Iron-export ferroxidase activity of ß-amyloid precursor protein is inhibited by zinc in Alzheimer's disease.

Alzheimer's Disease (AD) is complicated by pro-oxidant intraneuronal Fe(2+) elevation as well as extracellular Zn(2+) accumulation within amyloid plaque. We found that the AD ß-amyloid protein precursor (APP) possesses ferroxidase activity mediated by a conserved H-ferritin-like active site, which is inhibited specifically by Zn(2+). Like ceruloplasmin, APP catalytically oxidizes Fe(2+), loads Fe(3+) into transferrin, and has a major interaction with ferroportin in HEK293T cells (that lack ceruloplasmin) and in human cortical tissue. Ablation of APP in HEK293T cells and primary neurons induces marked iron retention, whereas increasing APP695 promotes iron export. Unlike normal mice, APP(-/-) mice are vulnerable to dietary iron exposure, which causes Fe(2+) accumulation and oxidative stress in cortical neurons. Paralleling iron accumulation, APP ferroxidase activity in AD postmortem neocortex is inhibited by endogenous Zn(2+), which we demonstrate can originate from Zn(2+)-laden amyloid aggregates and correlates with Aß burden. Abnormal exchange of cortical zinc may link amyloid pathology with neuronal iron accumulation in AD.

Loss-of-function and gain-of-function studies refute the hypothesis that tau protein is causally involved in the pathogenesis of Huntington's disease.

Tubulin-associated unit (Tau) is a microtubule-associated protein, whose abnormal phosphorylation and deposition in the brain characterizes a range of neurodegenerative diseases called tauopathies. Recent clinical (post-mortem) and pre-clinical evidence suggests that Huntington's disease (HD), an autosomal dominant neurodegenerative disorder, could be considered as a tauopathy. Studies have found the presence of hyperphosphorylated tau, altered tau isoform ratio and aggregated tau in HD brains. However, little is known about the implication of tau in the development of HD pathophysiology, which includes motor, cognitive and affective symptoms. To shine a light on the involvement of tau in HD, our present study aimed at (i) knocking out tau expression and (ii) expressing a transgene encoding mutant human tau in the R6/1 mouse model of HD. We hypothesized that expression of the mutant human tau transgene in HD mice would worsen the HD phenotype, while knocking out endogenous mouse tau in HD mice would improve some behavioral deficits displayed by HD mice. Our data suggest that neither the expression of a tau transgene nor the ablation of tau expression impacted the progression of the HD motor, cognitive and affective phenotypes. Supporting these behavioral findings, we also found that modulating tau expression had no effect on brain weights in HD mice. We also report that expression of the tau transgene increased the weight of WT and HD male mice, whereas tau ablation increased the weight of HD females only. Together, our results indicate that tau might not be as important in regulating the onset and progression of HD symptomatology as previously proposed.

Exploring the significance of lipids in Alzheimer's disease and the potential of extracellular vesicles.

Lipids play a significant role in maintaining central nervous system (CNS) structure and function, and the dysregulation of lipid metabolism is known to occur in many neurological disorders, including Alzheimer's disease. Here we review what is currently known about lipid dyshomeostasis in Alzheimer's disease. We propose that small extracellular vesicle (sEV) lipids may provide insight into the pathophysiology and progression of Alzheimer's disease. This stems from the recognition that sEV likely contributes to disease pathogenesis, but also an understanding that sEV can serve as a source of potential biomarkers. While the protein and RNA content of sEV in the CNS diseases have been studied extensively, our understanding of the lipidome of sEV in the CNS is still in its infancy.

Isolated rapid eye movement sleep behaviour disorder (iRBD) in the Island Study Linking Ageing and Neurodegenerative Disease (ISLAND) Sleep Study: protocol and baseline characteristics.

Isolated rapid eye movement (REM) sleep behaviour disorder (iRBD) is a sleep disorder that is characterised by dream enactment episodes during REM sleep. It is the strongest known predictor of α-synuclein-related neurodegenerative disease (αNDD), such that >80% of people with iRBD will eventually develop Parkinson's disease, dementia with Lewy bodies, or multiple system atrophy in later life. More research is needed to understand the trajectory of phenoconversion to each αNDD. Only five 'gold standard' prevalence studies of iRBD in older adults have been undertaken previously, with estimates ranging from 0.74% to 2.01%. The diagnostic recommendations for video-polysomnography (vPSG) to confirm iRBD makes prevalence studies challenging, as vPSG is often unavailable to large cohorts. In Australia, there have been no iRBD prevalence studies, and little is known about the cognitive and motor profiles of Australian people with iRBD. The Island Study Linking Ageing and Neurodegenerative Disease (ISLAND) Sleep Study will investigate the prevalence of iRBD in Tasmania, an island state of Australia, using validated questionnaires and home-based vPSG. It will also explore several cognitive, motor, olfactory, autonomic, visual, tactile, and sleep profiles in people with iRBD to better understand which characteristics influence the progression of iRBD to αNDD. This paper details the ISLAND Sleep Study protocol and presents preliminary baseline results.

Modification of Biodistribution and Brain Uptake of Copper Bis(thiosemicarbazonato) Complexes by the Incorporation of Amine and Polyamine Functional Groups.

The synthesis of new bis(thiosemicarbazonato)copper(II) complexes featuring polyamine substituents via selective transamination reactions is presented. Polyamines of different lengths, with different ionizable substituent groups, were used to modify and adjust the hydrophilic/lipophilic balance of the copper complexes. The new analogues were radiolabeled with copper-64 and their lipophilicities estimated using distribution coefficients. The cell uptake of the new polyamine complexes was investigated with preliminary in vitro biological studies using a neuroblastoma cancer cell line. The in vivo biodistribution of three of the new analogues was investigated in vivo in mice using positron-emission tomography imaging, and one of the new complexes was compared to [64Cu]Cu(atsm) in an A431 squamous cell carcinoma xenograft model. Modification of the copper complexes with various amine-containing functional groups alters the biodistribution of the complexes in mice. One complex, with a pendent ( N, N-dimethylamino)ethane functional group, displayed tumor uptake similar to that of [64Cu]Cu(atsm) but higher brain uptake, suggesting that this compound has the potential to be of use in the diagnostic brain imaging of tumors and neurodegenerative diseases.

Amyloid-ß Peptide Aß3pE-42 Induces Lipid Peroxidation, Membrane Permeabilization, and Calcium Influx in Neurons.

Pyroglutamate-modified amyloid-ß (pE-Aß) is a highly neurotoxic amyloid-ß (Aß) isoform and is enriched in the brains of individuals with Alzheimer disease compared with healthy aged controls. Pyroglutamate formation increases the rate of Aß oligomerization and alters the interactions of Aß with Cu(2+) and lipids; however, a link between these properties and the toxicity of pE-Aß peptides has not been established. We report here that Aß3pE-42 has an enhanced capacity to cause lipid peroxidation in primary cortical mouse neurons compared with the full-length isoform (Aß(1-42)). In contrast, Aß(1-42) caused a significant elevation in cytosolic reactive oxygen species, whereas Aß3pE-42 did not. We also report that Aß3pE-42 preferentially associates with neuronal membranes and triggers Ca(2+) influx that can be partially blocked by the N-methyl-d-aspartate receptor antagonist MK-801. Aß3pE-42 further caused a loss of plasma membrane integrity and remained bound to neurons at significantly higher levels than Aß(1-42) over extended incubations. Pyroglutamate formation was additionally found to increase the relative efficiency of Aß-dityrosine oligomer formation mediated by copper-redox cycling.

Characterization and Identification of Dityrosine Cross-Linked Peptides Using Tandem Mass Spectrometry.

The use of mass spectrometry coupled with chemical cross-linking of proteins has become a powerful tool for proteins structure and interactions studies. Unlike structural analysis of proteins using chemical reagents specific for lysine or cysteine residues, identification of gas-phase fragmentation patterns of endogenous dityrosine cross-linked peptides have not been investigated. Dityrosine cross-linking in proteins and peptides are clinical markers of oxidative stress, aging, and neurodegenerative diseases including Alzheimer's disease and Parkinson's disease. In this study, we investigated and characterized the fragmentation pattern of a synthetically prepared dityrosine cross-linked dimer of Aß(1-16) using ESI tandem mass spectrometry. We then detailed the fragmentation pattern of dityrosine cross-linked Aß(1-16), using collision induced dissociation (CID), higher-energy collision induced dissociation (HCD), electron transfer dissociation (ETD), and electron capture dissociation (ECD). Application of these generic fragmentation rules of dityrosine cross-linked peptides allowed for the identification of dityrosine cross-links in peptides of Aß and α-synuclein generated in vitro by enzymatic peroxidation. We report, for the first time, the dityrosine cross-linked residues in human hemoglobin and α-synuclein under oxidative conditions. Together these tools open up the potential for automated analysis of this naturally occurring post-translation modification in neurodegenerative diseases as well as other pathological conditions.

Stabilization of nontoxic Aß-oligomers: insights into the mechanism of action of hydroxyquinolines in Alzheimer's disease.

The extracellular accumulation of amyloid ß (Aß) peptides is characteristic of Alzheimer's disease (AD). However, formation of diffusible, oligomeric forms of Aß, both on and off pathways to amyloid fibrils, is thought to include neurotoxic species responsible for synaptic loss and neurodegeneration, rather than polymeric amyloid aggregates. The 8-hydroxyquinolines (8-HQ) clioquinol (CQ) and PBT2 were developed for their ability to inhibit metal-mediated generation of reactive oxygen species from Aß:Cu complexes and have both undergone preclinical and Phase II clinical development for the treatment of AD. Their respective modes of action are not fully understood and may include both inhibition of Aß fibrillar polymerization and direct depolymerization of existing Aß fibrils. In the present study, we find that CQ and PBT2 can interact directly with Aß and affect its propensity to aggregate. Using a combination of biophysical techniques, we demonstrate that, in the presence of these 8-HQs and in the absence of metal ions, Aß associates with two 8-HQ molecules and forms a dimer. Furthermore, 8-HQ bind Aß with an affinity of 1-10 µm and suppress the formation of large (>30 kDa) oligomers. The stabilized low molecular weight species are nontoxic. Treatment with 8-HQs also reduces the levels of in vivo soluble oligomers in a Caenorhabditis elegans model of Aß toxicity. We propose that 8-HQs possess an additional mechanism of action that neutralizes neurotoxic Aß oligomer formation through stabilization of small (dimeric) nontoxic Aß conformers.

Synthesis of Oxorhenium(V) and Oxotechnetium(V) Complexes That Bind to Amyloid-ß Plaques.

Alzheimer's disease is characterized by the presence of amyloid plaques in the brain. The primary constituents of the plaques are aggregated forms of the amyloid-ß (Aß) peptide. With the goal of preparing technetium-99(m) complexes that bind to Aß plaques with the potential to be diagnostic imaging agents for Alzheimer's disease, new tetradentate ligands capable of forming neutral and lipophilic complexes with oxotechentium(V) and oxorhenium(V) were prepared. Nonradioactive isotopes of technetium are not available so rhenium was used as a surrogate for exploratory chemistry. Two planar tetradentate N3O ligands were prepared that form charge-neutral complexes with oxorhenium(v) as well as a ligand featuring a styrylpyridyl functional group designed to bind to Aß plaques. All three ligands formed complexes with oxorhenium(V), and each complex was characterized by NMR spectroscopy, mass spectrometry, and X-ray crystallography. The oxorhenium(V) complex with a styrylpyridyl functional group binds to Aß plaques present in post-mortem human brain tissue. The chemistry was extrapolated to technetium-99(m) at the tracer level for two of the ligands. The resulting oxotechnetium(V) complexes were sufficiently lipophilic and charge-neutral to suggest that they have the potential to cross the blood-brain barrier but exhibited modest stability with respect to exchange with histidine. The chemistry presented here identifies a strategy to integrate styrylpyridyl functional groups into tetradentate ligands capable of forming complexes with [M═O](3+) cores (M = Re or Tc).

Oral treatment with Cu(II)(atsm) increases mutant SOD1 in vivo but protects motor neurons and improves the phenotype of a transgenic mouse model of amyotrophic lateral sclerosis.

Mutations in the metallo-protein Cu/Zn-superoxide dismutase (SOD1) cause amyotrophic lateral sclerosis (ALS) in humans and an expression level-dependent phenotype in transgenic rodents. We show that oral treatment with the therapeutic agent diacetyl-bis(4-methylthiosemicarbazonato)copper(II) [Cu(II)(atsm)] increased the concentration of mutant SOD1 (SOD1G37R) in ALS model mice, but paradoxically improved locomotor function and survival of the mice. To determine why the mice with increased levels of mutant SOD1 had an improved phenotype, we analyzed tissues by mass spectrometry. These analyses revealed most SOD1 in the spinal cord tissue of the SOD1G37R mice was Cu deficient. Treating with Cu(II)(atsm) decreased the pool of Cu-deficient SOD1 and increased the pool of fully metallated (holo) SOD1. Tracking isotopically enriched (65)Cu(II)(atsm) confirmed the increase in holo-SOD1 involved transfer of Cu from Cu(II)(atsm) to SOD1, suggesting the improved locomotor function and survival of the Cu(II)(atsm)-treated SOD1G37R mice involved, at least in part, the ability of the compound to improve the Cu content of the mutant SOD1. This was supported by improved survival of SOD1G37R mice that expressed the human gene for the Cu uptake protein CTR1. Improving the metal content of mutant SOD1 in vivo with Cu(II)(atsm) did not decrease levels of misfolded SOD1. These outcomes indicate the metal content of SOD1 may be a greater determinant of the toxicity of the protein in mutant SOD1-associated forms of ALS than the mutations themselves. Improving the metal content of SOD1 therefore represents a valid therapeutic strategy for treating ALS caused by SOD1.