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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Main Authors Grubman, A, James, S. A, James, J, Duncan, C, Volitakis, I, Hickey, J. L, Crouch, P. J, Donnelly, P. S, Kanninen, K. M, Liddell, J. R, Cotman, S. L, de Jonge, M. D, White, A. R
Format Journal Article
LanguageEnglish
Published 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
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Electronic supplementary information (ESI) available: Supplementary Fig. 1-7. See DOI
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  doi: Penner-Hahn
– issn: 2006
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  doi: Lakowicz
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  publication-title: The neuronal ceroid lipofuscinoses (batten disease)
  doi: Williams Mole Goebel
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