In-depth magnetometry and EPR analysis of the spin structure of human-liver ferritin: from DC to 9 GHz.

Ferritin, the major iron storage protein in organisms, stores iron in the form of iron oxyhydroxide most likely involving phosphorous as a constituent, the mineral form of which is not well understood. Therefore, the question of how the ca. 2000 iron atoms in the ferritin core are magnetically coupled is still largely open. The ferritin core, with a diameter of 5-8 nm, is encapsulated in a protein shell that also catalyzes the uptake of iron and protects the core from outside interactions. Neurodegenerative disease is associated with iron imbalance, generating specific interest in the magnetic properties of ferritin. Here we present 9 GHz continuous wave EPR and a comprehensive set of magnetometry techniques including isothermal remanent magnetization (IRM) and AC susceptibility to elucidate the magnetic properties of the core of human liver ferritin. For the analysis of the magnetometry data, a new microscopic model of the ferritin-core spin structure is derived, showing that magnetic moment is generated by surface-spin canting, rather than defects. The analysis explicitly includes the distribution of magnetic parameters, such as the distribution of the magnetic moment. This microscopic model explains some of the inconsistencies resulting from previous analysis approaches. The main findings are a mean magnetic moment of 337µB with a standard deviation of 0.947µB. In contrast to previous reports, only a relatively small contribution of paramagnetic and ferrimagnetic phases is found, in the order of maximally 3%. For EPR, the over 30 mT wide signal of the ferritin core is analyzed using the model of the giant spin system [Fittipaldi et al., Phys. Chem. Chem. Phys., 2016, 18, 3591-3597]. Two components are needed minimally, and the broadening of these components suggests a broad distribution of the magnetic resonance parameters, the zero-field splitting, D, and the spin quantum number, S. We compare parameters from EPR and magnetometry and find that EPR is particularly sensitive to the surface spins of the core, revealing the potential to use EPR as a diagnostic for surface-spin disorder.

Cortical iron accumulation in MAPT- and C9orf 72-associated frontotemporal lobar degeneration.

Neuroinflammation has been implicated in frontotemporal lobar degeneration (FTLD) pathophysiology, including in genetic forms with microtubule-associated protein tau (MAPT) mutations (FTLD-MAPT) or chromosome 9 open reading frame 72 (C9orf72) repeat expansions (FTLD-C9orf72). Iron accumulation as a marker of neuroinflammation has, however, been understudied in genetic FTLD to date. To investigate the occurrence of cortical iron accumulation in FTLD-MAPT and FTLD-C9orf72, iron histopathology was performed on the frontal and temporal cortex of 22 cases (11 FTLD-MAPT and 11 FTLD-C9orf72). We studied patterns of cortical iron accumulation and its colocalization with the corresponding underlying pathologies (tau and TDP-43), brain cells (microglia and astrocytes), and myelination. Further, with ultrahigh field ex vivo MRI on a subset (four FTLD-MAPT and two FTLD-C9orf72), we examined the sensitivity of T2*-weighted MRI for iron in FTLD. Histopathology showed that cortical iron accumulation occurs in both FTLD-MAPT and FTLD-C9orf72 in frontal and temporal cortices, characterized by a diffuse mid-cortical iron-rich band, and by a superficial cortical iron band in some cases. Cortical iron accumulation was associated with the severity of proteinopathy (tau or TDP-43) and neuronal degeneration, in part with clinical severity, and with the presence of activated microglia, reactive astrocytes and myelin loss. Ultra-high field T2*-weighted MRI showed a good correspondence between hypointense changes on MRI and cortical iron observed on histology. We conclude that iron accumulation is a feature of both FTLD-MAPT and FTLD-C9orf72 and is associated with pathological severity. Therefore, in vivo iron imaging using T2*-weighted MRI or quantitative susceptibility mapping may potentially be used as a noninvasive imaging marker to localize pathology in FTLD.

Off-resonance saturation as an MRI method to quantify mineral- iron in the post-mortem brain.

PURPOSE: To employ an off-resonance saturation method to measure the mineral-iron pool in the postmortem brain, which is an endogenous contrast agent that can give information on cellular iron status. METHODS: An off-resonance saturation acquisition protocol was implemented on a 7 Tesla preclinical scanner, and the contrast maps were fitted to an established analytical model. The method was validated by correlation and Bland-Altman analysis on a ferritin-containing phantom. Mineral-iron maps were obtained from postmortem tissue of patients with neurological diseases characterized by brain iron accumulation, that is, Alzheimer disease, Huntington disease, and aceruloplasminemia, and validated with histology. Transverse relaxation rate and magnetic susceptibility values were used for comparison. RESULTS: In postmortem tissue, the mineral-iron contrast colocalizes with histological iron staining in all the cases. Iron concentrations obtained via the off-resonance saturation method are in agreement with literature. CONCLUSIONS: Off-resonance saturation is an effective way to detect iron in gray matter structures and partially mitigate for the presence of myelin. If a reference region with little iron is available in the tissue, the method can produce quantitative iron maps. This method is applicable in the study of diseases characterized by brain iron accumulation and can complement existing iron-sensitive parametric methods.

MR imaging for the quantitative assessment of brain iron in aceruloplasminemia: A postmortem validation study.

AIMS: Non-invasive measures of brain iron content would be of great benefit in neurodegeneration with brain iron accumulation (NBIA) to serve as a biomarker for disease progression and evaluation of iron chelation therapy. Although magnetic resonance imaging (MRI) provides several quantitative measures of brain iron content, none of these have been validated for patients with a severely increased cerebral iron burden. We aimed to validate R2* as a quantitative measure of brain iron content in aceruloplasminemia, the most severely iron-loaded NBIA phenotype. METHODS: Tissue samples from 50 gray- and white matter regions of a postmortem aceruloplasminemia brain and control subject were scanned at 1.5 T to obtain R2*, and biochemically analyzed with inductively coupled plasma mass spectrometry. For gray matter samples of the aceruloplasminemia brain, sample R2* values were compared with postmortem in situ MRI data that had been obtained from the same subject at 3 T - in situ R2*. Relationships between R2* and tissue iron concentration were determined by linear regression analyses. RESULTS: Median iron concentrations throughout the whole aceruloplasminemia brain were 10 to 15 times higher than in the control subject, and R2* was linearly associated with iron concentration. For gray matter samples of the aceruloplasminemia subject with an iron concentration up to 1000 mg/kg, 91% of variation in R2* could be explained by iron, and in situ R2* at 3 T and sample R2* at 1.5 T were highly correlated. For white matter regions of the aceruloplasminemia brain, 85% of variation in R2* could be explained by iron. CONCLUSIONS: R2* is highly sensitive to variations in iron concentration in the severely iron-loaded brain, and might be used as a non-invasive measure of brain iron content in aceruloplasminemia and potentially other NBIA disorders.

Quantification of different iron forms in the aceruloplasminemia brain to explore iron-related neurodegeneration.

AIMS: Aceruloplasminemia is an ultra-rare neurodegenerative disorder associated with massive brain iron deposits, of which the molecular composition is unknown. We aimed to quantitatively determine the molecular iron forms in the aceruloplasminemia brain, and to illustrate their influence on iron-sensitive MRI metrics. METHODS: The inhomogeneous transverse relaxation rate (R2*) and magnetic susceptibility obtained from 7 T MRI were combined with Electron Paramagnetic Resonance (EPR) and Superconducting Quantum Interference Device (SQUID) magnetometry. The basal ganglia, thalamus, red nucleus, dentate nucleus, superior- and middle temporal gyrus and white matter of a post-mortem aceruloplasminemia brain were studied. MRI, EPR and SQUID results that had been previously obtained from the temporal cortex of healthy controls were included for comparison. RESULTS: The brain iron pool in aceruloplasminemia detected in this study consisted of EPR-detectable Fe3+ ions, magnetic Fe3+ embedded in the core of ferritin and hemosiderin (ferrihydrite-iron), and magnetic Fe3+ embedded in oxidized magnetite/maghemite minerals (maghemite-iron). Ferrihydrite-iron represented above 90% of all iron and was the main driver of iron-sensitive MRI contrast. Although deep gray matter structures were three times richer in ferrihydrite-iron than the temporal cortex, ferrihydrite-iron was already six times more abundant in the temporal cortex of the patient with aceruloplasminemia compared to the healthy situation (162 µg/g vs. 27 µg/g), on average. The concentrations of Fe3+ ions and maghemite-iron in the temporal cortex in aceruloplasminemia were within the range of those in the control subjects. CONCLUSIONS: Iron-related neurodegeneration in aceruloplasminemia is primarily associated with an increase in ferrihydrite-iron, with ferrihydrite-iron being the major determinant of iron-sensitive MRI contrast.

Effects of Alzheimer's disease and formalin fixation on the different mineralised-iron forms in the human brain.

Iron accumulation in the brain is a phenomenon common to many neurodegenerative diseases, perhaps most notably Alzheimer's disease (AD). We present here magnetic analyses of post-mortem brain tissue of patients who had severe Alzheimer's disease, and compare the results with those from healthy controls. Isothermal remanent magnetization experiments were performed to assess the extent to which different magnetic carriers are affected by AD pathology and formalin fixation. While Alzheimer's brain material did not show higher levels of magnetite/maghemite nanoparticles than corresponding controls, the ferrihydrite mineral, known to be found within the core of ferritin proteins and hemosiderin aggregates, almost doubled in concentration in patients with Alzheimer's pathology, strengthening the conclusions of our previous studies. As part of this study, we also investigated the effects of sample preparation, by performing experiments on frozen tissue as well as tissue which had been fixed in formalin for a period of 5 months. Our results showed that the two different preparations did not critically affect the concentration of magnetic carriers in brain tissue, as observable by SQUID magnetometry.

Iron, Myelin, and the Brain: Neuroimaging Meets Neurobiology.

Although iron is crucial for neuronal functioning, many aspects of cerebral iron biology await clarification. The ability to quantify specific iron forms in the living brain would open new avenues for diagnosis, therapeutic monitoring, and understanding pathogenesis of diseases. A modality that allows assessment of brain tissue composition in vivo, in particular of iron deposits or myelin content on a submillimeter spatial scale, is magnetic resonance imaging (MRI). Multimodal strategies combining MRI with complementary analytical techniques ex vivo have emerged, which may lead to improved specificity. Interdisciplinary collaborations will be key to advance beyond simple correlative analyses in the biological interpretation of MRI data and to gain deeper insights into key factors leading to iron accumulation and/or redistribution associated with neurodegeneration.

Quantitative comparison of different iron forms in the temporal cortex of Alzheimer patients and control subjects.

We present a quantitative study of different molecular iron forms found in the temporal cortex of Alzheimer (AD) patients. Applying the methodology we developed in our previous work, we quantify the concentrations of non-heme Fe(III) by Electron Paramagnetic Resonance (EPR), magnetite/maghemite and ferrihydrite by SQUID magnetometry, together with the MRI transverse relaxation rate [Formula: see text], to obtain a systematic view of molecular iron in the temporal cortex. Significantly higher values of [Formula: see text], a larger concentration of ferrihydrite, and a larger magnetic moment of magnetite/maghemite particles are found in the brain of AD patients. Moreover, we found correlations between the concentration of the iron detected by EPR, the concentration of the ferrihydrite mineral and the average iron loading of ferritin. We discuss these findings in the framework of iron dis-homeostasis, which has been proposed to occur in the brain of AD patients.

Human-brain ferritin studied by muon spin rotation: a pilot study.

Muon spin rotation is employed to investigate the spin dynamics of ferritin proteins isolated from the brain of an Alzheimer's disease (AD) patient and of a healthy control, using a sample of horse-spleen ferritin as a reference. A model based on the Néel theory of superparamagnetism is developed in order to interpret the spin relaxation rate of the muons stopped by the core of the protein. Using this model, our preliminary observations show that ferritins from the healthy control are filled with a mineral compatible with ferrihydrite, while ferritins from the AD patient contain a crystalline phase with a larger magnetocrystalline anisotropy, possibly compatible with magnetite or maghemite.

A novel approach to quantify different iron forms in ex-vivo human brain tissue.

We propose a novel combination of methods to study the physical properties of ferric ions and iron-oxide nanoparticles in post-mortem human brain, based on the combination of Electron Paramagnetic Resonance (EPR) and SQUID magnetometry. By means of EPR, we derive the concentration of the low molecular weight iron pool, as well as the product of its electron spin relaxation times. Additionally, by SQUID magnetometry we identify iron mineralization products ascribable to a magnetite/maghemite phase and a ferrihydrite (ferritin) phase. We further derive the concentration of magnetite/maghemite and of ferritin nanoparticles. To test out the new combined methodology, we studied brain tissue of an Alzheimer's patient and a healthy control. Finally, we estimate that the size of the magnetite/maghemite nanoparticles, whose magnetic moments are blocked at room temperature, exceeds 40-50 nm, which is not compatible with the ferritin protein, the core of which is typically 6-8 nm. We believe that this methodology could be beneficial in the study of neurodegenerative diseases such as Alzheimer's Disease which are characterized by abnormal iron accumulation in the brain.