Increased expression of BIN1 mediates Alzheimer genetic risk by modulating tau pathology.

Genome-wide association studies (GWAS) have identified a region upstream the BIN1 gene as the most important genetic susceptibility locus in Alzheimer's disease (AD) after APOE. We report that BIN1 transcript levels were increased in AD brains and identified a novel 3 bp insertion allele ∼28 kb upstream of BIN1, which increased (i) transcriptional activity in vitro, (ii) BIN1 expression levels in human brain and (iii) AD risk in three independent case-control cohorts (Meta-analysed Odds ratio of 1.20 (1.14-1.26) (P=3.8 × 10(-11))). Interestingly, decreased expression of the Drosophila BIN1 ortholog Amph suppressed Tau-mediated neurotoxicity in three different assays. Accordingly, Tau and BIN1 colocalized and interacted in human neuroblastoma cells and in mouse brain. Finally, the 3 bp insertion was associated with Tau but not Amyloid loads in AD brains. We propose that BIN1 mediates AD risk by modulating Tau pathology.

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.

MAPs, MARKs and microtubule dynamics.

Microtubules (MTs) serve as tracks for cellular transport, and regulate cell shape and polarity. Rapid transitions between stable and dynamic forms of MTs are central to these processes. This dynamic instability is regulated by a number of cellular factors, including the structural MT-associated proteins (MAPs), which in turn are regulated by phosphorylation. MT-affinity-regulating kinases (MARKs) are novel mammalian serine/threonine kinases that phosphorylate the tubulin-binding domain of MAPs and thereby cause their detachment from MTs and increased MT dynamics. Molecular cloning of MARKs revealed a family of four closely related protein kinases that share homology with genes from the nematode Caenorhabditis elegans and fission yeast that are involved in the generation of cell shape and polarity. Hence, MARKs might play a role in the regulation of MT stability during morphogenesis.

BRD4 bimodal binding at promoters and drug-induced displacement at Pol II pause sites associates with I-BET sensitivity.

BACKGROUND: Deregulated transcription is a major driver of diseases such as cancer. Bromodomain and extra-terminal (BET) proteins (BRD2, BRD3, BRD4 and BRDT) are chromatin readers essential for maintaining proper gene transcription by specifically binding acetylated lysine residues. Targeted displacement of BET proteins from chromatin, using BET inhibitors (I-BETs), is a promising therapy, especially for acute myeloid leukemia (AML), and evaluation of resistance mechanisms is necessary to optimize the clinical efficacy of these drugs. RESULTS: To uncover mechanisms of intrinsic I-BET resistance, we quantified chromatin binding and displacement for BRD2, BRD3 and BRD4 after dose response treatment with I-BET151, in sensitive and resistant in vitro models of leukemia, and mapped BET proteins/I-BET interactions genome wide using antibody- and compound-affinity capture methods followed by deep sequencing. The genome-wide map of BET proteins sensitivity to I-BET revealed a bimodal pattern of binding flanking transcription start sites (TSSs), in which drug-mediated displacement from chromatin primarily affects BRD4 downstream of the TSS and prolongs the pausing of RNA Pol II. Correlation of BRD4 binding and drug-mediated displacement at RNA Pol II pause sites with gene expression revealed a differential behavior of sensitive and resistant tumor cells to I-BET and identified a BRD4 signature at promoters of sensitive coding and non-coding genes. CONCLUSIONS: We provide evidence that I-BET-induced shift of Pol II pausing at promoters via displacement of BRD4 is a determinant of intrinsic I-BET sensitivity. This finding may guide pharmacological treatment to enhance the clinical utility of such targeted therapies in AML and potentially other BET proteins-driven diseases.

Phosphorylation of Ser262 strongly reduces binding of tau to microtubules: distinction between PHF-like immunoreactivity and microtubule binding.

Tau protein, a component of Alzheimer paired helical filaments, can be phosphorylated by several kinases. Of particular interest is the phosphorylation at Ser/Thr-Pro motifs because the resulting state of tau is similar to that found in Alzheimer's disease, as judged by its immunoreactivity. This state can be mimicked by a brain extract kinase activity and by MAP kinase. We have now studied the effect of these modes of phosphorylation on the interaction between tau and microtubules. Although MAP kinase efficiently phosphorylates many Ser/Thr-Pro motifs of tau, its effect on microtubule binding is only moderate. By contrast, phosphorylation of a single residue, Ser262, has a major effect on binding. Ser262 is not phosphorylated by MAP kinase or other proline-directed kinases, but is phosphorylated by a 35/41 kd kinase in brain, whose purification is described.

Glutathionyl(cysteine-374) actin forms filaments of low mechanical stability.

Rabbit muscle actin reacts with 2,4-dinitrophenylglutathionyldisulfide, forming a mixed disulfide in position 374. the product S-(cysteine-374)glutathionyl actin forms filaments which are easily disrupted under shearing stress. Even weak mechanical strain, as exerted, for example, during capillary viscometry or heating the solution to 37 degrees C, leads to considerable breakage of these filaments. Because of spontaneous repair which consumes ATP, the mechanically broken filaments exhibit an approx. 6-fold enhanced steady-state ATPase activity as compared to normal F-actin. Monomers of glutathionyl actin have a reduced affinity for their bound nucleotide and a slightly increased critical concentration. Disruption of the filaments and enhanced ATPase activity are reversed by the addition of KCl or the mushroom toxin phalloidin. By the large stabilizing effects of KCl and phalloidin on glutathionyl actin filaments we propose glutathionyl actin as a tool for detecting filament-stabilizing agents and for studying the different mechanisms of filament stabilization.

Microtubule-affinity regulating kinase (MARK) is tightly associated with neurofibrillary tangles in Alzheimer brain: a fluorescence resonance energy transfer study.

Paired helical filaments, the main structural components of the neurofibrillary tangles in Alzheimer disease, consist of phosphorylated tau protein. Because the levels and degree of phosphorylation are significantly higher in paired helical filament (PHF)-derived tau than in normal adult tau, and because phosphorylation of tau severely disrupts microtubule stability, it is postulated that tau phosphorylation is an important step in PHF formation. The kinases and/or phosphatases that act in vivo to help induce such a pathological state of tau, however, are not yet known. In this study we implicate the non-proline directed kinase MARK in PHF-tau phosphorylation, by virtue of its close intermolecular association with the phosphorylated Ser262 epitope on PHF-tau as assessed by fluorescence resonance energy transfer. Moreover, because this tight enzyme-substrate association is observed in neurofibrillary tangles in Alzheimer tissue, we suggest that PHF-tau phosphorylation may occur to some extent on assembled PHF filaments.

Tau domains, phosphorylation, and interactions with microtubules.

We consider the interactions of tau protein with microtubules from two points of view, phosphorylation and domain structure. Tau can be phosphorylated at many sites and by several kinases, notably by proline-directed kinases (MAPK, GSK-3, cdk5) which generate Alzheimer-like antibody epitopes. Other kinases phosphorylate Ser 262, a site that has a particularly pronounced influence on the affinity of tau for microtubules. All of these sites can be cleared by phosphatases PP-2a and calcineurin. The site Ser262 lies within the repeat domain of tau. However, when probing the domains of tau for their effects on microtubule binding, nucleation, assembly, or bundling, the repeat domain has only a weak influence. Whereas the repeat domain of tau binds to microtubules with low affinity, repeat-less tau binds strongly yet unproductively in terms of microtubule assembly. Productive binding of tau to microtubules depends on the combination of (some) repeats with the flanking regions, as if the flanking regions acted as "jaws" for the proper positioning of tau on the microtubule surface.

Cloning and sequencing of a cDNA encoding rat brain mitogen-activated protein (MAP) kinase activator.

The complete cDNA sequence for mitogen-activated protein kinase activator from rat brain was cloned. It encodes a protein kinase of 393 amino acids with a calculated M(r) of 43,465.

Interchain and intrachain crosslinking of actin thiols by a bifunctional thiol reagent.

From 1,9-nonylenedithiol and Ellman's Reagent the bifunctional asymmetric disulfide n-nonylene-1,9-bis-[5-dithio-(2-nitrobenzoic acid)] (NBDN) was prepared. By monovalent reaction with cysteine-374 the crosslinker could be introduced into monomeric actin, with release of one equivalent of yellow 2-nitro-5-thiobenzoate (NTB). From the monovalent actin derivative we prepared a crosslinked actin dimer (Cys-374-Cys-374') as well as a monomer with a crosslink between Cys-374 and Cys-10. Neither crosslinked actin species was able to polymerize the crosslinked monomer even in the presence of phalloidin. The crosslinked monomer polymerized on the addition of dithiothreitol, thus providing the first unpolymerizable actin species whose polymerizability can be restored under mild conditions. We suggest the use of NBDN as a thiol-specific crosslinker that reacts under spectrophotometric control and can be removed by the addition of thiols.

Probing the phalloidin binding site of actin.

Phallotoxins form tight complexes with filamentous actin and stabilize the polymer against shearing stress. In the present study a phalloidin derivative containing a thiol-capturing moiety was prepared and reacted with single thiol groups of monomeric muscle actin. Sites of attachment in the protein were Cys-374 next to the C-terminus and Cys-10, close to the N-terminus; the latter was recently shown to be uncovered during a slow but reversible conformational transition occurring in ADP-G-actin. Phalloidin bound to Cys-374 stabilizes filaments against shearing stress almost as effectively as free phalloidin, indicating that the phalloidin binding site cannot be far from the C-terminus of actin. Stabilization was also achieved when the phalloidin reagent was added to F-actin, however, the subsequent formation of a covalent linkage with Cys-374 was not observed, most likely due to a restricted mobility of the reactants. In contrast to the efficient stabilization of filaments by phalloidin linked to Cys-374 a destabilizing effect was observed when phalloidin was attached to Cys-10. It appears that phalloidin located close to the N-terminus is unable to bind to the normal binding site in its own filament. Pronounced gelification of this actin derivative suggests that the toxin is able to mediate crosslinking with neighbouring filaments. From these results we conclude that the phalloidin binding site of actin is distant from the N-terminus, but close to the C-terminus. Furthermore, the data provide evidence that binding of phalloidin reduces the mobility of the C-terminus.

Dephosphorylation of tau protein and Alzheimer paired helical filaments by calcineurin 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 microtubules [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.

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.

Structure, microtubule interactions, and phosphorylation of tau protein.

This paper summarizes recent structural and functional studies on tau protein, its interactions with microtubules, its self-assembly into paired helical filaments (PHF)-like fibers, and its modification by phosphorylation. The structure of tau in solution resembles that of a random coil. Both tau and Alzheimer PHFs have very little secondary structure, making it improbable that the assembly of tau into PHFs is based on interacting beta sheets. Tau's binding to microtubules can be described by a "jaws" effect. The domain containing the repeats binds very weakly, while the flanking regions (jaws) bind strongly, even without the repeats. However, only the combination of flanking regions and repeats makes binding productive in terms of microtubule nucleation and assembly. Although the majority of Alzheimer-like phosphorylation sites are outside the repeats they have only a weak influence on binding, whereas the phosphorylation at Ser262 inside the repeats inhibits binding and makes microtubules dynamically unstable. This site can be phosphorylated by kinases present in brain tissue, and it is uniquely phosphorylated in Alzheimer brain.

Microtubule-associated protein tau, paired helical filaments, and phosphorylation.

This paper summarizes our recent studies on microtubule-associated protein tau and its pathological state resembling that of the paired helical filaments of Alzheimer's disease. The Alzheimer-like state of tau protein can be identified and analyzed in terms of certain phosphorylation sites and phosphorylation-dependent antibody epitopes. It can be induced by protein kinases which tend to phosphorylate serine or threonine residues followed by a proline; this includes mitogen-activated protein kinase (MAPK) and glycogen-synthase kinase 3 (GSK-3). Both of these are tightly associated with microtubules as well as with paired helical filaments. Structurally, tau appears as a rod-like molecule; it tends to self-associate into dimers whose monomers are antiparallel. Constructs of truncated tau made up of antiparallel dimers of the microtubule binding domain can be assembled into paired helical filaments in vitro.

[The use of contrast media in computer tomography (author's transl)]. / Möglichkeiten der Kontrastmittelanwendung bei der Computertomographie.

There are a variety of applications for contrast media in CT. They can be used for better anatomic demonstration or for showing pathological processes. Nearly all available contrast media may be found useful. Intravenously injected renographic contrast media are most commonly employed. In addition to their inherent contrast, specific pharmaco-kinetic changes may be of diagnostic value. This adds some knowledge of function to the purely morphological information obtained by computer tomography. For this purpose, contrast has to be injected rapidly and a fast scanner must be used (so-called angio-CT).

Recurrent mutations, including NPM1c, activate a BRD4-dependent core transcriptional program in acute myeloid leukemia.

Recent evidence suggests that inhibition of bromodomain and extra-terminal (BET) epigenetic readers may have clinical utility against acute myeloid leukemia (AML). Here we validate this hypothesis, demonstrating the efficacy of the BET inhibitor I-BET151 across a variety of AML subtypes driven by disparate mutations. We demonstrate that a common 'core' transcriptional program, which is HOX gene independent, is downregulated in AML and underlies sensitivity to I-BET treatment. This program is enriched for genes that contain 'super-enhancers', recently described regulatory elements postulated to control key oncogenic driver genes. Moreover, our program can independently classify AML patients into distinct cytogenetic and molecular subgroups, suggesting that it contains biomarkers of sensitivity and response. We focus AML with mutations of the Nucleophosmin gene (NPM1) and show evidence to suggest that wild-type NPM1 has an inhibitory influence on BRD4 that is relieved upon NPM1c mutation and cytosplasmic dislocation. This leads to the upregulation of the core transcriptional program facilitating leukemia development. This program is abrogated by I-BET therapy and by nuclear restoration of NPM1. Finally, we demonstrate the efficacy of I-BET151 in a unique murine model and in primary patient samples of NPM1c AML. Taken together, our data support the use of BET inhibitors in clinical trials in AML.

Chemoproteomics-based kinome profiling and target deconvolution of clinical multi-kinase inhibitors in primary chronic lymphocytic leukemia cells.

The pharmacological induction of apoptosis in neoplastic B cells presents a promising therapeutic avenue for the treatment of chronic lymphocytic leukemia (CLL). We profiled a panel of clinical multi-kinase inhibitors for their ability to induce apoptosis in primary CLL cells. Whereas inhibitors targeting a large number of receptor and intracellular tyrosine kinases including c-KIT, FLT3, BTK and SYK were comparatively inactive, the CDK inhibitors BMS-387032 and flavopiridol showed marked efficacy similar to staurosporine. Using the kinobeads proteomics method, kinase expression profiles and binding profiles of the inhibitors to target protein complexes were quantitatively monitored in CLL cells. The targets most potently affected were CDK9, cyclin T1, AFF3/4 and MLLT1, which may represent four subunits of a deregulated positive transcriptional elongation factor (p-TEFb) complex. Albeit with lower potency, both drugs also bound the basal transcription factor BTF2/TFIIH containing CDK7. Staurosporine and geldanamycin do not affect these targets and thus seem to exhibit a different mechanism of action. The data support a critical role of p-TEFb inhibitors in CLL that supports their future clinical development.

Proteomics-based strategies in kinase drug discovery.

Studies of drug action classically assess biochemical activity in settings which typically contain the isolated target only. Recent technical advances in mass spectrometry-based analysis of proteins have enabled the quantitative analysis of sub-proteomes and entire proteomes, thus initiating a departure from the traditional single gene--single protein--single target paradigm. Here, we review chemical proteomics-based experimental strategies in kinase drug discovery to analyse quantitatively the interaction of small molecule compounds or drugs with a defined sub-proteome containing hundreds of protein kinases and related proteins. One novel approach is based on 'Kinobeads'--an affinity resin comprised of a cocktail of immobilized broad spectrum kinase inhibitors--to monitor quantitatively the differential binding of kinases and related nucleotide-binding proteins in the presence and absence of varying concentrations of a lead compound or drug of interest. Differential binding is detected by high throughput and sensitive mass spectroscopy techniques utilizing isobaric tagging reagents (iTRAQ), yielding quantitative and detailed target binding profiles. The method can be applied to the screening of compound libraries and to selectivity profiling of lead compounds directly against their endogenously expressed targets in a range of cell types and tissue lysates. In addition, the method can be used to map drug-induced changes in the phosphorylation state of the captured sub-proteome, enabling the analysis of signalling pathways downstream of target kinases.

Microtubule affinity-regulating kinase 2 functions downstream of the PAR-3/PAR-6/atypical PKC complex in regulating hippocampal neuronal polarity.

The PAR-3/PAR-6/atypical PKC (aPKC) complex is required for axon-dendrite specification of hippocampal neurons. However, the downstream effectors of this complex are not well defined. In this article, we report a role for microtubule affinity-regulating kinase (MARK)/PAR-1 in axon-dendrite specification. Knocking down MARK2 expression with small interfering RNAs induced formation of multiple axon-like neurites and promoted axon outgrowth. Ectopic expression of MARK2 caused phosphorylation of tau (S262) and led to loss of axons, and this phenotype was rescued by expression of PAR-3, PAR-6, and aPKC. In contrast, the polarity defects caused by an MARK2 mutant (T595A), which is not responsive to aPKC, were not rescued by the PAR-3/PAR-6/aPKC complex. Moreover, polarity was abrogated in neurons overexpressing a mutant of MARK2 with a deleted kinase domain but an intact aPKC-binding domain. Finally, suppression of MARK2 rescued the polarity defects induced by a dominant-negative aPKC mutant. These results suggest that MARK2 is involved in neuronal polarization and functions downstream of the PAR-3/PAR-6/aPKC complex. We propose that aPKC in complex with PAR-3/PAR-6 negatively regulates MARK(s), which in turn causes dephosphorylation of microtubule-associated proteins, such as tau, leading to the assembly of microtubules and elongation of axons.