Cytoplasmic aggregation of uranium in human dopaminergic cells after continuous exposure to soluble uranyl at non-cytotoxic concentrations

Abstract

Uranium exposure can lead to neurobehavioral alterations in particular of the monoaminergic system, even at non-cytotoxic concentrations. However, the mechanisms of uranium neurotoxicity after noncytotoxic exposure are still poorly understood. In particular, imaging uranium in neurons at low intracellular concentration is still very challenging. We investigated uranium intracellular localization by means of synchrotron X-ray fluorescence imaging with high spatial resolution (< 300 nm) and high analytical sensitivity (< 1 µg.g-1 per 300 nm pixel). Neuron-like SH-SY5Y human cells differentiated into a dopaminergic phenotype were continuously exposed, for seven days, to a non-cytotoxic concentration (10 μM) of soluble natural uranyl. Cytoplasmic submicron uranium aggregates were observed accounting on average for 62% of the intracellular uranium content. In some aggregates, uranium and iron were co-localized suggesting common metabolic pathways between uranium and iron storage. Uranium aggregates contained no calcium or phosphorous indicating that detoxification mechanisms in neuron-like cells are different from those described in bone or kidney cells. Uranium intracellular distribution was compared to fluorescently labeled organelles (lysosomes, early and late endosomes) and to fetuin-A, a high affinity uranium-binding protein. A strict correlation could not be evidenced between uranium and the labelled organelles, or with vesicles containing fetuin-A. Our results indicate a new mechanism of uranium cytoplasmic aggregation after non-cytotoxic uranyl exposure that could be involved in neuronal defense through uranium sequestration into less reactive species. The remaining soluble fraction of uranium would be responsible for protein binding and the resulting neurotoxic effects.

Native cryo-correlative light and synchrotron X-ray fluorescence imaging of proteins and essential metals in developing neurons

“…Many other commercially available fluorescent dyes can be used, for example for organelle labeling, which involves the expression of organelle-specific fluorescent proteins using a vector expressed in living cells (Bissardon et al, 2023). This approach can also be extended to any protein of interest by using transfection and expression of fluorescent protein constructs (Carmona et al, 2019;.…”

“…For example, livecell FLM of HeLa cells expressing a fluorescently tagged protein, huntingtin exon-1-mYFP, has been used to identify protein aggregates and compared with the location of the elemental content obtained by cryo-SXRF (Figure path 4) . Live-cell FLM was performed on HeLa cells expressing GFP-tagged SLC30A10 protein, a Mnefflux transporter, and an RFP protein targeting the Golgi apparatus followed by plunge freezing, freeze-drying and SXRF imaging at RT (Figure 5 path 5) (Carmona et al, 2019). The stable position of the Golgi-apparatus within the time frame of sample preparation allowed correlative visualization of Mn accumulation in this organelle.…”

“…SXRF spectrometry is a very powerful analytical tool offering high detection sensitivity, high spatial resolution on dedicated beamlines (<100 nm), and quantitative capability for metal mapping in biological samples (Pushie et al, 2022). Although SXRF cannot be performed on living cells due to cellular damage caused by intense irradiation, SXRF can be performed under cryogenic conditions, enabling imaging of the native intracellular distribution of elements (Kapishnikov et al, 2017;DeSamber et al, 2018;Carmona et al, 2019;Conesa et al;Rumancev et al, 2020). SXRF imaging can be combined with other microscopy or spectro-imaging techniques to complement the information acquired on metal distribution with biological indicators such as cell compartments and organelles (Roudeau et al, 2014;Kapishnikov et al, 2017;Yang et al, 2019;Conesa et al, 2020;Gramaccioni et al, 2020;Gustavsson et al, 2021;Tardillo, 2021;Lemelle, et al, 2022;Nagarajan et al, 2022).…”

“…To avoid this denaturing step of chemical fixation, protein labeling can be performed with fluorescent dyes developed for live-cell microcopy. We have designed correlative approaches for imaging proteins by fluorescence light microscopy (FLM) in living cells and subsequent imaging of chemical elements by SXRF (Carmona et al, 2019, Carmona et al, 2021. Live-cell FLM can be performed using epifluorescence, confocal or super-resolution microscopy, characterized by different spatial resolutions ranging from submicron to tenth-nanometer scale.…”

X-ray fluorescence imaging of uranium distribution in human dopaminergic cells

“…We applied a protocol for correlative light/SXRF microscopy 4 . Such method allowed to image intracellular uranium distribution and to check for the presence, or not, of uranium in specific cellular compartments by comparing and correlating the optical fluorescence images of marked organelles in live cells with SXRF elemental maps 5 .…”