E.-M. Mandelkow

Tau blocks traffic of organelles, neurofilaments, and APP vesicles in neurons and enhances oxidative stress

We studied the effect of microtubule-associated tau protein on trafficking of vesicles and organelles in primary cortical neurons, retinal ganglion cells, and neuroblastoma cells. Tau inhibits kinesin-dependent transport of peroxisomes, neurofilaments, and Golgi-derived vesicles into neurites. Loss of peroxisomes makes cells vulnerable to oxidative stress and leads to degeneration. In particular, tau inhibits transport of amyloid precursor protein (APP) into axons and dendrites, causing its accumulation in the cell body. APP tagged with yellow fluorescent protein and transfected by adenovirus associates with vesicles moving rapidly forward in the axon (∼80%) and slowly back (∼20%). Both movements are strongly inhibited by cotransfection with fluorescently tagged tau (cyan fluorescent protein–tau) as seen by two-color confocal microscopy. The data suggests a linkage between tau and APP trafficking, which may be significant in Alzheimers disease.

Clogging of axons by tau, inhibition of axonal traffic and starvation of synapses

Loss of synapses and dying back of axons are considered early events in brain degeneration during Alzheimers disease. This is accompanied by an aberrant behavior of the microtubule-associated protein tau (hyperphosphorylation, aggregation). Since microtubules are the tracks for axonal transport, we are testing the hypothesis that tau plays a role in the malfunctioning of transport. Experiments with various neuronal and non-neuronal cells show that tau is capable of reducing net anterograde transport of vesicles and cell organelles by blocking the microtubule tracks. Thus, a misregulation of tau could cause the starvation of synapses and enhanced oxidative stress, long before tau detaches from microtubules and aggregates into Alzheimer neurofibrillary tangles. In particular, the transport of amyloid precursor protein is retarded when tau is elevated, suggesting a possible link between the two key proteins that show abnormal behavior in Alzheimers disease.

Phosphorylation of MAP2c and MAP4 by MARK kinases leads to the destabilization of microtubules in cells

Microtubules serve as transport tracks in molecular mechanisms governing cellular shape and polarity. Rapid transitions between stable and dynamic microtubules are regulated by several factors, including microtubule-associated proteins (MAPs). We have shown that MAP/microtubule affinity regulating kinases (MARK) can phosphorylate the microtubule-associated-proteins MAP4, MAP2c, and tau on their microtubule-binding domain in vitro. This leads to their detachment from microtubules (MT) and an increased dynamic instability of MT. Here we show that MARK protein kinases phosphorylate MAP2 and MAP4 on their microtubule-binding domain in transfected CHO cells. In CHO cells expressing MARK1 or MARK2 under control of an inducible promoter, MARK2 phosphorylates an endogenous MAP4-related protein. Prolonged expression of MARK2 results in microtubule-disruption, detachment of cells from the substratum, and cell death. Concomitant with microtubule disruption, we also observed a breakdown of the vimentin network, whereas actin fibers remained unaffected. Thus, MARK seems to play an important role in controlling cytoskeletal dynamics.

Stepwise proteolysis liberates tau fragments that nucleate the Alzheimer-like aggregation of full-length tau in a neuronal cell model

Tau is a highly soluble protein, yet it aggregates abnormally in Alzheimers disease. Here, we address the question of proteolytic processing of tau and the nucleation of aggregates by tau fragments. We show in neuronal cell models that fragments of the repeat domain of tau containing mutations of FTDP17 (frontotemporal dementia with parkinsonism linked to chromosome 17), produced by endogenous proteases, can induce the aggregation of full-length tau. Fragments are generated by successive cleavages, first N-terminally between K257 and S258, then C-terminally around residues 353–364; conversely, when the N-terminal cleavage is inhibited, no fragmentation and aggregation takes place. The C-terminal truncation and the coaggregation of fragments with full-length tau depends on the propensity for β-structure. The aggregation is modulated by phosphorylation but does not depend on it. Aggregation but not fragmentation as such is toxic to cells; conversely, toxicity can be prevented by inhibiting either aggregation or proteolysis. The results reveal a novel pathway of abnormal tau aggregation in neuronal cells.

N-Phenylamine Derivatives as Aggregation Inhibitors in Cell Models of Tauopathy

Cell models of tauopathy were generated in order to study mechanisms of neurodegeneration involving abnormal changes of tau. They are based on neuroblastoma cell lines (N2a) that inducibly express different forms of the repeat domain of tau (tau(RD)), e.g. the 4-repeat domain of tau with the wild-type sequence, the repeat domain with the DeltaK280 mutation (pro-aggregation mutant), or the repeat domain with DeltaK280 and two proline point mutations (anti-aggregation mutant). The data indicate that the aggregation of tau(RD) is toxic, and that aggregation and toxicity can be prevented by low molecular weight compounds, notably compounds based on the N-phenylamine core. Thus the cell models are suitable for developing aggregation inhibitor drugs.

Signaling from MARK to Tau: Regulation, Cytoskeletal Crosstalk, and Pathological Phosphorylation

The hyperphosphorylation of tau is an early step in the degeneration of neurons in Alzheimer’s disease and other tauopathies. Of particular importance is the phosphorylation of tau in the repeat domain which detaches tau from microtubules. This makes microtubules dynamic for their role in differentiation and neurite outgrowth, and it controls the level of tau on the microtubule surface which keeps the tracks clear for axonal transport. However, the detachment of tau from microtubules can also initiate the reactions that lead to pathological aggregation into neurofibrillary tangles. Phosphorylation of tau in the repeat domain is achieved by the kinase MARK/Par-1, a member of the calcium/calmodulin-dependent protein kinase group of kinases. In this report, we focus on the modes of MARK regulation. MARK contains several domains which offer multiple ways of regulation by posttranslational modification (e.g. phosphorylation), interactions with scaffolding proteins and subcellular targeting (e.g. 14-3-3), and interactions with other proteins. We consider in particular the interactions between MARK and other kinases, notably MARKK/TAO-1 and PAK5. MARKK (a member of the Ste20 family of kinases) activates MARK by phosphorylating it at a critical threonine residue within the activation loop. Activated MARK in turn phosphorylates tau, causes its detachment from microtubules and renders them labile. PAK5 inactivates MARK, not by phosphorylation, but by binding to the catalytic domain. PAK5 contributes to microtubule stability by preventing the MARK-induced phosphorylation of tau; conversely, PAK5 contributes to actin dynamics, presumably through the activation of cofilin, an F-actin severing protein. Thus, MARK and its regulators MARKK and PAK5 appear to mediate the crosstalk between the actin and microtubule cytoskeleton in an antagonistic fashion.

Interaction of kinesin motors, microtubules, and MAPs.

Kinesins are a family of microtubule-dependent motor proteins that carry cargoes such as vesicles, organelles, or protein complexes along microtubules. Here we summarize structural studies of the “conventional” motor protein kinesin-1 and its interactions with microtubules, as determined by X-ray crystallography and cryo-electron microscopy. In particular, we consider the docking between the kinesin motor domain and tubulin subunits and summarize the evidence that kinesin binds mainly to β tubulin with the switch-2 helix close to the intradimer interface between α and β tubulin.

Spectroscopic Approaches to the Conformation of Tau Protein in Solution and in Paired Helical Filaments

The abnormal aggregation of the microtubule-associated protein tau into paired helical filaments is one the hallmarks of Alzheimer’s disease. This aggregation is based in the partial formation of β-structure. In contrast, the soluble protein shows a mostly random coil structure, as judged by circular dichroism, Fourier transform infrared, X-ray scattering and biochemical assays. Here, we review the basis of the natively unstructured character of tau, as well as recent studies of residual structure and long-range interactions between different domains of the protein. Analysis of the primary structure reveals a very low content of hydrophobic amino acids and a high content of charged residues, both of which tend to counteract a well-folded globular state of proteins. In the case of tau, the low overall hydrophobicity is sufficient to explain the lack of folding. This is in contrast to other proteins which also carry an excess charge at physiological pH. By tryptophan scanning mutagenesis and fluorimetry we found that most of the sequence is solvent exposed. Analysis of the hydrodynamic radii confirms a mostly random coil structure of various tau isoforms and tau domains. The proteins can be further expanded by denaturation with GdHCl which indicates some global folding. This was substantiated by a FRET-based approach where the distances between different domains of tau were determined. The combined data show that tau is mostly disordered and flexible but tends to assume a hairpin-like overall fold which may be important in the transition to a pathological aggregate.

Generation of Tau Aggregates and Clearance by Autophagy in an Inducible Cell Model of Tauopathy

We have studied the mechanism of aggregation in an inducible cell model of Tau pathology. When the repeat domain of human Tau (TauRD) carrying the FTDP-17 mutation ΔK280 is expressed, the cells develop aggregates, as seen by thioflavin S fluorescence, electron microscopy, and sarkosyl extraction methods. By contrast, mutants of TauRD that are unable to generate β-structure do not aggregate. Enhanced aggregation leads to enhanced toxicity, visible by live cell microscopy and LDH release assay. The aggregation process is initiated by the sequential cleavage of TauRD which yields highly amyloidogenic fragments. This cleavage occurs only with proaggregant TauRD, and not with the nonaggregating mutants, indicating that β-structure makes TauRD vulnerable to both proteolytic degradation and aggregation. Aggregation is reversed by switching off the expression of TauRD, by inhibitor compounds, and by certain protease inhibitors. In all cases, the enhanced toxicity is rescued. The clearance of the aggregates involves autophagy, whereas proteasomal degradation plays only a minor role.

Conformations of kinesin: solution vs. crystal structures and interactions with microtubules

Abstract Recently, the molecular structures of monomeric and dimeric kinesin constructs in complex with ADP have been determined by X-ray crystallography (Kull et al. 1996; Kozielski et al. 1997 a; Sack et al. 1997). The “motor” or “head” domains have almost identical conformations in the known crystal structures, yet the kinesin dimer is asymmetric: the orientation of the two heads relative to the coiled-coil formed by their neck regions is different. We used small angle solution scattering of kinesin constructs and microtubules decorated with kinesin in order to find out whether these crystal structures are of relevance for kinesins structure under natural conditions and for its interaction with microtubules. Our preliminary results indicate that the crystal structures of monomeric and dimeric kinesin are similar to their structures in solution, though in solution the center-of-mass distance between the motor domains of the dimer could be slightly greater. The crystal structure of dimeric kinesin can be interpreted as representing two equivalent conformations. Transitions between these or very similar conformational states may occur in solution. Binding of kinesin to microtubules has conformational effects on both, the kinesin and the microtubule. Solution scattering of kinesin decorated microtubules reveals a peak in intensity that is characteristic for the B-surface lattice and that can be used to monitor the axial repeat of the microtubules under various conditions. In decoration experiments, dimeric kinesin dissociates, at least partly, leading to a stoichiometry of 1:1 (one kinesin head per tubulin dimer; Thormählen et al. 1998 a) in contrast to the stoichiometry of 2:1 reported for dimeric ncd. This discrepancy is possibly due to the effect of steric hindrance between kinesin dimers on adjacent binding sites.