title: X-ray fluorescence imaging reveals subcellular biometal disturbances in a childhood neurodegenerative disorder.
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 homeostasisin 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 cerebellarCln6nclf(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 CbCln6nclfcells. Subtle differences in the zinc K-edge X-ray Absorption Near Edge Structure (XANES) spectra of control and CbCln6nclfcells suggested that impaired zinc homeostasis may be associated with an altered ligand set in CbCln6nclfcells. Importantly, a zinc-complex, ZnII(atsm), restored the nuclear-to-cytoplasmic zinc ratios in CbCln6nclfcells 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 CbCln6nclfcells. 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.
authors: Department of Pathology, University of Melbourne, Parkville 3010, Australia.
authors: A
authors: A
authors: Grubman
authors: Australian Synchrotron, Clayton 3168, Australia ; Materials Science and Engineering and the Preventative Health Flagship, CSIRO, Clayton 3168, Australia.
authors: S A
authors: SA
authors: James
authors: Department of Pathology, University of Melbourne, Parkville 3010, Australia.
authors: J
authors: J
authors: James
authors: Department of Pathology, University of Melbourne, Parkville 3010, Australia.
authors: C
authors: C
authors: Duncan
authors: The Florey Institute of Neuroscience and Mental Health, University of Melbourne, Parkville 3010, Australia.
authors: I
authors: I
authors: Volitakis
authors: School of Chemistry and Bio21 Institute for Molecular Science and Biotechnology, The University of Melbourne, Parkville 3010, Australia.
authors: J L
authors: JL
authors: Hickey
authors: Department of Pathology, University of Melbourne, Parkville 3010, Australia.
authors: P J
authors: PJ
authors: Crouch
authors: School of Chemistry and Bio21 Institute for Molecular Science and Biotechnology, The University of Melbourne, Parkville 3010, Australia.
authors: P S
authors: PS
authors: Donnelly
authors: A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, FI-70211, Finland.
authors: K M
authors: KM
authors: Kanninen
authors: Department of Pathology, University of Melbourne, Parkville 3010, Australia.
authors: J R
authors: JR
authors: Liddell
authors: Molecular Neurogenetics Unit, Center for Human Genetic Research, Massachusetts General Hospital, Boston, Massachusetts, United States of America.
authors: S L
authors: SL
authors: Cotman
authors: Australian Synchrotron, Clayton 3168, Australia.
authors: de Jonge
authors: Department of Pathology, University of Melbourne, Parkville 3010, Australia.
authors: A R
authors: AR
authors: White