Brain iron trafficking in health and disease : a systems approach

The study in this thesis sets to explore the techniques to study the iron trafficking into the brain, and more closely in the substantia nigra par compacta (SNpc) region, and also, the altered iron loading mechanism, with particular application in disorders where specific compartments show elevated iron concentration, such as Parkinson’s disease (PD). Iron is essential for numerous biochemical reactions in the brain, but excess iron may produce reactive oxygen species that can induce cell death. The increase in iron concentration in this area has been shown repeatedly over the years, and it has been accepted as one of the main characteristics of PD. Another main characteristic of PD is the loss of neuromelanin containing dopaminergic (DA) neurone in the SNpc. Neuromelanin binds to and stores iron. The disappearance of neuromelanin in the SNpc is another indication that iron is involved in the progression of PD. The detail of iron involvement in PD, however, is still unknown. Attempts have been made in this thesis to create tools to answer some of the questions by image analysis to examine post-mortem human brain tissue; and building a novel computational model of iron transport into the brain. Synchrotron-based experiments, scanning transmission X-ray microscopy (STXM) and synchrotron X-ray fluorescence (SXRF), on post-mortem tissue are described in this thesis to investigate the spatial distribution and relative concentration of iron in the DA neurone and the redox state of the iron. These methods do not require dyes or fixing of the sample, thus allowing the native chemistry of the tissue samples to be better preserved. The investigation of the chemical state of iron using STXM method is of interest because it shows the region-specific toxicity state of the iron in the tissue and the neuromelanin. SXRF mapping can produce a high-resolution map of iron distribution in the tissue, and also calculate the relative concentration of iron in the DA neurone compared to the extracellular concentration. SXRF mapping and processing are described in detail in this thesis to inform how SXRF could be an excellent tool to study the involvement of iron in the PD. SXRF maps from experiments performed before this project are analysed and presented here. The iron absorption spectra from STXM mapping of 200 nm resin-embedded tissues revealed the presence of redox-active iron in the PD case but not in the control. This result supports the hypothesis that the cell death in PD is induced by oxidative stress. Carbon Kedge examination of the neuromelanin in the tissue reveals a feature in the energy spectrum that is possibly unique to neuromelanin. However, further study needs to be done to confirm this finding. The result of such a study would allow label-free direct analysis of the chemical state of iron in the neuromelanin and to determine if the state changes in PD. Two computational models were built to create in silico representations of (1) iron transport into the interstitial fluid of the brain, and (2) iron transport into the DA neurones, using the modelling software COPASI. Model 1, the barrier systems model, was based on an existing well-developed concept from Drs Mitchell and Collingwood, and the original work in this thesis arose from testing and refining the model. Model 2, the DA neurone model, is completely original work, and designed so that it can be integrated with the barrier systems model in the longer term. The models are comprised from nonlinear ordinary differential equations which are used to characterise the kinetics of each chemical species incorporated. Model parameters values for compartmental volumes, and initial concentrations and rate constants for each species, were derived from experimental results from the literature. The simulations show that the regulatory activity of the brain barrier systems protects the brain against excessive iron loading, and a sensing mechanism may be required to prevent low brain iron concentration. Metabolic control analysis identified TfR as the key regulatory factor of iron concentration in the dopaminergic neurones and also the brain barrier systems. These new models are, to the best of our knowledge, the most comprehensive computational models of brain iron transport that have been developed to date. It is intended that they will provide in silico resources to explore the dysregulation of iron transport in PD and related disorders. The synchrotron-based mapping techniques and computational modelling presented in this thesis are excellent tools to study the implication of iron in PD. The SXRF mapping has the potential to produce a cell-specific concentration of iron as an input to the computational model, and the STXM mapping has the potential to reveal the basic knowledge for building the models, such as the compartmentalisation of the iron deposits in the neuromelanin. The synchrotron-based analyses produce results that are useful in the building of the computational model, and the computational models reveal the dynamic processes involved that cannot be observed from post-mortem analysis, so they are both critical. In combination, they offer a new approach to study unresolved questions in the field

Analysis of neuronal iron deposits in Parkinson's disease brain tissue by synchrotron x-ray spectromicroscopy

Neuromelanin-pigmented neurons, which are highly susceptible to neurodegeneration in the Parkinson’s disease substantia nigra, harbour elevated iron levels in the diseased state. Whilst it is widely believed that neuronal iron is stored in an inert, ferric form, perturbations to normal metal homeostasis could potentially generate more reactive forms of iron capable of stimulating toxicity and cell death. However, non-disruptive analysis of brain metals is inherently challenging, since use of stains or chemical fixatives, for example, can significantly influence metal ion distributions and/or concentrations in tissues

Biogenic metallic elements in the human brain?

The chemistry of copper and iron plays a critical role in normal brain function. A variety of enzymes and proteins containing positively charged Cu+, Cu2+, Fe2+, and Fe3+ control key processes, catalyzing oxidative metabolism and neurotransmitter and neuropeptide production. Here, we report the discovery of elemental (zero–oxidation state) metallic Cu0 accompanying ferromagnetic elemental Fe0 in the human brain. These nanoscale biometal deposits were identified within amyloid plaque cores isolated from Alzheimer’s disease subjects, using synchrotron x-ray spectromicroscopy. The surfaces of nanodeposits of metallic copper and iron are highly reactive, with distinctly different chemical and magnetic properties from their predominant oxide counterparts. The discovery of metals in their elemental form in the brain raises new questions regarding their generation and their role in neurochemistry, neurobiology, and the etiology of neurodegenerative disease

Impact of autophagy and ageing on iron load and ferritin in Drosophila brain

Biometals such as iron, copper, potassium and zinc are essential regulatory elements of several biological processes. The homeostasis of biometals is often affected in age-related pathologies. Notably, impaired iron metabolism has been linked to several neurodegenerative disorders. Autophagy, an intracellular degradative process dependent on the lysosomes, is involved in the regulation of ferritin and iron levels. Impaired autophagy has been associated with normal, pathological ageing and neurodegeneration. Non-mammalian model organisms such as Drosophila have proven to be appropriate for the investigation of age-related pathologies. Here, we show that ferritin is expressed in adult Drosophila brain and that iron and holoferritin accumulate with ageing. At whole-brain level we found no direct relationship between the accumulation of holoferritin and a deficit in autophagy in aged Drosophila brain. However, synchrotron X-ray spectromicroscopy revealed an additional spectral feature in the iron-richest region of autophagy-deficient fly brains, consistent with iron-sulphur. This potentially arises from iron-sulphur clusters associated with altered mitochondrial iron homeostasis

Illuminating the brain : revealing brain biochemistry with synchrotron x-ray spectromicroscopy

The synchrotron x-ray spectromicroscopy technique Scanning Transmission X-ray Microscopy (STXM) offers a powerful means to examine the underlying biochemistry of biological systems, owing to its combined chemical sensitivity and nanoscale spatial resolution. Here we introduce and demonstrate methodology for the use of STXM to examine the biochemistry of the human brain. We then discuss how this approach can help us better understand the biochemical changes that occur during the development of degenerative brain disorders, potentially facilitating the development of new therapies for disease diagnosis and treatment

Illuminating the brain: Revealing brain biochemistry with synchrotron X-ray spectromicroscopy

The synchrotron x-ray spectromicroscopy technique Scanning Transmission X-ray Microscopy (STXM) offers a powerful means to examine the underlying biochemistry of biological systems, owing to its combined chemical sensitivity and nanoscale spatial resolution. Here we introduce and demonstrate methodology for the use of STXM to examine the biochemistry of the human brain. We then discuss how this approach can help us better understand the biochemical changes that occur during the development of degenerative brain disorders, potentially facilitating the development of new therapies for disease diagnosis and treatment

Label-free nano-imaging of neuromelanin in the brain by soft x-ray spectromicroscopy

A hallmark of Parkinson's disease is the death of neuromelanin-pigmented neurons, but the role of neuromelanin is unclear. The in situ characterization of neuromelanin remains dependent on detectable pigmentation, rather than direct quantification of neuromelanin. We show that direct, label-free nanoscale visualization of neuromelanin and associated metal ions in human brain tissue can be achieved using synchrotron scanning transmission x-ray microscopy (STXM), through a characteristic feature in the neuromelanin x-ray absorption spectrum at 287.4 eV that is also present in iron-free and iron-laden synthetic neuromelanin. This is confirmed in consecutive brain sections by correlating STXM neuromelanin imaging with silver nitrate-stained neuromelanin. Analysis suggests that the 1s–σ* (C−S) transition in benzothiazine groups accounts for this feature. This method illustrates the wider potential of STXM as a label-free spectromicroscopy technique applicable to both organic and inorganic materials

Label-Free In Situ Chemical Characterization of Amyloid Plaques in Human Brain Tissues

The accumulation of amyloid plaques and increased brain redox burdens are neuropathological hallmarks of Alzheimer’s disease. Altered metabolism of essential biometals is another feature of Alzheimer’s, with amyloid plaques representing sites of disturbed metal homeostasis. Despite these observations, metal-targeting disease treatments have not been therapeutically effective to date. A better understanding of amyloid plaque composition and the role of the metals associated with them is critical. To establish this knowledge, the ability to resolve chemical variations at nanometer length scales relevant to biology is essential. Here, we present a methodology for the label-free, nanoscale chemical characterization of amyloid plaques within human Alzheimer’s disease tissue using synchrotron X-ray spectromicroscopy. Our approach exploits a C–H carbon absorption feature, consistent with the presence of lipids, to visualize amyloid plaques selectively against the tissue background, allowing chemical analysis to be performed without the addition of amyloid dyes that alter the native sample chemistry. Using this approach, we show that amyloid plaques contain elevated levels of calcium, carbonates, and iron compared to the surrounding brain tissue. Chemical analysis of iron within plaques revealed the presence of chemically reduced, low-oxidation-state phases, including ferromagnetic metallic iron. The zero-oxidation state of ferromagnetic iron determines its high chemical reactivity and so may contribute to the redox burden in the Alzheimer’s brain and thus drive neurodegeneration. Ferromagnetic metallic iron has no established physiological function in the brain and may represent a target for therapies designed to lower redox burdens in Alzheimer’s disease. Additionally, ferromagnetic metallic iron has magnetic properties that are distinct from the iron oxide forms predominant in tissue, which might be exploitable for the in vivo detection of amyloid pathologies using magnetically sensitive imaging. We anticipate that this label-free X-ray imaging approach will provide further insights into the chemical composition of amyloid plaques, facilitating better understanding of how plaques influence the course of Alzheimer’s disease