Necl-1/CADM3 regulates cone synapse formation in the mouse retina.

In vertebrates, retinal neural circuitry for visual perception is organized in specific layers. The outer plexiform layer is the first synaptic region in the visual pathway, where photoreceptor synaptic terminals connect with bipolar and horizontal cell processes. However, molecular mechanisms underlying cone synapse formation to mediate OFF pathways remain unknown. This study reveals that Necl-1/CADM3 is localized at S- and S/M-opsin-containing cones and dendrites of type 4 OFF cone bipolar cells (CBCs). In Necl-1-/- mouse retina, synapses between cones and type 4 OFF CBCs were dislocated, horizontal cell distribution became abnormal, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors were dislocated. Necl-1-/- mice exhibited aberrant short-wavelength-light-elicited signal transmission from cones to OFF CBCs, which was rescued by AMPA receptor potentiator. Additionally, Necl-1-/- mice showed impaired optokinetic responses. These findings suggest that Necl-1 regulates cone synapse formation to mediate OFF cone pathways elicited by short-wavelength light in mouse retina.

Heterogeneity of perivascular astrocyte endfeet depending on vascular regions in the mouse brain.

Astrocytes interact with not only synapses but also brain blood vessels through perivascular astrocyte endfeet (PV-AEF) to form the neurovascular unit (NVU). However, PV-AEF components have not been fully identified. Here, we biochemically isolated blood vessels from mouse brain homogenates and purified PV-AEF. The purified PV-AEF were observed in different sizes, similar to PV-AEF on brain blood vessels. Mass spectrometry analysis identified 9,762 proteins in the purified PV-AEF, including cell adhesion molecules, nectin-2δ, Kirrel2, and podoplanin. Immunofluorescence microscopic analysis revealed that nectin-2δ and podoplanin were concentrated mainly in arteries/arterioles and veins/venules of the mouse brain, whereas Kirrel2 was mainly in arteries/arterioles. Nectin-2α/δ, Kirrel2, and podoplanin were preferentially observed in large sizes of the purified PV-AEF. Furthermore, Kirrel2 potentially has cell adhesion activity of cultured astrocytes. Collectively, these results indicate that PV-AEF have heterogeneity in sizes and molecular components, implying different roles of PV-AEF in NVU function depending on vascular regions.

s-Afadin binds to MAGUIN/Cnksr2 and regulates the localization of the AMPA receptor and glutamatergic synaptic response in hippocampal neurons.

A hippocampal mossy fiber synapse implicated in learning and memory is a complex structure in which a presynaptic bouton attaches to the dendritic trunk by puncta adherentia junctions (PAJs) and wraps multiply branched spines. The postsynaptic densities (PSDs) are localized at the heads of each of these spines and faces to the presynaptic active zones. We previously showed that the scaffolding protein afadin regulates the formation of the PAJs, PSDs, and active zones in the mossy fiber synapse. Afadin has two splice variants: l-afadin and s-afadin. l-Afadin, but not s-afadin, regulates the formation of the PAJs but the roles of s-afadin in synaptogenesis remain unknown. We found here that s-afadin more preferentially bound to MAGUIN (a product of the Cnksr2 gene) than l-afadin in vivo and in vitro. MAGUIN/CNKSR2 is one of the causative genes for nonsyndromic X-linked intellectual disability accompanied by epilepsy and aphasia. Genetic ablation of MAGUIN impaired PSD-95 localization and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic (AMPA) receptor surface accumulation in cultured hippocampal neurons. Our electrophysiological analysis revealed that the postsynaptic response to glutamate, but not its release from the presynapse, was impaired in the MAGUIN-deficient cultured hippocampal neurons. Furthermore, disruption of MAGUIN did not increase the seizure susceptibility to flurothyl, a GABAA receptor antagonist. These results indicate that s-afadin binds to MAGUIN and regulates the PSD-95-dependent cell surface localization of the AMPA receptor and glutamatergic synaptic responses in the hippocampal neurons and that MAGUIN is not involved in the induction of epileptic seizure by flurothyl in our mouse model.

Necl2/3-mediated mechanism for tripartite synapse formation.

Ramified, polarized protoplasmic astrocytes interact with synapses via perisynaptic astrocyte processes (PAPs) to form tripartite synapses. These astrocyte-synapse interactions mutually regulate their structures and functions. However, molecular mechanisms for tripartite synapse formation remain elusive. We developed an in vitro co-culture system for mouse astrocytes and neurons that induced astrocyte ramifications and PAP formation. Co-cultured neurons were required for astrocyte ramifications in a neuronal activity-dependent manner, and synaptically-released glutamate and activation of astrocytic mGluR5 metabotropic glutamate receptor were likely involved in astrocyte ramifications. Astrocytic Necl2 trans-interacted with axonal Necl3, inducing astrocyte-synapse interactions and astrocyte functional polarization by recruiting EAAT1/2 glutamate transporters and Kir4.1 K+ channel to the PAPs, without affecting astrocyte ramifications. This Necl2/3 trans-interaction increased functional synapse number. Thus, astrocytic Necl2, synaptically-released glutamate and axonal Necl3 cooperatively formed tripartite glutamatergic synapses in vitro. Studies on hippocampal mossy fiber synapses in Necl3 knockout and Necl2/3 double knockout mice confirmed these previously unreported mechanisms for astrocyte-synapse interactions and astrocyte functional polarization in vivo.

Identification of lysophosphatidic acid in serum as a factor that promotes epithelial apical junctional complex organization.

The apical junctional complex (AJC) consists of adherens junctions (AJs) and tight junctions and regulates epithelial integrity and remodeling. However, it is unclear how AJC organization is regulated based on environmental cues. We found here using cultured EpH4 mouse mammary epithelial cells that fetal bovine serum (FBS) in a culture medium showed an activity to promote AJC organization and that FBS showed an activity to promote tight junction formation even in the absence of AJ proteins, such as E-cadherin, αE-catenin, and afadin. Furthermore, we purified the individual factor responsible for these functions from FBS and identified this molecule as lysophosphatidic acid (LPA). In validation experiments, purified LPA elicited the same activity as FBS. In addition, we found that the AJC organization-promoting activity of LPA was mediated through the LPA receptor 1/5 via diacylglycerol-novel PKC and Rho-ROCK pathway activation in a mutually independent, but complementary, manner. We demonstrated that the Rho-ROCK pathway activation-mediated AJC organization was independent of myosin II-induced actomyosin contraction, although this signaling pathway was previously shown to induce myosin II activation. These findings are in contrast to the literature, as previous results suggested an AJC organization-disrupting activity of LPA. The present results indicate that LPA in serum has an AJC organization-promoting activity in a manner dependent on or independent of AJ proteins.

Stimulatory role of nectin-4 and p95-ErbB2 in multilayered T47D cell proliferation.

Multilayered proliferation in an adherent culture as well as proliferation in a suspension culture is a characteristic feature of cancer cells. We previously showed using T47D human mammary cancer cells that nectin-4, upregulated in many cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2ΔEx16, and enhances DNA synthesis mainly through the PI3K-AKT pathway in an adherent culture. We showed here that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and ErbB2 or that of nectin-4 and ErbB2ΔEx16, cooperatively enhanced multilayered T47D cell proliferation through the Hippo pathway-mediated SOX2 gene expression in an adherent culture. T47D cells expressed the components of the apical junctional complex (AJC) consisting of adherens junctions (AJs) and tight junctions and cell polarity molecules, but not the AJ component afadin. The AJC and apicobasal polarity were disorganized in T47D cells in a monolayer and T47D cells stably expressing both nectin-4 and p95-ErbB2 in multilayers. These results indicate that nectin-4 and p95-ErbB2 play a stimulatory role in multilayered proliferation in an adherent culture.

Nectin-2 in general and in the brain.

Nectins are immunoglobulin-like cell adhesion molecules constituting a family with four members, nectin-1, nectin-2, nectin-3, and nectin-4. In the brain, nectin-2 as well as nectin-1 and nectin-3 are expressed whereas nectin-4 is hardly expressed. In the nervous system, physiological functions of nectin-1 and nectin-3, such as synapse formation, mossy fiber trajectory regulation, interneurite affinity, contextual fear memory formation, and stress-related mental disorders, have been revealed. Nectin-2 is ubiquitously expressed in non-neuronal tissues and various nectin-2 functions in non-nervous systems have been extensively investigated, but nectin-2 functions in the brain have not been revealed until recently. Recent findings have revealed that nectin-2 is expressed in the specific areas of the brain and plays important roles, such as homeostasis of astrocytes and neurons and the formation of synapses. Moreover, a single nucleotide polymorphism in the human NECTIN2 gene is associated with Alzheimer's disease. We here summarize recent progress in our understanding of nectin-2 functions in the brain.

A novel role for PRL in regulating epithelial cell density by inducing apoptosis at confluence.

Maintaining proper epithelial cell density is essential for the survival of multicellular organisms. Although regulation of cell density through apoptosis is well known, its mechanistic details remain elusive. Here, we report the involvement of membrane-anchored phosphatase of regenerating liver (PRL), originally known for its role in cancer malignancy, in this process. In epithelial Madin-Darby canine kidney cells, upon confluence, doxycycline-induced expression of PRL upregulated apoptosis, reducing cell density. This could be circumvented by artificially reducing cell density via stretching the cell-seeded silicon chamber. Moreover, small interfering RNA-mediated knockdown of endogenous PRL blocked apoptosis, leading to greater cell density. Mechanistically, PRL promoted apoptosis by upregulating the translation of E-cadherin and activating the TGF-ß pathway. Morpholino-mediated inhibition of PRL expression in zebrafish embryos caused developmental defects, with reduced apoptosis and increased epithelial cell density during convergent extension. Overall, this study revealed a novel role for PRL in regulating density-dependent apoptosis in vertebrate epithelia. This article has an associated First Person interview with the first author of the paper.

Nectins and Nectin-like molecules in synapse formation and involvement in neurological diseases.

Synapses are interneuronal junctions which form neuronal networks and play roles in a variety of functions, including learning and memory. Two types of junctions, synaptic junctions (SJs) and puncta adherentia junctions (PAJs), have been identified. SJs are found at all excitatory and inhibitory synapses whereas PAJs are found at excitatory synapses, but not inhibitory synapses, and particularly well developed at hippocampal mossy fiber giant excitatory synapses. Both SJs and PAJs are mediated by cell adhesion molecules (CAMs). Major CAMs at SJs are neuroligins-neurexins and Nectin-like molecules (Necls)/CADMs/SynCAMs whereas those at PAJs are nectins and cadherins. In addition to synaptic PAJs, extrasynaptic PAJs have been identified at contact sites between neighboring dendrites near synapses and regulate synapse formation. In addition to SJs and PAJs, a new type of cell adhesion apparatus different from these junctional apparatuses has been identified and named nectin/Necl spots. One nectin spot at contact sites between neighboring dendrites at extrasynaptic regions near synapses regulates synapse formation. Several members of nectins and Necls had been identified as viral receptors before finding their physiological functions as CAMs and evidence is accumulating that many nectins and Necls are related to onset and progression of neurological diseases. We review here nectin and Necls in synapse formation and involvement in neurological diseases.

Nectin-4 and p95-ErbB2 cooperatively regulate Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Nectin-4, upregulated in various cancer cells, cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, p95-ErbB2 and ErbB2∆Ex16, enhancing DNA synthesis through the PI3K-AKT signaling in human breast cancer T47D cells in an adherent culture. We found here that nectin-4 and p95-ErbB2, but not nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced SOX2 gene expression and cell proliferation in a suspension culture. This enhancement of T47D cell proliferation in a suspension culture by nectin-4 and p95-ErbB2 was dependent on the SOX2 gene expression. In T47D cells, nectin-4 and any one of p95-ErbB2, ErbB2, or ErbB2∆Ex16 cooperatively activated the PI3K-AKT signaling, known to induce the SOX2 gene expression, to similar extents. However, only a combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively enhanced the SOX2 gene expression. Detailed studies revealed that only nectin-4 and p95-ErbB2 cooperatively activated the Hippo signaling. YAP inhibited the SOX2 gene expression in this cell line and thus the MST1/2-LATS1/2 signaling-mediated YAP inactivation increased the SOX2 gene expression. These results indicate that only the combination of nectin-4 and p95-ErbB2, but not that of nectin-4 and either ErbB2 or ErbB2∆Ex16, cooperatively regulates the Hippo signaling-dependent SOX2 gene expression, enhancing anchorage-independent T47D cell proliferation.

Filopodium-derived vesicles produced by MIM enhance the migration of recipient cells.

Extracellular vesicles (EVs) are classified as large EVs (l-EVs, or microvesicles) and small EVs (s-EVs, or exosomes). S-EVs are thought to be generated from endosomes through a process that mainly depends on the ESCRT protein complex, including ALG-2 interacting protein X (ALIX). However, the mechanisms of l-EV generation from the plasma membrane have not been identified. Membrane curvatures are generated by the bin-amphiphysin-rvs (BAR) family proteins, among which the inverse BAR (I-BAR) proteins are involved in filopodial protrusions. Here, we show that the I-BAR proteins, including missing in metastasis (MIM), generate l-EVs by scission of filopodia. Interestingly, MIM-containing l-EV production was promoted by in vivo equivalent external forces and by the suppression of ALIX, suggesting an alternative mechanism of vesicle formation to s-EVs. The MIM-dependent l-EVs contained lysophospholipids and proteins, including IRS4 and Rac1, which stimulated the migration of recipient cells through lamellipodia formation. Thus, these filopodia-dependent l-EVs, which we named as filopodia-derived vesicles (FDVs), modify cellular behavior.

Recent advances in understanding tight junctions.

Tight junctions (TJs) are one type of cell-cell junction in epithelial cell types in vertebrates. They form a paracellular diffusion barrier and create the boundary between the apical and basolateral plasma membrane domains. The molecular constituents of TJs have mostly been identified, and now their cell biology has shifted to understanding of their formation, dynamics, and functional regulation as well as their relationship to the organization of epithelial cells. Accumulating novel findings are supported by new methods, including super-resolution microscopy, quantitative microscopy, biophysical measurements, and genome editing-mediated gene manipulation. As a conceptual breakthrough, liquid-liquid phase separation seems to be involved in the formation of TJs as super-molecular complexes. This short article summarizes seminal studies in the cell biology of TJs from the last three years.

CD112 Regulates Angiogenesis and T Cell Entry into the Spleen.

Junctional adhesion proteins play important roles in controlling angiogenesis, vascular permeability and leukocyte trafficking. CD112 (nectin-2) belongs to the immunoglobulin superfamily and was shown to engage in homophilic and heterophilic interactions with a variety of binding partners expressed on endothelial cells and on leukocytes. Recent in vitro studies suggested that CD112 regulates human endothelial cell migration and proliferation as well as transendothelial migration of leukocytes. However, so far, the role of CD112 in endothelial cell biology and in leukocyte trafficking has not been elucidated in vivo. We found CD112 to be expressed by lymphatic and blood endothelial cells in different murine tissues. In CD112-deficient mice, the blood vessel coverage in the retina and spleen was significantly enhanced. In functional in vitro studies, a blockade of CD112 modulated endothelial cell migration and significantly enhanced endothelial tube formation. An antibody-based blockade of CD112 also significantly reduced T cell transmigration across endothelial monolayers in vitro. Moreover, T cell homing to the spleen was significantly reduced in CD112-deficient mice. Overall, our results identify CD112 as a regulator of angiogenic processes in vivo and demonstrate a novel role for CD112 in T cell entry into the spleen.

Nectin-2α is localized at cholinergic neuron dendrites and regulates synapse formation in the medial habenula.

The medial habenula (MHb) receives afferents from the triangular septum and the medial septal complex, projects efferents to the interpeduncular nucleus (IPN) in the midbrain to regulate dopamine and serotonin levels, and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed that the cell adhesion molecule nectin-2α is localized at the boundary between adjacent somata of clustered cholinergic neurons and regulates the voltage-gated A-type K+ channel Kv4.2 localization at membrane specializations in the MHb. This adhesion apparatus, named nectin-2α spots, is not associated with the nectin-binding protein afadin or any classic cadherins and their binding proteins p120-catenin and ß-catenin. We showed here that nectin-2α was additionally localized at cholinergic neuron dendrites in synaptic regions of the MHb. The genetic ablation of nectin-2 reduced the number of synapses in the MHb without affecting their morphology. Nectin-2α was associated with afadin, cadherin-8, p120-catenin, ß-catenin, and αN-catenin, forming puncta adherentia junctions (PAJs). Nectin-2α was observed in the IPN, but not in the triangular septum or the medial septal complex. The genetic ablation of nectin-2 did not affect synapse formation in the IPN. These results indicate that nectin-2α forms two types of adhesion apparatus in the MHb, namely nectin-2α spots at neighboring somata and PAJs at neighboring dendrites, and that dendritic PAJs regulate synapse formation in the MHb.

Afadin regulates actomyosin organization through αE-catenin at adherens junctions.

Actomyosin-undercoated adherens junctions are critical for epithelial cell integrity and remodeling. Actomyosin associates with adherens junctions through αE-catenin complexed with ß-catenin and E-cadherin in vivo; however, in vitro biochemical studies in solution showed that αE-catenin complexed with ß-catenin binds to F-actin less efficiently than αE-catenin that is not complexed with ß-catenin. Although a "catch-bond model" partly explains this inconsistency, the mechanism for this inconsistency between the in vivo and in vitro results remains elusive. We herein demonstrate that afadin binds to αE-catenin complexed with ß-catenin and enhances its F-actin-binding activity in a novel mechanism, eventually inducing the proper actomyosin organization through αE-catenin complexed with ß-catenin and E-cadherin at adherens junctions.

Optimizing Nervous System-Specific Gene Targeting with Cre Driver Lines: Prevalence of Germline Recombination and Influencing Factors.

The Cre-loxP system is invaluable for spatial and temporal control of gene knockout, knockin, and reporter expression in the mouse nervous system. However, we report varying probabilities of unexpected germline recombination in distinct Cre driver lines designed for nervous system-specific recombination. Selective maternal or paternal germline recombination is showcased with sample Cre lines. Collated data reveal germline recombination in over half of 64 commonly used Cre driver lines, in most cases with a parental sex bias related to Cre expression in sperm or oocytes. Slight differences among Cre driver lines utilizing common transcriptional control elements affect germline recombination rates. Specific target loci demonstrated differential recombination; thus, reporters are not reliable proxies for another locus of interest. Similar principles apply to other recombinase systems and other genetically targeted organisms. We hereby draw attention to the prevalence of germline recombination and provide guidelines to inform future research for the neuroscience and broader molecular genetics communities.

Identification of Sox2 and NeuN Double-Positive Cells in the Mouse Hypothalamic Arcuate Nucleus and Their Reduction in Number With Aging.

The hypothalamus plays a central role in homeostasis and aging. The hypothalamic arcuate nucleus (ARC) controls homeostasis of food intake and energy expenditure and retains adult neural stem cells (NSCs)/progenitor cells. Aging induces the loss of NSCs and the enhancement of inflammation, including the activation of glial cells in the ARC, but aging-associated alterations of the hypothalamic cells remain obscure. Here, we identified Sox2 and NeuN double-positive cells in a subpopulation of cells in the mouse ARC. These cells were reduced in number with aging, although NeuN-positive neuronal cells were unaltered in the total number. Diet-induced obesity mice fed with high-fat diet presented a similar hypothalamic alteration to aged mice. This study provides a new insight into aging-induced changes in the hypothalamus.

Nectin-4 cis-interacts with ErbB2 and its trastuzumab-resistant splice variants, enhancing their activation and DNA synthesis.

Nectin-4 cell adhesion molecule and ErbB2 tyrosine kinase receptor are upregulated in many cancers, including breast cancer, and promote cancer cell proliferation and metastasis. Using human breast cancer cell lines T47D and SUM190-PT, in which both nectin-4 and ErbB2 were upregulated, we showed here that nectin-4 cis-interacted with ErB2 and enhanced its dimerization and activation, followed by the activation of the phosphoinositide 3-kinase-AKT signalling pathway for DNA synthesis. The third immunoglobulin-like domain of nectin-4 cis-interacted with domain IV of ErbB2. This region differs from the trastuzumab-interacting region but is included in the trastuzumab-resistant splice variants of ErbB2, p95-ErbB2 and ErbB2ΔEx16. Nectin-4 also cis-interacted with these trastuzumab-resistant splice variants and enhanced the activation of the phosphoinositide 3-kinase-AKT signalling pathway for DNA synthesis. In addition, nectin-4 enhanced the activation of the p95-ErbB2-induced JAK-STAT3 signalling pathway, but not the ErbB2- or ErbB2ΔEx16-induced JAK-STAT3 signalling pathway. These results indicate that nectin-4 cis-interacts with ErbB2 and its trastuzumab-resistant splice variants and enhances the activation of these receptors and downstream signalling pathways in a novel mechanism.

Interaction of nectin-2α with the auxiliary protein of the voltage-gated A-type K+ channel Kv4.2 dipeptidyl aminopeptidase-like protein at the boundary between the adjacent somata of clustered cholinergic neurons in the medial habenula.

The medial habenula (MHb) receives septal inputs and sends efferents to the interpeduncular nucleus and is implicated in stress, depression, memory, and nicotine withdrawal syndrome. We previously showed by immunofluorescence microscopy that the cell adhesion molecule nectin-2α is expressed in the cholinergic neurons in the developing and adult mouse MHbs and localized at the boundary between the adjacent somata of clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 is localized. We further showed by immunoelectron microscopy that Kv4.2 is localized at the membrane specializations (MSs) whereas nectin-2α is localized mostly outside of these MSs. In addition, we showed that genetic ablation of nectin-2 delays the localization of Kv4.2 at the MSs in the developing MHb. We investigated here how nectin-2α regulates this localization of Kv4.2 at the MSs. In vitro biochemical analysis revealed that nectin-2α interacted with the auxiliary protein of Kv4.2 dipeptidyl aminopeptidase-like protein 6 (DPP6), but not with Kv4.2 or another auxiliary protein Kv channel-interacting protein 1 (KChIP1). Immunofluorescence microscopy analysis showed that DPP6 was colocalized with nectin-2α at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. Immunoelectron microscopy analysis on this boundary revealed that DPP6 was localized both at the inside and the outside of the MSs. Genetic ablation of nectin-2 did not affect the localization of DPP6 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing and adult MHbs. These results indicate that nectin-2α interacts with DPP6 but regulates the localization of Kv4.2 at the MSs in a DPP6-independent manner.