Iron and neurodegeneration in the multiple sclerosis brain

Abstract

Objective: Iron may contribute to the pathogenesis and progression of multiple sclerosis (MS) due to its accumulation in the human brain with age. Our study focused on nonheme iron distribution and the expression of the iron-related proteins ferritin, hephaestin, and ceruloplasmin in relation to oxidative damage in the brain tissue of 33 MS and 30 control cases.

Methods: We performed (1) whole-genome microarrays including 4 MS and 3 control cases to analyze the expression of iron-related genes, (2) nonheme iron histochemistry, (3) immunohistochemistry for proteins of iron metabolism, and (4) quantitative analysis by digital densitometry and cell counting in regions representing different stages of lesion maturation.

Results: We found an age-related increase of iron in the white matter of controls as well as in patients with short disease duration. In chronic MS, however, there was a significant decrease of iron in the normal-appearing white matter (NAWM) corresponding with disease duration, when corrected for age. This decrease of iron in oligodendrocytes and myelin was associated with an upregulation of iron-exporting ferroxidases. In active MS lesions, iron was apparently released from dying oligodendrocytes, resulting in extracellular accumulation of iron and uptake into microglia and macrophages. Iron-containing microglia showed signs of cell degeneration. At lesion edges and within centers of lesions, iron accumulated in astrocytes and axons.

Interpretation: Iron decreases in the NAWM of MS patients with increasing disease duration. Cellular degeneration in MS lesions leads to waves of iron liberation, which may propagate neurodegeneration together with inflammatory oxidative burst.

Figure 1

Correlation of whole tissue iron with age (A) and disease duration (B). (A) Whole tissue iron, as quantified by digital optical densitometry, correlated with age (in years) at the nonlesioned subcortical white matter (WM). Lines represent separate linear regressions of controls and multiple sclerosis (MS) cases. (B) Whole tissue iron is subtracted from the iron levels predicted by the age-dependent linear regression equation derived from controls (continuous line in A). There is a significant correlation between the reduction of iron load and disease duration. Furthermore, a significant negative partial correlation between whole tissue iron and disease duration (in months) was found when pooling all data points and setting disease duration of controls to zero (r_partial = −0.497, p < 0.001). Analysis of whole tissue iron in the deep WM led to comparable results (not shown). AMS = acute MS; RRMS = relapsing–remitting MS.

Figure 2

Iron and the expression of iron-related proteins in the white matter (WM) of controls and multiple sclerosis (MS) patients. (A) Luxol fast blue–periodic acid Schiff myelin staining (blue) and (B) total nonheme iron (brown) of consecutive double-hemispheric slides of MS Subject 25. High iron load is visible in the basal ganglia and the leukocortical boundary. Multiple demyelinated WM lesions, which harbor less iron than the normal-appearing WM (NAWM), are indicated by arrows. (C–Q) Presence of total nonheme iron and iron-related proteins in the normal WM (NWM) of controls (left panel of images; C, F, I, L, O), in the NAWM of MS patients (middle panel; D, G, J, M, P), and in the periplaque WM (PPWM) of MS patients close to the edge of an active lesion (right panel; E, H, K, N, Q). (C–E) In the NWM (C), iron is mainly present in myelin and cells with oligodendrocytic morphology (inset in C) and is reduced in the NAWM of MS patients (D). Inset in D shows small round cells indicative of oligodendrocytes and a larger cell indicative of activated microglia morphology. Close to active plaques, iron is largely lost from oligodendrocytes and myelin, but is present in activated microglia cells (E). (F–H) Immunohistochemistry for ferritin (brown) shows a similar cellular distribution as iron; however, more cells are detected with ferritin immunohistochemistry than with total nonheme iron staining. (I–N) Hephaestin (blue) is expressed in the NWM in oligodendrocytes (I; oligodendrocyte marker TPPP/p25 = red in I–K) and in astrocytes (L; astrocyte marker glial fibrillary acidic protein = red in L–N). In the MS NAWM, both hephaestin⁺ oligodendrocytes (J) and astrocytes (M) are reduced. In the PPWM close to active lesions, hephaestin is upregulated in relation to the NAWM in oligodendrocytes (K), but not in astrocytes (N). (O–Q) Ceruloplasmin (brown) is mainly observed in astrocytes. Its expression is consistently low in the NWM (O), elevated in the NAWM of some MS patients (P), and consistently strong at the edge of active lesions (Q). AG = astroglia; OG = oligodendroglia; scale bars = 100μm; inset scale bars = 10μm (C, I–N) or 25μm (D–H).

Figure 3

Iron and iron-related molecules in different types of MS lesions. (A–F) There are 3 different types of MS lesions regarding iron content. (A, D) In slowly expanding lesions with demyelinating activity at the lesion edge, and less frequently and less pronounced in inactive lesions, a rim of iron within microglia and macrophages is seen at the lesion edge. The iron content within the lesion is reduced, but perivascular accumulation of iron is occasionally seen within lesions. (A) Proteolipid protein (PLP) immunohistochemistry (IHC). (D) Total nonheme iron staining. (B, E) Some classic late active MS lesions contain more iron compared to the normal-appearing white matter (NAWM). The iron is found within macrophages, astrocytes, and axons in these lesions. (B) Luxol fast blue–periodic acid Schiff myelin staining (blue). (E) Total nonheme iron staining. (C, F) In the majority of MS lesion, iron is reduced compared to the NAWM, and total nonheme iron staining closely matches the staining for myelin. (C) PLP IHC. (F) Total nonheme iron staining. (G–U) Iron and iron-related proteins are shown in early active lesions (left panel; G, J, M, P, S), late active lesions (middle panel; H, K, N, Q, T), and inactive lesions (right panel; I, L, O, R, U). The figure documents an extreme example (MS Subject 5, who developed acute MS at the age of 78 years; the lesions formed in a brain with a high age-related iron load). Lesional activity was defined according to Brück et al. In early active lesions, total nonheme iron (G, brown) is mainly seen in the cytoplasm of macrophages and microglia and to a lesser extent as fine extracellular granules. In contrast, ferrous nonheme iron (J, brown) is largely detected as extracellular granules or in lysosomes or endosomes of macrophages and microglia. Ferritin expression (M, brown) additionally reflects the massive macrophage and microglia activation. Few oligodendrocytes (P; TPPP/p25 = red in P–R) and many astrocytes (S; glial fibrillary acidic protein [GFAP] = red in S to U) express hephaestin (blue in P–U) in this early active lesion. In late active lesions, there is a shift of iron-containing macrophages toward the perivascular space (H). Extracellular total (H, brown) and ferrous nonheme iron (K, brown) are sparse in comparison to early active lesions. Ferritin (N; brown) is predominantly expressed in macrophages. Oligodendrocytes are largely lost (Q), and there is sparse hephaestin expression in astrocytes (T). In inactive lesions, total nonheme iron is seen in few scattered astrocytes (I), whereas ferrous nonheme iron (L) is not detectable. Ferritin expression (O; brown) is only seen in some cells, predominantly astrocytes. Oligodendrocytes are lost from the lesions (R), and there is profound fibrillary gliosis (U). Hephaestin expression is minor or absent (R, U). (V) Confocal laser microscopy shows the expression of hephaestin (green) in oligodendrocytes (carbonic anhydrase II = red) and astrocytes (GFAP = blue). In this area of MS periplaque white matter, hephaestin is detected predominantly in oligodendrocytes. In active lesions (W–Y), ceruloplasmin (green in W–Y) is detected mainly in astrocytes (W; GFAP = red in W) and axons (X). However, in many cells and axons, ceruloplasmin is colocalized with fibrin (red in X and Y), suggesting nonspecific uptake of soluble serum ceruloplasmin from the extracellular space under conditions of severe blood–brain barrier damage. Nonetheless, some cells with astrocyte morphology are stained for ceruloplasmin in the absence of fibrin reactivity, suggesting autochthonous expression of ceruloplasmin in at least some astrocytes. AG = astroglia; OG = oligodendroglia; scale bars = 4mm (A–F), 100μm (G–U); inset scale bars = 10μm.

Figure 4

Dystrophic microglia, axonal iron, and oxidized phospholipids in multiple sclerosis (MS) lesions. Iron-loaded microglia and macrophages in active MS lesions show signs of degeneration (dystrophy) with process beading, retraction, and fragmentation (A), which is also visible in microglia stained for ferritin light polypeptide (FTL; B, brown). At active lesion edges, total (C) and rarely also ferrous nonheme iron (D, brown) accumulates in axons. (E–H) Oxidized phospholipids (E06 reactivity; E and G; brown) are detected in lesions with high iron content (total nonheme iron staining in F and H). Scale bars = 20μm (A, B); 100μm (C, D); 200μm (E, F); 75μm (G, H).