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Imaging and speciation of iron and manganese in dopamine neurons

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Disturbance of Fe homeostasis may play a crucial role in the pathogenesis of Parkinson’s disease by induction of oxidative stress and/or promotion of alpha-synuclein aggregation. Thus, Parkinson’s disease represents the most common α-synucleinopathy and iron levels are greater in the substantia nigra of patients with Parkinson’s disease, but the potential interconnection between these two molecular changes is still poorly understood. Since α-synuclein can bind iron in vitro we have tested the hypothesis that iron content and distribution may be altered in cells over-expressing α-synuclein.

Using nano-SXRF and micro-PIXE chemical imaging methods, we quantified and described the distribution of iron at the subcellular level. We show that, in neurons exposed to an excess of iron, the overexpression of human α-synuclein leads to the increase of the level of intracellular iron and its redistribution in the cytoplasm, to the perinuclear region in the inclusions of α-synuclein (Figure 1).

Reproducible results were obtained on two different recombinant expression systems, on the rat midbrain primary neurons and on a rat neuroblastic cell line (PC12), infected with viral vectors expressing human α-synuclein. Our results link two specific molecular features of Parkinson’s disease, α-synuclein accumulation, and increased iron levels in substantia nigra (Ortega et al., 2016).

Figure 1. Nano-SXRF imaging of dopaminergic neurons exposed in vitro to excess iron without (higher panel) or with (lower panel) overexpression of alpha synuclein. In neurons over-expressing alpha-synuclein, iron accumulates in perinuclear inclusions rich in alpha-synuclein.

On the other hand, environmental exposure to neurotoxic metals such as manganese could be one of the risk factors causing Parkinson’s disease. Organometallic manganese compounds are used as pesticides (Maneb), or as additives in unleaded gasoline (MMT).

Using micro-SXRF methods (Synchrotron X -Ray Fluorescence) and micro-XAS (X -ray Absorption Spectroscopy) at the ESRF (European Synchrotron Radiation Facility), we highlighted the accumulation of manganese in the Golgi apparatus of dopaminergic cells (Figure 2).

This result, completely new, tells us about the detoxification mechanisms of this element and the possible link to Parkinson’s disease. Disruption of vesicular trafficking by alteration and fragmentation of the Golgi apparatus could explain the neurotoxic effects of Mn, especially on the critical dopaminergic system affected by neurodegeneration.

In addition we have shown that whatever the nature of environmental Mn compounds (inorganic, organometallic), the mechanism of toxicity is the same and involves an interaction of Mn2+ ions with the Golgi apparatus. Toxicity is proportional to the solubility of the compounds, as shown by speciation, joining the toxicity paradigm vs solubility described for cobalt oxide.

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Figure 2. Micro-SXRF imaging and micro-XAS spectroscopy of dopaminergic neurons exposed in vitro to Mn showing accumulation in the Golgi apparatus and speciation as Mn2+. Data obtained at the ESRF ID21 beamline. Scale bar : 10 µm.


α-Synuclein over-expression induces increased iron accumulation and redistribution in iron-exposed neurons
Ortega R., Carmona A., Roudeau S., Perrin L., Dučić T., Carboni E., Bohic S., Cloetens P., Lingor P. (2016), Molecular Neurobiology, 53, 1925-1934. [pubmed] [link]

Environmental manganese compounds accumulate as Mn(II) within the Golgi apparatus of dopamine cells : relationship between speciation, subcellular distribution, and cytotoxicity
Carmona A., Roudeau S., Perrin L., Veronesi G., Ortega R. (2014), Metallomics, 6, 822-832. [pubmed] [link]

Manganese accumulates within Golgi apparatus in dopaminergic cells as revealed by synchrotron X-Ray fluorescence nano-imaging
Carmona A., Devès G., Roudeau S., Cloetens P., Bohic S., Ortega R. (2010), ACS Chemical Neurosciences, 1, 194-203.[pubmed] [link]