The Endosome Localized Arf-GAP AGAP1 Modulates Dendritic Spine Morphology Downstream of the Neurodevelopmental Disorder Factor Dysbindin.

AGAP1 is an Arf1 GTPase activating protein that interacts with the vesicle-associated protein complexes adaptor protein 3 (AP-3) and Biogenesis of Lysosome Related Organelles Complex-1 (BLOC-1). Overexpression of AGAP1 in non-neuronal cells results in an accumulation of endosomal cargoes, which suggests a role in endosome-dependent traffic. In addition, AGAP1 is a candidate susceptibility gene for two neurodevelopmental disorders, autism spectrum disorder (ASD) and schizophrenia (SZ); yet its localization and function in neurons have not been described. Here, we describe that AGAP1 localizes to axons, dendrites, dendritic spines and synapses, colocalizing preferentially with markers of early and recycling endosomes. Functional studies reveal overexpression and down-regulation of AGAP1 affects both neuronal endosomal trafficking and dendritic spine morphology, supporting a role for AGAP1 in the recycling endosomal trafficking involved in their morphogenesis. Finally, we determined the sensitivity of AGAP1 expression to mutations in the DTNBP1 gene, which is associated with neurodevelopmental disorder, and found that AGAP1 mRNA and protein levels are selectively reduced in the null allele of the mouse ortholog of DTNBP1. We postulate that endosomal trafficking contributes to the pathogenesis of neurodevelopmental disorders affecting dendritic spine morphology, and thus excitatory synapse structure and function.

Modulation of dendritic spine development and plasticity by BDNF and vesicular trafficking: fundamental roles in neurodevelopmental disorders associated with mental retardation and autism.

The process of axonal and dendritic development establishes the synaptic circuitry of the central nervous system (CNS) and is the result of interactions between intrinsic molecular factors and the external environment. One growth factor that has a compelling function in neuronal development is the neurotrophin brain-derived neurotrophic factor (BDNF). BDNF participates in axonal and dendritic differentiation during embryonic stages of neuronal development, as well as in the formation and maturation of dendritic spines during postnatal development. Recent studies have also implicated vesicular trafficking of BDNF via secretory vesicles, and both secretory and endosomal trafficking of vesicles containing synaptic proteins, such as neurotransmitter and neurotrophin receptors, in the regulation of axonal and dendritic differentiation, and in dendritic spine morphogenesis. Several genes that are either mutated or deregulated in neurodevelopmental disorders associated with mental retardation have now been identified, and several mouse models of these disorders have been generated and characterized. Interestingly, abnormalities in dendritic and synaptic structure are consistently observed in human neurodevelopmental disorders associated with mental retardation, and in mouse models of these disorders as well. Abnormalities in dendritic and synaptic differentiation are thought to underlie altered synaptic function and network connectivity, thus contributing to the clinical outcome. Here, we review the roles of BDNF and vesicular trafficking in axonal and dendritic differentiation in the context of dendritic and axonal morphological impairments commonly observed in neurodevelopmental disorders associated with mental retardation.

Bdnf overexpression in hippocampal neurons prevents dendritic atrophy caused by Rett-associated MECP2 mutations.

The expression of the methylated DNA-binding protein MeCP2 increases during neuronal development, which suggests that this epigenetic factor is crucial for neuronal terminal differentiation. We evaluated dendritic and axonal development in embryonic day-18 hippocampal neurons in culture by measuring total length and counting branch point numbers at 4 days in vitro, well before synapse formation. Pyramidal neurons transfected with a plasmid encoding a small hairpin RNA (shRNA) to knockdown endogenous Mecp2 had shorter dendrites than control untransfected neurons, without detectable changes in axonal morphology. On the other hand, overexpression of wildtype (wt) human MECP2 increased dendritic branching, in addition to axonal branching and length. Consistent with reduced neuronal growth and complexity in Rett syndrome (RTT) brains, overexpression of human MECP2 carrying missense mutations common in RTT individuals (R106W or T158M) reduced dendritic and axonal length. One of the targets of MeCP2 transcriptional control is the Bdnf gene. Indeed, endogenous Mecp2 knockdown increased the intracellular levels of BDNF protein compared to untransfected neurons, suggesting that MeCP2 represses Bdnf transcription. Surprisingly, overexpression of wt MECP2 also increased BDNF levels, while overexpression of RTT-associated MECP2 mutants failed to affect BDNF levels. The extracellular BDNF scavenger TrkB-Fc prevented dendritic overgrowth in wt MECP2-overexpressing neurons, while overexpression of the Bdnf gene reverted the dendritic atrophy caused by Mecp2-knockdown. However, this effect was only partial, since Bdnf increased dendritic length only to control levels in mutant MECP2-overexpressing neurons, but not as much as in Bdnf-transfected cells. Our results demonstrate that MeCP2 plays varied roles in dendritic and axonal development during neuronal terminal differentiation, and that some of these effects are mediated by autocrine actions of BDNF.

Consensus nomenclature for the human ArfGAP domain-containing proteins.

At the FASEB summer research conference on "Arf Family GTPases", held in Il Ciocco, Italy in June, 2007, it became evident to researchers that our understanding of the family of Arf GTPase activating proteins (ArfGAPs) has grown exponentially in recent years. A common nomenclature for these genes and proteins will facilitate discovery of biological functions and possible connections to pathogenesis. Nearly 100 researchers were contacted to generate a consensus nomenclature for human ArfGAPs. This article describes the resulting consensus nomenclature and provides a brief description of each of the 10 subfamilies of 31 human genes encoding proteins containing the ArfGAP domain.

The neuronal Arf GAP centaurin alpha1 modulates dendritic differentiation.

Centaurin alpha1 is an Arf GTPase-activating protein (GAP) that is highly expressed in the nervous system. In the current study, we show that endogenous centaurin alpha1 protein is localized in the synaptosome fraction, with peak expression in early postnatal development. In cultured dissociated hippocampal neurons, centaurin alpha1 localizes to dendrites, dendritic spines and the postsynaptic region. siRNA-mediated knockdown of centaurin alpha1 levels or overexpression of a GAP-inactive mutant of centaurin alpha1 leads to inhibition of dendritic branching, dendritic filopodia and spine-like protrusions in dissociated hippocampal neurons. Overexpression of wild-type centaurin alpha1 in cultured hippocampal neurons in early development enhances dendritic branching, and increases dendritic filopodia and lamellipodia. Both filopodia and lamellipodia have been implicated in dendritic branching and spine formation. Following synaptogenesis in cultured neurons, wild-type centaurin alpha1 expression increases dendritic filopodia and spine-like protrusions. Expression of a GAP-inactive mutant diminishes spine density in CA1 pyramidal neurons within cultured organotypic hippocampal slice cultures. These data support the conclusion that centaurin alpha1 functions through GAP-dependent Arf regulation of dendritic branching and spines that underlie normal dendritic differentiation and development.

The arf6 GAP centaurin alpha-1 is a neuronal actin-binding protein which also functions via GAP-independent activity to regulate the actin cytoskeleton.

Centaurin alpha-1 is a high-affinity PtdIns(3,4,5)P3-binding protein enriched in brain. Sequence analysis indicates centaurin alpha-1 contains two pleckstrin homology domains, ankyrin repeats and an Arf GAP homology domain, placing it in the AZAP family of phosphoinositide-regulated Arf GAPs. Other members of this family are involved in actin cytoskeletal and focal adhesion organization. Recently, it was reported that centaurin alpha-1 expression diminishes cortical actin and decreases Arf6GTP levels consistent with it functioning as an Arf6 GAP in vivo. In the current report, we show that centaurin alpha-1 binds Arfs in vitro and colocalizes with Arf6 and Arf5 in vivo, further supporting an interaction with Arfs. Centaurin alpha-1 expression produces dramatic effects on the actin cytoskeleton, decreasing stress fibers, diminishing cortical actin, and enhancing membrane ruffles and filopodia. Expression of centaurin alpha-1 also enhances cell spreading and disrupts focal adhesion protein localization. The effects of centaurin alpha-1 on stress fibers and cell spreading are reminiscent of those of Arf6GTP. Consistent with this, we show that many of the centaurin alpha-1-induced effects on the actin cytoskeleton and actin-dependent activities do not require GAP activity. Thus, centaurin alpha-1 likely functions via both GAP-dependent and GAP-independent mechanisms to regulate the actin cytoskeleton. Furthermore, we demonstrate that in vitro, centaurin alpha-1 binds F-actin directly, with actin binding activity localized to the PtdIns(3,4,5)P3-binding PH domain. Our data suggest that centaurin alpha-1 may be a component of the neuronal PI 3-kinase cascade that leads to regulation of the neuronal actin cytoskeleton.

Regulation of inositol phospholipid binding and signaling through syndecan-4.

Syndecan-4 is a transmembrane heparan sulfate proteoglycan that can regulate cell-matrix interactions and is enriched in focal adhesions. Its cytoplasmic domain contains a central region unlike that of any other vertebrate or invertebrate syndecan core protein with a cationic motif that binds inositol phospholipids. In turn, lipid binding stabilizes the syndecan in oligomeric form, with subsequent binding and activation of protein kinase C. The specificity of phospholipid binding and its potential regulation are investigated here. Highest affinity of the syndecan-4 cytoplasmic domain was seen with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5P)(2)) and phosphatidylinositol 4-phosphate, and both promoted syndecan-4 oligomerization. Affinity was much reduced for 3-phosphorylated inositides while no binding of diacylglycerol was detected. Syndecan-2 cytoplasmic domain had negligible affinity for any lipid examined. Inositol hexakisphosphate, but not inositol tetrakisphosphate, also had high affinity for the syndecan-4 cytoplasmic domain and could compete effectively with PtdIns(4,5)P(2). Since inositol hexaphosphate binding to syndecan-4 does not promote oligomer formation, it is a potential down-regulator of syndecan-4 signaling. Similarly, phosphorylation of serine 183 in syndecan-4 cytoplasmic domain reduced PtdIns(4,5)P(2) binding affinity by over 100-fold, although interaction could still be detected by nuclear magnetic resonance spectroscopy. Only protein kinase Calpha was up-regulated in activity by the combination of syndecan-4 and PtdIns(4,5)P(2), with all other isoforms tested showing minimal response. This is consistent with the codistribution of syndecan-4 with the alpha isoform of protein kinase C in focal adhesions.

Functions of PI 3-kinase in development of the nervous system.

In the nervous system, receptor regulated phosphoinositide (PI) 3-kinases (PI 3-kinases) participate in fundamental cellular activities that underlie development. Activated by trophic factors, growth factors, neuregulins, cytokines, or neurotransmitters, PI 3-kinases have been implicated in neuronal and glial survival and differentiation. PI 3-kinases produce inositol lipid second messengers that bind to pleckstrin homology (PH) domains in diverse groups of signal transduction proteins, and control their enzymatic activities, subcellular membrane localization, or both. Downstream targets of the inositol lipid messengers include protein kinases and regulators of small GTPases. The kinase Akt/PKB functions as a key component of the PI 3-kinase dependent survival pathway through its phosphorylation and regulation of apoptotic proteins and transcription factors. Furthermore, since members of the Rho GTPase and Arf GTPase families have been implicated in regulation of the actin cytoskeleton, vesicular trafficking, and transcription, the downstream targets of PI 3-kinase that control these GTPases are excellent candidates to mediate aspects of PI 3-kinase dependent neuronal and glial differentiation.