g., nicotine and METH). There are two α 7 isoforms, α 7 nAChR and dupα 7 nAChR, that are present in human brain and innate immune system. Dupα 7 nAChR is a partially duplicated α 7 nAChR genetic variant 42 . It remains elusive whether and how Dupα 7 nAChR may contribute to the pathogenesis of HAND in a manner different from α 7 nAChR 42 . Therefore, the mechanisms underlying these pathogenicities are not well-defined until now. It is likely that a common set of genes and pathways regulated by the common receptor α 7 nAChR for HIV-1 gp120, METH and NT, leading to the balanced regulation of the proinflammatory and anti-inflammatory factors in a manner dependent on the cholinergic signaling pathway.
Taken together, the major finding of the present report is that chemical blockages of α 7 nAChR could significantly reduce HIV-1 gp120-, METH-and NT-induced BBB injury and CNS disorders by decreasing Aβ transport, leukocyte recruitment, cholinergic signaling, premature senescence of HBMECs and neuronal inflammation. Further insight into how HIV-1, METH and NT utilize the host cholinergic α 7 nAChR pathway to augment their virulence capacity will advance our understanding of the pathogenesis and therapeutics of CNS disorders caused by multiple comorbidities.

Methods and Materials
Ethics statement. This study was performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. Our protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of The Saban Research Institute of Children's Hospital Los Angeles (Permit number: A3276-01). All surgery was performed under anesthesia with ketamine and lidocaine, and all efforts were made to minimize suffering.
Brain endothelial cells and in vitro BBB model. HBMECs were isolated and cultured as described previously 17,43 . HBMECs were routinely cultured in RPMI 1640 medium (Mediatech, Herndon, VA) containing 10% heat inactivated fetal bovine serum, 10% Nu-serum, 2 mM glutamine, 1 mM sodium pyruvate, essential amino acids, vitamins, penicillin G (50 mg/ml) and streptomycin (100 mg/ml) at 37 °C in 5% CO 2 . All the phenotypes of HBMECs were confirmed as described previously 43 . HBMECs were used an in vitro BBB model to study Alzheimer's-like pathology, brain endothelial permeability and senescence. Cells were treated with HIV-1 virotoxin gp120, METH and NT.
Animal Model and Treatment Protocol. All animal experiments were performed using C57BL/6 J mice.
The mice (6 week-old) were divided into 6 groups (I: Control treated with PBS; II: NT; III: METH; IV: gp120; V: METH + gp120; VI: NT + gp120)(n = 4). Two groups (II and III) of animals were exposed to low dose (1.5 mg/ kg/day) of NT (oral delivery) for 3 days (twice per day) or gradually increased doses (2, 4, 6, 8, 10, 10, 10, 10, 10, 10 mg/kg from day 1 to day 10) of METH [intraperitoneal (i.p.) injection] for 10 days as described previously 10,11 . The animals in Group IV received daily injections from tail veins (50 ng/mouse) of endotoxin-free recombinant HIV-1 gp120 for 2 days as described previously 11 . The animals (V and VI) were exposed to METH or NT as described in groups (II and III), after METH or NT exposure, all mice received gp120 as described in group IV from Day 9 to Day 10. After perfusion through cardiac puncture with 20 ml PBS, the skull was opened. Brain tissues and CSF samples were collected as described previously 10,11 . The brain tissues were used to determine Aβ (17)(18)(19)(20)(21)(22)(23)(24) protein expression by Western blot. CSF Biomarker changes in neuronal injury (UCHL1) and neurodegenerative disorder (Tau) were determined by ELISA using antibodies and antigens from Protein Tech (Chicago, IL) (UCHL1) and Ana Spec Inc (Fremont, CA) (Tau protein).
Immunofluorescence microscopy. Mouse brains were harvested, fixed in 10% buffered formalin for 24 h, and embedded in paraffin. Sections with 5 mm thickness were prepared, deparaffinized with xylene and then rehydrated with graded ethanol and distilled water. Heat treatment in a microwave, blockage of endogenous peroxidase activity with 3% H 2 O 2 and incubation with 10% goat serum were carried out as described in detail previously 10 . The brain tissues were stained with FITC-conjugated antibodies against α 7 nAChR (Rabbit) and S100B (rabbit) 10 . The brain tissues were then mounted with mounting medium containing DAPI (from Vector).
Scientific RepoRts | 7:40467 | DOI: 10.1038/srep40467 The samples were examined under a Leica fluorescence microscope at the Congressman Dixon Cellular Imaging Core Facility, Children's Hospital Los Angeles. All pictures were taken using the same parameters to ensure that the fluorescence strength of each treatment could be compared and calculated.

Monocytes transmigration assays.
Monocytes suspensions in experimental medium were used for leukocyte transmigration assays as described previously 17,[46][47][48] . Briefly, the confluence of the HBMECs monolayers in transwell filters (6.0 μ m pore size, 6.5 mm diameter, BD Biosciences) coated with collagen was confirmed by light microscopy before the start of the assays. To test the inhibitory effects of the α 7 antagonist (MLA) and RAGE inhibitor (FPS-ZM1) on gp120-, METH-and NT-induced monocyte transmigration across HBMECs. The HBMECs monolayers were pre-incubated with different doses of MLA (0, 0.1 μ M, 1 μ M, 10 μ M) for 1 h or FPS-ZM1 (0, 1 nM, 10 nM, 100 nM) for 2 h and stimulated with gp120 (50 ng/ml) and METH (50 nM) in the upper chambers. Then, monocyte-like vitamin D3-differentiated HL60 cells (1 × 10 6 cells/ml) were freshly prepared, added to the upper chamber and allowed to migrate over for 4 h. MLA or FPS-ZM1 was present throughout the monocyte transmigration experiment. At the end of the incubation, migrated HL60 cells were collected from the lower chamber and counted in a blinded-fashion using a hemacytometer. Final results of monocyte transmigration were expressed as the percentage of HL60 cells across the HBMECs monolayers.
Senescence-associated β-galactosidase activity assay. HBMECs (1.0 × 10 4 /ml) were grown in chamber slides, MLA were added 1 h before treatment with gp120 (50 ng/ml), METH (50 nM) or NT (10 μ M), respectively and the control group was treated with PBS. Two days post-treatment, the cells were washed with 2 ml 1x PBS, fixed in 2 ml of 3% formaldehyde, washed, and incubated for 24 h at 37 °C with a solution containing 1 mg/ml of 5-bromo-4-chloro-3-indolyl B-D-galactoside (X-Gal), 40 mM citric acid/sodium phosphate at pH 6.0, 5 mM potassium ferrocyanide, 5 mM potassium ferricyanide, 150 mM NaCl, and 2 mM MgCl2. After washing, the cell staining was viewed with a fluorescent microscope, and photographed. Statistical analysis. The statistical analysis of the data from our study involved analysis of variance (ANOVA). Raw data were entered into Software GraphPad Prsim 5.0 and automatically converted to the compatible format for statistical analysis packages. ANOVA and co-variates were followed by a multiple comparison test such as the Newmann-Keuls test to determine the statistical significance between the control and treatment groups. P < 0.05 was considered to be significant.