Quantitative elemental bio-imaging of Mn, Fe, Cu and Zn in 6-hydroxydopamine induced Parkinsonism mouse models

This study demonstrates the application of quantitative elemental bio-imaging for the determination of the distribution Cu, Mn, Fe and Zn in Parkinsonism mouse model brains. Elevated concentrations of these metals within the substantia nigra (SN) are suspected to play a role on the development of Parkinson's disease. Elemental bio-imaging employs laser ablation inductively coupled mass spectrometry (LA-ICP-MS) to construct images of trace element distribution. Quantitative data was produced by ablating the standard tissue sections and recording the mean signal intensity calibrated against multi level matrix matched tissue standards. The concentrations of Fe within the substantia nigra of the lesioned animals increased significantly when compared against control animals. Furthermore, the data was compared against solution nebulisation ICP-MS in which the whole substantia nigra was excised. The trends were the same for both methods; however the elemental bio-imaging method returned significantly higher concentrations. This was caused by dilution from inclusion of surrounding tissue of the SN during the excision procedure. © The Royal Society of Chemistry 2009

Mechanisms of Copper Ion Mediated Huntington's Disease Progression

Huntington's disease (HD) is caused by a dominant polyglutamine expansion within the N-terminus of huntingtin protein and results in oxidative stress, energetic insufficiency and striatal degeneration. Copper and iron are increased in the striata of HD patients, but the role of these metals in HD pathogenesis is unknown. We found, using inductively-coupled-plasma mass spectroscopy, that elevations of copper and iron found in human HD brain are reiterated in the brains of affected HD transgenic mice. Increased brain copper correlated with decreased levels of the copper export protein, amyloid precursor protein. We hypothesized that increased amounts of copper bound to low affinity sites could contribute to pro-oxidant activities and neurodegeneration. We focused on two proteins: huntingtin, because of its centrality to HD, and lactate dehydrogenase (LDH), because of its documented sensitivity to copper, necessity for normoxic brain energy metabolism and evidence for altered lactate metabolism in HD brain. The first 171 amino acids of wild-type huntingtin, and its glutamine expanded mutant form, interacted with copper, but not iron. N171 reduced Cu(2+) in vitro in a 1∶1 copper∶protein stoichiometry indicating that this fragment is very redox active. Further, copper promoted and metal chelation inhibited aggregation of cell-free huntingtin. We found decreased LDH activity, but not protein, and increased lactate levels in HD transgenic mouse brain. The LDH inhibitor oxamate resulted in neurodegeneration when delivered intra-striatially to healthy mice, indicating that LDH inhibition is relevant to neurodegeneration in HD. Our findings support a role of pro-oxidant copper-protein interactions in HD progression and offer a novel target for pharmacotherapeutics

Regulation of insulin-regulated membrane aminopeptidase activity by its C-terminal domain

The development of inhibitors of insulin-regulated aminopeptidase (TRAP), a membrane-bound zinc metallopeptidase, is a promising approach for the discovery of drugs for the treatment of memory loss such as that associated with Alzheimer's disease. There is, however, no consensus in the literature about the mechanism by which inhibition occurs. Sequence alignments, secondary structure predictions, and homology models based on the structures of recently determined related metallopeptidases suggest that the extracellular region consists of four domains. Partial proteolysis and mass spectrometry reported here confirm some of the domain boundaries. We have produced purified recombinant fragments of human IRAP on the basis of these data and examined their kinetic and biochemical properties. Full-length extracellular constructs assemble as dimers with different nonoverlapping fragments dimerizing as well, suggesting an extended dimer interface. Only recombinant fragments containing domains 1 and 2 possess aminopeptidase activity and bind the radiolabeled hexapeptide inhibitor, angiotensin IV (Ang IV). However, fragments lacking domains 3 and 4 possess reduced activity, although they still bind a range of inhibitors with the same affinity as longer fragments. In the presence of Ang IV, IRAP is resistant to proteolysis, suggesting significant conformational changes occur upon binding of the inhibitor. We show that TRAP has a second Zn(2+) binding site, not associated with the catalytic region, which is lost upon binding Ang IV. Modulation of activity caused by domains 3 and 4 is consistent with a conformational change regulating access to the active site of IRAP

Intracellular amyloid formation in muscle cells of Aβ-transgenic Caenorhabditis elegans: determinants and physiological role in copper detoxification

Background: The amyloid β-peptide is a ubiquitous peptide, which is prone to aggregate forming soluble toxic oligomers and insoluble less-toxic aggregates. The intrinsic and external/environmental factors that determine Aβ aggregation in vivo are poorly understood, as well as the cellular meaning of this process itself. Genetic data as well as cell biological and biochemical evidence strongly support the hypothesis that Aβ is a major player in the onset and development of Alzheimer's disease. In addition, it is also known that Aβ is involved in Inclusion Body Myositis, a common myopathy of the elderly in which the peptide accumulates intracellularly. Results: In the present work, we found that intracellular Aβ aggregation in muscle cells of Caenorhabditis elegans overexpressing Aβ peptide is affected by two single amino acid substitutions, E22G (Arctic) and V18A (NIC). Both variations show decrease intracellular amyloidogenesis compared to wild type Aβ. We show that intracellular amyloid aggregation of wild type Aβ is accelerated by Cu2+ and diminished by copper chelators. Moreover, we demonstrate through toxicity and behavioral assays that Aβ-transgenic worms display a higher tolerance to Cu2+ toxic effects and that this resistance may be linked to the formation of amyloid aggregates. Conclusion: Our data show that intracellular Aβ amyloid aggregates may trap excess of free Cu2+ buffering its cytotoxic effects and that accelerated intracellular Aβ aggregation may be part of a cell protective mechanism

Rubidium and potassium levels are altered in Alzheimer's disease brain and blood but not in cerebrospinal fluid

Loss of intracellular compartmentalization of potassium is a biochemical feature of Alzheimer's disease indicating a loss of membrane integrity and mitochondrial dysfunction. We examined potassium and rubidium (a biological proxy for potassium) in brain tissue, blood fractions and cerebrospinal fluid from Alzheimer's disease and healthy control subjects to investigate the diagnostic potential of these two metal ions. We found that both potassium and rubidium levels were significantly decreased across all intracellular compartments in the Alzheimer's disease brain. Serum from over 1000 participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing (AIBL), showed minor changes according to disease state. Potassium and rubidium levels in erythrocytes and cerebrospinal fluid were not significantly different according to disease state, and rubidium was slightly decreased in Alzheimer's disease patients compared to healthy controls. Our data provides evidence that contrasts the hypothesized disruption of the blood-brain barrier in Alzheimer's disease, with the systemic decrease in cortical potassium and rubidium levels suggesting influx of ions from the blood is minimal and that the observed changes are more likely indicative of an internal energy crisis within the brain. These findings may be the basis for potential diagnostic imaging studies using radioactive potassium and rubidium tracers

Degradation of the Alzheimer disease amyloid β-peptide by metal-dependent up-regulation of metalloprotease activity

Biometals play an important role in Alzheimer disease, and recent reports have described the development of potential therapeutic agents based on modulation of metal bioavailability. The metal ligand clioquinol (CQ) has shown promising results in animal models and small phase clinical trials; however, the actual mode of action in vivo has not been determined. We now report a novel effect of CQ on amyloid β-peptide (Aβ) metabolism in cell culture. Treatment of Chinese hamster ovary cells overexpressing amyloid precursor protein with CQ and Cu2+ or Zn2+ resulted in an ∼85-90% reduction of secreted Aβ-(1-40) and Aβ-(1-42) compared with untreated controls. Analogous effects were seen in amyloid precursor protein-overexpressing neuroblastoma cells. The secreted Aβ was rapidly degraded through up-regulation of matrix metalloprotease (MMP)-2 and MMP-3 after addition of CQ and Cu2+. MMP activity was increased through activation of phosphoinositol 3-kinase and JNK. CQ and Cu2+ also promoted phosphorylation of glycogen synthase kinase-3, and this potentiated activation of JNK and loss of Aβ-(1-40). Our findings identify an alternative mechanism of action for CQ in the reduction of Aβ deposition in the brains of CQ-treated animals and potentially in Alzheimer disease patients

Mitochondrial Oxidative Stress Causes Hyperphosphorylation of Tau

Age-related neurodegenerative disease has been mechanistically linked with mitochondrial dysfunction via damage from reactive oxygen species produced within the cell. We determined whether increased mitochondrial oxidative stress could modulate or regulate two of the key neurochemical hallmarks of Alzheimer's disease (AD): tau phosphorylation, and ß-amyloid deposition. Mice lacking superoxide dismutase 2 (SOD2) die within the first week of life, and develop a complex heterogeneous phenotype arising from mitochondrial dysfunction and oxidative stress. Treatment of these mice with catalytic antioxidants increases their lifespan and rescues the peripheral phenotypes, while uncovering central nervous system pathology. We examined sod2 null mice differentially treated with high and low doses of a catalytic antioxidant and observed striking elevations in the levels of tau phosphorylation (at Ser-396 and other phospho-epitopes of tau) in the low-dose antioxidant treated mice at AD-associated residues. This hyperphosphorylation of tau was prevented with an increased dose of the antioxidant, previously reported to be sufficient to prevent neuropathology. We then genetically combined a well-characterized mouse model of AD (Tg2576) with heterozygous sod2 knockout mice to study the interactions between mitochondrial oxidative stress and cerebral Aß load. We found that mitochondrial SOD2 deficiency exacerbates amyloid burden and significantly reduces metal levels in the brain, while increasing levels of Ser-396 phosphorylated tau. These findings mechanistically link mitochondrial oxidative stress with the pathological features of AD

Risk prediction of late-onset Alzheimer’s disease implies an oligogenic architecture

Genetic association studies have identified 44 common genome-wide significant risk loci for late-onset Alzheimer's disease (LOAD). However, LOAD genetic architecture and prediction are unclear. Here we estimate the optimal P-threshold (P) of a genetic risk score (GRS) for prediction of LOAD in three independent datasets comprising 676 cases and 35,675 family history proxy cases. We show that the discriminative ability of GRS in LOAD prediction is maximised when selecting a small number of SNPs. Both simulation results and direct estimation indicate that the number of causal common SNPs for LOAD may be less than 100, suggesting LOAD is more oligogenic than polygenic. The best GRS explains approximately 75% of SNP-heritability, and individuals in the top decile of GRS have ten-fold increased odds when compared to those in the bottom decile. In addition, 14 variants are identified that contribute to both LOAD risk and age at onset of LOAD

Lithium suppression of tau induces brain iron accumulation and neurodegeneration

Lithium is a first-line therapy for bipolar affective disorder. However, various adverse effects, including a Parkinson-like hand tremor, often limit its use. The understanding of the neurobiological basis of these side effects is still very limited. Nigral iron elevation is also a feature of Parkinsonian degeneration that may be related to soluble tau reduction. We found that magnetic resonance imaging T2 relaxation time changes in subjects commenced on lithium therapy were consistent with iron elevation. In mice, lithium treatment lowers brain tau levels and increases nigral and cortical iron elevation that is closely associated with neurodegeneration, cognitive loss and parkinsonian features. In neuronal cultures lithium attenuates iron efflux by lowering tau protein that traffics amyloid precursor protein to facilitate iron efflux. Thus, tau- and amyloid protein precursor-knockout mice were protected against lithium-induced iron elevation and neurotoxicity. These findings challenge the appropriateness of lithium as a potential treatment for disorders where brain iron is elevated (for example, Alzheimer’s disease), and may explain lithium-associated motor symptoms in susceptible patients

A blood-based biomarker panel indicates IL-10 and IL-12/23p40 are jointly associated as predictors of β-amyloid load in an AD cohort

Alzheimer\u27s Disease (AD) is the most common form of dementia, characterised by extracellular amyloid deposition as plaques and intracellular neurofibrillary tangles of tau protein. As no current clinical test can diagnose individuals at risk of developing AD, the aim of this project is to evaluate a blood-based biomarker panel to identify individuals who carry this risk. We analysed the levels of 22 biomarkers in clinically classified healthy controls (HC), mild cognitive impairment (MCI) and Alzheimer\u27s participants from the well characterised Australian Imaging, Biomarker and Lifestyle (AIBL) study of aging. High levels of IL-10 and IL-12/23p40 were significantly associated with amyloid deposition in HC, suggesting that these two biomarkers might be used to detect at risk individuals. Additionally, other biomarkers (Eotaxin-3, Leptin, PYY) exhibited altered levels in AD participants possessing the APOE ϵ4 allele. This suggests that the physiology of some potential biomarkers may be altered in AD due to the APOE ϵ4 allele, a major risk factor for AD. Taken together, these data highlight several potential biomarkers that can be used in a blood-based panel to allow earlier identification of individuals at risk of developing AD and/or early stage AD for which current therapies may be more beneficial