HIV TAT-mediated microglial senescence: Role of SIRT3-dependent mitochondrial oxidative stress
Authors: Ming-Lei Guo
Ashutosh Tripathi
Seema Singh
Annadurai Thangaraj
Palsamy Periyasamy
Shilpa Buch
Fang Niu
Prakash Pillai
Ernest T Chivero
Journal: Redox Biology (2021)
Discipline: BIOLOGY & BIOCHEMISTRY
Keywords: HIV TAT; Microglial activation; Mitochondria stress; Neuroinflammation; SIRT3
Affiliations: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5880, USA., Division of Neurobiology, Department of Zoology, Faculty of Science, The M.S. University of Baroda, Vadodara, India., Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, 68198-5880, USA. Electronic address: .

Abstract:  The advent of combined antiretroviral treatment (cART) as a treatment for HIV-1 infection has not only resulted in a dramatic decrease in the peripheral viral load but has also led to increased life expectancy of the infected individuals. Paradoxically, increased lifespan is accompanied with higher prevalence of age-related comorbidities, including HIV-associated neurocognitive disorders (HAND). Present study was aimed at exploring the role of HIV TAT protein in mediating microglial mitochondrial oxidative stress, ultimately resulting in neuroinflammation and microglial senescence. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to HIV TAT protein resulted in a senescence-like phenotype, that was characterized by elevated expression of both p16 and p21 proteins, increased numbers of senescence-associated-β-galactosidase positive cells, augmented cell-cycle arrest, increased release of proinflammatory cytokines and decreased telomerase activity. Additionally, exposure of mPMs to HIV TAT also resulted downregulation of SIRT3 with a concomitant increase in mitochondrial oxidative stress. Dual luciferase reporter assay identified miR-505 as a novel target of SIRT3, which was upregulated in mPMs exposed to HIV TAT. Furthermore, transient transfection of mPMs with either the SIRT3 plasmid or miRNA-505 inhibitor upregulated the expression of SIRT3 and mitochondrial antioxidant enzymes, with a concomitant decrease in microglial senescence. These in vitro findings were also validated in the prefrontal cortices and striatum of HIV transgenic rats as well as cART-treated HIV-infected individuals. In summary, this study underscores a yet undiscovered novel mechanism(s) underlying HIV TAT-mediated induction of senescence phenotype in microglia, involving the miR-505-SIRT3 axis-mediated induction of mitochondrial oxidative stress. 

Retracted: No

PubPeer comments:
 Brought to my attention by a reader. Figure 5DE: * Red boxes: The right two lanes of the VDAC panel in Figure 5D have a similar shape and background spots as the right two lanes of the SIRT4 panel in Figure 5E. Other lanes in those panels have different shapes. ![file] , 
 Thank you for pointing out this error! After carefully going through our raw data, we realized that this was due to oversight: 1) The VDAC image in Fig 5D actually belonged to the VDAC corresponding to Fig. 5E (we erroneously included it in Fig 5D); 2) The current VDAC image in Fig. 5E above was actually not the VDAC but a non-specific band on the original blot, that was erroneously cropped (see raw data below in Raw Figure B). The original, corresponding raw data for all of these images are included below, along with the amended western blot images for Figure 5D and 5E (see below). The quantification remains unchanged since it was done with the correct, corresponding VDAC images. Please email us any further queries you may have at sbuch@unmc.edu, and we will be happy to respond. We will communicate with the Journal for the necessary corrigenda. **Amended Figure 5D-5E** ![file] ![file] ![file] In Figure D, we are showing a cut image of VDAC (in the red box) and not the whole membrane, since, during developing the VDAC image, we had to cover the upper portion of the membrane owing to the high non-specific background (Fig E above in the red box). , 
 Thank you, Dr. Buch for this update. , 
 SIRT4 is a 41 kDa protein not a 32 kDa protein. Why is no molecular weight markers visible in Raw figure A and B? Marker is visible in Raw figure C!! I trust that the authors have done the studies in triplicate, why not present the image from the other blots to clarify the issue. , 
 **Response:** For this study, we used SIRT4 antibody from Abcam (Cat No. ab124521), and as per the datasheet shown below, a predicted molecular weight of 32 kDa was stated for SIRT4. There are, however, several reports in the literature that claim the molecular weight of SIRT4 ranging from ~30 kDa to ~38 kDa (see references below). Validation of SIRT4 expression in most of the studies was done by comparing the expression in Wild-type and SIRT4 knockout mice. In our hands, we found SIRT4 expression around 32kDa. https: www.abcam.com sirt4-antibody-ab124521.html ![file] https: www.nature.com articles s41419-019-1541-1 SIRT4 (Proteintech, no cat number) 35 kDa https: doi.org 10.1016 j.cell.2014.11.046 SIRT4 (Santa Cruz; (sc-135053) below 38 kDa https: www.thno.org v10p9458.htm SIRT4 Abcam; ab10140) 38 kDa https: www.biorxiv.org content 10.1101 2020.02.17.940692v1.full SIRT4 (Sigma-Aldrich; SAB1407208) 34 kDa https: www.frontiersin.org articles 10.3389 fnagi.2014.00333 full SIRT4 (Sigma-Aldrich; S0948) 30 kDa https: onlinelibrary.wiley.com doi full 10.1111 jnc.12942 SIRT4 (Abcam; ab10140) below 37 kDa; also they used SIRT4 KO mice https: www.thno.org v10p2571.htm SIRT4 (Santa Cruz and Abcam; Didnu2019t provide cat number) 35 kDa https: www.spandidos-publications.com or 40 6 3635 SIRT4 (Abcam; ab124521) above 17 kDa https: journals.physiology.org doi full 10.1152 ajpendo.00465.2019 SIRT4 (Everest Biotech; No cat number) 37 kDa https: www.mdpi.com 2073-4409 9 9 1950 SIRT4 (Santa Cruz; (sc-135053) 34 kDa Regarding the question about the visibility of protein molecular weight markers, we want to emphasize that we used PageRuler Plus Prestained Protein Ladder-10 to 250 kDa (Cat No. 26620) as a protein molecular weight standard in all our experiments and used FluorChem M FM0243 instrument for image capturing (chemiluminescent). These protein molecular weight markers can be visualized clearly in the western blot membrane, however, in the developed image, they appear as a faint impression as white or black bands that are seen in the raw images that we presented previously. For the sake of clarification, in our previous blots, we emphasized these faint bands with red line in Raw Figure A and B. To respond to your query, we are now showing the faint protein ladder impression with a red mark on the side (left) of each membrane (as shown below). Note: to clearly view the protein marker s impression, we adjusted the brightness of the whole image. For further clarity, we are also showing another set of the SIRT4 and VDAC western blot images (New Raw Figure E and F). We hope this satisfies your concern about the SIRT4 protein molecular weight and molecular weight markers. Please email us any further queries you may have at sbuch@unmc.edu, and we will be happy to respond. ![file] ![file] , 
 Correction, March 2021 : https: www.sciencedirect.com science article pii S2213231721000574  The authors regret that two figures were erroneously published in Figure 5 of the original article. 1) The VDAC image in published Figure 5D actually belonged to the VDAC panel corresponding to Figure 5E; 2) The published VDAC image in Figure 5E was a non-specific band on the original blot, that was erroneously cropped, instead of the correct VDAC image being included. The correct western blot images are shown below. The quantification remains unchanged since it was done with the correct, corresponding VDAC images. The authors would like to apologise for any inconvenience caused. ,