Intrinsically Disordered Proteins in the Neurodegenerative Processes: Formation of Tau Protein Paired Helical Filaments and Their Analysis

1. Several intrinsically disordered proteins (IDPs) play principal role in the neurodegenerative processes of various types. Among them, α-synuclein is involved in Parkinson's disease, prion protein in transmissible spongiform encephalopathies, and tau protein in Alzheimer's disease (AD) and related tauopathies. Neuronal damage in AD is accompanied by the presence of tau protein fibrils composed of paired helical filaments (PHF).2. Tau protein represents a typical IDP. IDPs do not exhibit any stable secondary structure in the free form, but they are able to fold after binding to targets and contain regions with large propensity to adopt a defined type of secondary structure. Binding–folding event at tau protein leading to PHF generation is believed to happen in the course of tauopathies.3. Detailed molecular topology of PHF formation is unknown. There are evidences about the cross-beta structure in PHF core; however the precise arrangement of the tau polypeptide chain is unclear. In this review we summarize current attempts at in vitro PHF reconstruction and the development of methods for PHF structure determination. The emphasis is put on the monoclonal antibodies used as structural molecular probes for research on the role of IDPs in pathogenesis of neurodegenerative diseases.Dedicated to the late Peter Kontsek.     

The Abnormally Phosphorylated Tau Lesion of Early Alzheimer’s Disease

The perirhinal cortex (area 35) is well-known locus for neurofibrillary tangles (NFT) in initial Alzheimer’s disease (AD) and fully developed AD and may contain tau alterations in non-demented elderly. The topography and location of this vulnerable cortex, however, is difficult to appreciate because of its variable architecture and to deviations imposed by temporal sulcal patterns. We have immunostained human brains with a short duration of dementia using antibody AT8, which recognize abnormally hyperphosphorylated tau, calcium binding protein-parvalbumin and other phenotype markers to more fully appreciate the extent of area 35 before it is obscured by pathology. We have observed in the mildly affected AD tau immunoreactive lesion that extends from the temporopolar/insular region anteriorly to the posterior parahippocampal cortex. In its anterior–posterior course, it covers the medial bank of the collateral sulcus. Although the tau lesion encroaches slightly into the temporopolar cortex (area TG) anteriorly and medially and the ectorhinal cortex (area 36) laterally, area 35 is unambiguously defined. Ventromedial temporal pathology as revealed by AT8 suggests the presence of a relatively large lesion early in AD involving all of the perirhinal cortex and other non-isocortical areas. The present study demonstrated that the early stage AD patients exhibited AT8 immunoreactive cells in the temporopolar, hippocampus, perirhinal, entorhinal, and insular cortices. Special issue article in honor of Dr. George DeVries.     

Tau phosphorylation and aggregation in Alzheimer's disease pathology

In this article I shall review how tau phosphorylation and aggregation participates in Alzheimer's disease (AD) and other tauopathies. Tau, a microtubule associated protein, is the main component, in phosphorylated form, of the aberrant paired helical filaments found in AD. Tau is present in phosphorylated and aggregated form not only in AD, but in other pathologies (tauopathies). In this review, the phosphorylation of tau, its aggregation, and the possible relation between tau phosphorylation and aggregation is, briefly, described. Also, it is discussed the toxicity of modified tau. In addition, I propose a working model detailing the progression of tau pathologies.     

Molecular aging of tau: disulfide-independent aggregation and non-enzymatic degradation in vitro and in vivo

Smearing from high-molecular-mass regions to low-molecular-mass regions on western blot is the most striking observation of the tau making up paired helical filaments in brain tissues affected by Alzheimer's disease. Because our previous study showed site-specific deamidation/isomerization in the smeared tau in vivo, a feature of protein aging, recombinant tau was subjected to prolonged (up to 90 days) in vitro incubation. Carboxymethylated tau at approximately 50 kDa gradually disappeared and was converted to dimers and to high- and low-molecular-mass smearing. In addition, the same site-specific deamidation/isomerization as previously identified in the smeared tau in vivo emerged. Most importantly, tau was spontaneously degraded, generating fragments that start from bulky residues next to asparaginyl residues. This spontaneous degradation of tau probably represents non-enzymatic cleavage through the formation of succinimide intermediates. Similar degradation products starting from the bulky residues next to asparaginyl residues were found in the smeared tau in vivo partially purified from the homogenates from Alzheimer's disease brains.     

The role of cholesterol in pathogenesis of Alzheimer's disease: dual metabolic interaction between amyloid beta-protein and cholesterol

The implication that cholesterol plays an essential role in the pathogenesis of Alzheimer’s disease (AD) is based on the 1993 finding that the presence of apolipoprotein E (apoE) allele ε4 is a strong risk factor for developing AD. Since apoE is a regulator of lipid metabolism, it is reasonable to assume that lipids such as cholesterol are involved in the pathogenesis of AD. Recent epidemiological and biochemical studies have strengthened this assumption by demonstrating the association between cholesterol and AD, and by proving that the cellular cholesterol level regulates synthesis of amyloid β-protein (Aβ). Yet several studies have demonstrated that oligomeric Aβ affects the cellular cholesterol level, which in turn has a variety of effects on AD-related pathologies, including modulation of tau phosphorylation, synapse formation and maintenance of its function, and the neurodegenerative process. All these findings suggest that the involvement of cholesterol in the pathogenesis of AD is dualistic—it is involved in Aβ generation and in the amyloid cascade, leading to disruption of synaptic plasticity, promotion of tau phosphorylation, and eventual neurodegeneration. This review article describes recent findings that may lead to the development of a strategy for AD prevention by decreasing the cellular cholesterol level, and also focuses on the impact of Aβ on cholesterol metabolism in AD and mild cognitive impairment (MCI), which may result in promotion of the amyloid cascade at later stages of the AD process.     

Extracellular tau is toxic to neuronal cells

The degeneration of neurons in disorders such as Alzheimer's disease has an immediate consequence, the release of intracellular proteins into the extracellular space. One of these proteins, tau, has proven to be toxic when added to cultured neuronal cells. This toxicity varies according to the degree of protein aggregation. The addition of tau to cultured neuroblastoma cells provoked an increase in the levels of intracellular calcium, which is followed by cell death. We suggest that this phenomenon may be mediated by the interaction of tau with muscarinic receptors, which promotes the liberation of calcium from intracellular stores.     

The Role of Neurotrophins in Brain Aging: A Perspective in Honor of Regino Perez-Polo

During brain aging and progression of Alzheimer’s disease, the levels of Aβ and proinflammatory cytokines accumulate very early in the pathogenic process prior to any major degenerative changes. Accumulation of these molecules may impair with signal transduction pathways critical for neuronal health. Neurotrophin signaling is a critical mechanism involved in synaptic plasticity, learning and memory and neuronal health. We have recently shown that exposure to low levels of Aβ impairs BDNF trkB signal transduction, suppressing the Ras/ERK, and the PI3-K/Akt pathways but not the PLCγ pathway. As a result, downstream regulation of gene expression and neuronal viability are impaired. Recently, we have found that at least three agents – Aβ, TNFα, Il-1β – suppress TrkB signaling and act via a common and novel mechanism. These factors all regulate the docking proteins (e.g., IRS and Shc) that link the activated Trk receptor to downstream effectors. While this is a novel mechanism underlying regulation of Trk signaling, such a mechanism has been identified for the insulin/IGF-1 receptor in the presence of proinflammatory cytokines and is one of the mechanisms for insulin/IGF-resistance, which is a key risk factor for type II diabetes (1). We suggest that accumulation of AB and proinflammatory cytokines during aging generates in the brain a “neurotrophin resistance” state that places the brain at risk for cognitive decline and dementia.     

Dopaminergic Agonists: Possible Neurorescue Drugs Endowed with Independent and Synergistic Multisites of Action

Dopaminergic agonists have been usually used as adjunctive therapy for the cure of Parkinson’s disease (PD). It is generally believed that treatment with these drugs is symptomatic rather then curative and does not stop or delay the progression of neuronal degeneration. However, several DA agonists of the DA D2–receptor family (including D2, D3 and D4-subtypes) have recently been shown to possess neuroprotective properties in different in vitro and in vivo experimental PD models. Here we summarize some recent data from our and other groups underlining the wide pharmacological spectrum of DA agonists currently used for treating PD patients. In particular, the mechanism of action of different DA agonists does not appear to be restricted to the stimulation of selective DA receptor subtypes being these drugs endowed with intrinsic, independent, and peculiar antioxidant effects. This activity may represent an additional pharmacological property contributing to their clinical efficacy in PD. Special issue dedicated to Dr. Moussa Youdim.     

The role of exosomes in the processing of proteins associated with neurodegenerative diseases

Exosomes are small membranous vesicles secreted by a number of cell types and can be isolated from conditioned cell media or bodily fluids such as urine and plasma. Exosome biogenesis involves the inward budding of multivesicular bodies (MVB) to form intraluminal vesicles (ILV). When fused with the plasma membrane, the MVB releases the vesicles into the extracellular environment as exosomes. Proposed functions of these vesicles include roles in cell–cell signalling, removal of unwanted proteins, and the transfer of pathogens between cells, such as HIV-1. Another such pathogen which exploits this pathway is the prion, the infectious particle responsible for the transmissible neurodegenerative diseases such as Creutzfeldt-Jakob disease (CJD) of humans or bovine spongiform encephalopathy (BSE) of cattle. Interestingly, this work is mirrored by studies on another protein involved in neurodegenerative disease, the amyloid precursor protein (APP) which is associated with Alzheimer’s disease (AD). Recent work has found APP proteolytic fragments in association with exosomes, suggesting a common pathway previously unknown for proteins associated with neurodegenerative diseases. This review will be discussing the current literature regarding the role of exosomes in secretion of the proteins, PrP and APP, and the subsequent implications for neurodegenerative disease. Australian Society for Biophysics Special Issue: Metals and Membranes in Neuroscience.     

Genes and the Environment in Neurodegeneration

Neurodegenerative diseases are a heterogeneous group of pathologies which includes complex multifactorial diseases, monogenic disorders and disorders for which inherited, sporadic and transmissible forms are known. Factors associated with predisposition and vulnerability to neurodegenerative disorders may be described usefully within the context of gene–environment interplay. There are many identified genetic determinants for neurodegeneration, and it is possible to duplicate many elements of recognized human neurodegenerative disorders in animal models of the disease. However, there are similarly several identifiable environmental influences on outcomes of the genetic defects; and the course of a progressive neurodegenerative disorder can be greatly modified by environmental elements. In this review we highlight some of the major neurodegenerative disorders (Alzheimer’s disease, Parkinson’s disease, Amyotrophic lateral sclerosis, Huntington’s disease, and prion diseases.) and discuss possible links of gene–environment interplay including, where implicated, mitochondrial genes.     

The cyanobacterial alkaloid nostocarboline: an inhibitor of acetylcholinesterase and trypsin

Preselected cyanobacterial strains (available from culture collections and our own isolates), belonging primarily to the heterocystous cluster, were screened for inhibitors against butyrylcholinesterase. About one-half of the extracts exhibited inhibitory activity. Nostocarboline, the responsible metabolite in Nostoc 78–12A, was studied in more detail as an acetylcholinesterase (AChE) inhibitor. The compound showed potent activity against this enzyme (IC₅₀ = 5.3 μM), and the Michaelis-Menten kinetics indicated a non-competitive component in the inhibitory mechanism. In addition, nostocarboline turned out to be a potent inhibitor of trypsin (IC₅₀ = 2.8 μM), and thus is the first described cyanobacterial serine protease inhibitor of an alkaloid structure. The function of nostocarboline in aquatic ecosystems and its potential as a lead compound for the development of useful therapeutic AChE inhibitors is discussed.     

Degeneration of growth cones in a culture of embryonic neurons of mouse with presenilin 1 knockout

A comparative study of growth cone morphology in cultured embryonic neurons derived from wild type PS1(+/+) and knockout PS1(?/?) mice has been performed. Growth cones from wild type PS1(+/+) mice were well spread and usually formed radially continuous and regular lamellar extensions, short filopodia. In contrast, most growth cones from knockout PS1(?/?) mice collapsed after 3–4 days in culture. Summarizing these data, we suggest that PS1 plays an important role in growth cone structure by stabilizing the integrity of the cytoskeleton. The growth cone collapse may be the main reason of abnormal neuronal migration and impaired synaptic function in PS1(?/?) mice.     

Modeling synucleinopathies in genetically modified animals: Successes and failures

The synuclein family and particularly α-synuclein takes a central part in aetiology and pathogenesis of Parkinson’s disease—one of the most common human neurodegenerative diseases. The pathological changes in certain other neurodegenerative diseases are also linked to changes in the metabolism and function of α-synuclein, hence comprising a new group of diseases—synucleinopathies. The molecular and cellular mechanisms that are involved in the development of neurodegeneration in synucleinopathies are still largely unknown. As a result, the therapeutic approaches to the treatment of synucleinopathies are inadequately tampered. The development of models of neurodegenerative process in laboratory animals plays a crucial role in the study of these molecular mechanisms. Recently a special emphasis was placed on transgenic animal models with modified expression of genes, whose mutations are associated with inherited forms of human neurodegenerative diseases. The current review is devoted to the analysis of different models of synucleinopathies as a result of genetic modifications of α-synuclein expression.     

A comparison of the neuronal dysfunction caused by <Emphasis Type="Italic">Drosophila</Emphasis> tau and human tau in a <Emphasis Type="Italic">Drosophila</Emphasis> model of tauopathies

Hyperphosphorylation and aggregation of tau into tangles is a feature of disorders such as Alzheimer’s disease and other Tauopathies. To model these disorders in Drosophila melanogaster, human tau has been over-expressed and a variety of phenotypes have been observed including neurotoxicity, disrupted neuronal and synaptic function and locomotor impairments. Neuronal dysfunction has been seen prior to neuronal death and in the absence of tangle formation. The Drosophila tau protein shares a large degree of homology with human tau but differs in the crucial microtubule binding domains. Although like human tau Drosophila tau can induce neurotoxicity, little is known about its ability to disrupt neuronal function. In this study we demonstrate that like human tau, over-expression of Drosophila tau results in disrupted axonal transport, altered neuromuscular junction morphology and locomotor impairments. This indicates that like human tau, over-expression of Drosophila tau compromises neuronal function despite significant differences in microtubule binding regions.     

Hyperphosphorylation and Accumulation of Neurofilament Proteins in Transgenic Mice with Alzheimer Presenilin 1 Mutation

Neurofilaments (NFs) are hyperphosphorylated and accumulate in Alzheimer’s disease (AD) brains. In this study, employing the transgenic mouse model, we explored the effect of presenilin 1 (PS-1) mutation on the phosphorylation and distribution of NFs. Western blot analysis showed that there was a significant increase in the phosphorylation of NF-H and NF-M subunits with a concomitant increase in phosphorylated c-Jun N-terminal protein kinase 1/2 (JNK1/2) mitogen-activated protein kinase (MAPK) in hippocampus of PS-1 transgenic mice compared to that of wild-type littermates. Immunohistochemical analysis revealed that phosphorylated NFs accumulated throughout the hippocampal neurons of the transgenic mice. These findings suggest that PS-1 mutation may induce hyperphosphorylation and accumulation of NFs via a JNK1/2-involved mechanism.     

The Polyphenol Piceid Destabilizes Preformed Amyloid Fibrils and Oligomers In Vitro: Hypothesis on Possible Molecular Mechanisms

Alzheimer’s disease (AD) is characterized by deposits of amyloid in various tissues. The neuronal cytotoxicity of Aβ peptides is attributed not only to various mechanisms but also to amyloid fibrils and soluble oligomeric intermediates. Consequently, finding molecules to prevent or reverse the oligomerization and fibrillization of Aβ could be of therapeutic value in the treatment of AD. We show that piceid, a polyphenol of the stilbene family, destabilized fibrils and oligomers to give back monomers that are not neurotoxic molecules. The mechanism of this destabilization could be a dynamic interaction between the polyphenol and the Aβ that could open the hydrophobic zipper and shift the reversible equilibrium “random coil⇔β-sheet” to the disordered structure.     

Free Copper,Ferroxidase and SOD1 Activities,Lipid Peroxidation and NO<Subscript><Emphasis Type="Italic">x</Emphasis></Subscript> Content in the CSF. A Different Marker Profile in Four Neurodegenerative Diseases

The understanding of oxidative damage in different neurodegenerative diseases could enhance therapeutic strategies. Our objective was to quantify lipoperoxidation and other oxidative products as well as the activity of antioxidant enzymes and cofactors in cerebrospinal fluid (CSF) samples. We recorded data from all new patients with a diagnosis of either one of the four most frequent neurodegenerative diseases: Parkinson’s disease (PD), Alzheimer’s disease (AD), Huntington’s disease (HD) and lateral amyotrophic sclerosis (ALS). The sum of nitrites and nitrates as end products of nitric oxide (NO) were increased in the four degenerative diseases and fluorescent lipoperoxidation products in three (excepting ALS). A decreased Cu/Zn-dependent superoxide dismutase (SOD) activity characterized the four diseases. A significantly decreased ferroxidase activity was found in PD, HD and AD, agreeing with findings of iron deposition in these entities, while free copper was found to be increased in CSF and appeared to be a good biomarker of PD.