Colvin Robert A, Lai Barry, Holmes William R, Lee Daewoo
Department of Biological Sciences, Ohio University, Athens, OH 45701, USA.
Metallomics. 2015 Jul;7(7):1111-23. doi: 10.1039/c5mt00084j.
The purpose of this study was to demonstrate how single cell quantitative and subcellular metallomics inform us about both the spatial distribution and cellular mechanisms of metal buffering and homeostasis in primary cultured neurons from embryonic rat brain, which are often used as models of human disease involving metal dyshomeostasis. The present studies utilized synchrotron radiation X-ray fluorescence (SRXRF) and focused primarily on zinc and iron, two abundant metals in neurons that have been implicated in the pathogenesis of neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Total single cell contents for calcium, iron, zinc, copper, manganese, and nickel were determined. Resting steady state zinc showed a diffuse distribution in both soma and processes, best defined by the mass profile of the neuron with an enrichment in the nucleus compared with the cytoplasm. Zinc buffering and homeostasis was studied using two modes of cellular zinc loading - transporter and ionophore (pyrithione) mediated. Single neuron zinc contents were shown to statistically significantly increase by either loading method - ionophore: 160 million to 7 billion; transporter 160 million to 280 million atoms per neuronal soma. The newly acquired and buffered zinc still showed a diffuse distribution. Soma and processes have about equal abilities to take up zinc via transporter mediated pathways. Copper levels are distributed diffusely as well, but are relatively higher in the processes relative to zinc levels. Prior studies have observed iron puncta in certain cell types, but others have not. In the present study, iron puncta were characterized in several primary neuronal types. The results show that iron puncta could be found in all neuronal types studied and can account for up to 50% of the total steady state content of iron in neuronal soma. Although other metals can be present in iron puncta, they are predominantly iron containing and do not appear to be associated with ferritin cages or transferrin receptor endosomes. The iron content and its distribution in puncta were similar in all neuron types studied including primary dopaminergic neurons. In summary, quantitative measurements of steady state metal levels in single primary cultured neurons made possible by SRXRF analyses provide unique information on the relative levels of each metal in neuronal soma and processes, subcellular location of zinc loads, and have confirmed and extended the characterization of heretofore poorly understood cytoplasmic iron puncta.
本研究的目的是展示单细胞定量和亚细胞金属组学如何让我们了解来自胚胎大鼠脑的原代培养神经元中金属缓冲和稳态的空间分布及细胞机制，这些神经元常被用作涉及金属稳态失调的人类疾病模型。本研究利用同步辐射X射线荧光（SRXRF），主要聚焦于锌和铁，这两种在神经元中含量丰富的金属，它们与阿尔茨海默病和帕金森病等神经退行性疾病的发病机制有关。测定了钙、铁、锌、铜、锰和镍的单细胞总量。静息稳态锌在胞体和突起中均呈弥散分布，通过神经元的质量分布能最好地界定，与细胞质相比，细胞核中锌含量更高。使用两种细胞锌加载模式——转运体介导和离子载体（吡啶硫酮）介导，研究了锌缓冲和稳态。结果表明，通过任何一种加载方法，单个神经元的锌含量均有统计学显著增加——离子载体介导：每个神经元胞体从1.6亿个原子增加到70亿个原子；转运体介导：每个神经元胞体从1.6亿个原子增加到2.8亿个原子。新获取和缓冲的锌仍呈弥散分布。胞体和突起通过转运体介导途径摄取锌的能力大致相当。铜水平也呈弥散分布，但相对于锌水平，在突起中相对较高。先前研究在某些细胞类型中观察到了铁斑，但其他研究未观察到。在本研究中，对几种原代神经元类型中的铁斑进行了表征。结果表明，在所研究的所有神经元类型中都能发现铁斑，其在神经元胞体中可占铁稳态总量的50%。尽管铁斑中可能存在其他金属，但它们主要含铁，似乎与铁蛋白笼或转铁蛋白受体内体无关。在所研究的所有神经元类型（包括原代多巴胺能神经元）中，铁斑中的铁含量及其分布相似。总之，SRXRF分析实现的对单个原代培养神经元稳态金属水平的定量测量，提供了关于神经元胞体和突起中每种金属相对水平、锌负载的亚细胞定位的独特信息，并证实和扩展了对迄今了解甚少的细胞质铁斑的表征。
