Inhibition of Cathepsin B and SAPC Secreted by HIV-Infected Macrophages Reverses Common and Unique Apoptosis Pathways

Abstract

Human immunodeficiency virus 1 (HIV-1) infects blood monocytes that cross the blood–brain barrier to the central nervous system, inducing neuronal damage. This is prompted by the secretion of viral and neurotoxic factors by HIV-infected macrophages, resulting in HIV-associated neurocognitive disorders. One of these neurotoxic factors is cathepsin B (CATB), a lysosomal cysteine protease that plays an important role in neurodegeneration. CATB interacts with the serum amyloid P component (SAPC), contributing to HIV-induced neurotoxicity. However, the neuronal apoptosis pathways triggered by CATB and the SAPC remain unknown. We aimed to elucidate these pathways in neurons exposed to HIV-infected macrophage-conditioned media before and after the inhibition of CATB or the SAPC with antibodies using tandem mass tag proteomics labeling. Based on the significant fold change (FC) ≥ |2| and p-value < 0.05 criteria, a total of 10, 48, and 13 proteins were deregulated after inhibiting CATB, SAPC antibodies, and the CATB inhibitor CA-074, respectively. We found that neurons exposed to the CATB antibody and SAPC antibody modulate similar proteins (TUBA1A and CYPA/PPIA) and unique proteins (LMNA and HSPH1 for the CATB antibody) or (CFL1 and PFN1 for the SAPC antibody). CATB, SAPC, or apoptosis-related proteins could become potential targets against HIV-induced neuronal degeneration.

Introduction

Human immunodeficiency virus 1 (HIV-1) infection continues to be a worldwide problem,¹ and it is now categorized as a chronic disease since the combined antiretroviral therapy (CART) allows patients to live longer. HIV-1 infection can induce HIV-associated neurocognitive disorders (HANDs)^2,3 regardless of CART.⁴ HANDs can start as early as the HIV enters into the central nervous system (CNS) after peripheral infection.⁵ The development of HANDs is largely attributed to increased transmigration of HIV-infected monocytes across the blood–brain barrier to the CNS similar to the “Trojan Horse”,^6,7 facilitating viral spread, inflammation, and neuronal damage.

AIDS research is still driven toward find novel therapeutic approaches against HANDs since they prevail in 20–50% of HIV-infected patients.^4,8,9 HIV infection, monocyte transmigration, and inflammation lead to neuronal dysfunction and apoptosis, even though the virus does not productively infect neurons. The direct neurotoxic mechanism in HANDs involves the secretion of viral proteins from infected cells in the CNS.^10,11 In contrast, the indirect HIV-mediated neurotoxicity requires the response of non-neuronal cells to the viral infection.^12,13 Previous studies from our laboratory reported that during the in vitro HIV-1 infection of monocyte-derived macrophages (MDMs), secreted cathepsin B (CATB) can trigger neuronal apoptosis.^14,15 CATB is a cysteine protease of lysosomal origin that plays an important role in neurodegeneration¹⁶ and apoptosis.¹⁷ Furthermore, CATB secretion is potentiated when HIV-infected MDMs are exposed to cocaine, increasing neuronal cell death.¹⁸ In addition to CATB, the acute-phase protein serum amyloid P component (SAPC) is another neurotoxic factor¹⁹ present after an infection or inflammatory disease and can contribute to HANDs. The SAPC binds to fibrils in all types of amyloid deposits and facilitates amyloidosis that is a hallmark of Alzheimer’s disease (AD).²⁰ A more recent study from our laboratory links CATB to the SAPC as interacting partners secreted in HIV-infected MDMs and absent in uninfected cells, promoting neuronal apoptosis.²¹ However, their mechanisms of action are unknown.

HIV proteomics have emerged with a large number of novel approaches allowing the qualitative and the quantitative study of cellular proteins.²² Tandem mass tag (TMT) labeling is a quantitative proteomics method that allows identification and quantitation of proteins using mass spectrometry (MS). In comparison to other proteomics methods coupled to MS, TMT labeling enables to identify and determine the relative abundance of peptides simultaneously by using up to a 10plex isotope labeling kit that becomes a single sample for tandem MS analysis. In this study, we applied TMT proteomics analysis to fill the gaps on novel pathways that lead to neuronal apoptosis upon the addition of HIV-infected MDM supernatants containing CATB/SAPC interacting partners. We found that antibodies against CATB and the SAPC, as well as the CATB inhibitor CA-074, downregulated apoptosis-related proteins.

Materials and Methods

Isolation, Culture, and Infection of Primary Macrophages

Primary human macrophages were isolated from peripheral blood mononuclear cells (PBMCs) of healthy donors (n = 12) using the University of Puerto Rico Medical Sciences Campus Institutional Review Board (IRB) protocol #0720116 from the NIH/NIGMS SC1 project. Blood was collected in acid citrate dextrose (ACD) tubes and PBMCs isolated using Ficoll density gradient separation. Adherent monocytes were cultured in T-25 flasks at a concentration of 5 × 10⁶ cells/well and grown in Roswell Park Memorial Institute (RPMI) medium supplemented with 20% heat-inactivated fetal bovine serum (FBS), 10% heat-inactivated human serum, and 1% penicillin/streptomycin (Pen/Strep) (all from Sigma-Aldrich, St. Louis, MO). Seven days later, macrophages were differentiated by adherence and infected with HIV-1_ADA (University of Nebraska) at 0.1 MOI (18 to 24 h). Half of the medium was changed every 3 days for all cultures and incubated at 37 °C, 5% CO₂ until 12 days post-infection (dpi), collecting supernatants at 3, 6, 9, and 12 dpi.¹⁵ After the supernatant was collected at 12 dpi, the cells were washed with serum-free media and cultured in the serum-free media for 24 h. The 13 dpi serum-free supernatant, termed as the macrophage-conditioned medium (MCM), was collected and stored at −80 °C.

Detection of HIV Infection and CATB Secretion and Activity

An ELISA p24 kit (Express BioTech, Maryland, USA) was used to measure the levels of infection by the detection of HIV-1 p24 antigen levels from 3, 6, 9, and 12 dpi MDM supernatants. Secretion of total CATB (R & D Systems, STATE, USA) and the SAPC (Abcam, STATE, USA) was assessed using the 13 dpi serum-free MCM from uninfected and HIV-1-infected MDMs following the manufacturer’s instructions. CATB enzymatic activity was measured by using 13 dpi MCM serum-free supernatants and was assessed using a fluorescence substrate-based kit (Bio Vision, STATE, USA). The CATB assay was performed in a Varioskan (Thermo Fisher).

Human Neuroblastoma SK-N-SH Cell Cultures and Lysates

The human neuroblastoma cell line SK-N-SH (ATCC HTB-11) was cultured in T25 flasks using the essential modified Eagle’s medium (EMEM) supplemented with 10% FBS (Sigma-Aldrich, St. Louis, MO), 1% Pen/Strep, 1% nonessential amino acids (Sigma-Aldrich), and 1% sodium pyruvate (Sigma-Aldrich) and incubated at 37 °C, 5% CO₂. This neuronal cell line was chosen for this study as it has been used in previous studies and behaves similarly to primary neuronal cells with respect to CATB secretion and neurotoxicity.^13,15,18,21 The SK-N-SH culture medium was changed every 2–3 days until 80% confluence. The neuronal cells were exposed to 13 dpi serum-free MCM diluted at 1:4 in EMEM and treated with the monoclonal anti-CATB antibody (8.33 μg/mL; Sigma-Aldrich), monoclonal anti-SAPC (14.1 μg/mL; Abcam), or CA-074 CATB inhibitor (10 μM, Sigma-Aldrich) for 24 h at 37 °C, 5% CO₂. The treated neuronal cells were washed twice with sterile phosphate-buffered saline and detached by incubation using radioimmunoprecipitation assay buffer (Abcam, Cambridge). The cell lysates were collected and stored at −80 °C for TMT labeling experiments.

Neuronal Apoptosis Measurement by Terminal Deoxynucleotidyl Transferase dUTP Nick End Labeling Assay

The SK-N-SH (ATCCHTB-11) neuroblastoma cells were cultured and exposed to 13 dpi MCM at a 1:4 dilution with or without CATB, the SAPC antibody, or the CA-074 inhibitor for 24 h. Neurons were fixed using 4% of paraformaldehyde (PFA) for 1 h, permeabilized for 10 min with 0.1% of sodium citrate, and autofluorescence was quenched by using 3% hydrogen peroxide in methanol. In situ cell neuronal death was assessed by terminal deoxynucleotidyl transferase dUTP nick end labeling assay (TUNEL) assay (ROCHE) following the manufacturer’s instructions. The positive control consisted of neuronal cells exposed to DNase I (30 U/mL) for 10 min at room temperature to promote DNA fragmentation and a green fluorescence. The cells were observed using a Nikon Eclipse E400 microscope, camera SPOT Insight QE, and Fluorescence X-Cite Series 120. The software used with the microscope was Spot Imaging 5.1. The analysis and cell quantification were carried out using the cell counter tool of Image J 1.49 software; green fluorescence nuclei were divided by the total number of neuronal nuclei (DAPI in blue color).

Sample Preparation and Processing

The total protein concentration from the treated neuronal lysates was determined using the bicinchoninic acid (BCA) assay (DC, protein assay, Bio-Rad). TMT proteomics was performed for a total protein quantitation. After BCA analysis, 20 μg of protein per sample was aliquoted for TMT studies. A total of 48 samples (treatment of neurons from six different donors and under eight conditions) were briefly cleaned by performing acetone precipitation with 50 μL of 10% sodium dodecyl sulfate (SDS) incubated at 70 °C for 15 min. The samples were centrifuged, 1 mL of cold acetone was added, and they were incubated at −20 °C overnight. The next day, the samples were microcentrifuged at 10,000g for 10 min and the supernatant was discarded. To isolate the total protein, the samples were briefly dried to remove the remaining acetone and prepared for a short run on a fixed 12% SDS-PAGE gel (BioRad) for 15 min at 150V. This protocol was performed for neurons treated with the MCM from all six donors. All gel lanes containing protein bands from each condition were cut out manually into 1 mm cubes, avoiding contamination. The gel pieces were properly destained with 50 mM ammonium bicarbonate and 50% acetonitrile for 30 min. This process was repeated until the gel samples were clear and no longer showed blue color. The samples were reduced and alkylated by incubating with 25 mM dithiothreitol (DTT) at 55 °C and 10 mM iodoacetamide in the dark, both in 50 mM ammonium bicarbonate separately. The samples were dehydrated with 100% acetonitrile and hydrated with HPLC water. Protein digestion was achieved by resuspending the samples in a trypsin solution (in a 1:100 ratio of trypsin/protein), followed by incubation at 37 °C overnight for a maximum of 16 h. Peptide extracts were prepared using 50% acetonitrile, 2.5% formic acid, and then 100% acetonitrile. The extracts were dried, and the gel pieces were stored at −80 °C until TMT labeling and LC–MS/MS analysis.

TMT Labeling

Peptide labeling was performed using a total of six sets of TMT10plex mass tag labeling kit (Thermo Scientific); a representative illustration for the protocol can be seen in Figure 1 (this was performed on neurons treated with the MCM for all six donors with increased CATB secretion). An internal sample pool was prepared using 100 μL of each one of the samples before drying the extractions. The samples were resuspended in 100 mM triethyl ammonium bicarbonate buffer following the manufacturer’s instructions. A total of 41 μL of TMT labeling reagent was added to each sample and incubated for 1 h at room temperature. The TMT reaction was quenched with 5% hydroxylamine for 15 min. A total of six sample pool tests (one per kit) and ratio check analysis were performed to obtain the sample volume for the final pooling per kit. Ratio check was used to normalize the relative abundance of each reporter ion and determine the amount of each sample to be used for injection into the MS. Ratio check was performed using 2 μL of each labeled sample and analyzed using a Q-Exactive Plus (Thermo Fisher Scientific, IL, USA). After a proper ratio check, the samples were mixed, and the final pools were cleaned, dried, and resuspended in 0.1% formic acid in water for a 2 μL injection into the Q-Exactive for the MS/MS analysis. All samples were cleaned by using C18 spin columns (Thermo Fisher Scientific). The samples were passed through the column resin twice to ensure sample binding and centrifuged at 1500 × g for 1 min. An elution solution was added to the resin bed using 70% acetonitrile. All cleaned samples were dried in a Vacufuge Plus system before LC–MS/MS analysis.

Figure 1.

Measurement of CATB and SAPC secretion after HIV infection. The samples were analyzed by the stratification criteria of increased or decreased CATB secretion. (a) Total CATB levels measured at 13 dpi. Six donors that increased CATB. (b) Six donors with decreased CATB. (c) Levels of serum amyloid P after HIV infection of donors that increased CATB secretion. (d) SAPC secretion in the six donors with decreased CATB.

LC–MS/MS, Protein Identification, and Quantitative Analysis

The mass spectrometric analysis was performed by employing an Easy nLC 1200 coupled to a Q-Exactive Plus (Thermo Scientific). For peptide separation, a PicoChip H354 REPROSIL-Pur C18-AQ 3 μm 120 A (75 μm × 105 mm) chromatographic column (New Objective) was used. The configured chromatographic gradient started at 7% of 0.1% of formic acid in acetonitrile (buffer B) for 2 min, increasing at 7–25% for 100 min, further increasing at 25–60% of buffer B for 20 min, and 60–95% buffer B for 6 min, making the total gradient time 128 min at a flow rate of 300 nl/min and a maximum pressure of 300 bars.

The Q-Exactive Plus mass spectrometer (Thermo Fisher Scientific) was operated in the positive polarity mode and data-dependent mode. The full scan (MS1) was measured over the range of 375–1400 m/z with a resolution of 70,000, while the MS2 (MS/MS) analysis was configured to select the 10 most intense ions for fragmentation with a resolution of 35,000. A dynamic exclusion parameter was set for 30.0 s. Raw data was analyzed using Proteome Discoverer version 2.1 software (Thermo Fisher Scientific) for the protein identification against a human database from the Universal Protein Resource (UniProt) downloaded in November 2017. To search in the Sequest HT node, the following modifications were included: dynamic modification for the oxidation of +15.995 Da (M), a static modification of +57.021 Da (C), and static modifications from the TMT reagents of +229.163 Da (Any N Term, K). A concatenated target/decoy strategy was employed, and FDR targets were set from 0.01 (strict) to 0.05 (relaxed). The results are filtered for keratins, high confidence, and master proteins. The results from the MS abundances were exported to a Microsoft Excel file for further bioinformatics analysis. Excel data files containing the protein abundances were analyzed with Limma R-Bioconductor²³ software for statistical analysis as a single channel analysis between cases versus controls. The Limma software analysis results were based on two parameters, namely, fold change (FC) and p-value. Proteins were considered deregulated if their FC values were FC ≥ 2 or FC ≤ −2 (i.e., FC ≥ |2|), while the p-value was lower up to 0.05 (p-value < 0.05), that is, a 95% confidence. Deregulated proteins were considered downregulated if FC ≤ -2 and upregulated if FC x ≥ 2. Ingenuity pathway analysis (IPA) QIAGEN Inc. (https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) was used for determining the pathways in which the deregulated proteins were involved. In addition, we used IPA to identify relationships between protein networks in neuronal cells exposed to the HIV-1-infected MDM supernatant in the presence and absence of CATB or SAPC antibodies. A list of deregulated proteins was also used in IPA for mapping the data and to create a molecular network related to apoptosis and understand pathways related to HANDs.

Protein Validation by Western Blotting

Western blot was performed on neuronal lysates using 15 μg of protein. The protein samples were loaded on 4–20% TGX gels (Bio-Rad) at 150V for 1 h. All polyvinylidene fluoride membranes were probed with anti-Lamin A + C (Abcam; 1:200), tubulin 1a (Abcam 1), and vinculin (Abcam 1:10,000) as primary antibodies. Secondary antibodies used were goat antimouse or antirabbit, all coupled to horseradish peroxidase (1:10,000; Sigma-Aldrich). Protein selection for western blot validation was based on Limma statistics results (i.e., FC and p-value) and published literature regarding neurodegeneration and CATB. Analysis and quantification of western blot bands were performed by dividing the volume intensity of each protein band by the volume intensity of the loading control band. Cyclophilin B (Abcam, 1:5000) was used as the loading control (internal control). Restore PLUS Mild Western Blot Stripping Buffer (Thermo Fisher Scientific, MA, USA) was used to reprobe membranes for 30 min at 37 °C, followed by washing, blocking, and reprobing with a different antibody.

Statistics and Bioinformatics Analyses

Data was analyzed using the Graph Pad Prism software Inc. version 7.0 (La Jolla, California, USA) and Excel Microsoft 2007 program (Microsoft, USA). Shapiro–Wilk test was used for a parametric or nonparametric analysis based on the normality test. T-tests were used for nonparametric data to analyze SAPC ELISA and CATB ELISA results. For the TUNEL assay, we used two-way ANOVA with the Sidak post-test. All graphs are presented using the standard error mean. For all statistical analyses, a p value less than 0.05 was considered as significant (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). Bioinformatics analyses were performed on TMT data using Limma software, an R Bioconductor package.²³Limma that implements a linear regression model to correlate variances and apply empirical Bayesian methods to obtain variance estimators. Significant results were based on FC ≥ |2| and p value < 0.05.

IPA (QIAGEN Inc., https://www.qiagenbioinformatics.com/products/ingenuity-pathway-analysis) was used for protein network and pathway enrichment analysis. IPA is a web-based bioinformatics application that allows researchers to upload data analysis results from high-throughput experiments, such as proteomics, for functional analysis, integration, and further understanding. It also allows interactive building of networks and pathways that represent biological systems.

Results

Measurement of HIV-1 Infection in Macrophages

Quantitative measurement of HIV-1 p24 protein was used to assess viral replication. The HIV-1 p24 antigen was measured using MDM supernatants of 3, 6, 9, and 12 dpi from all 12 donors. HIV-1 p24 protein increased significantly over time at 12 dpi compared to uninfected controls (p < 0.001) (Figure S1a). A productive infection was observed in all 12 donors.

Total CATB Secretion Is Variable after HIV-1 Infection, While Activity Remains Unchanged

Total CATB secretion was measured in the 13 dpi MCM from HIV-1 infected and uninfected controls (n = 12). After HIV-1 infection, 50% of MDM donors showed increased CATB secretion (n = 6), while the other 50% presented decreased protein secretion (n = 6) (Figure S1b). Therefore, we stratified the proteomics analyses in two groups: those who showed a significant increase in CATB secretion (p < 0.05) (Figure 1a) and those who showed a significant decrease (p < 0.05) (Figure 1b). CATB activity after HIV-1 infection was not different in all donors, even after the stratification of groups (Figure S2). The SAPC concentration presented a trend toward differences when comparing HIV-1-infected and -uninfected controls even after stratification by increased CATB secretion levels (Figure 1c) or decreased CATB secretion levels (Figure 1d), which is quite interesting and deserves future studies. These results were expected since previous works demonstrate that SAPC co-immunoprecipitates with CATB in HIV-1-infected MDMs, but the infection does not have a significant effect on SAPC secretion.²¹

CATB Secretion Levels Regulate Neuronal Apoptosis, While SAPC Maintains Its Neurotoxic Potential Regardless of Secretion Levels

To assess the neurotoxic potential of each protein after stratification according to CATB secretion levels, human neuroblastoma SK-N-SH cells were treated with the 13 dpi MCM alone or in combination with CATB or SAPC antibodies using the TUNEL assay (Figure 2a). Our results revealed that the HIV-infected MCM with higher levels of CATB secretion induced a significant increase in neuronal apoptosis in comparison to uninfected controls (Figure 2b). On the other hand, the HIV-infected MCM with decreased CATB secretion levels after infection did not show differences in neuronal apoptosis levels in comparison to uninfected controls (Figure 2c,d). Neuronal apoptosis decreased significantly after the treatment of the HIV-infected MCM with each one of the following: CATB, SAPC antibodies, CATB inhibitor, and CA074, despite the significant differences in CATB levels between both groups of donors after HIV-1 infection. Subsequent experiments to determine the effect of CATB and SAPC Ab on neuronal proteins were focused on the group that increased CATB to determine the intracellular pathways associated with the effect of CATB inhibitors in decreasing neuronal apoptosis and to uncover proteins that could become potential targets in preventing the neurodegeneration observed in HANDs.

Figure 2.

Neuronal apoptosis measured by in situ cell death (TUNEL) assay in donors that increased and decreased CATB. The neuroblastoma cell line (HTB-11) was treated with the MCM alone or in the presence of one of the following: CATB antibody, SAPC antibody, or CA-074. Green fluorescence (TUNEL-positive) for neuronal apoptosis by DNA fragmentation. Blue color is representative of cell nuclei stained with DAPI. DNaseI was used as a positive control. TUNEL assay in donors with increased CATB secretion (a). Donors with increased CATB secretion showed increased apoptosis with HIV + MCM that was inhibited with antibodies against CATB, SAPC, and CA-074 (b). TUNEL assay in donors with decreased CATB secretion (c). Donors with decreased CATB secretion did not increase apoptosis with HIV + MCM but decreased further with CA-074, SAPC, and CATB Ab(c). Neuronal apoptosis was quantified by two-way ANOVA.

Quantitative Proteomics Analysis of Neuronal Cells Exposed to the MCM from HIV-1-Infected MDMs in the Presence or Absence of CATB and the SAPC

Neuronal SK-N-SH cell lysates were used to perform TMT labeling using 10plex in accordance with the stratification groups: those exposed to HIV-MCM from increased (n = 6) CATB secretion versus MCM from HIV-negative controls. A TMT kit was used to label the following groups of samples: HIV+ (126; HIV-infected MCM) versus HIV– (128C; uninfected), HIV + CATB Ab (127N; HIV-infected MCM with CATB antibody) versus HIV+ (126; HIV-infected MCM), and HIV + SAPC Ab (127C; HIV-infected MCM with SAPC antibody) versus HIV+ (126; HIV-infected MCM). Additional HIV-uninfected controls for TMT were (HIV–, HIV – CATB Ab and HIV – CA074) (Figure 3). TMT MS/MS raw data was processed and analyzed by Proteome Discoverer 2.1 software. Raw data files were searched with a human database from Uni Prot Limma software to determine the significant protein abundances, designing a single channel analysis between experimental cases versus controls. Limma analysis results of protein abundances were based on FCs and p-values. A FC ≥ |2| and a p-value < 0.05 were considered as statistically significant.

Figure 3.

Proteomics workflow: neuronal cell cultures were treated with the MCM from uninfected and HIV-infected MDMs with or without the CATB antibody in a 1:4 dilution for 24 h. Neuronal cells were lysed, and the protein concentration was determined by BCA. Acetone precipitation was performed for the elimination of unwanted substances, followed by gel electrophoresis. The gel bands were cut out and reduced with DTT, followed by alkylation with iodoacetamide and digestion with trypsin. TMT labeling was performed, followed by a LC–MS/MS analysis using a Thermo Q Exactive instrument. Proteomics analysis was performed using Proteome discoverer, Limma software, and IPA.

The Limma software results revealed only 10 proteins with statistically significant abundances for HIV + CATB Ab compared to HIV+, six upregulated and four downregulated. Treatment with HIV + SAPC Ab compared to HIV + control showed a total of 13 deregulated proteins: 3 upregulated and 10 downregulated. Finally, treatment with CA074 compared to HIV + control showed a total of 48 deregulated proteins, 26 upregulated and 22 downregulated proteins.

The deregulated proteins were represented in a Venn diagram with circles that denote the different comparisons of unique and common proteins between neurons treated with HIV + supernatants in the presence of the CATB antibody, SAPC antibody, or CA074. A total of 2 unique deregulated proteins for HIV + CATB Ab/HIV+, 2 unique proteins for HIV + SAPC, and 16 unique proteins for CA074. The Venn diagram presents only one protein in common for all three groups, that is, TUBA1A (Figure 4). A heat map denoting quantitative differences of the comparison groups is presented in Figure S3.

Figure 4.

Model of common deregulated proteins after exposure to SAPC, CATB antibodies, or CA074. Inhibition of these neurotoxic proteins using antibodies or CA074 inhibitor separately reduces neuronal apoptosis by downregulating a common protein: TUBA1. Red = upregulated and green = downregulated.

IPA Results

We performed an IPA analysis from the group that increased CATB secretion to investigate the pathways and protein–protein interactions observed in deregulated proteins in neurons exposed to the HIV-MCM in the presence of CATB and SAPC inhibitors. We analyzed the apoptosis pathway since it was the major route induced by CATB and SAPC, causing neuronal damage. All proteins deregulated were described and are listed in Tables S1–S3.

The deregulated protein pathways analyzed by IPA were the following: (1) apoptosis signaling pathway and relationship trees with a central node function for apoptosis, (2) literature evidence related to the function of interest (apoptosis), and (3) differential changes in FC and p-values among treatments after CATB/SAPC antibody treatment compared to untreated controls. All proteins related to apoptosis and neurodegeneration were taken into consideration in this study. Deregulated proteins for HIV + CATB Ab/HIV+ and apoptosis importance were proteasome subunit alpha type-5 (PSMA5), eukaryotic translation initiation factor 3 subunit A (EIF3A), Ras-related protein Rab-7a (RAB7A), heat shock protein 105 kDa (HSPH1), Lamin A, peptidyl-prolyl cis–trans isomerase A (PPIA), eukaryotic translation initiation factor 5a 1 (EIF5A), and tubulin alpha-1A chain (TUBA1A). Deregulated proteins were actin-related protein 3 (ACTR3), exportin-2 (CSE1L), proteasome subunit alpha type-1 (PSMA1), cofilin-1 (CFL1), profilin-1 (PFN1), small nuclear ribonucleoprotein-associated protein B (SNRPB), and TUBA1A (Figures S4, S5).

HIV + CATB Ab/HIV + denotes an upregulation of HSPH1 (FC = 17.4) and a downregulation of the following: LMNA, TUBA1A (FC = 23.2), PPIA (FC = 20.2), and EIF5A (FC = 20.4) (Figure 5a). The relationship tree for apoptosis for HIV + SAPC Ab/HIV + demonstrates an upregulation of CSE1L (FC = 11.3) and a downregulation of PPIA (FC = 13.5), TUBA1A (FC = 34.2), CFL1 (FC = 18.5), and PFN1 (FC = 18.5) (Figure 5b). The relationship tree for apoptosis for HIV + CA074/HIV + demonstrates a predominant upregulation of 12 proteins and a downregulation of 7 proteins (Figure 5c). A pathway analysis of interactions between LMNA and TUBA1A is shown in Figure S5.

Figure 5.

Deregulated apoptotic proteins in neuronal cells exposed to HIV-MCM treated with the CATB Ab, SAPC Ab b, and CATB inhibitor. Deregulated apoptotic proteins in neuronal cells exposed to the HIV-MCM treated with CATB Ab (a), SAPC Ab (b), and CATB inhibitor CA074 (c). Relationship of deregulated proteins are based on FC≥ |2| values. HIV + CATB antibody/HIV + deregulated proteins related to the apoptosis node. IPA. Legend: Red = upregulated proteins and green = downregulated proteins. IPA.

Western Blot of the Proteins Deregulated by CATB and SAPC in the Apoptosis Signaling Pathway

We selected LMNA and TUBA1A for verification by western blot based on the statistical analysis of relevant proteins with higher confidence. Relevant proteins were selected based on UniProt, common proteins in all three relationship trees of apoptosis, FC, and apoptotic function. Densitometry was determined between experimental (HIV-infected supernatants treated with the CATB antibody) and HIV-infected samples. HIV negative controls were included for comparison of normal levels. The results revealed a downregulation of LMNA by the CATB antibody by TMT analysis (FC = −13.05) but not by western blot. An unexpected significant decrease in LMNA occurred with the CA074 inhibitor that was not detected by TMT analyses and was not comparable to HIV negative controls (Figure 6a,b). TMT analysis revealed a downregulation of TUBA1A when neuronal cells were treated with CATB Ab (FC = −23.2), SAPC Ab (FC = −34.2), and CA074 (FC = −22.48) when compared to the untreated HIV+ (FC = 17.2). Western blot for TUBA1a protein confirmed results of TMT analyses showing a small decrease in TUBA1A with CATB antibody treatment compared to HIV+ and a significant decrease in TUBA1A with SAPC antibody and CA074 treatments (Figure 6c). The level of reduction was comparable to HIV negative controls. Original blots are shown in Figure S3a–c.

Figure 6.

Western blot validation of Lamin A and TUBA1a proteins for each of the treatments. (a) Densitometry analyses for volume intensity normalized against cyclophilin B (CYPB). Blots are the representative of uninfected (−) and HIV-1 infection (+) treatments (n = 6). All band intensities were normalized to CYPB, the internal control (n = 6). Densitometry analyses of LMNA (b) and TUBA1a (c). Western blot pictures have been cropped, but the original complete pictures are included in the Supporting Information.

The apoptosis-inducing factor (AIF) is well known to be responsible for the DNA fragmentation in the apoptosis process, specifically in the caspase-independent apoptosis pathway.²⁴ Previous studies in neurons demonstrate that AIF migrates from the mitochondria to the nuclei, leading to cell death upon interaction with PPIA, also known as cyclophilin A.²⁵ Neuronal studies on AIF/PPIA complex interaction demonstrate that this interaction leads to apoptosis, suggesting that the blockage of this interaction ameliorates apoptosis.²⁶

An illustrative picture of the relevant apoptotic proteins that appear to be reversed by treatment with CATB and SAPC antibodies is included in Figure 7. Both TUBA1 and PPIA were downregulated in neurons after exposure to HIV-infected macrophage supernatants with the treatment of specific antibodies. HIV induces oxidative stress and mitochondrial dysfunction. This results in migration of the AIF to the nuclei and PPIA/AIF interaction that leads to apoptosis. A downregulation of these proteins occurs with CATB and SAPC antibodies, suggesting that the interaction between the AIF and PPIA its decrease causes reduction of apoptosis.

Figure 7.

Model of common deregulated proteins after exposure to SAPC or CATB antibodies or the CA074 inhibitor. HIV-infected MDMs secreted CATB and SAPC that cause neuronal apoptosis. CATB and SAPC have been shown to be secreted in extracellular vesicles and internalized by neurons and induce caspase 3 activation.²⁷ This internalization can promote the migration of the AIF to the nuclei and PPIA and AIF interaction that causes apoptosis. A downregulation of the AIF and PPIA proteins occurs with CATB and SAPC antibodies, suggesting that the interaction between the AIF and PPIA and its decrease result in the reduction of apoptosis.²⁶

Discussion

HANDs continue to be a challenge for this viral infection that gains access to the CNS. Neuronal damage occurs due to the release of viral proteins, inflammatory cytokines, and neurotoxins from macrophages, microglia, and astrocytes. Previous studies from our laboratory have demonstrated that macrophages can secrete increased CATB after HIV-1 infection, decreasing interaction with its inhibitor cystatin B and resulting in a new interaction with SAPC, a protein related to increased amyloid deposition.²⁷ In this study, we aimed to identify the intracellular neuronal pathways deregulated specifically by CATB and SAPC proteins secreted by HIV-infected macrophages. As in previous studies, we found that the levels of CATB secretion vary among donors.²⁸ In contrast, SAPC did not show differences in secretion. However, the inhibition of CATB and SAPC with antibodies and the CATB chemical inhibitor decreased neurotoxicity. In order to understand the effect of CATB secretion levels from MDMs in inducing neuronal apoptosis, the TUNEL assay was performed with the stratification of groups according to the levels of CATB. The TUNEL assay confirmed previous studies that high levels of CATB secretion by HIV-infected MDMs resulted in high levels of neuronal apoptosis that can be reverted after treatment with CATB or SAPC antibodies.^13,15,18 Interestingly, in those donors who showed decreased CATB secretion from HIV-infected MDMs there was no difference in neuronal apoptosis when compared with uninfected control. However, despite the CATB secretion levels, apoptosis was decreased by CATB and SAPC antibodies. These results revealed that high levels of CATB are neurotoxic and that CATB and SAPC antibodies are protective against neurotoxicity, regardless of the amount of CATB or SAPC present in the HIV-infected MCMs.

Quantitative proteomic analyses depicted by the Venn diagrams indicated that neurons exposed to the CATB antibody and SAPC antibody modulate similar proteins (tubulin 1A, TUBA1A, and PPIA) and unique proteins (laminin A, LMNA, heat shock protein 1, and HSPH1) for the CATB antibody or (cofilin 1, CFL1, profilin 1, and PFN1 for the SAPC antibody). Even though unique proteins were deregulated in neurons exposed to HIV + CATB Ab compared to HIV + SAPC Ab, there are common proteins related to apoptosis pathways. These results suggest that MDM-derived CATB and SAPC complexes could trigger apoptosis in neurons through shared mechanisms, which according to the TMT analyses include TUBA1A. IPA as an exploratory apoptosis signaling pathway of significant proteins demonstrated the downregulation of LMNA and TUBA1A in neurons exposed to HIV-1-infected macrophage supernatants, while the CATB antibody reversed this effect. The nuclear lamina is composed of a network of lamins in association with the inner nuclear membrane. The possible downregulation of LMNA by TMT suggests that CATB induces apoptosis by disrupting the nuclear membrane and causing cell shrinkage, which was reverted by the addition of the CATB antibody. Previous studies demonstrate that CATB can trigger apoptosis by a caspase 3-dependent mechanism²⁹ This result might suggest an alternate mechanism of apoptosis induced by CATB dependent on caspase 6 since the LMNA substrate is exclusively cleaved by effector caspase 6.³⁰⁻³² CATB inhibitor CA074 also upregulated TUB1A, which suggests that the CATB antibody or CA074 inhibitor affected the same protein pathway. However, CA074 also upregulated 5 additional proteins and downregulated 12 proteins. Ten of these upregulated proteins were related to apoptosis mechanisms, suggesting that CA074 inhibits additional pathways. Unfortunately, western blots could not confirm the upregulation of LMNA or TUB1A in neurons exposed to HIV-infected supernatants and CATB or SAPC-antibodies compared to controls. This technique was difficult for apoptotic cells as there was limited selection of antibodies as loading controls that could be stable in the apoptotic neurons.

CATB has been linked to the degradation of β-amyloid peptides (Aβ), protecting against AD.³³ An opposed mechanism described in the literature of CATB is that it can also act as a β-secretase by cleaving the amyloid precursor protein (APP) into Aβ peptides, promoting AD.³⁴ Our results revealed that the proteasome subunit alpha type5 (PSMA5) protein had the highest FC and was upregulated in the presence of CATB Ab but not SAPC Ab. As a subunit of the proteasome, PSMA5 downregulation can lead to impairment and apoptosis mediated by the overexpression of APP.³⁵ Interestingly, our data indicates that an upregulation of PSMA5 by the pretreatment of the HIV-infected MCM with CATB Ab may reduce APP intraneuronal accumulation and could prevent the development of HANDs. Since recent studies demonstrate that HIV infection induces extracellular CATB uptake and neuronal damage,²⁷ this hypothesis needs further exploration, and it confirms previous studies that support the role of CATB as a B secretase increasing Aβ production and AD.^36,37

Overexpression of HSP105 suppressed the activation of caspase 3 and caspase 9 by preventing apoptosis.^38,39 Our proteomics results reveal that CATB Ab upregulates HSP105, which might be another mechanism to decrease apoptosis. The proteins PPIA and TUBA1A were both downregulated after CATB and SAPC antibody treatment. These proteins appear to be activated by oxidative stress and mitochondrial toxicity. Additional proteins, such as cofilin (CFL1) and profilin (PFN1), increased apoptosis, which were downregulated after SAPC antibody treatment. This is supported by previous literature, indicating that CFL is important in apoptosis⁴⁰ and that the reduction of cofilin protein inhibits apoptosis,⁴¹ while increased PFN1 upregulates p53, inducing the apoptosis pathway.⁴² In light of these results and from recent results of our laboratory, CATB appears to be internalized by neurons and triggers apoptosis by a caspase-dependent mechanism.²⁷

Future studies will focus on the understanding of common apoptotic pathways triggered by neurotoxic proteins CATB and the SAPC. Overall, this study not only confirms that the secretion of the CATB/SAPC complex activates several apoptotic mechanisms in neurons but also that these can be reverted using CATB or SAPC antibodies. Therefore, blocking of CATB and the SAPC shows potential for therapeutic agents against HANDs by reducing neuronal apoptosis mediated by HIV-MDM-secreted interacting proteins CATB/SAPC.

Proteomics Data

Proteomics data are available via ProteomeXchange with the identifier PXD024636. Project Name: Inhibition of CATB and SAPC secreted by HIV-infected macrophages reverses common and unique apoptosis pathways. Project accession: PXD024636 Project DOI: 10.6019/PXD024636.

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jproteome.1c00187.

• HIV replication levels in MDM and stratification in accordance to CATB secretion levels, measurement of the CATB activity of MDM supernatants, heat map of different comparison groups, apoptosis signaling pathways, comparison of HIV + CA074 and HIV+ and comparison of HIV + CATB Ab and HIV+, pathway analysis of interactions between LMNA and TUBA1, deregulated proteins from HIV + CATB Ab treatment with a role related to apoptosis identified by IPA, deregulated proteins from HIV + SAPC Ab treatment with an apoptosis role identified by IPA, deregulated proteins from HIV + CA074 with an apoptosis role identified by IPA, original western blots of uninfected (−) and HIV-1-infected (+) alone or in combination with the CATB Ab, SAPC Ab, or CA074 inhibitor, and Coomassie-stained gels (PDF)

The authors declare no competing financial interest.