Study population
For the present study, we evaluated brain tissues from a total of 33 HIV+ donors (Table 1) from the National NeuroAIDS Tissue Consortium (NNTC) (Institutional Review Board [IRB] #080323). All studies adhered to the ethical guidelines of the National Institutes of Health and the University of California, San Diego. These cases had neuromedical and neuropsychological examinations within a median of 12 months before death. Subjects were excluded if they had a history of CNS opportunistic infections or non-HIV-related developmental, neurologic, psychiatric, or metabolic conditions that might affect CNS functioning (e.g., loss of consciousness exceeding 30 minutes, psychosis, etc). HAND diagnoses were determined from a comprehensive neuropsychological test battery administered according to standardized protocols [13].
Neuromedical and neuropsychological evaluation
Participants underwent a comprehensive neuromedical evaluation that included assessment of medical history, structured medical and neurological examinations, and the collection of blood, cerebrospinal fluid (CSF), and urine samples, as previously described[13, 14]. Clinical data (plasma viral load [VL], postmortem interval, CD4 count, global, learning and motor deficit scores [GDS, LDS, and MDS]) were collected for the HAND donor cohorts.
Neuropsychological evaluation was performed, and HAND diagnoses were determined via a comprehensive neuropsychological test battery, which was constructed to maximize sensitivity to neurocognitive deficits associated with HIV infection [see [13] for a list of tests]. Raw tests scores were transformed into demographically adjusted T-scores, including adjustments for age, education, gender and race. These demographically adjusted T-scores were converted to clinical ratings to determine presence and degree of neurocognitive impairment on seven neurocognitive domains, as previously described [13]. As part of the neuropsychological battery, participants also completed self-report questionnaires of everyday functioning (i.e., Lawton and Brody Activities of Daily Living questionnaire; [15], and/or Patient’s Assessment of Own Functioning; PAOFI; [16, 17]). Participant’s performance on the neuropsychological test battery and their responses to the everyday functioning questionnaires were utilized to assign HAND diagnoses following established criteria [18], i.e., HIV-associated asymptomatic neurocognitive impairment (ANI), HIV-associated mild neurocognitive disorder (MND), and HIV-associated dementia (HAD).
ImmunoBlot of human brain specimens
Frontal cortex tissues from human brains were homogenized in lysis buffer (1.0 mmol/L HEPES; Gibco, cat. no. 15630-080), 5.0 mmol/L benzamidine, 2.0 mmol/L 2-mercaptoethanol (Gibco, cat. no. 21985), 3.0 mmol/L EDTA (Omni pur, cat. no. 4005), 0.5 mmol/L magnesium sulfate, 0.05% sodium azide; final pH 8.8). In brief, as previously described [19], tissues from human brain samples (0.1 g) were homogenized by sonication for 15 seconds in 0.7 ml of lysis buffer containing phosphatase and protease inhibitor cocktails (Calbiochem, cat. no. 524624 and 539131). Samples were precleared by centrifugation at 2000 × g for 5 min at room temperature. The supernatant was collected as representing the whole lysate.
After determination of the protein content of all samples by bicinchoninic acid assay (Thermo Fisher Scientific, cat. no. 23225) and denaturation in lamellae sample buffer, samples were loaded (20 μg total protein/lane) on 4–12% Bis-Tris gels (Invitrogen, cat. no. WG1402BX10) and electrophoresed in 5% HEPES running buffer and transferred onto PVDF membrane with iBlot transfer stacks (Invitrogen, cat. no. IB24001) using NuPage transfer buffer (ThermoFisher Scientific, cat. no NP0006). The membranes were blocked in 5% BSA in phosphate-buffered saline-tween 20 (PBST) for 1 h. Membranes were incubated overnight at 4 °C with primary antibody. Following visualization, blots were stripped and probed with a mouse monoclonal antibody against b-actin (ACTB; Sigma Aldrich, cat. no. A5441) diluted 1:2000 in blocking buffer as a loading control. All blots were then washed in PBST, and then incubated with species-specific IgG conjugated to HRP (American Qualex, cat. no. A102P5) diluted 1:5000 in PBST and visualized with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, cat. no. 34096). Images were obtained, and semi-quantitative analysis was performed with the VersaDoc gel imaging system and Quantity One software (Bio-Rad).
Immunohistochemistry and double immunofluorescence
Free-floating 40 μm thick vibratome sections of human brains were washed with phosphate buffered saline (PBS) 3 times, pre-treated for 20 min in 3% H2O2, and blocked with 2.5% horse serum (Vector Laboratories, cat. no. S-2012) for 1 h at room temperature. Sections were incubated at 4 °C overnight with the primary antibody, C/EBPb (Santa Cruz Biotechnology; C-150) diluted in blocking buffer. Sections were then incubated in secondary antibody, Immpress HRP Anti-rabbit IgG (Vector, cat. no. MP-7401) for 30 min, followed by peroxidase (HRP) substrate made with DAB Peroxidase (HRP) Substrate Kit as per manufacturer’s instructions (Vector, cat. no. SK-4800). Control experiments consisted of incubation with secondary antibody only. Tissues were mounted on Superfrost plus slides (Fisherbrand, cat. no. 12-550-15) and coverslipped with cytoseal (Richard Allen Scientific, cat. no. 8310-16). Immunostained sections were imaged with a digital Olympus microscope to identify C/EBPb immunoreactivity.
Double immunolabelling studies were performed as previously described[20] to determine the cellular localization of C/EBPb. For this purpose, vibratome sections of human brains were immunostained with antibodies against C/EBPb with GFAP (Cell Signaling Technology; catalog #3670), and MAP2 (Santa Cruz Biotechnologies, cat# sc-32791). Sections were then reacted with fluorescent secondary antibodies, goat anti mouse IgG 488 (Invitrogen, cat. no. A11011) and goat anti rabbit IgG 568 (Invitrogen, cat. no. A11036). Sections were mounted on superfrost plus slides and cover-slipped with vectasheild (Vector, cat. no. 1000). Sections were imaged with a Zeiss 63X (N.A. 1.4) objective on an Axiovert 35 microscope (Zeiss) with an attached MRC1024 laser scanning confocal microscope system (BioRad, Hercules, CA). An examiner blinded to sample identification analyzed all immunostaining. Ten fields of view were analyzed and a minimum of 20 cells (astrocytes and neurons) per slide were examined to analyze colocalization. The percent colocalization was determined using Image J software with the SQASSH plug-in, as previously described. Double immunolabelling of C/EBPb with the microglial marker IBA1 was excluded due to technical difficulties and high level of non-specific signal when using the two antibodies simultaneously. Future investigation will focus on identifying levels of C/EBPb in microglial cells.
In vitro studies of human astrocytes and neuronal cells
In vitro models for human astrocytes generated from fetal tissue are important for controlling for differences in genetic background, which cannot be controlled for by using cell lines. Moreover, astrocytes from human tissue are directly relatable to human disease, unlike astrocytes generated from rodents. The cell model for astrocytes was approved by the University of California San Diego Human Research Protections Program and the National Institutes of Health as part of a grant actively funded by the National Institute for Mental Health. Astrocytes were isolated from fetal human brain tissue from elective terminated pregnancy between 12 and 16 weeks of gestation, acquired from Advanced Bioscience Resources. Donors gave written informed consent for research-use of the cells and tissue. Tissue was fragmented and mechanically dissociated using a scalpel and washed 3 times with HBSS holding media (Gibco, cat. no.14175-095) with 1 mM Glutamax (Gibco, cat. no. 35050-061), 20 μg/mL Gentamicin (Gibco, cat. no. 15710-064) and 5 mM HEPES (Gibco, cat. no. 15630-080). The tissue was homogenized with the addition of 15 mL of 0.25% trypsin EDTA (Gibco, cat. no. 25200-056) for 5 min in a 37 °C incubator. After 5 min, 1 mL of a trypsin inhibitor (Roche, cat. no. 10109) and 24 mL of DMEM media (Gibco, cat. no. 11960-044) with human serum (Corning, cat. no. 35-060-cl) was added. The mixture was then centrifuged for 5 min at 4 °C to pellet the cells. Supernatant was removed and discarded, and the cells were resuspended in 5 ml of DMEM media and strained with a 70 μM strainer (Falcon, cat. no. 352350). The cell suspension was underlaid with 7 ml of a solution of filtered 8% BSA in PBS and cells were centrifuged at 1 x 104 rpm at 4 °C for 10 min. The supernatant was removed, and the cells were resuspended in DMEM media with human serum. Astrocytes were plated at a density of 1 x 107/T75 flask and cultured as adherent monolayers. After 1 week, the astroglia DMEM media with human serum was replaced with DMEM media with 10% fetal bovine serum (FBS) (Gibco, cat. no. 16000044) and 1% penicillin/ streptomycin (P/S) (Corning, cat. no. 30-001-CI-1). Every 3 days, a half media exchange was performed on each cell type. Astrocytes were routinely tested purity and consistently found to be >95% pure by immunostaining for GFAP.
As previously described[21], B103 cells (rat neuroblastoma) were cultured at 37 and 5% CO2. B103 rat neuroblastoma cells were used here for the cholinergic and GABAergic phenotypes[22], both of which are implicated in frontal cortex and basal ganglia function[23, 24] and relevant to HAND[25, 26]. B103 cultures were grown in DMEM with 5% FBS.
Transfection of astroglia with pC/EBPb
Astrocytes were split into 12 well plates at 500,000 cells/well on the day prior to transfection. Astrocytes were transfected using Lipofectamine 3000 (Thermo Fischer Scientific, cat. no. L3000075). Lipofectamine 3000 and an empty lentiviral expression plasmid (p) as a control or pC/EBPb (OriGene Technologies, Rockville, MD; CAT. SC319561) (1mg) with p3000 were diluted separately in Opti-Mem Media and then mixed together at a 1:1 ratio and left to incubate for 15 minutes at room temperature. After 15 minutes, the plasmid-lipid complexes were added to the cells. Three days after transfection, RNA was isolated from astroglia. Genes were selected for validation by real-time polymerase chain reaction (rt2PCR) based on bioinformatic analyses of transcriptome of the PWH indicating they were potential targets of C/EBPb transcription in astrocytes. Moreover, all transcripts tested are implicated in HAND, neuroinflammation, or both.
RNA isolation and TaqMan® human inflammation array and real-time reverse transcription polymerase chain reaction
Astroglia were split into 12 well plates at 5 x 105 cells/well for RNA isolation. Three days after transfection, media were removed, and the cells were washed once with PBS. RNA was extracted with RNeasy plus mini kit (Qiagen, cat. no. 74136) according to manufacturer’s instructions and analyzed for purity and concentration with a spectrophotometer. RNA was reverse transcribed into cDNA with a high capacity cDNA Reverse Transcription Kit (Life technologies, cat. no. 4358813) as per manufacturers instructions. Taqman gene expression assays were performed using the StepOnePlus sequence-detection system (Life Technologies), using primers specific to DNM1L (Taqman, cat. no. hs00174131), IRAK1 (Taqman, cat. no. hs001155570), BCL11B (Taqman, cat. no. hs01102259), PINK1 (Taqman, cat. no. hs00260868), and ActB (Applied Biosystems, cat. no.1612290). A master mix was made using 5 ml of 2x Fast advanced master mix (Thermofisher Scientific, cat. no. 4444557), 0.5 ml of 20X primers, and 2 ml of water per reaction well. To each well of a microamp fast optical plate (Applied Biosystems, cat. no. 4346907), 8 ml of master mix and 2 ml of cDNA was added (The reactions were carried out at 48 OC for 30 min and 95 OC for 10 min, followed by 40 cycles of 95 OC for 15 s and 60 OC for 1 min). Samples were analyzed in duplicate. Fold changes were calculated using the comparative CT method.
Exposure of astrocytes and neurons to HIV recombinant proteins
Cells (Astrocytes or B103 neuronal cells) were plated on 12 well plates at 800,000 cells per well and treated for 24 or 96 hours with Vehicle or recombinant gp120 (100 ng/mL; clade E, cat# 2968), nef (100 ng/ml; cat# 11478), or Tat (10 ng/ml; cat# 2222). Total RNA and cytosolic and and nuclear enriched lysate fractions were isolated as described.
Immunoblot of cell lysates
Recombinant proteins were acquired from the NIH AIDS Reagents program. Following treatment with recombinant proteins or transfection with plasmids, cells were washed with sterile PBS and detached using 0.25% trypsin EDTA (Gibco, cat. no. 25200-056). Cells were collected using DMEM media with 10% fetal bovine serum (FBS) (Gibco, cat. no. 16000044) and 1% penicillin/ streptomycin (P/S) (Corning, cat. no. 30-001-CI-1). Cells were then centrifuged at 10,000 rpm for 30 seconds. The supernatant was discarded, and cells were washed with 700 μl of PBS followed by centrifugation at 10,000 rpm for 30 seconds. The PBS was removed, and cells were lysed using a solution of 0.1% Triton-X in PBS with the addition of protease inhibitors. Cells were then centrifuged again at 10,000rpm for 30 seconds. The supernatant was retained as the cytosolic-enriched fraction and the pellet, the nuclear-enriched fraction, was resuspended in lysis buffer and sonicated. After protein concentration was determined using bicinchoninic acid assay (Thermo Fisher Scientific, cat. no. 23225) and samples were denatured in lamellae sample buffer (Bio Rad, cat. no. 1610747), cytosolic and nuclear fractions were loaded (10 μg total protein/lane) on 4–15% Criterion TGX stain free gels (Bio Rad, cat. no. 5678085) and electrophoresed in Tris/Glycine/SDS running buffer (Bio Rad, cat. no. 161-0772) and transferred onto LF PVDF membrane with Bio Rad transfer stacks and transfer buffer (Bio Rad, cat. no 1704275) using Bio Rad Trans Blot Turbo transfer system. After the transfer, total protein was imaged using Bio Rad ChemiDoc imager under the stain free blot setting for normalization purposes. The membranes were then blocked in 1% casein in tris-buffered saline (TBS) (Bio Rad, cat. no. 1610782) for 1 h. Membranes were incubated overnight at 4 °C with primary antibodies, C/EBPb, DNM1L and ACTB, diluted in blocking buffer. All blots were then washed in PBST, and then incubated with species-specific IgG conjugated to HRP (American Qualex, cat. no. A102P5) diluted 1:5000 in PBST and visualized with SuperSignal West Femto Maximum Sensitivity Substrate (ThermoFisher Scientific, cat. no. 34096).
Antibodies
The following antibodies were used in immunoblot, immunohistochemistry, or both: C/EBPβ (Santa Cruz Biotechnology Inc. [SCBT]; catalog #sc-7962/ #sc150), GFAP (Cell Signaling Technology; catalog #3670), MAP2 (SCBT, catalogue #sc-32791), DNM1L (SCBT; catalog #sc-32898), and β-actin (ACTB; Sigma-Aldrich; catalog #A2228).
Statistical analysis
All the analyses of images were conducted on coded samples blinded to the examiner. After the results were obtained, the code was broken, and data were analyzed with Prism software. Comparisons among groups were performed with one-way ANOVA with posthoc Fisher test and unpaired Student’s T test where appropriate. All results were expressed as mean ±SEM. The differences were considered to be significant if p values were <0.05.
Gene expression analysis
Total RNA was isolated from 50 mg of postmortem brain tissues from Broddmann Area 46 using the Qiagen RNeasy Lipid Tissue Kit per manufacturer instructions (Qiagen; cat# 74804). The mRNA libraries were generated by the UC San Diego Institute for Genomic Medicine. RNA-seq data (75 bp single end reads with coverage of 20 million) was obtained from RNA extracted from the frontal cortex of MND and HIV+ cognitive normal subjects (CNHIV+). The quality of the raw FASTQ files were assessed using FASTQC v0.11.8. Adapters and low-quality reads were trimmed using a kmer approach as implemented in BBDuk v38.62. Transcripts were quantified using quasi-mapping mode of Salmon v0.14.1[27] and summarized to gene counts for downstream analysis using the tximport v1.10.0[28] package. Genes were retained in the analysis if they achieved counts per million (cpm) > 1 in at least half of the brain samples sample. Effective library sizes were estimated by TMM scale-normalization prior to analysis to estimate observational weights[29, 30]. Surrogate variables representing latent noise were estimated using sva v package[31]. For the log-transformed expression data with precision weights, per-gene linear regression models were fit to account for the effects of cognitive impairment status after adjustment for unmodeled variation sources. Test for statistical significance was achieved by implementation of a Bayesian strategy of Lönnstedt and Speed as implemented in R package limma v3.38.3[32]. Significance was defined by using an adjusted p-value cut-off of 0.05 after multiple testing correction using a moderated t-static in limma.
To identify the underlying biological functions enriched in frontal cortex of MND relative to cognitive normal, Gene Set Enrichment Analysis (GSEA) was implemented which identifies the enrichment of functionally defined gene sets using a modified Kolmogorov-Smirnov statistic[33] and the Molecular Signature Database (MSigDb v6.0). Statistical significance after adjusting for multiple testing is defined at FDR < 0.05. Gene set-based permutation test of 1000 permutations was applied. Hypergeometric test was utilized to test the statistical significance of the enriched biological process and pathways identified for the unique differential expressed genes for each group[34]. Overrepresentation enrichment analysis was conducted using the full set of detected genes as the reference gene set, corrected for multiple testing using the Benjamini-Hochberg procedure and FDR < 0.05 was considered significant. For the identified transcriptional factors (TF) dysregulated in MND, differentially regulated targets were obtained using the experimentally validated TF binding profiles from the ChEA and ENCODE databases[35, 36]. A database of gene expression in mature human astrocytes was created[37] with threshold gene expression set at 1 FPKM per sample. The astrocyte marker genes were ranked according to the overall expression across all samples. Astrocyte marker genes were identified from C/EBPβ targets and hypergeometric test was used to analyze the functional pathway. All analyses were completed on R statistical software (v3.6.1)[38]