HIV-1 TAT-mediated microglial activation: role of mitochondrial dysfunction and defective mitophagy
Authors: Palsamy Periyasamy
Shannon Callen
Venkata Sunil Bendi
Gurudutt Pendyala
Shilpa Buch
Ke Liao
Annadurai Thangaraj
Journal: Autophagy (2018)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: HIV-1 TAT; PINK1; PRKN; microglial activation; mitochondria
Affiliations: a Department of Pharmacology and Experimental Neuroscience , University of Nebraska Medical Center , Omaha , NE , USA., b Department of Anesthesiology , University of Nebraska Medical Center , Omaha , NE , USA.

Abstract:  While the advent of combination antiretroviral therapy (cART) has dramatically increased the life expectancy of HIV-1 infected individuals, paradoxically, however, the prevalence of HIV-1-associated neurocognitive disorders is on the rise. Based on the premise that the cytotoxic HIV-1 protein, transactivator of transcription (TAT), a known activator of glial cells that is found to persist in the central nervous system (CNS) despite cART, we sought to explore the role of defective mitophagy in HIV-1 TAT-mediated microglial activation. Our results demonstrated that exposure of mouse primary microglia to HIV-1 TAT resulted in cellular activation involving altered mitochondrial membrane potential that was accompanied by accumulation of damaged mitochondria. Exposure of microglia to HIV-1 TAT resulted in increased expression of mitophagy signaling proteins, such as PINK1, PRKN, and DNM1L, with a concomitant increase in the formation of autophagosomes, as evidenced by increased expression of BECN1 and MAP1LC3B-II. Intriguingly, exposure of cells to HIV-1 TAT also resulted in increased expression of SQSTM1, signifying thereby a possible blockade of the mitophagy flux, leading, in turn, to the accumulation of mitophagosomes. Interestingly, HIV-1 TAT-mediated activation of microglia was associated with decreased rate of extracellular acidification and mitochondrial oxygen consumption and increased expression of proinflammatory cytokines, such as Tnf, Il1b, and Il6. HIV-1 TAT-mediated defective mitophagy leading to microglial activation was further validated in vivo in the brains of HIV-1 transgenic rats. In conclusion, HIV-1 TAT activates microglia by increasing mitochondrial damage via defective mitophagy. Abbreviations: 3-MA: 3-methyladenine; Δψm: mitochondrial membrane potential; ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATP: adenosine triphosphate; BAF: bafilomycin A1; BECN1: beclin 1, autophagy related; cART: combined antiretroviral therapy; CNS: central nervous system; DNM1L: dynamin 1 like; DMEM: Dulbecco modified Eagle medium; DAPI: 4,6-diamidino-2-phenylindole‎; ECAR: extracellular acidification rate; FBS: fetal bovine serum; FCCP: trifluoromethoxy carbonylcyanide phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; HAND: HIV-1-associated neurocognitive disorders; HIV-1 TAT: human immunodeficiency virus-1 transactivator of transcription; IL1B: interleukin 1, beta; IL6: interleukin 6; ITGAM: integrin subunit alpha M; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mPMs: mouse primary microglial cells; MRC: maximal respiratory capacity; mt-CO1: mitochondrially encoded cytochrome c oxidase; mt-ND6: mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 6; NFKB1: nuclear factor kappa B subunit 1; NLRP3: NLR family pyrin domain containing 3; OCR: oxygen consumption rate; PBS: phosphate-buffered saline; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TNF: tumor necrosis factor. 

Retracted: No

PubPeer comments:
 -For the bands delimited by green squares we made a mistake (Figure 5 Carcinogenesis 30 vs. Figure 5 Mol Cancer Res 7; Figure 9A Breast Cancer Res Treat 129 vs. Figure 2A BBA 1843). -The comments for the Figures 1B Exp Cell Res 319, 1B BBA 1843 and Fig 5 Mol Cancer Res 7 are difficult to explain: we did not make mosaics of bands because we did not need it. It does not make sense to add 1 lane to a series. , 
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 This paper, Biochim Biophys Acta. 2014 Apr;1843(4):815-26, is in the North East. ![file] , 
 #3  For the bands delimited by green squares we made a mistake (Figure 5 Carcinogenesis 30 vs. Figure 5 Mol Cancer Res 7; Figure 9A Breast Cancer Res Treat 129 vs. Figure 2A BBA 1843).  Could the authors correct their mistake? , 
 Comparing *[left]* Fig 5E with *[right]* cytosolic extracts panel of Fig 4A, [Maroni et al (2015)] . ![file] Apparent overlap between the vinculin loading controls (after a horizontal flip). , 
 Figure 3B. Another library loading control that possibly needs its own diagram to pull all the manifestations together. ![file] Here flipped horizontally for comparison with two panels of Fig 8D from [Maroni et al (2014)] . ![file] See also [Bendinelli et al (2017)] : ![] [Bendinelli et al (2015)] : ![] Fig 4B from [Matteucci et al (2013)] ![] , 
 #7 Fig 3B again. Here compared with Fig 7B from [Matteucci et al (2016)] . ![file] I do not begrudge the authors the repeated publication of the Snail band across papers, but when a **completely different** Vinculin band is chosen to normalise it (and the results are the same), I have to wonder what is going on. , 
 Figre 1D: a Matrigen invasion assay. ![file] I have not tried to identify **all** the duplicated or triplicated cells. , 
 Figure 2B (two upper panels). ![file] The photocloned cells extend across both panels, as if they are cut from a single strip of wallpaper. , 
 Fig 3A. ![file] Another Matrigel invasion assay. , 
 Adventures in Loading Controls. Above here, Fig 2A. Middle left, Fig 4A from [Bendinelli et al (2013)] . At middle right, Fig 3B from [Bendinelli et al (2011)] . At the bottom, Fig 9A [Hypoxia] from [Maroni et al (2011)] . ![file] The Vinculin band is versatile. , 
 The matrigel invasion assay images (in Fig. 1D, 2B, 3A) additionally do not appear to be made using the methods described in the paper. These assays are performed using a porous membrane, where cells that have migrated through pores and remain attached to the membrane are stained and imaged using standard phase-contrast microscopy. In the resulting images, the pores should be visible as small circles randomly distributed throughout the field. For example, see this image in from the [product literature] for Corning Biocoat Matrigel invasion chambers: ![file] I am not aware of an imaging technique that can show Diff-Quick stained cells attached to a porous membrane, without also showing the pores. ,