Abstract
In the pigmented dopaminergic neurons of the human substantia nigra pars compacta the system relevant in iron storage is the polymer neuromelanin (NM). Although in most cells this function is mainly accomplished by ferritin, this protein complex appears not to be expressed in NM-containing neurons. Nevertheless the conceivable presence of iron-storing proteins as part of the NM granules has recently been discussed on the basis of Mössbauer spectroscopy and synchrotron x-ray microspectroscopy. Intriguingly by combining subcellular fractionation of NM granules, peptide sequencing via tandem mass spectrometry, and the additional confirmation by multiple reaction monitoring and immunogold labeling for electron microscopy, L-ferritin could now be unambiguously identified and localized in NM granules for the first time. This finding not only supports direct evidence for a regulatory role of L-ferritin in neuroectodermal cell pigmentation but also integrates a new player within a complicated network governing iron homeostasis in the dopamine neurons of the human substantia nigra. Thus our finding entails far reaching implications especially when considering etiopathogenetic aspects of Parkinson disease.
Highlights
In the pigmented dopaminergic neurons of the human substantia nigra pars compacta the system relevant in iron storage is the polymer neuromelanin (NM)
We report for the first time the identification of L-ferritin as a component of NM granules, pointing to a ferritin-based iron storage mechanism in the NM-containing neurons of the substantia nigra pars compacta (SN), by using an approach combining onedimensional (1-D) SDS-PAGE, reversed-phase nano-HPLC electrospray ionization tandem mass spectrometry (nano-LCESI-MS/MS and nano-LC-ESI-multiple reaction monitoring (MRM)-MS/MS), Western blot analysis, and immunotransmission electron microscopy
NM granules were isolated from the human SN by a topdown density gradient approach that yields specimens virtually free of contaminants, e.g. from glia and organelles [24]
Summary
In the pigmented dopaminergic neurons of the human substantia nigra pars compacta the system relevant in iron storage is the polymer neuromelanin (NM). By combining subcellular fractionation of NM granules, peptide sequencing via tandem mass spectrometry, and the additional confirmation by multiple reaction monitoring and immunogold labeling for electron microscopy, L-ferritin could be unambiguously identified and localized in NM granules for the first time This finding supports direct evidence for a regulatory role of L-ferritin in neuroectodermal cell pigmentation and integrates a new player within a complicated network governing iron homeostasis in the dopamine neurons of the human substantia nigra. NM appears in greatest quantities in the human brain and in lesser amounts in some other non-human primates but is absent from the brain of many lower species Interest in this pigment has seen a resurgence in recent years because of a hypothesized link between NM and the especial vulnerability of NM-containing neurons of the substantia nigra pars compacta (SN) for cell death in Parkinson disease (PD) [2, 3]. The main iron storage protein, is primarily located in glia rather than in neurons [23], it seems unlikely that it could regulate neuronal iron levels, and until today the exact iron storing mechanism in the NM-containing neurons of the SN was unknown
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