The synaptic vesicle protein Mover/TPRG1L is associated with lipid droplets in astrocytes.

Crucial brain functions such as neurotransmission, myelination, and signaling pose a high demand for lipids. Lipid dysregulation is associated with neuroinflammation and neurodegeneration. Astrocytes protect neurons from lipid induced damage by accumulating and metabolizing toxic lipids in organelles called lipid droplets (LDs). LDs have long been considered as lipid storage compartments in adipocytes, but less is known about their biogenesis and composition in the brain. In particular, proteins covering the LD surface are not yet fully identified. Here, we report that the presynaptic protein Mover/TPRG1L, which regulates the probability of neurotransmitter release in neurons, is a component of the LD coat in astrocytes. Using conventional and super-resolution microscopy, we demonstrate that Mover surrounds naive and oleic acid induced astrocytic LDs. We confirm the identity of astrocytic LDs using the neutral lipid stains Bodipy and LipidTox, as well as immunofluorescence for perilipin-2, a known component of the LD coat. In astrocytes, recombinant Mover was sufficient to induce an accumulation of LDs. Furthermore, we identified point mutations that abolish targeting to LDs and show similarities in the required binding sequences for association to the presynapse and LDs. Our results show that Mover is not only a presynaptic protein but also a candidate for LD regulation. This highlights the dual role of Mover in synaptic transmission and regulation of astrocytic LDs, which may be particularly important in the context of lipid-related neurological disorders.

Imaging the structural organization of chemical elements in growth cones of developing hippocampal neurons.

During neurodevelopment, neurons form growth cones, F-actin rich extensions located at the distal end of the neurites. Growth cones allow dendrites and axons to build synaptic connections through a process of neurite guidance whose mechanisms have not been fully elucidated. Calcium is an important element in this process by inducing F-actin reorganization. We hypothesized that other biologically active elements might be involved in the growth cone-mediated neurite guidance mechanisms. We performed super resolution and confocal microscopy of F-actin, followed by synchrotron X-ray fluorescence microscopy of phosphorous, sulfur, chlorine, potassium, calcium, iron and zinc on growth cones from primary rat hippocampal neurons. We identified two main patterns of element organization. First, active growth cones presenting an asymmetric distribution of Ca co-localized with the cytoskeleton protein F-actin. In active growth cones, we found that the distributions of P, S, Cl, K, and Zn are correlated with Ca. This correlation is lost in the second pattern, quiescent growth cones, exhibiting a spread elemental distribution. These results suggest that Ca is not the only element required in the F-actin rich active regions of growth cones. In addition, highly concentrated Fe spots of submicrometer size were observed in calcium-rich areas of active growth cones. These results reveal the need for biological active elements in growth cones during neural development and may help explain why early life deficiencies of elements, such as Fe or Zn, induce learning and memory deficits in children.

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

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 non-cytotoxic 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 labeled 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 for the resulting neurotoxic effects.

Correlating STED and synchrotron XRF nano-imaging unveils cosegregation of metals and cytoskeleton proteins in dendrites.

Zinc and copper are involved in neuronal differentiation and synaptic plasticity but the molecular mechanisms behind these processes are still elusive due in part to the difficulty of imaging trace metals together with proteins at the synaptic level. We correlate stimulated-emission-depletion microscopy of proteins and synchrotron X-ray fluorescence imaging of trace metals, both performed with 40 nm spatial resolution, on primary rat hippocampal neurons. We reveal the co-localization at the nanoscale of zinc and tubulin in dendrites with a molecular ratio of about one zinc atom per tubulin-αß dimer. We observe the co-segregation of copper and F-actin within the nano-architecture of dendritic protrusions. In addition, zinc chelation causes a decrease in the expression of cytoskeleton proteins in dendrites and spines. Overall, these results indicate new functions for zinc and copper in the modulation of the cytoskeleton morphology in dendrites, a mechanism associated to neuronal plasticity and memory formation.

Manganese levels in infant formula and young child nutritional beverages in the United States and France: Comparison to breast milk and regulations.

Exposure to high levels of manganese (Mn) in children has recently been associated with adverse neurodevelopmental effects. Current infant formula regulations for Mn content were set between 1981 (United States), 2006 (European Union, France), and 2007 (Codex Alimentarius) prior to the publication of much of the growing body of research on the developmental neurotoxicity of Mn. In this study, we sought to measure the concentrations of Mn in some infant formulas and young child nutritional beverages available on the United States (US) and French markets using ion beam analysis by particle induced X-ray emission (PIXE) spectrometry and then compare the analytical results to concentrations reported in the literature for breast milk and applicable infant formula regulations and guidelines. We were particularly interested in measuring Mn concentrations in product types for which there is very little data from previous surveys, especially soy-based, rice-based, goat-milk based, chocolate-flavored, and nutritional beverages for young children that are not regulated as infant or follow-on formulas (e.g. "toddler formulas" and "toddler powders"). We purchased 44 infant formulas and young child nutritional beverage products in the US and France with varying protein sources (cow-milk, goat-milk, soy, rice) labelled for birth to 3 years. We selected these samples using maximum variation sampling to explore market extremes to facilitate comparisons to regulatory limits. Since this sampling method is non-probabilistic, other inferences cannot be made beyond this set of samples to the overall markets. We used ion beam analysis to measure the concentrations of Mn in each product. The range of measured Mn concentrations in the products is 160-2,800 µg/L, substantially higher than the 3-6 µg/L mean Mn concentration reported in human breast milk. All products satisfied national and Codex Alimentarius Commission (CAC) international standards for minimum Mn content in infant formulas; however, 7/25 of the products purchased in the US exceeded the CAC Guidance Upper Level of 100 µg Mn/kcal for infant formula.

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 ß-tubulin content in dendrites. Copper supplementation impaired the ß-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.