George S. Bloom

Regulation of the Phosphorylation State and Microtubule-Binding Activity of Tau by Protein Phosphatase 2A

Recently, we reported that a pool of protein phosphatase 2A (PP2A) is associated with microtubules. Here, we demonstrate that specific isoforms of PP2A bind and dephosphorylate the neuronal microtubule-associated protein tau. Coexpression of tau and SV40 small t, a specific inhibitor of PP2A, in CV-1, NIH 3T3, or NT2 cells induced the phosphorylation of tau at multiple sites, including Ser-199, Ser-202, Thr-205, Ser-396, and Ser-404. Immunofluorescent and biochemical analyses revealed that hyperphosphorylation correlated with dissociation of tau from microtubules and a loss of tau-induced microtubule stabilization. Taken together, these results support the hypothesis that PP2A controls the phosphorylation state of tau in vivo.

Submolecular domains of bovine brain kinesin identified by electron microscopy and monoclonal antibody decoration

Kinesin is a microtubule-activated ATPase thought to transport membrane-bounded organelles along MTs. To illuminate the structural basis for this function, EM was used to locate submolecular domains on bovine brain kinesin. Rotary shadowed kinesin appeared rod-shaped and approximately 80 nm long. One end of each molecule contained a pair of approximately 10 x 9 nm globular domains, while the opposite end was fan-shaped. Monoclonal antibodies against the approximately 124 kd heavy chains of kinesin decorated the globular structures, while those specific for the approximately 64 kd light chains labeled the fan-shaped end. Quick-freeze, deep-etch EM was used to analyze MTs polymerized from tubulin and cross-linked to latex microspheres by kinesin. Microspheres frequently attached to MTs by arm-like structures, 25-30 nm long. The MT attachment sites often appeared as one or two approximately 10 nm globular bulges. Morphologically similar cross-links were observed by quick-freeze, deep-etch EM between organelles and MTs in the neuronal cytoskeleton in vivo. These collective observations suggest that bovine brain kinesin binds to MTs by globular domains that contain the heavy chains, and that the attachment sites for organelles are at the opposite, fan-shaped end of kinesin, where the light chains are located.

Amyloid-β and tau: the trigger and bullet in Alzheimer disease pathogenesis.

The defining features of Alzheimer disease (AD) include conspicuous changes in both brain histology and behavior. The AD brain is characterized microscopically by the combined presence of 2 classes of abnormal structures, extracellular amyloid plaques and intraneuronal neurofibrillary tangles, both of which comprise highly insoluble, densely packed filaments. The soluble building blocks of these structures are amyloid-β (Aβ) peptides for plaques and tau for tangles. Amyloid-β peptides are proteolytic fragments of the transmembrane amyloid precursor protein, whereas tau is a brain-specific, axon-enriched microtubule-associated protein. The behavioral symptoms of AD correlate with the accumulation of plaques and tangles, and they are a direct consequence of the damage and destruction of synapses that mediate memory and cognition. Synapse loss can be caused by the failure of live neurons to maintain functional axons and dendrites or by neuron death. During the past dozen years, a steadily accumulating body of evidence has indicated that soluble forms of Aβ and tau work together, independently of their accumulation into plaques and tangles, to drive healthy neurons into the diseased state and that hallmark toxic properties of Aβ require tau. For instance, acute neuron death, delayed neuron death following ectopic cell cycle reentry, and synaptic dysfunction are triggered by soluble, extracellular Aβ species and depend on soluble, cytoplasmic tau. Therefore, Aβ is upstream of tau in AD pathogenesis and triggers the conversion of tau from a normal to a toxic state, but there is also evidence that toxic tau enhances Aβ toxicity via a feedback loop. Because soluble toxic aggregates of both Aβ and tau can self-propagate and spread throughout the brain by prionlike mechanisms, successful therapeutic intervention for AD would benefit from detecting these species before plaques, tangles, and cognitive impairment become evident and from interfering with the destructive biochemical pathways that they initiate.

Molecular Interactions among Protein Phosphatase 2A, Tau, and Microtubules IMPLICATIONS FOR THE REGULATION OF TAU PHOSPHORYLATION AND THE DEVELOPMENT OF TAUOPATHIES

Hyperphosphorylated forms of the neuronal microtubule (MT)-associated protein tau are major components of Alzheimer’s disease paired helical filaments. Previously, we reported that ABαC, the dominant brain isoform of protein phosphatase 2A (PP2A), is localized on MTs, binds directly to tau, and is a major tau phosphatase in cells. We now describe direct interactions among tau, PP2A, and MTs at the submolecular level. Using tau deletion mutants, we found that ABαC binds a domain on tau that is indistinguishable from its MT-binding domain. ABαC binds directly to MTs through a site that encompasses its catalytic subunit and is distinct from its binding site for tau, and ABαC and tau bind to different domains on MTs. Specific PP2A isoforms bind to MTs with distinct affinities in vitro, and these interactions differentially inhibit the ability of PP2A to dephosphorylate various substrates, including tau and tubulin. Finally, tubulin assembly decreases PP2A activity in vitro, suggesting that PP2A activity can be modulated by MT dynamics in vivo. Taken together, these findings indicate how structural interactions among ABαC, tau, and MTs might control the phosphorylation state of tau. Disruption of these normal interactions could contribute significantly to development of tauopathies such as Alzheimer’s disease.

Prion-like behaviour and tau-dependent cytotoxicity of pyroglutamylated amyloid-β

Extracellular plaques of amyloid-β and intraneuronal neurofibrillary tangles made from tau are the histopathological signatures of Alzheimer’s disease. Plaques comprise amyloid-β fibrils that assemble from monomeric and oligomeric intermediates, and are prognostic indicators of Alzheimer’s disease. Despite the importance of plaques to Alzheimer’s disease, oligomers are considered to be the principal toxic forms of amyloid-β. Interestingly, many adverse responses to amyloid-β, such as cytotoxicity, microtubule loss, impaired memory and learning, and neuritic degeneration, are greatly amplified by tau expression. Amino-terminally truncated, pyroglutamylated (pE) forms of amyloid-β are strongly associated with Alzheimer’s disease, are more toxic than amyloid-β, residues 1–42 (Aβ1–42) and Aβ1–40, and have been proposed as initiators of Alzheimer’s disease pathogenesis. Here we report a mechanism by which pE-Aβ may trigger Alzheimer’s disease. Aβ3(pE)–42 co-oligomerizes with excess Aβ1–42 to form metastable low-n oligomers (LNOs) that are structurally distinct and far more cytotoxic to cultured neurons than comparable LNOs made from Aβ1–42 alone. Tau is required for cytotoxicity, and LNOs comprising 5% Aβ3(pE)–42 plus 95% Aβ1–42 (5% pE-Aβ) seed new cytotoxic LNOs through multiple serial dilutions into Aβ1–42 monomers in the absence of additional Aβ3(pE)–42. LNOs isolated from human Alzheimer’s disease brain contained Aβ3(pE)–42, and enhanced Aβ3(pE)–42 formation in mice triggered neuron loss and gliosis at 3 months, but not in a tau-null background. We conclude that Aβ3(pE)–42 confers tau-dependent neuronal death and causes template-induced misfolding of Aβ1–42 into structurally distinct LNOs that propagate by a prion-like mechanism. Our results raise the possibility that Aβ3(pE)–42 acts similarly at a primary step in Alzheimer’s disease pathogenesis.

IQGAP1, a Novel Vascular Endothelial Growth Factor Receptor Binding Protein, Is Involved in Reactive Oxygen Species—Dependent Endothelial Migration and Proliferation

Endothelial cell (EC) proliferation and migration are important for reendothelialization and angiogenesis. We have demonstrated that reactive oxygen species (ROS) derived from the small GTPase Rac1-dependent NAD(P)H oxidase are involved in vascular endothelial growth factor (VEGF)–mediated endothelial responses mainly through the VEGF type2 receptor (VEGFR2). Little is known about the underlying molecular mechanisms. IQGAP1 is a scaffolding protein that controls cellular motility and morphogenesis by interacting directly with cytoskeletal, cell adhesion, and small G proteins, including Rac1. In this study, we show that IQGAP1 is robustly expressed in ECs and binds to the VEGFR2. A pulldown assay using purified proteins demonstrates that IQGAP1 directly interacts with active VEGFR2. In cultured ECs, VEGF stimulation rapidly promotes recruitment of Rac1 to IQGAP1, which inducibly binds to VEGFR2 and which, in turn, is associated with tyrosine phosphorylation of IQGAP1. Endogenous IQGAP1 knockdown by siRNA shows that IQGAP1 is involved in VEGF-stimulated ROS production, Akt phosphorylation, endothelial migration, and proliferation. Wound assays reveal that IQGAP1 and phosphorylated VEGFR2 accumulate and colocalize at the leading edge in actively migrating ECs. Moreover, we found that IQGAP1 expression is dramatically increased in the VEGFR2-positive regenerating EC layer in balloon-injured rat carotid artery. These results suggest that IQGAP1 functions as a VEGFR2-associated scaffold protein to organize ROS-dependent VEGF signaling, thereby promoting EC migration and proliferation, which may contribute to repair and maintenance of the functional integrity of established blood vessels.

Tau-dependent microtubule disassembly initiated by prefibrillar β-amyloid

Alzheimers Disease (AD) is defined histopathologically by extracellular β-amyloid (Aβ) fibrils plus intraneuronal tau filaments. Studies of transgenic mice and cultured cells indicate that AD is caused by a pathological cascade in which Aβ lies upstream of tau, but the steps that connect Aβ to tau have remained undefined. We demonstrate that tau confers acute hypersensitivity of microtubules to prefibrillar, extracellular Aβ in nonneuronal cells that express transfected tau and in cultured neurons that express endogenous tau. Prefibrillar Aβ42 was active at submicromolar concentrations, several-fold below those required for equivalent effects of prefibrillar Aβ40, and microtubules were insensitive to fibrillar Aβ. The active region of tau was localized to an N-terminal domain that does not bind microtubules and is not part of the region of tau that assembles into filaments. These results suggest that a seminal cell biological event in AD pathogenesis is acute, tau-dependent loss of microtubule integrity caused by exposure of neurons to readily diffusible Aβ.

The Mechanism for Regulation of the F-actin Binding Activity of IQGAP1 by Calcium/Calmodulin ,

IQGAP1 colocalizes with actin filaments in the cell cortex and binds in vitro to F-actin and several signaling proteins, including calmodulin, Cdc42, Rac1, and β-catenin. It is thought that the F-actin binding activity of IQGAP1 is regulated by its reversible association with these signaling molecules, but the mechanisms have remained obscure. Here we describe the regulatory mechanism for calmodulin. Purified adrenal IQGAP1 was found to consist of two distinct protein pools, one of which bound F-actin and lacked calmodulin, and the other of which did not bind F-actin but was tightly associated with calmodulin. Based on this finding we hypothesized that calmodulin negatively regulates binding of IQGAP1 to F-actin. This hypothesis was tested in vitro using recombinant wild type and mutated IQGAP1s and in live cells that transiently expressed IQGAP1-YFP. In vitro, the affinity of wild type IQGAP1 for F-actin decreased with increasing concentrations of calmodulin, and this effect was dramatically enhanced by Ca2+ and required the IQ domains of IQGAP1. In addition, we found that calmodulin bound wild type IQGAP1 much more efficiently in the presence of Ca2+ than EGTA, and all 8 IQ motifs in each IQGAP1 dimer could bind calmodulin simultaneously. In live cells, IQGAP1-YFP localized to the cell cortex, but elevation of intracellular Ca2+ reversibly induced the fluorescent fusion protein to become diffusely distributed. Taken together, these results support a model in which a rise in free intracellular Ca2+ promotes binding of calmodulin to IQGAP1, which in turn inhibits IQGAP1 from binding to cortical actin filaments.

IQGAP1 regulates cell motility by linking growth factor signaling to actin assembly

IQGAP1 has been implicated as a regulator of cell motility because its overexpression or underexpression stimulates or inhibits cell migration, respectively, but the underlying mechanisms are not well understood. Here, we present evidence that IQGAP1 stimulates branched actin filament assembly, which provides the force for lamellipodial protrusion, and that this function of IQGAP1 is regulated by binding of type 2 fibroblast growth factor (FGF2) to a cognate receptor, FGFR1. Stimulation of serum-starved MDBK cells with FGF2 promoted IQGAP1-dependent lamellipodial protrusion and cell migration, and intracellular associations of IQGAP1 with FGFR1 – and two other factors – the Arp2/3 complex and its activator N-WASP, that coordinately promote nucleation of branched actin filament networks. FGF2 also induced recruitment of IQGAP1, FGFR1, N-WASP and Arp2/3 complex to lamellipodia. N-WASP was also required for FGF2-stimulated migration of MDBK cells. In vitro, IQGAP1 bound directly to the cytoplasmic tail of FGFR1 and to N-WASP, and stimulated branched actin filament nucleation in the presence of N-WASP and the Arp2/3 complex. Based on these observations, we conclude that IQGAP1 links FGF2 signaling to Arp2/3 complex-dependent actin assembly by serving as a binding partner for FGFR1 and as an activator of N-WASP.

Cytoskeletal Pathologies of Alzheimer Disease

The histopathological hallmarks of Alzheimer disease are the extracellular amyloid plaques, composed principally of the amyloid beta peptide, and the intracellular neurofibrillary tangles, composed of paired helical filaments of the microtubule-associated protein, tau. Other histopathological structures involving actin and the actin-binding protein, cofilin, have more recently been recognized. Here we review new findings about these cytoskeletal pathologies, and, emphasize how plaques, tangles, the actin-containing inclusions and their respective building blocks may contribute to Alzheimer pathogenesis and the primary behavioral symptoms of the disease. Cell Motil. Cytoskeleton, 2009. (c) 2009 Wiley-Liss, Inc.