Neural Abelson-related gene-binding protein 2 (nArgBP2) was originally identified as a protein that directly interacts with synapseassociated 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 spinesynapse 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. nArgBP2 | dendritic spines | excitatory synapse | actin | bipolar disorder T he postsynaptic enriched adaptor protein neural Abelsonrelated gene-binding protein 2 (nArgBP2), a neural-specific splice variant of the ubiquitous ArgBP2, was originally identified as a binding partner of synapse-associated protein 90/postsynaptic density protein 95-associated protein 3 (SAPAP3) (1). It belongs to a family of adaptor proteins that are involved in the regulation of cell adhesion, actin cytoskeleton organization, and growth factor receptor signaling (2). This protein family is characterized by a sorbin homology (SoHo) domain in the NH 2 -terminal region and three Src homology 3 (SH3) domains in the COOH-terminal region (3). Although the SoHo domain remains poorly characterized, the SH3 domains bind signaling protein kinases, the ubiquitin ligase, and protein involved in the regulation of focal adhesions and adhering junctions (1, 4-6). The NH 2 -terminal region of nArgBP2, which contains the SoHo domain, interacts with spectrin, whereas the COOH-terminal SH3 domains bind dynamin, synaptojanin, Wiskott-Aldrich syndrome protein-family verprolin homologous protein (WAVE) isoforms, and WAVE regulatory proteins (3), all of which participate in the regulation of the actin cytoskeleton. We also found that the down-re...

Despite being a highly enriched synaptic vesicle (SV) protein and a candidate gene for autism, the physiological function of SCAMP5 remains mostly enigmatic. Here, using optical imaging and electrophysiological experiments, we demonstrate that SCAMP5 plays a critical role in release site clearance at the active zone. Truncation analysis revealed that the 2/3 loop domain of SCAMP5 directly interacts with adaptor protein 2, and this interaction is critical for its role in release site clearance. Knockdown (KD) of SCAMP5 exhibited pronounced synaptic depression accompanied by a slower recovery of the SV pool. Moreover, it induced a strong frequency-dependent short-term depression of synaptic release, even under the condition of sufficient release-ready SVs. Super-resolution microscopy further proved the defects in SV protein clearance induced by KD. Thus, reduced expression of SCAMP5 may impair the efficiency of SV clearance at the active zone, and this might relate to the synaptic dysfunction observed in autism.

Proper sorting of exocytosed synaptic vesicle (SV) proteins into individual SVs during endocytosis is of the utmost importance for the fidelity of subsequent neurotransmission. Recent studies suggest that each SV protein is sorted into individual SVs by its own dedicated adaptors as well as by association between SV proteins. The SH3-containing GRB2-like protein 3-interacting protein 1 (SGIP1), an ortholog of Fer/Cip4 homology domain-only (FCHo) proteins, contains a μ-homology domain (μHD) and binds AP-2 and Eps15, thus functioning as an endocytic regulator of clathrin-mediated endocytosis (CME). Its longest isoform SGIP1α is predominantly expressed in the brain but the functional significance of SGIP1 in SV recycling remains unknown. Here, we found that SGIP1α, a brain-specific long isoform of SGIP1 binds synaptotagmin1 (Syt1) via its μHD and promotes the internalization of Syt1 on the neuronal surface. The small hairpin RNA (shRNA)-mediated knockdown (KD) of SGIP1α caused selective impairment of Syt1 internalization at hippocampal synapses and it was fully rescued by coexpression of the shRNA-resistant form of SGIP1α in KD neurons. We further found that the μHD of SGIP1α is structurally similar to those of AP-2 and stonin2, and mutations at Trp771 and Lys781, which correspond to Syt1-recognition motifs of AP-2 and stonin2, to Ala bound less efficiently to Syt1 and failed to rescue the endocytic defect of Syt1 caused by KD. Our results indicate that SGIP1α is an endocytic adaptor dedicated to the retrieval of surface-stranded Syt1. Since endocytic sorting of Syt1 is also mediated by the overlapping activities of synaptic vesicle glycoprotein 2A/B (SV2A/B) and stonin2, our results suggest that complementary fail-safe mechanism by these proteins ensures high fidelity of Syt1 retrieval.

AbstractnArgBP2, a candidate gene for intellectual disability, is a postsynaptic protein critical for dendritic spine development and morphogenesis, and its knockdown (KD) in developing neurons severely impairs spine-bearing excitatory synapse formation. Surprisingly, nArgBP2 KD in mature neurons did not cause morphological defects in the existing spines at rest, raising questions of how it functions in mature neurons. We found that unlike its inaction at rest, nArgBP2 KD completely inhibited the enlargement of dendritic spines during chemically induced long-term potentiation (cLTP) in mature neurons. We further found that nArgBP2 forms condensates in dendritic spines and that these condensates are dispersed by cLTP, which spatiotemporally coincides with spine head enlargement. Condensates with CaMKII phosphorylation-deficient mutant or CaMKII inhibition are neither dispersed nor accompanied by spine enlargement during cLTP. We found that nArgBP2 condensates in spines exhibited liquid-like properties, and in heterologous and in vitro expression systems, nArgBP2 undergoes liquid-liquid phase separation via multivalent intermolecular interactions between SH3 domains and proline-rich domains. It also forms coacervates with CaMKIIα, which is rapidly dissembled by calcium/CaMKIIα-dependent phosphorylation. We further showed that the interaction between nArgBP2 and WAVE1 competes with nArgBP2 phase separation and that blocking the nArgBP2-WAVE1 interaction prevents spine enlargement during cLTP. Together, our results suggest that nArgBP2 at rest is confined to the condensates but is released by CaMKIIα-mediated phosphorylation during synaptic plasticity, which regulates its timely interaction with WAVE1 to induce spine head enlargement in mature neurons.

Na+(K+)/H+ exchanger 6 (NHE6) on synaptic vesicle (SV) is critical for the presynaptic regulation of quantal size at the glutamatergic synapses by converting the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane. We recently found that NHE6 directly interacts with secretory carrier membrane protein 5 (SCAMP5), and SCAMP5-dependent recruitment of NHE6 to SVs controls the strength of synaptic transmission by modulation of quantal size of glutamate release at rest. It is, however, unknown whether NHE6 recruitment by SCAMP5 plays a role during synaptic plasticity. Here, we found that the number of NHE6-positive presynaptic boutons was significantly increased by the chemical long-term potentiation (cLTP). Since cLTP involves new synapse formation, our results indicated that NHE6 was recruited not only to the existing presynaptic boutons but also to the newly formed presynaptic boutons. Knock down of SCAMP5 completely abrogated the enhancement of NHE6 recruitment by cLTP. Interestingly, despite an increase in the number of NHE6-positive boutons by cLTP, the quantal size of glutamate release at the presynaptic terminals remained unaltered. Together with our recent results, our findings indicate that SCAMP5-dependent recruitment of NHE6 plays a critical role in manifesting presynaptic efficacy not only at rest but also during synaptic plasticity. Since both are autism candidate genes, reduced presynaptic efficacy by interfering with their interaction may underlie the molecular mechanism of synaptic dysfunction observed in autism.

Amyloid-β impairs synaptic function in neurons by inhibiting intersynaptic vesicular trafficking.

Glutamate uptake into synaptic vesicles (SVs) depends on cation/H+ exchange activity, which converts the chemical gradient (ΔpH) into membrane potential (Δψ) across the SV membrane at the presynaptic terminals. Thus, the proper recruitment of cation/H+ exchanger to SVs is important in determining glutamate quantal size, yet little is known about its localization mechanism. Here, we found that secretory carrier membrane protein 5 (SCAMP5) interacted with the cation/H+ exchanger NHE6, and this interaction regulated NHE6 recruitment to glutamatergic presynaptic terminals. Protein–protein interaction analysis with truncated constructs revealed that the 2/3 loop domain of SCAMP5 is directly associated with the C-terminal region of NHE6. The use of optical imaging and electrophysiological recording showed that small hairpin RNA–mediated knockdown (KD) of SCAMP5 or perturbation of SCAMP5/NHE6 interaction markedly inhibited axonal trafficking and the presynaptic localization of NHE6, leading to hyperacidification of SVs and a reduction in the quantal size of glutamate release. Knockout of NHE6 occluded the effect of SCAMP5 KD without causing additional defects. Together, our results reveal that as a key regulator of axonal trafficking and synaptic localization of NHE6, SCAMP5 could adjust presynaptic strength by regulating quantal size at glutamatergic synapses. Since both proteins are autism candidate genes, the reduced quantal size by interrupting their interaction may underscore synaptic dysfunction observed in autism.

Synaptic strength and reliability are determined by the number of vesicles released per action potential and the availability of release-competent vesicles in the readily releasable pool (RRP). Compared with release of a single vesicle (univesicular release), multivesicular release (MVR) would speed up RRP depletion, yet whether the RRP is refilled differently during the two different release modes has not been investigated. Here, we address this question by quantitative optical imaging with an axon-targeting glutamate sensor, iGluSnFRpre. We found that hippocampal synapses preferentially release multiple vesicles per action potential at high extracellular calcium or by paired-pulse stimulation. When MVR prevails, the RRP is recovered very rapidly with a time constant of 430 ms. This rapid recovery is mediated by dynamin-dependent endocytosis followed by direct reuse of retrieved vesicles. Furthermore, our simulation proved that the portion of retrieved vesicles that directly refill the RRP increases dramatically (.70%) in MVR compared with that in univesicular release (,10%). These results suggest that the contribution of rapid and direct recruitment of retrieved vesicle to the RRP changes dynamically with release mode at the level of individual synapses, which suggests a form of presynaptic homeostatic plasticity for reliable synaptic transmission during various synaptic activity.