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E M Mandelkow

Publications and source records attributed to E M Mandelkow.

At least 19 recordsLinked to original sources
PubMed1992

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 phosphorylatable epitopes at Ser-Pro motifs, as well as a 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.

Alzheimer Disease↗
PubMed1992

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 tau's 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.

Alzheimer Disease↗
PubMed1992

Phosphorylation-dependent epitopes of neurofilament antibodies on tau protein and relationship with Alzheimer tau.

We have studied the phosphorylation of tau protein from Alzheimer paired helical filaments, of tau from normal human brain, and of recombinant tau isoforms. As a tool we used monoclonal antibodies against neurofilament protein [Sternberger, N., Sternberger, L. & Ulrich, J. (1985) Proc. Natl. Acad. Sci. USA 82, 4274-4276] that crossreact with tau in a phosphorylation-dependent manner. This allowed us to deduce the state of phosphorylation in normal and pathological tau, as well as antibody epitopes. The epitope of antibody SMI33 is at the first Lys-Ser-Pro sequence motif (residues 234-236) and requires an unphosphorylated Ser-235. Antibody SMI31 binds between Ser-396 (in the second Lys-Ser-Pro motif) and Ser-404, both of which must be phosphorylated. SMI34 has a conformational epitope that depends on the interaction between regions on either side of the microtubule-binding region; it also requires phosphorylation. The phosphorylatable serines detected by the SMI antibodies are part of Ser-Pro motifs and can be phosphorylated by a protein kinase activity that can be used to induce a paired helical filament-like state in human brain tau in vitro. The phosphates are incorporated in several stages that can be identified by antibody reactivity and gel shift. This suggests a role for the phosphorylation sites in Alzheimer disease, as well as the involvement of a Ser-Pro-directed protein kinase.

Alzheimer Disease↗
PubMed1992

The juvenile microtubule-associated protein MAP2c is a rod-like molecule that forms antiparallel dimers.

We have developed a procedure to isolate the microtubule-associated protein 2c (MAP2c), a juvenile form of MAP2 occurring in mammalian brain. The shape, size, self-association, and antibody interactions of MAP2c were studied. Monomeric MAP2c is an elongated molecule with a length approximately 48 nm, considerably shorter than the higher molecular weight forms MAP2a or b of adult brain. Two monoclonal antibodies whose epitopes are near the N or C terminus, respectively, are located close to the opposite ends of the MAP2c rods. This places constraints on the types of internal folding of the molecule. MAP2c self-associates into dimers and fibrous aggregates. The dimers are predominantly antiparallel and nearly in register, as judged by antibody labeling.

Animals↗
PubMed1992

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 Alzheimer's 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.

Alzheimer Disease↗
PubMed1992

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 Alzheimer's 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.

Alzheimer Disease↗
PubMed1992

Sabeluzole, a memory-enhancing molecule, increases fast axonal transport in neuronal cell cultures.

Morphological rearrangements, such as synapse number changes, have been observed in the adult mammalian brain after various experimental paradigms of learning and behavioral experience. The role of axonal transport in the physical translocation of material during this form of brain plasticity has not been fully appreciated. We show here by quantitative video microscopy that sabeluzole (R58735), a new memory-enhancing drug in humans, effectively increases fast axonal transport in rat neuronal cell cultures. Long-term incubation (24 hr) with sabeluzole in the concentration range between 0.1 and 1 microM increases both velocity and jump length of saltatory movements maximally by 20-30% in embryonic hippocampal neurons. Acute treatment only increases the velocity by 15-20%. Furthermore, the inhibition of axonal transport by 0.1 mM vanadate in N4 neuroblastoma cells is reversed by 1 microM sabeluzole. Observations on the kinesin-induced microtubule mobility in a reconstituted system show a 10% enhancement by sabeluzole at an optimal concentration of 2 microM, but no increase in kinesin ATPase activity. To our knowledge, this is the first pharmacological compound shown to increase fast axonal transport. The mechanism of fast axonal transport enhancement is discussed as a rationale for new therapeutic treatment in neuropathology.

Animals↗
PubMed1992

Alzheimer-like paired helical filaments and antiparallel dimers formed from microtubule-associated protein tau in vitro.

Recent evidence from several laboratories shows that the paired helical filaments of Alzheimer's disease brains consist mainly of the protein tau in an abnormally phosphorylated form, but the mode of assembly is not understood. Here we use EM to study several constructs derived from human brain tau and expressed in Escherichia coli. All constructs or tau isoforms are rodlike molecules with a high tendency to dimerize in an antiparallel fashion, as shown by antibody labeling and chemical crosslinking. The length of the rods is largely determined by the region of internal repeats that is also responsible for microtubule binding. One unit length of the repeat domain (three or four repeats) is around 22-25 nm, comparable to the cross-section of Alzheimer PHF cores. Constructs corresponding roughly to the repeat region of tau can form synthetic paired helical filaments resembling those from Alzheimer brain tissue. A similar self-assembly occurs with the chemically cross-linked dimers. In both cases there is no need for phosphorylation of the protein.

Alzheimer Disease↗
PubMed1991

Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study.

Microtubules display the unique property of dynamic instability characterized by phase changes between growth and shrinkage, even in constant environmental conditions. The phases can be synchronized, leading to bulk oscillations of microtubules. To study the structural basis of dynamic instability we have examined growing, shrinking, and oscillating microtubules by time-resolved cryo-EM. In particular we have addressed three questions which are currently a matter of debate: (a) What is the relationship between microtubules, tubulin subunits, and tubulin oligomers in microtubule dynamics?; (b) How do microtubules shrink? By release of subunits or via oligomers?; and (c) Is there a conformational change at microtubule ends during the transitions from growth to shrinkage and vice versa? The results show that (a) oscillating microtubules coexist with a substantial fraction of oligomers, even at a maximum of microtubule assembly; (b) microtubules disassemble primarily into oligomers; and (c) the ends of growing microtubules have straight protofilaments, shrinking microtubules have protofilaments coiled inside out. This is interpreted as a transition from a tense to a relaxed conformation which could be used to perform work, as suggested by some models of poleward chromosome movement during anaphase.

Animals↗
PubMed1991

Effect of MAP2, MAP2c, and tau on kinesin-dependent microtubule motility.

By making use of DIC video microscopy to monitor microtubule motility we have studied the effect of several MAPs (MAP2, MAP2c, tau) on microtubule-kinesin interactions and microtubule gliding. Of the three MAPs tested, MAP2 interferes most strongly with kinesin-dependent microtubule motility.

Adenosine Triphosphatases↗
PubMed1990

Microtubule oscillations. Role of nucleation and microtubule number concentration.

Microtubules are capable of performing synchronized oscillations of assembly and disassembly which has been explained by reaction mechanisms involving tubulin subunits, oligomers, microtubules, and GTP. Here we address the question of how microtubule nucleation or their number concentration affects the oscillations. Assembly itself requires a critical protein concentration (Cc), but oscillations require in addition a critical microtubule number concentration (CMT). In spontaneous assembly this can be achieved with protein concentrations Cos well above the critical concentration Cc because this enhances the efficiency of nucleation. Seeding with microtubules can either generate oscillations or suppress them, depending on how the seeds alter the effective microtubule number concentration. The relative influence of microtubule number and total protein concentrations can be varied by the rate at which assembly conditions are induced (e.g. by a temperature rise): Fast T-jumps induce oscillations because of efficient nucleation, slow ones do not. Oscillations become damped for several reasons. One is the consumption of GTP, the second is a decrease in microtubule number, and the third is that the ratio of microtubules in the two phases (growth-competent and shrinkage-competent) approach a steady state value. This ratio can be perturbed, and the oscillations restarted, by a cold shock, addition of seeds, addition of GTP, or fragmentation. Each of these is equivalent to a change in the effective microtubule number concentration.

Guanosine Diphosphate↗
PubMed1990

Phosphorylation of microtubule-associated protein tau: identification of the site for Ca2(+)-calmodulin dependent kinase and relationship with tau phosphorylation in Alzheimer tangles.

The microtubule array in neuronal cells undergoes extensive growth, dynamics and rearrangements during neurite outgrowth. While little is known about how these changes are regulated, microtubule-associated proteins (MAPs) including tau protein are likely to perform an important role. Tau is one of the MAPs in mammalian brain. When isolated it is usually a mixture of several isoforms containing between 341 and 441 residues that arise from alternative splicing. Tau can be phosphorylated by several protein kinases. Phosphorylation at certain sites results in major structural and functional changes, as seen by changes in electrophoretic mobility, interaction with microtubules, molecular length and elasticity. Here we show that the sites of phosphorylation by four kinases (PKA, PKC, CK and CaMK) all lie in the C-terminal microtubule-binding half of tau, but only the phosphorylation by CaM kinase shows the pronounced shift in electrophoretic mobility characteristic for tau from Alzheimer neurofibrillary tangles. By using a combination of limited proteolysis, protein sequencing and protein engineering we show that a single phosphorylation site is responsible for this shift, located at Ser 405 in the C-terminal tail of the protein outside the region of internal repeats. Phosphorylation at this site not only reduces the electrophoretic mobility of tau, it also makes the protein long and stiff, as shown earlier. The site is likely to be phosphorylated in tau from Alzheimer neurofibrillary tangles.

Alzheimer Disease↗