Stéphane Roudeau

Manganese Accumulates within Golgi Apparatus in Dopaminergic Cells as Revealed by Synchrotron X-ray Fluorescence Nanoimaging

Chronic exposure to manganese results in neurological symptoms referred to as manganism and is identified as a risk factor for Parkinsons disease. In vitro, manganese induces cell death in the dopaminergic cells, but the mechanisms of manganese cytotoxicity are still unexplained. In particular, the subcellular distribution of manganese and its interaction with other trace elements needed to be assessed. Applying synchrotron X-ray fluorescence nanoimaging, we found that manganese was located within the Golgi apparatus of PC12 dopaminergic cells at physiologic concentrations. At increasing concentrations, manganese accumulates within the Golgi apparatus until cytotoxic concentrations are reached resulting in a higher cytoplasmic content probably after the Golgi apparatus storage capacity is exceeded. Cell exposure to manganese and brefeldin A, a molecule known to specifically cause the collapse of the Golgi apparatus, results in the striking intracellular redistribution of manganese, which accumulates in the cytoplasm and the nucleus. These results indicate that the Golgi apparatus plays an important role in the cellular detoxification of manganese. In addition manganese exposure induces a decrease in total iron content, which could contribute to the overall neurotoxicity.

Evaluation of sample preparation methods for single cell quantitative elemental imaging using proton or synchrotron radiation focused beams

Particle induced X-ray emission with a focused beam (μPIXE) and synchrotron-based X-ray micro-fluorescence (μSXRF) are used to determine the distribution and contents of trace elements in single cells. A proper sample preparation method is required to ensure that the elemental distribution is preserved spatially and quantitatively. The aim of this study was to establish an optimal sample preparation method for single whole cell microanalysis, compatible with both μPIXE and μSXRF techniques. To find the most efficient method, we used PC12 cells as the cellular model and compared four widely applied protocols using a combination of rinsing solutions (phosphate buffered saline or ammonium acetate) and fixation methods (cryofixation or chemical fixation with 3% paraformaldehyde or methanol). The results showed a loss of diffusible elements K and Mg and an increase in Na, S, Cl and Zn concentrations in chemically fixed cells compared to cryofixed cells. In addition, K/Na and Cl/K cellular ratios indicated a good preservation of the chemical and structural integrity of cryofixed cells but not those of chemically fixed ones. The disturbance of elemental distributions after chemical fixation was also observed on rat brain tissue sections. Our results suggest that the optimal sample preparation method to study elemental distribution in single whole cells prepared for X-ray microanalysis is achieved when cells are rinsed with ammonium acetate, quickly frozen by plunging into liquid nitrogen–chilled cryogenic fluid and freeze-dried at low temperatures. This protocol was also successfully validated on rat primary hippocampal neurons, a delicate in vitro neuronal model.

α-Synuclein Over-Expression Induces Increased Iron Accumulation and Redistribution in Iron-Exposed Neurons

Parkinson’s disease is the most common α-synucleinopathy, and increased levels of iron are found in the substantia nigra of Parkinson’s disease patients, but the potential interlink between both molecular changes has not been fully understood. Metal to protein binding assays have shown that α-synuclein can bind iron in vitro; therefore, we hypothesized that iron content and iron distribution could be modified in cellulo, in cells over-expressing α-synuclein. Owing to particle-induced X-ray emission and synchrotron X-ray fluorescence chemical nano-imaging, we were able to quantify and describe the iron distribution at the subcellular level. We show that, in neurons exposed to excess iron, the mere over-expression of human α-synuclein results in increased levels of intracellular iron and in iron redistribution from the cytoplasm to the perinuclear region within α-synuclein-rich inclusions. Reproducible results were obtained in two distinct recombinant expression systems, in primary rat midbrain neurons and in a rat neuroblastic cell line (PC12), both infected with viral vectors expressing human α-synuclein. Our results link two characteristic molecular features found in Parkinson’s disease, the accumulation of α-synuclein and the increased levels of iron in the substantia nigra.

Correlative organelle fluorescence microscopy and synchrotron X-ray chemical element imaging in single cells

AbstractX-ray chemical element imaging has the potential to enable fundamental breakthroughs in the understanding of biological systems because chemical element interactions with organelles can be studied at the sub-cellular level. What is the distribution of trace metals in cells? Do some elements accumulate within sub-cellular organelles? What are the chemical species of the elements in these organelles? These are some of the fundamental questions that can be addressed by use of X-ray chemical element imaging with synchrotron radiation beams. For precise location of the distribution of the elements, identification of cellular organelles is required; this can be achieved, after appropriate labelling, by use of fluorescence microscopy. As will be discussed, this approach imposes some limitations on sample preparation. For example, standard immunolabelling procedures strongly modify the distribution of the elements in cells as a result of the chemical fixation and permeabilization steps. Organelle location can, however, be performed, by use of a variety of specific fluorescent dyes or fluorescent proteins, on living cells before cryogenic fixation, enabling preservation of element distribution. This article reviews the methods used for fluorescent organelle labelling and X-ray chemical element imaging and speciation of single cells. Selected cases from our work and from other research groups are presented to illustrate the potential of the combination of the two techniques. FigureSynchrotron X-ray fluorescence distribution maps of Ca, P and S in yeast cells. Elemental distribution maps (green color scale) were combined with the image of vacuoles labeled with Arg-CMAC (red color scale). The yellow signal of superposed images shows that Ca and P are preferentially located within the vacuole.

Multimodal analysis of metals in copper-zinc superoxide dismutase isoforms separated on electrophoresis gels.

It has been suggested that copper-zinc superoxide dismutase (CuZnSOD) isoforms of distinct isoelectric point (pI) could result from differences in their metallation state. Our aim was then to develop and validate analytical methods for the determination and understanding of metallation states in human CuZnSOD isoforms. To avoid metal losses during sample preparation steps, CuZnSOD isoforms were separated according to their pI using non-denaturing isoelectric focusing (IEF) gel electrophoresis. Metal quantification was directly performed in-gel. Cu/Zn ratios of CuZnSOD isoforms were quantified by Particle-Induced X-ray Emission (PIXE) and Laser Ablation-Inductively Coupled Plasma-Mass Spectrometry (LA-ICP-MS). Cu/Zn ratios were measured close to the value of 1 as expected from the known stoichiometry of CuZnSOD with slight, but statistically significant, differences between acidic and basic isoforms. Overall, this study demonstrates that metal quantification can be performed directly on metalloproteins separated on electrophoresis gels.

Amyotrophic lateral sclerosis-like superoxide dismutase 1 proteinopathy is associated with neuronal loss in Parkinson’s disease brain

Neuronal loss in numerous neurodegenerative disorders has been linked to protein aggregation and oxidative stress. Emerging data regarding overlapping proteinopathy in traditionally distinct neurodegenerative diseases suggest that disease-modifying treatments targeting these pathological features may exhibit efficacy across multiple disorders. Here, we describe proteinopathy distinct from classic synucleinopathy, predominantly comprised of the anti-oxidant enzyme superoxide dismutase-1 (SOD1), in the Parkinson’s disease brain. Significant expression of this pathology closely reflected the regional pattern of neuronal loss. The protein composition and non-amyloid macrostructure of these novel aggregates closely resembles that of neurotoxic SOD1 deposits in SOD1-associated familial amyotrophic lateral sclerosis (fALS). Consistent with the hypothesis that deposition of protein aggregates in neurodegenerative disorders reflects upstream dysfunction, we demonstrated that SOD1 in the Parkinson’s disease brain exhibits evidence of misfolding and metal deficiency, similar to that seen in mutant SOD1 in fALS. Our data suggest common mechanisms of toxic SOD1 aggregation in both disorders and a potential role for SOD1 dysfunction in neuronal loss in the Parkinson’s disease brain. This shared restricted proteinopathy highlights the potential translation of therapeutic approaches targeting SOD1 toxicity, already in clinical trials for ALS, into disease-modifying treatments for Parkinson’s disease.

Zinc and Copper Effects on Stability of Tubulin and Actin Networks in Dendrites and Spines of Hippocampal Neurons

Zinc and copper ions can modulate the activity of glutamate receptors. However, labile zinc and copper ions likely represent only the tip of the iceberg and other neuronal functions are suspected for these metals in their bound state. We performed synchrotron X-ray fluorescence imaging with 30 nm resolution to image total biometals in dendrites and spines from hippocampal neurons. We found that zinc is distributed all along the dendrites while copper is mainly pinpointed within the spines. In spines, zinc content is higher within the spine head while copper is higher within the spine neck. Such specific distributions suggested metal interactions with cytoskeleton proteins. Zinc supplementation induced the increase of β-tubulin content in dendrites. Copper supplementation impaired the β-tubulin and F-actin networks. Copper chelation resulted in the decrease of F-actin content in dendrites, drastically reducing the number of F-actin protrusions. These results indicate that zinc is involved in microtubule stability whereas copper is essential for actin-dependent stability of dendritic spines, although copper excess can impair the dendritic cytoskeleton.

Multicellular Consortia Preserved in Biogenic Ductile-Plastic Nodules of Okondja Basin (Gabon) by 2.1 Ga

We highlight complex ductile nodules found in the FB2 formation of the Okondja francevillian Basin (Gabon) dated to 2.1 Ga, during the Great Oxygenation Event (GOE). More than 500 specimens were collected from unmetamorphosed pelites in an excellent state of preservation. These nodules are divided into two groups: globular and elongated forms, one to three cm in diameter and over six cm long along the axis of elongation. They are characterized by two hemispheres separated by a central zone. They have a remarkable radial structure in spheroidal forms, and often have a polyphased structure in elongated forms. Chemical and microscopic analyses indicate that these nodules are formed over 80% of a fabric of micro-quartz, fossilizing calcite grains (rounded grains and remnants of biofilms) and clay channels. These slightly pyritized nodules also contain iron in the form of hematite and goethite. There are several types of micro-organisms, several biomorphic iron particles (generally less than 300 μm in size) among which are preserved some multicellular clusters measuring between 50 and 250 microns, and some larger biomorphs. Their organization is very complex with fibro-radial and polyphased internal fabric and a discrete external peripheral system. The very low values of δ13C carb in calcite (-17 and -26‰) suggest precipitation of calcite from decomposition of organic matter in anoxic photosynthesis conditions. The morphological, petrographic, geochemical and isotopic characteristics of these nodules indicate a biological origin such as microbial/algal consortia, associated with eukaryotic organisms. They lived on the seafloor, buried just below the surface of the sediment, in calm, shallow and oxido-reducing environment.

Reduced net charge and heterogeneity of pI isoforms in familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase.

Familial cases of amyotrophic lateral sclerosis (fALS) are related to mutations of copper/zinc superoxide dismutase 1 (SOD1). Aggregation of SOD1 plays a central role in the pathogenesis of fALS and altered metallation of SOD1 mutants could be involved in this process. Using IEF gel electrophoresis under non‐denaturating conditions and particle induced X‐ray emission (PIXE) analysis, we studied the pI distribution and metallation status of fALS SOD1 mutants (A4V, G93A, D125H) compared to human wild‐type (hWT). SOD1 fALS mutants are characterized by a variable number of isoforms and higher pI compared to hWT, reflecting a reduced net charge that might explain their greater propensity to precipitation and aggregation. Cu/Zn ratios were slightly different for the predominant expressed isoforms of A4V, G93A, and D125H mutants compared to hWT. Differences in metallation were observed within each genotype, the more basic isoforms exhibiting lower Cu/Zn ratios. Moreover, we revealed the existence of a pool of fALS mutants SOD1 pI isoforms, slightly expressed (<10%), with a low Cu/Zn ratio and high pI values. Overall, IEF‐PIXE results suggest that the toxicity of SOD1 mutants should be studied at the pI isoform level with a particular attention to the species with the lowest charges.

Fluorine microimaging and quantification using nuclear reaction analysis: A tool for validating tissue distribution of positron emission tomography tracers

An application of ion beam microprobe analysis has been developed for the quantitative imaging of fluorine compounds in biological samples on a micrometer scale. The difficulty of imaging fluorine at trace level concentration in biological tissues has been addressed by using a combination of nuclear reaction analysis and proton resonant backscattering spectrometry. With this method, the limit of detection of fluorine in thin biological samples can be as low as 3 μg/g. Quantitative imaging of fluorodeoxyglucose distribution, the most widely used tracer for detection of solid tumors by positron emission tomography, is shown as an example of this application.