Raquel Cuadros

Extracellular tau is toxic to neuronal cells

The degeneration of neurons in disorders such as Alzheimers 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.

Phosphorylated, but not native, tau protein assembles following reaction with the lipid peroxidation product, 4-hydroxy-2-nonenal

A correlation between hyperphosphorylation of tau protein and its aberrant assembly into paired helical filaments has lead to suggestions that phosphorylation controls assembly, but lacked a mechanistic basic. In this work, we have found that phosphorylated, but not native, tau protein is able to form polymers after the reaction with 4‐hydroxy‐2‐nonenal, a highly toxic product of lipid peroxidation. Phosphorylation of tau by both proline or non‐proline directed kinases, was able to assemble it into polymers.

Regulation of GSK3 isoforms by phosphatases PP1 and PP2A

Dephosphorylation of phospho GSK3 isoforms, from COS-7 cells, was determined in vitro and in cultured cells in the absence or the presence of okadaic acid and lithium. Our results indicate a preferential dephosphorylation of phospho GSK3α by PP2A phosphatase, whereas dephosphorylation of phospho GSK3β mainly takes place by PP1 phosphatase.

Zeta 14-3-3 protein favours the formation of human tau fibrillar polymers.

Tau protein can aggregate, in an aberrant way, in Alzheimers disease and other tauopathies. The formation of those aggregates could take place in vitro by the addition of different compounds like polyanions or fatty acids and their derivates. Now, we found that a protein, zeta 14-3-3, facilitates the assembly of tau as well as a tau peptide containing the self-assembly region of tau molecule and a site for PKA phosphorylation. Also, we have found that tau and tau peptide polymerization are reduced, but not abolished upon PKA phosphorylation. The involvement of a scaffolding protein like 14-3-3 in the generation of tau filaments in tauopathies, like AD, is suggested.

GSK-3 dependent phosphoepitopes recognized by PHF-1 and AT-8 antibodies are present in different tau isoforms.

It is widely known that the tau protein that forms the aggregates found in tauopathies like Alzheimers disease (AD) is hyperphosphorylated. Many of the sites that are hyperphosphorylated in AD can also be found phosphorylated in non-pathological control brains, although to a lesser extend. Among the different kinases that are able to phosphorylate tau in these sites, GSK-3 has emerged as a key effector of AD pathogenesis in view of its interaction with many of the proteins involved in the ethiology of AD. In this work, we have tested if control samples show only a decrease in the amount of phosphorylated tau molecules, or if the phosphorylation at different sites occurs in different tau isoforms, whereas in the pathological situation a single tau isoform is modified simultaneously at the different sites. Our results indicate that the second possibility takes place and that the differences in the phosphorylation of different tau isoforms could be due to a different subcellular distribution of these different tau isoforms in a neuron.

The role of the VQIVYK peptide in tau protein phosphorylation.

Although it remains unclear whether they are related to one another, tau aggregation and phosphorylation are the main pathological hallmarks of the neuronal disorders known as tauopathies. The capacity to aggregate is impaired in a variant of the tau 3R isoform that lacks residues 306–311 (nomenclature for the largest CNS tau isoform) and hence, we have taken advantage of this feature to study how phosphorylation and aggregation may be related as well as the role of this six amino acid peptide (VQIVYK). Through these analyses, we found that the phosphorylation of the tau variant was higher than that of the complete tau protein and that not only the deletion of these residues, but also the interaction of these residues, in tau 3R, with thioflavin‐S augmented tau phosphorylation by glycogen synthase kinase 3. In addition, the binding of the peptide containing the residues 306–311 to the whole tau protein provoked an increase in tau phosphorylation. This observation could be physiologically relevant as may suggest that tau–tau interactions, through those residues, facilitate tau phosphorylation. In summary, our data indicate that deletion of residues VQIVYK, in tau protein produces an increase in tau phosphorylation, without tau aggregation, because the VQIVYK peptide, that favors aggregation, is missing. On the other hand, when the whole tau protein interacts with thioflavin‐S or the peptide VQIVYK, an increase in both aggregation and phosphorylation occurs.

Thermodynamics of the Interaction between Alzheimer's Disease Related Tau Protein and DNA

Tau hyperphosphorylation can be considered as one of the hallmarks of Alzheimers disease and other tauophaties. Besides its well-known role as a microtubule associated protein, Tau displays a key function as a protector of genomic integrity in stress situations. Phosphorylation has been proven to regulate multiple processes including nuclear translocation of Tau. In this contribution, we are addressing the physicochemical nature of DNA-Tau interaction including the plausible influence of phosphorylation. By means of surface plasmon resonance (SPR) we measured the equilibrium constant and the free energy, enthalpy and entropy changes associated to the Tau-DNA complex formation. Our results show that unphosphorylated Tau binding to DNA is reversible. This fact is in agreement with the protective role attributed to nuclear Tau, which stops binding to DNA once the insult is over. According to our thermodynamic data, oscillations in the concentration of dephosphorylated Tau available to DNA must be the variable determining the extent of Tau binding and DNA protection. In addition, thermodynamics of the interaction suggest that hydrophobicity must represent an important contribution to the stability of the Tau-DNA complex. SPR results together with those from Tau expression in HEK cells show that phosphorylation induces changes in Tau protein which prevent it from binding to DNA. The phosphorylation-dependent regulation of DNA binding is analogous to the Tau-microtubules binding inhibition induced by phosphorylation. Our results suggest that hydrophobicity may control Tau location and DNA interaction and that impairment of this Tau-DNA interaction, due to Tau hyperphosphorylation, could contribute to Alzheimers pathogenesis.

Tau Protein Provides DNA with Thermodynamic and Structural Features which are Similar to those Found in Histone-DNA Complex

Tau protein has been proposed as a trigger of Alzheimers disease once it is hyperphosphorylated. However, the role that native tau forms play inside the neuronal nucleus remains unclear. In this work we present results concerning the interaction of tau protein with double-stranded DNA, single-stranded DNA, and also with a histone-DNA complex. The tau-DNA interaction results in a structure resembling the beads-on-a-string form produced by the binding of histone to DNA. DNA retardation assays show that tau and histone induce similar DNA retardation. A surface plasmon resonance study of tau-DNA interaction also confirms the minor groove of DNA as a binding site for tau, similarly to the histone binding. A residual binding of tau to DNA in the presence of Distamycin A, a minor groove binder, suggests the possibility that additional structural domains on DNA may be involved in tau interaction. Finally, DNA melting experiments show that, according to the Zipper model of helix-coil transition, the binding of tau increases the possibility of opening the DNA double helix in isolated points along the chain, upon increasing temperature. This behavior is analogous to histones and supports the previously reported idea that tau could play a protective role in stress situations. Taken together, these results show a similar behavior of tau and histone concerning DNA binding, suggesting that post-translational modifications on tau might impair the role that, by modulating the DNA function, might be attributable to the DNA-tau interaction.

Secretion of full-length tau or tau fragments in a cell culture model

Tau is a microtubule-associated protein that plays an important role in the pathogenesis of neurodegenerative diseases such as Alzheimers disease. Several studies have suggested that tau may be secreted to extracellular medium and may be responsible of spreading of neurodegeneration. The overexpression of tau in cultured non-neuronal cells leads to the secretion of this protein. The proline-rich region of tau may serve as a membrane-binding site during the secretion of the full-length tau molecule. Tau fragments lacking this proline-region are either not secreted or are secreted in a distinct manner to the full-length molecule.

Argyrophilic Grain Pathology as a Natural Model of Tau Propagation

Argyrophilic grain disease (AGD) is a sporadic 4 R tauopathy that usually presents in combination with other sporadic tauopathies or with Alzheimers disease (AD) pathology, and may contribute to dementia in older age patients. In previous studies, a detailed analysis of AGD pathology in the medial temporal lobe has been hampered by the common presence of concurrent AD changes. With the objective to assess the potentiality of AGD in research on tau propagation, here we present a study of a series of AGD postmortem cases (n = 53). The total series was divided in a subgroup of cases with Braak-stage ≤ II (n = 23) and a subgroup with Braak-stage>II or indeterminate (n = 30) in order to minimize interference with AD pathology. A detailed neuropathological evaluation of the medial temporal lobe was performed at three coronal levels with Gallyas stain, and immunostains with p62, AT8, and AT100 antibodies. Western blot analysis of the entorhinal and hippocampal cortex was performed in 8 cases with a panel of anti-tau antibodies. Cases were genotyped for APOE polymorphism and for H1/H2 alleles of the MAPT gene. All cases, and particularly lower Braak-stage cases, displayed a highly homogeneous pattern of involvement by argyrophilic grains and pretangles between connected regions (primarily basolateral nuclei of the amygdala, entorhinal/transentorhinal cortex, and hippocampal cortex). Staging of cases reveals progression of pathology along well-established neuroanatomical pathways. Western blot studies yielded a specific pattern of isoforms with a characteristic predominant band at 64 kDa. Genetic analysis showed a strong association with the H1 allele of the MAPT gene. AGD may thus be an optimal natural disease model for testing hypotheses related to tau propagation in human tissue.