Sunghoe Chang

LRRK2 regulates synaptic vesicle endocytosis

The leucine-rich repeat kinase 2 (LRRK2) has been identified as the defective gene at the PARK8 locus causing the autosomal dominant form of Parkinsons disease (PD). Although several LRRK2 mutations were found in familial as well as sporadic PD patients, its physiological functions are not clearly defined. In this study, using yeast two-hybrid screening, we report the identification of Rab5b as an LRRK2-interacting protein. Indeed, our GST pull down and co-immunoprecipitation assays showed that it specifically interacts with LRRK2. In addition, subcellular fractionation and immunocytochemical analyses confirmed that a fraction of both proteins co-localize in synaptic vesicles. Interestingly, we found that alteration of LRRK2 expression by either overexpression or knockdown of endogenous LRRK2 in primary neuronal cells significantly impairs synaptic vesicle endocytosis. Furthermore, this endocytosis defect was rescued by co-expression of functional Rab5b protein, but not by its inactive form. Taken together, we propose that LRRK2, in conjunction with its interaction with Rab5b, plays an important role in synaptic function by modulating the endocytosis of synaptic vesicles.

Delayed reentry of recycling vesicles into the fusion-competent synaptic vesicle pool in synaptojanin 1 knockout mice

Synaptojanin 1 is a polyphosphoinositide phosphatase implicated in synaptic vesicle recycling. We used FM1-43 imaging and electron microscopy in cultured cortical neurons from control and synaptojanin 1 knockout mice to study how the absence of this protein affects specific steps of the synaptic vesicle cycle. Exo/endocytosis after a moderate stimulus was unchanged. However, during prolonged stimulation, the regeneration of fusion-competent synaptic vesicles was severely impaired. In stimulated nerve terminals, there was a persistent accumulation of clathrin-coated vesicles and a backup of newly reformed vesicles in the cytomatrix-rich area around the synaptic vesicle cluster. These findings demonstrate that synaptojanin 1 function is needed for the progression of recycling vesicles to the functional synaptic vesicle pool.

SNX9 regulates tubular invagination of the plasma membrane through interaction with actin cytoskeleton and dynamin 2.

Dynamic membrane remodeling during intracellular trafficking is controlled by the intricate interplay between lipids and proteins. BAR domains are modules that participate in endocytic processes by binding and deforming the lipid bilayer. Sorting nexin 9 (SNX9), which functions in clathrin-mediated endocytosis, contains a BAR domain, however, the properties of this domain are not well understood. Here we show that SNX9 shares many properties with other BAR domain-containing proteins, such as amphiphysin and endophilin. SNX9 is able to deform the plasma membrane, as well as liposomes, into narrow tubules and recruit N-WASP and dynamin 2 to these tubules via its SH3 domain. SNX9-induced tubulation is antagonized by N-WASP and dynamin 2 while it is enhanced by perturbation of actin dynamics. However, SNX9 also has several unique properties. The tubulating activity requires the BAR and PX domains, as well as the low-complexity (LC) domain, which binds the Arp2/3 complex. SNX9 also binds to PtdIns(4)P-5-kinases via its PX domain and its tubulating activity is regulated by phosphoinositides. In addition, the kinase activity of PtdIns(4)P-5-kinases is stimulated by interaction with SNX9, suggesting a positive feedback interaction between SNX9 and PtdIns(4)P-5-kinases. These results suggest that SNX9 functions in the coordination of membrane remodeling and fission via interactions with actin-regulating proteins, endocytic proteins and PtdIns(4,5)P2-metabolizing enzymes.

Sorting Nexin 9 Interacts with Dynamin 1 and N-WASP and Coordinates Synaptic Vesicle Endocytosis

Sorting nexin 9 (SNX9) is a member of the sorting nexin family of proteins, each of which contains a characteristic Phox homology domain. SNX9 is widely expressed and plays a role in clathrin-mediated endocytosis, but it is not known if it is present in neuronal cells. We report that SNX9 is expressed in the presynaptic compartment of cultured hippocampal neurons, where it binds to dynamin-1 and N-WASP. Overexpression of full-length SNX9 or a C-terminal truncated version caused severe defects in synaptic vesicle endocytosis during, as well as after, stimulation. Knockdown of SNX9 with short interfering RNA also reduced synaptic vesicle endocytosis, and the W39A mutation of SNX9 abolished the inhibitory effect of SNX9 on endocytosis. Rescue experiments showed that most of the effect of SNX9 on endocytosis results from its interaction with dynamin 1, although its interaction with N-WASP contributes in some degree. We further showed that SNX9 dimerizes through its C-terminal domain, suggesting that it may interact simultaneously with dynamin 1 and N-WASP. We propose that SNX9 interacts with dynamin-1 and N-WASP in presynaptic terminals, where it links actin dynamics and synaptic vesicle endocytosis.

A novel pathway for presynaptic mitogen-activated kinase activation via AMPA receptors

AMPA-type glutamate receptors play a key role in mediating postsynaptic responses of excitatory neurotransmitters. It is now well accepted that AMPA receptors are also present at the presynapse, where they are thought to modulate neurotransmitter release. However, the mechanisms through which they control synaptic vesicle traffic have remained elusive. We used cultured hippocampal neurons and growth cone particles prepared from fetal rat brain to investigate the functional role of presynaptic AMPA receptors. We show here that stimulation of presynaptic AMPA receptors induces activation of mitogen-activated protein kinase (MAPK) through a nonreceptor tyrosine kinase-dependent and Na+/Ca2+-independent mechanism. This pathway is activated predominantly in axonal growth cones compared with the somatodendritic compartment. After stimulation of presynaptic AMPA receptors, synapsin I is phosphorylated at MAPK-specific sites. These events are paralleled by a prominent increase in evoked synaptic vesicle recycling that is blocked by the specific MAPK inhibitor 2-(2-amino-3-methoxyphenyl)-4H-1-benzopyran-4-one. Similarly, in synaptosomes isolated from adult brain, AMPA stimulation induces MAPK activation and phosphorylation of synapsin I at MAPK-dependent sites and enhances significantly synaptic vesicle recycling. These results reveal a novel pathway for activation of presynaptic MAPK and suggest a role of this pathway in the regulation of short-term presynaptic plasticity.

Dazl can bind to dynein motor complex and may play a role in transport of specific mRNAs.

Male germ cell development includes mitotic and meiotic cell divisions that are followed by dramatic morphological changes resulting in the production of spermatozoa. Genetic evidence has indicated that the DAZ family genes are critical for successful male germ cell development in diverse animals as well as humans. In the present study, we investigated the cellular functions of Dazl in the mouse male germ cells. We identified a specific interaction of Dazl with the dynein light chain, a component of the dynein–dynactin motor complex. The subcellular distribution of Dazl was microtubule‐dependent and a selected number of Dazl‐bound mRNAs could accumulate in the perinuclear area. Based on these results, we propose that Dazl may play a role in transport of specific mRNAs via dynein motor complex. The Dazl‐bound mRNAs may be stored at specific sites and would be available for future developmental processes. Our study revealed the presence of an active mRNA transport system in mouse male germ cells.

δ-Catenin Induced Dendritic Morphogenesis: An Essential Role of p190RhoGEF Interaction through Akt1 Mediated Phosphorylation

δ-Catenin was first identified through its interaction with Presenilin-1 and has been implicated in the regulation of dendrogenesis and cognitive function. However, the molecular mechanisms by which δ-catenin promotes dendritic morphogenesis were unclear. In this study, we demonstrated δ-catenin interaction with p190RhoGEF, and the importance of Akt1-mediated phosphorylation at Thr-454 residue of δ-catenin in this interaction. We have also found that δ-catenin overexpression decreased the binding between p190RhoGEF and RhoA, and significantly lowered the levels of GTP-RhoA but not those of GTP-Rac1 and -Cdc42. δ-Catenin T454A, a defective form in p190RhoGEF binding, did not decrease the binding between p190RhoGEF and RhoA. δ-Catenin T454A also did not lower GTP-RhoA levels and failed to induce dendrite-like process formation in NIH 3T3 fibroblasts. Furthermore, δ-catenin T454A significantly reduced the length and number of mature mushroom shaped spines in primary hippocampal neurons. These results highlight signaling events in the regulation of δ-catenin-induced dendrogenesis and spine morphogenesis.

Regulation of transferrin recycling kinetics by PtdIns[4,5]P2 availability

Phosphatidylinositol 4,5‐bisphosphate (PtdIns[4,5]P2) is a phosphoinositide involved in a variety of cellular functions, including signal transduction, organelle trafficking, and actin dynamics. Although the role of PtdIns[4,5]P2 in endocytosis is well established, the precise trafficking steps relying on normal PtdIns[4,5]P2 balance in the endosomal pathway have not yet been elucidated. Here we show that decrease in intracellular PtdIns[4,5]P2 levels achieved by the overexpression of the 5‐phosphatase domain of synaptojanin 1 or by siRNA knock‐down of PIP5Ks expression lead to severe defects in the internalization of transferrin as well as in the recycling of internalized transferrin back to the cell surface in COS‐7 cells. These defects suggest that PtdIns[4,5]P2 participates in multiple trafficking and/or sorting events during endocytosis. Coexpression of the PtdIns[4,5]P2 synthesizing enzyme, PIP5KI, was able to rescue these endocytic defects. Furthermore, decreased levels of PtdIns[4,5]P2 caused delays in rapid and slow membrane recycling pathways as well as a severe backup of endocytosed membrane. Taken together, our results demonstrate that PtdIns[4,5]P2 availability regulates multiple steps in the endocytic cycle in non‐neuronal cells.—Kim, S., Kim, H., Chang, B., Ahn, N., Hwang, S., Di Paolo, G., Chang, S. Regulation of transferrin recycling kinetics by PtdIns[4,5]P2 availability. FASEB J. 20, E1753–E1762 (2006)

The adhesion protein IgSF9b is coupled to neuroligin 2 via S-SCAM to promote inhibitory synapse development

IgSF9b forms a novel subsynaptic domain for adhesion that links to the gephyrin- and GABAA receptor–containing domain to promote inhibitory synaptic development.

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.