X-ray fluorescence analysis of iron and manganese distribution in primary dopaminergic neurons.
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ABSTRACT:
SUBMITTER: Ducic T
PROVIDER: S-EPMC3563009 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:Increasing evidence suggests that metal dyshomeostasis plays an important role in human neurodegenerative diseases. Although distinctive metal distributions are described for mature hippocampus and cortex, much less is known about metal levels and intracellular distribution in individual hippocampal neuronal somata. To solve this problem, we conducted quantitative metal analyses utilizing synchrotron radiation X-Ray fluorescence on frozen hydrated primary cultured neurons derived from rat embryonic cortex (CTX) and two regions of the hippocampus: dentate gyrus (DG) and CA1. Comparing average metal contents showed that the most abundant metals were calcium, iron, and zinc, whereas metals such as copper and manganese were less than 10% of zinc. Average metal contents were generally similar when compared across neurons cultured from CTX, DG, and CA1, except for manganese that was larger in CA1. However, each metal showed a characteristic spatial distribution in individual neuronal somata. Zinc was uniformly distributed throughout the cytosol, with no evidence for the existence of previously identified zinc-enriched organelles, zincosomes. Calcium showed a peri-nuclear distribution consistent with accumulation in endoplasmic reticulum and/or mitochondria. Iron showed 2-3 distinct highly concentrated puncta only in peri-nuclear locations. Notwithstanding the small sample size, these analyses demonstrate that primary cultured neurons show characteristic metal signatures. The iron puncta probably represent iron-accumulating organelles, siderosomes. Thus, the metal distributions observed in mature brain structures are likely the result of both intrinsic neuronal factors that control cellular metal content and extrinsic factors related to the synaptic organization, function, and contacts formed and maintained in each region.
| S-EPMC4951041 | biostudies-literature
Project description:Chronic exposure to manganese results in neurological symptoms referred to as manganism and is identified as a risk factor for Parkinson's 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.
| S-EPMC3368660 | biostudies-literature
Project description:Manganese (Mn) is both an essential biological cofactor and neurotoxicant. Disruption of Mn biology in the basal ganglia has been implicated in the pathogenesis of neurodegenerative disorders, such as parkinsonism and Huntington's disease. Handling of other essential metals (e.g. iron and zinc) occurs via complex intracellular signaling networks that link metal detection and transport systems. However, beyond several non-selective transporters, little is known about the intracellular processes regulating neuronal Mn homeostasis. We hypothesized that small molecules that modulate intracellular Mn could provide insight into cell-level Mn regulatory mechanisms. We performed a high throughput screen of 40,167 small molecules for modifiers of cellular Mn content in a mouse striatal neuron cell line. Following stringent validation assays and chemical informatics, we obtained a chemical 'toolbox' of 41 small molecules with diverse structure-activity relationships that can alter intracellular Mn levels under biologically relevant Mn exposures. We utilized this toolbox to test for differential regulation of Mn handling in human floor-plate lineage dopaminergic neurons, a lineage especially vulnerable to environmental Mn exposure. We report differential Mn accumulation between developmental stages and stage-specific differences in the Mn-altering activity of individual small molecules. This work demonstrates cell-level regulation of Mn content across neuronal differentiation.
| S-EPMC4210885 | biostudies-literature
Project description:Manganese (Mn) intoxication results in neurological conditions similar, but not identical, to idiopathic Parkinson's disease. While the mechanism(s) by which Mn exposure leads to neurotoxic effects remains unclear, studies by magnetic resonance imaging demonstrate a high Mn accumulation in the hippocampal formation (HPCf) of the brain. Metal quantification using this method is not possible. Using X-ray fluorescence imaging, we measured the distribution of Mn in the HPCf for a rodent model of chronic Mn exposure and quantitatively compared it with distributions of other biologically relevant metals. We found considerable increases in average Mn concentrations in all analyzed areas and we identified the dentate gyrus (DG) and the cornus ammonis 3 (CA3) layer as areas accumulating the highest Mn content (?1.2 ?g Mn per g tissue). The DG is significantly enriched with iron (Fe), while the CA3 layer has high zinc (Zn) content. Additionally, significant spatial correlations were found for Mn-Zn concentrations across the HPCf substructures and for Mn-Fe concentrations in the DG. Combined results support that at least two mechanisms may be responsible for Mn transport and/or storage in the brain, associated with either Fe or Zn. Subcellular resolution images of metal distribution in cells of the CA3 show diffuse Mn distributions consistent with Mn localization in both the cytoplasm and nucleus. Mn was not increased in localized intracellular Fe or copper accumulations. A consistent Mn-Zn correlation both at the tissue (40 ?m × 40 ?m) and cellular (0.3 ?m × 0.3 ?m) levels suggests that a Zn transport/storage mechanism in the HPCf is likely associated with Mn accumulation.
| S-EPMC3892963 | biostudies-literature
Project description:The neurotoxic effect of manganese (Mn) establishes itself in a condition known as manganism or Mn induced parkinsonism. While this condition was first diagnosed about 170 years ago, the mechanism of the neurotoxic action of Mn remains unknown. Moreover, the possibility that Mn exposure combined with other genetic and environmental factors can contribute to the development of Parkinson's disease has been discussed in the literature and several epidemiological studies have demonstrated a correlation between Mn exposure and an elevated risk of Parkinson's disease. Here, we introduce X-ray fluorescence imaging as a new quantitative tool for analysis of the Mn distribution in the brain with high spatial resolution. The animal model employed mimics deficits observed in affected human subjects. The obtained maps of Mn distribution in the brain demonstrate the highest Mn content in the globus pallidus, the thalamus, and the substantia nigra pars compacta. To test the hypothesis that Mn transport into/distribution within brain cells mimics that of other biologically relevant metal ions, such as iron, copper, or zinc, their distributions were compared. It was demonstrated that the Mn distribution does not follow the distributions of any of these metals in the brain. The majority of Mn in the brain was shown to occur in the mobile state, confirming the relevance of the chelation therapy currently used to treat Mn intoxication. In cells with accumulated Mn, it can cause neurotoxic action by affecting the mitochondrial respiratory chain. This can result in increased susceptibility of the neurons of the globus pallidus, thalamus, and substantia nigra pars compacta to various environmental or genetic insults. The obtained data is the first demonstration of Mn accumulation in the substantia nigra pars compacta, and thus, can represent a link between Mn exposure and its potential effects for development of Parkinson's disease.
| S-EPMC3501493 | biostudies-literature
Project description:BackgroundMany metals are essential for plants and humans. Knowledge of metal distribution in plant tissues in vivo contributes to the understanding of physiological mechanisms of metal uptake, accumulation and sequestration. For those studies, X-rays are a non-destructive tool, especially suited to study metals in plants.ResultsWe present microfluorescence imaging of trace elements in living plants using a customized benchtop X-ray fluorescence machine. The system was optimized by additional detector shielding to minimize stray counts, and by a custom-made measuring chamber to ensure sample integrity. Protocols of data recording and analysis were optimised to minimise artefacts. We show that Zn distribution maps of whole leaves in high resolution are easily attainable in the hyperaccumulator Noccaea caerulescens. The sensitivity of the method was further shown by analysis of micro- (Cu, Ni, Fe, Zn) and macronutrients (Ca, K) in non-hyperaccumulating crop plants (soybean roots and pepper leaves), which could be obtained in high resolution for scan areas of several millimetres. This allows to study trace metal distribution in shoots and roots with a wide overview of the object, and thus avoids making conclusions based on singular features of tiny spots. The custom-made measuring chamber with continuous humidity and air supply coupled to devices for imaging chlorophyll fluorescence kinetic measurements enabled direct correlation of element distribution with photosynthesis. Leaf samples remained vital even after 20 h of X-ray measurements. Subtle changes in some of photosynthetic parameters in response to the X-ray radiation are discussed.ConclusionsWe show that using an optimized benchtop machine, with protocols for measurement and quantification tailored for plant analyses, trace metal distribution can be investigated in a reliable manner in intact, living plant leaves and roots. Zinc distribution maps showed higher accumulation in the tips and the veins of young leaves compared to the mesophyll tissue, while in the older leaves the distribution was more homogeneous.
| S-EPMC7278123 | biostudies-literature
Project description:Chronic exposure to high levels of manganese (Mn) leads to manganism, a neurological disorder with similar symptoms to those inherent to Parkinson's disease. However, the underlying mechanisms of this pathological condition have yet to be established. Since the human excitatory amino acid transporter 2 (EAAT2) (glutamate transporter 1 in rodents) is predominantly expressed in astrocytes and its dysregulation is involved in Mn-induced excitotoxic neuronal injury, characterization of the mechanisms that mediate the Mn-induced impairment in EAAT2 function is crucial for the development of novel therapeutics against Mn neurotoxicity. Repressor element 1-silencing transcription factor (REST) exerts protective effects in many neurodegenerative diseases. But the effects of REST on EAAT2 expression and ensuing neuroprotection are unknown. Given that the EAAT2 promoter contains REST binding sites, the present study investigated the role of REST in EAAT2 expression at the transcriptional level in astrocytes and Mn-induced neurotoxicity in an astrocyte-neuron coculture system. The results reveal that astrocytic REST positively regulates EAAT2 expression with the recruitment of an epigenetic modifier, cAMP response element-binding protein-binding protein/p300, to its consensus binding sites in the EAAT2 promoter. Moreover, astrocytic overexpression of REST attenuates Mn-induced reduction in EAAT2 expression, leading to attenuation of glutamate-induced neurotoxicity in the astrocyte-neuron coculture system. Our findings demonstrate that astrocytic REST plays a critical role in protection against Mn-induced neurotoxicity by attenuating Mn-induced EAAT2 repression and the ensuing excitotoxic dopaminergic neuronal injury. This indicates that astrocytic REST could be a potential molecular target for the treatment of Mn toxicity and other neurological disorders associated with EAAT2 dysregulation.
| S-EPMC8626589 | biostudies-literature
Project description:Generation of induced dopaminergic (iDA) neurons may provide a significant step forward towards cell replacement therapy for Parkinson's disease (PD). To study and compare transcriptional programs of induced cells versus primary DA neurons is a preliminary step towards characterizing human iDA neurons. We have optimized a protocol to efficiently generate iDA neurons from human pluripotent stem cells (hPSCs). We then sequenced the transcriptomes of iDA neurons derived from 6 different hPSC lines and compared them to that of primary midbrain (mDA) neurons. We identified a small subset of genes with altered expression in derived iDA neurons from patients with Parkinson's Disease (PD). We also observed that iDA neurons differ significantly from primary mDA neurons in global gene expression, especially in genes related to neuron maturation level. Results suggest iDA neurons from patient iPSCs could be useful for basic and translational studies, including in vitro modeling of PD. However, further refinement of methods of induction and maturation of neurons may better recapitulate full development of mDA neurons from hPSCs.
| S-EPMC4740755 | biostudies-literature
Project description:Multilevel interactions among nutrients occur in the soil-plant system. Among them, Fe and Zn homeostasis in plants are of great relevance because of their importance for plant and human nutrition. However, the mechanisms underlying the interplay between Fe and Zn in plants are still poorly understood. In order to elucidate how Zn interacts with Fe homeostasis, it is crucial to assess Zn distribution either in the plant tissues or within the cells. In this study, we investigated the subcellular Zn distribution in Fe-deficient leaf cells of cucumber plants by using two different approaches: cellular fractionation coupled with inductively coupled plasma mass spectrometry (ICP/MS) and nanoscopic synchrotron X-ray fluorescence imaging. Fe-deficient leaves showed a strong accumulation of Zn as well as a strong alteration of the organelles' ultrastructure at the cellular level. The cellular fractionation-ICP/MS approach revealed that Zn accumulates in both chloroplasts and mitochondria of Fe deficient leaves. Nano-XRF imaging revealed Zn accumulation in chloroplast and mitochondrial compartments, with a higher concentration in chloroplasts. Such results show that (i) both approaches are suitable to investigate Zn distribution at the subcellular level and (ii) cellular Fe and Zn interactions take place mainly in the organelles, especially in the chloroplasts.
| S-EPMC6085429 | biostudies-literature
Project description:Manganese serves as a cofactor to a variety of proteins necessary for proper bodily development and function. However, an overabundance of Mn in the brain can result in manganism, a neurological condition resembling Parkinson's disease (PD). Bulk sample measurement techniques have identified the globus pallidus and thalamus as targets of Mn accumulation in the brain, however smaller structures/cells cannot be measured. Here, X-ray fluorescence microscopy determined the metal content and distribution in the substantia nigra (SN) of the rodent brain. In vivo retrograde labeling of dopaminergic cells (via FluoroGold™) of the SN pars compacta (SNc) subsequently allowed for XRF imaging of dopaminergic cells in situ at subcellular resolution. Chronic Mn exposure resulted in a significant Mn increase in both the SN pars reticulata (>163%) and the SNc (>170%) as compared to control; no other metal concentrations were significantly changed. Subcellular imaging of dopaminergic cells demonstrated that Mn is located adjacent to the nucleus. Measured intracellular manganese concentrations range between 40-200 ?M; concentrations as low as 100 ?M have been observed to cause cell death in cell cultures. Direct observation of Mn accumulation in the SNc could establish a biological basis for movement disorders associated with manganism, specifically Mn caused insult to the SNc. Accumulation of Mn in dopaminergic cells of the SNc may help clarify the relationship between Mn and the loss of motor skills associated with manganism.
| S-EPMC4594222 | biostudies-literature