EFA6D (guanine nucleotide exchange factor for ADP-ribosylation factor 6 [Arf6]D) is also known as EFA6R, Psd3, and HCA67. It is the fourth member of the EFA6 family with guanine nucleotide exchange activity for Arf6, a small guanosine triphosphatase (GTPase) that regulates endosomal trafficking and actin cytoskeleton remodeling. We propose a classification and nomenclature of 10 EFA6D variants deposited in the GenBank database as EFA6D1a, 1b, 1c, 1d, 1s, 2a, 2b, 2c, 2d, and 2s based on the combination of N-terminal and C-terminal insertions. Polymerase chain reaction analysis showed the expression of all EFA6D variants except for variants a and d in the adult mouse brain. Immunoblotting analysis with novel variant-specific antibodies showed the endogenous expression of EFA6D1b, EFA6D1c, and EFA6D1s at the protein level, with the highest expression being EFA6D1s, in the brain. Immunoblotting analysis of forebrain subcellular fractions showed the distinct subcellular distribution of EFA6D1b/c and EFA6D1s. The immunohistochemical analysis revealed distinct but overlapping immunoreactive patterns between EFA6D1b/c and EFA6D1s in the mouse brain. In immunoelectron microscopic analyses of the hippocampal CA3 region, EFA6D1b/c was present predominantly in the mossy fiber axons of dentate granule cells, whereas EFA6D1s was present abundantly in the cell bodies, dendritic shafts, and spines of hippocampal pyramidal cells. These results provide the first anatomical evidence suggesting the functional diversity of EFA6D variants, particularly EFA6D1b/c and EFA6D1s, in neurons. J. Comp. Neurol. 524:2531-2552, 2016. © 2016 Wiley Periodicals, Inc.

The data is related to the research article entitled "Arf6 mediates Schwann cell differentiation and myelination" [1]. To further investigate the role of Arf6 in promoting myelination by Schwann cells in vivo, we have characterized an another line (#2) of small-hairpin (sh)RNA transgenic mice targeting Arf6. The number of transgenes per one allele in this line was very low (2 transgenes), comparing with high copies in the previous line (#1, 20 transgenes) [1]. In 4 days of neonatal age, transgenic mice exhibited decreased myelin thickness; however, decreased levels were not as much as those in the line #1, likely depending on transgene copy number. In 60-day-old mice, the difference became smaller. On the other hand, transgene׳s effect was not related to cell proliferation and apoptosis. These data support the key role of Arf6 in Schwann cell myelination, especially in the initiation.

Vascular endothelial growth factor (VEGF) is one of the most potent angiogenesis stimulators. VEGF binds to VEGF receptor 1 (VEGFR1), inducing angiogenesis through the receptor's tyrosine kinase domain (TK), but the mechanism is not well understood. We investigated the role of VEGFR1 tyrosine kinase signaling in angiogenesis using the ischemic hind limb model. Relative to control mice, blood flow recovery was significantly impaired in mice treated with VEGFA-neutralizing antibody. VEGFR1 tyrosine kinase knockout mice (TK-/-) had delayed blood flow recovery from ischemia and impaired angiogenesis, and this phenotype was unaffected by treatment with a VEGFR2 inhibitor. Compared to wild type mice (WT), TK-/- mice had no change in the plasma level of VEGF, but the plasma levels of stromal-derived cell factor 1 (SDF-1) and stem cell factor, as well as the bone marrow (BM) level of pro-matrix metalloproteinase-9 (pro-MMP-9), were significantly reduced. The recruitment of cells expressing VEGFR1 and C-X-C chemokine receptor type 4 (CXCR4) into peripheral blood and ischemic muscles was also suppressed. Furthermore, WT transplanted with TK-/- BM significantly impaired blood flow recovery more than WT transplanted with WT BM. These results suggest that VEGFR1-TK signaling facilitates angiogenesis by recruiting CXCR4+VEGFR1+ cells from BM.

Charcot-Marie-Tooth (CMT) disease is composed of a heterogeneous group of hereditary peripheral neuropathies. The peripheral nervous system primarily comprises two types of cells: neuronal cells and myelinating glial Schwann cells. CMT2 N is an autosomal dominant disease and its responsible gene encodes alanyl-tRNA synthetase (AARS), which is a family of cytoplasmic aminoacyl-tRNA synthetases. CMT2 N is associated with the mutation, including a missense mutation, which is known to decrease the enzymatic activity of AARS, but whether and how its mutation affects AARS localization and neuronal process formation remains to be understood. First, we show that the AARS mutant harboring Asn71-to-Tyr (N71Y) is not localized in cytoplasm. The expression of AARS mutant proteins in COS-7 cells mainly leads to localization into lysosome, whereas the wild type is indeed localized in cytoplasm. Second, in N1E-115 cells as the neuronal cell model, cells expressing the N71Y mutant do not have the ability to grow processes. Third, pretreatment with antiepileptic valproic acid reverses the inhibitory effect of the N71Y mutant on process growth. Taken together, the N71Y mutation of AARS leads to abnormal intracellular localization, inhibiting process growth, yet this inhibition is reversed by valproic acid.

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.

Glycine is an inhibitory neurotransmitter in the brainstem and spinal cord. Glycine transporter 2 (GLYT2) is responsible for the uptake of extracellular glycine. GLYT2 is specifically expressed in glycinergic neurons and thus has been used as a marker of glycinergic neurons. Here, we generated GLYT2 promotor-driven Cre recombinase (Cre)-expressing mice (GLYT2-Cre knock-in mice) to develop a tool for manipulating gene expression in glycinergic neurons. Cre activity was examined by crossing the GLYT2-Cre knock-in mice with a Cre reporter mouse line, R26R, which express β-galactosidase (β-gal) in a Cre-dependent manner. X-gal staining of GLYT2-Cre/R26R double transgenic mouse brains and spinal cords revealed that the Cre activity was primarily distributed in the brainstem, cerebellum, and spinal cord. These areas are rich in glycinergic neurons. Furthermore, we performed immunohistochemistry for β-gal combined with in situ hybridization for GLYT2 in the GLYT2-Cre/R26R double transgenic mouse brains to determine whether Cre activity is specifically localized to glycinergic neurons. The β-gal protein and GLYT2 mRNAs were colocalized in the cerebellar Golgi cells, dorsal cochlear nucleus, gigantocellular reticular nucleus, spinal trigeminal nucleus, nucleus of the trapezoid body, and lateral lemniscus. More than 98% of the GLYT2 mRNA-expressing cells in these brain regions also expressed β-gal, whereas 90-98% of the β-gal-positive cells expressed the GLYT2 mRNAs. Thus, Cre activity is specifically localized to glycinergic neurons with high fidelity in the GLYT2-Cre knock-in mice. The GLYT2-Cre knock-in mouse line will be a useful tool for studying glycinergic neurons and neurotransmission.

Fatty acids and their metabolites have recently been shown to modulate various functions of dendritic cells (DCs) including their differentiation and cytokine production, although the mechanisms underlying their cellular functions are not fully understood. In view of our previous finding that epidermal-type fatty acid binding protein (E-FABP) was exclusively expressed in splenic DCs among FABP family, we examined the phenotype of E-FABP-null mutant mice in order to elucidate the functional significance of E-FABP expression in DCs. Although E-FABP-null mutant mice showed no apparent abnormalities in the population density and subset distribution of DCs as well as the microscopic morphology in the spleen, DCs isolated from E-FABP-null spleen showed enhanced production of IL-12p70, a key cytokine for innate immune responses, in response to appropriate stimuli as compared with wild-type. In real-time PCR, the expression level of IL-12p35 mRNA after LPS stimuli was much higher in mutant DCs when compared with wild-type, while no apparent change of IL-12p40 mRNA level was detected. Phosphorylated forms of p38 mitogen-activated protein kinase (p38MAPK) and IkappaB-alpha, molecules critical for IL-12 production, were detected at higher levels in E-FABP-null-mutant DCs after LPS stimuli when compared with wild-type counterparts. Collectively, it is suggested that E-FABP may be a novel negative regulator of IL-12 production in DCs, and this regulation may be exerted via its involvement in the p38MAPK-mediated transcription of IL-12p35.

Calcium-calmodulin dependent protein kinase IV (CaMKIV) is a protein kinase that activates the transcription factor CREB, the cyclic AMP-response element binding protein. CREB is a key transcription factor in synaptic plasticity and memory consolidation. To elucidate the behavioral effects of CaMKIV deficiency, we subjected CaMKIV knockout (CaMKIV KO) mice to a battery of behavioral tests. CaMKIV KO had no significant effects on locomotor activity, motor coordination, social interaction, pain sensitivity, prepulse inhibition, attention, or depression-like behavior. Consistent with previous reports, CaMKIV KO mice exhibited impaired retention in a fear conditioning test 28 days after training. In contrast, however, CaMKIV KO mice did not show any testing performance deficits in passive avoidance, one of the most commonly used fear memory paradigms, 28 days after training, suggesting that remote fear memory is intact. CaMKIV KO mice exhibited intact spatial reference memory learning in the Barnes circular maze, and normal spatial working memory in an eight-arm radial maze. CaMKIV KO mice also showed mildly decreased anxiety-like behavior, suggesting that CaMKIV is involved in regulating emotional behavior. These findings indicate that CaMKIV might not be essential for fear memory or spatial memory, although it is possible that the activities of other neural mechanisms or signaling pathways compensate for the CaMKIV deficiency.

The expression of immediate early genes (IEGs) is thought to be an essential molecular basis of neuronal plasticity for higher brain function. Many IEGs contain serum response element in their transcriptional regulatory regions and their expression is controlled by serum response factor (SRF). SRF is known to play a role in concert with transcriptional cofactors. However, little is known about how SRF cofactors regulate IEG expression during the process of neuronal plasticity. We hypothesized that one of the SRF-regulated neuronal IEGs, activity-regulated cytoskeleton-associated protein (Arc; also termed Arg3.1), is regulated by an SRF coactivator, megakaryoblastic leukemia (MKL). To test this hypothesis, we initially investigated which binding site of the transcription factor or SRF cofactor contributes to brain-derived neurotrophic factor (BDNF)-induced Arc gene transcription in cultured cortical neurons using transfection and reporter assays. We found that BDNF caused robust induction of Arc gene transcription through a cAMP response element, binding site of myocyte enhancer factor 2, and binding site of SRF in an Arc enhancer, the synaptic activity-responsive element (SARE). Regardless of the requirement for the SRF-binding site, the binding site of a ternary complex factor, another SRF cofactor, did not affect BDNF-mediated Arc gene transcription. In contrast, chromatin immunoprecipitation revealed occupation of MKL at the SARE. Furthermore, knockdown of MKL2, but not MKL1, significantly decreased BDNF-mediated activation of the SARE. Taken together, these findings suggest a novel mechanism by which MKL2 controls the Arc SARE in response to BDNF stimulation.

Arf6 (ADP ribosylation factor 6), activated by Arf-GEF (guanine nucleoside exchange factor), is involved in the membrane trafficking and actin-remodeling which are critical for maintenance of cell organization and activity and for fusion of myoblasts to form myotubes/myofibers. EFA6A (exchange factor for Arf6 type A) and BRAG2 (brefeldin A-resistant Arf-GEF 2) represent members of discrete subfamilies of Arf-GEF, while PIP5Kγ (phosphatidylinositol4-phosphate5-kinase γ) produces PI 4,5-bisphosphate (PIP2) and it is target for Arf6. In the present study, immunoreactive bands for Arf6, EFA6A, BRAG2 and PIP5Kγ were detected in immunoblots of skeletal muscle homogenates of mice at E18D (embryonic day 18), while the bands for Arf6, EFA6A and PIP5Kγ were reduced in density and no significant bands for BRAG2 were discerned at P1D (postnatal 1 day). No immunoblot bands for any of the molecules were eventually detected in skeletal fibers of adult mice. Immunoreactivities for endogenous Arf6, EFA6A and PIP5Kγ were visualized using immuno-light microscopy localized as periodic striations running perpendicular to the longitudinal axes of skeletal muscle fibers of mice at E18D and P1D. All the striations were co-immunoreactive for β-actin in double immunofluorescence microscopy, and the immunoreactivities were confined to thin myofilaments at sarcomeric I-domains in immuno-electron microscopy. On the other hand, immunoreactivities for Arf6, BRAG2 and PIP5Kγ were conspicuous on plasmalemma of myoblasts at E14D, while immunoreactivity for EFA6A was already distinct in striations perpendicular to myofibrils in myotubes at E14D. The present findings suggest three possibilities: involvement of EFA6A-activated Arf6 together with PIP5Kγ in maturation of myofibrils, movement of Arf6 and PIP5Kγ from the plasmalemma of myoblasts to myofibrils of myotubes, and that of BRAG2 to the cytoplasm of myotubes; and further a function of EFA6A independent of the activation of Arf6 in immature myofibrils. In addition, the involvement of Arf6, BRAG2 and PIP5Kγ in the fusion of myoblasts into myotubes was supported by the present finding.

IQSEC3, a gephyrin-binding GABAergic (gamma-aminobutyric acidergic) synapse-specific guanine nucleotide exchange factor, was recently reported to regulate activity-dependent GABAergic synapse maturation, but the underlying signaling mechanisms remain incompletely understood.

Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ) acts as a regulatory kinase that phosphorylates and activates multiple downstream kinases including CaMKI, CaMKIV, 5'AMP-activated protein kinase (AMPK) and protein kinase B (PKB), resulting in regulation of wide variety of Ca2+-dependent physiological responses under normal and pathological conditions. CaMKKβ is regulated by Ca2+/calmodulin-binding, autophosphorylation, and transphosphorylation by multiple protein kinases including cAMP-dependent protein kinase (PKA). In this report, we found that phosphorylation of CaMKKβ is dynamically regulated by protein phosphatase/kinase system in HeLa cells. Global phosphoproteomic analysis revealed the constitutive phosphorylation at 8 Ser residues including Ser128, 132, and 136 in the N-terminal regulatory domain of rat CaMKKβ in unstimulated HeLa cells as well as inducible phosphorylation of Thr144 in the cells treated with a phosphatase inhibitor, okadaic acid (OA). Thr144 phosphorylation in CaMKKβ has shown to be rapidly induced by OA treatment in a time- and dose-dependent manner in transfected HeLa cells, indicating that Thr144 in CaMKKβ is maintained unphosphorylated state by protein phosphatase(s). We confirmed that in vitro dephosphorylation of pThr144 in CaMKKβ by protein phosphatase 2A and 1. We also found that the pharmacological inhibition of protein phosphatase(s) significantly induces CaMKKβ-phosphorylating activity (at Thr144) in HeLa cell lysates as well as in intact cells; however, it was unlikely that this activity was catalyzed by previously identified Thr144-kinases, such as AMPK and PKA. Taken together, these results suggest that the phosphorylation and dephosphorylation of Thr144 in CaMKKβ is dynamically regulated by multiple kinases/phosphatases signaling resulting in fine-tuning of the enzymatic property.

Metabotropic glutamate receptors (mGluRs) couple to G-proteins to modulate slow synaptic transmission via intracellular second messengers. The first cloned mGluR, mGluR1, regulates motor coordination, synaptic plasticity and synapse elimination. mGluR1 undergoes alternative splicing giving rise to four translated variants that differ in their intracellular C-terminal domains. Our current knowledge about mGluR1 relates almost entirely to the long mGluR1α isoform, whereas little is known about the other shorter variants. To study the expression of mGluR1γ, we have generated by means of the CRISPR/Cas9 system a new knock-in (KI) mouse line in which the C-terminus of this variant carries two short tags. Using this mouse line, we could establish that mGluR1γ is either untranslated or in amounts that are undetectable in the mouse cerebellum, indicating that only mGluR1α and mGluR1β are present and active at cerebellar synapses. The trafficking and function of mGluR1 appear strongly influenced by adaptor proteins such as long Homers that bind to the C-terminus of mGluR1α. We generated a second transgenic (Tg) mouse line in which mGluR1α carries a point mutation in its Homer binding domain and studied whether disruption of this interaction influenced mGluR1 subcellular localization at cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses by means of the freeze-fracture replica immunolabeling technique. These Tg animals did not show any overt behavioral phenotype, and despite the typical mGluR1 perisynaptic distribution was not significantly changed, we observed a higher probability of intrasynaptic diffusion suggesting that long Homers regulate the lateral mobility of mGluR1. We extended our ultrastructural analysis to other mouse lines in which only one mGluR1 variant was reintroduced in PC of mGluR1-knock out (KO) mice. This work revealed that mGluR1α preferentially accumulates closer to the edge of the postsynaptic density (PSD), whereas mGluR1β has a less pronounced perijunctional distribution and, in the absence of mGluR1α, its trafficking to the plasma membrane is impaired with an accumulation in intracellular organelles. In conclusion, our study sets several firm points on largely disputed matters, namely expression of mGluR1γ and role of the C-terminal domain of mGluR1 splice variants on their perisynaptic clustering.

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 Ras-GTPase activating protein synGAP is a brain-specific protein of approximately 130 kDa and is a negative regulator of Ras. We previously reported 5 C-terminal isoforms of synGAP (alpha1, alpha2, beta1/2, beta 3/4 and gamma) [Li et al., 2001, J. Biol. Chem. 276: 21417-21424]. In this study, we investigated the expression profiles of the two major isoforms, synGAP alpha1 and synGAP beta, in the adult rat brain and cultured neurons of the rat hippocampus. Examination of pepsin-pretreated brain sections demonstrated that both isoforms were expressed mainly in the forebrain structures, which suggests their association with postsynaptic density. The distribution of the synGAP alpha1 and beta (beta1-4) isoforms in the adult rat brain was clearly different in cerebellum, hippocampus, cerebral cortex, septum and olfactory bulb. In particular, synGAP alpha1 was specifically localized to the cerebellar glomeruli, dense synaptic sites. From the analysis using cultured neurons, unique expression of synGAP beta was found in a neuron with a sea urchin-like morphology, possibly a star pyramidal neuron, in which the synGAP beta expression was relatively high, in particular, at the distal part of its processes. SynGAP alpha1 was mostly or specifically localized to excitatory postsynaptic sites, whereas synGAP beta was present at both excitatory and inhibitory postsynaptic sites. Finally, there are more non-synaptic clusters in dendrites in the case of synGAP beta than synGAP alpha1. Thus, the two synGAP isoforms, alpha1 and beta, distribute differently in neuronal cells and the brain.

During neural development, endosomal trafficking controls cell shape and motility through the polarized transport of membrane proteins related to cell-cell and cell-extracellular matrix interactions. ADP ribosylation factor 6 (Arf6) is a critical small GTPase that regulates membrane trafficking between the plasma membrane and endosomes. We herein demonstrated that the knockdown of endogenous Arf6 in mouse cerebral cortices led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin and syntaxin12 in migrating neurons. Rescue experiments with separation-of-function Arf6 mutants identified Rab11 family-interacting protein 3 (FIP3)/Arfophilin-1, a dual effector for Arf6 and Rab11, as a downstream effector of Arf6 in migrating neurons. The knockdown of FIP3 led to impaired neuronal migration in the intermediate zone and cytoplasmic retention of N-cadherin in migrating neurons, similar to that of Arf6, which could be rescued by the coexpression of wild-type FIP3 but not FIP3 mutants lacking the binding site for Arf6 or Rab11. These results suggest that Arf6 regulates cortical neuronal migration in the intermediate zone through the FIP3-dependent endosomal trafficking.

Based on a previous study by others reporting that PIP5Kγ (phosphatidylinositol 4-phosphate 5-kinase γ) and its product, phosphatidylinositol 4,5 bisphosphate (PIP2 ), are involved in the regulation of nociception, the present immunohistochemical study examined the localization of PIP5Kγ-immunoreactivity in dorsal root ganglia (DRG) and their peripheral and central terminal fields. PIP5Kγ-immunoreactivity was localized for the first time in the muscle spindles, in which it was found in I-bands of polar regions of intrafusal muscle fibers and also in sensory nerve terminals abutting on equatorial regions of the muscle fibers. This finding indicates the involvement of PIP5Kγ in the proprioception and suggests somehow complicated mechanisms of its involvement because of its heterogeneous localization in intra-I-band structures. In DRG, on the other hand, PIP5Kγ-immunoreactivity was shown to be localized heterogeneously, but not evenly, over apposed plasma membranes of both neurons and ganglionic satellite cells in immune electron microscopy. In addition, no peripheral nerve terminals of DRG showing its distinct immunoreactivity were found in most peripheral fields of nociception and any other sensory perception except for the proprioception through muscle spindles. In contrast, numerous central terminals of DRG in the spinal posterior horn were immunoreactive for it. This finding leads us to consider the possibility that the regulation by PIP5Kγ of nociception is dominantly exerted in DRG and sensory neural tracts central, rather than peripheral, to DRG.

Oligodendroglial cells (oligodendrocytes) differentiate to form the myelin that wraps neuronal axons in the central nervous system (CNS). This myelin sheath supports the propagation of saltatory conduction and protects axons from physical stresses. When oligodendrocytes do not normally differentiate to myelinate axons, their key functions as oligodendrocytes in the CNS are severely impaired. The molecular mechanics that control differentiation still remain to be clarified. Arf6 belongs to the small GTPase family and is known to be a positive regulator of oligodendrocyte differentiation. Here, we show that the phospholipase D (PLD) and phosphatidylinositol-4-phosphate 5-kinase 1 (PIP5K1) molecules, the major effectors of Arf6, are involved in the regulation of oligodendrocyte differentiation. Knockdown of PLD1 or PIP5K type 1γ (PIP5K1C) by their respective specific siRNAs in mouse oligodendroglial FBD-102b cells inhibited morphological differentiation into structures bearing myelin-like processes; this finding is consistent with the concurrent changes in expression of differentiation and myelin marker proteins. Treatment with VU0155069 or UNC3230, specific inhibitors of PLD and PIP5K1, respectively, blunted morphological differentiation and decreased expression of myelin and differentiation marker proteins. Similar results have been obtained in studies using primary oligodendrocytes. These results suggest that the major Arf6 effector molecules PLD and PIP5K1 are among the molecules involved in the regulation of morphological differentiation in oligodendrocytes prior to myelination.

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.

During the development of the cerebral cortex, N-cadherin plays a crucial role in facilitating radial migration by enabling cell-to-cell adhesion between migrating neurons and radial glial fibers or Cajar-Reztius cells. ADP ribosylation factor 4 (Arf4) and Arf5, which belong to the Class II Arf small GTPase subfamily, control membrane trafficking in the endocytic and secretory pathways. However, their specific contribution to cerebral cortex development remains unclear. In this study, we sought to investigate the functional involvement of Class II Arfs in radial migration during the layer formation of the cerebral cortex using mouse embryos and pups. Our findings indicate that knock-down of Arf4, but not Arf5, resulted in the stalling of transfected neurons with disorientation of the Golgi in the upper intermediate zone (IZ) and reduction in the migration speed in both the IZ and cortical plate (CP). Migrating neurons with Arf4 knock-down exhibited cytoplasmic accumulation of N-cadherin, along with disturbed organelle morphology and distribution. Furthermore, supplementation of exogenous N-cadherin partially rescued the migration defect caused by Arf4 knock-down. In conclusion, our results suggest that Arf4 plays a crucial role in regulating radial migration via N-cadherin trafficking during cerebral cortical development.