COPENHAGEN, Denmark — A recent postmortem study finds a global reduction of iron in the nonlesioned white matter of brains from patients with multiple sclerosis (MS) that correlates with disease duration.

"MS leads to a global iron loss in the brain white matter," the researchers, with first author Simon Hametner, MD, from the Center for Brain Research, Medical University of Vienna, Austria, conclude. "Demyelination in MS leads to waves of iron liberation from intracellular stores which may promote oxidative damage."

The researchers further found that in active lesions, iron appeared to be liberated from damaged oligodendrocytes and taken up into microglia and macrophages. These iron-containing microglia, in turn, showed signs of cell degeneration, which leads to leads to microglia loss in old burnt-out MS plaques.

"Regarding iron in the nonlesioned white matter of MS brains, we observed 2 opposing effects," Dr. Hametner told Medscape Medical News. "The first is the age-dependent accumulation of iron, as observed in controls, and the second is a disease duration-dependent loss of iron in the nonlesioned white matter. In progressive MS, we find significantly reduced iron content in myelin and the oligodendrocytes."

They propose this could represent a preconditioning mechanism because oligodendrocytes have been shown in previous in vitro studies by the group of James R. Connor to be more prone to cell death when they are iron-loaded, he said.

Their findings were presented here at the Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS). The main findings were also published online July 19 in Annals of Neurology.

Dr. Simon Hametner

Iron Shuttling

In the healthy human central nervous system, iron is stored within oligodendrocytes and myelin. Excess iron can promote oxidative damage and has been implicated in various neurodegenerative disorders, including Alzheimer's disease and Parkinson's disease, they note.

The aim of this study was to look at the effect of liberation of iron from within oligodendrocytes and myelin due to the demyelination and destruction of oligodendrocytes that occurs in the setting of MS. Iron also accumulates with age, Dr. Hametner noted, with a peak at about 40 years of age in some brain regions, 50 years in other areas, the same age at which many patients with MS convert from a relapsing-remitting disease course to a progressive one.

"Therefore we suspected that the higher iron load of older patients in MS would facilitate the conversion from relapsing remitting MS to secondary progressive MS, or would even facilitate the onset of primary progressive MS, which is also later than relapsing-remitting MS," he said.

In this study, formalin-fixed, paraffin-embedded autopsy brain tissue from 33 MS cases were studied, including 7 acute MS cases and 30 control cases.

They used diaminobenzidine-enhanced Turnbull blue staining to detect nonheme iron. Immunohistochemistry was used to detect the iron storage protein ferritin, the microglia markers Iba-1 and CD68, the ferroxidases hephaestin and ceruloplasmin, and oxidized phospholipids as markers of oxidative stress.

Microarray analysis showed upregulation of cytoplasmic ferritin, mitochondrial ferritin, and ferroxidases, as well as downregulation of the mitochondrial iron exporter frataxin in acute MS lesions, suggesting cellular and mitochondrial stress related to iron, the researchers note.

Compared with controls, MS brains showed a global reduction of iron in nonlesioned white matter oligodendrocytes, and this reduction was significantly correlated with the duration of disease. Together with this observation, they found upregulation of an iron exporter protein called hephaestin in oligodendrocytes, "so we suggest that hephaestin upregulation might be one of the factors leading to iron efflux driven by inflammation," Dr. Hametner said.

In early active lesions, they observed a shift of iron storage and ferritin expression from oligodendrocytes to microglia and macrophages, as well as extracellular ferrous iron in a few patients who had onset of MS at an old age.

In a subset of 4 acute MS cases and 3 control cases, they performed whole-genome microarrays to analyze messenger RNA expression levels of iron-related genes. They found upregulation of iron importers, especially in the white matter around active lesions. They found upregulation of ferritins in the cytosol and in the mitochondria especially of established lesions, he said.

"We also found a profound downregulation of the mitochondrial protein frataxin, which could be linked to mitochondrial oxidative stress, and seen together with mitochondrial ferritin upregulation, which could prevent the oxidative stress; so, a rescue mechanism," he noted.

In the slowly expanding lesions that dominate in the progressive phase, there was still some iron in the normal-appearing white matter, iron accumulation at the active lesion edge, and iron loss in the demyelinated lesion center.

Iron in the microglia led to microglial dystrophy, which would eventually lead to cell death, Dr. Hametner noted. Many iron-loaded microglia were found at the edge of slow-expanding lesions, so they speculate many of these microglia degenerate due to iron.

Another observation was a significant loss of microglia in normal-appearing white matter and in demyelinated lesion center. "Taken together, we think the oxidative stress exerted by iron in these microglial cells leads to dystrophy and subsequent microglial loss in the inactive lesion cores of such slowly-expanding progressive lesions," Dr. Hametner said. "We also found sometimes ferrous iron within axons at such lesion edges."

He said it is unlikely that these observations would have therapeutic implications at this time, although he noted that they may be instructive with regard to the proposed role of chronic cerebrospinal venous insufficiency in MS.

They do see iron staining around blood vessels in brain slices, he said, "and this could be misinterpreted to be due to iron leakage and vascular or venous hypertension problems and iron drainage into the brain. But…we see it in the late active demyelination state and not in the early, so when we see this perivascular iron, things have happened already. Myelin already has been damaged, myelin already has been phagocytosed by macrophages, and these cells actually are more likely in our interpretation—of course we cannot prove it—but are more likely to leave the CNS [central nervous system] than to go into it."

They are focusing on glial iron metabolism in MS and its animal model, experimental autoimmune encephalomyelitis (EAE), in collaboration with Dr. Juan Zarruk and Dr. Samuel David from McGill University, Montreal, Quebec, Canada. "We want to know how our findings in MS are reflected in the model disease EAE, an approach which may deepen our understanding of glial iron metabolism under inflammatory conditions in general," Dr. Hametner concluded.

Role of Iron "Remains a Mystery"

Asked for comment on these findings, Bruce F. Bebo Jr, PhD, associate vice president of discovery research at the National Multiple Sclerosis Society, pointed out that previous studies have shown that iron is deposited in some white matter lesions in MS, "but it remains a mystery what the source of this iron is and what role it may or may not play in the disease process."

This study, by a well-known group of researchers, provides some evidence of where the iron is coming from and what it might be doing, Dr. Bebo told Medscape Medical News. Iron is a critical cofactor for the function of enzymes in cells of the central nervous system, he adds, and is released when cells are damaged. This happens as a natural part of the aging process but appears to be more prevalent in people with MS, and may indicate accelerated damage to the nervous system.

"What this presentation and paper have shown is that iron is released from the myelin-producing cells — oligodendrocytes — that are dying in the white matter as a consequence of the immune attack associated with MS," Dr. Bebo said. The iron is subsequently picked up by other brain cells, such as microglia, astrocytes, and even nerve axons, he notes. The authors also showed some evidence that the iron causes cellular degeneration.

"These data support a hypothesis that the immune damage of oligodendrocytes caused by the first waves of inflammation release many substances, including iron, that may trigger the more progressive and neurodegenerative phase of MS," Dr. Bebo said. "It argues for early and aggressive treatment to stop or limit inflammation as a strategy for reducing progression."

This knowledge, he says, could help researchers develop new imaging techniques to detect the transition to the progressive phase of the disease. "It could also yield targets for drugs (or other strategies) that limit the damage that triggers progression."

The study was funded by the Austrian Science Fund. The authors have disclosed no relevant financial relationships.

Congress of the European Committee for Treatment and Research in Multiple Sclerosis (ECTRIMS). Abstract #50. Presented October 2, 2013.

Ann Neurol. Published online July 19, 2013. Abstract

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