Brooks, Jake (2020) Advancing synchrotron spectromicroscopy to investigate metal dysregulation in neurodegenerative disorders. PhD thesis, University of Warwick.

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Abstract

Metal dysregulation and aberrant protein aggregation in the brain are associated with many neurodegenerative diseases, including Alzheimer’s disease (AD) and Parkinson’s disease (PD). Whilst metals are essential to normal brain function, perturbations to normal metal homeostasis may contribute towards toxicity and cell death. Disrupted brain iron and copper concentrations have been widely reported in neurodegenerative disorders, and importantly have been included in mechanistic interpretations due to their redox properties. Calcium imbalance has also been implicated in disrupted neuronal signalling. Non-disruptive analysis of brain metals is inherently challenging, since conventional use of stains or chemical fixatives can significantly influence the native chemistry of tissue samples.

The primary aim of this work was to apply label-free, multi-modal synchrotron techniques to the characterisation of metal ion deposits and their local environment in pathological regions from AD and PD brain tissue. Efforts were focussed on examining the distribution and speciation of iron in vulnerable melanised nigral neurons from PD and neurologically healthy controls, and in amyloid plaque pathology isolated from AD brain tissue, where copper and calcium deposits were also characterised. Method development was supported by application of high energy (hard) x-ray techniques to in vitro protein:metal complexes and physiologically relevant models.

The potential for the peptide amyloid-beta to alter the oxidation state of iron and copper was demonstrated using x-ray absorption near-edge spectroscopy (XANES), also revealing a smaller ferriductase effect for alpha-synuclein. Results support a role for these pathological proteins in generating reactive species in vivo. Synchrotron xray fluorescence (SXRF) mapping was employed for sensitive multi-element mapping in mouse and human brain tissue at a wide range of length scales, revealing signature biometal profiles for specific structures. Combined SXRF and XANES supported complementary speciation of metal-rich deposits.

Use of lower energy (soft) x-rays granted signal access from both organics and inorganics, providing context for metal ion distributions. Scanning transmission x-ray microscopy (STXM) was used to develop an original method for label-free visualisation of neuromelanin, a biopolymer central to the characteristic depigmentation and loss of dopaminergic neurons in PD. This novel approach uniquely does not depend on staining or visible pigmentation, relying instead on a characteristic feature in the neuromelanin x-ray absorption spectrum at 287.4 eV. The method was validated using new histology protocols adapted for ultrathin sections. Neuromelanin clusters were shown to harbour iron in mixed oxidation states in PD substantia nigra neurons, where intranuclear iron was also observed. In amyloid plaque cores, novel STXM analysis revealed the presence of iron, copper and calcium in various chemical forms. Variation in the mineral form of calcium suggests that calcification of plaques may occur in vivo. Redox metals iron and copper were evidenced in both oxidised and chemically reduced chemical states, including the highly reactive zero valent metallic forms of these metals. Discovery of metallic iron and copper in human-derived AD amyloid pathology strongly supports links between the formation of amyloid plaques and chemical reduction of metal ions. Magnetic characterisation using x-ray magnetic circular dichroism confirmed identification of metallic iron, also revealing the presence of magnetic iron mineral phase, magnetite/maghemite. The outstanding sensitivity, specificity, and spatial resolution of soft x-ray spectromicroscopy was exploited to highlight variation in metal chemistry over sub-micron length scales, indicating the potential for redox cycling of metal ions. This may raise oxidative burden in vivo, which has been implicated in neurodegeneration.

Elucidating the native biochemistry within key areas of disease pathology is critical to developing new treatment strategies. It is demonstrated here that complementary synchrotron methods offer unique insight to meet this challenge.

Item Type:	Thesis (PhD)
Subjects:	Q Science > QC Physics Q Science > QH Natural history Q Science > QP Physiology R Medicine > RC Internal medicine
Library of Congress Subject Headings (LCSH):	Synchrotrons, Microscopy, Nervous system -- Degeneration, Alzheimer's disease, Parkinson's disease, Brain
Official Date:	September 2020
Dates:	Date Event September 2020 Published
Institution:	University of Warwick
Theses Department:	School of Engineering
Thesis Type:	PhD
Publication Status:	Unpublished
Supervisor(s)/Advisor:	Collingwood, Joanna
Sponsors:	Engineering and Physical Sciences Research Council
Format of File:	pdf
Extent:	xxv, 225, 26, 12, 11991, 6, 11796 leaves: illustrations, plates, charts.
Language:	eng

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