EFA6A, an Exchange Factor for Arf6, Regulates NGF-Dependent TrkA Recycling From Early Endosomes and Neurite Outgrowth in PC12 Cells.

Endosomal trafficking of TrkA is a critical process for nerve growth factor (NGF)-dependent neuronal cell survival and differentiation. The small GTPase ADP-ribosylation factor 6 (Arf6) is implicated in NGF-dependent processes in PC12 cells through endosomal trafficking and actin cytoskeleton reorganization. However, the regulatory mechanism for Arf6 in NGF signaling is largely unknown. In this study, we demonstrated that EFA6A, an Arf6-specific guanine nucleotide exchange factor, was abundantly expressed in PC12 cells and that knockdown of EFA6A significantly inhibited NGF-dependent Arf6 activation, TrkA recycling from early endosomes to the cell surface, prolonged ERK1/2 phosphorylation, and neurite outgrowth. We also demonstrated that EFA6A forms a protein complex with TrkA through its N-terminal region, thereby enhancing its catalytic activity for Arf6. Similarly, we demonstrated that EFA6A forms a protein complex with TrkA in cultured dorsal root ganglion (DRG) neurons. Furthermore, cultured DRG neurons from EFA6A knockout mice exhibited disturbed NGF-dependent TrkA trafficking compared with wild-type neurons. These findings provide the first evidence for EFA6A as a key regulator of NGF-dependent TrkA trafficking and signaling.

C-terminal truncations in IQSEC2: implications for synaptic localization, guanine nucleotide exchange factor activity, and neurological manifestations.

IQSEC2 gene on chromosome Xq11.22 encodes a member of guanine nucleotide exchange factor (GEF) protein that is implicated in the activation of ADP-ribosylation factors (Arfs) at the postsynaptic density (PSD), and plays a crucial role in synaptic transmission and dendritic spine formation. Alterations in IQSEC2 have been linked to X-linked intellectual developmental disorders including epilepsy and behavioral abnormalities. Of interest, truncating variants at the C-terminus of IQSEC2 can cause severe phenotypes, akin to truncating variants located in other regions. Here, we present a 5-year-old boy with severe intellectual disability and progressive epilepsy. The individual carried a nonsense variant p.Q1227* in the last exon of the IQSEC2 gene that was supposed to escape nonsense-mediated mRNA decay, thereby leading to a translation of C-terminus truncated IQSEC2 protein with residual activity. The functional analyses showed that the GEF activity of IQSEC2 Q1227* was compromised, and that the IQSEC2 Q1227* lacked preferential synaptic localization due to the absence of functional domains for binding to scaffolding proteins in the PSD. The impaired GEF activity and disrupted synaptic localization of the mutant IQSEC2 protein could impact dendritic and spine development in neurons, potentially explaining the patient's severe neurological manifestations. Our findings indicate that C-terminal truncations in IQSEC2, previously not well-characterized, may have significant pathogenic implications.

Physiological and Pathological Roles of the Cytohesin Family in Neurons.

The cytohesin proteins, consisting of four closely related members (cytohesins-1, -2, -3, and -4), are a subfamily of the Sec7 domain-containing guanine nucleotide exchange factors for ADP ribosylation factors (Arfs), which are critical regulators of membrane trafficking and actin cytoskeleton remodeling. Recent advances in molecular biological techniques and the development of a specific pharmacological inhibitor for cytohesins, SecinH3, have revealed the functional involvement of the cytohesin-Arf pathway in diverse neuronal functions from the formation of axons and dendrites, axonal pathfinding, and synaptic vesicle recycling, to pathophysiological processes including chronic pain and neurotoxicity induced by proteins related to neurodegenerative disorders, such as amyotrophic lateral sclerosis and Alzheimer's disease. Here, we review the physiological and pathological roles of the cytohesin-Arf pathway in neurons and discuss the future directions of this research field.

BRAG2a Mediates mGluR-Dependent AMPA Receptor Internalization at Excitatory Postsynapses through the Interaction with PSD-95 and Endophilin 3.

Brefeldin A-resistant ArfGEF 2 (BRAG2) [or Iqsec1 (IQ motif and Sec7 domain-containing protein 1)] is a guanine nucleotide exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes. BRAG2 regulates Arf6-dependent endocytosis of AMPA receptors (AMPARs) through the direct interaction during the hippocampal long-term depression. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Herein, using mouse brains of both sexes, we demonstrated that BRAG2a, an alternative isoform with a long C-terminal insert containing a proline-rich domain and type I PDZ-binding motif, was selectively localized to the excitatory postsynaptic density (PSD). Using yeast two-hybrid screening, we identified PSD-95 and endophilin 1/3 as BRAG2a-binding partners in the brain. The interaction with PSD-95 was required for synaptic targeting of BRAG2a. In cultured hippocampal neurons, stimulation of group I metabotropic glutamate receptors (mGluRs) increased the interaction of BRAG2a with endophilin 3 and concomitant Arf6 activation in a time-dependent manner. Knockdown of BRAG2 in cultured hippocampal neurons blocked the mGluR-dependent decrease in surface AMPAR levels, which was rescued by introducing wild-type BRAG2a, but not wild-type BRAG2b or BRAG2a mutants lacking the ability to activate Arf6 or to interact with endophilin 3 or PSD-95. Further postembedding immunoelectron microscopic analysis revealed the preorganized lateral distribution of BRAG2a, Arf6, and endophilin 3 for efficient endocytosis at the postsynaptic membrane. Together, the present findings unveiled a novel molecular mechanism by which BRAG2a links AMPARs to the clathrin-dependent endocytic pathway through its interaction with PSD-95 and endophilin 3.SIGNIFICANCE STATEMENT BRAG2/Iqsec1 is a GDP/GTP exchange factor for ADP ribosylation factor 6 (Arf6), a small GTPase implicated in the membrane trafficking between the plasma membrane and endosomes, and regulates Arf6-dependent endocytosis of AMPARs through direct interaction during hippocampal long-term depression, one of the mechanisms of synaptic plasticity related to learning and memory. However, the molecular mechanism by which the BRAG2-Arf6 pathway links AMPARs to the endocytic machinery remains elusive. Here, we identified isoform-specific mechanisms of BRAG2-mediated AMPAR internalization. We demonstrated that the interaction of BRAG2a isoform with PSD-95 and endophilin 3 was required for the mGluR-dependent decrease in surface AMPARs in hippocampal neurons. These results unveiled a novel molecular mechanism by which BRAG2 links AMPARs to the clathrin-mediated endocytic machinery at postsynaptic sites.

Cytohesin-2 mediates group I metabotropic glutamate receptor-dependent mechanical allodynia through the activation of ADP ribosylation factor 6 in the spinal cord.

Group I metabotropic glutamate receptors (mGluRs), mGluR1 and mGluR5, in the spinal cord are implicated in nociceptive transmission and plasticity through G protein-mediated second messenger cascades leading to the activation of various protein kinases such as extracellular signal-regulated kinase (ERK). In this study, we demonstrated that cytohesin-2, a guanine nucleotide exchange factor for ADP ribosylation factors (Arfs), is abundantly expressed in subsets of excitatory interneurons and projection neurons in the superficial dorsal horn. Cytohesin-2 is enriched in the perisynapse on the postsynaptic membrane of dorsal horn neurons and forms a protein complex with mGluR5 in the spinal cord. Central nervous system-specific cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia in inflammatory and neuropathic pain models. Pharmacological blockade of cytohesin catalytic activity with SecinH3 similarly reduced mechanical allodynia and inhibited the spinal activation of Arf6, but not Arf1, in both pain models. Furthermore, cytohesin-2 conditional knockout mice exhibited reduced mechanical allodynia and ERK1/2 activation following the pharmacological activation of spinal mGluR1/5 with 3,5-dihydroxylphenylglycine (DHPG). The present study suggests that cytothesin-2 is functionally associated with mGluR5 during the development of mechanical allodynia through the activation of Arf6 in spinal dorsal horn neurons.

Pallidin is a novel interacting protein for cytohesin-2 and regulates the early endosomal pathway and dendritic formation in neurons.

Cytohesin-2 is a member of the guanine nucleotide exchange factors for ADP ribosylation factor 1 (Arf1) and Arf6, which are small GTPases that regulate membrane traffic and actin dynamics. In this study, we first demonstrated that cytohesin-2 localized to the plasma membrane and vesicles in various subcellular compartment in hippocampal neurons by immunoelectron microscopy. Next, to understand the molecular network of cytohesin-2 in neurons, we conducted yeast two-hybrid screening of brain cDNA libraries using cytohesin-2 as bait and isolated pallidin, a component of the biogenesis of lysosome-related organelles complex 1 (BLOC-1) involved in endosomal trafficking. Pallidin interacted specifically with cytohesin-2 among cytohesin family members. Glutathione S-transferase pull-down and immunoprecipitation assays further confirmed the formation of a protein complex between cytohesin-2 and pallidin. Immunofluorescence demonstrated that cytohesin-2 and pallidin partially colocalized in various subsets of endosomes immunopositive for EEA1, syntaxin 12, and LAMP2 in hippocampal neurons. Knockdown of pallidin or cytohesin-2 reduced cytoplasmic EEA1-positive early endosomes. Furthermore, knockdown of pallidin increased the total dendritic length of cultured hippocampal neurons, which was rescued by co-expression of wild-type pallidin but not a mutant lacking the ability to interact with cytohesin-2. In contrast, knockdown of cytohesin-2 had the opposite effect on total dendritic length. The present results suggested that the interaction between pallidin and cytohesin-2 may participate in various neuronal functions such as endosomal trafficking and dendritic formation in hippocampal neurons. Cover Image for this issue: doi: 10.1111/jnc.14197.

FIP4/Arfophilin-2 plays overlapping but distinct roles from FIP3/Arfophilin-1 in neuronal migration during cortical layer formation.

The class II Rab11 family-interacting proteins, FIP3 and FIP4, also termed Arfophilin-1 and Arfophilin-2, respectively, are endosomal proteins that function as dual effector proteins for Rab11 and ADP ribosylation factor (Arf) small GTPases. In the present study, we examined the expression and role of FIP4 in neuronal migration during cerebral layer formation. FIP4 mRNA was first weakly detected in post-mitotic migrating neurons in the upper intermediate zone, and expression was markedly increased in the cortical layer. Exogenously expressed FIP4 protein was localized to subpopulations of EEA1- and syntaxin 12-positive endosomes in migrating neurons, and was partially colocalized with FIP3. Knockdown of FIP4 by in utero electroporation significantly stalled transfected neurons in the lower cortical layer and decreased the speed of neuronal migration in the upper intermediate zone and in the cortical plate compared with control small hairpin RNA (shRNA)-transfected neurons. Furthermore, co-transfection of shRNA-resistant wild-type FIP4, but not wild type FIP3 or FIP4 mutants lacking the binding region for Rab11 or Arf, significantly improved the disturbed cortical layer formation caused by FIP4 knockdown. Collectively, our findings suggest that FIP4 and FIP3 play overlapping but distinct roles in neuronal migration downstream of Arf and Rab11 during cortical layer formation.

Cell-Type-Specific Spatiotemporal Expression of Creatine Biosynthetic Enzyme S-adenosylmethionine:guanidinoacetate N-methyltransferase in Developing Mouse Brain.

Creatine is synthesized by S-adenosylmethionine:guanidinoacetate N-methyltransferase (GAMT), and the creatine/phosphocreatine shuttle system mediated by creatine kinase (CK) is essential for storage and regeneration of high-energy phosphates in cells. Although the importance of this system in brain development is evidenced by the hereditary nature of creatine deficiency syndrome, the spatiotemporal cellular expression patterns of GAMT in developing brain remain unknown. Here we show that two waves of high GAMT expression occur in developing mouse brain. The first involves high expression in mitotic cells in the ventricular zone of the brain wall and the external granular layer of the cerebellum at the embryonic and neonatal stages. The second was initiated by striking up-regulation of GAMT in oligodendrocytes during the second and third postnatal weeks (i.e., the active myelination stage), which continued to adulthood. Distinct temporal patterns were also evident in other cell types. GAMT was highly expressed in perivascular pericytes and smooth muscle cells after birth, but not in adults. In neurons, GAMT levels were low to moderate in neuroblasts residing in the ventricular zone, increased during the second postnatal week when active dendritogenesis and synaptogenesis occur, and decreased to very low levels thereafter. Moderate levels were observed in astrocytes throughout development. The highly regulated, cell type-dependent expression of GAMT suggests that local creatine biosynthesis plays critical roles in certain phases of neural development. In accordance with this idea, we observed increased CK expression in differentiating neurons; this would increase creatine/phosphocreatine shuttle system activity, which might reflect increased energy demand.

TARP γ-2 and γ-8 Differentially Control AMPAR Density Across Schaffer Collateral/Commissural Synapses in the Hippocampal CA1 Area.

The number of AMPA-type glutamate receptors (AMPARs) at synapses is the major determinant of synaptic strength and varies from synapse to synapse. To clarify the underlying molecular mechanisms, the density of AMPARs, PSD-95, and transmembrane AMPAR regulatory proteins (TARPs) were compared at Schaffer collateral/commissural (SCC) synapses in the adult mouse hippocampal CA1 by quantitative immunogold electron microscopy using serial sections. We examined four types of SCC synapses: perforated and nonperforated synapses on pyramidal cells and axodendritic synapses on parvalbumin-positive (PV synapse) and pravalbumin-negative interneurons (non-PV synapse). SCC synapses were categorized into those expressing high-density (perforated and PV synapses) or low-density (nonperforated and non-PV synapses) AMPARs. Although the density of PSD-95 labeling was fairly constant, the density and composition of TARP isoforms was highly variable depending on the synapse type. Of the three TARPs expressed in hippocampal neurons, the disparity in TARP γ-2 labeling was closely related to that of AMPAR labeling. Importantly, AMPAR density was significantly reduced at perforated and PV synapses in TARP γ-2-knock-out (KO) mice, resulting in a virtual loss of AMPAR disparity among SCC synapses. In comparison, TARP γ-8 was the only TARP expressed at nonperforated synapses, where AMPAR labeling further decreased to a background level in TARP γ-8-KO mice. These results show that synaptic inclusion of TARP γ-2 potently increases AMPAR expression and transforms low-density synapses into high-density ones, whereas TARP γ-8 is essential for low-density or basal expression of AMPARs at nonperforated synapses. Therefore, these TARPs are critically involved in AMPAR density control at SCC synapses. SIGNIFICANCE STATEMENT: Although converging evidence implicates the importance of transmembrane AMPA-type glutamate receptor (AMPAR) regulatory proteins (TARPs) in AMPAR stabilization during basal transmission and synaptic plasticity, how they control large disparities in AMPAR numbers or densities across central synapses remains largely unknown. We compared the density of AMPARs with that of TARPs among four types of Schaffer collateral/commissural (SCC) hippocampal synapses in wild-type and TARP-knock-out mice. We show that the density of AMPARs correlates with that of TARP γ-2 across SCC synapses and its high expression is linked to high-density AMPAR expression at perforated type of pyramidal cell synapses and synapses on parvalbumin-positive interneurons. In comparison, TARP γ-8 is the only TARP expressed at nonperforated type of pyramidal cell synapses, playing an essential role in low-density or basal AMPAR expression.

CDKL5 controls postsynaptic localization of GluN2B-containing NMDA receptors in the hippocampus and regulates seizure susceptibility.

Mutations in the Cyclin-dependent kinase-like 5 (CDKL5) gene cause severe neurodevelopmental disorders accompanied by intractable epilepsies, i.e. West syndrome or atypical Rett syndrome. Here we report generation of the Cdkl5 knockout mouse and show that CDKL5 controls postsynaptic localization of GluN2B-containing N-methyl-d-aspartate (NMDA) receptors in the hippocampus and regulates seizure susceptibility. Cdkl5 -/Y mice showed normal sensitivity to kainic acid; however, they displayed significant hyperexcitability to NMDA. In concordance with this result, electrophysiological analysis in the hippocampal CA1 region disclosed an increased ratio of NMDA/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor-mediated excitatory postsynaptic currents (EPSCs) and a significantly larger decay time constant of NMDA receptor-mediated EPSCs (NMDA-EPSCs) as well as a stronger inhibition of the NMDA-EPSCs by the GluN2B-selective antagonist ifenprodil in Cdkl5 -/Y mice. Subcellular fractionation of the hippocampus from Cdkl5 -/Y mice revealed a significant increase of GluN2B and SAP102 in the PSD (postsynaptic density)-1T fraction, without changes in the S1 (post-nuclear) fraction or mRNA transcripts, indicating an intracellular distribution shift of these proteins to the PSD. Immunoelectron microscopic analysis of the hippocampal CA1 region further confirmed postsynaptic overaccumulation of GluN2B and SAP102 in Cdkl5 -/Y mice. Furthermore, ifenprodil abrogated the NMDA-induced hyperexcitability in Cdkl5 -/Y mice, suggesting that upregulation of GluN2B accounts for the enhanced seizure susceptibility. These data indicate that CDKL5 plays an important role in controlling postsynaptic localization of the GluN2B-SAP102 complex in the hippocampus and thereby regulates seizure susceptibility, and that aberrant NMDA receptor-mediated synaptic transmission underlies the pathological mechanisms of the CDKL5 loss-of-function.

Cellular and subcellular localization of ADP-ribosylation factor 6 in mouse peripheral tissues.

ADP-ribosylation factor 6 (Arf6) is a small GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. In the present study, we comprehensively examined the cellular and subcellular localization of Arf6 in adult mouse peripheral tissues by immunofluorescence and immunoelectron microscopy using the heat-induced antigen retrieval method with Tris-EDTA buffer (pH 9.0). Marked immunolabeling of Arf6 was observed particularly in epithelial cells of several tissues including the esophagus, stomach, small and large intestines, trachea, kidney, epididymis, oviduct, and uterus. In most epithelial cells of simple or pseudostratified epithelia, Arf6 exhibited predominant localization to the basolateral membrane and a subpopulation of endosomes. At an electron microscopic level, Arf6 was localized along the basolateral membrane, with dense accumulation at interdigitating processes and infoldings. Arf6 was present in a ring-like appearance at intercellular bridges in spermatogonia and spermatocytes in the testis and at the Flemming body of cytokinetic somatic cells in the ovarian follicle, thymus, and spleen. The present study provides anatomical clues to help understand the physiological roles of Arf6 at the whole animal level.

Dynamin-Related Protein 1 Deficiency Leads to Receptor-Interacting Protein Kinase 3-Mediated Necroptotic Neurodegeneration.

Mitochondria are dynamic organelles that divide and fuse to modulate their number and shape. We have previously reported that the loss of dynamin-related protein 1 (Drp1), which mediates mitochondrial division, leads to the degeneration of cerebellar Purkinje cells in mice. Because Drp1 has been shown to be important for apoptosis and necroptosis, it is puzzling how Purkinje neurons die in the absence of Drp1. In this study, we tested whether neurodegeneration involves necrotic cell death by generating Purkinje cell-specific Drp1-knockout (KO) mice that lack the receptor-interacting protein kinase 3 (Rip3), which regulates necroptosis. We found that the loss of Rip3 significantly delays the degeneration of Drp1-KO Purkinje neurons. In addition, before neurodegeneration, mitochondrial tubules elongate because of unopposed fusion and subsequently become large spheres as a result of oxidative damage. Surprisingly, Rip3 loss also helps Drp1-KO Purkinje cells maintain the elongated morphology of the mitochondrial tubules. These data suggest that Rip3 plays a role in neurodegeneration and mitochondrial morphology in the absence of mitochondrial division.

The active zone protein CAST regulates synaptic vesicle recycling and quantal size in the mouse hippocampus.

Synaptic efficacy is determined by various factors, including the quantal size, which is dependent on the amount of neurotransmitters in synaptic vesicles at the presynaptic terminal. It is essential for stable synaptic transmission that the quantal size is kept within a constant range and that synaptic efficacy during and after repetitive synaptic activation is maintained by replenishing release sites with synaptic vesicles. However, the mechanisms for these fundamental properties have still been undetermined. We found that the active zone protein CAST (cytomatrix at the active zone structural protein) played pivotal roles in both presynaptic regulation of quantal size and recycling of endocytosed synaptic vesicles. In the CA1 region of hippocampal slices of the CAST knockout mice, miniature excitatory synaptic responses were increased in size, and synaptic depression after prolonged synaptic activation was larger, which was attributable to selective impairment of synaptic vesicle trafficking via the endosome in the presynaptic terminal likely mediated by Rab6. Therefore, CAST serves as a key molecule that regulates dynamics and neurotransmitter contents of synaptic vesicles in the excitatory presynaptic terminal in the central nervous system.

Interaction of serologically defined colon cancer antigen-3 with Arf6 and its predominant expression in the mouse testis.

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. Here, we identified the serologically defined colon antigen-3 (SDCCAG3) as an Arf6-interacting protein by yeast two-hybrid screening with a constitutively active Arf6 mutant. SDCCAG3 interacts specifically with Arf6 among the Arf family members through its 101  C-terminal amino acids. SDCCAG3 is expressed most intensely in the testis at the mRNA and protein levels. In the testis, SDCCAG3 is expressed in spermatocytes and spermatids. We also show that full-length SDCCAG3, but not a mutant lacking the ability to interact with Arf6, is recruited to the midbody during cytokinesis when expressed exogenously in HeLa cells. These findings suggest that SDCCAG3 might function in endosomal trafficking downstream of Arf6.

The synaptic targeting of mGluR1 by its carboxyl-terminal domain is crucial for cerebellar function.

The metabotropic glutamate receptor subtype 1 (mGluR1, Grm1) in cerebellar Purkinje cells (PCs) is essential for motor coordination and motor learning. At the synaptic level, mGluR1 has a critical role in long-term synaptic depression (LTD) at parallel fiber (PF)-PC synapses, and in developmental elimination of climbing fiber (CF)-PC synapses. mGluR1a, a predominant splice variant in PCs, has a long carboxyl (C)-terminal domain that interacts with Homer scaffolding proteins. Cerebellar roles of the C-terminal domain at both synaptic and behavior levels remain poorly understood. To address this question, we introduced a short variant, mGluR1b, which lacks this domain into PCs of mGluR1-knock-out (KO) mice (mGluR1b-rescue mice). In mGluR1b-rescue mice, mGluR1b showed dispersed perisynaptic distribution in PC spines. Importantly, mGluR1b-rescue mice exhibited impairments in inositol 1,4,5-trisphosphate receptor (IP3R)-mediated Ca(2+) release, CF synapse elimination, LTD induction, and delay eyeblink conditioning: they showed normal transient receptor potential canonical (TRPC) currents and normal motor coordination. In contrast, PC-specific rescue of mGluR1a restored all cerebellar defects of mGluR1-KO mice. We conclude that the long C-terminal domain of mGluR1a is required for the proper perisynaptic targeting of mGluR1, IP3R-mediated Ca(2+) release, CF synapse elimination, LTD, and motor learning, but not for TRPC currents and motor coordination.

Opposing role of NMDA receptor GluN2B and GluN2D in somatosensory development and maturation.

Development of correct topographical connections between peripheral receptors and central somatosensory stations requires activity-dependent synapse refinement, in which the NMDA type of glutamate receptors plays a key role. Here we compared functional roles of GluN2B (GluRε2 or NR2B) and GluN2D (GluRε4 or NR2D), two major regulatory subunits of neonatal NMDA receptors, in development of whisker-related patterning at trigeminal relay stations. Compared with control littermates, both the appearance of whisker-related patterning and the termination of the critical period, as assessed by unilateral infraorbital nerve transection, were delayed by nearly a day in the somatosensory cortex of GluN2B(+/-) mice but advanced by nearly a day in GluN2D(-/-) mice. Similar temporal shifts were found at subcortical relay stations in the thalamus and brainstem of GluN2B(+/-) and GluN2D(-/-) mice. In comparison, the magnitude of lesion-induced critical period plasticity in the somatosensory cortex, as assessed following row-C whisker removal, was normal in both mutants. Thus, GluN2B and GluN2D play counteractive roles in temporal development and maturation of somatosensory maps without affecting the magnitude of critical period plasticity. To understand the opposing action, we then examined neuronal and synaptic expressions of the two subunits along the trigeminal pathway. At each trigeminal station, GluN2B was predominant at asymmetrical synapses of non-GABAergic neurons, whereas GluN2D was selective to asymmetrical synapses of GABAergic neurons. Together, our findings suggest that GluN2B expressed at glutamatergic synapses on glutamatergic projection neurons facilitates refinement of ascending pathway synapses directly, whereas GluN2D expressed at glutamatergic synapses on GABAergic interneurons delays it indirectly.

Point mutation in syntaxin-1A causes abnormal vesicle recycling, behaviors, and short term plasticity.

Syntaxin-1A is a t-SNARE that is involved in vesicle docking and vesicle fusion; it is important in presynaptic exocytosis in neurons because it interacts with many regulatory proteins. Previously, we found the following: 1) that autophosphorylated Ca(2+)/calmodulin-dependent protein kinase II (CaMKII), an important modulator of neural plasticity, interacts with syntaxin-1A to regulate exocytosis, and 2) that a syntaxin missense mutation (R151G) attenuated this interaction. To determine more precisely the physiological importance of this interaction between CaMKII and syntaxin, we generated mice with a knock-in (KI) syntaxin-1A (R151G) mutation. Complexin is a molecular clamp involved in exocytosis, and in the KI mice, recruitment of complexin to the SNARE complex was reduced because of an abnormal CaMKII/syntaxin interaction. Nevertheless, SNARE complex formation was not inhibited, and consequently, basal neurotransmission was normal. However, the KI mice did exhibit more enhanced presynaptic plasticity than wild-type littermates; this enhanced plasticity could be associated with synaptic response than did wild-type littermates; this pronounced response included several behavioral abnormalities. Notably, the R151G phenotypes were generally similar to previously reported CaMKII mutant phenotypes. Additionally, synaptic recycling in these KI mice was delayed, and the density of synaptic vesicles was reduced. Taken together, our results indicated that this single point mutation in syntaxin-1A causes abnormal regulation of neuronal plasticity and vesicle recycling and that the affected syntaxin-1A/CaMKII interaction is essential for normal brain and synaptic functions in vivo.

EFA6A, a guanine nucleotide exchange factor for Arf6, interacts with sorting nexin-1 and regulates neurite outgrowth.

The membrane trafficking and actin cytoskeleton remodeling mediated by ADP ribosylation factor 6 (Arf6) are functionally linked to various neuronal processes including neurite formation and maintenance, neurotransmitter release, and receptor internalization. EFA6A is an Arf6-specific guanine nucleotide exchange factor that is abundantly expressed in the brain. In this study, we identified sorting nexin-1 (SNX1), a retromer component that is implicated in endosomal sorting and trafficking, as a novel interacting partner for EFA6A by yeast two-hybrid screening. The interaction was mediated by the C-terminal region of EFA6A and a BAR domain of SNX1, and further confirmed by pull-down assay and immunoprecipitation from mouse brain lysates. In situ hybridization analysis demonstrated the widespread expression of SNX1 in the mouse brain, which overlapped with the expression of EFA6A in the forebrain. Immunofluorescent analysis revealed the partial colocalization of EFA6A and SNX1 in the dendritic fields of the hippocampus. Immunoelectron microscopic analysis revealed the overlapping subcellular localization of EFA6A and SNX1 at the post-synaptic density and endosomes in dendritic spines. In Neuro-2a neuroblastoma cells, expression of either EFA6A or SNX1 induced neurite outgrowth, which was further enhanced by co-expression of EFA6A and SNX1. The present findings suggest a novel mechanism by which EFA6A regulates Arf6-mediated neurite formation through the interaction with SNX1.

Accumulation of α-synuclein triggered by presynaptic dysfunction.

Pathological examination of dementia with Lewy bodies patients identified the presence of abnormal α-synuclein (αSyn) aggregates in the presynaptic terminals. αSyn is involved in the regulation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex. Importantly, αSyn-transgenic mouse and postmortem examination of patients with Parkinson's disease have demonstrated the abnormal distribution of SNARE protein in presynaptic terminals. In this study, we investigated the effects of SNARE dysfunction on endogenous αSyn using Snap25(S187A/S187A) mutant mice. These mice have homozygous knock-in gene encoding unphosphorylatable S187A-substituted synaptosomal-associated protein of 25 kDa (SNAP-25). The mice displayed a significant age-dependent change in the distribution of αSyn and its Ser(129)-phosphorylated form in abnormally hypertrophied glutamatergic nerve terminals in the striatum. Electron-microscopic analysis revealed the abnormally condensed synaptic vesicles with concomitant mislocalization of αSyn protein to the periactive zone in the glutamatergic nerve terminals. However, the Snap25(S187A/S187A) mutant mouse harbored no abnormalities in the nigrostriatal dopaminergic neurons. Our present results suggest that SNARE dysfunction is the initial trigger of mislocalization and accumulation of αSyn, and probably is an important pathomechanism of α-synucleinopathies.

Type I phosphatidylinositol 4-phosphate 5-kinase γ is required for neuronal migration in the mouse developing cerebral cortex.

Type I phosphatidylinositol 4-phosphate 5-kinase (PIP5KI)γ is one of the phosphoinositide kinases that produce phosphatidylinositol 4,5-bisphosphate, which is a critical regulator of cell adhesion formation, actin dynamics and membrane trafficking. Here, we examined the functional roles of PIP5KIγ in radial neuronal migration during cortical formation. Reverse transcription-polymerase chain reaction analysis revealed that PIP5KIγ_v2/v6 and PIP5KIγ_v3 were expressed throughout cortical development with distinct expression patterns. In situ hybridisation analysis showed that PIP5KIγ mRNA was expressed throughout the cortical layers. Immunohistochemical analysis revealed that PIP5KIγ was localised in a punctate manner in the radial glia and migrating neuroblasts. Knockdown of PIP5KIγ using in utero electroporation disturbed the radial neuronal migration and recruitment of talin and focal adhesion kinase to puncta beneath the plasma membrane. The same inhibitory effect on neuronal migration was observed by overexpression of a catalytically inactive mutant of PIP5KIγ_v2 but not PIP5KIγ_v1 or PIP5KIγ_v3. These findings suggest an essential role of PIP5KIγ, particularly PIP5KIγ_i2, in neuronal migration, possibly through recruitment of adhesion components to the plasma membrane.