Death by iron: how hypoxic microglia kill oligodendrocytes (A comment on the article by Rathnasamy et al.,)
Alexandra B. Chung and Joseph M. Antony*
Introduction
Periventricular white matterDeath by iron: how hypoxic microglia kill oligodendrocytes (A comment on the article by Rathnasamy et al.,)
Alexandra B. Chung and Joseph M. Antony*
Introduction
Periventricular white matter injury (PWMI) refers to a spectrum of cerebral injuries that range from focal cystic necrotic lesions to diffuse myelination disturbances. Its major cause is hypoxia and is usually presented as symmetric lesions localized adjacent to both lateral ventricles [1]. It is the main cause of cerebral palsy in pre-term and neonatal infants. As well, globally, between 4 and 9 million newborns suffer birth asphyxia each year, leading to about 1.2 million deaths and an equal number of individuals with disabilities (National Center for Health Statistics, USA). Thus, there is a great need to understand the mechanism of PWMI and develop therapeutic interventions for this disease.
Pathological studies have shown astrogliosis and microgliosis in lesions of PWMI in human subjects [2]. Since microglia/macrophage activation is the first cellular event in and around the lesion following the insult, the role of microglia/macrophages in periventricular white matter neuropathogenesis needs to be elucidated. Microglia are the resident macrophages of the central nervous system (CNS) and their numbers can range from 5% to 20% depending on the region of the brain [3]. Their functions include immune-surveillance and -response and phagocytosis in the nervous system. Microglia arise in the developing embryonic brain prior to vascularization. Fate mapping and lineage tracing studies showed that microglia are derived from primitive myeloid precursors in the yolk sac just before E8 [4]. Precursors enter the developing brain and differentiate into microglia which guide invading vasculature to establish blood circulation in the developing CNS in addition to its other functions [3].
Microglial progenitor cells that colonize the CNS during development might differentiate into amoeboid microglia [3]. Amoeboid microglia refers to microglial cells that penetrate the brain during early development, express surface antigens similar to, and share morphology and function with activated macrophages [3]. They can phagocytose cellular debris that arise during brain development and trim axonal projections and synapses; the latter process termed synaptic stripping [5], as part of a normal structural development of the CNS. Amoeboid microglia perhaps also transform into a ramified form with long processes as development proceeds, where the decrease in number of amoeboid microglia correlates to the increase in ramified microglia [3].
Ramified and amoeboid microglia could have distinct functions and responses to hypoxia. In this context, the paper by Rathnasamy et al., [6] describes the unique role played by amoeboid microglia during hypoxia in causing tissue damage mediated by iron. They demonstrated that hypoxia caused an increase in iron concentration and that the iron was specifically found to accumulate in lectin-positive microglia in the periventricular white matter of neonatal rat brain. Macrophages are able to regulate iron homeostasis by recovering iron from old red blood cells and returning it to circulation for binding to transferrin, a glycoprotein that binds to iron tightly but reversibly. Iron retention within activated macrophages is due to increased iron uptake and decreased iron export [7]. Iron is bound to transferrin and the hydrophilic nature of this complex would normally prevent its passage into the brain. However, brain capillary endothelial cells express transferrin receptor 1 (TfR1), which facilitates iron movement into the brain. As shown in Rathnasamy et al., [6], under normal conditions, microglia contain low levels of iron, which dramatically increase under hypoxic conditions. On the other hand, subtypes of tissue macrophages regulate iron homeostasis differently, where alternatively activated macrophages are better at iron export than classically activated macrophages [7]. Alternatively activated macrophages (M2 polarization) function to dampen the inflammatory response, control growth and allow tissue repair. Classically activated macrophages (M2 polarization) are important drivers of the inflammatory response. M1 macrophages are activated by microbes and/or Th1 cytokines and are associated with the production of free radicals and pro-inflammatory cytokines. Thus, it has been shown that various tissue macrophages exert different functions depending on the stimuli. Cellular iron homeostasis is regulated by two cytosolic proteins, the iron regulatory proteins (IRPs 1 & 2) and the dysregulation of these proteins has been shown to be responsible for various neuropathologies. It has been shown that increased levels of IRP2 expression in M2 macrophages leads to upregulation of TfR1, which is associated with iron release [7]. However, Rathnasamy et al., demonstrated that amoeboid microglia (possibly inflammatory as evidenced by increased expression of TNF and IL-1) upregulate IRPs and TfR as a consequence of hypoxia, and is associated with iron retention by the microglia [6], suggesting that the effect of upregulation of IRPs and TfR is context-dependent. Furthermore, since differences in iron levels are associated with distinct phenotypical heterogeneity of M1/M2 macrophage populations, it could be speculated that similar heterogeneity might exist among microglia.
In the CNS, myelination and inflammatory processes involve iron. Traditionally, the view has been that oligodendrocytes, the myelin-producing cells of the CNS, possess high concentrations of iron, due to the enzymes involved in lipid production that utilize iron as a component of their catalytic center [8]. However, during hypoxia, amoeboid microglia have higher iron content than that found in oligodendrocytes. Iron is primarily present in microglia during the first 2 weeks of post-natal development but in the adult CNS, these iron stores shift to oligodendrocytes to meet the requirements of myelination. Quite naturally, during CNS pathology, disruption of iron metabolism and homeostasis leads to oxidative damage in oligodendrocytes [9]. Rathnasamy et al., [6] show that hypoxia reduces the levels of the antioxidant enzyme, glutathione (GSH) in oligodendrocytes making them vulnerable to hypoxia-induced death by free radical-mediated mechanisms. Along with death of oligodendrocytes, conditioned medium from hypoxic microglia also blocked oligodendrocyte proliferation, due to the pro-inflammatory cytokine, IL-1 [6]. Indeed, high levels of iron have been suggested to promote neuropathogenesis of Multiple Sclerosis (MS), which prompted clinical trials involving the therapeutic compound Desferal (also known as deferoxamine) that was administered to MS patients. Results, however, were inconclusive and larger double-blind trials need to be performed to resolve the question of whether Desferal, indeed acts to suppress MS neuropathogenesis by limiting iron-catalyzed free-radical tissue damage, demonstrated in pre-clinical mouse models [8]. Unfortunately, contrasting results in mouse models also contributed to the discrepancies and currently there seem to be no active clinical trials with Desferal. The paper by Rathnasamy et al., [6] provides a mechanistic approach to the problem of iron overload in the CNS, which has also been suggested to be the instigator of MS neuropathogenesis. Recent controversial treatments for MS, such as balloon angioplasty is based on the hypothesis that MS is caused due to constriction of the neck veins leading to increase in iron load in the CNS [10].
In the light of these findings in MS patients, the study by Rathnasamy et al., [6] sheds light on iron-mediated neuropathogenesis since microglia seem to be the culprits, wherein iron from hypoxic microglia cause neonatal rat periventricular white matter damage through the production of pro-inflammatory cytokines and reactive oxygen/nitrogen species. In fact, in this study, it was found that the levels of iron in oligodendrocytes was low and did not contribute to the pathogenesis. Conditioned medium from hypoxic microglia contained a toxic cocktail of cytokines and free radicals that killed oligodendrocytes but was rescued by the iron chelator, deferoxamine. While this adverse effect can be prevented by deferoxamine treatment in vitro, it is not known if deferoxamine administered to neonatal rats can prevent hypoxia-induced PWMI. Another aspect that remains unknown is whether a similar mechanism contributes to the axonal damage seen due to hypoxia. The lack of myelin sheaths on axons and loss of trophic support due to death of oligodendrocytes causes axonal damage but it is reasonable to speculate that free radicals and inflammatory cytokines produced by hypoxic microglia can damage axonal processes of neurons. Also, another interesting aspect of this study is the potential role of astrocytes in mediating oligodendrocyte death. Astrocytes are known to mediate oligodendrocyte death through a free radical-mediated pathway. Since hypoxic microglia are also known to induce release of TNF and IL-1 by astrocytes, it is possible that in PWMI, microglia and astrocytes synergistically contribute to oligodendrocyte death (Figure 1).
Thus, the finding that amoeboid microglia accumulate iron during hypoxia and produce free radicals and pro-inflammatory cytokines causing oligodendrocyte death may underlie the mechanism of PWMI [6]. This study has important implications not only for the role of microglia in iron-mediated neuropathogenesis but also has far reaching implications in neurodegenerative diseases of the white matter.
References
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