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Eileen M. Lafer - One of the best experts on this subject based on the ideXlab platform.

  • dynamic interactions between Clathrin and locally structured elements in a disordered Protein mediate Clathrin lattice Assembly
    Journal of Molecular Biology, 2010
    Co-Authors: Yue Zhuo, Udayar Ilangovan, Virgil Schirf, Borries Demeler, Rui J Sousa, Andrew P Hinck, Eileen M. Lafer
    Abstract:

    Abstract Assembly of Clathrin lattices is mediated by Assembly/adaptor Proteins that contain domains that bind lipids or membrane-bound cargo Proteins and Clathrin binding domains (CBDs) that recruit Clathrin. Here, we characterize the interaction between Clathrin and a large fragment of the CBD of the Clathrin Assembly Protein AP180. Mutational, NMR chemical shift, and analytical ultracentrifugation analyses allowed us to precisely define two Clathrin binding sites within this fragment, each of which is found to bind weakly to the N-terminal domain of the Clathrin heavy chain (TD). The locations of the two Clathrin binding sites are consistent with predictions from sequence alignments of previously identified Clathrin binding elements and, by extension, indicate that the complete AP180 CBD contains ∼ 12 degenerate repeats, each containing a single Clathrin binding site. Sequence and circular dichroism analyses have indicated that the AP180 CBD is predominantly unstructured and our NMR analyses confirm that this is largely the case for the AP180 fragment characterized here. Unexpectedly, unlike the many Proteins that undergo binding-coupled folding upon interaction with their binding partners, the AP180 fragment is similarly unstructured in its bound and free states. Instead, we find that this fragment exhibits localized β-turn-like structures at the two Clathrin binding sites both when free and when bound to Clathrin. These observations are incorporated into a model in which weak binding by multiple, pre-structured Clathrin binding elements regularly dispersed throughout a largely unstructured CBD allows efficient recruitment of Clathrin to endocytic sites and dynamic Assembly of the Clathrin lattice.

  • probing the phosphoinositide binding site of the Clathrin Assembly Protein ap 2 with photoaffinity labels
    Archives of Biochemistry and Biophysics, 1998
    Co-Authors: Adam A Profit, Eileen M. Lafer, Kondury Prasad, Jian Chen, Anu Chaudhary, Glenn D Prestwich
    Abstract:

    The relative binding specificities of the subunitsof bovine Assembly Protein AP-2 for the phosphatidylinositol polyphosphates (PtdInsPn) and inositol polyphosphates (InsPn) were determined by photoaffinitylabeling. Three types of benzophenone-containing photoprobes were employed: (i) the water-solubleP-1- or P-2-tethered p-benzoyldihydrocinnamoyl-InsPn (BZDC-InsPn) analogs, (ii) P-1-linked phosphotriester PtdInsPn analogs that sampled the interface between the water and lipid phases, and (iii) sn-1-O-acyl-linked PtdInsPn analogs that interacted with Proteins penetrating the bilayer. The InsPn and PtdInsPn probes bind with highest selectivity and affinity to the two alpha subunit isoforms, with certain probes and conditions resulting in strong labeling of the 50-kDa mu subunit. Three main conclusions were reached: (i) head group recognition predominated over acyl chain recognition, (ii) the PtdInsPn binding site of alpha-AP-2 prefers more highly phosphorylated species, and (iii) the Protein-acyl chain interactions showed high capacity but low selectivity.

  • Clathrin binding and Assembly activities of expressed domains of the synapse specific Clathrin Assembly Protein ap 3
    Journal of Biological Chemistry, 1995
    Co-Authors: Eileen M. Lafer
    Abstract:

    Abstract We separately expressed the 58-kDa C-terminal, 42-kDa middle, 16-kDa C-terminal, and 33-kDa N-terminal regions of AP-3 (also called F1-20, AP180, NP185, and pp155), and determined their Clathrin binding and Assembly properties. The 58-kDa C-terminal region of AP-3 is able to bind to Clathrin triskelia and assemble them into a homogeneous population of Clathrin cages and will also bind to preassembled Clathrin cages. The 42-kDa central region of AP-3 can bind to both Clathrin triskelia and to Clathrin cages, but cannot assemble Clathrin triskelia into Clathrin cages. The 16-kDa C-terminal region of AP-3 can bind to Clathrin cages, but cannot bind to Clathrin triskelia or assemble Clathrin triskelia into Clathrin cages. The Clathrin binding activities of the 42-kDa central region and 16-kDa C-terminal region are weaker than the corresponding activity of either the 58-kDa C-terminal region or full-length AP-3. Previous efforts had mapped a Clathrin binding site within the N-terminal 33 kDa of AP-3 (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Morris, S. A., Schroder, S., Plessmann, U., Weber, K., and Ungewickell, E. (1993) EMBO J. 12, 667-675). However, although the N-terminal 33 kDa of AP-3 is able to bind to Clathrin triskelia (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) [Abstract] J. Neurosci. Res. 41, 15-26), it does not promote their Assembly into Clathrin cages (Murphy, J. E., Pleasure, I. T., Puszkin, S., Prasad, K., and Keen, J. H. (1991) J. Biol. Chem. 266, 4401-4408; Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26) or bind to preassembled Clathrin cages (Ye, W., and Lafer, E. M. (1995) J. Neurosci. Res. 41, 15-26). It appears that the smallest functional unit that carries out all of the reported Clathrin binding and Assembly properties of AP-3, essentially as well as the full-length Protein, is the 58-kDa C-terminal region.

  • inhibition of Clathrin Assembly by high affinity binding of specific inositol polyphosphates to the synapse specific Clathrin Assembly Protein ap 3
    Journal of Biological Chemistry, 1995
    Co-Authors: Nawab Ali, Michael E Bembenek, Stephen B Shears, Eileen M. Lafer
    Abstract:

    Bacterially expressed synapse-specific Clathrin Assembly Protein, AP-3 (F1-20/AP180/NP185/pp155), bound with high affinity both inositol hexakisphosphate (InsP6) (Kd = 239 nM) and diphosphoinositol pentakisphosphate (PP-InsP5) (Kd = 22 nM). The specificity of this ligand binding was demonstrated by competitive displacement of bound [3H]InsP6. IC50 values were as follows: PP-InsP5 = 50 nM, InsP6 = 240 nM, inositol-1,2,4,5,6-pentakisphosphate (Ins(1,2,4,5,6)P5) = 2.2 μM, inositol-1,3,4,5,6-pentakisphosphate (Ins(1,3,4,5,6)P5) = 5 μM, inositol-1,3,4,5-tetrakisphosphate (Ins(1,3,4,5)P4) > 10 μM, inositol-1,4,5-trisphosphate (Ins(1,4,5)P3) > 10 μM. Moreover, 10 μM inositol hexasulfate (InsS6) displaced only 15% of [3H]InsP6. The physiological significance of this binding is the ligand-specific inhibition of Clathrin Assembly (PP-InsP5 > InsP6 > Ins(1,2,4,5,6)P5); Ins(1,3,4,5,6)P5 and InsS6 did not inhibit Clathrin Assembly. We also observed high affinity binding of InsP6 to purified bovine brain AP-3. We separately expressed the 33-kDa amino terminus and the 58-kDa carboxyl terminus, and it was the former that contained the high affinity inositol polyphosphate binding site. These studies suggest that specific inositol polyphosphates may play a role in the regulation of synaptic function by interacting with the synapse-specific Clathrin Assembly Protein AP-3.

  • the synapse specific phosphoProtein f1 20 is identical to the Clathrin Assembly Protein ap 3
    Journal of Biological Chemistry, 1993
    Co-Authors: Shibin Zhou, Nancy H Tannery, Jun Yang, Saul Puszkin, Eileen M. Lafer
    Abstract:

    F1-20 and AP-3 are independently described, synapse-associated, developmentally regulated phosphoProteins with similar apparent molecular masses on SDS-polyacrylamide gel electrophoresis (PAGE). F1-20 was cloned and characterized because of its synapse specificity. AP-3 was purified and studied biochemically because of its function as a Clathrin Assembly Protein. Here we present evidence that establishes the identity of F1-20 and AP-3. Monoclonal antibodies against F1-20 and AP-3 both specifically recognize a single Protein from mouse brain with an apparent molecular mass of 190 kDa on SDS-PAGE. These monoclonal antibodies also specifically recognize the cloned F1-20 Protein expressed in Escherichia coli. The anti-F1-20 monoclonal antibody (mAb) stains a bovine Protein with an apparent molecular mass on SDS-PAGE of 190 kDa that copurifies with brain Clathrin-coated vesicles (CCVs) and that can be extracted from the brain CCVs under conditions that extract AP-3. The anti-F1-20 and anti-AP-3 mAbs specifically recognize the same spot on a two-dimensional gel run on a bovine brain Clathrin-coated vesicle extract. AP-3 purified from bovine brain CCVs is recognized by both the anti-F1-20 and anti-AP-3 mAbs. Purified preparations of bovine AP-3 and bacterially expressed mouse F1-20 give identical patterns of protease digestion with bromelain and subtilisin. Sequence analyses reveal that F1-20 has an essentially neutral 30-kDa NH2-terminal domain with an amino acid composition typical of a globular structure and an acidic COOH-terminal domain rich in proline, serine, threonine, and alanine. This is consistent with proteolysis experiments that suggested that AP-3 could be divided into a 30-kDa globular uncharged Clathrin-binding domain and an acidic, anomalously migrating domain.

James H Keen - One of the best experts on this subject based on the ideXlab platform.

  • Clathrin Assembly Protein ap 2 induces aggregation of membrane vesicles a possible role for ap 2 in endosome formation
    Journal of Cell Biology, 1992
    Co-Authors: Kenneth A Beck, Michael Chang, Frances M Brodsky, James H Keen
    Abstract:

    We have examined the in vitro behavior of Clathrin-coated vesicles that have been stripped of their surface coats such that the majority of the Clathrin is removed but substantial amounts of Clathrin Assembly Proteins (AP) remain membrane-associated. Aggregation of these stripped coated vesicles (s-CV) is observed when they are placed under conditions that approximate the pH and ionic strength of the cell interior (pH 7.2, approximately 100 mM salt). This s-CV aggregation reaction is rapid (t1/2 < or = 0.5 min), independent of temperature within a range of 4-37 degrees C, and unaffected by ATP, guanosine-5'-O-(3-thiophosphate), and in particular EGTA, distinguishing it from Ca(2+)-dependent membrane aggregation reactions. The process is driven by the action of membrane-associated AP molecules since partial proteolysis results in a full loss of activity and since aggregation is abolished by pretreatment of the s-CVs with a monoclonal antibody that reacts with the alpha subunit of AP-2. However, vesicle aggregation is not inhibited by PPPi, indicating that the previously characterized polyphosphate-sensitive AP-2 self-association is not responsible for the reaction. The vesicle aggregation reaction can be reconstituted: liposomes of phospholipid composition approximating that found on the cytoplasmic surfaces of the plasma membrane and of coated vesicles (70% L-alpha-phosphatidylethanolamine (type I-A), 15% L-alpha-phosphatidyl-L-serine, and 15% L-alpha-phosphatidylinositol) aggregated after addition of AP-2, but not of AP-1, AP-3 (AP180), or pure Clathrin triskelions. Aggregation of liposomes is abolished by limited proteolysis of AP-2 with trypsin. In addition, a highly purified AP-2 alpha preparation devoid of beta causes liposome aggregation, whereas pure beta subunit does not, consistent with results obtained in the s-CV assay which also indicate the involvement of the alpha subunit. Using a fluorescence energy transfer assay we show that AP-2 does not cause fusion of liposomes under physiological solution conditions. However, since the fusion of membranes necessarily requires the close opposition of the two participating bilayers, the AP-2-dependent vesicle aggregation events that we have identified may represent an initial step in the formation and fusion of endosomes that occur subsequent to endocytosis and Clathrin uncoating in vivo.

  • inositol hexakisphosphate receptor identified as the Clathrin Assembly Protein ap 2
    Biochemical and Biophysical Research Communications, 1992
    Co-Authors: Susan M Voglmaier, James H Keen, Jo Ellen Murphy, Christopher D Ferris, Glenn D Prestwich, Solomon H Snyder, Anne B Theibert
    Abstract:

    To clarify the function of the receptor binding Protein for inositol hexakisphosphate (IP6), we obtained a partial amino acid sequence from the purified Protein and a partial nucleotide sequence from a cDNA clone of the gene. The sequences are essentially identical to those of the alpha-subunit of the Clathrin Assembly Protein AP-2. The IP6 receptor Protein analyzed by SDS-PAGE contains a series of subunits which are the same as those of AP-2. Antibodies to AP-2 react with the IP6 receptor Protein in immunoblot analysis.

  • interaction of Assembly Protein ap 2 and its isolated subunits with Clathrin
    Biochemistry, 1991
    Co-Authors: Kondury Prasad, James H Keen
    Abstract:

    The Clathrin Assembly Protein complex AP-2 is a multimeric subunit complex consisting of two 100-115-kDa subunits known as alpha and beta and 50- and 16-kDa subunits. The subunits have been dissociated and separated by ion-exchange chromatography in 7.5 M urea. Fractions highly enriched in either the alpha or beta subunit were obtained. The alpha fraction interacted with Clathrin as evidenced by its ability to bind to preassembled Clathrin cages. It also reacted with dissociated Clathrin trimers under conditions that favor Assembly of coat structures, but did not yield discrete Clathrin polygonal lattices. The enriched beta fraction (containing small amounts of alpha) reacted with Clathrin to yield intact coats with the incorporation of approximately equivalent amounts of alpha and beta subunits into the polymerized species; excess free beta subunit was unreactive. The AP-2 complex was also completely dissociated in a highly denaturing solvent, 6 M Gdn.HCl, and the constituent subunits of 100-115, 50, and 16 kDa were separated by gel filtration. In a coAssembly assay with Clathrin, the Clathrin polymerizing activity was exclusively associated with the 100-kDa subunit fraction with stoichiometric incorporation of both alpha and beta subunits of 100 kDa into the polymerized coats, and with no requirement for 50- or 16-kDa subunits. These observations demonstrate that the Assembly activity of the complex is associated with the alpha and beta subunits and suggest that both subunits, through independent interactions with Clathrin, are required for expression of complete lattice Assembly activity.

  • self association of the plasma membrane associated Clathrin Assembly Protein ap 2
    Journal of Biological Chemistry, 1991
    Co-Authors: Kenneth A Beck, James H Keen
    Abstract:

    Abstract A self-association reaction involving the plasma membrane-associated Clathrin Assembly Protein AP-2 has been detected by incubating AP-2 alone under solution conditions that would favor the Assembly of complete coat structures if Clathrin were present. Self-association was rapid, unaffected by nonionic detergents, readily reversible, and gave rise to sedimentable aggregates. Only the AP subtype AP-2 exhibited self-association: the structurally or functionally related Assembly Proteins AP-1 and AP-3 and unrelated Proteins neither self-associated nor were incorporated into the AP-2 aggregate. AP-2 interactions responsible for self-association were of high affinity, with an apparent Kd of approximately 10(-8)M. By proteolytic dissection, the self-association domain was localized to the core of the molecule containing the intact 50- and 16-kDa polypeptides in association with the truncated 60-66-kDa moieties of the parent alpha/beta polypeptides. Self-association of the intact AP-2 molecule was pH-dependent, exhibiting an apparent pKa approximately 7.4. While it is unlikely that the large AP-2 aggregates formed in solution are themselves biologically relevant structures, the AP-2 interactions involved in their formation have properties consistent with their occurrence in intact cells and thus may be important in cellular functions of the plasma membrane-localized Assembly Protein.

  • interaction of phosphoinositide cycle intermediates with the plasma membrane associated Clathrin Assembly Protein ap 2
    Journal of Biological Chemistry, 1991
    Co-Authors: Kenneth A Beck, James H Keen
    Abstract:

    Several components of the phosphoinositide cycle have been found to interact specifically and at physiological concentrations with the plasma membrane-associated Clathrin Assembly (adaptor) Protein AP-2. These include phosphatidylinositol 4,5-bisphosphate and inositol 1,4,5-trisphosphate, which are present at the plasma membrane, as well as other polyphosphoinositols. ATP and other polyphosphate molecules complete with the polyphosphoinositols, however, they are at least 80-fold less potent. Also, the effect of ATP, unlike the polyphosphoinositols, is blocked by physiological concentrations of Mg2+. Photoaffinity labeling of AP-2 by [α-32P]8-azidoadenosine 5'-triphosphate and its competition by polyphosphoinositols has been used to identify the α subunit of the AP-2 complex as the site of specific interaction with the polyphosphoinositols and to confirm direct ultrafiltration binding experiments. Proteolytic dissection of the labeled AP-2 demonstrated that binding occurred exclusively on the N-terminal portion of the α subunit. Interaction of purified AP-2 with sub-microM concentrations of polyphosphoinositols has inhibitory effects on a novel AP-2 self-association described in the accompanying paper (Beck, K. A., and Keen, J. H., J. Biol. Chem. 266, 4437-4441), and at higher concentrations on the binding of AP-2 to dissociated Clathrin trimers as well as AP-2-mediated Clathrin coat Assembly. Review of the literature shows that several physiological stimuli that are known to result in increased coat pit formation in intact cells correlate with increased phosphoinositide turnover. These in vivo correlations and the in vitro observations reported here suggest that coated membrane and phosphoinositide cycles may be interdependent within cells.

Pamela J. Yao - One of the best experts on this subject based on the ideXlab platform.

  • CALM, a Clathrin Assembly Protein, influences cell surface GluR2 abundance.
    NeuroMolecular Medicine, 2011
    Co-Authors: Asaff Harel, Mark P. Mattson, Pamela J. Yao
    Abstract:

    The Clathrin Assembly Protein, CALM, promotes the Assembly of Clathrin-coated vesicles. In a previous study, we showed that CALM controls the level of the synaptic vesicle Protein VAMP2 at the plasma membrane by regulating VAMP2 endocytosis. Here, we provide evidence that CALM also influences the cell surface level of the AMPA receptor subunit GluR2. Although mechanistic details as well as the physiological relevance of CALM and GluR2 in the neuron have yet to be established, CALM-mediated trafficking could function as a component of a dedicated system for controlling postsynaptic abundance of GluR2.

  • Neuronal activity and the expression of Clathrin-Assembly Protein AP180.
    Biochemical and Biophysical Research Communications, 2010
    Co-Authors: Mark P. Mattson, Pamela J. Yao
    Abstract:

    The Clathrin-Assembly Protein AP180 is known to promote the Assembly of Clathrin-coated vesicles in the neuron. However, it is unknown whether the expression of AP180 is influenced by neuronal activity. In this study, we report that chronic depolarization results in a reduction of AP180 from hippocampal neurons, while acute depolarization causes a dispersed synaptic distribution of AP180. Activity-induced effects are observed only for AP180, but not for the structurally-related Clathrin-Assembly Proteins CALM, epsin1, or HIP1. These findings suggest that AP180 levels and synaptic distribution are highly sensitive to neuronal activity.

  • The Clathrin Assembly Protein AP180 regulates the generation of amyloid-β peptide
    Biochemical and biophysical research communications, 2009
    Co-Authors: Yasuji Matsuoka, Mark P. Mattson, Pamela J. Yao
    Abstract:

    Abstract The overproduction and extracellular buildup of amyloid-β peptide (Aβ) is a critical step in the etiology of Alzheimer’s disease. Recent data suggest that intracellular trafficking is of central importance in the production of Aβ. Here we use a neuronal cell line to examine two structurally similar Clathrin Assembly Proteins, AP180 and CALM. We show that RNA interference-mediated knockdown of AP180 reduces the generation of Aβ1–40 and Aβ1–42, whereas CALM knockdown has no effect on Aβ generation. Thus AP180 is among the traffic controllers that oversee and regulate amyloid precursor Protein processing pathways. Our results also suggest that AP180 and CALM, while similar in their domain structures and biochemical properties, are in fact dedicated to separate trafficking pathways in neurons.

  • Clathrin Assembly Protein AP180 and CALM Differentially Control Axogenesis and Dendrite Outgrowth in Embryonic Hippocampal Neurons
    The Journal of neuroscience : the official journal of the Society for Neuroscience, 2008
    Co-Authors: Ittai Bushlin, Mark P. Mattson, Ronald S. Petralia, Asaff Harel, Mohamed R. Mughal, Pamela J. Yao
    Abstract:

    Emerging data suggest that, much like epithelial cells, the polarized growth of neurons requires both the secretory and endocytic pathways. The Clathrin Assembly Proteins AP180 and CALM (Clathrin Assembly lymphoid myeloid Protein) are known to be involved in Clathrin-mediated endocytosis, but their roles in mammalian neurons and, in particular, in developmental processes before synaptogenesis are unknown. Here we provide evidence that AP180 and CALM play critical roles in establishing the polarity and controlling the growth of axons and dendrites in embryonic hippocampal neurons. Knockdown of AP180 primarily impairs axonal development, whereas reducing CALM levels results in dendritic dystrophy. Conversely, neurons that overexpress AP180 or CALM generate multiple axons. Ultrastructural analysis shows that CALM affiliates with a wider range of intracellular trafficking organelles than does AP180. Functional analysis shows that endocytosis is reduced in both AP180-deficient and CALM-deficient neurons. Additionally, CALM-deficient neurons show disrupted secretory transport. Our data demonstrate previously unknown functions for AP180 and CALM in intracellular trafficking that are essential in the growth of neurons.

  • Immunohistochemical characterization of Clathrin Assembly Protein AP180 and synaptophysin in human brain
    Neurobiology of aging, 2003
    Co-Authors: Pamela J. Yao, Timothy M O’herron, Paul D. Coleman
    Abstract:

    Neurons rely on Clathrin-mediated endocytosis for retrieving synaptic vesicles (SVs) at the presynaptic compartment after the release of neurotransmitters. The Clathrin Assembly Protein AP180 is shown to be a regulator for this Clathrin-dependent SV recycling pathway. AP180 is efficient in facilitating the formation of Clathrin-coated vesicles and regulating their size, but its exact location in synapse is not clear. In this study, we compared the expression of AP180 with synaptophysin in the aged human brain using confocal immunofluorescence microscopy. Synaptophysin is well characterized for its association with SVs and therefore a commonly used presynaptic marker. We achieved satisfactory immunofluorescent labeling by using an autofluorescence blocker Sudan Black B and more photostable Alexa Fluor dyes. Although we found that AP180 had an overall expression similar to synaptophysin, the immunoreactivity for the two Proteins did not always co-localize.

Paul D. Coleman - One of the best experts on this subject based on the ideXlab platform.

  • Immunohistochemical characterization of Clathrin Assembly Protein AP180 and synaptophysin in human brain
    Neurobiology of aging, 2003
    Co-Authors: Pamela J. Yao, Timothy M O’herron, Paul D. Coleman
    Abstract:

    Neurons rely on Clathrin-mediated endocytosis for retrieving synaptic vesicles (SVs) at the presynaptic compartment after the release of neurotransmitters. The Clathrin Assembly Protein AP180 is shown to be a regulator for this Clathrin-dependent SV recycling pathway. AP180 is efficient in facilitating the formation of Clathrin-coated vesicles and regulating their size, but its exact location in synapse is not clear. In this study, we compared the expression of AP180 with synaptophysin in the aged human brain using confocal immunofluorescence microscopy. Synaptophysin is well characterized for its association with SVs and therefore a commonly used presynaptic marker. We achieved satisfactory immunofluorescent labeling by using an autofluorescence blocker Sudan Black B and more photostable Alexa Fluor dyes. Although we found that AP180 had an overall expression similar to synaptophysin, the immunoreactivity for the two Proteins did not always co-localize.

  • High-resolution localization of Clathrin Assembly Protein AP180 in the presynaptic terminals of mammalian neurons.
    The Journal of comparative neurology, 2002
    Co-Authors: Pamela J. Yao, Paul D. Coleman, David J. Calkins
    Abstract:

    Synaptic vesicles (SVs) assemble at the presynaptic compartment through a Clathrin-dependent mechanism that involves one or more Assembly Proteins (APs). The Assembly Protein AP180 is especially efficient at facilitating Clathrin cage formation, but its precise ultrastructural localization in neurons is unknown. Using immunoelectron microscopy, we demonstrate the presynaptic localization of AP180 in axon terminals of rat cerebellar neurons. In contrast, the Assembly Protein AP2 was associated with both the presynaptic plasma membrane and the cytosolic side of the membrane at postsynaptic and extrasynaptic sites. Furthermore, ultrastructural analysis of primate retina showed that AP180 immunoreactivity was preferentially and highly enriched at ribbon synapses, where glutamate is released tonically at high levels and rapid vesicle turnover is essential. To maintain functional synaptic transmission, neurotransmitter-filled SVs must be readily available, and this requires proper reAssembly of new vesicles. The expression of AP180, in addition to AP-2, in the Clathrin-mediated endocytic pathway might add another level of control to SV reformation for efficient Assembly of Clathrin, effectively controlling the size of assembled vesicles and faithfully recovering SV-specific components. J. Comp. Neurol. 447:152–162, 2002. © 2002 Wiley-Liss, Inc.

  • Clathrin Assembly Protein ap 2 is detected in both neurons and glia and its reduction is prominent in layer ii of frontal cortex in alzheimer s disease
    Neurobiology of Aging, 2000
    Co-Authors: Jill Weimer, Timothy M Oherron, Paul D. Coleman
    Abstract:

    Abstract There are several adaptor Proteins associated with Clathrin coated vesicles. Among them are AP180 and AP-2. We and others have previously described synaptic localization of AP180. AP180 immunoreactivity is altered in both the superior frontal gyrus and hippocampus in Alzheimer’s disease (AD). We here investigate the location and alteration of another adaptor Protein, AP-2. In contrast to AP180, we have found that AP-2 is expressed by both neurons and glia. Furthermore, the only noticeable change of AP-2 in AD is a loss of its immunoreactivity in layer II of the superior frontal gyrus.

  • Changes in synaptic expression of Clathrin Assembly Protein AP180 in Alzheimer's disease analysed by immunohistochemistry
    Neuroscience, 1999
    Co-Authors: Pamela J. Yao, R. Morsch, Linda M. Callahan, Paul D. Coleman
    Abstract:

    Clathrin Assembly Protein AP180 plays a regulatory role in Clathrin-mediated synaptic vesicle recycling in synapses. Previously, using immunoblot analysis, we observed a significant reduction of AP180 Protein in Alzheimer's disease neocortex. In this study, we examined immunohistochemically the expression of AP180 in post mortem brains with Alzheimer's disease (n = 5) in comparison with neurologically normal controls (n = 5). Overall, AP180 was revealed as immunoreactive punctate granules located in the neuropil, and around neuronal cell bodies and their processes, consistent with the typical expression of synaptic Proteins. Reduced density of AP180 immunoreactive puncta was seen throughout all layers of the superior frontal gyrus in Alzheimer's disease, but the loss of AP180 immunoreactivity was not as prominent in the cerebellum. This regional difference is in agreement with our previous results from immunoblot analyses. In the hippocampus, cell body AP180 immunoreactivity normally seen in the hilus and the CA3 regions of control brains was completely lost in Alzheimer's disease. In addition, AP180 immunoreactivity in the molecular layer of the dentate gyrus showed several changes in Alzheimer's disease. These appeared to be expansion of the inner molecular layer and relative changes in immunoreactivity that resulted in clearer delineation of the inner and outer molecular layers. These results provide anatomical and spatial information on AP180 expression in Alzheimer's disease brains. The variations in altered AP180 immunoreactivity in different brain regions of Alzheimer's disease may underlie the dysfunction of the corresponding synapses.

  • Reduced O-glycosylated Clathrin Assembly Protein AP180: implication for synaptic vesicle recycling dysfunction in Alzheimer's disease
    Neuroscience letters, 1998
    Co-Authors: Pamela J. Yao, Paul D. Coleman
    Abstract:

    Synapse loss is one of the neuropathologies in Alzheimer's disease (AD) that may play a crucial role in the mechanism of its distinct cognitive impairment and dementia. In a previous study [18], a significant reduction of O-glycosylated Clathrin Assembly Protein AP180 was observed in neocortex of AD. The reduction correlated with the density of neurofibrillary tangles. In this study we further determine that the O-GlcNAc/AP180 ratio is not changed, but the level of AP180 Protein decreases in AD. Furthermore, whereas the level of neurofilament (NF-M) remains relatively unchanged, another Clathrin Assembly Protein, AP-2, is also reduced in AD along with a small loss of synaptophysin. Our findings suggest that synaptic vesicle recycling dysfunction may be involved in the pathology of synapse loss in AD.

P Amouyel - One of the best experts on this subject based on the ideXlab platform.

  • the role of clusterin complement receptor 1 and phosphatidylinositol binding Clathrin Assembly Protein in alzheimer disease risk and cerebrospinal fluid biomarker levels
    Archives of General Psychiatry, 2011
    Co-Authors: Britmaren M Schjeide, Jeancharles Lambert, Cathrin Schnack, Christina M Lill, Julia Kirchheiner, Hayrettin Tumani, Markus Otto, Rudolph E Tanzi, Hans Lehrach, P Amouyel
    Abstract:

    Context: Two recent and simultaneously published genome-wide association studies independently implicated clusterin (CLU), complement receptor 1 (CR1), and phosphatidylinositol binding Clathrin Assembly Protein (PICALM) as putative novel Alzheimer disease (AD) risk loci. Despite their strong statistical support, all 3 signals emerged from heterogeneous case-control populations and lack replication in different settings. Objective: To determine whether genetic variants in CLU, CR1, and PICALM confer risk for AD in independent data sets (n=4254) and to test the impact of these markers on cerebrospinal fluid (CSF)―Aβ42 and totaltau Protein levels (n=425). Design: Genetic association study using family-based and case-control designs. Setting: Ambulatory or hospitalized care. Participants: Family samples originate from mostly multiplex pedigrees recruited at different centers in the United States (1245 families, 2654 individuals with AD, and 1175 unaffected relatives). Unrelated case-control subjects originate from 1 clinical center in Germany (214 individuals with AD and 211 controls). All subjects were of European descent. Main Outcome Measures: The association between 5 genetic variants in CLU, CR1, and PICALM and risk for AD, and the correlation between these 5 genetic variants and CSF-Aβ42 and tau levels. Results: All 3 investigated loci showed significant associations between risk for AD (1-tailed P values ranging from <.001 to .02) and consistent effect sizes and direction. For each locus, the overall evidence of association was substantially strengthened on meta-analysis of all available data (2-tailed P values ranging from 1.1 × 10 ―16 to 4.1 × 10 ―7 ). Of all markers tested, only rs541458 in PICALM was shown to have an effect on CSF Protein levels, suggesting that the AD risk allele is associated with decreased CSF Aβ42 levels (2-tailed P=.002). Conclusions: This study provides compelling independent evidence that genetic variants in CLU, CR1, and PICALM are genetically associated with risk for AD. Furthermore, the CSF biomarker analyses provide a first insight into the potentially predominant pathogenetic mechanism(s) underlying the association between AD risk and PICALM.

  • the role of clusterin complement receptor 1 and phosphatidylinositol binding Clathrin Assembly Protein in alzheimer disease risk and cerebrospinal fluid biomarker levels
    Archives of General Psychiatry, 2011
    Co-Authors: Britmaren M Schjeide, Jeancharles Lambert, Cathrin Schnack, Christina M Lill, Julia Kirchheiner, Hayrettin Tumani, Markus Otto, Rudolph E Tanzi, Hans Lehrach, P Amouyel
    Abstract:

    Context Two recent and simultaneously published genome-wide association studies independently implicated clusterin (CLU), complement receptor 1 (CR1), and phosphatidylinositol binding Clathrin Assembly Protein (PICALM) as putative novel Alzheimer disease (AD) risk loci. Despite their strong statistical support, all 3 signals emerged from heterogeneous case-control populations and lack replication in different settings. Objective To determine whether genetic variants inCLU,CR1, andPICALMconfer risk for AD in independent data sets (n = 4254) and to test the impact of these markers on cerebrospinal fluid (CSF)–Aβ42 and total-tau Protein levels (n = 425). Design Genetic association study using family-based and case-control designs. Setting Ambulatory or hospitalized care. Participants Family samples originate from mostly multiplex pedigrees recruited at different centers in the United States (1245 families, 2654 individuals with AD, and 1175 unaffected relatives). Unrelated case-control subjects originate from 1 clinical center in Germany (214 individuals with AD and 211 controls). All subjects were of European descent. Main Outcome Measures The association between 5 genetic variants inCLU,CR1, andPICALMand risk for AD, and the correlation between these 5 genetic variants and CSF-Aβ42 and tau levels. Results All 3 investigated loci showed significant associations between risk for AD (1-tailedPvalues ranging from Conclusions This study provides compelling independent evidence that genetic variants inCLU,CR1, andPICALMare genetically associated with risk for AD. Furthermore, the CSF biomarker analyses provide a first insight into the potentially predominant pathogenetic mechanism(s) underlying the association between AD risk andPICALM.