Richard Ortega

Iron Storage within Dopamine Neurovesicles Revealed by Chemical Nano-Imaging

Altered homeostasis of metal ions is suspected to play a critical role in neurodegeneration. However, the lack of analytical technique with sufficient spatial resolution prevents the investigation of metals distribution in neurons. An original experimental setup was developed to perform chemical element imaging with a 90 nm spatial resolution using synchrotron-based X-ray fluorescence. This unique spatial resolution, combined to a high brightness, enables chemical element imaging in subcellular compartments. We investigated the distribution of iron in dopamine producing neurons because iron-dopamine compounds are suspected to be formed but have yet never been observed in cells. The study shows that iron accumulates into dopamine neurovesicles. In addition, the inhibition of dopamine synthesis results in a decreased vesicular storage of iron. These results indicate a new physiological role for dopamine in iron buffering within normal dopamine producing cells. This system could be at fault in Parkinsons disease which is characterized by an increased level of iron in the substancia nigra pars compacta and an impaired storage of dopamine due to the disruption of vesicular trafficking. The re-distribution of highly reactive dopamine-iron complexes outside neurovesicles would result in an enhanced death of dopaminergic neurons.

Quantitative micro-analysis of metal ions in subcellular compartments of cultured dopaminergic cells by combination of three ion beam techniques

Quantification of the trace element content of subcellular compartments is a challenging task because of the lack of analytical quantitative techniques with adequate spatial resolution and sensitivity. Ion beam micro-analysis, using MeV protons or alpha particles, offers a unique combination of analytical methods that can be used with micrometric resolution for the determination of chemical element distributions. This work illustrates how the association of three ion beam analytical methods, PIXE (particle induced X-ray emission), BS (backscattering spectrometry), and STIM (scanning transmission ion spectrometry), allows quantitative determination of the trace element content of single cells. PIXE is used for trace element detection while BS enables beam-current normalization, and STIM local mass determination. These methods were applied to freeze-dried cells, following a specific cryogenic protocol for sample preparation which preserves biological structures and chemical distributions in the cells. We investigated how iron accumulates into dopaminergic cells cultured in vitro. We found that the iron content increases in dopaminergic cells exposed to an excess iron, with marked accumulation within distal ends, suggesting interaction between iron and dopamine within neurotransmitter vesicles. Increased iron content of dopaminergic neurons is suspected to promote neurodegeneration in Parkinson’s disease.

Nano-imaging of trace metals by synchrotron X-ray fluorescence into dopaminergic single cells and neurite-like processes

The metallome has been defined as the distribution of metals and metalloids among the different species and cell compartments. The detection of trace elements at the subcellular level is a challenging task that requires sophisticated analytical developments. In this study, we report how chemical element imaging was performed in subcellular compartments of dopaminergic cells at high spatial resolution using the X-ray fluorescence nanoprobe recently developed at the European Synchrotron Radiation Facility. High spatial resolution is obtained using the concept of a secondary source focused to a 90 nm probe by multilayer mirrors bent in Kirkpatrick–Baez geometry. This original setup was applied for trace metal mapping of single dopaminergic cells, chosen as an in vitro model of degenerative cells involved in Parkinsons disease. This cellular model is able to differentiate upon exposure to nerve growth factor and to extend neurite-like processes. Two important results were obtained. First, iron is distributed in a granular form into dopamine vesicles, found mainly in primary neurite outgrowths and distal ends. Second, thin neurite-like processes produced by differentiated cells accumulate copper, zinc, and to a minor extent lead. Overall, the high resolution imaging of single neuronal cells offers unique information to understand the role of trace metals in neurochemistry.

Low-solubility particles and a Trojan-horse type mechanism of toxicity: the case of cobalt oxide on human lung cells

BackgroundThe mechanisms of toxicity of metal oxide particles towards lung cells are far from being understood. In particular, the relative contribution of intracellular particulate versus solubilized fractions is rarely considered as it is very challenging to assess, especially for low-solubility particles such as cobalt oxide (Co3O4).MethodsThis study was possible owing to two highly sensitive, independent, analytical techniques, based on single-cell analysis, using ion beam microanalysis, and on bulk analysis of cell lysates, using mass spectrometry.ResultsOur study shows that cobalt oxide particles, of very low solubility in the culture medium, are readily incorporated by BEAS-2B human lung cells through endocytosis via the clathrin-dependent pathway. They are partially solubilized at low pH within lysosomes, leading to cobalt ions release. Solubilized cobalt was detected within the cytoplasm and the nucleus. As expected from these low-solubility particles, the intracellular solubilized cobalt content is small compared with the intracellular particulate cobalt content, in the parts-per-thousand range or below. However, we were able to demonstrate that this minute fraction of intracellular solubilized cobalt is responsible for the overall toxicity.ConclusionsCobalt oxide particles are readily internalized by pulmonary cells via the endo-lysosomal pathway and can lead, through a Trojan-horse mechanism, to intracellular release of toxic metal ions over long periods of time, involving specific toxicity.

X-ray absorption spectroscopy of biological samples. A tutorial

X-ray absorption spectroscopy (XAS) is an element specific spectroscopy sensitive to the local chemical and structural order of the absorber element. XAS is nowadays increasingly used for the speciation analysis of chemical elements owing to the development of new synchrotron radiation facilities worldwide. XAS can be divided into X-ray absorption near edge structure (XANES), which provides information primarily about the geometry and oxidation state, and extended X-ray absorption fine structure (EXAFS), which provides information about metal site ligation. The main advantages of the XAS method are its subatomic (angstrom) resolution, the ability to analyze almost any type of samples including amorphous (non-crystalline) materials, the possibility to analyze such materials in situ requiring minor or no sample preparation. The main limitations of XAS are its sensitivity in the mM (or μg g−1) range, the difficulty to deconvolute the bulk data when the sample is composed of a mixture of structures of the absorber element, and the limited chemical selectivity of ligands to within one row of the periodic table. This tutorial will discuss the strengths and limitations of XAS and compare them to those of alternative or complementary methods such as X-ray diffraction and X-ray photoelectron spectroscopy. The tutorial will also present and discuss the specific needs in terms of sample preparation and preservation all along the process of storage and analysis, and discuss the importance of the use of cryogenic methods when XAS is applied to biological samples. Applications in life sciences are reviewed, not exhaustively, with a special emphasis on some characteristic examples. The article ends with some perspectives on future trends of XAS: micro- and nano-XAS, time-resolved XAS, and high energy resolution XAS.

Direct speciation analysis of inorganic elements in single cells using X-ray absorption spectroscopy

Up to now, X-ray absorption spectroscopy (XAS), which includes XANES (X-ray absorption near edge structure) and EXAFS (Extended X-ray absorption fine structure), was mainly used with unfocused beams. The recent development of X-ray focusing optics, with spatial resolution down to the micrometre level while maintaining high photon fluxes, has opened the field of XAS application to single cell analysis. Micro-XAS enables the characterization of the local structure of the elements within subcellular structures in terms of oxidation state, site ligation, and coordination. It has been mainly applied to the determination of trace metal oxidation states using micro-XANES, and in some favorable cases to identify the molecules binding to the metals in cells. Due to the minute quantity of analytes in subcellular compartments, micro-EXAFS is still more challenging to perform than micro-XANES as it requires higher signal to noise ratios. Micro-XAS has proven to be useful in various fields of research such as metal-based neuro-degeneration, cellular pharmacology, trace element physiology and metal toxicology. Micro-XAS presents unique capabilities over other speciation methods because it can be performed in situ, directly in subcellular compartments, without cell fractionation which is prone to modify the chemical element species. However, the stability of chemical element species all along the analytical procedure, from sample preparation to storage and analysis should be tested systematically.

Cobalt chloride speciation, mechanisms of cytotoxicity on human pulmonary cells, and synergistic toxicity with zinc

Cobalt is used in numerous industrial sectors, leading to occupational diseases, particularly by inhalation. Cobalt-associated mechanisms of toxicity are far from being understood and information that could improve knowledge in this area is required. We investigated the impact of a soluble cobalt compound, CoCl(2)·6H(2)O, on the BEAS-2B lung epithelial cell line, as well as its impact on metal homeostasis. Cobalt speciation in different culture media, in particular soluble and precipitated cobalt species, was investigated via theoretical and analytical approaches. The cytotoxic effects of cobalt on the cells were assessed. Upon exposure of BEAS-2B cells to cobalt, intracellular accumulation of cobalt and zinc was demonstrated using direct in situ microchemical analysis based on ion micro-beam techniques and analysis after cell lysis by inductively coupled plasma mass spectrometry (ICP-MS). Microchemical imaging revealed that cobalt was rather homogeneously distributed in the nucleus and in the cytoplasm whereas zinc was more abundant in the nucleus. The modulation of zinc homeostasis led to the evaluation of the effect of combined cobalt and zinc exposure. In this case, a clear synergistic increase in toxicity was observed as well as a substantial increase in zinc content within cells. Western blots performed under the same coexposure conditions revealed a decrease in ZnT1 expression, suggesting that cobalt could inhibit zinc release through the modulation of ZnT1. Overall, this study highlights the potential hazard to lung function, of combined exposure to cobalt and zinc.

Direct speciation of metals in copper-zinc superoxide dismutase isoforms on electrophoresis gels using X-ray absorption near edge structure

X-Ray absorption near edge structure (XANES) can be used for the speciation analysis of inorganic elements bound to proteins, after separation using polyacrylamide gel electrophoresis. In this paper, we report new methodological developments for the speciation of oxidation states in copper–zinc superoxide dismutase (CuZnSOD) isoforms separated in non-denaturing conditions according to their isoelectric point. The nature of CuZnSOD isoforms is still elusive, although differences in their metallation state have been suggested. A non-denaturing protocol for isoelectrofocusing electrophoresis was applied to preserve the metallation state of the proteins. XANES has been performed to study the copper and zinc oxidation states. As expected, zinc is present in its Zn(II) oxidation state in all analysed isoforms. Copper is present in the Cu(II) oxidation state in the main acidic isoform, while it is found in both Cu(II) and Cu(I) states in the main basic isoform. These methodological developments enable the direct speciation analysis of metals in CuZnSOD without any protein purification step that could modify their oxidation state.

Coupling of native IEF and extended X‐ray absorption fine structure to characterize zinc‐binding sites from pI isoforms of SOD1 and A4V pathogenic mutant

Extended X‐ray absorption fine structure (EXAFS) has already provided high‐resolution structures of metal‐binding sites in a wide variety of metalloproteins. Usually, EXAFS is performed on purified metalloproteins either in solution or crystallized form but purification steps are prone to modify the metallation state of the protein. We developed a protocol to couple EXAFS analysis to metalloprotein separation using native gel electrophoresis. This coupling opens a large field of applications as metalloproteins can be characterized in their native state avoiding purification steps. Using native isoelectric focusing, the method enables the EXAFS analysis of metalloprotein pI isoforms. We applied this methodology to SOD1, wild‐type, and Ala4Val mutant (A4V), a mutation found in amyotrophic lateral sclerosis (ALS) because decreased Zn affinity to SOD1 mutants is suggested to be involved in the pathogenesis of this neurodegenerative disease. We observed similar coordination structures for Zn in wild‐type and mutant proteins, in all measured pI isoforms, demonstrating the feasibility of EXAFS on electrophoresis gels and suggesting that the Zn‐binding site is not structurally modified in A4V SOD1 mutant.

Overview of chemical imaging methods to address biological questions

Chemical imaging offers extensive possibilities for better understanding of biological systems by allowing the identification of chemical components at the tissue, cellular, and subcellular levels. In this review, we introduce modern methods for chemical imaging that can be applied to biological samples. This work is mainly addressed to the biological sciences community and includes the bases of different technologies, some examples of its application, as well as an introduction to approaches on combining multimodal data.