Estudios sobre la fosforilación y agregación de la proteína tau y su posible relación con la enfermedad de Alzheimer

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 28-03-2008The appearance of aberrant structures, in Alzheimer disease, like neurofibrillary tangles, senile plaques or Hirano bodies has been described. Although the etiology of Alzheimer’s disease is still incompletely understood it is thought that oxidative stress could play an important role in the onset and the progression of the disease. In addition to the oxidative stress promoted by amyloid β aggregates, mitochondria generate most of the reactive oxygen species produced during disease. Coenzyme Q is an important cofactor of mitochondrial bioenergetics and it has been directly involved, together with secondary metabolites obtained from xenobiotics metabolism, with aging related energetic changes. Quinones represent a class of intermediary metabolites that may produce cellular damage. The toxicity of quinones may take place through arylation reactions of important biomolecules like DNA or proteins. Alternatively, quinones, depending on the redox state, could exert prooxidant or antioxidant effects. On the other hand, it has been described that compounds like 4-hydroxi-2-nonenal, which are products of lipid peroxidation, could favour the assembly of aberrant structures like paired helical filaments present in neurofibrillary tangles. Compounds that are produced as a consequence of oxidative stress also may affect other microtubular proteins like tubulin. Since it has been described the connection between oxidative stress and the assembly of aberrant structures, we wanted to study if quinones also could have the same effect on protein tau aberrant assembly and Hirano bodies formation. First, we have identified the presence of quinones both in paired helical filaments and Hirano bodies. Once we have stated quinones may be involved in the formation of these aberrant structures that appear in Alzheimer disease, we have studied the effect of this molecules on tau polymerization or aggregation both in vitro and in cell cultures. On the other hand we have also tested if quinones could facilitate the formation of Hirano-like bodies in vitro. Additionally, since microtubule network has been found affected in the onset and progression of Alzheimer disease we have studied the effect of quinones on microtubule network in cell cultures. We have observed that quinones could react with tubulin hampering microtubule polimerization. Thus, we propose that quinones produced by oxidative stress generated during Alzheimer disease, favour polymerization or aggregation of tau protein and prevent microtubule polymerization causing both disturbance on axonal transport and synaptic dysfunction. Other well known inducers of tau aggregation are high molecular weight sulphated glycosaminoglycans, which also are known to favour the Aβ peptide aggregation. Nowadays pharmaceutical companies are developing low molecular weight structural analogs of sulphated glycosaminoglycans to avoid the formation of Aβ aggregates. One of these compounds is Tramiprosate (3-APS). This compound is able to interact with Aβ peptide preventing its aggregation but until now, any research group was studied the effect of this small molecule on tau aggregation. Thus, and finally we have analyzed this interaction demonstrating that 3-APS facilitates aggregation of tau. In this way, also we have tested he effect of its structural analog, taurine, which may promote tau aggregation but in less extent. In summary, our work demonstrates that tau protein modifications provoked by oxidative stress could explain, in part, what is occurring in Alzheimer disease and other tauopathies: its hyperphosphorylation and aggregationIsmael Santa Maria Perez ha recibido financiación del Ministerio de Educación y Ciencia dentro del programa de Formación de Personal Investigador (BES-2004-5505). El trabajo del laboratorio ha sido financiado en parte por el Ministerio de Educación y Ciencia (SAF2003-02697) y por la Fundación LillyPeer reviewe

Quinones Facilitate the Self-Assembly of the Phosphorylated Tubulin Binding Region of Tau into Fibrillar Polymers.

The fragment of tau containing the first and third tubulin-binding motifs, involved in self- assembly of tau, was phosphorylated by protein kinase A (PKA). In the presence of hydroxynonenal (HNE) or in the presence of quinones such as juglone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q0 or DMM), or menadione, the polymerization of this phosphorylated tau fragment is catalyzed, whereas polymerization of the unmodified fragment takes place in a lesser extent. The quinones coenzyme Q0 and menadione are found in every cell, including neural cells, and may interact with tau protein to facilitate its assembly into filamentous structures. These tau filaments, assembled in the presence of quinones, have a fibrillar morphology very similar to that of paired helical filaments present in the brains of patients with Alzheimer’s disease.pre-print862 K

In vitro tau fibrillization: Mapping protein regions

AbstractWe have investigated the propensity to form fibrillar aggregates of a variety of fragments and variants of the tau protein under the influence of a tau fibrillization inducer: coenzyme Q0. To better identify fibrillization hotspots, we compare the polymerization propensity of tau fragments containing the sequence of putative hotspots with that of tau variants with that same sequence deleted. We also investigate the effects of biologically occurring modifications such as phosphorylation and deamidation. We found that residues 305 to 335 are essential for in vitro tau fibrillization. Residues 306 to 311 facilitate in vitro assembly, but are not sufficient to mimic the in vivo fibrillization of tau. Furthermore, the propensity of the 306–311 sequence to form fibrils is highly decreased by chemical modifications of tyrosine 310 that are commonly found in vivo

Looking at the periphery—new hypothesis to look for new targets for Alzheimer’s disease therapy

Currently, the predominant targets for the treatment of Alzheimer’s disease (AD) are the main components of the two pathological structures: senile plaques (composed of amyloid beta peptide aggregates) or neurofibrillary tangles (constructed of tau protein polymers). However, the existence of adequate disease modifiers based on such targets is discussed. In this special issue, it has been suggested to search for new possible targets for AD therapy. This contribution tries to analyze non-neuronal tissues (periphery) to identify potential factors (target) involved in the development of AD

Tau – an inhibitor of deacetylase HDAC6 function

Analysis of brain microtubule protein from patients with Alzheimer’s disease showed decreased alpha tubulin levels along with increased acetylation of the alpha tubulin subunit, mainly in those microtubules from neurons containing neurofibrillary tau pathology. To determine the relationship of tau protein and increased tubulin acetylation, we studied the effect of tau on the acetylation-deacetylation of tubulin. Our results indicate that tau binds to the tubulin-deacetylase, histone deacetylase 6 (HDAC6), decreasing its activity with a consequent increase in tubulin acetylation. As expected, increased acetylation was also found in tubulin from wild-type mice compared with tubulin from mice lacking tau because of the tau-mediated inhibition of the deacetylase. In addition, we found that an excess of tau protein, as a HDAC6 inhibitor, prevents induction of autophagy by inhibiting proteasome function.This work was supported by grants from Spanish Plan Nacional, Comunidad de Madrid, Fundación Botín, CIBERNED, and an institutional grant Fundación Areces.Peer reviewe

Near barrier scattering of 8He on 208Pb

The exotic nucleus 8He is investigated by means of the measurement of the angular distributions of the elastic channel and the 6He and 4He fragment yields produced in the collision with a 208Pb target at two energies around the Coulomb barrier, 16 and 22 MeV. The experiment was performed at the GANIL-SPIRAL facility, with the aim of extracting information about the structure of 8He and the relevant reaction mechanisms. In this contribution, details of the experimental setup and preliminary data on elastic cross sections are reporte

Differential gene expression analysis of human entorhinal cortex support a possible role of some extracellular matrix proteins in the onset of Alzheimer disease

The onset of Alzheimer's disease (AD) has been associated with the specific vulnerability of neurons in the upper layers of the entorhinal cortex. To define the molecular characteristics of those neurons, we have used microarrays to define the gene expression in that region. In this way, we identified several genes that are expressed distinctly in the upper and lower layers of the entorhinal cortex. These include the genes encoding the matrix Gla protein, collagen type 1α2, reelin, semaphorin 3C or the relaxin receptor, all related to the extracellular matrix. Thus, differences in the extracellular matrix components between the upper and lower layers of the entorhinal cortex may in part explain the vulnerability of neurons present in the upper layers of this brain region in disorders like AD.This work was supported by grants from the Spanish Plan Nacional, the Fundación CIEN, CIBERNED, the Comunidad de Madrid and the Fundación M. Botin.Peer reviewe

Taurine, an inducer for tau polymerization and a weak inhibitor for amyloid-β-peptide aggregation

Taurine is an abundant aminoacid present in brain. Its concentration is decreased in the brain of Alzheimer's disease (AD) patients. The chemical structure of taurine is similar to 3-amino-1-propanesulfonic acid, a known compound which interferes with beta-amyloid peptide aggregation. Here, we have tested if taurine show similar properties. Taurine slightly decreases beta-amyloid peptide aggregation at a milimolar concentration. At that concentration, taurine favours the assembly of tau protein into fibrillars polymers. Thus, it is proposed that the negative charge present in taurine may be involved in the binding to tau protein, facilitating its assembly. In addition, the possible role of taurine in Alzheimer disease is commented.This work was supported by grants from Plan Nacional (Ministerio de Educación y Ciencia, Spain), Comunidad de Madrid (NEURODEGMODELS-CM), Fundación Botin, and by an Institucional Grant of Fundación R. Areces. Also, this work is part to our contribution to CIBER Enfermedades Neurodegenerativas (CIBERNED) (Ministerio de Sanidad y Consumo).Peer reviewe

Quinones facilitate the self-assembly of the phosphorylated tubulin binding region of tau into fibrillar polymers

The fragment of tau containing the first and third tubulin-binding motifs, involved in self-assembly of tau, was phosphorylated by protein kinase A (PKA). In the presence of hydroxynonenal (HNE) or in the presence of quinones such as juglone, 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q0 or DMM), or menadione, the polymerization of this phosphorylated tau fragment is catalyzed, whereas polymerization of the unmodified fragment takes place in a lesser extent. The quinones coenzyme Q0 and menadione are found in every cell, including neural cells, and may interact with tau protein to facilitate its assembly into filamentous structures. These tau filaments, assembled in the presence of quinones, have a fibrillar morphology very similar to that of paired helical filaments present in the brains of patients with Alzheimer's disease.This work was supported by grants from CICYT, Comunidad de Madrid, Fundación Lilly, and Neuropharma and by an Institutional Grant of Fundación R. Areces.Peer reviewe

Characteristics of the binding of thioflavin S to tau paired helical filaments

Binding of histological benzothiazole dye thioflavin S (ThS) to protein aggregates has been related with the presence of amyloid β sheet structure in those protein aggregates. Paired helical filaments (PHF) from Alzheimer's disease (AD) patients, (whose main component is the microtubule associated protein, tau) bind to thioflavins. By using a novel immunofluorescence method, the binding of ThS to isolated tau filaments was tested. Also, the characteristics of this binding of ThS to PHF or to the in vitro assembled tau filaments, have been analyzed. Our results suggests that ThS binds to PHF with a higher affinity than to the straight filaments (SF), also found in AD.Peer reviewe