Direct demonstration of abnormal phosphorylation of Tau microtubular proteins in Alzheimer's disease

Two polyclonal antibodies, the first raised against Alzheimer's disease PHF and the second raised against human native Tau proteins, led us to find two Tau proteins with an abnormal molecular weight of 64 and 69 kDa in Alzheimer brain cortices. Tau 64 and Tau 69 were never detected in control brains. The molecular weight of Tau 64 and 69 dramatically decreased after dephosphorylation by the alkaline phosphatase, showing that they are abnormally phosphorylated. This is the first report demonstrating their specific presence in brain regions having the Alzheimer pathology. They could be a very useful tool for the study of the early events that lead to neuronal death.     

The regulatory effect of Tau protein on polymerization of MCF7 microtubules in vitro

Growing evidence continues to point toward the critical role of beta tubulin isotypes in regulating some intracellular functions. Changes that were observed in the microtubules’ intrinsic dynamics, the way they interact with some chemotherapeutic agents, or differences on translocation specifications of some molecular motors along microtubules, were associated to their structural uniqueness in terms of beta tubulin isotype distributions. These findings suggest that the effects of microtubule associated proteins (MAPs) may also vary on structurally different microtubules. Among different microtubule associated proteins, Tau proteins, which are known as neuronal MAPs, bind to beta tubulin, stabilize microtubules, and consequently promote their polymerizations.In this study, in a set of well controlled experiments, the direct effect of Tau proteins on the polymerization of two structurally different microtubules, porcine brain and breast cancer (MCF7), were tested and compared. Remarkably, we found that in contrast with the promoted effect of Tau proteins on brain microtubules’ polymerization, MCF7 expressed a demoted polymerization while interacting with Tau proteins. This finding can potentially be a novel insight into the mechanism of drug resistance in some breast cancer cells.It has been reported that microtubules show destabilizing behavior in some MCF7 cells with overexpression of Tau protein when treated with a microtubules’ stabilizing agent, Taxol. This behavior has been classified by others as drug resistance, but it may instead be potentially caused by a competition between the destabilizing effect of the Tau protein and the stabilizing effect of the drug on MCF7 microtubules. Also, we quantified the polarization coefficient of MCF7 microtubules in the presence and absence of Tau proteins by the electro-orientation method and compared the values. The two significantly different values obtained can possibly be one factor considered to explain the effect of Tau proteins on the polymerization of MCF7 microtubules.     

Identification of Stk25 as a genetic modifier of Tau phosphorylation in Dab1-mutant mice

Hyperphosphorylation of the microtubule binding protein Tau is a feature of a number of neurodegenerative diseases, including Alzheimer's disease. Tau is hyperphosphorylated in the hippocampus of dab1-null mice in a strain-dependent manner; however, it has not been clear if the Tau phosphorylation phenotype is a secondary effect of the morbidity of these mutants. The dab1 gene encodes a docking protein that is required for normal brain lamination and dendritogenesis as part of the Reelin signaling pathway. We show that dab1 gene inactivation after brain development leads to Tau hyperphosphorylation in anatomically normal mice. Genomic regions that regulate the phospho Tau phenotype in dab1 mutants have previously been identified. Using a microarray gene expression comparison between dab1-mutants from the high-phospho Tau expressing and low-phospho Tau expressing strains, we identified Stk25 as a differentially expressed modifier of dab1-mutant phenotypes. Stk25 knockdown reduces Tau phosphorylation in embryonic neurons. Furthermore, Stk25 regulates neuronal polarization and Golgi morphology in an antagonistic manner to Dab1. This work provides insights into the complex regulation of neuronal behavior during brain development and provides insights into the molecular cascades that regulate Tau phosphorylation.     

Qualitative and quantitative comparison of brain proteins in Alzheimer's disease

In human brain extracts, most proteins of pathological interest in Alzheimer's disease are insoluble and their analysis is often performed on denatured and reduced samples by immunoblotting after electrophoresis on polyacrylamide gel in presence of sodium dodecyl sulfate. Because we needed to accurately compare the concentration of several proteins in brain extracts to investigate the etiology of the disease, the quantitative aspect of immunoblotting was assessed and the results compared for a soluble component with those obtained by electroimmunoassay. Glial fibrillary acidic protein (GFAP) and Tau proteins were analysed by immunoblotting in brain homogenates treated with the Laemmli sample buffer from 10 control and 25 Alzheimer's disease brains. The linearity of densitometric measures of dilutions for one given sample was demonstrated. A 8 to 16-fold GFAP increase in Alzheimer brain was established. With regard to Tau proteins it was possible to show the presence of two pathological Tau variants (Tau 64 and 69) in all the Alzheimer brain homogenates, furthermore, the amount of Tau 64 and 69 was proportional to the presence of neurofibrillary degeneration. As far as alpha 1-antichymotrypsin is concerned, we showed, in a second set of brain samples (14 control and 12 Alzheimer brains), discrepancies between the results obtained by immunoblotting and by electroimmunoassay while for a given sample linearity of immunoblotting measures of dilutions of this sample was demonstrated. Quantitation by immunoblotting of such components which can be quantified using other procedures is uncertain whereas the interest of immunoblotting is undoubted for the insoluble proteins in the brain extracts.     

Abnormal tau species are produced during Alzheimer's disease neurodegenerating process

Tau proteins were detected in human brain using two polyclonal antibodies: anti-paired helical filaments and anti-human native tau proteins. Both antisera detected identically the normal set of tau proteins in control brains. Moreover they detected two abnormal tau variants of 64 and 69 kDa exclusively in brain areas showing neurofibrillary tangles and senile plaques. Tau 64 and 69 were abnormally phosphorylated as revealed by the decrease in their molecular mass observed after alkaline phosphatase treatment. Therefore, tau 64 and 69 are specific markers of the neurofibrillary degeneration of the Alzheimer type and might be useful tools for studying the first pathological events that lead to neuronal death.     

Tau protein as a differential biomarker of tauopathies

Microtubule-associated Tau proteins are the basic component of intraneuronal and glial inclusions observed in many neurological disorders, the so-called tauopathies. Many etiological factors, phosphorylation, splicing, and mutations, relate Tau proteins to neurodegeneration. Molecular analysis has revealed that hyperphosphorylation and abnormal phosphorylation might be one of the important events in the process leading to tau intracellular aggregation. Specific set of pathological tau proteins exhibiting a typical biochemical pattern, and a different regional and laminar distribution, could characterize five main classes of tauopathies. A direct correlation has been established between the regional brain distribution of tau pathology and clinical symptoms; for instance progressive involvement of neocortical areas is well correlated to the severity of dementia in Alzheimer's disease, overall suggesting that pathological tau proteins are reliable marker of the neurodegenerative process. Recent discovery of tau gene mutations in frontotemporal dementia with parkinsonism linked to chromosome 17 has reinforced the predominant role attributed to tau proteins in the pathogenesis of neurodegenerative disorders, and underlined the fact that distinct sets of tau isoforms expressed in different neuronal populations could lead to different pathologies. Overall, a better knowledge of the etiological factors responsible for the aggregation of tau proteins in brain diseases is essential for development of future differential diagnosis and therapeutic strategies. They would hopefully find their application against Alzheimer's disease but also in all neurological disorders for which a dysfunction of Tau biology has been identified.     

Docosahexaenoic Acid Promotes Axon Outgrowth by Translational Regulation of Tau and Collapsin Response Mediator Protein 2 Expression

n-3 PUFAs are essential for neuronal development and brain function. However, the molecular mechanisms underlying their biological effects remain unclear. Here we examined the mechanistic action of docosahexaenoic acid (DHA), the most abundant n-3 polyunsaturated fatty acids in the brain. We found that DHA treatment of cortical neurons resulted in enhanced axon outgrowth that was due to increased axon elongation rates. DHA-mediated axon outgrowth was accompanied by the translational up-regulation of Tau and collapsin response mediator protein 2 (CRMP2), two important axon-related proteins, and the activation of Akt and p70 S6 kinase. Consistent with these findings, rapamycin, a potent inhibitor of mammalian target of rapamycin (mTOR), prevented DHA-mediated axon outgrowth and up-regulation of Tau and CRMP2. In addition, DHA-dependent activation of the Akt-mTOR-S6K pathway enhanced 5′-terminal oligopyrimidine tract-dependent translation of Tau and CRMP2. Therefore, our results revealed an important role for the Akt-mTOR-S6K pathway in DHA-mediated neuronal development.     

Tau蛋白在阿尔茨海默病中的作用

阿尔茨海默病(AD)是非常普遍的神经变性性疾病并且是老年人痴呆的主要原因。AD患者的症状特点包括进行性的认知障碍、记忆丧失和行为障碍,与大脑中的病理变化密切相关。AD现成为全球最严重的健康和社会经济问题。在AD患者脑中神经纤维网或神经营养障碍的过程中存在tau蛋白的异常。tau蛋白丧失其促微管组装的生物学功能,导致细胞骨架的破坏、丝状物形成和神经缠结,轴突运输损害,进而导致突触蛋白失去功能和神经退行性病变。其数量和结构的改变将会影响其功能而且会出现异常聚集。调节Tau蛋白的异常聚集的分子机制主要是一些翻译后修饰使其结构及构象发生变化。因此,异常磷酸化和截断的tau蛋白作为tau蛋白病理过程的关键机制而引起学者关注。本文描述了tau蛋白的结构和功能及其在AD中的主要病理变化,同时在本文中还涉及到磷酸化的tau蛋白是神经元对氧化应激的代偿反应这一观点。对tau蛋白进行更加全面的解读。     

Tau story: from frontotemporal dementia to other tauopathies

Tau proteins belong to the family of microtubule-associated proteins. They are mainly expressed in neurons where they play an important role in the assembly of tubulin monomers into microtubules to constitute the neuronal microtubules network. Tau proteins are translated from a single gene located on chromosome 17. Their expression is developmentally regulated by an alternative splicing mechanism and six different isoforms exist in the human adult brain. Tau proteins are the major constituents of fibrillar lesions described in Alzheimer's disease and numerous neurodegenerative disorders referred to as 'tauopathies'. Molecular analysis has revealed that an abnormal phosphorylation might be one of the important events in the process leading to their aggregation. Moreover, a specific set of pathological tau proteins exhibiting a typical biochemical pattern, and a different regional and laminar distribution could characterize each of these disorders. Finally, the recent discovery of tau gene mutations in fronto-temporal dementia with parkinsonism linked to chromosome 17 has reinforced the direct role attributed to tau proteins in the pathogenesis of neurodegenerative disorders, and underlined the fact that distinct sets of tau isoforms expressed in different neuronal populations could lead to different pathologies. Conversely, recent data in myotonic dystrophy has demonstrated that indirect effect (CTG repeat expansion) leading to variations in tau alternative splicing also produce neurofibrillary degeneration.     

The Rate of Tau Synthesis Is Differentially Regulated During Postnatal Development in Mouse Cerebellum

1. Tau, which is a microtubule-associated protein, with mRNA targeted to the axon and growth cone, is involved in axonal elongation. During postnatal development in mouse, Tau expression in cerebellar granule cells is reduced after the second postnatal week. The aim of this work was to study the regulation of the rate of the synthesis of Tau protein during the period of granule cell axonal growth in mouse cerebellum.2. We found four [³⁵S]methionine-labeled isoforms of Tau synthesized postnataly. Their levels remain constant from postnatal day 9 to 12 (P9–P12), and decreased by P20.3. The rate of Tau synthesis showed differences with the rate of synthesis of total proteins. They also differ from proteins phosphatases 2A and 2B, both associated with the regulation of Tau function. In addition, the turnover of newly synthesized Tau increased at P20, compared with P9 and P12.4. These results imply a specific developmental regulation of mRNA translation of Tau, and indicate that, after the period of synapse formation is complete, and therefore axonal growth has finished (P20), only a limited number of new Tau molecules are synthesized. This might reflect that, after synapse formation is complete, newly synthesized Tau molecules are not longer needed.     

Tau protein and neurodegeneration

Many of the human neurodegenerative conditions involve a reorganization of the neuronal cytoskeleton. The way in which the cytoskeleton is reorganized may provide a clue to the nature of the insult causing the neurodegeneration. The most common of these conditions is Alzheimer's disease, in which microtubules are lost from neurites that fill up with filamentous structures. One component of the filamentous structures is the microtubule-associated protein (MAP), tau. The tau protein is the product of a single gene expressed predominantly in neurons. The tau gene undergoes complex alternative splicing that is regulated both by development, and by the particular neuronal cell population in which it is expressed. Tau protein can be further modified, following its translation by phosphorylation at several sites. Much of the recent interest in the transition of tau to an abnormal state within a tangle-bearing neuron has focused on phosphorylation. A group of proteins that migrate slightly more slowly than tau, designated PHF-tau, are found in regions of the Alzheimer brain rich in dystrophic neurites, are hyperphosphorylated, fail to bind to microtubules, have distinct solubility properties, and can be derived from fractions of paired helical filaments (PHF).     

Changes with aging in the levels of amino acids in rat CNS structural elements II. Taurine and small neutral amino acids

Taurine (Tau) and the small neutral amino acids glycine (Gly), serine (Ser), threonine (Thr), and alanine (Ala) were measured in 53 brain areas of 3- and 29-month-old male Fisher 344 rats. The ratio of highest to lowest level was 34 for Tau, 9.1 for Thr, 7.6 for Gly and Ser, and 6.5 for Ala. The heterogeneity was found in numerous areas; for example, Tau levels were more than 90 nmol/mg protein in 6 areas, and less than 20 nmol/mg protein in 10 areas. Similar heterogeneity was found with the other amino acids. The relative distribution of the small neutral amino acids showed several similarities; Tau distribution was different. With age, four amino acids decreased in 10–18 areas, and increased in only 1–3, while Thr increased in more areas than it decreased. The five amino acids of this paper, and the four of the previous paper, are among the amino acids at highest level in the brain; the sequence in their levels shows considerable regional heterogeneity.     

Developmental regulation of tau phosphorylation, tau kinases, and tau phosphatases

Tau is a neuronal microtubule-associated protein. Its hyperphosphorylation plays a critical role in Alzheimer disease (AD). Expression and phosphorylation of tau are regulated developmentally, but its dynamic regulation and the responsible kinases or phosphatases remain elusive. Here, we studied the developmental regulation of tau in rats during development from embryonic day 15 through the age of 24 months. We found that tau expression increased sharply during the embryonic stage and then became relatively stable, whereas tau phosphorylation was much higher in developing brain than in mature brain. However, the extent of tau phosphorylation at seven of the 14 sites studied was much less in developing brain than in AD brain. Tau phosphorylation during development matched the period of active neurite outgrowth in general. Tau phosphorylation at various sites had different topographic distributions. Several tau kinases appeared to regulate tau phosphorylation collectively at overlapping sites, and the decrease of overall tau phosphorylation in adult brain might be due to the higher levels of tau phosphatases in mature brain. These studies provide new insight into the developmental regulation of site-specific tau phosphorylation and identify the likely sites required for the abnormal hyperphosphorylation of tau in AD.     

Pinning down phosphorylated tau and tauopathies

Neurofibrillary tangles (NFTs) are prominent neuronal lesions in a large subset of neurodegenerative diseases, including Alzheimer's disease (AD). NFTs are mainly composed of insoluble Tau that is hyperphosphorylated on many serine or threonine residues preceding proline (pSer/Thr-Pro). Tau hyperphosphorylation abolishes its biological function to bind microtubules and promotes microtubule assembly and precedes neurodegeneration. Not much is known about how tau is further regulated following phosphorylation. Notably, we have recently shown that phosphorylated Ser/Thr-Pro motifs exist in two distinct conformations. The conversion between two conformations in some proteins is catalyzed by the prolyl isomerase Pin1. Pin1 binds to tau phosphorylated specifically on the Thr231-Pro site and probably catalyzes cis/trans isomerization of pSer/Thr-Pro motif(s), thereby inducing conformational changes in tau. Such conformational changes can directly restore the ability of phosphorylated Tau to bind microtubules and promote microtubule assembly and/or facilitate tau dephosphorylation by its phosphatase PP2A, as PP2A activity is conformation-specific. Furthermore, Pin1 expression inversely correlates with the predicted neuronal vulnerability in normally aged brain and also with actual neurofibrillary degeneration in AD brain. Moreover, deletion of the gene encoding Pin1 in mice causes progressive age-dependent neuropathy characterized by motor and behavioral deficits, tau hyperphosphorylation, tau filament formation and neuronal degeneration. Distinct from all other mouse models where transgenic overexpression of specific proteins elicits tau-related pathologies, Pin1 is the first protein whose depletion causes age-dependent neurodegeneration and tau pathologies. Thus, Pin1 is pivotal in maintaining normal neuronal function and preventing age-dependent neurodegeneration. This could represent a promising interventive target to prevent neurodegenerative diseases.     

人类神经Tau蛋白结构与功能研究进展

Ｔａｕ属微管结合蛋白,对神经细胞的生长发育,物质运输及信息传导起着重要作用。该分子不具有明显的二级结构,其生物学功能的调节功能主要是通过磷酸化和去磷酸化来实现。目前的研究表明,早老性痴呆等疾病与Ｔａｕ分子高级结构的异常改变和功能丧失有关。     

肝性脑病患者脑脊液Tau 蛋白水平测定及其临床意义

目的:探讨肝性脑病患者脑脊液中总Tau蛋白(t-Tau)和磷酸化Tau蛋白(p-Tau)水平的变化及其与疾病严重程度的相关性。方法:采集26例肝性脑病患者及31例健康对照的脑脊液标本,采用酶联免疫吸附法(Elisa)检测脑脊液中的t-Tau和p-Tau水平,分析其与Child-Pugh评分和West Haven分级的相关性。结果:(1)肝性脑病患者脑脊液t-Tau和p-Tau水平显著高于健康对照组(P0.01)。(2)肝性脑病患者脑脊液t-Tau(γ=0.876,P0.01;γ=0.952,P0.01)、p-Tau(γ=0.808,P0.01;γ=0.808,P0.01)水平与Child-Pugh评分及West-Haven分级呈正相关。结论:由于脑脊液t-Tau和p-Tau水平为神经元损害的标志物,同时反应脑内应激状况,本研究证实肝性脑病患者脑内处于应激状态并有神经元损伤。     

Use of proteomics for the identification of novel drug targets in brain diseases

In spite of the rapid advances in the development of the new proteomic technologies, there are, to date, relatively fewer studies aiming to explore the neuronal proteome. One of the reasons is the complexity of the brain, which presents high cellular heterogeneity and a unique subcellular compartmentalization. Therefore, tissue fractionation of the brain to enrich proteins of interest will reduce the complexity of the proteomics approach leading to the production of manageable and meaningful results. In this review, general considerations and strategies of proteomics, the advantages and challenges to exploring the neuronal proteome are described and summarized. In addition, this article presents an overview of recent advances of proteomic technologies and shows that proteomics can serve as a valuable tool to globally explore the changes in brain proteome during various disease states. Understanding the molecular basis of brain function will be extremely useful in identifying novel targets for the treatment of brain diseases.     

Differential phosphorylation of tau proteins during kitten brain development and Alzheimer's disease

Differential distribution and phosphorylation of tau proteins were studied in developing kitten brain by using several antibodies, and was compared to phosphorylation in Alzheimer's disease. Several antibodies demonstrated the presence of phosphorylated tau proteins during kitten brain development and identified pathological structures in human brain tissue. Antibody AD2, recognized tau in kittens and adult cats, but reacted in Alzheimer's tissue only with a pathological tau form. Antibody AT8 was prominent in developing kitten neurons and was found in axons and dendrites. After the first postnatal month this phosphorylation type disappeared from axons. Furthermore, dephosphorylation of kitten tau with alkaline phosphatase abolished immunoreactivity of AT8, but not that of AD2, pointing to a protection of the AD2 epitope in cats. Tau proteins during early cat brain development are phosphorylated at several sites that are also phosphorylated in paired helical filaments during Alzheimer's disease. In either event, phosphorylation of tau may play a crucial role to modulate microtubule dynamics, contributing to increased microtubule instability and promoting growth of processes during neuronal development or changing dynamic properties of the cytoskeleton and contributing to the formation of pathological structures in neurodegenerative diseases.     

Effects of neonatal hypothyroidism on rat brain gene expression.

To define at the molecular biological level the effects of thyroid hormone on brain development we have examined cDNA clones of brain mRNAs and identified several whose expression is altered in hypothyroid animals during the neonatal period. Clones were identified with probes prepared by subtractive or differential hybridization, and those corresponding to mRNAs altered in hypothyroidism were further studied by Northern blot analysis. Using RNA prepared from whole brains, no effect of hypothyroidism was found on the expression of the astroglial gene coding for glial fibrillary acidic protein. Among genes of neuronal expression, no significant alterations were found in the steady state levels of mRNAs coding for neuron-specific enolase, microtubule-associated protein-2, Tau, or nerve growth factor. N-CAM mRNA increased slightly in hypothyroid brains. In contrast a 2- to 3-fold decrease was found in the mRNA coding for a novel neuronal gene, RC3. This is the first neuronal gene known to be significantly altered at the mRNA level by thyroid hormone deprivation. The abundance of the mRNAs for the major myelin proteins proteolipid protein, myelin basic protein, and myelin-associated glycoprotein, expressed by oligodendrocytes, were also decreased in hypothyroid brains. Developmental studies on RC3 and myelin-associated glycoprotein expression indicated that the corresponding mRNAs accumulate in the brain of normal rats during the first 15-20 days of neonatal life. A similar accumulation occurred in hypothyroid brains, but at much reduced levels. The results demonstrate that thyroid hormone controls the steady state levels of particular mRNAs during brain development.