Yoonju Kim

SNX18 shares a redundant role with SNX9 and modulates endocytic trafficking at the plasma membrane

SNX18 and SNX9 are members of a subfamily of SNX (sorting nexin) proteins with the same domain structure. Although a recent report showed that SNX18 and SNX9 localize differently in cells and appear to function in different trafficking pathways, concrete evidence regarding whether they act together or separately in intracellular trafficking is still lacking. Here, we show that SNX18 has a similar role to SNX9 in endocytic trafficking at the plasma membrane, rather than having a distinct role. SNX18 and SNX9 are expressed together in most cell lines, but to a different extent. Like SNX9, SNX18 interacts with dynamin and stimulates the basal GTPase activity of dynamin. It also interacts with neuronal Wiskott-Aldrich syndrome protein (N-WASP) and synaptojanin, as does SNX9. SNX18 and SNX9 can form a heterodimer and colocalize in tubular membrane structures. Depletion of SNX18 by small hairpin RNA inhibited transferrin uptake. SNX18 successfully compensates for SNX9 deficiency during clathrin-mediated endocytosis and vice versa. Total internal reflection fluorescence microscopy in living cells shows that a transient burst of SNX18 recruitment to clathrin-coated pits coincides spatiotemporally with a burst of dynamin and SNX9. Taken together, our results suggest that SNX18 functions with SNX9 in multiple pathways of endocytosis at the plasma membrane and that they are functionally redundant.

Interaction of SPIN90 with Dynamin I and Its Participation in Synaptic Vesicle Endocytosis

SH3 protein interacting with Nck, 90 kDa (SPIN90) is an Nck-binding protein that contains one Src homology 3 (SH3) domain, three proline-rich domains (PRDs), a serine/threonine-rich region, and a hydrophobic C-terminal region. Previously, we have shown that SPIN90 plays roles in the sarcomere assembly in cardiac muscles and in the formation of focal contacts in HeLa cells. Besides in heart, SPIN90 is also highly expressed in the brain, but its role in the neuronal system is completely unknown. Here, we found that SPIN90 is expressed in the presynaptic compartment in which it binds dynamin I, a key component of the endocytic machinery, and that it participates in synaptic vesicle endocytosis. Pull-down analysis and coimmunoprecipitation proved the associations of SPIN90 with dynamin I through SH3-PRD interaction. Overexpression of SPIN90 or knocking down SPIN90 by small interfering RNA impaired synaptic vesicle endocytosis. We further confirmed by the rescue experiments that the endocytic defects by SPIN90 expression come from its interaction with dynamin I. Exocytosis kinetics was not affected by SPIN90 expression. Together, our findings suggest that SPIN90 could modulate the interactions of dynamin I with other endocytic proteins that cooperate in the coated vesicle formation, thus regulating synaptic vesicle endocytosis.

Ever-expanding network of dynamin-interacting proteins

Clathrin-mediated endocytosis is a major cellular pathway for internalization of proteins and lipids and for recycling of synaptic vesicles. The GTPase dynamin plays a key role in this process, and the proline-rich domain of dynamin participates in various protein-protein interactions to ensure a proper coordination of endocytic processes. Although dynamin is not directly associated with actin, several dynamin-binding proteins can interact with actin or with proteins that regulate actin assembly, thereby coordinately regulating actin assembly and trafficking events. This article summarizes dynamin interactions with various Src homology 3-containing proteins, many of which are actin-binding proteins. It also discusses the recently identified two new dynamin binding proteins, SH3 protein interacting with Nck, 90 kDa/Wiskott-Aldrich syndrome protein interacting with SH3 protein (SPIN90/WISH) and sorting nexin 9, and outlines their potential role as a link between endocytosis and actin dynamics.

Overexpression of Dyrk1A Causes the Defects in Synaptic Vesicle Endocytosis

Trisomy 21-linked Dyrk1A (dual-specificity tyrosine phosphorylation-regulated kinase 1A) overexpression is implicated in pathogenic mechanisms underlying mental retardation in Down syndrome (DS). It is known to phosphorylate multiple substrates including endocytic proteins in vitro, but the functional consequence of Dyrk1A-mediated phosphorylation on endocytosis has never been investigated. Here, we show that overexpression of Dyrk1A causes defects in clathrin-mediated endocytosis and specifically, in the recruitment of endocytic proteins to clathrin-coated pits in fibroblasts. Synaptic vesicle endocytosis also significantly slowed down as a result of Dyrk1A overexpression in cultured hippocampal neurons. These effects are dependent on Dyrk1A kinase activity. The inhibitory effect of Dyrk1A on synaptic vesicle endocytosis was confirmed in neuronal cultures derived from transgenic mice overexpressing Dyrk1A at levels found in DS. Pharmacological blockade of Dyrk1A with epigallocatechin gallate rescued the endocytic phenotypes found in transgenic neurons. Together, our results suggest that aberrant Dyrk1A-mediated phosphorylation of the endocytic machinery perturbs synaptic vesicle endocytosis, which may contribute to synaptic dysfunctions and cognitive deficits associated with DS.

ADP-ribosylation factor 6 (ARF6) bidirectionally regulates dendritic spine formation depending on neuronal maturation and activity.

Background: Conflicting results regarding the role of ARF6 in dendritic spine development have not been answered. Results: ARF6-mediated Rac1 or RhoA activation via PLD pathway either positively or negatively regulates spine formation. Conclusion: The key factor underlying conversion of the ARF6 effect during development is neuronal activity. Significance: Activity dependence of ARF6-mediated spine formation may play a role in structural plasticity of mature neurons. Recent studies have reported conflicting results regarding the role of ARF6 in dendritic spine development, but no clear answer for the controversy has been suggested. We found that ADP-ribosylation factor 6 (ARF6) either positively or negatively regulates dendritic spine formation depending on neuronal maturation and activity. ARF6 activation increased the spine formation in developing neurons, whereas it decreased spine density in mature neurons. Genome-wide microarray analysis revealed that ARF6 activation in each stage leads to opposite patterns of expression of a subset of genes that are involved in neuronal morphology. ARF6-mediated Rac1 activation via the phospholipase D pathway is the coincident factor in both stages, but the antagonistic RhoA pathway becomes involved in the mature stage. Furthermore, blocking neuronal activity in developing neurons using tetrodotoxin or enhancing the activity in mature neurons using picrotoxin or chemical long term potentiation reversed the effect of ARF6 on each stage. Thus, activity-dependent dynamic changes in ARF6-mediated spine structures may play a role in structural plasticity of mature neurons.

SCAMP5 Plays a Critical Role in Synaptic Vesicle Endocytosis during High Neuronal Activity

Secretory carrier membrane protein 5 (SCAMP5), a recently identified candidate gene for autism, is brain specific and highly abundant in synaptic vesicles (SVs), but its function is currently unknown. Here, we found that knockdown (KD) of endogenous SCAMP5 by SCAMP5-specific shRNAs in cultured rat hippocampal neurons resulted in a reduction in total vesicle pool size as well as in recycling pool size, but the recycling/resting pool ratio was significantly increased. SCAMP5 KD slowed endocytosis after stimulation, but impaired it severely during strong stimulation. We also found that KD dramatically lowered the threshold of activity at which SV endocytosis became unable to compensate for the ongoing exocytosis occurring during a stimulus. Reintroducing shRNA-resistant SCAMP5 reversed these endocytic defects. Therefore, our results suggest that SCAMP5 functions during high neuronal activity when a heavy load is imposed on endocytosis. Our data also raise the possibility that the reduction in expression of SCAMP5 in autistic patients may be related to the synaptic dysfunction observed in autism.

SNX26, a GTPase-activating Protein for Cdc42, Interacts with PSD-95 Protein and Is Involved in Activity-dependent Dendritic Spine Formation in Mature Neurons

Background: The role of SNX26, a brain-enriched RhoGAP, in mature neurons remains unknown. Results: SNX26 interacts with PSD-95 and regulates formation of dendritic spines. Conclusion: SNX26 has a role in the activity-dependent structural change of dendritic spines in mature neurons. Significance: Dynamic regulation of SNX26 plays a pivotal role in dendritic arborization of mature neurons. SNX26, a brain-enriched RhoGAP, plays a key role in dendritic arborization during early neuronal development in the neocortex. In mature neurons, it is localized to dendritic spines, but little is known about its role in later stages of development. Our results show that SNX26 interacts with PSD-95 in dendritic spines of cultured hippocampal neurons, and as a GTPase-activating protein for Cdc42, it decreased the F-actin content in COS-7 cells and in dendritic spines of neurons. Overexpression of SNX26 resulted in a GTPase-activating protein activity-dependent decrease in total protrusions and spine density together with dramatic inhibition of filopodia-to-spine transformations. Such effects of SNX26 were largely rescued by a constitutively active mutant of Cdc42. Consistently, an shRNA-mediated knockdown of SNX26 significantly increased total protrusions and spine density, resulting in an increase in thin or stubby type spines at the expense of the mushroom spine type. Moreover, endogenous expression of SNX26 was shown to be bi-directionally modulated by neuronal activity. Therefore, we propose that in addition to its key role in neuronal development, SNX26 also has a role in the activity-dependent structural change of dendritic spines in mature neurons.

SNX14 is a bifunctional negative regulator for neuronal 5-HT6 receptor signaling.

The 5‐hydroxytryptamine (5‐HT, also known as serotonin) subtype 6 receptor (5‐HT6R, also known as HTR6) plays roles in cognition, anxiety and learning and memory disorders, yet new details concerning its regulation remain poorly understood. In this study, we found that 5‐HT6R directly interacted with SNX14 and that this interaction dramatically increased internalization and degradation of 5‐HT6R. Knockdown of endogenous SNX14 had the opposite effect. SNX14 is highly expressed in the brain and contains a putative regulator of G‐protein signaling (RGS) domain. Although its RGS domain was found to be non‐functional as a GTPase activator for G&agr;s, we found that it specifically bound to and sequestered G&agr;s, thus inhibiting downstream cAMP production. We further found that protein kinase A (PKA)‐mediated phosphorylation of SNX14 inhibited its binding to G&agr;s and diverted SNX14 from G&agr;s binding to 5‐HT6R binding, thus facilitating the endocytic degradation of the receptor. Therefore, our results suggest that SNX14 is a dual endogenous negative regulator in 5‐HT6R‐mediated signaling pathway, modulating both signaling and trafficking of 5‐HT6R.

nArgBP2 regulates excitatory synapse formation by controlling dendritic spine morphology

Significance Recent studies have implicated postsynaptic scaffolding protein synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3) or Shank3 in mood disorders such as bipolar disorder, autism, and obsessive-compulsive disorders. Neural Abelson-related gene-binding protein 2 (nArgBP2) is a binding partner of both SAPAP3 and Shank3, and its genetic deletion in mice leads to manic/bipolar-like behavior resembling symptoms of bipolar disorder. Remarkably, nothing is known about the synaptic function of nArgBP2 or its connection with the synaptic alterations associated with bipolar disorder. Here, we provide compelling evidence that nArgBP2 regulates spine morphogenesis and that its ablation causes a robust and selective inhibition of excitatory synapse formation by controlling actin dynamics. Our results revealed the underlying mechanism for the synaptic dysfunction caused by nArgBP2 down-regulation that may be associated with human bipolar disorder. Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that directly interacts with synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3), a postsynaptic scaffolding protein critical for the assembly of glutamatergic synapses. Although genetic deletion of nArgBP2 in mice leads to manic/bipolar-like behaviors resembling many aspects of symptoms in patients with bipolar disorder, the actual function of nArgBP2 at the synapse is completely unknown. Here, we found that the knockdown (KD) of nArgBP2 by specific small hairpin RNAs (shRNAs) resulted in a dramatic change in dendritic spine morphology. Reintroducing shRNA-resistant nArgBP2 reversed these defects. In particular, nArgBP2 KD impaired spine-synapse formation such that excitatory synapses terminated mostly at dendritic shafts instead of spine heads in spiny neurons, although inhibitory synapse formation was not affected. nArgBP2 KD further caused a marked increase of actin cytoskeleton dynamics in spines, which was associated with increased Wiskott–Aldrich syndrome protein-family verprolin homologous protein 1 (WAVE1)/p21-activated kinase (PAK) phosphorylation and reduced activity of cofilin. These effects of nArgBP2 KD in spines were rescued by inhibiting PAK or activating cofilin combined with sequestration of WAVE. Together, our results suggest that nArgBP2 functions to regulate spine morphogenesis and subsequent spine-synapse formation at glutamatergic synapses. They also raise the possibility that the aberrant regulation of synaptic actin filaments caused by reduced nArgBP2 expression may contribute to the manifestation of the synaptic dysfunction observed in manic/bipolar disorder.

Neuronal specific βPix-b stimulates actin-dependent processes via the interaction between its PRD and WH1 domain of N-WASP

βPix, a Pak‐interacting nucleotide exchange factor (Cool‐1/p85SPR), is a Cdc42/Rac1‐specific guanine nucleotide exchange factor (GEF) involved in various actin‐related processes. Many previous studies have focused on ubiquitously expressed βPix‐a, while the role of the neuronal‐specific isoform βPix‐b is still unknown, especially whether its role is distinct from or similar to βPix‐a. Here we show that unlike βPix‐a, overexpression of βPix‐b stimulates actin‐dependent comet formation in BHK21 cells. This effect is attributed to the interaction between its proline‐rich domain (PRD) and the WH1 domain of N‐WASP. In addition, we show that overexpression of βPix‐b stimulates actin‐dependent dendritic spine formation in rat hippocampal neurons in culture, a formation that is blocked by co‐expression of the WH1 domain of N‐WASP or the PRD of βPix‐b. Knocking‐down endogenous expression of βPix‐b by shRNA reduced the number of dendritic spines, which were rescued only by PRD‐containing βPix‐b mutants. GEF activity of βPix‐b is also required for these effects. The results show that neuronal‐specific βPix‐b stimulates actin‐dependent processes in cells via the interaction between its PRD and the WH1 domain of N‐WASP. Our results identify N‐WASP as the first protein shown to interact with the PRD of βPix‐b, raising the possibility that, as an N‐WASP WH1‐binding protein, βPix‐b may regulate N‐WASPs activity in cells. J. Cell. Physiol. 227: 1476–1484, 2012.