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NAMPT-Derived NAD+ Fuels PARP1 to Promote Skin Inflammation Through

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NAMPT-Derived NAD+ Fuels PARP1 to Promote Skin Inflammation Through bioRxiv preprint doi: https://doi.org/10.1101/2021.02.19.431942; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 1 NAMPT-derived NAD+ fuels PARP1 to promote skin inflammation 2 through parthanatos 3 4 5 Francisco J. Martínez-Morcillo1,2, Joaquín Cantón-Sandoval1,2, Francisco J. Martínez- 6 Navarro1,2, Isabel Cabas1,2, Idoya Martínez-Vicente2,3, Joy Armistead4, Julia Hatzold4, 7 Azucena López-Muñoz1,2, Teresa Martínez-Menchón2,5, Raúl Corbalán-Vélez2,5, Jesús 8 Lacal6, Matthias Hammerschmidt4, José C. García-Borrón2,3, Alfonsa García-Ayala1,2, 9 María L. Cayuela2,5, Ana B. Pérez-Oliva1,2, Diana García-Moreno1,2, Victoriano 10 Mulero1,2,* 11 12 1Departmento de Biología Celular e Histología, Facultad de Biología, Universidad de 13 Murcia, Spain 14 2Instituto Murciano de Investigación Biosanitaria (IMIB)-Arrixaca, Murcia, Spain. 15 3Departamento de Bioquímica y Biología Molecular A e Inmmunología, Facultad de 16 Medicina, Universidad de Murcia, Murcia, Spain. 17 4Institute of Zoology, Cologne Excellence Cluster on Cellular Stress Responses in 18 Aging-Associated Diseases and Center for Molecular Medicine Cologne, University of 19 Cologne, Cologne, Germany. 20 5Hospital Clínico Universitario Virgen de la Arrixaca, Murcia, Spain. 21 6 Departamento de Microbiología y Genética, Facultad de Biología, Universidad de 22 Salamanca, Spain. 23 24 *To whom correspondence should be sent at: [email protected]. 25 26 1 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.19.431942; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 27 Summary 28 Several studies have revealed a correlation between chronic inflammation and 29 NAD+ metabolism but the precise mechanism involved is unknown. Here we report that 30 the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase 31 (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, 32 reduced oxidative stress, inflammation, and keratinocyte DNA damage, 33 hyperproliferation and cell death in zebrafish models of chronic skin inflammation, 34 while all these effects were reversed by NAD+ supplementation. Similarly, genetic and 35 pharmacological inhibition of poly ADP-ribose (PAR) polymerase 1 (Parp1), 36 overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, 37 inhibition of NADPH oxidases and reactive oxygen species (ROS) scavenging, all 38 phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH 39 oxidases/NAMPT/PARP/AIFM1 axis decreased expression of pathology-associated 40 genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant 41 induction of both NAMPT amounts and PARP activity was observed in lesional skin 42 from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS- 43 induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation 44 through parthanatos cell death. 45 Keywords: inflammation, psoriasis, neutrophils, NAD+, PARP1, NAMPT, ROS, 46 NADPH oxidases, DNA damage, parthanatos. 47 Highlights 48 • NAMPT inhibition alleviates inflammation in zebrafish and human epidermis 49 organoid models of psoriasis. 50 • NADPH oxidase-derived ROS mediates keratinocyte DNA damage and Parp1 51 overactivation. 52 • Inhibition of parthanatos cell death phenocopies the effects of NAMPT 53 inhibition in zebrafish and human psoriasis models. 54 • NAMPT and PAR metabolism is altered in psoriasis patients. 55 2 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.19.431942; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 56 Introduction 57 Psoriasis is a non-contagious chronic inflammatory skin disease with global 58 prevalence of 0.1-3 % (Guttman-Yassky and Krueger, 2017). Despite being a relapsing 59 and disabling disease that affects both physical and mental health, is not usually life 60 threatening. However, the cytokines and chemokines produced in the lesion may reach 61 the blood and consequently cause comorbidities (Furue and Kadono, 2017). Although 62 the etiology is still undetermined, external agents trigger inflammation in genetically 63 predisposed epithelium (Greb et al., 2016; Weidinger et al., 2018). Cytokines released 64 by keratinocytes stimulate dendritic/Langerhans cells that drive specific Th17 cell 65 immune response and additional cytokines and chemokines close the inflammatory 66 feed-back loop that result in the skin lesion (Dainichi et al., 2018). 67 Nicotinamide adenine dinucleotide (NAD+) is the most important hydrogen 68 carrier in redox reactions in the cell, participating in vital cellular processes such as 69 mitochondrial function and metabolism, immune response, inflammation and DNA 70 repair, among others (Rajman et al., 2018). NAD+ levels are tightly regulated by Preiss- 71 Handler, de novo and salvage pathways (Canto et al., 2015). Different tissues 72 preferentially employ a distinct pathway regarding available precursors. The most 73 important NAD+ precursor is dietary niacin (also known as vitamin B3), consisting of 74 nicotinamide (NAM), nicotinic acid (NA) and NAM riboside (NR) (Houtkooper et al., 75 2010). NAM is also the product of NAD+-consuming enzymes, that is why most 76 mammalian tissues rely on NAM to maintain the NAD+ pool, via the NAD+ salvage 77 pathway (Canto et al., 2015; Rajman et al., 2018). The rate-limiting enzyme in the 78 NAD+ salvage pathway is NAM phosphoribosyltransferase (NAMPT) that converts 79 NAM into nicotinamide mononucleotide (NMN). After that, NMN adenylyltransferases 80 (NMNAT 1-3) transform NMN into NAD+ (Houtkooper et al., 2010). NAMPT has been 81 associated with oxidative stress and inflammation (Garten et al., 2015), being identified 82 as a universal biomarker of chronic inflammation, including psoriasis (Mesko et al., 83 2010). FK-866 is a noncompetitive highly specific NAMPT pharmacological inhibitor 84 that induces a progressive NAD+ depletion (Hasmann and Schemainda, 2003). FK-866 85 has demonstrated anti-inflammatory effects in different experimental settings, including 86 murine models of colitis and collagen-induced arthritis (Busso et al., 2008; Gerner et al., 87 2018). 88 Several enzymes depend on NAD+ to accomplish their biological functions. 89 Poly(ADP-Ribose) (PAR) polymerases (PARPs) are major NAD+-consuming enzymes 3 bioRxiv preprint doi: https://doi.org/10.1101/2021.02.19.431942; this version posted February 22, 2021. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY 4.0 International license. 90 which transfer ADP-ribose molecules (linear or branching PAR) to proteins or itself 91 (auto-PARylation) (Qi et al., 2019). PARPs are implicated in DNA repair and chromatin 92 organization, gene transcription, inflammation and cell death or stress responses, among 93 others (Canto et al., 2015; Qi et al., 2019). However, the main PARP biological function 94 is to orchestrate the spatio-temporal repair of DNA damage; that is why PARP1 is 95 predominantly localized in the nucleus (Canto et al., 2015), being responsible for 96 approximately 90 % of PAR biosynthesis (Qi et al., 2019). PARP1-3 are recruited and 97 activated upon single- and double-strand DNA breaks (ssBs and dsBs) (Qi et al., 2019). 98 Several PARP inhibitors have been developed, such as olaparib, niraparib, rucaparib, 99 talazoparib and veliparib (Kukolj et al., 2017; Nesic et al., 2018). Once PARP1 is 100 recruited to a ssB, the inhibitors prevent PARP enzymatic activity, entrapping and 101 accumulating inactive PARP on DNA and triggering the collapse of replication forks 102 resulting in dsB generation during replication (Kukolj et al., 2017). 103 Under physiological conditions, DNA damage provoked by cellular metabolism 104 is successfully handled by PARP1. However, alkylating DNA damage, oxidative stress, 105 hypoxia, hypoglycemia or activation of inflammatory pathways can trigger PARP1 106 hyperactivation. Excessive PARylation depletes cellular NAD+ and ATP stores, 107 although it does not directly imply cell death. However, the accumulation of PAR 108 polymers and PARylated proteins reach the mitochondria causing depolarization of the 109 membrane potential and apoptosis-inducing factor (AIFM1) release into the cytosol 110 (Fatokun et al., 2014; Galluzzi et al., 2018). AIFM1 then recruits macrophage migration 111 inhibitory factor (MIF) to the nucleus where AIFM1-MIF nuclease activity executes a 112 large-scale DNA fragmentation resulting in a cell death pathway known as parthanatos 113 (Wang et al., 2016). 114 In this work, we report a critical role played by NAD+ and PAR metabolism in 115 skin oxidative stress and inflammation. We found that hyperactivation of PARP1 in 116 response to ROS-induced DNA damage, and fuelled by NAMPT-derived NAD+, 117 mediates inflammation
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