9940 • The Journal of Neuroscience, December 11, 2019 • 39(50):9940–9953 Cellular/Molecular Iron Metabolism in the Peripheral Nervous System: The Role of DMT1, Ferritin, and Transferrin Receptor in Schwann Cell Maturation and Myelination X Diara A. Santiago Gonza´lez,* XVeronica T. Cheli,* Rensheng Wan, and XPablo M. Paez Hunter James Kelly Research Institute, Department of Pharmacology and Toxicology, Jacobs School of Medicine and Biomedical Sciences, State University of New York, University at Buffalo, Buffalo, New York 14203 Iron is an essential cofactor for many cellular enzymes involved in myelin synthesis, and iron homeostasis unbalance is a central component of peripheral neuropathies. However, iron absorption and management in the PNS are poorly understood. To study iron metabolism in Schwann cells (SCs), we have created 3 inducible conditional KO mice in which three essential proteins implicated in iron uptake and storage, the divalent metal transporter 1 (DMT1), the ferritin heavy chain (Fth), and the transferrin receptor 1 (Tfr1), were postnatally ablated specifically in SCs. Deleting DMT1, Fth, or Tfr1 in vitro significantly reduce SC proliferation, maturation, and the myelination of DRG axons. This was accompanied by an important reduction in iron incorporation and storage. When these proteins were KO in vivo during the first postnatal week, the sciatic nerve of all 3 conditional KO animals displayed a significant reduction in the synthesis of myelin proteins and in the percentage of myelinated axons. Knocking out Fth produced the most severe phenotype, followed by DMT1 and, last, Tfr1. Importantly, DMT1 as well as Fth KO mice showed substantial motor coordination deficits. In contrast, deleting these proteins in mature myelinating SCs results in milder phenotypes characterized by small reductions in the percentage of myelinated axons and minor changes in the g-ratio of myelinated axons. These results indicate that DMT1, Fth, and Tfr1 are critical proteins for early postnatal iron uptake and storage in SCs and, as a consequence, for the normal myelination of the PNS. Key words: DMT1; ferritin; iron; myelination; Schwann cells; transferrin receptor Significance Statement To determine the function of the divalent metal transporter 1, the transferrin receptor 1, and the ferritin heavy chain in Schwann cell (SC) maturation and myelination, we created 3 conditional KO mice in which these proteins were postnatally deleted in Sox10-positive SCs. We have established that these proteins are necessary for normal SC iron incorporation and storage, and, as a consequence, for an effective myelination of the PNS. Since iron is indispensable for SC maturation, understanding iron metab- olism in SCs is an essential prerequisite for developing therapies for demyelinating diseases in the PNS. Introduction required for myelin production as a cofactor for enzymes in- In the CNS, iron is essential for myelin synthesis and mainte- volved in cholesterol and lipid synthesis (Lange and Que, 1998; nance (Todorich et al., 2009; Cheli et al., 2018). Iron is directly Beard et al., 2003), and oligodendrocytes are the cells with the highest iron levels in the brain which is linked to their highly metabolic needs associated with the process of myelination Received June 17, 2019; revised Oct. 9, 2019; accepted Oct. 29, 2019. (LeVine and Macklin, 1990; Connor and Menzies, 1996). The Author contributions: D.A.S.G., V.T.C., and P.M.P. designed research; D.A.S.G., V.T.C., and R.W. performed re- PNS has also an elevated iron requirement (Levi and Taveggia, search;D.A.S.G.,V.T.C.,R.W.,andP.M.P.analyzeddata;D.A.S.G.,V.T.C.,andP.M.P.wrotethefirstdraftofthepaper; 2014), and Schwann cell (SC) development has been associated D.A.S.G., V.T.C., and P.M.P. edited the paper; D.A.S.G., V.T.C., and P.M.P. wrote the paper. This work was supported in part by the National Institutes of Health/National Institute of Neurological Disorders with iron uptake (Salis et al., 2002, 2012). Moreover, iron defi- andStrokeGrant5R01NS078041.WethankDr.LucianaFrick,Dr.EmmaWilson,Dr.ChelseyReed,andGustavoDella ciency is involved in the pathogenesis of several peripheral neu- Flora Nunes (Hunter James Kelly Research Institute) for assistance with DRG explant cultures preparation and ropathies (Kabakus et al., 2002; Salas et al., 2010), and some analysis. neurodegenerative diseases characterized with brain iron accu- The authors declare no competing financial interests. mulation show phenotypic alterations in the PNS (Levi and Tav- *D.A.S.G. and V.T.C. contributed equally to this work. Correspondence should be addressed to Pablo M. Paez at [email protected]. eggia, 2014). Nevertheless, the physiological role of iron in the https://doi.org/10.1523/JNEUROSCI.1409-19.2019 PNS and particularly in SC maturation and myelination is poorly Copyright © 2019 the authors understood. Santiago Gonza´lez, Cheli et al. • Iron and Schwann Cell Maturation J. Neurosci., December 11, 2019 • 39(50):9940–9953 • 9941 Several proteins have been shown to play a role in maintaining mice of either sex were used. Transgenic mice were genotyped by PCR. cell iron homeostasis by regulating iron uptake and storage. The genomic DNA was isolated, and PCRs were performed using the Transferrin receptor 1 (Tfr1) mediates cellular iron uptake Terra PCR Direct Polymerase Mix (Takara). The following primers were Ј through receptor-mediated endocytosis and is highly expressed used for detection of floxed alleles: DMT1: 5 -ATGGGCGAGTTAGA Ј Ј Ј by neurons and oligodendrocytes in the CNS (Connor and Fine, GGCTTT-3 (sense) and 5 -CCTGCATGTCAGAACCAATG-3 (anti- sense); Fth: 5Ј-CCATCAACCGCCAGATCAAC-3Ј (sense) and 5Ј-CG 1986; Giometto et al., 1990). Tfr1 is also present in the PNS, and CCATACTCCAGGAGGAAC-3Ј (antisense); Tfr1: 5Ј-TTCAGTTCCCA it has been implicated in SC iron uptake after nerve injury (Raiv- GTGACCACA-3Ј (sense) and 5Ј-TCCTTTCTGTGCCCAGTTCT-3Ј ich et al., 1991). The iron incorporated by the Tfr1 is transported (antisense); and Sox10-CreER T2:5Ј-TTGCGATGGGAGAGTCTGAC- from endosomes into the cytosol by the divalent metal trans- 3Ј (sense) and 5Ј-AGGTACAGGAGGTAGTCCCT-3Ј (antisense). porter 1 (DMT1) (Fleming et al., 1998). DMT1 is a proton-driven Mice treatments. A tamoxifen stock solution (2.5 mg/ml) was prepared metal transporter primarily responsible for iron transport from by dissolving and sonicating tamoxifen in autoclaved vegetable oil. To endosomes as well as for diet iron uptake in the gut (Gruenheid et delete DMT1, Fth, or Tfr1 in Sox10-positive SCs, P2 DMT1 KO Ϫ Ϫ al., 1995; Veuthey and Wessling-Resnick, 2014). DMT1 is ex- (DMT1 f/f, Sox10Cre Cre/ ), Fth KO (Fth f/f, Sox10Cre Cre / ), Tfr KO (Tfr1 f/f, Sox10Cre Cre / Ϫ), and control (Cre-negative) littermates pressed by neurons (Skjørringe et al., 2015), astrocytes (Erikson f/f f/f f/f Ϫ / Ϫ and Aschner, 2006; Song et al., 2007), and oligodendrocytes in the (DMT1 , Fth , or Tfr1 , Sox10Cre ) were injected intraperitone- brain (Burdo et al., 2001, Cheli et al., 2018); and by SCs in the PNS ally once a day for 5 consecutive days with 25 mg/kg of tamoxifen, and sciatic nerves were collected at P15 and P30. Additionally, P60 DMT1 KO, (Vivot et al., 2013). Fth KO,orTfrKO and control littermates were injected once a day for 5 Ferritin is the major iron storage protein; it is a heteropolymer consecutive days with 100 mg/kg of tamoxifen (20 mg/ml stock solu- composed of 24 subunits of heavy (Fth) and light (Ftl) types, tion), and sciatic nerves were collected at P90. which can store up to 4500 atoms of iron (Harrison et al., 1967). DRG explant cultures. DRGs were extracted from the spinal cord of The Fth subunit contains ferroxidase activity, which is required mouse embryos at embryonic day 13.5. Twenty DRGs were collected per for converting soluble ferrous into ferric iron, which is then de- embryo in 1 ml of Leibovitz’s-15 medium (L-15) (Invitrogen), centri- posited inside the ferritin core (Levi et al., 1988; Harrison and fuged at 700 rpm for 5 min, and resuspended in 500 ␮l of 0.25% trypsin Arosio, 1996). In the CNS, the highest ferritin expression levels solution without EDTA (Invitrogen). DRGs were then incubated for 45 ␮ are in oligodendrocytes, which express equal amounts of both Fth min at 37°C, after which 500 l of L-15 medium containing 10% FBS and Ftl subunits (Connor, 1994). Microglia cells express ferritin (Invitrogen) was added to each tube. After 10 min centrifugation at 1000 rpm, the cell pellet was triturated and resuspended in C-medium: MEM enriched in Ftl subunits, whereas ferritin in neurons is mainly (Invitrogen) supplemented with D-glucose (4 g/L), FBS (10%), L-glu- composed of Fth subunits (Connor, 1994). Despite the extensive tamine (2 mM), NGF (50 ng/ml), and gentamycin; 150 ␮l of cell suspen- literature regarding ferritin in the CNS (Friedman et al., 2011), sion was transferred to the center of a coverslip (15 mm) coated with very little is known on this protein in the PNS. collagen. The cells were then incubated at 37°C with 5% CO2 overnight. Much effort has been paid to understand the molecular mech- The following day, the culture medium was replaced for Neurobasal anism involved in CNS iron homeostasis. This has been stimu- (Invitrogen) supplemented with B27 (Invitrogen), D-glucose (4 g/L), lated by the evidence that abnormal iron homeostasis can result L-glutamine (2 mM), NGF (50 ng/ml), and gentamycin. After 2 d, the cells in CNS pathological conditions. Excessive iron levels have been were transferred to C-medium, which was replaced every other day. Cre associated with neurodegenerative diseases (Zecca et al., 2004; activity and recombination were induced by treating the cells with 4-OH Molina-Holgado et al., 2007), whereas iron deficiency during tamoxifen for 4 consecutive days starting at day 4 after plating.