Plaques that characterize Alzheimer’s disease accumulate over 20 years as a result of decreased clearance of amyloid-β peptides. Such long-lived peptides are subjected to multiple post-translational modifications, in particular isomerization. Using liquid chromatography ion mobility separations mass spectrometry, we characterized the most common isomerized amyloid-β peptides present in the temporal cortex of sporadic Alzheimer’s disease brains. Quantitative assessment of amyloid-β N-terminus revealed that > 80% of aspartates (Asp-1 and Asp-7) in the N-terminus was isomerized, making isomerization the most dominant post-translational modification of amyloid-β in Alzheimer’s disease brain. Total amyloid-β1-15 was ∼ 85% isomerized at Asp-1 and/or Asp-7 residues, with only 15% unmodified amyloid-β1-15 left in Alzheimer’s disease. While amyloid-β4-15 the next most abundant N-terminus found in Alzheimer’s disease brain, was only ∼ 50% isomerized at Asp-7 in Alzheimer’s disease. Further investigations into different biochemically defined amyloid-β-pools indicated a distinct pattern of accumulation of extensively isomerized amyloid-β in the insoluble fibrillar plaque and membrane associated pools, while the extent of isomerization was lower in peripheral membrane/vesicular and soluble pools. This pattern correlated with the accumulation of aggregation prone amyloid-β42 in Alzheimer’s disease brains. Isomerization significantly alters the structure of the amyloid-β peptide, which not only has implications for its degradation, but also for oligomer assembly, and the binding of therapeutic antibodies that directly target the N-terminus, where these modifications are located.
A dysregulation in the homeostasis of metals such as copper, iron and zinc is speculated to be involved in the pathogenesis of tauopathies, which includes Alzheimer's disease (AD).
Glucose, in the presence of reactive oxygen species (ROS), acts as an as an oxidative agent and drives deleterious processes in Diabetes Mellitus. We have studied the mechanism and the toxicological effects of glucose-dependent glycoxidation reactions driven by copper and ROS, using a model peptide based on the exposed sequence of Human Serum Albumin (HSA) and containing a lysine residue susceptible to copper complexation. The main products of these reactions are Advanced Glycation End-products (AGEs). Carboxymethyl lysine and pyrraline condensed on the model peptide, generating a Modified Peptide (MP). These products were isolated, purified, and tested on cultured motor neuron cells. We observed DNA damage, enhancement of membrane roughness, and formation of domes. We evaluated nuclear abnormalities by the cytokinesis-blocked micronucleus assay and we measured cytostatic and cytotoxic effects, chromosomal breakage, nuclear abnormalities, and cell death. AGEs formed by glycoxidation caused large micronucleus aberrations, apoptosis, and large-scale nuclear abnormalities, even at low concentrations.
The presence of the bicarbonate/carbon dioxide pair is known to accelerate the transition metal ion-catalysed oxidation of various biotargets. It has been shown that stable Cu(II) complexes formed with imine ligands that allow redox cycling between Cu(I) and Cu(II) display diverse apoptotic effects on cell cultures. It is also reported that Cu(II)-tetraglycine can form a stable Cu(III) complex. In the present study, radical generation from H(2)O(2) and H(2)O(2)/HCO(3)(-) in the presence of these two different classes of Cu(II) complexes was evaluated by monitoring the oxidation of dihydrorhodamine 123 and NADH and by the quantitative determination of thiobarbituric acid reactive substances (TBARs method). Cu(II)-imine complexes produced low levels of reactive species whereas Cu(II)-Gly-derived complexes, as well as the free Cu(II) ion, produced oxygen-derived radicals in significantly larger amounts. The effects of these two classes of complexes on mammalian tumour cell viability were equally distinct, in that Cu(II)-imine complexes caused apoptosis, entered in cell and remained almost unaffected in high levels whilst, at the same concentrations, Cu(II)-Gly peptide complexes and Cu(II) sulphate stimulated cell proliferation, with the cell managing copper efficiently. Taken together, these results highlight the different biological effects of Cu(II) complexes, some of which have been recently studied as anti-tumour drugs and radical system generators, and also update the effects of reactive oxygen species generation on cell cycle control.
Background: Abnormally hyperphosphorylated tau is a defining pathological feature of tauopathies, such as Alzheimer’s disease (AD), and accumulating evidence suggests a role for iron in mediating tau pathology that may lead to cognitive decline in these conditions. The metal chelator deferiprone (DFP), which has a high affinity for iron, is currently in clinical trials for AD and Parkinson’s disease. However, the effect of DFP on tau pathology remains underexplored. Objective: We aimed to investigate the impact of chronic DFP treatment on tau pathology using a well-characterized mouse model of tauopathy (rTg(tauP301L)4510). Methods: Animals were treated daily with DFP (100 mg/kg) via oral gavage for 16 weeks. After 14 weeks, mice were tested in the Y-maze, open field, Morris water maze, and rotorod. At the end of the study, brain tissue was collected to examine metal levels (using inductively coupled plasma-mass spectrometry) and for western blot analysis of DFP on tau and iron associated pathways. Results: DFP significantly reduced anxiety-like behavior, and revealed a trend toward improved cognitive function. This was accompanied by a decrease in brain iron levels and sarkosyl-insoluble tau. Our data also showed downregulation of the tau kinases glycogen synthase kinase 3β and cyclin dependent kinase-5 in DFP treated mice and an increase in the methylation of the catalytic subunit of protein phosphatase 2A. Conclusion: These data support the hypothesis that suggests that iron plays a neurotoxic role in tauopathies and may be a potential therapeutic target for this class of disorders.
The accumulation of neurofibrillary tangles (NFTs), which is composed of abnormally hyperphosphorylated tau aggregates, is the classic neuropathology associated with cognitive dysfunction in tauopathies such as Alzheimer's disease (AD). However, there is an emerging theory suggesting that dysregulation in cerebral iron may contribute to NFT formation. Iron is speculated to bind to tau and induce conformational changes of the protein, potentially leading to subsequent aggregation and cognitive decline. Deferiprone (DFP) is a clinically available iron chelator, which has demonstrated potential therapeutic advantages of chelating iron in neurodegenerative disorders, and is currently in clinical trials for AD. However, its effect on tau pathology remains unclear. Here, we report the effects of short-term DFP treatment (4 weeks, 100 mg/kg/daily, via oral gavage) in a mixedgender cohort of the rTg (tauP301L) 4510 mouse model of tauopathy. Our results revealed that DFP improved Y-maze and open field performance, accompanied by a 28% decrease in brain iron levels, measured by inductively coupled plasma mass spectrometry (ICP-MS) and reduced AT8-labeled p-tau within the hippocampus in transgenic tau mice. This data supports the notion that iron may play a neurotoxic role in tauopathies and may be a potential therapeutic target for this class of disorders that can be modulated by the clinically available metal chelator DFP.
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