In vitro assembly of tau protein: mapping the regions involved in filament formation.

Unraveling the mechanism of self-assembly of the protein tau into paired helical filaments (PHFs) is a crucial step toward the understanding of Alzheimer's and other neuropathological diseases at the molecular level. In an effort to map the role of different regions of tau in the mechanism of self-assembly, we have studied the polymerization ability of different tau fragments using an in vitro assay. Our results indicate that the N-terminal domain interferes with tau's ability to polymerize in vitro. The effect seems to be size dependent. Particularly, an isoform of tau from the peripheral nervous system, which has a much larger N-terminal domain, was found unable to form filaments in our in vitro assay. This finding can explain why in Alzheimer's patients PHFs only accumulate in the neurons from the central nervous system. We also report that a short segment of tau located in the third microtubule binding repeat (residues 317 to 335, peptide 1/2R) is probably the minimal segment of that region able to grow into filaments in vitro and in the presence of heparin. In contrast with whole peptide 1/2R, peptides corresponding to either the N-terminal or C-terminal halves of this segment were unable to form filaments. Finally, our polymerization studies of peptides from the C-terminal domain reveal a short sequence spanning residues 391 to 407 that grows into filaments in vitro. This tau segment forms filaments regardless of whether is incubated with heparin. Moreover, such filaments differ in diameter and morphology, suggesting a different mechanism of self-assembly.

The marine compound spisulosine, an inhibitor of cell proliferation, promotes the disassembly of actin stress fibers.

Spisulosine is a novel antiproliferative (antitumoral) compound of marine origin. In this work the molecular target for this toxic agent has been analyzed. In the presence of spisulosine, cultured cells change their morphology, first acquiring a fusiform morphology, and later becoming rounded without focal adhesions. Analysis of the cytoskeleton of treated cells indicate the absence of actin stress fibers.

Polymerization of tau into filaments in the presence of heparin: the minimal sequence required for tau-tau interaction.

Paired helical filaments isolated from the brains of patients with Alzheimer's disease are composed of a major protein component, the microtubule-associated protein termed tau, together with other nonprotein components, including heparan, a glycosaminoglycan, the more extensively sulfated form of which is heparin. As some of these nonprotein components may modulate the assembly of tau into filamentous structures, we have analyzed the ability of the whole tau protein or some of its fragments to self-assemble in the presence of heparin. Different tau fragments, all of them containing some sequences of the tubulin-binding motif, can assemble in vitro into filaments. We have also found formation of polymers with the 18-residue-long peptide corresponding to the third tubulin-binding motif of tau. This suggests that the ability of tau for self-assembly could be localized in a short sequence of amino acids present in the tubulin-binding repeats of the tau molecule.

Protein kinases involved in the phosphorylation of human tau protein in transfected COS-1 cells.

Human tau phosphorylation has been studied in transfected COS-1 cells. Treatment with okadaic acid alters the electrophoretic mobility of human tau protein transiently expressed in transfected cells, due to an increase in the level of phosphorylation. Treatment with okadaic acid also results in an increased phosphorylation of Alzheimer's disease-type phosphoepitopes. Tau phosphorylation within COS-1 cells is partially inhibited by in vivo treatment with DRB, a protein kinase inhibitor. Double treatment of transfected cells with okadaic acid and DRB reveals that phosphorylation of tau protein at the AT8 epitope is achieved by a DRB-resistant protein kinase which is different from that responsible for tau phosphorylation at the SMI-31 epitope, which appears to be sensitive to DRB.

Glycogen synthase kinase 3 phosphorylates recombinant human tau protein at serine-262 in the presence of heparin (or tubulin).

Tau protein, the major component of the aberrant structures termed paired helical filaments (PHFs) present in the brain of Alzheimer's disease patients, is pathologically phosphorylated in sites in and around the tubulin-binding sites. A single protein kinase, glycogen synthase kinase 3 (GSK 3), is able to phosphorylate tau at the flanking regions and, additionally, at the tubulin-binding motifs if heparin or tubulin is present. Serines-262 and -324 have been found to be modified at the tubulin-binding region of tau protein by GSK 3 in the presence of heparin or tubulin.

The role of tau phosphorylation in transfected COS-1 cells.

Tau cDNAs from each of the six human isoforms were transfected into COS-1 cells and, in every case, more than one peptide was observed. The diversity of expressed isoforms was due to different levels of tau phosphorylation. Tau phosphorylation results in a decrease of the protein electrophoretic mobility. The major contribution to this mobility shift is due to the phosphorylation at the at the C-terminus of the molecule, as inferred from the expression of tau fragments. Phosphorylation takes place in some of the sites modified in neural cells and in the basis of AD patients. Copolymerization studies indicate that the level of phosphorylation, as well as the localization of the modified residues, may affect the binding of the protein to microtubules. These results indicate that phosphorylation regulates tau function inside the cell.

Microtubule-associated protein MAP1B showing a fetal phosphorylation pattern is present in sites of neurofibrillary degeneration in brains of Alzheimer's disease patients.

Alzheimer's disease results in the appearance of cytoskeletal disorders yielding pathological structures such a neurofibrillary tangles or dystrophic neurites. It has been previously described that the microtubule-associated protein, tau, modified by phosphorylation in serines adjacent to prolines, is a major component of these structures. Here, we show that another microtubule associated protein, MAP1B, aberrantly phosphorylated by a proline-dependent protein kinase, is a component of these previously mentioned structures. Thus, a possible common phosphorylation of axonal MAPs such as tau or MAP1B may correlate with their association with those aberrant cytoskeletal structures present in AD.

Overexpression of tau protein in COS-1 cells results in the stabilization of centrosome-independent microtubules and extension of cytoplasmic processes.

COS-1 cells were transfected with cDNAs coding for different human tau isoforms. Expressed tau isoforms bind to cellular microtubules in vivo, preferentially at the distal regions of microtubules nucleated by the centrosome, leading to their stabilization. Eventually, tau-coated microtubules without any association with the centrosome were observed. A major difference between tau isoforms containing three tubulin-binding motifs and tau isoforms containing four tubulin-binding motifs is the greater ability of the latter in inducing the formation of long cytoplasmic processes.

Differentiation of neuroblastoma cells correlates with an altered splicing pattern of tau RNA.

Morphological differentiation of N2A neuroblastoma cells is associated with an altered splicing of the gene of the microtubule-associated protein, tau. Two populations of RNA (coding for tau proteins containing three or four tubulin-binding motifs) are present in a similar proportion in undifferentiated neuroblastoma cells while in differentiated cells the proportion is changed in favour of that population coding for tau protein containing four tubulin-binding motifs. An increase in a high molecular weight tau isoforms correlates with the increase in the RNA population coding for four tubulin-binding motifs. A possible consequence of expressing a higher proportion of the tau protein containing four tubulin-binding motifs could be an increase in microtubule stability of differentiated neuroblastoma cells.

Measurement of distinct immunochemical presentations of tau protein in Alzheimer disease.

The tau protein is a microtubule-associated protein that is normally located in nerve axons. In Alzheimer disease, it is a constituent of paired helical filaments (PHFs), which are the principal fibrous component of the characteristic neurofibrillary tangles. The tau protein, therefore, is abnormally sequestered in an insoluble form in PHFs in the cell body and dendrites in Alzheimer disease. We have used two monoclonal antibodies (mAbs) to selectively measure the levels of normal, soluble tau protein and of PHF-associated tau protein in the brain. mAb 423 binds to PHFs and recognizes a 12-kDa fragment of tau protein released by formic acid treatment of PHFs, but it does not recognize normal tau protein. In contrast, mAb 7.51 recognizes normal tau protein as well as the PHF core-derived tau fragment, but its epitope is concealed in the PHF-bound form. The differential binding properties for these two mAbs have enabled us in this study to quantify insoluble PHF-associated tau protein in the somatodendritic compartment as well as normal soluble tau protein in its predominantly axonal location. Our findings demonstrate that a distinct immunochemical presentation of tau protein recognized by mAb 423, a PHF-specific marker, can be used to quantify neurofibrillary pathology in Alzheimer disease independently of the presence of normal tau proteins.

Collagenous structures present in brain contain epitopes shared by collagen and microtubule-associated protein tau.

A novel type of collagenous fibers has been isolated from human brain and characterized by electron microscopy and optical diffraction. It was found that the morphology of the fibers is similar, but not identical, to that of skin collagen. Also, the collagenous fibers show some similarities with the paracrystals that could be assembled in vitro from purified microtubule-associated protein tau. Immunological analyses indicated the presence of epitopes in these collagenous fibers which react with antibodies against collagen and tau.

Characterization of tau protein present in microtubules and paired helical filaments of Alzheimer's disease patient's brain.

Two proteins immunologically related to porcine tau protein are found in the brain of Alzheimer's disease patients. One is bound to microtubules and, after isolation by co-polymerization with tubulin, shows a size and tryptic peptide map, similar to the microtubule-associated tau protein, present in the brain of non-demented patients. The other tau-related protein is present as the major protein of a purified fraction of paired helical filaments. The paired helical filament-associated protein shows smaller molecular weight (33,000) than microtubule-associated tau; however, this 33,000 mol. wt protein reacts with a monospecific anti-tau antibody and with an antibody to a 19-amino acid peptide corresponding to amino acids 228-246 of human tau. Furthermore, the 33,000 mol. wt protein and the tau protein have similar tryptic peptide maps. These results suggest that the paired helical filament protein is a modified form of the microtubule-associated tau protein.

Altered levels of microtubule proteins in brains of Alzheimer's disease patients.

The amount of microtubule protein present in the total soluble protein from brains of Alzheimer's disease patients and from brains of non-Alzheimer age-matched controls, were determined by radioimmunoassay. No differences were found in the amount of tubulin or microtubule-associated protein MAP2 present in either group. However, the amount of tau protein or MAP1 from the brains of Alzheimer's disease patients was about half of that present in their control counterparts.

Tau factor polymers are similar to paired helical filaments of Alzheimer's disease.

Tau factor, upon urea treatment, is able to polymerize in vitro. These polymers are composed of tau factor as shown by immunogold staining. The structure of tau polymers is very similar to that of paired helical filaments (PHFs) of Alzheimer's disease in their dimensions as well as in their periodicity. Metal shadowing of both polymers shows a similar twisting. Also, similar peptide maps were found for tau factor and a 33 kDa protein that is the main component of our PHF preparations.

A modified form of microtubule-associated tau protein is the main component of paired helical filaments.

Paired helical filaments, which are present in the brain of Alzheimer's disease patients have been isolated and characterized. Treatment of the filaments with reducing agents and detergents extracts several proteins from these structures. The remaining filaments are composed mainly of a protein with molecular weight of 33 kDa suggesting that this protein is the core component of these filaments. Peptide mapping using trypsin and endoproteinase Arg-C revealed that the 33 kDa protein, was a modified form of tau protein present in normal human brain.

In vitro conditions for the self-polymerization of the microtubule-associated protein, tau factor.

One of the microtubule associated proteins, tau factor, that appears associated to the paired helical filaments of Alzheimer's disease presents, by itself, after urea treatment, the ability of polymerizing in vitro as tested by immunoelectronmicroscopy. These polymers resemble in their width and appearance those of the paired helical filaments. The conditions required for this assembly have been studied and determined the protein concentration needed, the influence of salt concentration and pH as well as the possible modifications (deamination, acylation) which may be implied in such in vitro polymerization.

Self assembly of microtubule associated protein tau into filaments resembling those found in Alzheimer disease.

Microtubule associated protein tau factor self-assembles into filamentous structures resembling the paired helical filaments found in Alzheimer disease. Tau polymerization requires of a previous modification; conversion of glutamine into glutamic acid by deamination.

The interaction between a Na+-channel toxin and brain microtubule proteins in vitro.

The aim of this work was to characterize the interaction of the Na+-channel toxin, purified from venom of the scorpion Leiurus quinquestriatus, with microtubule proteins in vitro. The toxin enhanced microtubule assembly, causing the formation of microtubule 'bundles'. It interacted with the Na+-channel 270 kDa subunit and was subsequently found to be unrelated to high molecular weight microtubule-associated protein with respect to apparent molecular weight and toxin binding. However, the radiolabelled toxin bound to tubulin, although with a much lower affinity than that published for the reconstituted Na+-channel. This binding appears to occur in the carboxy terminal 4 kDa region of tubulin. These results may reflect a secondary action of the toxin involved in binding to its receptors in the neural plasma membrane.

Quantitation and characterization of tau factor in porcine tissues.

Using a monospecific antibody against brain tau factor purified by affinity chromatography, we have studied the distribution of tau factor or related polypeptides in different cells. The presence of tau in all cell types tested was demonstrated by a radioimmunoassay. Tau factor-related proteins were found in liver, spleen, pancreas, kidney and lung, although at much lower levels than that found in neural cells. In all cases, they copolymerized with tubulin and were heat-resistant. When the distribution of tau factor-related proteins was studied by Western blotting, tau factor antiserum reacted against peptides with an electrophoretic mobility that was similar to those of brain tau factor peptides. Immunofluorescence studies have also been performed with the same antibody to determine the distribution of tau factor-related peptides in PK15 cells. Our results indicated that these peptides were associated to the microtubule network.

Localization of the tubulin binding site for tau protein.

Limited proteolysis of tubulin with subtilisin resulted in the removal of the carboxyl-terminal moiety of tubulin subunits. The remaining peptides from both alpha and beta tubulin lacking the carboxyl terminal did not bind to tau factor nor to MAP2 or MAP1. The carboxyl-terminal fragments bind to tau factor and MAP2 and both compete for the same binding sites in the tubulin molecule. Our results suggest that the carboxyl-terminal region of tubulin is a regulatory domain for the assembly of tubulin and the site for interaction with MAPs.