cells Article The Chemically-Modified Tetracycline COL-3 and Its Parent Compound Doxycycline Prevent Microglial Inflammatory Responses by Reducing Glucose-Mediated Oxidative Stress Nilson Carlos Ferreira Junior 1,2,3, Maurício dos Santos Pereira 1,2,3 , Nour Francis 1, Paola Ramirez 1, Paula Martorell 1, Florencia González-Lizarraga 4, Bruno Figadère 5, Rosana Chehin 4, Elaine Del Bel 2,3, Rita Raisman-Vozari 1,* and Patrick Pierre Michel 1,* 1 Sorbonne Université, Paris Brain Institute-ICM, Inserm, CNRS, APHP, Hôpital de la Pitié Salpêtrière, 75013 Paris, France; [email protected] (N.C.F.J.); [email protected] (M.d.S.P.); [email protected] (N.F.); [email protected] (P.R.); [email protected] (P.M.) 2 Department of Basic and Oral Biology, FORP, Campus USP, University of São Paulo, Av. Café, s/no, Ribeirão Preto 14040-904, Brazil; [email protected] 3 USP, Center for Interdisciplinary Research on Applied Neurosciences (NAPNA), São Paulo 05508-220, Brazil 4 Instituto de Investigación en Medicina Molecular y Celular Aplicada (IMMCA) (CONICET-UNT-SIPROSA), CP 4000 Tucumán, Argentina; mfl[email protected] (F.G.-L.); [email protected] (R.C.) Citation: Ferreira Junior, N.C.; dos 5 BioCIS, CNRS, Université Paris-Saclay, 92290 Châtenay-Malabry, France; [email protected] Santos Pereira, M.; Francis, N.; * Correspondence: [email protected] (R.R.-V.); [email protected] (P.P.M.); Ramirez, P.; Martorell, P.; Tel.: +33-(0)157274550 (R.R.-V.); +33-(0)157274534 (P.P.M.) González-Lizarraga, F.; Figadère, B.; Chehin, R.; Del Bel, E.; Abstract: We used mouse microglial cells in culture activated by lipopolysaccharide (LPS) or α- Raisman-Vozari, R.; et al. The synuclein amyloid aggregates (αSa) to study the anti-inflammatory effects of COL-3, a tetracycline Chemically-Modified Tetracycline derivative without antimicrobial activity. Under LPS or αSa stimulation, COL-3 (10, 20 µM) efficiently COL-3 and Its Parent Compound repressed the induction of the microglial activation marker protein Iba-1 and the stimulated-release Doxycycline Prevent Microglial of the pro-inflammatory cytokine TNF-α. COL-30s inhibitory effects on TNF-α were reproduced Inflammatory Responses by by the tetracycline antibiotic doxycycline (DOX; 50 µM), the glucocorticoid dexamethasone, and Reducing Glucose-Mediated apocynin (APO), an inhibitor of the superoxide-producing enzyme NADPH oxidase. This last Oxidative Stress. Cells 2021, 10, 2163. observation suggested that COL-3 and DOX might also operate themselves by restraining oxidative https://doi.org/10.3390/ stress-mediated signaling events. Quantitative measurement of intracellular reactive oxygen species cells10082163 (ROS) levels revealed that COL-3 and DOX were indeed as effective as APO in reducing oxidative Academic Editor: Dominique Debanne stress and TNF-α release in activated microglia. ROS inhibition with COL-3 or DOX occurred together with a reduction of microglial glucose accumulation and NADPH synthesis. This suggested that Received: 29 June 2021 COL-3 and DOX might reduce microglial oxidative burst activity by limiting the glucose-dependent Accepted: 13 August 2021 synthesis of NADPH, the requisite substrate for NADPH oxidase. Coherent with this possibility, Published: 22 August 2021 the glycolysis inhibitor 2-deoxy-D-glucose reproduced the immunosuppressive action of COL-3 and DOX in activated microglia. Overall, we propose that COL-3 and its parent compound DOX Publisher’s Note: MDPI stays neutral exert anti-inflammatory effects in microglial cells by inhibiting glucose-dependent ROS production. with regard to jurisdictional claims in These effects might be strengthened by the intrinsic antioxidant properties of DOX and COL-3 in published maps and institutional affil- a self-reinforcing manner. iations. Keywords: COL-3; glucose metabolism; microglia; NADPH oxidase; neuroinflammation; oxidative stress; tetracyclines Copyright: © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article 1. Introduction distributed under the terms and Tetracyclines are a family of antibiotics that inhibit bacterial protein synthesis by conditions of the Creative Commons Attribution (CC BY) license (https:// attaching to the ribosomal subunit and consequently blocking the binding of aminoacyl- creativecommons.org/licenses/by/ tRNAs to the ribosome A site [1]. Although the effects of tetracyclines as antibiotics have 4.0/). long been known, there has been a renewed interest in this family of molecules. Indeed, in Cells 2021, 10, 2163. https://doi.org/10.3390/cells10082163 https://www.mdpi.com/journal/cells Cells 2021, 10, x FOR PEER REVIEW 2 of 16 1. Introduction Tetracyclines are a family of antibiotics that inhibit bacterial protein synthesis by at- taching to the ribosomal subunit and consequently blocking the binding of aminoacyl- Cells 2021, 10, 2163 tRNAs to the ribosome A site [1]. Although the effects of tetracyclines as antibiotics have2 of 16 long been known, there has been a renewed interest in this family of molecules. Indeed, in addition to their antibacterial activity, they can exert pharmacological effects that may have clinical applications in various disease states, including cancer [2], inflammation- relatedaddition disorders to their antibacterial[3], and CNS activity, neurodegenerative they can exert pathologies pharmacological [4,5]. effects that may have clinicalAs an applications illustration in of various that, doxycycline disease states, (DOX), including an old cancertetracycline [2], inflammation-related antibiotic currently disorders [3], and CNS neurodegenerative pathologies [4,5]. used for the treatment of skin problems [6], combines anti-tumoral [7], anti-inflammatory As an illustration of that, doxycycline (DOX), an old tetracycline antibiotic currently [8], and neuroprotective [9,10] activities. In particular, a number of studies reported neu- used for the treatment of skin problems [6], combines anti-tumoral [7], anti-inflammatory [8], roprotective effects of DOX in toxin-induced animal models of Parkinson’s disease (PD) and neuroprotective [9,10] activities. In particular, a number of studies reported neuropro- [9,10]. DOX was also found to decrease the expression of several inflammation markers in tective effects of DOX in toxin-induced animal models of Parkinson’s disease (PD) [9,10]. microglial cultures activated with the bacterial inflammogen lipopolysaccharide (LPS) [8], DOX was also found to decrease the expression of several inflammation markers in mi- which indicates that DOX’s neuroprotective effects may be due in part to its ability to limit croglial cultures activated with the bacterial inflammogen lipopolysaccharide (LPS) [8], inflammation-related events [9,10]. The anti-inflammatory properties of DOX may also which indicates that DOX’s neuroprotective effects may be due in part to its ability to explain why this tetracycline provided relief against L-DOPA-induced dyskinesia in a PD limit inflammation-related events [9,10]. The anti-inflammatory properties of DOX may rat model [11]. Interestingly, DOX was also found capable of preventing amyloid aggre- also explain why this tetracycline provided relief against L-DOPA-induced dyskinesia in gation of α Synuclein (αS) and tau, two seeding-prone proteins involved in PD [12,13] and a PD rat model [11]. Interestingly, DOX was also found capable of preventing amyloid Alzheimer’saggregation ofdiseaseα Synuclein [14] pathologies, (αS) and tau, respectively, two seeding-prone suggesting proteins that involvedthis tetracycline in PD [12 has,13] theand potential Alzheimer’s of a multimod disease [14al] pathologies,neuroprotective respectively, drug. suggesting that this tetracycline has the potentialCOL-3, also of a multimodalnamed chemically neuroprotective modified drug. tetracycline 3 (CMT-3), 4-dedimethyla- minosancyclineCOL-3, also, or named incyclinide chemically, belongs modified to a group tetracycline of modified 3 (CMT-3), tetracyclines 4-dedimethylamin- that have beenosancycline, structurally or incyclinide, rearranged to belongs suppress to atheir group antibacterial of modified properti tetracyclineses. Removal that haveof the been an- timicrobialstructurally activity rearranged obtained to suppress by eliminating their antibacterial the dimethylamino properties. group Removal from of carbon the antimi- 4 in thecrobial A ring activity of the obtainedtetracycline by eliminatingcore structure the (Figure dimethylamino 1) was carried group out from with carbon the principle 4 in the ofA preserving ring of the other tetracycline activities core of structureinterest [15,16]. (Figure COL1) was-3 has carried been outtested with in theclinical principle trials ofin cancerpreserving patients other [17,18], activities and its of interestcapacity [of15 ,inhibiting16]. COL-3 matrix has been metalloproteinases tested in clinical offers trials the in possibilitycancer patients to reduce [17,18 the], and excessive its capacity breakdown of inhibiting of connective matrix metalloproteinasestissue in many pathological offers the conditionspossibility [19 to– reduce21]. COL the-3 excessive is highly breakdownlipophilic, so of it connective can cross the tissue blood in many–brain pathologicalbarrier and exertconditions CNS effects [19–21 [3,22].]. COL-3 In isthat highly respect, lipophilic, systemic so administration it can cross the of blood–brain COL-3 was barrier reported and toexert inhibit CNS brain effects microglial [3,22].