Iron is essential for oligodendrocyte genesis following intraspinal macrophage activation

https://doi.org/10.1016/j.expneurol.2009.04.005Get rights and content

Abstract

Progenitor proliferation and differentiation are necessary for oligodendrocyte replacement. Previously, we showed that intraspinal activation of microglia and macrophages with the TLR4 agonist lipopolysaccharide (LPS) induced robust oligodendrocyte genesis. In this study we investigated whether this process involves iron since LPS can alter macrophage regulation of iron and its storage protein ferritin, and oligodendrocytes require iron for proper development and myelination. Further, activated macrophages can sequester and release iron and ferritin. We first examined whether iron or ferritin was present following LPS microinjection. Using Perl's stain, we noted a slight increase in iron at 1d, and peak iron levels 3d post-injection coincident with maximal macrophage activation. Ferritin+ cells were prevalent by 3d and included macrophages and NG2 cells (putative oligodendrocyte progenitors). At 7d, ferritin was mainly expressed by new oligodendrocytes prevalent throughout the lesions. Because of the timing and distribution of iron and ferritin after LPS, we next used an iron chelator to test whether free iron was necessary for maximal LPS-induced oligodendrocyte genesis. Chelating iron by Deferasirox (Exjade®) after LPS microinjection significantly reduced the number of proliferating NG2 cells and new oligodendrocytes. Of the remaining oligodendrocytes, there was a 2-fold decrease in those expressing ferritin, revealing that the number of oligodendrocytes with high iron stores was reduced. Collectively, these results establish that iron accumulates after intraspinal TLR4 activation and is required for maximal TLR4-induced oligodendrogenesis. Since TLR4 agonists are abundant in CNS injury/disease sites, these results suggest that iron may be essential for macrophage/oligodendrocyte communication and adult glial replacement.

Introduction

Iron plays an important role in central nervous system (CNS) myelination and inflammation. During development, a spatial–temporal relationship exists involving iron accumulation by microglia and oligodendrocytes. Most iron is present within microglia during the first two weeks after birth; subsequently iron stores shift to oligodendrocytes, which become the major iron-containing cells in the adult CNS (Connor et al., 1995). Coincident with iron uptake by oligodendrocytes is the onset of myelination, which is impaired in iron deficient animals (Connor and Menzies, 1996, Beard et al., 2003, Ortiz et al., 2004). The high metabolic activity required to produce and maintain myelin, a process that relies on iron-dependent enzymes, likely contributes to the high iron content of oligodendrocytes (for review, see Connor and Menzies, 1996).

Iron homeostasis is often disrupted by CNS pathology, which can negatively impact oligodendrocytes. When iron levels become abnormally high, as occurs in multiple sclerosis, Parkinson's disease, Alzheimer's disease and CNS trauma (Levine, 1997, Smith et al., 1997, Mehindate et al., 2001, Liu et al., 2003, Rathore et al., 2008), oligodendrocytes are particularly vulnerable to death by oxidative damage (for review, see McTigue and Tripathi, 2008). When iron levels are too low, oligodendrogenesis and remyelination can be impaired. Thus, proper iron balance is essential for oligodendrocyte sparing and replacement following CNS injury.

Activated microglia and macrophages can greatly influence iron balance and therefore indirectly affect oligodendrocyte function. For instance, macrophages attempt to prevent the spread of pathogenic infection by sequestering extracellular iron and by removing iron from within other cells (for reviews, see Crichton et al., 2002, Weiss, 2002). Macrophages also engulf extravasated red blood cells thereby removing potentially toxic iron-containing heme (Finch and Huebers, 1982, Huebers and Finch, 1987, Moura et al., 1998). Through cytokine release, activated macrophages can alter levels of iron and the iron storage protein ferritin (Torti et al., 1988, Rogers, 1996, Pinero et al., 2000, Ludwiczek et al., 2003). In certain circumstances, activated macrophages also secrete ferritin, which can be actively taken up by oligodendrocytes and function to promote their survival (Hulet et al., 2000, Zhang et al., 2006). Thus, microglia and macrophages may promote oligodendrocyte survival by removing excess iron and by secreting ferritin, but the same process of iron removal may impair oligodendrocyte replacement and myelination.

Our previous work showed an interesting link between macrophage activation and oligodendrocyte replacement. Following intraspinal microinjection of the TLR4 agonist LPS, microglia/macrophage activation was accompanied by an initial loss of oligodendrocytes followed by robust proliferation and differentiation of new oligodendrocyte lineage cells (Schonberg et al., 2007). Work by others has shown that LPS injected into the intact brain causes iron and ferritin accumulation within macrophages (Zhang et al., 2005). Thus, we hypothesized that iron may play a role in the progenitor proliferation and differentiation occurring after intraspinal LPS injection. Therefore, the present study investigated whether iron or ferritin was present in macrophages, NG2+ progenitors and/or oligodendrocytes following intraspinal LPS injection. Then, to determine if iron is required for LPS-induced oligodendrocyte generation, we treated LPS-injected animals with the highly lipophilic iron chelator Exjade, which can chelate extracellular and intracellular iron pools (Neufeld, 2006, Cappellini, 2007). Our results show that iron chelation significantly impaired TLR4-induced progenitor proliferation and new oligodendrocyte formation revealing that iron is a required reagent for maximum oligodendrocyte formation in LPS lesions. Current studies are underway to resolve the specific steps and/or processes regulated by iron in this model. Since TLR4 ligands are prevalent in sites of CNS pathology (Pasinetti et al., 1993, Aravalli et al., 2007, Goos et al., 2007), this work has significance for understanding macrophage-mediated repair mechanisms in multiple CNS injury sites.

Section snippets

Microinjections

Adult female Sprague–Dawley rats (230–250 g; n = 4/group) were anesthetized with ketamine (80 mg/kg, ip) and xylazine (10 mg/kg, ip) and given prophylactic antibiotics (Gentocin, 5 mg/kg, sc). Using aseptic technique, a laminectomy was performed at the T8 vertebral level. After removing the dura covering the exposed cord, a glass micropipette (custom pulled and beveled to an external tip diameter of 30–40 μm) was inserted 0.7 mm lateral to midline and 1.1 mm ventral to the surface of the spinal

Intraspinal iron levels increase following LPS microinjection

Microinjections were targeted to the lateral white matter of the intact adult rat spinal cord. Vehicle injection caused no microglial activation and only baseline levels of iron were visible (Figs. 1A, F). As in our previous work (Schonberg et al., 2007), LPS injected into the lateral funiculus induced pathology in white matter and adjacent gray matter. In LPS lesions, a significant increase in activated microglia and macrophages was present as early as 1d post-injection (Figs. 1B, K). At this

Discussion

Our previous data revealed that the TLR4 agonist LPS injected into adult rat spinal cords evokes moderate oligodendrocyte loss by 3d followed by dramatic progenitor proliferation, oligodendrogenesis and a rise in overall oligodendrocyte numbers by 7d after injection (Schonberg et al., 2007). Currently, we examined whether iron is involved in this process since iron is necessary for oligodendrocyte development and is upregulated in the brain by LPS (Zhang et al., 2005). Specifically, the current

Summary

Iron is essential for normal cell functions. Too much iron, however, is dangerous and leads to free radical formation and cellular destruction. Consequently, iron levels are tightly regulated in the CNS and throughout the body. Our data reveal that TLR4-induced intraspinal macrophage activation leads to a significant increase in local iron concentration, which appears essential for oligodendrocyte precursor turnover and subsequent new oligodendrocyte formation. Elevated iron and ferritin have

Acknowledgments

The authors gratefully acknowledge Drs. Phillip Popovich and Kristina Kigerl for critical review of the manuscript and Ping Wei and A. Todd Lash for excellent technical assistance. The BrdU antibody developed by S.J. Kaufman was obtained from the Developmental Studies Hybridoma bank, developed under the auspices of the NICHD and maintained by the University of Iowa, Department of Biological Sciences, Iowa City, IA 552242. This work was funded by NINDS NS043494, NS059776 and P30-NS045758.

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