Altered theta and beta oscillatory synchrony in a genetic mouse model of pathological anxiety.

While the neural circuits mediating normal, adaptive defensive behaviors have been extensively studied, substantially less is currently known about the network mechanisms by which aberrant, pathological anxiety is encoded in the brain. Here we investigate in mice how deletion of Neuroligin-2 (Nlgn2), an inhibitory synapse-specific adhesion protein that has been associated with pathological anxiety and other psychiatric disorders, alters the communication between key brain regions involved in mediating defensive behaviors. To this end, we performed multi-site simultaneous local field potential (LFP) recordings from the basolateral amygdala (BLA), centromedial amygdala (CeM), bed nucleus of the stria terminalis (BNST), prefrontal cortex (mPFC) and ventral hippocampus (vHPC) in an open field paradigm. We found that LFP power in the vHPC was profoundly increased and was accompanied by an abnormal modulation of the synchrony of theta frequency oscillations particularly in the vHPC-mPFC-BLA circuit. Moreover, deletion of Nlgn2 increased beta and gamma frequency synchrony across the network, and this increase was associated with increased center avoidance. Local deletion of Nlgn2 in the vHPC and BLA revealed that they encode distinct aspects of this avoidance phenotype, with vHPC linked to immobility and BLA linked to a reduction in exploratory activity. Together, our data demonstrate that alterations in long-range functional connectivity link synaptic inhibition to abnormal defensive behaviors, and that both exaggerated activation of normal defensive circuits and recruitment of fundamentally distinct mechanisms contribute to this phenotype. Nlgn2 knockout mice therefore represent a highly relevant model to study the role of inhibitory synaptic transmission in the circuits underlying anxiety disorders.

Defective Reelin/Dab1 signaling pathways associated with disturbed hippocampus development of homozygous yotari mice.

Homozygous Dab1 yotari mutant mice, Dab1yot (yot/yot) mice, have an autosomal recessive mutation of Dab1 and show reeler-like phenotype including histological abnormality of the cerebellum, hippocampus, and cerebral cortex. We here show abnormal hippocampal development of yot/yot mice where granule cells and pyramidal cells fail to form orderly rows but are dispersed diffusely in vague multiplicative layers. Possibly due to the positioning failure of granule cells and pyramidal cells and insufficient synaptogenesis, axons of the granule cells did not extend purposefully to connect with neighboring regions in yot/yot mice. We found that both hippocampal granule cells and pyramidal cells of yot/yot mice expressed proteins reactive with the anti-Dab1 antibody. We found that Y198- phosphorylated Dab1 of yot/yot mice was greatly decreased. Accordingly the downstream molecule, Akt was hardly phosphorylated. Especially, synapse formation was defective and the distribution of neurons was scattered in hippocampus of yot/yot mice. Some of neural cell adhesion molecules and hippocampus associated transcription factors of the neurons were expressed aberrantly, suggesting that the Reelin-Dab1 signaling pathway seemed to be importantly involved in not only neural migration as having been shown previously but also neural maturation and/or synaptogenesis of the mice. It is interesting to clarify whether the defective neural maturation is a direct consequence of the dysfunctional Dab1, or alternatively secondarily due to the Reelin-Dab1 intracellular signaling pathways.

SPARCL1 Promotes Excitatory But Not Inhibitory Synapse Formation and Function Independent of Neurexins and Neuroligins.

Emerging evidence supports roles for secreted extracellular matrix proteins in boosting synaptogenesis, synaptic transmission, and synaptic plasticity. SPARCL1 (also known as Hevin), a secreted non-neuronal protein, was reported to increase synaptogenesis by simultaneously binding to presynaptic neurexin-1α and to postsynaptic neuroligin-1B, thereby catalyzing formation of trans-synaptic neurexin/neuroligin complexes. However, neurexins and neuroligins do not themselves mediate synaptogenesis, raising the question of how SPARCL1 enhances synapse formation by binding to these molecules. Moreover, it remained unclear whether SPARCL1 acts on all synapses containing neurexins and neuroligins or only on a subset of synapses, and whether it enhances synaptic transmission in addition to boosting synaptogenesis or induces silent synapses. To explore these questions, we examined the synaptic effects of SPARCL1 and their dependence on neurexins and neuroligins. Using mixed neuronal and glial cultures from neonatal mouse cortex of both sexes, we show that SPARCL1 selectively increases excitatory but not inhibitory synapse numbers, enhances excitatory but not inhibitory synaptic transmission, and augments NMDAR-mediated synaptic responses more than AMPAR-mediated synaptic responses. None of these effects were mediated by SPARCL1-binding to neurexins or neuroligins. Neurons from triple neurexin-1/2/3 or from quadruple neuroligin-1/2/3/4 conditional KO mice that lacked all neurexins or all neuroligins were fully responsive to SPARCL1. Together, our results reveal that SPARCL1 selectively boosts excitatory but not inhibitory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.SIGNIFICANCE STATEMENT Emerging evidence supports roles for extracellular matrix proteins in boosting synapse formation and function. Previous studies demonstrated that SPARCL1, a secreted non-neuronal protein, promotes synapse formation in rodent and human neurons. However, it remained unclear whether SPARCL1 acts on all or on only a subset of synapses, induces functional or largely inactive synapses, and generates synapses by bridging presynaptic neurexins and postsynaptic neuroligins. Here, we report that SPARCL1 selectively induces excitatory synapses, increases their efficacy, and enhances their NMDAR content. Moreover, using rigorous genetic manipulations, we show that SPARCL1 does not require neurexins and neuroligins for its activity. Thus, SPARCL1 selectively boosts excitatory synaptogenesis and synaptic transmission by a novel mechanism that is independent of neurexins and neuroligins.

Angiocrine Hepatocyte Growth Factor Signaling Controls Physiological Organ and Body Size and Dynamic Hepatocyte Proliferation to Prevent Liver Damage during Regeneration.

Liver sinusoidal endothelial cells (LSECs) control organ functions, metabolism, and development through the secretion of angiokines. LSECs express hepatocyte growth factor (Hgf), which is involved in prenatal development, metabolic homeostasis, and liver regeneration. This study aimed to elucidate the precise contribution of LSEC-derived Hgf in physiological homeostasis and liver regeneration. Stab2-iCretg/wt;Hgffl/fl (HgfΔLSEC) mice were generated to abrogate Hgf expression selectively in LSECs from early fetal development onwards, to study global development, metabolic and endothelial zonation, and organ functions as well as liver regeneration in response to 70% partial hepatectomy (PH). Although zonation and liver/body weight ratios were not altered, total body weight and total liver weight were reduced in HgfΔLSEC. Necrotic organ damage was more marked in HgfΔLSEC mice, and regeneration was delayed 72 hours after PH. This was associated with decreased hepatocyte proliferation at 48 hours after PH. Molecularly, HgfΔLSEC mice showed down-regulation of Hgf/c-Met signaling and decreased expression of Deptor in hepatocytes. In vitro knockdown of Deptor was associated with decreased proliferation. Therefore, angiocrine Hgf controls hepatocyte proliferation and susceptibility to necrosis after partial hepatectomy via the Hgf/c-Met axis involving Deptor to prevent excessive organ damage.

Role of Reelin in cell positioning in the cerebellum and the cerebellum-like structure in zebrafish.

The cerebellum and the cerebellum-like structure in the mesencephalic tectum in zebrafish contain multiple cell types, including principal cells (i.e., Purkinje cells and type I neurons) and granule cells, that form neural circuits in which the principal cells receive and integrate inputs from granule cells and other neurons. It is largely unknown how these cells are positioned and how neural circuits form. While Reelin signaling is known to play an important role in cell positioning in the mammalian brain, its role in the formation of other vertebrate brains remains elusive. Here we found that zebrafish with mutations in Reelin or in the Reelin-signaling molecules Vldlr or Dab1a exhibited ectopic Purkinje cells, eurydendroid cells (projection neurons), and Bergmann glial cells in the cerebellum, and ectopic type I neurons in the tectum. The ectopic Purkinje cells and type I neurons received aberrant afferent fibers in these mutants. In wild-type zebrafish, reelin transcripts were detected in the internal granule cell layer, while Reelin protein was localized to the superficial layer of the cerebellum and the tectum. Laser ablation of the granule cell axons perturbed the localization of Reelin, and the mutation of both kif5aa and kif5ba, which encode major kinesin I components in the granule cells, disrupted the elongation of granule cell axons and the Reelin distribution. Our findings suggest that in zebrafish, (1) Reelin is transported from the granule cell soma to the superficial layer by axonal transport; (2) Reelin controls the migration of neurons and glial cells from the ventricular zone; and (3) Purkinje cells and type I neurons attract afferent axons during the formation of the cerebellum and the cerebellum-like structure.

Metabotropic glutamate type 5 receptor requires contactin-associated protein 1 to control memory formation.

The hippocampus is a key brain region for memory formation. Metabotropic glutamate type 5 receptors (mGlu5R) are strongly expressed in CA1 pyramidal neurons and fine-tune synaptic plasticity. Accordingly, mGlu5R pharmacological manipulation may represent an attractive therapeutic strategy to manage hippocampal-related neurological disorders. Here, by means of a membrane yeast two-hybrid screening, we identified contactin-associated protein 1 (Caspr1), a type I transmembrane protein member of the neurexin family, as a new mGlu5R partner. We report that mGlu5R and Caspr1 co-distribute and co-assemble both in heterologous expression systems and in rat brain. Furthermore, downregulation of Caspr1 in rat hippocampal primary cultures decreased mGlu5R-mediated signaling. Finally, silencing Caspr1 expression in the hippocampus impaired the impact of mGlu5R on spatial memory. Our results indicate that Caspr1 plays a pivotal role controlling mGlu5R function in hippocampus-dependent memory formation. Hence, this new protein-protein interaction may represent novel target for neurological disorders affecting hippocampal glutamatergic neurotransmission.

The Role of Ninjurin1 and Its Impact beyond the Nervous System.

Ninjurin1 (Ninj1) is a double-transmembrane cell surface protein that could promote nerve regeneration in the process of the peripheral nervous system injury and repairment. Nonetheless, the accurate function of Ninj1 in the central nervous system and outside the nervous system is not completely clear. According to the recent studies, we found that Ninj1 is also aberrantly expressed in various pathophysiological processes in vivo, including inflammation, tumorigenesis, and vascular, bone, and muscle homeostasis. These findings suggest that Ninj1 may play an influential role during these pathophysiological processes. Our review summarizes the diverse roles of Ninj1 in multiple pathophysiological processes inside and outside the nervous system. Ninj1 should be considered as an important and novel therapeutic target in certain diseases, such as inflammatory diseases and ischemic diseases. Our study provided a better understanding of Ninj1 in different pathophysiological processes and thereby provided the theoretical support for further research.

Morphological and Physiological Characteristics of Ebf2-EGFP-Expressing Cajal-Retzius Cells in Developing Mouse Neocortex.

Cajal-Retzius (CR) cells are one of the earliest populations of neurons in the cerebral cortex of rodents and primates, and they play a critical role in corticogenesis and cortical lamination during neocortical development. However, a comprehensive morphological and physiological profile of CR cells in the mouse neocortex has not yet been established. Here, we systematically investigated the dynamic development of CR cells in Tg(Ebf2-EGFP)58Gsat/Mmcd mice. The morphological complexity, membrane activities and presynaptic inputs of CR cells coordinately increase and reach a plateau at P5-P9 before regressing. Using 3D reconstruction, we delineated a parallel-stratification pattern of the axonal extension of CR cells. Furthermore, we found that the morphological structure and presynaptic inputs of CR cells were disturbed in Reelin-deficient mice. These findings confirm that CR cells undergo a transient maturation process in layer 1 before disappearing. Importantly, Reelin deficiency impairs the formation of synaptic connections onto CR cells. In conclusion, our results provide insights into the rapid maturation and axonal stratification of CR cells in layer 1. These findings suggest that both the electrophysiological activities and the morphology of CR cells provide vital guidance for the modulation of early circuits, in a Reelin-dependent manner.

Stigmasterol promotes neuronal migration via reelin signaling in neurosphere migration assays.

Stigmasterol (ST) is a multifunctional phytosterol and is found in diverse food. In our previous transcriptomics study, we found ST upregulated migration-related genes. In the present study, we carried out in vitro neurosphere migration assays to investigate the effects of ST on neuronal migration. For this purpose, neurospheres were produced by culturing rat (Sprague-Dawley) E14 cortical neurons. The addition of ST (75 µM) to culture medium increased not only the numbers of migratory neurons by 15% but the distance of movement up to 120 µm from the centers of neurospheres as compared to vehicle cultures. Immunocytochemistry and immunoblotting showed ST upregulated the expressions of Reelin (Reln) and its downstream signaling molecules like phospho-JNK (c-Jun N-terminal kinase), doublecortin (DCX) and dynein heavy chain (DHC) in migratory neurons. Furthermore, in silico molecular docking simulation indicated that ST interacts with Relin receptor ApoER2 by forming a hydrogen bond with Lys2467 and other van der Waals interactions. Taken together, our study shows that ST upregulates Reln signaling and promotes neuronal migration and suggests that ST supplementation is considered as a potential means of treating migration-related CNS disorders.

Reelin Promotes Cisplatin Resistance by Induction of Epithelial-Mesenchymal Transition via p38/GSK3ß/Snail Signaling in Non-Small Cell Lung Cancer.

BACKGROUND Emerging evidence suggests the involvement of Reelin in chemoresistance in various cancers. However, its function in cisplatin (DDP) sensitivity of non-small cell lung cancer (NSCLC) needs to be investigated. MATERIAL AND METHODS Reelin expression in cisplatin-sensitive A549 cells and cisplatin-resistant NSCLC (A549/DDP) cells was analyzed by western blot analysis. qRT-PCR, western blotting, immunofluorescence, CCK-8 assays, Annexin V/propidium iodide apoptosis assay, and Transwell migration assays were carried out to determine the function of Reelin on DDP resistance. RESULTS Reelin was markedly increased in A549/DDP cells relative to A549 cells. Knockdown of Reelin enhanced DDP chemosensitivity of A549/DDP cells, whereas overexpression of Reelin enhanced DDP resistance of A549, H1299, and H460 cells. Reelin induced DDP resistance in NSCLC cells via facilitating epithelial-mesenchymal transition (EMT). Furthermore, Reelin modulated p38/GSK3ß signal transduction and promoted Snail (EMT-associated transcription factor) expression. Suppression of p38/Snail reversed Reelin-induced EMT and resistance of NSCLC cells to DDP. CONCLUSIONS These data indicated that Reelin induces DDP resistance of NSCLC by regulation of the p38/GSK3ß/Snail/EMT signaling pathway and provide evidence that Reelin suppression can be an effective strategy to suppress DDP resistance in NSCLC.

Reelin transiently promotes N-cadherin-dependent neuronal adhesion during mouse cortical development.

Reelin is an essential glycoprotein for the establishment of the highly organized six-layered structure of neurons of the mammalian neocortex. Although the role of Reelin in the control of neuronal migration has been extensively studied at the molecular level, the mechanisms underlying Reelin-dependent neuronal layer organization are not yet fully understood. In this study, we directly showed that Reelin promotes adhesion among dissociated neocortical neurons in culture. The Reelin-mediated neuronal aggregation occurs in an N-cadherin-dependent manner, both in vivo and in vitro. Unexpectedly, however, in a rotation culture of dissociated neocortical cells that gradually reaggregated over time, we found that it was the neural progenitor cells [radial glial cells (RGCs)], rather than the neurons, that tended to form clusters in the presence of Reelin. Mathematical modeling suggested that this clustering of RGCs could be recapitulated if the Reelin-dependent promotion of neuronal adhesion were to occur only transiently. Thus, we directly measured the adhesive force between neurons and N-cadherin by atomic force microscopy, and found that Reelin indeed enhanced the adhesiveness of neurons to N-cadherin; this enhanced adhesiveness began to be observed at 30 min after Reelin stimulation, but declined by 3 h. These results suggest that Reelin transiently (and not persistently) promotes N-cadherin-mediated neuronal aggregation. When N-cadherin and stabilized ß-catenin were overexpressed in the migrating neurons, the transfected neurons were abnormally distributed in the superficial region of the neocortex, suggesting that appropriate regulation of N-cadherin-mediated adhesion is important for correct positioning of the neurons during neocortical development.

Detrimental Role of Nerve Injury-Induced Protein 1 in Myeloid Cells under Intestinal Inflammatory Conditions.

Nerve injury-induced protein 1 (Ninjurin1, Ninj1) is a cell-surface adhesion molecule that regulates cell migration and attachment. This study demonstrates the increase in Ninj1 protein expression during development of intestinal inflammation. Ninj1-deficient mice exhibited significantly attenuated bodyweight loss, shortening of colon length, intestinal inflammation, and lesser pathological lesions than wild-type mice. Although more severe inflammation and serious lesions are observed in wild-type mice than Ninj1-deficient mice, there were no changes in the numbers of infiltrating macrophages in the inflamed tissues obtained from WT and Ninj1-deficient mice. Ninj1 expression results in activation of macrophages, and these activated macrophages secrete more cytokines and chemokines than Ninj1-deficient macrophages. Moreover, mice with conditional deletion of Ninj1 in myeloid cells (Ninj1fl/fl; Lyz-Cre+) alleviated experimental colitis compared with wild-type mice. In summary, we propose that the Ninj1 in myeloid cells play a pivotal function in intestinal inflammatory conditions.

The Adhesion-GPCR BAI1 Promotes Excitatory Synaptogenesis by Coordinating Bidirectional Trans-synaptic Signaling.

Excitatory synapses are specialized cell-cell contacts located on actin-rich dendritic spines that mediate information flow and storage in the brain. The postsynaptic adhesion-G protein-coupled receptor (A-GPCR) BAI1 is a critical regulator of excitatory synaptogenesis, which functions in part by recruiting the Par3-Tiam1 polarity complex to spines, inducing local Rac1 GTPase activation and actin cytoskeletal remodeling. However, a detailed mechanistic understanding of how BAI1 controls synapse and spine development remains elusive. Here, we confirm that BAI1 is required in vivo for hippocampal spine development, and we identify three distinct signaling mechanisms mediating BAI1's prosynaptogenic functions. Using in utero electroporation to sparsely knock down BAI1 expression in hippocampal pyramidal neurons, we show that BAI1 cell-autonomously promotes spinogenesis in the developing mouse brain. BAI1 appears to function as a receptor at synapses, as its extracellular N-terminal segment is required for both its prospinogenic and prosynaptogenic functions. Moreover, BAI1 activation with a Stachel-derived peptide, which mimics a tethered agonist motif found in A-GPCRs, drives synaptic Rac1 activation and subsequent spine and synapse development. We also reveal, for the first time, a trans-synaptic function for BAI1, demonstrating in a mixed-culture assay that BAI1 induces the clustering of presynaptic vesicular glutamate transporter 1 (vGluT1) in contacting axons, indicative of presynaptic differentiation. Finally, we show that BAI1 forms a receptor complex with the synaptogenic cell-adhesion molecule Neuroligin-1 (NRLN1) and mediates NRLN1-dependent spine growth and synapse development. Together, these findings establish BAI1 as an essential postsynaptic A-GPCR that regulates excitatory synaptogenesis by coordinating bidirectional trans-synaptic signaling in cooperation with NRLN1.SIGNIFICANCE STATEMENT Adhesion-G protein-coupled receptors are cell-adhesion receptors with important roles in nervous system development, function, and neuropsychiatric disorders. The postsynaptic adhesion-G protein-coupled receptor BAI1 is a critical regulator of dendritic spine and excitatory synapse development. However, the mechanism by which BAI1 controls these functions remains unclear. Our study identifies three distinct signaling paradigms for BAI1, demonstrating that it mediates forward, reverse, and lateral signaling in spines. Activation of BAI1 by a Stachel-dependent mechanism induces local Rac1 activation and subsequent spinogenesis/synaptogenesis. BAI1 also signals trans-synaptically to promote presynaptic differentiation. Furthermore, BAI1 interacts with the postsynaptic cell-adhesion molecule Neuroligin-1 (NRLN1) and facilitates NRLN1-dependent spine growth and excitatory synaptogenesis. Thus, our findings establish BAI1 as a functional synaptogenic receptor that promotes presynaptic and postsynaptic development in cooperation with synaptic organizer NRLN1.

Altered Auditory Processing, Filtering, and Reactivity in the Cntnap2 Knock-Out Rat Model for Neurodevelopmental Disorders.

Sensory processing, and auditory processing in particular, is altered in individuals with neurodevelopmental disorders such as autism spectrum disorders (ASDs). The typical maturation of the auditory system is perturbed in these individuals during early development, which may underlie altered auditory reactivity that persists in later life. Of the many genes that regulate the auditory system development, loss-of-function mutations in the CNTNAP2 gene are strongly associated with language processing deficits and ASD. Therefore, using a novel Cntnap2 knock-out rat model, we tested the impact of Cntnap2 loss on auditory processing, filtering, and reactivity throughout development and young adulthood in male and female animals. Although hearing thresholds were not altered in Cntnap2 knock-out animals, we found a reduction in response amplitudes and a delay in response latency of the auditory brainstem response (ABR) in juvenile Cntnap2 knock-out rats compared with age-matched controls. Amplitudes and latency of the ABR largely normalized by adulthood, indicating a delayed maturation of auditory processing pathways in Cntnap2 knock-out rats. Despite the reduced ABR amplitudes, adolescent Cntnap2 knock-out animals displayed increased startle reactivity accompanied by disruptions in sensory filtering and sensorimotor gating across various conditions, most of which persisted in adulthood. All of these observations show striking parallels to disruptions reported in ASD. Our results also imply that developmental disruptions of sensory signal processing are associated with persistent changes in neural circuitries responsible for implicit auditory evoked behavior, emphasizing the need for interventions that target sensory processing disruptions early during development in ASD.SIGNIFICANCE STATEMENT This is the first study of brainstem auditory processing in a novel knock-out rat model with very high construct and face validity for autism spectrum disorders. Electrophysiological and behavioral measures of implicit auditory-evoked responses were systematically taken across developmental stages. Auditory processing, filtering, and reactivity disruptions show striking similarities to observations in autism. We also show for the first time that, whereas auditory brainstem responses normalize by adulthood, disruptions in brainstem-mediated auditory-evoked behavior persist. This indicates that early developmental perturbations in sensory processing can cause permanent maladaptive changes in circuitries responsible for auditory reactivity, underlining the importance for interventions early during development aiming at normalizing sensory processing.

Trajectory Analysis Unveils Reelin's Role in the Directed Migration of Granule Cells in the Dentate Gyrus.

Reelin controls neuronal migration and layer formation. Previous studies in reeler mice deficient in Reelin focused on the result of the developmental process in fixed tissue sections. It has remained unclear whether Reelin affects the migratory process, migration directionality, or migrating neurons guided by the radial glial scaffold. Moreover, Reelin has been regarded as an attractive signal because newly generated neurons migrate toward the Reelin-containing marginal zone. Conversely, Reelin might be a stop signal because migrating neurons in reeler, but not in wild-type mice, invade the marginal zone. Here, we monitored the migration of newly generated proopiomelanocortin-EGFP-expressing dentate granule cells in slice cultures from reeler, reeler-like mutants and wild-type mice of either sex using real-time microscopy. We discovered that not the actual migratory process and migratory speed, but migration directionality of the granule cells is controlled by Reelin. While wild-type granule cells migrated toward the marginal zone of the dentate gyrus, neurons in cultures from reeler and reeler-like mutants migrated randomly in all directions as revealed by vector analyses of migratory trajectories. Moreover, live imaging of granule cells in reeler slices cocultured to wild-type dentate gyrus showed that the reeler neurons changed their directions and migrated toward the Reelin-containing marginal zone of the wild-type culture, thus forming a compact granule cell layer. In contrast, directed migration was not observed when Reelin was ubiquitously present in the medium of reeler slices. These results indicate that topographically administered Reelin controls the formation of a granule cell layer.SIGNIFICANCE STATEMENT Neuronal migration and the various factors controlling its onset, speed, directionality, and arrest are poorly understood. Slice cultures offer a unique model to study the migration of individual neurons in an almost natural environment. In the present study, we took advantage of the expression of proopiomelanocortin-EGFP by newly generated, migrating granule cells to analyze their migratory trajectories in hippocampal slice cultures from wild-type mice and mutants deficient in Reelin signaling. We show that the compartmentalized presence of Reelin is essential for the directionality, but not the actual migratory process or speed, of migrating granule cells leading to their characteristic lamination in the dentate gyrus.

The Claudin-like Protein HPO-30 Is Required to Maintain LAChRs at the C. elegans Neuromuscular Junction.

Communications across chemical synapses are primarily mediated by neurotransmitters and their postsynaptic receptors. There are diverse molecular systems to localize and regulate the receptors at the synapse. Here, we identify HPO-30, a member of the claudin superfamily of membrane proteins, as a positive regulator for synaptic localization of levamisole-dependent AChRs (LAChRs) at the Caenorhabditis elegans neuromuscular junction (NMJ). The HPO-30 protein localizes at the NMJ and shows genetic and physical association with the LAChR subunits LEV-8, UNC-29, and UNC-38. Using genetic and electrophysiological assays in the hermaphrodite C. elegans, we demonstrate that HPO-30 functions through Neuroligin at the NMJ to maintain postsynaptic LAChR levels at the synapse. Together, this work suggests a novel function for a tight junction protein in maintaining normal receptor levels at the NMJ.SIGNIFICANCE STATEMENT Claudins are a large superfamily of membrane proteins. Their role in maintaining the functional integrity of tight junctions has been widely explored. Our experiments suggest a critical role for the claudin-like protein, HPO-30, in maintaining synaptic levamisole-dependent AChR (LAChR) levels. LAChRs contribute to <20% of the acetylcholine-mediated conductance in adult Caenorhabditis elegans; however, they play a significant functional role in worm locomotion. This study provides a new perspective in the study of LAChR physiology.

Neuronal growth regulator 1-deficient mice show increased adiposity and decreased muscle mass.

BACKGROUND: Neuronal growth regulator 1 (NEGR1) is a glycosylphosphatidylinositol-anchored membrane protein that mediates neural cell communication and synapse formation. Multiple genome-wide association studies have reported that variations in NEGR1 are associated with human body weight control. Recently, we found that NEGR1 is involved in intracellular cholesterol trafficking, suggesting that it performs a non-central nervous system (CNS) function associated with human obesity. METHODS: We compared peripheral tissues such as the adipose, liver, and skeletal muscle tissues of Negr1-/- and Negr1+/+ (wild-type [WT]) C57BL/6 mice (n = 5-14). Intracellular lipid content was measured, and lipid accumulation was visualized by staining tissue cross-sections with lipid-specific stains. Muscle capacity of the WT and Negr1-/- mice was determined by performing a treadmill endurance test, and muscle fiber size was examined. Plasma glucose and insulin levels were measured, and glucose and insulin tolerance tests were performed. RESULTS: The Negr1-/- mice showed a significant increase in fat mass (~1.5-fold increase in the epididymal white adipose tissue, p = 0.000002), with abnormally enlarged adipose cells, compared with the WT mice. Primary adipocytes of the Negr1-/- mice contained enlarged cytosolic lipid droplets (p = 0.049). Moreover, these mice showed significant hepatic lipid accumulation (~2.3-fold increase, p = 0.043). Although the Negr1-/- mice did not show a significant change in plasma lipoprotein level, they showed a >1.3-fold increase in a serum glucose (p = 0.0002) and insulin (p = 0.016) levels. Moreover, the Negr1-/- mice showed decreased muscle capacity, as indicated by a decrease in muscle mass (p = 0.000003). CONCLUSION: These results indicate that NEGR1 deficiency induces abnormal fat deposition in various peripheral cells, especially fat and liver tissue cells, and suggest that NEGR1 is a potential molecular target for designing anti-obesity drugs to regulate body weight both centrally and peripherally.

Structural basis for ligand capture and release by the endocytic receptor ApoER2.

Apolipoprotein E receptor 2 (ApoER2) is a close homologue of low-density lipoprotein receptor (LDLR) that mediates the endocytosis of ligands, including LDL particles. LDLR family members have been presumed to explore a large conformational space to capture ligands in the extended conformation at the cell surface. Ligands are subsequently released through a pH-titrated structural transition to a self-docked, contracted-closed conformation. In addition to lipoprotein uptake, ApoER2 is implicated in signal transduction during brain development through capture of the extracellular protein reelin. From crystallographic analysis, we determine that the full-length ApoER2 ectodomain adopts an intermediate contracted-open conformation when complexed with the signaling-competent reelin fragment, and we identify a previously unappreciated auxiliary low-affinity binding interface. Based on mutational analyses, we propose that the pH shift during endocytosis weakens the affinity of the auxiliary interface and destabilizes the ligand-receptor complex. Furthermore, this study elucidates that the contracted-open conformation of ligand-bound ApoER2 at neutral pH resembles the contracted-closed conformation of ligand-unbound LDLR at acidic pH in a manner suggestive of being primed for ligand release even prior to internalization.

NEGR1 and FGFR2 cooperatively regulate cortical development and core behaviours related to autism disorders in mice.

Autism spectrum disorders are neurodevelopmental conditions with diverse aetiologies, all characterized by common core symptoms such as impaired social skills and communication, as well as repetitive behaviour. Cell adhesion molecules, receptor tyrosine kinases and associated downstream signalling have been strongly implicated in both neurodevelopment and autism spectrum disorders. We found that downregulation of the cell adhesion molecule NEGR1 or the receptor tyrosine kinase fibroblast growth factor receptor 2 (FGFR2) similarly affects neuronal migration and spine density during mouse cortical development in vivo and results in impaired core behaviours related to autism spectrum disorders. Mechanistically, NEGR1 physically interacts with FGFR2 and modulates FGFR2-dependent extracellular signal-regulated kinase (ERK) and protein kinase B (AKT) signalling by decreasing FGFR2 degradation from the plasma membrane. Accordingly, FGFR2 overexpression rescues all defects due to Negr1 knockdown in vivo. Negr1 knockout mice present phenotypes similar to Negr1-downregulated animals. These data indicate that NEGR1 and FGFR2 cooperatively regulate cortical development and suggest a role for defective NEGR1-FGFR2 complex and convergent downstream ERK and AKT signalling in autism spectrum disorders.

Stabilin-1 expression defines a subset of macrophages that mediate tissue homeostasis and prevent fibrosis in chronic liver injury.

Macrophages are key regulators of fibrosis development and resolution. Elucidating the mechanisms by which they mediate this process is crucial for establishing their therapeutic potential. Here, we use experimental models of liver fibrosis to show that deficiency of the scavenger receptor, stabilin-1, exacerbates fibrosis and delays resolution during the recovery phase. We detected a subset of stabilin-1(+) macrophages that were induced at sites of cellular injury close to the hepatic scar in mouse models of liver fibrosis and in human liver disease. Stabilin-1 deficiency abrogated malondialdehyde-LDL (MDA-LDL) uptake by hepatic macrophages and was associated with excess collagen III deposition. Mechanistically, the lack of stabilin-1 led to elevated intrahepatic levels of the profibrogenic chemokine CCL3 and an increase in GFAP(+) fibrogenic cells. Stabilin-1(-/-) macrophages demonstrated a proinflammatory phenotype during liver injury and the normal induction of Ly6C(lo) monocytes during resolution was absent in stabilin-1 knockouts leading to persistence of fibrosis. Human stabilin-1(+) monocytes efficiently internalized MDA-LDL and this suppressed their ability to secrete CCL3, suggesting that loss of stabilin-1 removes a brake to CCL3 secretion. Experiments with cell-lineage-specific knockouts revealed that stabilin-1 expression in myeloid cells is required for the induction of this subset of macrophages and that increased fibrosis occurs in their absence. This study demonstrates a previously unidentified regulatory pathway in fibrogenesis in which a macrophage scavenger receptor protects against organ fibrosis by removing fibrogenic products of lipid peroxidation. Thus, stabilin-1(+) macrophages shape the tissue microenvironment during liver injury and healing.