Altered regulation of iron transport and storage in Parkinson's disease

Parkinson's disease (PD) is characterized by the death of dopaminergic neurons in the substantia nigra. This neuronal degeneration is associated with a strong microglial activation and iron accumulation in the affected brain structures. The increased iron content may result from an increased iron penetration into the brain parenchyma due to a higher expression of lactoferrin and lactoferrin receptors at the level of the blood vessels and dopaminergic neurons in the substantia nigra in PD. Iron may also accumulate in microglial cells after phagocytosis of dopaminergic neurons. These effects may be reinforced by a lack of up-regulation of the iron storage protein ferritin, as suggested by an absence of change in iron regulatory protein 1 (IRP-1) control of ferritin mRNA translation in PD. Thus, a dysregulation of the labile iron pool may participate in the degenerative process affecting dopaminergic neurons in PD.     

Mouse brains deficient in H-ferritin have normal iron concentration but a protein profile of iron deficiency and increased evidence of oxidative stress

Several neurodegenerative disorders such as Parkinson's Disease (PD) and Alzheimer's Disease (AD) are associated with elevated brain iron accumulation relative to the amount of ferritin, the intracellular iron storage protein. The accumulation of more iron than can be adequately stored in ferritin creates an environment of oxidative stress. We developed a heavy chain (H) ferritin null mutant in an attempt to mimic the iron milieu of the brain in AD and PD. Animals homozygous for the mutation die in utero but the heterozygotes (+/-) are viable. We examined heterozygous and wild-type (wt) mice between 6 and 8 months of age. Macroscopically, the brains of +/- mice were well formed and did not differ from control brains. There was no evidence of histopathology in the brains of the heterozygous mice. Iron levels in the brain of the +/- and wild-type (+/+) mice were similar, but +/- mice had less than half the levels of H-ferritin. The other iron management proteins transferrin, transferrin receptor, light chain ferritin, Divalent Metal Transporter 1, ceruloplasmin, were increased in the +/- mice compared to +/+ mice. The relative amounts of these proteins in relation to the iron concentration are similar to that found in AD and PD. Thus, we hypothesized that the brains of the heterozygote mice should have an increase in indices of oxidative stress. In support of this hypothesis, there was a decrease in total superoxide dismutase (SOD) activity in the heterozygotes coupled with an increase in oxidatively modified proteins. In addition, apoptotic markers Bax and caspase-3 were detected in neurons of the +/- mice but not in the wt. Thus, we have developed a mouse model that mimics the protein profile for iron management seen in AD and PD that also shows evidence of oxidative stress. These results suggest that this mouse may be a model to determine the role of iron mismanagement in neurodegenerative disorders and for testing antioxidant therapeutic strategies.     

Three-dimensional tomographic imaging and characterization of iron compounds within Alzheimer's plaque core material

Although it has been known for over 50 years that abnormal concentrations of iron are associated with virtually all neurodegenerative diseases, including Alzheimer's disease, its origin, nature and role have remained a mystery. Here, we use high-resolution transmission electron microscopy (HR-TEM), energy dispersive X-ray (EDX) spectroscopy and electron energy-loss spectroscopy (EELS), electron tomography, and electron diffraction to image and characterize iron-rich plaque core material - a hallmark of Alzheimer's disease pathology - in three dimensions. In these cores, we unequivocally identify biogenic magnetite and/or maghemite as the dominant iron compound. Our results provide an indication that abnormal iron biomineralization processes are likely occurring within the plaque or the surrounding diseased tissue and may play a role in aberrant peptide aggregation. The size distribution of the magnetite cores implies formation from a ferritin precursor, implicating a malfunction of the primary iron storage protein in the brain.     

Imaging of iron oxide nanoparticles by MR and light microscopy in patients with malignant brain tumours

OBJECTIVE: Ferumoxtran-10 (Combidex), a dextran-coated iron oxide nanoparticle, provides enhancement of intracranial tumours by magnetic resonance (MR) for more than 24 h and can be imaged histologically by iron staining. Our goal was to compare ferumoxtran imaging and histochemistry vs. gadolinium enhancement in malignant brain tumours on preoperative and postoperative MR. METHODS: Seven patients with primary and metastatic malignant tumours underwent MR imaging with gadolinium and ferumoxtran both pre- and postoperatively. Normalized signal intensities on the ferumoxtran-enhanced scans were determined in representative regions of interest. Resected tissue from six ferumoxtran patients and from three patients who did not receive ferumoxtran was assessed for localization of iron in tumour and reactive brain. RESULTS: All malignant tumours (all of which enhanced by gadolinium MR) showed ferumoxtran accumulation with T1 and T2 signal changes, even using a 0.15 T intraoperative MR unit in one patient. Iron staining was predominantly in reactive cells (reactive astrocytes and macrophages) and not tumour cells. In five of the seven patients, including two patients who showed additional lesions, areas enhancing with ferumoxtran but not with gadolinium were observed. Comparison of the pre- and postoperative MR revealed residual ferumoxtran-enhancing areas in four of seven cases. CONCLUSION: In malignant tumours, ferumoxtran may show areas of enhancement, even with a 0.15 T intraoperative MR, that do not enhance with gadolinium. Ferumoxtran-enhancing lesions have persistent increased T1 signal intensity for 2-5 days, which may provide advantages over gadolinium for postoperative imaging. Histochemistry for iron shows uptake of ferumoxtran in reactive cells (astrocytes and macrophages) rather than tumour cells.     

Ferritin binding in the developing mouse brain follows a pattern similar to myelination and is unaffected by the jimpy mutation

We have previously provided evidence that ferritin binds selectively to white matter tracts in adult mouse and human brains. In cell culture experiments, ferritin binding is specifically localized to oligodendrocytes. The goal of the present study is to test the hypothesis that the developmental pattern for ferritin binding will coincide with the onset and progression of myelination. The first evidence of ferritin binding in the mouse brain is at 12 days of age and occurs within the brainstem. Ferritin binding persisted in the brainstem and expanded to the corpus callosum by 15-16 days of age. By 23-24 days of age ferritin binding had further extended to the striatal white matter. By adulthood, ferritin binding was strongly and selectively expressed throughout all white matter tracts. To begin to identify which factors may be involved in the induction of ferritin-binding proteins on oligodendrocytes, brains from the myelin mutant jimpy mice and unaffected littermates were examined at postnatal days 16-18. Jimpy mice were chosen because their oligodendrocytes fail to produce myelin or accumulate iron. Thus, using jimpy mice would elucidate whether these factors are necessary for ferritin-binding protein expression. Both the jimpy mutants and their controls exhibited saturable ferritin binding with similar binding densities and dissociation constants. Dissociation constants for ferritin binding in the unaffected littermates and jimpy mutant mice were 0.38 +/- 0.04 and 0.32 +/- 0.06 nM, respectively and binding densities were similar (1.1 +/- 0.09 and 0.96 +/- 0.12 fmol/mg, respectively). Our results demonstrate that expression of ferritin binding is dependent on the age of the oligodendrocytes and not dependent upon iron accumulation by oligodendrocytes or myelin production. We propose that iron delivery to oligodendrocytes is predominantly via ferritin and this method of iron uptake is unique to oligodendrocytes in the brain.     

Neurochemical investigations of dopamine neuronal systems in iron-regulatory protein 2 (IRP-2) knockout mice

Abnormal iron accumulations are frequently observed in the brains of patients with Parkinson's disease and in normal aging. Iron metabolism is regulated in the CNS by iron regulatory proteins (IRP-1 and IRP-2). Mice engineered to lack IRP-2 develop abnormal motoric behaviors including tremors at rest, abnormal gait, and bradykinesia at middle to late age (18 to 24 months). To further characterize the dopamine (DA) systems of IRP-2 -/- mice, we harvested CNS tissue from age-matched wild type and IRP-2 -/- (16-19 months) and analyzed the protein levels of tyrosine hydroxylase (TH), dopamine transporter (DAT), vesicular monoamine transporter (VMAT2), and DA levels in dorsal striatum, ventral striatum (including the core and shell of nucleus accumbens), and midbrain. We further analyzed the phosphorylation of TH in striatum at serine 40, serine 31, and serine 19. In both dorsal and ventral striatum of IRP-2 knockout mice, there was a 20-25% loss of TH protein and accompanied by a approximately 50% increase in serine 40 phosphorylation above wild-type levels. No change in serine 31 phosphorylation was observed. In the ventral striatum, there was also a significant loss (approximately 40%) of DAT and VMAT2. Levels of DA were decreased (approximately 20%) in dorsal striatum, but turnover of DA was also elevated ( approximately 30%) in dorsal striatum of IRP-2 -/- mice. We conclude that iron misregulation associated with the loss of IRP-2 protein affects DA regulation in the striatum. However, the modest loss of DA and DA-regulating proteins does not reflect the pathology of PD or animal models of PD. Instead, these observations support that the IRP-2 -/- genotype may enable neurobiological events associated with aging.     

Brain iron and ferritin in Parkinson's and Alzheimer's diseases

Summary Semiquantitative histological evaluation of brain iron and ferritin in Parkinson's (PD) and Alzheimer's disease (DAT) have been performed in paraffin sections of brain regions which included frontal cortex, hippocampus, basal ganglia and brain stem. The results indicate a significant selective increase of Fe³⁺ and ferritin in substantia nigra zona compacta but not in zona reticulata of Parkinsonian brains, confirming the biochemical estimation of iron. No such changes were observed in the same regions of DAT brains. The increase of iron is evident in astrocytes, macrophages, reactive microglia and non-pigmented neurons, and in damaged areas devoid of pigmented neurons. In substantia nigra of PD and PD/DAT, strong ferritin reactivity was also associated with proliferated microglia. A faint iron staining was seen occasionally in peripheral halo of Lewy bodies. By contrast, in DAT and PD/DAT, strong ferritin immunoreactivity was observed in and around senile plaques and neurofibrillary tangles. The interrelationship between selective increase of iron and ferritin in PD requires further investigation, because both changes could participate in the induction of oxidative stress and neuronal dath, due to their ability to promote formation of oxygen radicals.Ferritin antisera were kindly provided by Dr. J. G. Joshi, Department of Biochemistry, University of Tennessee, Knoxville, TN, U.S.A.     

Systemic iron metabolism: a review and implications for brain iron metabolism

Mammalian cells and organisms coordinate to regulate expression of numerous proteins involved in the uptake, sequestration, and export of iron. When cells in the systemic circulation are depleted of iron, they increase synthesis of the transferrin receptor and decrease synthesis of the iron sequestration protein, ferritin. In iron-depleted animals, expression of duodenal iron transporters markedly increases and intestinal iron uptake increases accordingly. The major proteins of iron metabolism in the systemic circulation are also expressed in the central nervous system. However, the mechanisms by which iron is transported and distributed throughout the central nervous system are not well understood. Iron accumulation in specific regions of the brain is observed in several neurodegenerative diseases. It is likely that misregulation of iron metabolism is important in the pathophysiology of several human neurodegenerative diseases.     

MRI measures of corpus callosum iron and myelin in early Huntington's disease

Increased iron in subcortical gray matter (GM) structures of patients with Huntington's disease (HD) has been suggested as a causal factor in neuronal degeneration. But how iron content is related to white matter (WM) changes in HD is still unknown. For example, it is not clear whether WM changes share the same physiopathology (i.e. iron accumulation) with GM or whether there is a different mechanism. The present study used MRI to examine iron content in premanifest gene carriers (PreHD, n = 25) and in early HD patients (n = 25) compared with healthy controls (n = 50). 3T MRI acquisitions included high resolution 3D T1, EPI sequences for diffusion tensor imaging (DTI) as an indirect measure of tissue integrity, and T2*‐weighted gradient echo‐planar imaging for MR‐based relaxometry (R2*), which provides an indirect measure of ferritin/iron deposition in the brain. Myelin breakdown starts in the PreHD stage, but there is no difference in iron content values. Iron content reduction manifests later, in the early HD stage, in which we found a lower R2* parameter value in the isthmus. The WM iron reduction in HD is temporally well‐defined (no iron differences in PreHD subjects and iron differences only in early HD patients). Iron level in callosal WM may be regarded as a marker of disease state, as iron does not differentiate PreHD subjects from controls but distinguishes between PreHD and HD. Hum Brain Mapp 35:3143–3151, 2014. © 2013 Wiley Periodicals, Inc .     

Brain iron uptake in hypotransferrinemic mice: influence of systemic iron status

The relationship of the heterogeneity of iron concentrations in the brain with the regulation of iron uptake into specific brain regions remains unresolved. We used hypotransferrinemic mice and an iron-deficient or control diet to explore whether plasma transferrin (Tf), transferrin saturation, and plasma iron levels influence the uptake of (59)Fe and whether there was brain region specificity. Weaning wild-type (+/+) and heterozygotic mice (+/hpx), were sorted randomly to either a iron-deficient diet or a control iron diet for 8 weeks, whereas homozygous mice (hpx/hpx) ate the control diet for 8 weeks before (59)Fe uptake studies. Iron-deficient heterozygous and wild-type mice both had significantly greater plasma Tf levels (37.5 and 42.5 microM) than control mice had (heterozygous and wild-type controls were 20 and 32.5 microM) and far more than homozygous mice (<0.2 microM) had, thus providing five distinct levels of plasma Tf concentrations. After intravenous injection of (59)Fe, brains of iron-deficient wild-type mice took up significantly more (59)Fe (0.15% dose) compared to control wild-type mice (0.056%) at 2 hr, a treatment effect that persisted through 24 hr. In contrast, diet had no effect in heterozygous mice. Importantly, homozygous mice had equivalent uptake to other groups (0.089% dose) by 24 hr. Early brain radioactivity varied by regions (hypothalamus and prefrontal cortex approximately 10-18% brain uptake > cerebellum, pons, thalamus, and striatum approximately 7-12% > cortex, hippocampus, and substantia nigra approximately 6-8%). This distribution of radioactivity changed over 24 hr in the hypothalamus of heterozygous mice, homozygous mice, and iron-deficient wild-type mice. Homozygous mice also showed higher uptake (13-15%) in some regions (hypothalamus and cerebellum) than in other regions. In wild-type and heterozygous mice, (59)Fe uptake was inversely related to brain Tf and was independent of regional brain iron concentrations and plasma Tf levels or saturation. These experimental data suggest that brain iron uptake may be constitutive and independent of plasma Tf, transferrin saturation, or regional brain iron concentration. The proteins and mechanisms responsible for additional iron uptake into specific regions, or perhaps the redistribution are unclear though the data are supportive of a non-transferrin-bound iron uptake pathway.