Active zones are specialized regions of the presynaptic plasma membrane designed for the efficient and repetitive release of neurotransmitter via synaptic vesicle (SV) exocytosis. Piccolo is a high molecular weight component of the active zone that is hypothesized to participate both in active zone formation and the scaffolding of key molecules involved in SV recycling. In this study, we use interference RNAs to eliminate Piccolo expression from cultured hippocampal neurons to assess its involvement in synapse formation and function. Our data show that Piccolo is not required for glutamatergic synapse formation but does influence presynaptic function by negatively regulating SV exocytosis. Mechanistically, this regulation appears to be calmodulin kinase II–dependent and mediated through the modulation of Synapsin1a dynamics. This function is not shared by the highly homologous protein Bassoon, which indicates that Piccolo has a unique role in coupling the mobilization of SVs in the reserve pool to events within the active zone.

Neurabin I, a neuronal actin-binding protein, binds protein phosphatase 1 (PP1) and p70 ribosomal S6 protein kinase (p70S6K), both proteins implicated in cytoskeletal dynamics. We expressed wild-type and mutant neurabins fused to green fluorescent protein in Cos7, HEK293, and hippocampal neurons. Biochemical and cellular studies showed that an N-terminal F-actin-binding domain dictated neurabin I localization at actin cytoskeleton and promoted disassembly of stress fibers. Deletion of the C-terminal coiled-coil and sterile alpha motif domains abolished neurabin I dimerization and induced filopodium extension. Immune complex assays showed that neurabin I recruited an active PP1 via a PP1-docking sequence, 457 Cross talk between protein kinases and phosphatases regulates synaptic strength and information processing in mammalian brain (33). Prior studies identified protein phosphatase 1 (PP1) as a key regulator of activity-dependent changes in synaptic function underlying the two major forms of plasticity known as long-term potentiation (3) and long-term depression (LTD) extensively studied for hippocampal neurons (19). LTD-inducing stimuli promoted distribution of PP1 to dendritic spines (18), where it associated with the actin-rich cytoskeletal structure known as the postsynaptic density or PSD (30). This localization was critical for effective dephosphorylation of PP1 substrates such as Ca 2ϩ /calmodulin-dependent protein kinase II (30) and DL-␣-amino-3-hydroxy-5-methylisoxazolepropionic acid receptors (34) and down-regulation of synaptic function. This has focused attention on the cellular mechanisms that target PP1 to the neuronal actin cytoskeleton (32).Neurabin I (NrbI), identified by F-actin binding (20), shares structural homology with spinophilin (1) or neurabin II (NrbII) (25), which was isolated as a PP1-binding protein. PP1 complexes containing both neurabins have been demonstrated in extracts from rat brain (16). In addition, the PDZ (PSD-95/ Dlg/ZO-1 homology) domain in NrbI recruited p70 ribosomal S6 protein kinase (p70S6K) (4) and kalirin-7 (24), a GTPexchange factor, molecules that have been implicated in the control of neuronal morphology. The C terminus of NrbI contained coiled-coil and SAM (sterile alpha motif) domains, shown to mediate homodimerization and heterodimerization of other proteins (14, 27), which may contribute to the in vitro actin-cross-linking or bundling activity of neurabins (20). The C terminus of NrbI also bound the trans-Golgi protein TGN38 (28) and suggested that NrbI was a multifunctional protein scaffold that regulated both membrane and cytoskeletal functions.By immunohistochemistry, NrbII was localized to dendritic spines and thus called spinophilin (1). In contrast, NrbI was present in both spines and growth cones (20). Subcellular fractionation showed that both neurabins are present in highly purified preparations of PSD (25, 32) and growth cones (R. T. Terry-Lorenzo and S. Shenolikar, unpublished observations). Ectopic expression of NrbI (20) and NrbII (25) in cul...

Vesicular trafficking of presynaptic and postsynaptic components is emerging as a general cellular mechanism for the delivery of scaffold proteins, ion channels, and receptors to nascent and mature synapses. However, the molecular mechanisms leading to the selection of cargos and their differential transport to subneuronal compartments are not well understood, in part because of the mixing of cargos at the plasma membrane and/or within endosomal compartments. In the present study, we have explored the cellular mechanisms of active zone precursor vesicle assembly at the Golgi in dissociated hippocampal neurons of Rattus norvegicus. Our studies show that Piccolo, Bassoon, and ELKS2/CAST exit the trans-Golgi network on a common vesicle that requires Piccolo and Bassoon for its proper assembly. In contrast, Munc13 and synaptic vesicle proteins use distinct sets of Golgi-derived transport vesicles, while RIM1␣ associates with vesicular membranes in a post-Golgi compartment. Furthermore, Piccolo and Bassoon are necessary for ELKS2/CAST to leave the Golgi in association with vesicles, and a core domain of Bassoon is sufficient to facilitate formation of these vesicles. While these findings support emerging principles regarding active zone differentiation, the cellular and molecular analyses reported here also indicate that the Piccolo-Bassoon transport vesicles leaving the Golgi may undergo further changes in protein composition before arriving at synaptic sites.

Neurabins are protein phosphatase-1 (PP1) targeting subunits that are highly concentrated in dendritic spines and post-synaptic densities. Immunoprecipitation of neurabin I and neurabin II/spinophilin from rat brain extracts sedimented PP1␥1 and PP1␣ but not PP1␤. In vitro studies showed that recombinant peptides representing central regions of neurabins also preferentially bound PP1␥1 and PP1␣ from brain extracts and associated poorly with PP1␤. Analysis of PP1 binding to chimeric neurabins suggested that sequences flanking a conserved PP1-binding motif altered their selectivity for PP1␤ and their activity as regulators of PP1 in vitro. Assays using recombinant PP1 catalytic subunits and a chimera of PP1 and protein phosphatase-2A indicated that the C-terminal sequences unique to the PP1 isoforms contributed to their recognition by neurabins. Collectively, the results from several different in vitro assays established the rank order of PP1 isoform selection by neurabins to be PP1␥1 > PP1␣ > PP1␤. This PP1 isoform selectivity was confirmed by immunoprecipitation of neurabin I and II from brain extracts from wild type and mutant PP1␥ null mice. In the absence of PP1␥1, both neurabins showed enhanced association with PP1␣ but not PP1␤. These studies identified some of the structural determinants in PP1 and neurabins that together contribute to preferential targeting of PP1␥1 and PP1␣ to the mammalian synapse.Protein phosphatase-1 (PP1), 1 a major eukaryotic protein serine/threonine phosphatase, is encoded by multiple genes in both plants and animals. Disruption of PP1 genes in fungi (1), fruit flies (2), and mice (3) suggested that PP1 isoforms encoded by individual genes control distinct but overlapping physiological functions. Three mammalian isoforms, PP1␣, PP1␤, and PP1␥1, are expressed in all tissues (4) with PP1␥2, an alternately spliced product of the PP1␥ gene, present predominantly in testes (5, 6). Immunocytochemistry using isoform-specific antibodies suggested that expression of PP1 isoforms varied in different brain regions where they are also localized to different subcellular compartments (6, 7). For example, PP1␤ was the predominant isoform associated with microtubules in the neuronal cell body, whereas PP1␥1 and PP1␣ were preferentially concentrated in dendritic spines (6,8). Furthermore, by analyzing endogenous PP1 movement during the cell cycle, Andreassen et al. (9) showed that the distribution of PP1 isoforms in cells was highly dynamic. This placed new emphasis on understanding the mechanisms that target individual PP1 isoforms to cellular organelles.Isolation of PP1 bound to skeletal muscle glycogen (10) and myosin (11) established the paradigm that regulatory or targeting subunits bound to PP1 catalytic subunits dictate the subcellular localization, substrate recognition, and hormonal control of PP1. The search for PP1 regulators that control functions as diverse as protein synthesis, gene expression, cell division, and motility has thus far yielded more than 50 PP1-binding proteins (12). Wh...

The majority of excitatory synapses in the mammalian brain form on filopodia and spines, actin-rich membrane protrusions present on neuronal dendrites. The biochemical events that induce filopodia and remodel these structures into dendritic spines remain poorly understood. Here, we show that the neuronal actin- and protein phosphatase-1-binding protein, neurabin-I, promotes filopodia in neurons and nonneuronal cells. Neurabin-I actin-binding domain bundled F-actin, promoted filopodia, and delayed the maturation of dendritic spines in cultured hippocampal neurons. In contrast, dimerization of neurabin-I via C-terminal coiled-coil domains and association of protein phosphatase-1 (PP1) with neurabin-I through a canonical KIXF motif inhibited filopodia. Furthermore, the expression of a neurabin-I polypeptide unable to bind PP1 delayed the maturation of neuronal filopodia into spines, reduced the synaptic targeting of AMPA-type glutamate (GluR1) receptors, and decreased AMPA receptor-mediated synaptic transmission. Reduction of endogenous neurabin levels by interference RNA (RNAi)-mediated knockdown also inhibited the surface expression of GluR1 receptors. Together, our studies suggested that disrupting the functions of a cytoskeletal neurabin/PP1 complex enhanced filopodia and impaired surface GluR1 expression in hippocampal neurons, thereby hindering the morphological and functional maturation of dendritic spines.

Inhibitor-2 (I-2) bound protein phosphatase-1 (PP1) and several PP1-binding proteins from rat brain extracts, including the actin-binding proteins, neurabin I and neurabin II. Neurabins from rat brain lysates were sedimented by I-2 and its structural homologue, I-4. The central domain of both neurabins bound PP1 and I-2, and mutation of a conserved PP1-binding motif abolished neurabin binding to both proteins. Microcystin-LR, a PP1 inhibitor, also attenuated I-2 binding to neurabins. Immunoprecipitation of neurabin I established its association with PP1 and I-2 in HEK293T cells and suggested that PP1 mediated I-2 binding to neurabins. The C terminus of I-2, although not required for PP1 binding, facilitated PP1 recruitment by neurabins, which also targeted I-2 to polymerized F-actin. Mutations that attenuated PP1 binding to I-2 and neurabin I suggested distinct and overlapping sites for these two proteins on the PP1 catalytic subunit. Immunocytochemistry in epithelial cells and cultured hippocampal neurons showed that endogenous neurabin II and I-2 colocalized at actin-rich structures, consistent with the ability of neurabins to target the PP1.I-2 complex to actin cytoskeleton and regulate cell morphology.

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The NMDAR (N-methyl-D-aspartate receptor) is a central regulator of synaptic plasticity and learning and memory. hDAAO (human D-amino acid oxidase) indirectly reduces NMDAR activity by degrading the NMDAR co-agonist D-serine. Since NMDAR hypofunction is thought to be a foundational defect in schizophrenia, hDAAO inhibitors have potential as treatments for schizophrenia and other nervous system disorders. Here, we sought to identify novel chemicals that inhibit hDAAO activity. We used computational tools to design a focused, purchasable library of compounds. After screening this library for hDAAO inhibition, we identified the structurally novel compound, ‘compound 2’ [3-(7-hydroxy-2-oxo-4-phenyl-2H-chromen-6-yl)propanoic acid], which displayed low nM hDAAO inhibitory potency (Ki=7 nM). Although the library was expected to enrich for compounds that were competitive for both D-serine and FAD, compound 2 actually was FAD uncompetitive, much like canonical hDAAO inhibitors such as benzoic acid. Compound 2 and an analog were independently co-crystalized with hDAAO. These compounds stabilized a novel conformation of hDAAO in which the active-site lid was in an open position. These results confirm previous hypotheses regarding active-site lid flexibility of mammalian D-amino acid oxidases and could assist in the design of the next generation of hDAAO inhibitors.