Changes in cortical protein markers of iron transport with gender, major depressive disorder and suicide.

Objectives: The objective of this study was to determine whether a breakdown in proteins regulating cortical iron homeostasis could be involved in the pathophysiology of mood disorders.Methods: Levels of select proteins responsible for cortical iron transport were quantitated by Western blotting of Brodmann's (BA) areas 6 and 10 from patients with major depressive disorder (n = 13), bipolar disorder (n = 12) and age/sex matched controls (n = 13).Results: We found the inactive form of ceruloplasmin was lower in BA 6 from males compared to females. Levels of copper containing ceruloplasmin was lower in BA 6 from suicide completers whilst levels of amyloid precursor protein, TAU and transferrin were higher in BA 10 from those individuals. The level of prion protein was lower in BA 6 from subjects with major depressive disorder.Conclusions: Our data suggests that perturbation in cortical iron transport proteins is not prevalent in mood disorders. By contrast, our data suggests changes in iron transport proteins in BA 6 and BA 10 are present after suicide completion. If these changes were present before death, they could have had a role in the genesis of the contemplation and completion of suicide.

Cortical biometals: Changed levels in suicide and with mood disorders.

BACKGROUND: Changes in levels of metals have been suggested to contribute to the pathophysiologies of several neurodegenerative disorders but to our knowledge this is the first metallomic study in CNS from patients with mood disorders. The focus of this study was on cortical regions affected by the pathophysiologies of bipolar disorders and major depressive disorders. METHODS: Levels of metals were measured using inductively coupled plasma mass spectrometry in Brodmann's areas (BA) 6, 10 and 17 from patients with major depressive disorders (n = 13), bipolar disorders (n = 12) and age / sex matched controls (n = 13). RESULTS: There were lower levels of cortical strontium (BA 6 & 10), ruthenium (BA 6 & 17) and cadmium (BA 10) from patients with major depressive disorder as well as lower levels of strontium in BA 10 from patients with bipolar disorders. Unexpectedly, there were changes in levels of 16 metals in the cortex, mainly BA 6, from suicide completers compared to those who died of other causes. LIMITATIONS: Cohort sizes were relatively small but comparable with many studies using human postmortem CNS. Like all studies on non-treatment naïve patients, drug treatment was a potential confound in our experiments. CONCLUSIONS: Our exploratory study suggests changes in levels of metals in bipolar disorders and major depressive disorders could be affecting cortical oxidative balance in patients with mood disorders. Our data raises the possibility that measuring levels of specific biometals in the blood could be used as a biomarker for increased risk of suicide.

Oxidation of Iron under Physiologically Relevant Conditions in Biological Fluids from Healthy and Alzheimer's Disease Subjects.

Ferroxidase activity has been reported to be altered in various biological fluids in neurodegenerative disease, but the sources contributing to the altered activity are uncertain. Here we assay fractions of serum and cerebrospinal fluid with a newly validated triplex ferroxidase assay. Our data indicate that while ceruloplasmin, a multicopper ferroxidase, is the predominant source of serum activity, activity in CSF predominantly derives from a <10 kDa component, specifically from polyanions such as citrate and phosphate. We confirm that in human biological samples, ceruloplasmin activity in serum is decreased in Alzheimer's disease, but in CSF a reduction of activity in Alzheimer's disease originates from the polyanion component.

A comparison of ceruloplasmin to biological polyanions in promoting the oxidation of Fe(2+) under physiologically relevant conditions.

BACKGROUND: Iron oxidation is thought to be predominantly handled enzymatically in the body, to minimize spontaneous combustion with oxygen and to facilitate cellular iron export by loading transferrin. This process may be impaired in disease, and requires more accurate analytical assays to interrogate enzymatic- and auto-oxidation within a physiologically relevant environment. METHOD: A new triplex ferroxidase activity assay has been developed that overcomes the previous assay limitations of measuring iron oxidation at a physiologically relevant pH and salinity. RESULTS: Revised enzymatic kinetics for ceruloplasmin (Vmax≈35µMFe(3+)/min/µM; Km≈15µM) are provided under physiological conditions, and inhibition by sodium azide (Ki for Ferric Gain 78.3µM, Ki for transferrin loading 8.1×10(4)µM) is quantified. We also used this assay to characterize the non-enzymatic oxidation of iron that proceeded linearly under physiological conditions. CONCLUSIONS AND GENERAL SIGNIFICANCE: These findings indicate that the requirement of an enzyme to oxidize iron may only be necessary under conditions of adverse pH or anionic strength, for example from hypoxia. In a normal physiological environment, Fe(3+) incorporation into transferrin would be sufficiently enabled by the biological polyanions that are prevalent within extracellular fluids.

Ceruloplasmin and ß-amyloid precursor protein confer neuroprotection in traumatic brain injury and lower neuronal iron.

Traumatic brain injury (TBI) is in part complicated by pro-oxidant iron elevation independent of brain hemorrhage. Ceruloplasmin (CP) and ß-amyloid protein precursor (APP) are known neuroprotective proteins that reduce oxidative damage through iron regulation. We surveyed iron, CP, and APP in brain tissue from control and TBI-affected patients who were stratified according to time of death following injury. We observed CP and APP induction after TBI accompanying iron accumulation. Elevated APP and CP expression was also observed in a mouse model of focal cortical contusion injury concomitant with iron elevation. To determine if changes in APP or CP were neuroprotective we employed the same TBI model on APP(-/-) and CP(-/-) mice and found that both exhibited exaggerated infarct volume and iron accumulation postinjury. Evidence supports a regulatory role of both proteins in defence against iron-induced oxidative damage after TBI, which presents as a tractable therapeutic target.

Iron accumulates in Huntington's disease neurons: protection by deferoxamine.

Huntington's disease (HD) is a progressive neurodegenerative disorder caused by a polyglutamine-encoding CAG expansion in the huntingtin gene. Iron accumulates in the brains of HD patients and mouse disease models. However, the cellular and subcellular sites of iron accumulation, as well as significance to disease progression are not well understood. We used independent approaches to investigate the location of brain iron accumulation. In R6/2 HD mouse brain, synchotron x-ray fluorescence analysis revealed iron accumulation as discrete puncta in the perinuclear cytoplasm of striatal neurons. Further, perfusion Turnbull's staining for ferrous iron (II) combined with transmission electron microscope ultra-structural analysis revealed increased staining in membrane bound peri-nuclear vesicles in R6/2 HD striatal neurons. Analysis of iron homeostatic proteins in R6/2 HD mice revealed decreased levels of the iron response proteins (IRPs 1 and 2) and accordingly decreased expression of iron uptake transferrin receptor (TfR) and increased levels of neuronal iron export protein ferroportin (FPN). Finally, we show that intra-ventricular delivery of the iron chelator deferoxamine results in an improvement of the motor phenotype in R6/2 HD mice. Our data supports accumulation of redox-active ferrous iron in the endocytic / lysosomal compartment in mouse HD neurons. Expression changes of IRPs, TfR and FPN are consistent with a compensatory response to an increased intra-neuronal labile iron pool leading to increased susceptibility to iron-associated oxidative stress. These findings, together with protection by deferoxamine, support a potentiating role of neuronal iron accumulation in HD.

Tau deficiency induces parkinsonism with dementia by impairing APP-mediated iron export.

The microtubule-associated protein tau has risk alleles for both Alzheimer's disease and Parkinson's disease and mutations that cause brain degenerative diseases termed tauopathies. Aggregated tau forms neurofibrillary tangles in these pathologies, but little is certain about the function of tau or its mode of involvement in pathogenesis. Neuronal iron accumulation has been observed pathologically in the cortex in Alzheimer's disease, the substantia nigra (SN) in Parkinson's disease and various brain regions in the tauopathies. Here we report that tau-knockout mice develop age-dependent brain atrophy, iron accumulation and SN neuronal loss, with concomitant cognitive deficits and parkinsonism. These changes are prevented by oral treatment with a moderate iron chelator, clioquinol. Amyloid precursor protein (APP) ferroxidase activity couples with surface ferroportin to export iron, but its activity is inhibited in Alzheimer's disease, thereby causing neuronal iron accumulation. In primary neuronal culture, we found loss of tau also causes iron retention, by decreasing surface trafficking of APP. Soluble tau levels fall in affected brain regions in Alzheimer's disease and tauopathies, and we found a similar decrease of soluble tau in the SN in both Parkinson's disease and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model. These data suggest that the loss of soluble tau could contribute to toxic neuronal iron accumulation in Alzheimer's disease, Parkinson's disease and tauopathies, and that it can be rescued pharmacologically.