X-ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons

Parkinson's disease (PD) is the most frequent neurodegenerative movement disorder with a prevalence of about 1% in a population older than 70 years and about 3% in individuals older than 80 years (Strickland and Bertoni 2004). Pathological hallmarks include, but are not confined to, the preferential loss of dopaminergic (DAergic) neurons within the substantia nigra pars compacta and the presence of intracytoplasmic inclusions containing α-synuclein and ubiquitin, so-called Lewy bodies (Braak et al. 2003). Next to the generation of free radicals in the course of dopamine metabolism, the increased presence of iron in the midbrain of PD patients has been assumed as a contributing pathogenic factor (Berg and Hochstrasser 2006). Iron participates in metabolic processes by undergoing oxidation–reduction reactions, a common property among transition metals, which allows this metal to undergo interconversion between the divalent cationic or ferrous (Fe2+), and trivalent cationic or ferric (Fe3+) states. These electron exchange processes can also lead to significant oxidative damage via free radical production within the brain when excess iron is present. In DAergic neurons during the dopamine catabolism, hydrogen peroxide is produced that in presence of iron favors Fenton reactions and oxidative stress [reviewed in (Papanikolaou and Pantopoulos 2005)]. Other transition metals such as Mn or Zn have been suggested to contribute to the pathogenesis of PD. For example, exposure of workers to high concentrations of manganese is an established risk factor for the development of a Parkinsonian syndrome, which clinically greatly mimics idiopathic Parkinson's disease [reviewed in (Guilarte 2010)]. Similar symptoms have been observed in psychostimulant drug abusers after repetitive intravenous injection of manganese-containing substances (Sikk 2011). Excessive levels of brain manganese have been linked to the loss of dopamine in the striatum, death of non-DAergic neurons in the globus pallidus, and damage of other neuronal pathways such as glutamatergic and GABAergic projections, all of which contribute to altered behavior, motor dysfunction, and cognition deficit (Erikson 2002). Manganese elevates intracellular H2O2 and related peroxides (HaMai et al. 2001) and reduces tyrosine hydroxylase activity and intracellular antioxidant levels (GSH, thiols, catalase) in DAergic neurons (Migheli 1999). Iron and manganese are known to pass across the blood–brain barrier [reviewed in (Yokel 2009; Moos 2007)]. Because of their chemical similarity, both metals share and compete for transport proteins in organisms ranging from bacteria to mammals. As such, during conditions of low iron, abnormal manganese accumulation occurs. Conversely, when manganese concentrations are altered, the homeostasis and deposition of iron and other transition metals are disrupted. However, the knowledge on the subcellular allocation of trace elements upon exposure to manganese or iron remains incomplete. Indirect methods, like radioactive 54Mn tracing after cellular extraction, are able to quantify manganese in cell extracts, but provide only limited spatial information (Kalia et al. 2008). The principle of X-ray fluorescence (XRF) is based on the irradiation of sample atoms with X-rays with energy sufficient to eject an electron from one of the atom's inner shells. The relaxation process is accompanied by the emission of a fluorescence photon. The energy of the emitted X-rays is characteristic of the excited element enabling the identification of the composition of the atoms constitutive of the sample. Beside element composition, X-ray spectromicroscopy performed using an X-ray nanoprobe beam is a unique method to determine the oxidation state of elements in cells and tissues (Qin 2011). X-rays can penetrate thick cells and tissues, eliminating the need of invasive preparation and sectioning of the specimen. Several studies have thus recently demonstrated the use of X-rays for trace element mapping of different cell types (Ortega 2007; Bacquart 2007; Ducic 2011). This study was designed to investigate the subcellular distribution of manganese and iron in primary midbrain neurons and to compare this distribution in the DAergic and non-DAergic subpopulations by X-ray fluorescence microscopy. Previous studies have been limited to cell lines, post-mortem tissue, or did not take into account changes in the oxidative state during sample processing (Bacquart 2007; Szczerbowska-Boruchowska 2007). In this study, we visualized the transition metal distribution upon exposure in primary midbrain neurons and used the cryo-preserving (so-called cryo-embedding) technique to preserve oxidative states of the metal ions, which, to the best of our knowledge, is the first description exploiting primary midbrain neurons culture model with X-ray imaging techniques.