Jacek Biernat

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.

The switch of tau protein to an Alzheimer-like state includes the phosphorylation of two serine-proline motifs upstream of the microtubule binding region

The paired helical filaments (PHFs) of Alzheimers disease consist mainly of the microtubule‐associated protein tau. PHF tau differs from normal human brain tau in that it has a higher Mr and a special state of phosphorylation. However, the protein kinase(s) involved, the phosphorylation sites on tau and the resulting conformational changes are only poorly understood. Here we show that a new monoclonal antibody, AT8, records the PHF‐like state of tau in vitro, and we describe a kinase activity that turns normal tau into a PHF‐like state. The epitope of AT8 is around residue 200, outside the region of internal repeats and requires the phosphorylation of serines 199 and/or 202. Both of these are followed by a proline, suggesting that the kinase activity belongs to the family of proline‐directed kinases. The epitope of AT8 is nearly coincident with that of another phosphorylation‐dependent antibody, TAU1 [Binder, L.I., Frankfurter, A. and Rebhun, L. (1985) J. Cell Biol., 101, 1371–1378], but the two are complementary since TAU1 requires a dephosphorylated epitope.

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.

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.

Structural Polymorphism of 441-Residue Tau at Single Residue Resolution

Alzheimer disease is characterized by abnormal protein deposits in the brain, such as extracellular amyloid plaques and intracellular neurofibrillary tangles. The tangles are made of a protein called tau comprising 441 residues in its longest isoform. Tau belongs to the class of natively unfolded proteins, binds to and stabilizes microtubules, and partially folds into an ordered β-structure during aggregation to Alzheimer paired helical filaments (PHFs). Here we show that it is possible to overcome the size limitations that have traditionally hampered detailed nuclear magnetic resonance (NMR) spectroscopy studies of such large nonglobular proteins. This is achieved using optimal NMR pulse sequences and matching of chemical shifts from smaller segments in a divide and conquer strategy. The methodology reveals that 441-residue tau is highly dynamic in solution with a distinct domain character and an intricate network of transient long-range contacts important for pathogenic aggregation. Moreover, the single-residue view provided by the NMR analysis reveals unique insights into the interaction of tau with microtubules. Our results establish that NMR spectroscopy can provide detailed insight into the structural polymorphism of very large nonglobular proteins.

Inducible Expression of Tau Repeat Domain in Cell Models of Tauopathy AGGREGATION IS TOXIC TO CELLS BUT CAN BE REVERSED BY INHIBITOR DRUGS

We generated several cell models of tauopathy in order to study the mechanisms of neurodegeneration in diseases involving abnormal changes of tau protein. N2a neuroblastoma cell lines were created that inducibly express different variants of the repeat domain of tau (tauRD) when exposed to doxycycline (Tet-On system). The following three constructs were chosen: (i) the repeat domain of tau that coincides with the core of Alzheimer paired helical filaments; (ii) the repeat domain with the deletion mutation ΔK280 known from frontotemporal dementia and highly prone to spontaneous aggregation; and (iii) the repeat domain with ΔK280 and two proline point mutations that inhibit aggregation. The comparison of wild-type, pro-aggregation, and anti-aggregation mutants shows the following. (a) Aggregation of tauRD is toxic to cells. (b) The degree of aggregation and toxicity depends on the propensity for β-structure. (c) Soluble mutants of tauRD that cannot aggregate are not toxic. (d) Aggregation is preceded by fragmentation. (e) Fragmentation of tauRD in cells is initially due to a thrombin-like protease activity. (f) Phosphorylation of tauRD (at KXGS motifs) precedes aggregation but is not correlated with the degree of aggregation. (g) Aggregates of tauRD disappear when the expression is silenced, showing that aggregation is reversible. (h) Aggregation can be prevented by drugs and even pre-formed aggregates can be dissolved again by drugs. Thus, the cell models open up new insights into the relationship between the structure, expression, phosphorylation, aggregation, and toxicity of tauRD that can be used to test current hypotheses on tauopathy and to develop drugs that prevent the aggregation and degeneration of cells.

MARK/PAR1 kinase is a regulator of microtubule-dependent transport in axons

Microtubule-dependent transport of vesicles and organelles appears saltatory because particles switch between periods of rest, random Brownian motion, and active transport. The transport can be regulated through motor proteins, cargo adaptors, or microtubule tracks. We report here a mechanism whereby microtubule associated proteins (MAPs) represent obstacles to motors which can be regulated by microtubule affinity regulating kinase (MARK)/Par-1, a family of kinases that is known for its involvement in establishing cell polarity and in phosphorylating tau protein during Alzheimer neurodegeneration. Expression of MARK causes the phosphorylation of MAPs at their KXGS motifs, thereby detaching MAPs from the microtubules and thus facilitating the transport of particles. This occurs without impairing the intrinsic activity of motors because the velocity during active movement remains unchanged. In primary retinal ganglion cells, transfection with tau leads to the inhibition of axonal transport of mitochondria, APP vesicles, and other cell components which leads to starvation of axons and vulnerability against stress. This transport inhibition can be rescued by phosphorylating tau with MARK.

The Potential for β-Structure in the Repeat Domain of Tau Protein Determines Aggregation, Synaptic Decay, Neuronal Loss, and Coassembly with Endogenous Tau in Inducible Mouse Models of Tauopathy

We describe two new transgenic mouse lines for studying pathological changes of Tau protein related to Alzheimers disease. They are based on the regulatable expression of the four-repeat domain of human Tau carrying the FTDP17 (frontotemporal dementia and parkinsonism linked to chromosome 17) mutation ΔK280 (TauRD/ΔK280), or the ΔK280 plus two proline mutations in the hexapeptide motifs (TauRD/ΔK280/I277P/I308P). The ΔK280 mutation accelerates aggregation (“proaggregation mutant”), whereas the proline mutations inhibit Tau aggregation in vitro and in cell models (“antiaggregation mutant”). The inducible transgene expression was driven by the forebrain-specific CaMKIIα (calcium/calmodulin-dependent protein kinase IIα) promoter. The proaggregation mutant leads to Tau aggregates and tangles as early as 2–3 months after gene expression, even at low expression (70% of endogenous mouse Tau). The antiaggregation mutant does not aggregate even after 22 months of gene expression. Both mutants show missorting of Tau in the somatodendritic compartment and hyperphosphorylation in the repeat domain [KXGS motifs, targets of the kinase MARK (microtubule affinity regulating kinase)]. This indicates that these changes are related to Tau expression rather than aggregation. The proaggregation mutant causes astrogliosis, loss of synapses and neurons from 5 months of gene expression onward, arguing that Tau toxicity is related to aggregation. Remarkably, the human proaggregation mutant TauRD coaggregates with mouse Tau, coupled with missorting and hyperphosphorylation at multiple sites. When expression of proaggregation TauRD is switched off, soluble and aggregated exogenous TauRD disappears within 1.5 months. However, tangles of mouse Tau, hyperphosphorylation, and missorting remain, suggesting an extended lifetime of aggregated wild-type Tau once a pathological conformation and aggregation is induced by a proaggregation Tau species.