SynArfGEF is a guanine nucleotide exchange factor for Arf6 and localizes preferentially at post-synaptic specializations of inhibitory synapses.

SynArfGEF, also known as BRAG3 or IQSEC3, is a member of the brefeldin A-resistant Arf-GEF/IQSEC family and was originally identified by screening for mRNA species associated with the post-synaptic density fraction. In this study, we demonstrate that synArfGEF activates Arf6, using Arf pull down and transferrin incorporation assays. Immunohistochemical analysis reveals that synArfGEF is present in somata and dendrites as puncta in close association with inhibitory synapses, whereas immunoelectron microscopic analysis reveals that synArfGEF localizes preferentially at post-synaptic specializations of symmetric synapses. Using yeast two-hybrid and pull down assays, we show that synArfGEF is able to bind utrophin/dystrophin and S-SCAM/MAGI-2 scaffolding proteins that localize at inhibitory synapses. Double immunostaining reveals that synArfGEF co-localizes with dystrophin and S-SCAM in cultured hippocampal neurons and cerebellar cortex, respectively. Both ß-dystroglycan and S-SCAM were immunoprecipitated from brain lysates using anti-synArfGEF IgG. Taken together, these findings suggest that synArfGEF functions as a novel regulator of Arf6 at inhibitory synapses and associates with the dystrophin-associated glycoprotein complex and S-SCAM.

Vezatin, a potential target for ADP-ribosylation factor 6, regulates the dendritic formation of hippocampal neurons.

ADP-ribosylation factor 6 (ARF6) is a small GTPase that regulates neuronal morphogenesis processes such as axonal, dendritic, and spine formation possibly through the actin cytoskeleton and membrane trafficking. In an attempt to define the molecular mechanisms that regulate neuronal morphogenesis by ARF6, we identified vezatin as a novel binding partner of active GTP-bound ARF6 using yeast two-hybrid screening. Vezatin was able to bind specifically to GTP-ARF6 among the ARF family. In the adult mouse brain, vezatin exhibited widespread gene expression with high levels in the hippocampus and medial habenular nucleus. In hippocampal neurons, vezatin was localized at dendrites as well as cell bodies. Knockdown of endogenous vezatin significantly reduced total dendritic length and arborization of cultured hippocampal neurons, while overexpression of vezatin increased dendritic length. Our present study suggests that vezatin may regulate dendritic formation as a downstream effector of ARF6.

DY-9760e inhibits endothelin-1-induced cardiomyocyte hypertrophy through inhibition of CaMKII and ERK activities.

Ca(2+)/calmodulin-dependent protein kinase II (CaMKII) and extracellular signal-regulated kinase (ERK) have pivotal roles in endothelin-1 (ET-1)-induced cardiomyocyte hypertrophy. We here tested whether a novel CaM antagonist, DY-9760e inhibits ET-1-induced hypertrophy through inhibition of CaMKII and ERK activities. We first confirmed that Ca(2+) oscillation induced by ET-1 treatment elicits transient activation of CaMKII and ERK in cultured cardiomyocytes. DY-9760e treatment with 3 microM totally and partially inhibited the ET-1-induced CaMKII and ERK activation, respectively. The ET-1-induced ERK activation was also partially blocked by a CaMKII inhibitor, KN93. To confirm involvement of CaMKII activity in the ERK activation by ET-1 and A23187, cultured cardiomyocytes were transfected with a constitutively active CaMKII. The transfection with the active CaMKII elicited ERK activation in cultured cardiomyocytes and cotransfection with dominant negative CaMKII eliminated its ERK activation. Consistent with inhibitory actions of DY-9760e on the ET-1-induced CaMKII and ERK activation, induction of hypertrophy-related genes including atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) was significantly inhibited by DY-9760e treatment. Combination treatment with DY-9760e and U0126, a MEK inhibitor, totally blocked the ET-1-induced ANP and BNP expression. DY-9760e treatment (3 microM) significantly inhibited the ET-1-induced hypertrophy and combination treatment with DY-9760e and U0126 totally blocked the ET-1-induced hypertrophy in cultured cardiomyocytes. These results suggest that DY-9760e elicits antihypertrophic action on ET-1-induced cardiac hypertrophy through inhibition of CaMKII and ERK activation and that CaMKII activity in part mediates ET-1-induced ERK activation.

The postsynaptic density protein, IQ-ArfGEF/BRAG1, can interact with IRSp53 through its proline-rich sequence.

IQ-ArfGEF/BRAG1, a guanine nucleotide exchange factor for Arf1 and Arf6, is localized at the postsynaptic density (PSD) and interacts with PSD-95. In this study, we identified a novel interaction of IQ-ArfGEF/BRAG1 with insulin receptor tyrosine kinase substrate of 53 kDa (IRSp53), also known as brain-specific angiogenesis inhibitor 1-associated protein 2. The interaction was mediated by the binding of the C-terminal proline-rich sequence of IQ-ArfGEF/BRAG1 to the SH3 domain of IRSp53. IQ-ArfGEF/BRAG1 and IRSp53 were colocalized at the PSD of excitatory synapses of certain neuronal populations. Our present findings suggest that IQ-ArfGEF/BRAG1 may play roles downstream of NMDA receptors through the interaction with multivalent PSD proteins such as IRSp53 and PSD-95.

Predominant localization of EFA6A, a guanine nucleotide exchange factor for ARF6, at the perisynaptic photoreceptor processes.

EFA6A is a guanine nucleotide exchange factor that is highly expressed in the nervous system with the ability to activate ADP ribosylation factor 6 (ARF6). In this study, we demonstrated the immunohistochemical localization of EFA6A in the adult mouse retina. Strong immunoreactivity for EFA6A was detected predominantly in the outer plexiform layer (OPL), where EFA6A was partially overlapped with dystrophin and synaptophysin. Immunoelectron microscopic analysis revealed that EFA6A was localized predominantly at the perisynaptic processes of photoreceptor terminals without association with synaptic ribbons. These findings suggest that EFA6A-ARF6 pathway may play a specific role at a subcompartment of perisynaptic photoreceptor processes.

Distinct developmental expression of two isoforms of Ca2+/calmodulin-dependent protein kinase kinases and their involvement in hippocampal dendritic formation.

Ca(2+)/calmodulin-dependent protein kinase kinases (CaMKKs) are upstream protein kinases that phosphorylate and activate CaMKI and CaMKIV, both of which are involved in a variety of neuronal functions. Here, we first demonstrated that the two isoforms of CaMKK were differentially expressed during neural development by in situ hybridization. We also demonstrated that both dominant negative and pharmacological interference with CaMKK inhibitor, STO-609 resulted in a significant decrease in the number of primary dendrites of cultured hippocampal neurons. Our present findings provide the detailed anatomical information on the developmental expression of CaMKKs and the functional involvement of CaMKK in the formation of primary dendrites.

Spatiotemporal expression of four isoforms of Ca2+/calmodulin-dependent protein kinase I in brain and its possible roles in hippocampal dendritic growth.

Among multifunctional Ca(2+)/calmodulin-dependent protein kinases (CaMKs), CaMKI has been shown to comprise a family of four structurally related isoforms (alpha, beta, gamma, and delta) encoded by separate genes with abundant expression in mature brain. In this study, we first examined the developmental gene expression of the four isoforms of CaMKI in mouse brain with special attention to the hippocampal formation by in situ hybridization analysis. The four isoforms of CaMKI were found to exhibit distinct spatiotemporal expression during neuronal development. We also examined the functional involvement of CaMKI in the dendritic formation of cultured hippocampal neurons. The overexpression of kinase-dead mutants of CaMKI reduced the average dendritic length of the transfected neurons without any significant effects on the number of primary dendrites and the branching index. Our present findings provide the detailed anatomical information on the developmental expression of the four isoforms of CaMKI in mouse brain, which represents the possible functional involvement of CaMKI in the basal dendritic growth of hippocampal neurons.

Distinct spatiotemporal expression of EFA6D, a guanine nucleotide exchange factor for ARF6, among the EFA6 family in mouse brain.

The EFA6 family is a member of guanine nucleotide exchange factors (GEFs) that can activate ARF6 specifically in vitro. In this study, we determined the complete primary sequence of mouse EFA6D encoding a protein of 1004 amino acids with a calculated molecular weight of 111,207 Da. In ARF pull-down assay, EFA6D showed a preferential GEF activity toward ARF6. RT-PCR analysis revealed the widespread tissue distribution of EFA6D and the high expression of EFA6A, C and D in the brain. In situ hybridization analysis demonstrated a distinct spatiotemporal expression pattern of EFA6D from those of EFA6A and C in mouse brain. Furthermore, immunoblot analysis revealed that EFA6D was highly concentrated in the postsynaptic density fraction. These findings suggest differential spatiotemporal regulation of ARF6 by three members of the EFA6 family in the brain.

Identification of the isoforms of Ca2+/calmodulin-dependent protein kinase II and expression of brain-derived neurotrophic factor mRNAs in the substantia nigra.

Ca2+/calmodulin-dependent protein kinase (CaMK)II is highly expressed in the CNS and mediates activity-dependent neuronal plasticity. Four CaMKII isoforms, alpha, beta, gamma and delta, have a large number of splicing variants. Here we identified isoforms of CaMKII in the rat substantia nigra (SN). Northern blot and RT-PCR analyses revealed that the gamma and delta isoform mRNAs with several splicing variants were predominantly expressed in SN. Immunoblot analysis indicated that the major isoforms were gammaA, gammaC, delta1 and delta3. An immunohistochemical study also confirmed the preferential localization of gamma and delta isoforms in SN dopaminergic neurons. In dopaminergic neurons, immunoreactivity against anti-CaMKIIdelta1-4 antibody was detected in both nucleus and cytoplasm, in contrast to the predominant expression of gamma isoforms in the cytoplasm. Furthermore, we showed expression of brain-derived neurotrophic factor (BDNF) mRNAs with exons II and IV in SN. Taken together with our previous observations, the results suggest that the CaMKIIdelta3 isoform is involved in the expression of BDNF in the SN.

Prominent expression and activity-dependent nuclear translocation of Ca2+/calmodulin-dependent protein kinase Idelta in hippocampal neurons.

Multifunctional Ca2+/calmodulin-dependent protein kinases (CaMKs) including CaMKI, II and IV, are thought to regulate a variety of neuronal functions. Unlike CaMKII, which is regulated by autophosphorylation, CaMKI as well as CaMKIV are activated by CaMKK. In this study, we examined the cellular and subcellular localization of CaMKIdelta, a recently identified fourth isoform of CaMKI, in the mature brain. In situ hybridization analysis demonstrated wide expression of CaMKIdelta mRNA in the adult mouse brain with prominent expression in the hippocampal pyramidal cells. FLAG-tagged CaMKIdelta was localized at the cytoplasm and neurites without nuclear immunoreactivity in approximately 80% of the transfected primary hippocampal neurons. The stimulation with either KCl depolarization or glutamate triggered the nuclear localization of FLAG-tagged CaMKIdelta by two-fold with a peak at 1 min. In contrast, the catalytically inactive mutants of CaMKIdelta remained cytoplasmic without nuclear translocation during KCl depolarization, indicating the requirement of its activation for the nuclear translocation. Furthermore, we showed that immunoprecipitated CaMKIdelta could phosphorylate cAMP response element binding protein (CREB)alphain vitro and that the over-expression of CaMKIdelta enhanced GAL4-CREB-luciferase activity in PC12 cells stimulated by KCl depolarization. Our present study provides the first evidence for the possible involvement of CaMKIdelta in nuclear functions through its nuclear translocation in response to stimuli that trigger intracellular Ca2+ influx.

Functional assay of EFA6A, a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (ARF6), in dendritic formation of hippocampal neurons.

EFA6A is a guanine nucleotide exchange factor (GEF) that can activate ADP-ribosylation factor 6 (ARF6) in vitro, with prominent expression in the forebrain including the hippocampal formation. In this section, we describe the neuronal transfection method and show that the overexpression of a catalytically inactive mutant of EFA6A induces a prominent dendritic formation of the primary hippocampal neurons, suggesting the intimate involvement of EFA6A in the regulation of neuronal dendritic development. This reliable and consistent neuronal transfection method will also be applicable for the vector-based RNA interference method.