Mercury(II) promotes the in vitro aggregation of tau fragment corresponding to the second repeat of microtubule‐binding domain: Coordination and conformational transition

The loss of metal homeostasis and the toxic effect of metal ion are important events in neurodegenerative and age‐related diseases, such as Alzheimer's disease (AD). For the first time, we investigated the impacts of mercury(II) ions on the folding and aggregation of Alzheimer's tau fragment R2 (residues 275‐305: VQIIN KKLDL SNVQS KCGSK DNIKH VPGGGS), corresponding to the second repeat unit of the microtubule‐binding domain, which was believed to be pivotal to the biochemical properties of full tau protein. By ThS fluorescence assay and electron microscopy, we found that mercury(II) dramatically promoted heparin‐induced aggregation of R2 at an optimum molar ratio of 1: 2 (metal: protein), and the resulting R2 filaments became smaller. Isothermal titration calorimetry (ITC) experiment revealed that the strong coordination of mercury(II) with R2 was an enthalpy‐controlled, entropy‐decreased thermodynamic process. The exceptionally large magnitude of heat release (ΔH₁ = ?34.8 Kcal mol^?1) suggested that the most possible coordinating site on the R2 peptide chain was the thiol group of cysteine residue (Cys291), and this was further confirmed by a control experiment using Cys291 mutated R2. Circular dichroism spectrum demonstrated that this peptide underwent a significant conformational change from random coil to β‐turn structure upon its binding to mercury(II) ion. This study was undertaken to better understand the mechanism of tau aggregation, and evaluate the possible role of mercury(II) in the pathogenesis of AD. © 2010 Wiley Periodicals, Inc. Biopolymers 93: 1100–1107, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Defective Protein Folding and Aggregation as the Basis of Neurodegenerative Diseases: The Darker Aspect of Proteins

Abstract  

The ability of a polypeptide to fold into a unique, functional, and three-dimensional structure depends on the intrinsic properties of the amino acid sequence, function of the molecular chaperones, proteins, and enzymes. Every polypeptide has a finite tendency to misfold and this forms the darker side of the protein world. Partially folded and misfolded proteins that escape the cellular quality control mechanism have the high tendency to form inter-molecular hydrogen bonding between the same protein molecules resulting in aggregation. This review summarizes the underlying and universal mechanism of protein folding. It also deals with the factors responsible for protein misfolding and aggregation. This article describes some of the consequences of such behavior particularly in the context of neurodegenerative conformational diseases such as Alzheimer’s, Parkinson’s, Huntington’s, amyotrophic lateral sclerosis and other non-neurodegenerative conformational diseases such as cancer and cystic fibrosis etc. This will encourage a more proactive approach to the early diagnosis of conformational diseases and nutritional counseling for patients.     

Impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau fragment corresponding to the third repeat of microtubule-binding domain

Environmental exposure to some heavy metals such as cadmium appears to be a risk factor for Alzheimer's disease (AD), however, definite mechanism of their toxicity in AD remains to be elucidated. Previous studies largely focused on the metal ions binding to beta-amyloid, however, very few papers concerned the interaction between tau and metal ions. For the first time, we investigated the impacts of Cd(II) on the conformation and self-aggregation of Alzheimer's tau peptide R3, corresponding to the third repeat of microtubule-binding domain. The initial state of R3 was proven to be dimeric linked by intermolecular disulfide bond, in the non-reducing buffer (Tris-HCl buffer pH7.5, containing no reducing reagent). In this paper, we show that Cd(II) can accelerate heparin-induced aggregation of R3 or independently induce the aggregation of R3, as monitored by ThS fluorescence. In the presence of Cd(II), the resulting R3 filaments became much smaller, as revealed by electron microscopy. Binding to the Cd(II) ion, the dimeric R3 partially lost its random coil, and converted to alpha-helix structure, as revealed by CD and Raman spectrum. Stoichiometric analysis of CD signal against the ratio of [Cd(II)]/[R3] suggested that the coordination intermediate consisted of two R3 dimers binding to a central cadmium ion. As the seed, the coordination intermediate could extensively accelerate the self-aggregation of R3 via promoting the nucleation step. On the other hand, gain in alpha-helix structure on the peptide chain, by coordinating with Cd(II), could be a critical role to promote self-aggregation, as revealed by Raman spectrum. These results provide a further insight into the mechanism of tau filament formation and emphasize the possible involvement of Cd(II) in the pathogenesis of AD.     

Extracellular chaperones modulate the effects of Alzheimer’s patient cerebrospinal fluid on Aβ<Subscript>1-42</Subscript> toxicity and uptake

Alzheimer’s disease is characterised by the inappropriate death of brain cells and accumulation of the Aβ peptide in the brain. Thus, it is possible that there are fundamental differences between Alzheimer’s disease patients and healthy individuals in their abilities to clear Aβ from brain fluid and to protect neurons from Aβ toxicity. In the present study, we examined (1) the cytotoxicity of Alzheimer’s disease cerebrospinal fluid (CSF) compared to control CSF, (2) the ability of Alzheimer’s disease and control CSF to protect cells from Aβ toxicity and to promote cell-mediated clearance of Aβ and lastly (3) the effects of extracellular chaperones, normally found in CSF, on these processes. We show that the Alzheimer’s disease CSF samples tested were more toxic to cultured neuroblastoma cells than normal CSF. In addition, the Alzheimer’s disease CSF samples tested were less able to protect cells from Aβ-induced toxicity and less efficient at promoting macrophage-like cell uptake when compared to normal CSF. The addition of physiologically relevant concentrations of the extracellular chaperones, clusterin, haptoglobin and α₂-macroglobulin into CSF protected neuroblastoma cells from Αβ_1-42 toxicity and promoted Αβ_1-42 uptake in macrophage-like cells. These results suggest that extracellular chaperones are an important element of a system of extracellular protein folding quality control that protects against Aβ toxicity and accumulation.     

Sugar-metal ion interaction. Synthesis,spectroscopic and structural analysis of Zn(II), Cd(II), and Hg(II) sugar complexes containing l-arabinose

Interaction between l-arabinose and the zinc group metal-ion salts has been studied in aqueous solution and solid complexes of the type M(l-arabinose)X₂·nH₂O, where M = Zn(II), Cd(II), and Hg(II) ions, X = Cl⁻ or Br⁻, and n = 0–2 have been isolated and characterized. On comparison with the structurally known Ca(l-arabinose) Cl₂·4H₂O and the corresponding magnesium compounds, it is concluded that the Zn(II) and Cd(II) ions are six-coordinated, binding to two arabinose moieties via 03, 04 of the first and 01, 05 of the second sugar molecule as well as to two H₂O molecules. The Hg(II) ion binds only to two sugar molecules in a similar fashion to zinc and cadmium ions, resulting in a four coordination around the mercury ion. The strong intermolecular hydrogen bonding network of the free arabinose is rearranged to that of the sugar OH...H₂O...halide system upon metalation. The β-anomer sugar conformation is predominant in the free sugar, while the α-anomer conformation is preferred by the alkaline earth and Zn(II), Cd(II), and Hg(II) cations.     

Ubiquitin/proteasome pathway impairment in neurodegeneration: therapeutic implications

The ubiquitin/proteasome pathway is the major proteolytic quality control system in cells. In this review we discuss the impact of a deregulation of this pathway on neuronal function and its causal relationship to the intracellular deposition of ubiquitin protein conjugates in pathological inclusion bodies in all the major chronic neurodegenerative disorders, such as Alzheimer’s, Parkinson’s and Huntington’s diseases as well as amyotrophic lateral sclerosis. We describe the intricate nature of the ubiquitin/proteasome pathway and discuss the paradox of protein aggregation, i.e. its potential toxic/protective effect in neurodegeneration. The relations between some of the dysfunctional components of the pathway and neurodegeneration are presented. We highlight possible ubiquitin/proteasome pathway-targeting therapeutic approaches, such as activating the proteasome, enhancing ubiquitination and promoting SUMOylation that might be important to slow/treat the progression of neurodegeneration. Finally, a model time line is presented for neurodegeneration starting at the initial injurious events up to protein aggregation and cell death, with potential time points for therapeutic intervention.     

Compensatory mechanisms to heal neuroplasticity impairment under Alzheiemr’s disease neurodegeneration. I: The role of amyloid beta and its’ precursor protein

In-depth scholar literature analysis of Alzheimer’s disease neurodegenerative features of amyloid beta protein neurochemistry modification and excessive phosphorylation of tau protein (and associated neuronal cytoskeleton rearrangements) are secondary phenomena. At early disease stage these neurobiochemical mechanisms are reversible and serve to heal an impairment of biophysical properties of neuronal membranes, neurotransmission, basic neuronal function and neuroplasticity, while preserving anatomical and functional brain fields. Aβ and tau could well serve to biochemically restore physico-chemical properties of neual membranes due to a role these proteins play in lipid metabolism. Under such scenario therapeutic block of aggregation and plaque formation of Aβ and inhibition of tau phosphorylation, as well as pharmaceutical modification of other secondary neurodegenerative features (such as a cascade of oxidative stress reactions) are unable to provide an effective cure of Alzheimer’s disease and related pathologies of the Central and peripheral nervous systems, because they are not arraying primary pathagenetic cause. We review the role of Aβ in compensatory mechanisms of neuroplasticity restoration under normal physiological condition and Alzheimer’s disease.     

The Study of Golgi Apparatus in Alzheimer’s Disease

Alzheimer’s disease is an irreversible, progressive neurodegenerative disorder leading invariably to death, usually within 7–10 years after diagnosis and is the leading cause of dementia in the elderly. Not only is Alzheimer’s disease a tragic disease in which people suffer from neurodegeneration in the years to come, it also becomes an incredible burden on the public health system. However, there is currently no effective treatment to halt the progression or prevent the onset of Alzheimer’s disease. This is partly due to the fact that the complex pathophysiology of Alzheimer’s disease is not yet completely understood. Recently, Golgi apparatus is found to play an important role in Alzheimer’s disease. In this review, we discuss the changes of Golgi apparatus during clinical progression and pathological development of Alzheimer’s disease. First, changes of Golgi apparatus size in Alzheimer’s disease are summarized. We then address the role of Golgi apparatus in the neuropathology of Alzheimer’s disease. Finally, the role of Golgi apparatus in the pathogenesis of Alzheimer’s disease is discussed. Understanding the contribution of Golgi apparatus dysfunction to Alzheimer’s disease and its pathophysiological basis will significantly impact our ability to develop more effective therapies for Alzheimer’s disease.     

Peptide Fragment Approach to Prion Misfolding: The Alpha-2 Domain

The mechanism that underlies a multitude of human disorders, including type II diabetes, Parkinson’s, Huntington’s and Alzheimer’s, and the prion encephalopathies, is β-structure expansion through a pathogenic aggregation-prone monomeric form. β-sheet expansion disorders share intermolecular association as a common determinant, being therefore collectively identified as conformational diseases, but little is known about the underlying mechanism. Transmissible spongiform encephalopathies, also known as prion diseases, are all characterised by progressive neuronal degeneration associated to marked extracellular accumulation of an amyloidogenic conformer of the normal cellular prion protein (PrP^C), referred to as the scrapie isoform (PrP^Sc), which is thought to be responsible for the disease symptoms. PrP^C is a ubiquitous 231-amino acid glycoprotein, whose physiological role is still elusive. It is organised as an N-terminal disordered region and a compact C-terminal domain, where secondary structure elements consist of three α-helices (α1, α2 and α3), with an α2-α3 disulphide bridge, and two short β-strands (β1 and β2). Evidence accumulated so far suggests that the protein possesses one or several ‘spots’ of intrinsic conformational weakness, which may trigger generic folding, leading the whole architecture to adopt aggregation-prone conformations. One of such spots is suspected to be the C-terminal side of the α-helix 2, which has recently gained the attention of several investigations because it gathers several disease-associated point mutations, can be strongly fibrillogenic and toxic to neuronal cells, and possesses chameleon conformational behaviour. This paper briefly reviews recent literature on α-2 domain-derived model peptides.     

Molecular modeling of zinc and copper binding with Alzheimer’s amyloid β-peptide

Aggregation of the amyloid β-peptide (Aβ) into insoluble fibrils is a key pathological event in Alzheimer’s disease. Cu(II) and Zn(II) ions were reported to be able to induce Aβ aggregation at nearly physiological concentrations in vitro. In this study, the binding modes of Cu(II) and Zn(II) in this process were explored by molecular modeling. In the pre-associated Aβ, Nτ atom of imidazole ring of His14, O atom of carbonyl of main-chain and two O atoms of water occupied the four ligand positions of the complex. While in the aggregated form of Aβ, the His13(N)–Metals–His14(N) bridges were formed through metal cross-linking action. These results would be helpful to put insight on revealing the formation mechanism of pathogenic Aβ aggregates in brain.     

Functional genomics approaches to neurodegenerative diseases

Many of the neurodegenerative diseases that afflict humans are characterised by the protein aggregation in neurons. These include complex diseases like Alzheimer’s disease and Parkinson’s disease, and Mendelian diseases caused by polyglutamine expansion mutations [like Huntington’s disease (HD) and various spinocerebellar ataxias (SCAs), like SCA3]. A range of functional genomic strategies have been used to try to elucidate pathways involved in these diseases. In this minireview, I focus on how modifier screens in organisms from yeast to mice may be of value in helping to elucidate pathogenic pathways.     

Cerebral amyloid plaques in Alzheimer’s disease but not in scrapie-affected mice are closely associated with a local inflammatory process

Complement proteins of the classical pathway can be immunohistochemically identified in cerebral amyloid plaques in Alzheimer’s disease. Microglial cells in and around amyloid plaques express class II major histocompatibility (MHC) antigens and complement receptors CR3 and CR4. Negative immunostaining for immunoglobulins and for T-cell subsets in the brain parenchyma demonstrates a lack of evidence for the involvement of specific immune responses (such as an immune complex-mediated complement activation or a cell-mediated immune response) in cerebral amyloid deposits in Alzheimer’s disease. Cerebral amyloid plaques in scrapie-affected mice (slow-virus induced encephalopathy) do not contain complement factors C1q and C3c and are not clustered with microglial cells expressing MHC class II molecules or complement receptor CR3. The data presented suggest the induction of a reactive inflammatory process by β/A4 amyloid in the human brain, but not by scrapie-induced PrP amyloid in mice. Our findings do not support the hypothesis that the immune system is involved in the generation of amyloid plaques in Alzheimer’s disease. This study was partly supported by a grant from the Praeventiefonds, project 28–1945     

Heat Shock Proteins and Amateur Chaperones in Amyloid-Beta Accumulation and Clearance in Alzheimer’s Disease

The pathologic lesions of Alzheimer’s disease (AD) are characterized by accumulation of protein aggregates consisting of intracellular or extracellular misfolded proteins. The amyloid-β (Aβ) protein accumulates extracellularly in senile plaques and cerebral amyloid angiopathy, whereas the hyperphosphorylated tau protein accumulates intracellularly as neurofibrillary tangles. “Professional chaperones”, such as the heat shock protein family, have a function in the prevention of protein misfolding and subsequent aggregation. “Amateur” chaperones, such as apolipoproteins and heparan sulfate proteoglycans, bind amyloidogenic proteins and may affect their aggregation process. Professional and amateur chaperones not only colocalize with the pathological lesions of AD, but may also be involved in conformational changes of Aβ, and in the clearance of Aβ from the brain via phagocytosis or active transport across the blood–brain barrier. Thus, both professional and amateur chaperones may be involved in the aggregation, accumulation, persistence, and clearance of Aβ and tau and in other Aβ-associated reactions such as inflammation associated with AD lesions, and may, therefore, serve as potential targets for therapeutic intervention.     

The cerebral proteopathies

The abnormal assembly and deposition of specific proteins in the brain is the probable cause of most neurodegenerative disease afflicting the elderly. These “cerebral proteopathies” include Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), prion diseases, and a variety of other disorders. Evidence is accumulating that the anomalous aggregation of the proteins, and not a loss of protein function, is central to the pathogenesis of these diseases. Thus, therapeutic strategies that reduce the production, accumulation, or polymerization of pathogenic proteins might be applicable to a wide range of some of the most devastating diseases of old age.     

Formation and participation of nano-amyloids in pathogenesis of Alzheimer’s disease and other amyloidogenic diseases

Studies of neurodegenerative disorders attract much attention of the world scientific community due to increasing dissemination of Alzheimer’s disease. The reason for such pathologies consists in transition of a “healthy” molecule or peptide from its native conformation into a very stable “pathological” form. During this process, molecules existing in the “pathological” conformation aggregate and form amyloid fibrils that can undergo an uncontrolled increase. Novel knowledge is required on sporadic forms of Alzheimer’s disease, on the nature of triggering mechanisms of the conformational transitions of beta-amyloid fragments from normally functioning proteins into new structure, nano-beta-amyloids, that escape of neuronal and whole-body control resulted in the loss of neurons. This review summarized results of studies on the formation of amyloid fibrils and their role in pathogenesis of amyloid diseases.     

Carbohydrate adducts with zinc-group-metal ions. Interactions of β-d-fructose with Zn(II), Cd(II), and Hg(II) cations,and the effects of metal-ion coordination on the sugar isomer binding

Interaction of β-d-fructose with hydrated salts of zinc-group-metal has been studied in aqueous solution and solid adducts of the type M(d-fructose)X₂·nH₂O, where M = Zn(II), Cd(II), and Hg(II) ions, X = Cl⁻ or Br⁻, and n = 0–2, have been isolated, and characterized by means of F.t.-i.r. spectroscopy, X-ray powder diffraction, and molar conductivity measurements. The marked spectral similarities observed with the Mg(d-fructose)X₂·4 H₂O (X = Cl⁻ or Br⁻) compounds indicated that the Zn(II) and Cd(II) ions are six-coordinated, binding to two d-fructose molecules through O-2, O-3 of the first d-fructose, and O-4, O-5 of the second, as well as to two H₂O. The Hg(II) ion binds to two sugar moieties in the same fashion as do the Zn(II) and Cd(II) ions, resulting in four-coordination geometry around the mercury ion. The crystalline sugar is in the β-d-fructopyranose form, and the coordination of the of the Ca(II) ion takes place through the β-d-fructopyranose isomer, whereas the binding of the Mg(II), Zn(II), Cd(II), Hg(II), and UO²⁺₂ cations could be via the β-d-fructopyranose and the β-d-fructofuranose structures.     

Theoretical study of the experimental coordination behavior of N-(2-aminophenyl)-D-glycero-D-gulo-heptonamide to Hg(II) ion

The reactivity of N-(2-aminophenyl)-d-glycero-d-gulo-heptonamide (adgha), with the group 12 cations, Zn(II), Cd(II), and Hg(II), was studied in DMSO-d₆ solution. The studied system showed a selective coordination to Hg(II), and the products formed were characterized by ¹H and ¹³C NMR in DMSO-d₆ solution and fast atom bombardment (FAB⁺) mass spectra. The expected coordination compounds, [Hg(adgha)](NO₃)₂ and [Hg(adgha)₂](NO₃)₂, were observed as unstable intermediates that decompose to bis-[2-(d-glycero-d-gulo-hexahydroxyhexyl)-benzimidazole-κN]mercury(II) dinitrate, [Hg(ghbz)₂](NO₃)₂. The chemical transformation of the complexes was followed by NMR experiments, and the nature of the species formed is sustained by a theoretical study done using DFT methodology. From this study, we propose the structure of the complexes formed in solution, the relative stability of the species formed, and the possible role of the solvent in the observed transformations.     

Non-cholinergic Effects of Huperzine A: Beyond Inhibition of Acetylcholinesterase

The use of acetylcholinesterase inhibitors to decrease the breakdown of the neurotransmitter acetylcholine has been the main symptomatic therapy for mild to moderate Alzheimer’s patients, though the etiology of Alzheimer’s disease remains unclear and seems to involve multiple factors. Further evidence has indicated that some of these acetylcholinesterase inhibitors also have non-cholinergic functions on the pathogenesis of Alzheimer’s disease including the formation and deposition of β-amyloid. Huperzine A, a potent and reversible inhibitor of acetylcholinesterase that was initially isolated from a Chinese herb, has been found to improve cognitive deficits in a broad range of animal models and has been used for Alzheimer’s disease treatment in China. The novel neuroprotective effects of huperzine A might yield beneficial effects in Alzheimer’s disease therapy and provide a potential template for the design of new selective and powerful anti-Alzheimer’s drugs. The present paper gives an overview on the neuroprotective effects of huperzine A beyond its acetylcholinesterase inhibition. These effects include regulating β-amyloid precursor protein metabolism, protecting against β-amyloid-mediated oxidative stress and apoptosis. The structure–function relationship of huperzine A is also discussed.     

Chemical Hypoxia Facilitates Alternative Splicing of EAAT2 in Presymptomatic APP23 Transgenic Mice

Hypoxia is one of the major common components of vascular risk factors for pathogenesis of Alzheimer’s disease. This study investigated the possible relationship between hypoxia and alternative splicing of the excitatory amino acid transporter 2 (EAAT2) in a transgenic model for Alzheimer’s disease. We used an APP23 mouse model prior to amyloid deposition and subjected it to chemical hypoxia treatment as induced by 3-nitropropionic acid. One hour after administration of 3-nitropropionic acid changes in the expression of the 5′-splice forms mEAAT2/5UT3, mEAAT2/5UT4, and mEAAT2/5UT5 were found in the frontal cortex, hippocampus and cerebellum of the APP23 model. In untreated APP23 animals the expression of EAAT2 splice variants was unchanged. Our results demonstrate that hypoxia facilitates alternative splicing of EAAT2 in the APP23 model. This may be a molecular mechanism linking vascular factors to early pathophysiology of Alzheimer’s disease.     

Metals ions and neurodegeneration

Neurodegenerative disorders include a variety of pathological conditions, which share similar critical metabolic processes such as protein aggregation and oxidative stress, both of which are associated with the involvement of metal ions. In this review Alzheimer’s disease and Parkinson’s disease are mainly discussed, with the aim of identifying common trends underlying these neurological conditions. Chelation therapy could be a valuable therapeutic approach, since metals are considered to be a pharmacological target for the rationale design of new therapeutic agents directed towards the treatment of neurodegeneration.