Structural basis of SALM3 dimerization and synaptic adhesion complex formation with PTPσ

bioRxiv: doi: https://doi.org/10.1101/2020.01.09.893701Synaptic adhesion molecules play an important role in the formation, maintenance and refinement of neuronal connectivity. Recently, several leucine rich repeat (LRR) domain containing neuronal adhesion molecules have been characterized including netrin G-ligands, SLITRKs and the synaptic adhesion-like molecules (SALMs). Dysregulation of these adhesion molecules have been genetically and functionally linked to various neurological disorders. Here we investigated the molecular structure and mechanism of ligand interactions for the postsynaptic SALM3 adhesion protein with its presynaptic ligand, receptor protein tyrosine phosphatase sigma (PTP sigma). We solved the crystal structure of the dimerized LRR domain of SALM3, revealing the conserved structural features and mechanism of dimerization. Furthermore, we determined the complex structure of SALM3 with PTP sigma using small angle X-ray scattering, revealing a 2:2 complex similar to that observed for SALM5. Solution studies unraveled additional flexibility for the complex structure, but validated the uniform mode of action for SALM3 and SALM5 to promote synapse formation. The relevance of the key interface residues was further confirmed by mutational analysis with cellular binding assays and artificial synapse formation assays. Collectively, our results suggest that SALM3 dimerization is a pre-requisite for the SALM3-PTP sigma complex to exert synaptogenic activity.Peer reviewe

Re-evaluation of the optimum dietary protein level for maximum growth of juvenile barred knifejaw Oplegnathus fasciatus reared in cages

Abstract We determined the optimum dietary protein level in juvenile barred knifejaw Oplegnathus fasciatus in cages. Five semi-purified isocaloric diets were formulated with white fish meal and casein-based diets to contain 35, 40, 45, 50, and 60 % crude protein (CP). Fish with an initial body weight of 7.1 ± 0.06 g (mean ± SD) were randomly distributed into 15 net cages (each size: 60 cm × 40 cm × 90 cm, W × L × H) as groups of 20 fish in triplicates. The fish were fed at apparent satiation level twice a day. After 8 weeks of feeding, the weight gain (WG) of fish fed 45, 50, and 60 % CP diets were significantly higher than those of fish fed 35 and 40 % CP diets. However, there were no significant differences in WG among fish fed 45, 50, and 60 % CP diets. Generally, feed efficiency (FE) and specific growth rate (SGR) showed a similar trend as WG. However, the protein efficiency ratio (PER) was inversely related to dietary protein levels. Energy retention efficiency increased with the increase of dietary protein levels by protein sparing from non-protein energy sources. Blood hematocrit content was not affected by dietary protein levels. However, a significantly lower amount of hemoglobin was found in fish fed 35 % CP than in fish fed 40, 45, 50, and 60 % CP diets. Fish fed 60 % CP showed the lowest survival rate than the fish fed 35, 40, 45, and 50 % CP diets. Broken-line analysis of WG showed the optimum dietary protein level was 45.2 % with 18.8 kJ/g diet for juvenile barred knifejaw. This study has potential implication for the successful cage culture of barred knifejaw

Slitrk2 controls excitatory synapse development via PDZ-mediated protein interactions

Members of the Slitrk (Slit- and Trk-like protein) family of synaptic cell-adhesion molecules control excitatory and inhibitory synapse development through isoform-dependent extracellular interactions with leukocyte common antigen-related receptor protein tyrosine phosphatases (LAR-RPTPs). However, how Slitrks participate in activation of intracellular signaling pathways in postsynaptic neurons remains largely unknown. Here we report that, among the six members of the Slitrk family, only Slitrk2 directly interacts with the PDZ domain-containing excitatory scaffolds, PSD-95 and Shank3. The interaction of Slitrk2 with PDZ proteins is mediated by the cytoplasmic COOH-terminal PDZ domain-binding motif (Ile-Ser-Glu-Leu), which is not found in other Slitrks. Mapping analyses further revealed that a single PDZ domain of Shank3 is responsible for binding to Slitrk2. Slitrk2 forms in vivo complexes with membrane-associated guanylate kinase (MAGUK) family proteins in addition to PSD-95 and Shank3. Intriguingly, in addition to its role in synaptic targeting in cultured hippocampal neurons, the PDZ domain-binding motif of Slitrk2 is required for Slitrk2 promotion of excitatory synapse formation, transmission, and spine development in the CA1 hippocampal region. Collectively, our data suggest a new molecular mechanism for conferring isoform-specific regulatory actions of the Slitrk family in orchestrating intracellular signal transduction pathways in postsynaptic neurons. © 2019, The Author(s).1

MDGA1 negatively regulates amyloid precursor protein-mediated synapse inhibition in the hippocampus

Abstract Balanced synaptic inhibition, controlled by multiple synaptic adhesion proteins, is critical for proper brain function. MDGA1 (meprin, A-5 protein, and receptor protein-tyrosine phosphatase mu [MAM] domain-containing glycosylphosphatidylinositol anchor protein 1) suppresses synaptic inhibition in mammalian neurons, yet the molecular mechanisms underlying MDGA1-mediated negative regulation of GABAergic synapses remain unresolved. Here, we show that the MDGA1 MAM domain directly interacts with the extension domain of amyloid precursor protein (APP). Strikingly, MDGA1-mediated synaptic disinhibition requires the MDGA1 MAM domain and is prominent at distal dendrites of hippocampal CA1 pyramidal neurons. Down-regulation of APP in presynaptic GABAergic interneurons specifically suppressed GABAergic, but not glutamatergic, synaptic transmission strength and inputs onto both the somatic and dendritic compartments of hippocampal CA1 pyramidal neurons. Moreover, APP deletion manifested differential effects in somatostatin- and parvalbumin-positive interneurons in the hippocampal CA1, resulting in distinct alterations in inhibitory synapse numbers, transmission, and excitability. The infusion of MDGA1 MAM protein mimicked postsynaptic MDGA1 gain-of-function phenotypes that involve the presence of presynaptic APP. The overexpression of MDGA1 wild type or MAM, but not MAM-deleted MDGA1, in the hippocampal CA1 impaired novel object-recognition memory in mice. Thus, our results establish unique roles of APP-MDGA1 complexes in hippocampal neural circuits, providing unprecedented insight into trans-synaptic mechanisms underlying differential tuning of neuronal compartment-specific synaptic inhibition.Peer reviewe

The small GTPase ARF6 regulates GABAergic synapse development

ADP ribosylation factors (ARFs) are a family of small GTPases composed of six members (ARF1-6) that control various cellular functions, including membrane trafficking and actin cytoskeletal rearrangement, in eukaryotic cells. Among them, ARF1 and ARF6 are the most studied in neurons, particularly at glutamatergic synapses, but their roles at GABAergic synapses have not been investigated. Here, we show that a subset of ARF6 protein is localized at GABAergic synapses in cultured hippocampal neurons. In addition, we found that knockdown (KD) of ARF6, but not ARF1, triggered a reduction in the number of GABAergic synaptic puncta in mature cultured neurons in an ARF activity-dependent manner. ARF6 KD also reduced GABAergic synaptic density in the mouse hippocampal dentate gyrus (DG) region. Furthermore, ARF6 KD in the DG increased seizure susceptibility in an induced epilepsy model. Viewed together, our results suggest that modulating ARF6 and its regulators could be a therapeutic strategy against brain pathologies involving hippocampal network dysfunction, such as epilepsy. © 2020 The Author(s).1

Loss of IQSEC3 Disrupts GABAergic Synapse Maintenance and Decreases Somatostatin Expression in the Hippocampus

Gephyrin interacts with various GABAergic synaptic proteins to organize GABAergic synapse development. Among the multitude of gephyrin-binding proteins is IQSEC3, a recently identified component at GABAergic synapses that acts through its ADP ribosylation factor-guanine nucleotide exchange factor (ARF-GEF) activity to orchestrate GABAergic synapse formation. Here, we show that IQSEC3 knockdown (KD) reduced GABAergic synaptic density in vivo, suggesting that IQSEC3 is required for GABAergic synapse maintenance in vivo. We further show that IQSEC3 KD in the dentate gyrus (DG) increases seizure susceptibility and triggers selective depletion of somatostatin (SST) peptides in the DG hilus in an ARF-GEP activity-dependent manner. Strikingly, selective introduction of SST into SST interneurons in DG-specific IQSEC3-KD mice reverses GABAergic synaptic deficits. Thus, our data suggest that IQSEC3 is required for linking gephyrin-GABAA receptor complexes with ARF-dependent pathways to prevent aberrant, runaway excitation and thereby contributes to the integrity of SST interneurons and proper GABAergic synapse maintenance. © 2020 The Author(s) In this study, Kim et al. investigate the effect of loss of function of IQSEC3, a gephyrin-binding GABAergic synapse-specific ARF-GEF, using hippocampal dentate gyrus (DG)-specific IQSEC3-knockdown (KD) mice. Strikingly, IQSEC3 KD causes a massive reduction of somatostatin (SST) expression. The restricted SST expression in SST+ interneurons reverses the pathological phenotypes. © 2020 The Author(s)1

Npas4 regulates IQSEC3 expression in hippocampal somatostatin interneurons to mediate anxiety-like behavior

Activity-dependent GABAergic synapse plasticity is important for normal brain functions, but the underlying molecular mechanisms remain incompletely understood. Here, we show that Npas4 (neuronal PAS-domain protein 4) transcriptionally regulates the expression of IQSEC3, a GABAergic synapse-specific guanine nucleotide-exchange factor for ADP-ribosylation factor (ARF-GEF) that directly interacts with gephyrin. Neuronal activation by an enriched environment induces Npas4-mediated upregulation of IQSEC3 protein specifically in CA1 stratum oriens layer somatostatin (SST)-expressing GABAergic interneurons. SST+ interneuron-specific knockout (KO) of Npas4 compromises synaptic transmission in these GABAergic interneurons, increases neuronal activity in CA1 pyramidal neurons, and reduces anxiety behavior, all of which are normalized by the expression of wild-type IQSEC3, but not a dominant-negative ARF-GEF-inactive mutant, in SST+ interneurons of Npas4-KO mice. Our results suggest that IQSEC3 is a key GABAergic synapse component that is directed by Npas4 and ARF activity, specifically in SST+ interneurons, to orchestrate excitation-to-inhibition balance and control anxiety-like behavior.1

Calsyntenin-3 Interacts With Both α- And β-Neurexins in the Regulation of Excitatory Synaptic Innervation in Specific Schaffer Collateral Pathways

Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, using LC-MS/MS-based protein analysis, confocal microscopy, RNAscope assays, and electrophysiological recordings, we show that β-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive β-Nrxn variants, but not insert-negative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo Neuron-specific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits, including Schaffer collateral afferents. © 2020 Kim et al.1

Controlling the stiffness of bistable kirigami surfaces via spatially varying hinges

Kirigami, the art of paper cutting, has offered a versatile way of constructing a reconfigurable structure from a 2D planar configuration into a target 3D shape. While several strategies for its design have been reported, little attention has been paid to the mechanical properties when deployed. Here, we investigate how to control the stiffness of bistable kirigami surfaces in the deployed configuration while maintaining the target 3D shape. We develop a computational procedure that can be used to design various bistable kirigami surfaces. Their deployed 3D shape and stability as well as the nonlinear stiffness can be successfully predicted as validated with experiments. Using the proposed approach, we found that the range of stiffness for deployed bistable kirigami surfaces can be significantly broadened by using spatially varying hinges without altering the shape and stability of deployed structures. We expect our study would advance the design of reconfigurable structures having identical shapes but different mechanical properties

Residual Life Prediction for Induction Furnace by Sequential Encoder with s-Convolutional LSTM

Induction furnaces are widely used for melting scrapped steel in small foundries and their use has recently become more frequent. The maintenance of induction furnaces is usually based on empirical decisions of the operator and an explosion can occur through operator error. To prevent an explosion, previous studies have utilized statistical models but have been unable to generalize the problem and have achieved a low accuracy. Herein, we propose a data-driven method for induction furnaces by proposing a novel 2D matrix called a sequential feature matrix(s-encoder) and multi-channel convolutional long short-term memory (s-ConLSTM). First, the sensor data and operation data are converted into sequential feature matrices. Then, N-sequential feature matrices are imported into the convolutional LSTM model to predict the residual life of the induction furnace wall. Based on our experimental results, our method outperforms general neural network models and enhances the safe use of induction furnaces