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.

ADP Ribosylation Factor 4 (Arf4) Regulates Radial Migration through N-Cadherin Trafficking during Cerebral Cortical Development.

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.

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.

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.

GluA2 overexpression in oligodendrocyte progenitors promotes postinjury oligodendrocyte regeneration.

Oligodendrocyte precursor cells (OPCs) are essential for developmental myelination and oligodendrocyte regeneration after CNS injury. These progenitors express calcium-permeable AMPA receptors (AMPARs) and form direct synapses with neurons throughout the CNS, but the roles of this signaling are unclear. To enable selective alteration of the properties of AMPARs in oligodendroglia, we generate mice that allow cell-specific overexpression of EGFP-GluA2 in vivo. In healthy conditions, OPC-specific GluA2 overexpression significantly increase their proliferation in an age-dependent manner but did not alter their rate of differentiation into oligodendrocytes. In contrast, after demyelinating brain injury in neonates or adults, higher GluA2 levels promote both OPC proliferation and oligodendrocyte regeneration, but do not prevent injury-induced initial cell loss. These findings indicate that AMPAR GluA2 content regulates the proliferative and regenerative behavior of adult OPCs, serving as a putative target for better myelin repair.

Telencephalon-specific Alkbh1 conditional knockout mice display hippocampal atrophy and impaired learning.

AlkB homolog 1 (ALKBH1) is responsible for the biogenesis of 5-formylcytidine (f5 C) on mitochondrial tRNAMet and essential for mitochondrial protein synthesis. The brain, especially the hippocampus, is highly susceptible to mitochondrial dysfunction; hence, the maintenance of mitochondrial activity is strongly required to prevent disorders associated with hippocampal malfunction. To study the role of ALKBH1 in the hippocampus, we generated dorsal telencephalon-specific Alkbh1 conditional knockout (cKO) mice in inbred C57BL/6 background. These mice showed reduced activity of the respiratory chain complex, hippocampal atrophy, and CA1 pyramidal neuron abnormalities. Furthermore, performances in the fear-conditioning and Morris water maze tests in cKO mice indicated that the hippocampal abnormalities led to impaired hippocampus-dependent learning. These findings indicate critical roles of ALKBH1 in the hippocampus.

The Vps52 subunit of the GARP and EARP complexes is a novel Arf6-interacting protein that negatively regulates neurite outgrowth of hippocampal neurons.

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.

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.

Mice lacking EFA6C/Psd2, a guanine nucleotide exchange factor for Arf6, exhibit lower Purkinje cell synaptic density but normal cerebellar motor functions.

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.

Hyperactivation of mTORC1 disrupts cellular homeostasis in cerebellar Purkinje cells.

Mammalian target of rapamycin (mTOR) is a central regulator of cellular metabolism. The importance of mTORC1 signaling in neuronal development and functions has been highlighted by its strong relationship with many neurological and neuropsychiatric diseases. Previous studies demonstrated that hyperactivation of mTORC1 in forebrain recapitulates tuberous sclerosis and neurodegeneration. In the mouse cerebellum, Purkinje cell-specific knockout of Tsc1/2 has been implicated in autistic-like behaviors. However, since TSC1/2 activity does not always correlate with clinical manifestations as evident in some cases of tuberous sclerosis, the intriguing possibility is raised that phenotypes observed in Tsc1/2 knockout mice cannot be attributable solely to mTORC1 hyperactivation. Here we generated transgenic mice in which mTORC1 signaling is directly hyperactivated in Purkinje cells. The transgenic mice exhibited impaired synapse elimination of climbing fibers and motor discoordination without affecting social behaviors. Furthermore, mTORC1 hyperactivation induced prominent apoptosis of Purkinje cells, accompanied with dysregulated cellular homeostasis including cell enlargement, increased mitochondrial respiratory activity, and activation of pseudohypoxic response. These findings suggest the different contributions between hyperactivated mTORC1 and Tsc1/2 knockout in social behaviors, and reveal the perturbations of cellular homeostasis by hyperactivated mTORC1 as possible underlying mechanisms of neuronal dysfunctions and death in tuberous sclerosis and neurodegenerative diseases.

New Features on the Expression and Trafficking of mGluR1 Splice Variants Exposed by Two Novel Mutant Mouse Lines.

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 mGluR1ß 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 mGluR1ß 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.

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.

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.

PTEN negatively regulates the cell lineage progression from NG2+ glial progenitor to oligodendrocyte via mTOR-independent signaling.

Oligodendrocytes (OLs), the myelin-forming CNS glia, are highly vulnerable to cellular stresses, and a severe myelin loss underlies numerous CNS disorders. Expedited OL regeneration may prevent further axonal damage and facilitate functional CNS repair. Although adult OL progenitors (OPCs) are the primary players for OL regeneration, targetable OPC-specific intracellular signaling mechanisms for facilitated OL regeneration remain elusive. Here, we report that OPC-targeted PTEN inactivation in the mouse, in contrast to OL-specific manipulations, markedly promotes OL differentiation and regeneration in the mature CNS. Unexpectedly, an additional deletion of mTOR did not reverse the enhanced OL development from PTEN-deficient OPCs. Instead, ablation of GSK3ß, another downstream signaling molecule that is negatively regulated by PTEN-Akt, enhanced OL development. Our results suggest that PTEN persistently suppresses OL development in an mTOR-independent manner, and at least in part, via controlling GSK3ß activity. OPC-targeted PTEN-GSK3ß inactivation may benefit facilitated OL regeneration and myelin repair.

Reduced expression of Na+/Ca2+ exchangers is associated with cognitive deficits seen in Alzheimer's disease model mice.

Na+/Ca2+ exchangers (NCXs) are expressed primarily in the plasma membrane of most cell types, where they mediate electrogenic exchange of one Ca2+ for three Na+ ions, depending on Ca2+ and Na+ electrochemical gradients across the membrane. Three mammalian NCX isoforms (NCX1, NCX2, and NCX3) are each encoded by a distinct gene. Here, we report that NCX2 and NCX3 protein and mRNA levels are relatively reduced in hippocampal CA1 of APP23 and APP-KI mice. Likewise, NCX2+/- or NCX3+/- mice exhibited impaired hippocampal LTP and memory-related behaviors. Moreover, relative to controls, calcium/calmodulin-dependent protein kinase II (CaMKII) autophosphorylation significantly decreased in NCX2+/- mouse hippocampus but increased in hippocampus of NCX3+/- mice. NCX2 or NCX3 heterozygotes displayed impaired maintenance of hippocampal LTP, a phenotype that in NCX2+/- mice was correlated with elevated calcineurin activity and rescued by treatment with the calcineurin (CaN) inhibitor FK506. Likewise, FK506 treatment significantly restored impaired hippocampal LTP in APP-KI mice. Moreover, Ca2+ clearance after depolarization following high frequency stimulation was slightly delayed in hippocampal CA1 regions of NCX2+/- mice. Electron microscopy revealed relatively decreased synaptic density in CA1 of NCX2+/- mice, while the number of spines with perforated synapses in CA1 significantly increased in NCX3+/- mice. We conclude that memory impairment seen in NCX2+/- and NCX3+/- mice reflect dysregulated hippocampal CaMKII activity, which alters dendritic spine morphology, findings with implications for memory deficits seen in Alzheimer's disease model mice.

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.

SNAP-25 phosphorylation at Ser187 regulates synaptic facilitation and short-term plasticity in an age-dependent manner.

Neurotransmitter release is mediated by the SNARE complex, but the role of its phosphorylation has scarcely been elucidated. Although PKC activators are known to facilitate synaptic transmission, there has been a heated debate on whether PKC mediates facilitation of neurotransmitter release through phosphorylation. One of the SNARE proteins, SNAP-25, is phosphorylated at the residue serine-187 by PKC, but its physiological significance has been unclear. To examine these issues, we analyzed mutant mice lacking the phosphorylation of SNAP-25 serine-187 and found that they exhibited reduced release probability and enhanced presynaptic short-term plasticity, suggesting that not only the release process, but also the dynamics of synaptic vesicles was regulated by the phosphorylation. Furthermore, it has been known that the release probability changes with development, but the precise mechanism has been unclear, and we found that developmental changes in release probability of neurotransmitters were regulated by the phosphorylation. These results indicate that SNAP-25 phosphorylation developmentally facilitates neurotransmitter release but strongly inhibits presynaptic short-term plasticity via modification of the dynamics of synaptic vesicles in presynaptic terminals.

BRAG2a, a Guanine Nucleotide Exchange Factor for Arf6, Is a Component of the Dystrophin-Associated Glycoprotein Complex at the Photoreceptor Terminal.

Purpose: Mutations in genes encoding the dystrophin-associated glycoprotein complex (DGC) can cause muscular dystrophy and disturb synaptic transmission in the photoreceptor ribbon synapse. However, the molecular composition and specific functions of the photoreceptor DGC remain unknown. Brefeldin A-resistant Arf-GEF 2 (BRAG2), also known as IQSEC1, is a guanine nucleotide exchange factor for ADP-ribosylation factor 6 (Arf6), a critical GTPase that regulates endosomal trafficking and actin cytoskeleton remodeling. In the present study, we characterized the expression of BRAG2a, an alternative splicing isoform of BRAG2, in the adult mouse photoreceptor. Methods: Immunofluorescence and immunoelectron microscopic analyses of adult mouse retinas were performed using a novel anti-BRAG2a antibody. Pull-down, immunoprecipitation, and in situ proximity ligation assays were performed to examine the interaction between BRAG2a and the DGC in vivo. Results: Immunofluorescence demonstrated punctate colocalization of BRAG2a with ß-dystroglycan in the outer plexiform layer. Immunoelectron microscopy revealed the localization of BRAG2a at the plasma membrane of lateral walls and processes of photoreceptor terminals within the synaptic cavity. Pull-down and immunoprecipitation assays using retinal lysates demonstrated the protein complex formation between BRAG2a with the DGC. In situ proximity ligation assays further detected a close spatial relationship between BRAG2a and ß-dystroglycan in the outer plexiform layer. Conclusions: The present study provided evidence that BRAG2a is a novel component of the photoreceptor DGC, suggesting functional involvement of the BRAG2a-Arf6 pathway downstream of the DGC.

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.