X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder

A. Grubman, S. A. James, J. James, C. Duncan, I. Volitakis, J. L. Hickey, P. J. Crouch, P. S. Donnelly, K. M. Kanninen, J. R. Liddell, S. L. Cotman, M. D. de Jonge, A. R. White

Research output: Contribution to journalArticleResearchpeer-review

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 Cln6nclf (CbCln6nclf) 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 CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6 nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in CbCln6nclf 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. This journal is
Original languageEnglish
Pages (from-to)2503-2516
Number of pages14
JournalChemical Science
Volume5
Issue number6
DOIs
Publication statusPublished - 7 Apr 2014
Externally publishedYes

Cite this

Grubman, A., James, S. A., James, J., Duncan, C., Volitakis, I., Hickey, J. L., ... White, A. R. (2014). X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder. Chemical Science, 5(6), 2503-2516. https://doi.org/10.1039/c4sc00316k
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. / X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder. In: Chemical Science. 2014 ; Vol. 5, No. 6. pp. 2503-2516.
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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 Cln6nclf (CbCln6nclf) 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 CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6 nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in CbCln6nclf 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. This journal is",
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Grubman, A, James, SA, James, J, Duncan, C, Volitakis, I, Hickey, JL, Crouch, PJ, Donnelly, PS, Kanninen, KM, Liddell, JR, Cotman, SL, de Jonge, MD & White, AR 2014, 'X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder' Chemical Science, vol. 5, no. 6, pp. 2503-2516. https://doi.org/10.1039/c4sc00316k

X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder. / 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.

In: Chemical Science, Vol. 5, No. 6, 07.04.2014, p. 2503-2516.

Research output: Contribution to journalArticleResearchpeer-review

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T1 - X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder

AU - Grubman, A.

AU - James, S. A.

AU - James, J.

AU - Duncan, C.

AU - Volitakis, I.

AU - Hickey, J. L.

AU - Crouch, P. J.

AU - Donnelly, P. S.

AU - Kanninen, K. M.

AU - Liddell, J. R.

AU - Cotman, S. L.

AU - de Jonge, M. D.

AU - White, A. R.

PY - 2014/4/7

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N2 - 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 Cln6nclf (CbCln6nclf) 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 CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6 nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in CbCln6nclf 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. This journal is

AB - 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 Cln6nclf (CbCln6nclf) 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 CbCln6nclf cells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6 nclf cells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclf cells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclf cells via nuclear zinc delivery, and restored the relationship between subcellular zinc and calcium levels to that observed in healthy control cells. ZnII(atsm) treatment also resulted in a reduction in the number of calcium-rich puncta observed in CbCln6nclf 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. This journal is

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