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. 2012 Aug 30;3(8):e381.
doi: 10.1038/cddis.2012.114.

Exosome-mediated shuttling of microRNA-29 regulates HIV Tat and morphine-mediated neuronal dysfunction

G Hu  1 , H Yao, A D Chaudhuri, M Duan, S V Yelamanchili, H Wen, P D Cheney, H S Fox, S Buch
Affiliations

Exosome-mediated shuttling of microRNA-29 regulates HIV Tat and morphine-mediated neuronal dysfunction

G Hu et al. Cell Death Dis. .

Abstract

Neuronal damage is a hallmark feature of HIV-associated neurological disorders (HANDs). Opiate drug abuse accelerates the incidence and progression of HAND; however, the mechanisms underlying the potentiation of neuropathogenesis by these drugs remain elusive. Opiates such as morphine have been shown to enhance HIV transactivation protein Tat-mediated toxicity in both human neurons and neuroblastoma cells. In the present study, we demonstrate reduced expression of the tropic factor platelet-derived growth factor (PDGF)-B with a concomitant increase in miR-29b in the basal ganglia region of the brains of morphine-dependent simian immunodeficiency virus (SIV)-infected macaques compared with the SIV-infected controls. In vitro relevance of these findings was corroborated in cultures of astrocytes exposed to morphine and HIV Tat that led to increased release of miR-29b in exosomes. Subsequent treatment of neuronal SH-SY5Y cell line with exosomes from treated astrocytes resulted in decreased expression of PDGF-B, with a concomitant decrease in viability of neurons. Furthermore, it was shown that PDGF-B was a target for miR-29b as evidenced by the fact that binding of miR-29 to the 3'-untranslated region of PDGF-B mRNA resulted in its translational repression in SH-SY5Y cells. Understanding the regulation of PDGF-B expression may provide insights into the development of potential therapeutic targets for neuronal loss in HIV-1-infected opiate abusers.

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Figures

Figure 1
Figure 1
SIV and morphine alter miRNA profiling in basal ganglia of macaques. (a) Unbiased heat map correlating the expression of various miRNAs in the basal ganglia of SIV-infected macaques with and without morphine dependence, with colors indicating relative expression levels. (b) Real-time PCR demonstrating increased miR-29b expression in basal ganglia of SIV-infected and morphine-dependent macaques. (c) In situ hybridization demonstrating increased miR-29b expression in neurons adjacent to astrocytes (arrows) in the basal ganglia of morphine-dependent SIV-infected macaques. Scale bar=10 μm
Figure 2
Figure 2
Downregulation of PDGF-BB protein in basal ganglia of SIV-infected macaques with opiate abuse. (a) Western blot analysis of PDGF-BB expression in basal ganglia of untreated or SIV-infected macaques with and without morphine dependence. (b) Real-time PCR analysis of PDGF-B mRNA expression in basal ganglia of SIV-infected macaques with and without morphine dependence. Bars represent the mean±S.D. from three independent experiments. *P<0.05 versus untreated group
Figure 3
Figure 3
Conditioned media from HIV-1 Tat-and morphine-treated astrocytes downregulated PDGF-BB expression in neurons. (a, c and e) Western blot analysis of PDGF-BB protein expression in neurons treated with astrocyte conditioned medium. (b, d and f) Real-time PCR analysis of PDGF-B chain expression in neurons treated with astrocyte conditioned medium. Bars represent the mean±S.D. from three independent experiments. NCM, nonculture medium; CACM, control astrocyte CM; MACM, morphine-treated astrocyte CM; TACM, Tat-treated astrocyte CM; MTACM, morphine and TACM
Figure 4
Figure 4
Exosomes released from astrocytes treated with morphine and Tat contain miR-29b that can be taken up by neurons. (a) Electron micrograph of A172 exosomes. The image shows small vesicles of approximately 50–80 nm in diameter. Scale bar=100 nm. (b) Western blot characterization of astrocyte exosomes. Exosome protein released from astrocytes was separated on SDS-PAGE and electroblotted to nitrocellulose membrane. Blots were probed with exosome marker antibody against Tsg101. (c) Rat primary astrocytes and A172 cells were pre-treated with exosome inhibitor (DMA) or lipid-raft pathway inhibitor (MβCD) for 1 h (to inhibit exosome release), followed by morphine and Tat treatment exposure for 24 h. Total RNA from exosomes was then analyzed by real-time PCR for miR-29b. Bars represent the mean±S.D. from three independent experiments. *P<0.05 versus control. (d) MiR-29 was increased in morphine and Tat-treated A172 released exosomes, as assessed by virtual northern blot. Cells were exposed to morphine and Tat for 24 h. Exosomes were isolated and followed by virtual northern blot for miR-29. Small nuclear RNA RNU6B (U6) was blotted to ensure equal loading. (e) SH-SY5Y cells were incubated with exosomes released from control or morphine- and Tat-treated A172 cells for 24 h, followed by western blot for PDGF-BB. (f) Detection of miR-29 transferred into SH-SY5Y cells using FAM-labeled microRNA mimic (miR-29). SH-SY5Y cells were incubated for 3 h at 37 °C with exosomes isolated from A172 cells transfected with FAM-labeled miR-29 mimic (exosome FAM miR-29). The uptake of exosomes was evaluated by confocal microscopy. MiRNAs incorporated within SH-SY5Y cells were detected as green fluorescence signal of FAM fluorophores (original magnification × 400). Scale bar=20 μm
Figure 5
Figure 5
MiR-29b targets PDGF-B 3′-UTR resulting in translational suppression. (a) PDGF-B mRNA shows one potential binding site in the 3′-UTR for miR-29. (b) Binding of miR-29b to the potential binding site in the PDGF-B 3′-UTR resulted in translational suppression. SH-SY5Y cells were cotransfected with the reporter construct and miR-29b precursors anti-miR-29b for 24 h. Luc activity was then measured and normalized to the control β-gal levels. (c) SH-SY5Y cells were transfected with either miR-29b precursor or anti-miR-29b for 24 h followed by western blotting for PDGF-BB. Bars represent the mean±S.D. from three independent experiments. *P<0.05 versus control; #P<0.05 versus treated control
Figure 6
Figure 6
MiR-29b is involved in neuronal death induced by morphine and Tat astrocyte conditioned media. (a) Cell viability of rat primary neurons and SH-SY5Y cells (± retinoic acid) treated with the indicated conditioned media using the MTT assay after 48 h. (b) MAP-2 staining in rat primary neurons treated with indicated conditioned media. Scale bar=10 μm. (c) Cell viability of SH-SY5Y cells transfected with miR-29b precursors with or without PDGF-B protein (100 ng/ml) or anti-miRs for 48 h using MTT assay. (d) Cell viability of SH-SY5Y cells treated with indicated exosomes for 48 h followed using MTT assay. Data are mean±S.D. of three independent experiments performed in triplicate. *P<0.05 versus control group; #P<0.05 versus treated control
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