Iron-binding characteristics of neuromelanin of the human substantia nigra

Abstract

The vulnerability of the dopaminergic neurons of the substantia nigra (SN) in Parkinson’s disease has been related to the presence of the pigment neuromelanin (NM) in these neurons. It is hypothesised that NM may act as an endogenous storage molecule for iron, an interaction suggested to influence free radical production. The current study quantified and characterised the interaction between NM and iron. Iron-binding studies demonstrated that both NM and synthetically-produced dopamine melanin contain equivalent numbers of high and low-affinity binding sites for iron but that the affinity of NM for iron is higher than that of synthetic melanin. Quantification of the total iron content in iron-loaded NM and synthetic melanin demonstrated that the iron-binding capacity of NM is 10-fold greater than that of the model melanin. This data was in agreement with the larger iron cluster size demonstrated by Mössbauer spectroscopy in the native pigment compared with the synthetic melanin. These findings are consistent with the hypothesis that NM may act as an endogenous iron-binding molecule in dopaminergic neurons of the SN in the human brain. The interaction between NM and iron has implications for disorders such as Parkinson’s disease where an increase in iron in the SN is associated with increased indices of oxidative stress.

Introduction

In recent years the oxidative stress hypothesis of neurodegenerative diseases has been intensively examined. Oxidative stress is suggested to be important in PD, either as a primary causal factor or alternatively as a secondary contributory factor. Pivotal to this hypothesis is the finding that iron homeostasis is changed in PD.

Neurochemical, physical, histochemical and imaging techniques have demonstrated increased iron levels in PD patients and in the parkinsonian SN post-mortem [4], [6], [11], [15], [32], [36]. The reason for this localised increase in iron is unknown but it is hypothesised that iron may increase oxidative load because of its ability to stimulate free radical production [5], [15], [23], [37]. SN neurons in the human are characterised by the presence of a dark polymer pigment NM, the presence of which has been directly related to their fate in PD [19], [22], [24]. Melanins in other body tissues actively protect against oxidative damage [25], [29], [33], [34], possibly as a consequence of their ability to bind reactive molecules, such as transition metals. The formation of an apparently similar pigment in the SN, a tissue which suffers a high intrinsic oxidative load [17], suggests a parallel function in the brain. In support of this hypothesis NM and DAM attenuate oxidative membrane damage in vitro[5], [12]. In the presence of high concentrations of iron, however, DAM acts as an effective pro-oxidant, rather than as an antioxidant [5], [31], [38]. This effect is attributed to the binding of iron to melanin and the subsequent reduction of bound ferric iron to a more weakly bound ferrous state, stimulating hydroxyl radical production [31]. Further, incubation of human NM or DAM with iron in vitro stimulates, rather than decreases, oxidative tissue damage [5], [13], [28], [30]. NM has been suggested to act as an iron storage molecule in vivo but this function might also increase the potential for oxidative damage within the vulnerable dopaminergic neurons if tissue iron levels are increased, for example in PD [5], [20], [23], [27]. We have previously shown that DAM binds iron in a quantifiable manner in vitro[5] but we have also shown that DAM differs structurally to NM [14]; thus it is unclear if this synthetic molecule is an adequate model of the native pigment. NM-bound iron has been quantified in the post-mortem brain [20], [23] but the iron-binding characteristics of NM cannot be determined in post-mortem tissue. The aim of the current study was to characterise for the first time the binding of iron to human NM.