X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorderElectronic supplementary information (ESI) available: Supplementary Fig. 1-7. See DOI: 10.1039/c4sc00316k
Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload,...
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Format | Journal Article |
Language | English |
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07.05.2014
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Abstract | Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload, deficiency or mislocalization. A major roadblock to understanding the impact of altered biometal homeostasis in neurodegenerative disease is the lack of rapid, specific and sensitive techniques capable of providing quantitative subcellular information on biometal homeostasis
in situ
. Recent advances in X-ray fluorescence detectors have provided an opportunity to rapidly measure biometal content at subcellular resolution in cell populations using X-ray Fluorescence Microscopy (XFM). We applied this approach to investigate subcellular biometal homeostasis in a cerebellar cell line isolated from a natural mouse model of a childhood neurodegenerative disorder, the CLN6 form of neuronal ceroid lipofuscinosis, commonly known as Batten disease. Despite no global changes to whole cell concentrations of zinc or calcium, XFM revealed significant subcellular mislocalization of these important biological second messengers in cerebellar
Cln6
nclf
(Cb
Cln6
nclf
) cells. XFM revealed that nuclear-to-cytoplasmic trafficking of zinc was severely perturbed in diseased cells and the subcellular distribution of calcium was drastically altered in Cb
Cln6
nclf
cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and Cb
Cln6
nclf
cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in Cb
Cln6
nclf
cells. Importantly, a zinc-complex, Zn
II
(atsm), restored the nuclear-to-cytoplasmic zinc ratios in Cb
Cln6
nclf
cells
via
nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. Zn
II
(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in Cb
Cln6
nclf
cells. This study highlights the complementarities of bulk and single cell analysis of metal content for understanding disease states. We demonstrate the utility and broad applicability of XFM for subcellular analysis of perturbed biometal metabolism and mechanism of action studies for novel therapeutics to target neurodegeneration.
XFM approach detects subcellular zinc and calcium mishandling in a fatal neurodegenerative disease, that is corrected by delivery of bioavailable zinc. |
---|---|
AbstractList | Biometals such as zinc, iron, copper and calcium play key roles in diverse physiological processes in the brain, but can be toxic in excess. A hallmark of neurodegeneration is a failure of homeostatic mechanisms controlling the concentration and distribution of these elements, resulting in overload, deficiency or mislocalization. A major roadblock to understanding the impact of altered biometal homeostasis in neurodegenerative disease is the lack of rapid, specific and sensitive techniques capable of providing quantitative subcellular information on biometal homeostasis
in situ
. Recent advances in X-ray fluorescence detectors have provided an opportunity to rapidly measure biometal content at subcellular resolution in cell populations using X-ray Fluorescence Microscopy (XFM). We applied this approach to investigate subcellular biometal homeostasis in a cerebellar cell line isolated from a natural mouse model of a childhood neurodegenerative disorder, the CLN6 form of neuronal ceroid lipofuscinosis, commonly known as Batten disease. Despite no global changes to whole cell concentrations of zinc or calcium, XFM revealed significant subcellular mislocalization of these important biological second messengers in cerebellar
Cln6
nclf
(Cb
Cln6
nclf
) cells. XFM revealed that nuclear-to-cytoplasmic trafficking of zinc was severely perturbed in diseased cells and the subcellular distribution of calcium was drastically altered in Cb
Cln6
nclf
cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and Cb
Cln6
nclf
cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in Cb
Cln6
nclf
cells. Importantly, a zinc-complex, Zn
II
(atsm), restored the nuclear-to-cytoplasmic zinc ratios in Cb
Cln6
nclf
cells
via
nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. Zn
II
(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in Cb
Cln6
nclf
cells. This study highlights the complementarities of bulk and single cell analysis of metal content for understanding disease states. We demonstrate the utility and broad applicability of XFM for subcellular analysis of perturbed biometal metabolism and mechanism of action studies for novel therapeutics to target neurodegeneration.
XFM approach detects subcellular zinc and calcium mishandling in a fatal neurodegenerative disease, that is corrected by delivery of bioavailable zinc. |
Author | James, S. A Duncan, C James, J Grubman, A White, A. R Hickey, J. L de Jonge, M. D Liddell, J. R Kanninen, K. M Crouch, P. J Donnelly, P. S Cotman, S. L Volitakis, I |
AuthorAffiliation | CSIRO Materials Science and Engineering and the Preventative Health Flagship A.I. Virtanen Institute for Molecular Sciences The University of Melbourne Massachusetts General Hospital Australian Synchrotron University of Eastern Finland The Florey Institute of Neuroscience and Mental Health Department of Pathology School of Chemistry and Bio21 Institute for Molecular Science and Biotechnology University of Melbourne Molecular Neurogenetics Unit Center for Human Genetic Research |
AuthorAffiliation_xml | – name: University of Eastern Finland – name: Massachusetts General Hospital – name: Materials Science and Engineering and the Preventative Health Flagship – name: School of Chemistry and Bio21 Institute for Molecular Science and Biotechnology – name: CSIRO – name: Center for Human Genetic Research – name: Department of Pathology – name: Australian Synchrotron – name: The University of Melbourne – name: The Florey Institute of Neuroscience and Mental Health – name: University of Melbourne – name: Molecular Neurogenetics Unit – name: A.I. Virtanen Institute for Molecular Sciences |
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References_xml | – issn: 1969 publication-title: Compilation of X-Ray Cross Sections doi: McMaster Kerr del Grande Mallett Hubbell – issn: 2013 issue: vol. 12 end-page: p 15-40 publication-title: Technologies for Detecting Metals in Single Cells doi: Penner-Hahn – issn: 2006 publication-title: Principles of Fluorescence Spectroscopy doi: Lakowicz – issn: 2011 publication-title: The neuronal ceroid lipofuscinoses (batten disease) doi: Williams Mole Goebel |
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Title | X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorderElectronic supplementary information (ESI) available: Supplementary Fig. 1-7. See DOI: 10.1039/c4sc00316k |
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