Eva Maria Mandelkow

Mitogen activated protein (MAP) kinase transforms tau protein into an Alzheimer-like state.

The microtubule‐associated protein tau is a major component of the paired helical filaments (PHFs) observed in Alzheimers disease brains. The pathological tau is distinguished from normal tau by its state of phosphorylation, higher apparent M(r) and reaction with certain antibodies. However, the protein kinase(s) have not been characterized so far. Here we describe a protein kinase from brain which specifically induces the Alzheimer‐like state in tau protein. The 42 kDa protein belongs to the family of mitogen activated protein kinases (MAPKs) and is activated by tyrosine phosphorylation. It is capable of phosphorylating Ser‐Pro and Thr‐Pro motifs in tau protein (approximately 14–16 P1 per tau molecule). By contrast, other proline directed Ser/Thr kinases such as p34(cdc2) combined with cyclin A or B have only minor effects on tau phosphorylation. We propose that MAP kinase is abnormally active in Alzheimer brain tissue, or that the corresponding phosphatases are abnormally passive, due to a breakdown of the normal regulatory mechanisms.

Glycogen synthase kinase‐3 and the Alzheimer‐like state of microtubule‐associated protein tau

The Alzheimer‐like state of tau protein includes phosphorylation by a proline‐directed Ser/Thr kinase present in normal or pathological human brain. Extending earlier results on MAP kinase, we show here that the proline‐directed kinase, GSK3, can induce an Alzheimer‐like immune response involving several distinct and phoshorylatable epitopes at Ser—Pro motifs, as well as gel mobility shift, similar to MAP kinase. Both kinases behave like microtubule‐associated proteins in that they co‐purify through cycles of assembly and disassembly, and both kinases are directly associated with paired helical filaments.

Abnormal Alzheimer-like phosphorylation of tau-protein by cyclin-dependent kinases cdk2 and cdk5

We have shown earlier that certain proline‐directed kinases such as MAP kinase or GSK‐3 can phosphorylate tau protein in an abnormal manner reminiscent of tau from Alzheimer paired helical filaments [Drewes et al. (1992); Mandelkow et al. (1992)]. Both kinases are abundant in brain tissue and associate physically with microtubules through several cycles of assembly and disassembly. In this report we show that cdk2/cyclinA incorporates ≈5 P, into recombinant tau, and that it also induces the M r shift and antibody reactivity typical of Alzheimer tau. However, since there is no cdk2 in brain [Meyerson et al. (1992)] we looked for other members of this family of kinases. Using an antibody against the conserved N‐terminus we isolated a cdk‐like kinase from brain which was capable of inducing the Alzheimer‐like characteristics in tau by phosphorylation. Its size (31 kDa), target specificity (proline‐directed), Chromatographic behavior, and abundance in brain suggest that this kinase is similar or identical to the neuronal cdc2‐like kinase nclk alias PSSARLE or cdk5 [Hellmich et al. (1992); Meyerson et al. (1992); Xiong et al. (1992); Tsai et al. (1993)]. This was confirmed by an antibody specific for cdk5. Like MAP kinase and GSK‐3, this kinase is physically associated with microtubules and can be enriched by cycles of microtubule assembly and disassembly. Thus, cdk5 should be regarded as another kinase that could be held responsible for the changes in tau protein during Alzheimer disease progression.

Tau fragmentation, aggregation and clearance: the dual role of lysosomal processing

Aggregation and cleavage are two hallmarks of Tau pathology in Alzheimer disease (AD), and abnormal fragmentation of Tau is thought to contribute to the nucleation of Tau paired helical filaments. Clearance of the abnormally modified protein could occur by the ubiquitin-proteasome and autophagy-lysosomal pathways, the two major routes for protein degradation in cells. There is a debate on which of these pathways contributes to clearance of Tau protein and of the abnormal Tau aggregates formed in AD. Here, we demonstrate in an inducible neuronal cell model of tauopathy that the autophagy-lysosomal system contributes to both Tau fragmentation into pro-aggregating forms and to clearance of Tau aggregates. Inhibition of macroautophagy enhances Tau aggregation and cytotoxicity. The Tau repeat domain can be cleaved near the N terminus by a cytosolic protease to generate the fragment F1. Additional cleavage near the C terminus by the lysosomal protease cathepsin L is required to generate Tau fragments F2 and F3 that are highly amyloidogenic and capable of seeding the aggregation of Tau. We identify in this work that components of a selective form of autophagy, chaperone-mediated autophagy, are involved in the delivery of cytosolic Tau to lysosomes for this limited cleavage. However, F1 does not fully enter the lysosome but remains associated with the lysosomal membrane. Inefficient translocation of the Tau fragments across the lysosomal membrane seems to promote formation of Tau oligomers at the surface of these organelles which may act as precursors of aggregation and interfere with lysosomal functioning.

Mutations of tau protein in frontotemporal dementia promote aggregation of paired helical filaments by enhancing local beta-structure

The microtubule-associated protein tau is a natively unfolded protein in solution, yet it is able to polymerize into the ordered paired helical filaments (PHF) of Alzheimers disease. In the splice isoforms lacking exon 10, this process is facilitated by the formation of β-structure around the hexapeptide motif PHF6 (306VQIVYK311) encoded by exon 11. We have investigated the structural requirements for PHF polymerization in the context of adult tau isoforms containing four repeats (including exon 10). In addition to the PHF6 motif there exists a related PHF6* motif (275VQIINK280) in the repeat encoded by the alternatively spliced exon 10. We show that this PHF6* motif also promotes aggregation by the formation of β-structure and that there is a cross-talk between the two hexapeptide motifs during PHF aggregation. We also show that two of the tau mutations found in hereditary frontotemporal dementias, ΔK280 and P301L, have a much stronger tendency for PHF aggregation which correlates with their high propensity for β-structure around the hexapeptide motifs.

RNA stimulates aggregation of microtubule‐associated protein tau into Alzheimer‐like paired helical filaments

The microtubule‐associated protein tau is the main component of the paired helical filaments (PHFs) of Alzheimers disease, the most common senile dementia. To understand the origin of taus abnormal assembly we have studied the influence of other cytosolic components. Here we report that PHF assembly is strongly enhanced by RNA. The RNA‐induced assembly of PHFs is dependent on the formation of intermolecular disulfide bridges involving Cys322 in the third repeat of tau, and it includes the dimerization of tau as an early intermediate. Three‐repeat constructs polymerize most efficiently, two repeat constructs are the minimum number required for assembly, and even all six full‐length isoforms of tau can be induced to form PHFs by RNA.

In Vivo Microdialysis Reveals Age-Dependent Decrease of Brain Interstitial Fluid Tau Levels in P301S Human Tau Transgenic Mice

Although tau is a cytoplasmic protein, it is also found in brain extracellular fluids, e.g., CSF. Recent findings suggest that aggregated tau can be transferred between cells and extracellular tau aggregates might mediate spread of tau pathology. Despite these data, details of whether tau is normally released into the brain interstitial fluid (ISF), its concentration in ISF in relation to CSF, and whether ISF tau is influenced by its aggregation are unknown. To address these issues, we developed a microdialysis technique to analyze monomeric ISF tau levels within the hippocampus of awake, freely moving mice. We detected tau in ISF of wild-type mice, suggesting that tau is released in the absence of neurodegeneration. ISF tau was significantly higher than CSF tau and their concentrations were not significantly correlated. Using P301S human tau transgenic mice (P301S tg mice), we found that ISF tau is fivefold higher than endogenous murine tau, consistent with its elevated levels of expression. However, following the onset of tau aggregation, monomeric ISF tau decreased markedly. Biochemical analysis demonstrated that soluble tau in brain homogenates decreased along with the deposition of insoluble tau. Tau fibrils injected into the hippocampus decreased ISF tau, suggesting that extracellular tau is in equilibrium with extracellular or intracellular tau aggregates. This technique should facilitate further studies of tau secretion, spread of tau pathology, the effects of different disease states on ISF tau, and the efficacy of experimental treatments.

The Alzheimer-like phosphorylation of tau protein reduces microtubule binding and involves Ser-Pro and Thr-Pro motifs

Tau protein can be transformed into an Alzheimer‐like state by phosphorylation with a kinase activity from brain [Biernat et al. (1992) EMBO J. 11, 1593–1597]. Here we show that the phosphorylation at Ser‐Pro motifs strongly decreases taus affinity for Microtubules. The major reduction occurs during the first of the three main stages of phosphorylation. The data explain the lower Stability of microtubules resulting from the pathological tau phosphorylation.

Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcmeurin and phosphatase-2A

We have shown previously that brain tissue contains protein kinases which can phosphorylate tau protein to a state reminiscent of the pathological state of Alzheimer paired helical filaments (PHFs); these include proline‐directed kinases which phosphorylate SP or TP motifs (such as MAP kinase and GSK‐3) [Drewes et al. (1992); Mandelkow et al. (1992)], as well as a novel kinase which phosphorylates S262 of tau protein and thereby strongly reduces the binding of tau to imcrotubules [Biernat et al. (1993)]. Here we report on the corresponding phosphatases in brain which normally keep the ‘pathological’ sites free of phosphate. The major phosphatases acting on tau are calcineurin and PP‐2A, but not PP‐1. Both are present and active in brain extracts, they can dephosphorylate recombinant tau after prior phosphorylation with either MAP kinase, GSK‐3, or brain extract, and the course of dephosphorylation can be monitored with antibodies diagnostic of the pathological state of tau. Both phosphatases also act directly on PHF tau isolated from Alzheimer brains.

Phosphorylation of tau protein by recombinant GSK-3β: pronounced phosphorylation at select Ser/Thr-Pro motifs but no phosphorylation at Ser262 in the repeat domain

Glycogen synthase kinase‐3β (GSK‐3β) has been described as a proline‐directed kinase which phosphorylates tau protein at several sites that are elevated in Alzheimer paired helical filaments. However, it has been claimed that GSK‐3β can also phosphorylate the non‐proline‐directed KXGS motifs in the presence of heparin, including Ser262 in the repeat domain of tau, which could induce the detachment of tau from microtubules. We have analyzed the activity of recombinant GSK‐3β and of GSK‐3β preparations purified from tissue, using two‐dimensional phosphopeptide mapping, immunoblotting with phosphorylation‐sensitive antibodies, and phosphopeptide sequencing. The most prominent phosphorylation sites on tau are Ser396 and Ser404 (PHF‐1 epitope), Ser46 and Thr50 in the first insert, followed by a less efficient phosphorylation of other Alzheimer phosphoepitopes (antibodies AT‐8, AT‐270, etc). We also show that the non‐proline‐directed activity at KXGS motifs is not due to GSK‐3β itself, but to kinase contaminations in common GSK‐3β preparations from tissues which are activated upon addition of heparin.