The Journal of Quality Research in Dementia, Issue 3

Dr Joanna F Collingwood

Alzheimer's Society Research Fellow, Institute of Science and Technology in Medicine, Keele University, Staffordshire ST4 7QB Telephone 01782 554253. Fax 01782 717079. Email j.f.collingwood@keele.ac.uk

Professor Jon Dobson

Professor of Biophysics and Biomedical Engineering, Institute of Science and Technology in Medicine, Keele University, Staffordshire ST4 7QB Telephone 01782 554 253. Fax 01782 717 079. Email: jdobson@keele.ac.uk

Abstract

The study of iron in the human brain is particularly important in the context of Alzheimer's disease. Iron is both essential for healthy brain function and is implicated as a factor in neurodegeneration. The chemical form of the iron is particularly critical, as this affects its toxicity and disrupted iron metabolism is linked to regional iron accumulation and pathological hallmarks, such as senile plaques and neurofibrillary tangles.

This review aims to clarify the forms in which iron is present in order to gain an improved understanding of iron's role in disease pathogenesis. Aspects of disrupted iron metabolism may also be helpful: iron has been identified as a potential MRI biomarker for early detection and diagnosis, while iron chelation therapies are under development.

Brain iron metabolism

Iron is the most abundant transition metal in the human brain. We depend on it, not only for oxygen transport, but also for the underlying formation and maintenance of the neuronal network, as well as for numerous aspects of DNA and enzyme processes, including neurotransmitter synthesis. Iron is present in vivo in both the ferrous (Fe2+) and ferric (Fe3+) valence states. The ease with which iron converts between Fe2+ and Fe3+ is critical for metabolism. Its uptake and transport across membranes, and release from transporter proteins such as transferrin (Tf), is dependent on reduction to Fe2+. As Fe2+ is highly redoxactive (and thus more toxic), the majority of non-haem iron that is not immediately utilised is prevented from participating in harmful reactions through uptake, oxidation, and storage in the iron-storage protein, ferritin. Some Fe2+ is thought to remain as free iron in the 'labile iron pool', which to an extent defines cellular iron levels. Free iron is readily bound by Tf, and the 'supply and demand' response to the labile iron pool is thought to determine the expression of Tf receptors and ferritin to maintain iron homeostasis within the brain.

Highly specialised proteins are involved in brain iron metabolism. Tf transports iron into the brain across the blood-brain-barrier via endocytosis, and ferritin sequesters iron in the cytosol. Ferritin is the primary iron storage protein and consists of a spherical cage containing a maximum of 4,500 iron atoms as a ferrihydrite-like core, up to 8nm in diameter. Additional iron transport proteins such as DMT1 (a transmembrane iron transporter observed in mammals) and iron stores (for example, haemosiderin) have been demonstrated. The extent to which Tf and ferritin dominate brain iron transport and storage is a matter of ongoing investigation.

Disrupted brain iron metabolism

While being essential to maintain a healthy brain, iron can play a toxic role. It exacerbates damage to brain tissue following processes such as stroke or trauma. Regional increase of iron within Alzheimer's diseased brains, compared with healthy controls, is considered a key factor in neuronal atrophy.[1] In stroke and trauma, the iron implicated in tissue damage is predominantly thought to be from haemoglobin, but this is not necessarily the case for iron-mediated damage in neurodegenerative disease. Regional accumulation and deposition of iron within the brain; altered iron transportand storage-protein regulation and the association of iron with neuropathology are evident in neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, Huntington's disease and several others.[2,3,4]

Iron in the Alzheimer's brain

In Alzheimer's disease, regions exhibiting extensive lesions and plaque-related pathology show iron accumulation. The cellular distribution of iron accumulations is yet to be fully determined, although there is evidence for increased intra-neuronal levels of iron in the ageing mammalian brain,[5] raising the possibility that some aspect of disrupted iron metabolism is responsible for a decrease in the capacity of neurones to export iron. What is clear is that the homeostasis of iron, and of the respective ironbinding proteins, is significantly altered in the brains of people with Alzheimer's disease.[6]

Iron accumulations occur in the cerebral cortex, the hippocampus and the basal nucleus of Meynert, colocalising with lesions, neurofibrillary tangles and plaques. These are particularly important areas in the clinical picture of Alzheimer's disease, being associated with the centres of memory and thought processes that are gradually lost as the disease progresses. Various studies have shown altered levels of ferritin and transferrin in affected regions of the Alzheimer's brain. There is evidence that the ratio of the ferritin protein to iron decreases in affected regions,[7] implying either increased loading of the ferritin core or failure to store the iron.

Iron concentrations are most apparent in the regions affected by Alzheimer's disease pathology, and are thought to be involved in the generation of an excess of reactive radical species, leading to the observed cell and tissue damage. One possibility is that iron is not properly taken up and oxidised in the ferritin core, leading to an excess of Fe2+, either as 'free iron' or as a component of the ferritin mineral core. Given that Fe2+ is highly reactive, an excess of Fe2+ may stimulate the overproduction of reactive chemical species, such as the hydroxyl radical (OH·). Such free radicals are responsible for oxidative stress, considered to be a primary contributing factor to neurodegeneration.[8,9]

What do we know about unusual iron accumulations in the brain?

While iron is present in both soluble and insoluble forms in the normal human brain, certain insoluble deposits may be indicative of disrupted iron metabolism. In addition to the normal ferrihydrite-like ferritin core, iron is also found as insoluble deposits of haemosiderin. Results from various studies suggest that haemosiderin may be a degradation product of intracellular ferritin agglomerations [10,11]. There is also evidence for mixedvalence species in brain tissue in the form of magnetite (a ferrimagnetic cubic iron oxide more commonly associated with magnetotactic bacteria) that contains alternating lattices of Fe2+ and Fe3+ and was first found in human brain tissue by Kirschvink and co-workers.[12] It has been suggested that the presence of magnetite may indicate a failure to fully oxidise Fe2+ in the ferritin core.[13] Its chemical and magnetic properties make it a candidate for mediating free radical generation, and therefore oxidative stress damage, via both Fenton chemistry and the low field triplet state stabilisation.[14]

How could this information be applied?

We do not know whether, or to what extent, iron plays a role in Alzheimer's disease pathogenesis, but the accumulation of iron related to Alzheimer's disease may provide a mechanism for early detection of disease.[11,13] Iron has been identified as a potential MRI biomarker, which may aid screening, early detection, and diagnosis for a wide variety of neurodegenerative disorders, including Alzheimer's disease.[11,15,16] A key objective is to identify altered regional iron concentrations and to differentiate between iron compounds in vivo. To do this it is first necessary to establish which iron compounds are present, both in healthy tissue and in neurodegenerative disorders. In our research, we are exploring ways to quantify, map and characterise various iron accumulations associated with Alzheimer's disease.

Why don't we have this information already?

Histochemical iron staining methods and various microprobe techniques have been used to identify regions of iron overload and the distribution of non-haeme iron, [17,18] to obtain certain information about the redox state of the iron, [8,19] and to quantify elemental concentrations of iron and other metal ions in brain tissue samples, including Alzheimer's.[20] These approaches can only provide limited information about the state of precipitated iron. Much remains to be understood about the precise location, form and role of iron accumulations in pathogenesis, despite more than five decades of research in this area.

What techniques are we using?

SQUID magnetometry

To quantify various iron oxides in brain tissue, we are using superconducting quantum interference device (SQUID) magnetometry, a technique that is very sensitive to tiny quantities of magnetic material. The magnetic properties of iron in brain tissue depend on the structural form of the iron that is present: for example, the magnetic properties of magnetite are distinct from the ferrihydrite-like mineral present in normal ferritin. Appropriate combinations of measurements can be used to quantify components in freeze-dried tissue, including normal ferritin, superparamagnetic ferrimagnetic material and larger magnetically blocked particles.[21] This approach has been applied to brain tissue from human Alzheimer's disease cases and age-matched controls,[22] where preliminary results indicated a possible correlation between the amount of nanoscale biogenic magnetite in diseased brain tissue and the onset and progression of Alzheimer's disease. They also demonstrated that magnetite is a contributing factor to the elevated levels of iron observed in Alzheimer's disease tissue.

It is necessary to extract these iron-rich particles from tissue to determine their morphology and enable detailed subsequent examination by transmission electron microscopy. But while these approaches are useful for quantifying various iron oxides and determining their crystal structure and size distribution, information about the relationship between unusual iron accumulations and extra/intracellular structures is not available. For this, we turn to alternative methods.

Synchrotron X-ray spectroscopy

We have developed a synchrotron X-ray approach that allows us both to locate and characterise iron compounds in autopsy tissue sections at sub-cellular resolution.[23,24,25] This builds on previous research involving both microfocus X-ray fluorescence mapping techniques [26] and X-ray absorption near edge spectroscopy (XANES) to look at the valence state of iron in neurodegenerative tissue.[27] It is the combination of these techniques with an intense microfocused beam that allows the distribution and chemical species to be discovered in conjunction with anatomical structure and pathology. An essential component of the mapping and characterisation technique has been developing sample preparation and mounting techniques that minimise disruption of the iron oxidation state.[23]

We can now map large areas (to the order of a few cm2) of tissue in a matter of hours to identify dispersed nanoscale iron particles. The composition of the iron compounds located in low-resolution scans is determined by collecting a XANES spectrum from the region of interest with a beam microfocused to ~ 3 mm x 3 mm, and fitting the spectrum with pre-measured standards for the various biogenic and synthetic iron compounds, such as ferrihydrite, magnetite, haemosiderin, haeme iron, metallic iron and so forth. A more detailed outline of this technique, along with a discussion of the information that can be obtained about metal ion accumulations associated with pathological hallmarks, can be found elsewhere. [24,25] Interestingly, many of the iron-rich spectra obtained during synchrotron mapping and characterisation work with autopsy tissue sections exhibit co-localised ferritin-like ferrihydrite and magnetite at the micron resolution limit of the technique. [24] Continued research in this area should provide insights concerning the formation and role of various iron oxides in the human brain.

Conclusion

The application of combined techniques from the physical sciences, including SQUID magnetometry and synchrotron X-ray spectroscopy, is allowing progress to be made in locating and identifying the nature of iron accumulations in Alzheimer's disease. This research should contribute to our understanding of the role of iron in Alzheimer's disease and to the development of early detection and diagnosis, while also providing clues as to the potential impact of iron chelation treatment on affected regions of the brain.

References

[1] Moos T and Morgan EH. The metabolism of neuronal iron and its pathogenic role in neurological disease: review. Ann. NY Acad. Sci. 2004; 1012-1014.

[2] Goodman L. Alzheimer's disease - a clinicopathologic analysis of 23 cases with a theory on pathogenesis, J. Nerv. Ment. Dis. 1953; 118, 97-130.

[3] Bartzokis G and Tishler TA. MRI evaluation of basal ganglia ferritin iron and neurotoxicity in Alzheimer's and Huntington's disease, Cell. Molec. Biol. 2000; 46 821- 834.

[4] Floor E. Iron as a vulnerability factor in nigrostriatal degeneration in aging and Parkinson's disease, Cell. Molec. Biol. 2000; 46 709-720.

[5] Benkovic SA, Connor JR. Ferritin, transferrin, and iron in selected regions of the adult and aged rat brain. J. Comp. Neurol. 1993; 338 97-113.

[6] Lovell MA, Robertson JD, Teesdale WJ, et al. Copper, iron and zinc in Alzheimer's disease senile plaques, J. Neurol. Sci. 1998; 158 47-52.

[7] JR Connor, BS Snyder, P Arosio, et al. A quantitative analysis of isoferritins in selected regions of aged, parkinsonian, and Alzheimer's diseased brains. J. Neurochem. 1995; 65 717- 24.

[8] Smith MA, Harris PLR, Sayre LM et al. Iron accumulation in Alzheimer disease is a source of redoxgenerated free radicals, Proc. Natl. Acad. Sci. USA 1997; 94 9866-9868.

[9] Casadesus G, Smith MA, Zhu X, et al. Alzheimer disease: evidence for a central pathogenic role of ironmediated reactive oxygen species, J. Alz. Dis. 2004; 6 165- 169.

[10] Andrews SC, Treffry A and Harrison PM. Siderosomal ferritin. The missing link between ferritin and haemosiderin? Biochem. J. 1987; 245 439-446.

[11] Schenck JF, and Zimmerman EA. High-field magnetic resonance imaging of brain iron: birth of a biomarker? NMR Biomed. 2004; 17 433-445.

[12] Kirschvink JL, Kobayashi-Kirschvink A and Woodford BJ. Magnetite biomineralization in the human brain, Proc. Natl.Acad. Sci. USA 1992; 89 7683- 7687.

[13] Dobson J. Nanoscale biogenic iron oxides and neurodegenerative disease, FEBS Lett. 2001; 496 1-5.

[14] Timmel CR, Till U, Brocklehurst B, et al. Effects of weak magnetic fields on free radical recombination reactions, Molec. Phys. 1998; 9571-89.

[15] Drayer B, Burger P, Darwin R, et al. MRI of Brain Iron, Am. J. Roentgenol. 1986; 147 103-110.

[16] Haacke EM, Cheng NY, House MJ, et al. Imaging iron stores in the brain using magnetic resonance imaging, Magn. Reson. Imaging 2005; 23 1-25.

[17] Perl PP and Good PF. Comparative techniques for determination of cellular iron distribution in brain tissue. Ann. Neurol. 1992; 32 S76-S81.

[18] Morris CM, Candy JM, Oakley AE, et al. Histochemical distribution of non-haem iron in the human brain, Acta. Anat. (Basel) 1992; 144 235-257.

[19] Sayre LM, Perry G, Harris PLR, et al. In situ oxidative catalysis by neurofibrillary tangles and senile plaques in Alzheimer's disease: A central role for bound transition metals, J. Neurochem. 2000; 74 270-279.

[20] Watt F. Nuclear microscope analysis in Alzheimer's and Parkinson's disease: A review, Cell. Mol. Biol. (Noisyle- grand) 1996; 42 17-26.

[21] Hautot D, Pankhurst QA and Dobson J. Superconducting quantum interference device measurements of dilute magnetic materials in biological samples, Rev. Sci. Instrum. 2005; 76 045101.

[22] Hautot D, Pankhurst QA, Khan N, et al. Preliminary evaluation of nanoscale biogenic magnetite and Alzheimer's disease, Proc. Royal Soc. Lon. B: Biol. Lett. 2003; 270 S62-S64.

[23] Mikhaylova A, Davidson M, Channel JET, et al. Detection, identification and mapping of iron anomalies in brain tissue using x-ray absorption spectroscopy. J. Royal Soc. Int. 2005; 2 33-37.

[24] Collingwood JF, Mikhaylova A, Davidson M, et al. In situ Characterization and Mapping of Iron Compounds in Alzheimer's Tissue, J. Alz. Dis. 2005; 7 267-272.

[25] Collingwood JF, Mikhaylova A, Davidson M, et al. Studying anomalous iron compounds in neurodegenerative brain tissue using x-ray absorption spectroscopy. J. Phys. Conf. Ser. 2005; 17 54-60.

[26] Szczerbowska-Boruchowska M, Lankosz M, Ostachowicz J, et al. Application of synchrotron radiation for elemental microanalysis of human central nervous system tissue, J. Phys. IV France 2003; 2 325.

[27] Yoshida S, Ektessabi A and Fujisawa S. XANES spectroscopy of a single neuron from a patient with Parkinson's disease, J. Synch. Rad. 2001; 8 998- 1000.