Fig 1.
Distinct miRNA profile in brains of HAND patients.
12 up-regulated (red) and 5 down-regulated (blue) miRNAs were identified in brains of HAND (n = 20) compared to nonHAND (n = 10) patients based on Gene Spring RMA normalization method. miRNAs that were down-regulated (bottom right) cluster together while up-regulated miRNAs (top right) form another cluster. Also, those from HAND and non-HAND patient samples form separate clusters.
Table 1.
Potential targets of miRNAs differentially expressed in HAND brain tissue.
Fig 2.
A subset of HAND-associated miRNAs negatively regulate expression of PEX mRNAs.
HEK293T cells were co-transfected with luciferase reporter plasmids (pMIR-REPORT-Luciferase) containing 3’-UTRs from PEX2 (A), PEX7 (B), PEX11B (C) and PEX13 (D) in forward (5’-3’) or reverse orientations (3’-5’), a transfection control reporter plasmid (pMIR-REPORT-β-gal) and miRNA mimics for miR-500a-5p, miR-34c-3p, miR-93-3p, miR-381-3p and miR-344-3p. After 48 hours, cell lysates were subjected to luciferase and β-gal assays. N = 3. Bars represent standard error of the mean. Key to plasmids: Vec = pMIR-REPORT-Luciferase; KLF4 = pMIR-REPORT-Luciferase with 3’ UTR of KLF4 downstream from luciferase cassette; PEX2 = pMIR-REPORT-Luciferase with 3’ UTR of PEX2 downstream from luciferase cassette; PEX7 = pMIR-REPORT-Luciferase with 3’ UTR of PEX7 downstream from luciferase cassette; PEX11B = pMIR-REPORT-Luciferase with 3’ UTR of PEX11B downstream from luciferase cassette; PEX13 = pMIR-REPORT-Luciferase with 3’ UTR of PEX13 downstream from luciferase cassette. As a positive control, miR-344-3p is shown to downregulate expression of luciferase under the control of the 3’UTR of KLF4 mRNA.
Fig 3.
A subset of HAND-associated miRNAs reduces expression of peroxisomal proteins and alters peroxisome abundance and/or morphology.
A. A549 cells were transfected with mimics (30 nM) for miR-NS, miR-500a-5p, miR-34c-3p, miR-93-3p or miR-381-3p. Forty-eight hours later, cell lysates were subjected to immunoblot analyses. PMP70 and actin were detected using primary mouse monoclonal antibodies and secondary donkey anti-mouse IgG conjugated to Alexa Fluor 680. PEX2, PEX7, PEX11B, PEX13 and catalase were detected using primary rabbit antibodies and secondary goat anti-rabbit IgG conjugated to Alexa Fluor 680. Relative peroxisomal protein levels (normalized to actin) in mock- and miRNA-transfected cells from three independent experiments are shown. Bars represent standard error of the mean. B. A549 cells were transfected with 30 nM of mimics for miR-NS, miR-500a-5p, miR-34c-3p, miR-93-3p or miR-381-3p for 38 hours after which they were processed for super resolution microscopy. Peroxisomes were identified using a mouse monoclonal antibody to PMP70 and donkey anti-mouse IgG conjugated to Alexa Fluor 488. Nuclei were stained with DAPI. Images were acquired and reconstructed using a DeltaVision OMX structured illumination microscope. Size bar is 10 μM. The relative numbers of peroxisomes in cells transfected with each miRNA were determined using Volocity image analyses software from three independent experiments (minimum of 20 cells). Bars represent standard error of the mean. *, p<0.05.
Fig 4.
HIV-1 infection causes loss of peroxisomal proteins and reduces the abundance of peroxisomes.
A. Hela CD4+ cells (clone 1022) were infected with HIV-1 (pYu2, MOI = 10.0) for 72 hours and then processed for indirect immunofluorescence and confocal microscopy. Peroxisomes were detected with a rabbit polyclonal antibody to peroxisomal targeting signal SKL and donkey anti-rabbit IgG conjugated to Alexa Fluor 546. HIV-infected cells were detected with a mouse monoclonal antibody to HIV-1 p24 protein and donkey anti-mouse IgG conjugated to Alexa Fluor 488. Nuclei were stained using DAPI. Images were obtained using spinning disc confocal microscopy. The numbers of peroxisomes (SKL-positive structures) in mock- and HIV-infected cells were determined using Volocity image analyses software. Averages were calculated from three independent experiments in which a minimum of 10 cells for each sample were analyzed. The average number in mock-treated cells was normalized to 1.0. Bars represent standard error of the mean. * p<0.05. B. Primary monocyte-derived macrophages (MDM) were infected with HIV (pYu2, MOI = 2.0) for 5 days and then were subjected to immunoblot analyses with antibodies to Catalase, PEX2, PEX7, PEX11B, PEX13, HIV p24 and actin. The relative levels of peroxisomal proteins (compared to actin) from 3 independent experiments (3 donors) were averaged and plotted. Error bars represent standard error of the mean. * p<0.05.
Fig 5.
HIV-1 induces loss of peroxisomal proteins in brain tissue.
A. Lysates from brain tissue from HIV negative (Neg-1-3), HIV positive (HIV), HIV positive with encephalitis (HIVE-1-2) and HAND patients (HAND-1-3) were subjected to immunoblotting with antibodies to catalase, PEX2, PEX7, PEX11B, PEX13 and actin. The relative levels of peroxisomal proteins (compared to actin) were averaged and plotted. N = 3 (triplicate from same sample). Error bars represent standard deviation of the mean. * p<0.05. B. Immunodetection peroxisome proteins in frontal lobe material from uninfected (HIV[-]) and HIV-infected (HIV[+]) patients. Peroxisomes were labeled with rabbit antibodies PEX13 or thiolase and microglia were detected using rabbit anti-Iba-1. Most of the cells that stain intensely for thiolase and PEX 13 immunopositive cells are astrocytes (arrows) but some could be oligodendrocytes or microglia. Confocal microscopy shows labeled astrocytes (green) and PEX immunoreactivity (scarlet) and DAPI-labeled nuclei. Slides from 4–5 patients per group were reviewed; all HIV+ patients were AIDS-defined and not receiving therapy at the time of death. (Size bar = 20μm).
Fig 6.
HIV-1 infection upregulates expression level of multiple miRNAs that target PEX mRNAs.
A. Primary monocyte-derived macrophages (MDM) from 3 donors were infected with HIV (pYu2, MOI = 2.0) for 5 days and relative levels of miRNAs were determined by RT-PCR from total RNA extracted from the samples. The average relative levels of miRNAs (normalized to snRNU6) from 3 independent experiments were determined. Error bars represent standard error of the mean. B. Hela CD4+ cells were infected with HIV-1 (pYu2, MOI = 10.0) for 48 hours and relative levels of miRNAs were determined as described in panel A. N = 3. Error bars represent standard error of the mean. * p<0.05.
Fig 7.
HIV-induced loss of peroxisomal proteins and peroxisomes is abrogated by blocking the function of miR-500a-5p.
A. HEK293T cells were transfected with a plasmid encoding HIV-1 provirus (pYU2) for 12 hours after which cells were transfected with anti-miRs that are complementary to the HAND-associated PEX-specific miRNAs. Cell lysates were collected 36 hours later and then subjected to immunoblot analyses with antibodies to PEX2, PEX7, PEX11B, PEX13, HIV-1 p24 and actin. The relative levels of peroxisomal proteins (normalized to actin) from 3 independent experiments were determined. Error bars represent standard error of the mean. * p<0.05. B. Hela CD4+ cells (clone 1022) were infected with HIV-1 (MOI = 10) for 16 hours and then transfected with anti-miR-500a-5p. Forty-eight hours later, cells were processed for indirect immunofluorescence and confocal microscopy. Peroxisomes were detected with a rabbit polyclonal antibody to the peroxisomal targeting signal SKL and donkey anti-rabbit IgG conjugated to Alexa Fluor 488. HIV-infected cells was detected with a mouse monoclonal antibody to HIV-1 p24 and donkey anti-mouse IgG conjugated to Alexa Fluor 546. Nuclei were stained using DAPI. Images were obtained using spinning disc confocal microscopy. Size bar is 10 μM. The relative numbers of peroxisomes (SKL-positive structures) in mock and HIV-infected cells transfected with or without anti-miR-500a-5p were determined using Volocity image analyses software. The average numbers of peroxisomes/cell were calculated from three independent experiments in which a minimum of 10 cells for each sample were analyzed. * p<0.05.
Fig 8.
Transfection of miRNA mimics that target PEX mRNAs leads to increased levels of innate immune mRNAs.
A549 cells were transfected with miRNA mimics (30 nM) for miR-NS, miR-500a-5p, miR-34c-3p, miR-93-3p or miR-381-3p. Forty-eight hours later, total RNA was extracted from the cells for use in RT-PCR. Relative levels of innate immune mRNAs (Viperin (A), IFI6 (B), IFIT2 (C), IRF1 (D), and OAS1 (E)) from 3 independent experiments were determined by RT-PCR from total RNA extracted from the samples. Error bars represent standard error of the mean.
Table 2.
Oligonucleotide primers.