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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. [ABSTRACT FROM AUTHOR]

The structural maintenance of neural circuits is critical for higher brain functions in adulthood. Although several molecules have been identified as regulators for spine maintenance in hippocampal and cortical neurons, it is poorly understood how Purkinje cell (PC) spines are maintained in the mature cerebellum. Here we show that the calcium channel type 1 inositol trisphosphate receptor (IP3R1) in PCs plays a crucial role in controlling the maintenance of parallel fiber (PF)-PC synaptic circuits in the mature cerebellum in vivo. Significantly, adult mice lacking IP3R1 specifically in PCs (L7-Cre;Itpr1flox/flox) showed dramatic increase in spine density and spine length of PCs, despite having normal spines during development. In addition, the abnormally rearranged PF-PC synaptic circuits in mature cerebellum caused unexpectedly severe ataxia in adult L7-Cre;Itprlflox/flox mice. Our findings reveal a specific role for IP3R1 in PCs not only as an intracellular mediator of cerebellar synaptic plasticity induction, but also as a critical regulator of PF-PC synaptic circuit maintenance in the mature cerebellum in vivo; this mechanism may underlie motor coordination and learning in adults. [ABSTRACT FROM AUTHOR]

Abstract: It is generally accepted that the dopamine system in the nucleus accumbens is activated and is involved in avoidance and escape behavior under aversive conditions. This study shows that the central dopamine system is involved in the jumping escape behavior in mice exposed to heat. In this study, the dopamine catabolite ratio in the nucleus accumbens was increased and dopamine 2 (D2) antagonists, chlorpromazine and haloperidol, inhibited the jumping escape behavior in mice exposed to 38.5°C. Chlorpromazine increased hyperthermia in mice exposed to 38.5°C, while haloperidol had no effect on rectal temperature in mice exposed to 38.5°C. These results indicate that D2 antagonists inhibit the jumping escape behavior in mice exposed to heat and the inhibition mechanism of D2 antagonists is independent of the disturbance of autonomic thermoregulation. [Copyright &y& Elsevier]

Abstract: A new heat escape behavior was revealed in mice (ddY mice) under acute heat stress conditions. Mice in a fully covered cage were exposed to 24, 34, 37 and 38.5 °C for 60 min. Rectal temperature increased in conditions above 34 °C. Furthermore, serum osmolality and body weight loss also increased in conditions above 37 °C. At above 37 °C, a large number of mice attempted to escape from the partially covered cage, and so exhibited jumping behavior during a period of 60 min. However, mice exposed to 24 and 34 °C did not exhibit such behavior. These results indicated that acute heat stress above 37 °C induced evaporative water loss and jumping escape behavior in mice. [Copyright &y& Elsevier]

Abstract Background Ca2+/calmodulin-dependent protein kinase kinase (CaMKK) is a pivotal activator of CaMKI, CaMKIV and 5'-AMP-activated protein kinase (AMPK), controlling Ca2+-dependent intracellular signaling including various neuronal, metabolic and pathophysiological responses. Recently, we demonstrated that CaMKKβ is feedback phosphorylated at Thr144 by the downstream AMPK, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. However, the regulatory phosphorylation of CaMKKβ at Thr144 in intact cells and in vivo remains unclear. Methods Anti-phosphoThr144 antibody was used to characterize the site-specific phosphorylation of CaMKKβ in immunoprecipitated samples from mouse cerebellum and in transfected mammalian cells that were treated with various agonists and protein kinase inhibitors. CaMKK activity assay and LC-MS/MS analysis were used for biochemical characterization of phosphorylated CaMKKβ. Results Our data suggest that the phosphorylation of Thr144 in CaMKKβ is rapidly induced by cAMP/cAMP-dependent protein kinase (PKA) signaling in CaMKKβ-transfected HeLa cells, that is physiologically relevant in mouse cerebellum. We confirmed that the catalytic subunit of PKA was capable of directly phosphorylating CaMKKβ at Thr144 in vitro and in transfected cells. In addition, the basal phosphorylation of CaMKKβ at Thr144 in transfected HeLa cells was suppressed by AMPK inhibitor (compound C). PKA-catalyzed phosphorylation reduced the autonomous activity of CaMKKβ in vitro without significant effect on the Ca2+/CaM-dependent activity, resulting in the conversion of CaMKKβ into Ca2+/CaM-dependent enzyme. Conclusion cAMP/PKA signaling may confer Ca2+-dependency to the CaMKKβ-mediated signaling pathway through direct phosphorylation of Thr144 in intact cells. General significance Our results suggest a novel cross-talk between cAMP/PKA and Ca2+/CaM/CaMKKβ signaling through regulatory phosphorylation. Graphical abstract Unlabelled Image Highlights • cAMP/PKA signal enhances phosphorylation of CaMKKβ at Thr144 in HeLa cells. • PKA directly phosphorylates CaMKKβ at Thr144 in cultured cells and in vitro. • Phosphorylation at Thr144 by PKA converts CaMKKβ into Ca2+/CaM-dependent enzyme. • CaMKKβ is phosphorylated at Thr144 in mouse cerebellum. [ABSTRACT FROM AUTHOR]

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. FIP4mRNA 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. Family of Rab11‐interacting protein 4 (FIP4)/Arfophilin‐2 regulates neuronal migration in the upper intermediate zone and cortical plate through Arf‐ and Rab11‐dependent endosomal trafficking during cortical layer formation. [ABSTRACT FROM AUTHOR]

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. • Cytohesin-2 shows preferential perisynaptic localization in dorsal horn neurons. • Cytohesin-2 forms a complex with mGluR5 in dorsal horn. • Cytohesin-2 cKO mice exhibit reduced mechanical allodynia in CFA and CCI models. • Cytohesin inhibitor SecinH3 blocks allodynia and Arf6 activation in pain models. • Cytohesin-2 cKO mice exhibit reduced DHPG-induced allodynia and ERK1/2 activation. [ABSTRACT FROM AUTHOR]

Hyperthermia and dehydration were important physiological phenomena in heat stress. But, the degrees of these phenomena were changed by heat stress conditions, and the distinction between both phenomena is necessary for investigation of response for individual phenomenon. Heat stress at 34 °C for 60 min increased rectal temperature, and heat stress at 38.5 °C for 60 min further increased rectal temperature and increased osmolality in mice. We investigated the activated region in hypothalamus, which played a role in thermoregulation, fluid regulation and so on, using immunostaining for Fos protein under these conditions in conscious mice. At 34 °C, Fos-positive neurons increased in the median preoptic nucleus, lateral preoptic area and anterior hypothalamic area, which were known to be the thermoregulatory center, and the dorsomedial hypothalamic nucleus, which was known to control eating. At 38.5 °C, Fos-positive neurons further increased in the regions mentioned above and appeared in the lateral septal nucleus, medial preoptic area, lateral hypothalamic area and zona incerta, which were thought to be involved in thermoregulation and/or fluid regulation, and the paraventricular hypothalamic nucleus, supraoptic nucleus and supraoptic nucleus in the retrochiasmatic part, which were known to be involved in neuroendocrine effector systems. These results support that the activated regions in hypothalamus differed with heat stress conditions, which induced only hyperthermia and both hyperthermia and dehydration. [Copyright &y& Elsevier]

ADP ribosylation factor 6 (Arf6) is a small GTPase that regulates various neuronal events including formation of the axon, dendrites and dendritic spines, and synaptic plasticity through actin cytoskeleton remodeling and endosomal trafficking. EFA6C, also known as Psd2, is a guanine nucleotide exchange factor for Arf6 that is preferentially expressed in the cerebellar cortex of adult mice, particularly in Purkinje cells. However, the roles of EFA6C in cerebellar development and functions remain unknown. In this study, we generated global EFA6C knockout (KO) mice using the CRISPR/Cas9 system and investigated their cerebellar phenotypes by histological and behavioral analyses. Histological analyses revealed that EFA6C KO mice exhibited normal gross anatomy of the cerebellar cortex, in terms of the thickness and cellularity of each layer, morphology of Purkinje cells, and distribution patterns of parallel fibers, climbing fibers, and inhibitory synapses. Electron microscopic observation of the cerebellar molecular layer revealed that the density of asymmetric synapses of Purkinje cells was significantly lower in EFA6C KO mice compared with wild-type control mice. However, behavioral analyses using accelerating rotarod and horizontal optokinetic response tests failed to detect any differences in motor coordination, learning or adaptation between the control and EFA6C KO mice. These results suggest that EFA6C plays ancillary roles in cerebellar development and motor functions. [ABSTRACT FROM AUTHOR]

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. Cytohesin‐2 is a guanine nucleotide exchange factor that is implicated in various neuronal functions through the activation of ADP ribosylation factors 1 (Arf1) and Arf6. In this study, we identified pallidin as a novel interacting protein specific for cytohesin‐2 among the cytohesin family. In cultured hippocampal neurons, knockdown of either cytohesin‐2 or pallidin similarly affected the endocytic pathway. Furthermore, knockdown of pallidin had a stimulatory effect on dendritic extension via its coiled coil domain that was responsible for the interaction with cytohesin‐2. These results provide the first evidence for a functional relationship between cytohesin‐2 and pallidin in neurons. Cover Image for this issue: doi: 10.1111/jnc.14197. [ABSTRACT FROM AUTHOR]

• Vps52, a core subunit of GARP and EARP complexes, interacts with Arf6. • Vps52 localizes to the TGN and vesicular endomembranes in hipppocampal neurons. • Vps52 knockdown increases axonal and dendritic length. ADP ribosylation factor 6 (Arf6) is a small GTP-binding protein implicated in neuronal morphogenesis through endosomal trafficking and actin remodeling. In this study, we identified Vps52, a core subunit of the Golgi-associated retrograde protein (GARP) and endosome-associated recycling protein (EARP) complexes, as a novel Arf6-binding protein by yeast two-hybrid screening. Vps52 interacted specifically with GTP-bound Arf6 among the Arf family. Immunohistochemical analyses of hippocampal pyramidal cells revealed that fine punctate immunolabeling for Vps52 was distributed throughout neuronal compartments, most densely in the cell body and dendritic shafts, and was largely associated with trans-Golgi network and vesicular endomembranes. In cultured hippocampal neurons, knockdown of Vps52 increased total length of axons and dendrites; these phenotypes were completely restored by co-expression of shRNA-resistant full-length Vps52. However, co-expression of a Vps52 mutant lacking the ability to interact with Arf6 restored only the Vps52-knockdown phenotype of the dendritic length. The present findings suggest that Vps52 is a novel Arf6-interacting protein that regulates neurite outgrowth in hippocampal neurons. [ABSTRACT FROM AUTHOR]