RESUMEN
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.
Asunto(s)
Proteínas de Microfilamentos , Receptores AMPA , Sinapsis , Animales , Ratones , Homólogo 4 de la Proteína Discs Large/metabolismo , Flurotilo , Hipocampo/metabolismo , Proteínas de Microfilamentos/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Receptores AMPA/metabolismo , Sinapsis/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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.
Asunto(s)
Neuronas Colinérgicas/metabolismo , Habénula/metabolismo , Nectinas/metabolismo , Canales de Potasio Shal/metabolismo , Aminopeptidasas/metabolismo , Animales , Membrana Celular/fisiología , Proteínas de Interacción con los Canales Kv/metabolismo , Potenciales de la Membrana/fisiología , Ratones Endogámicos C57BLRESUMEN
A hippocampal mossy fiber synapse has a complex structure in which presynaptic boutons attach to the dendritic trunk by puncta adherentia junctions (PAJs) and wrap multiply-branched spines, forming synaptic junctions. It was previously shown that afadin regulates the formation of the PAJs cooperatively with nectin-1, nectin-3, and N-cadherin. Afadin is a nectin-binding protein with two splice variants, l-afadin and s-afadin: l-afadin has an actin filament-binding domain, whereas s-afadin lacks it. It remains unknown which variant is involved in the formation of the PAJs or how afadin regulates it. We showed here that re-expression of l-afadin, but not s-afadin, in the afadin-deficient cultured hippocampal neurons in which the PAJ-like structure was disrupted, restored this structure as estimated by the accumulation of N-cadherin and αΝ-catenin. The l-afadin mutant, in which the actin filament-binding domain was deleted, or the l-afadin mutant, in which the αΝ-catenin-binding domain was deleted, did not restore the PAJ-like structure. These results indicate that l-afadin, but not s-afadin, regulates the formation of the hippocampal synapse PAJ-like structure through the binding to actin filaments and αN-catenin. We further found here that l-afadin bound αN-catenin, but not γ-catenin, whereas s-afadin bound γ-catenin, but hardly αN-catenin. These results suggest that the inability of s-afadin to form the hippocampal synapse PAJ-like structure is due to its inability to efficiently bind αN-catenin.
Asunto(s)
Uniones Adherentes/metabolismo , Proteínas de Microfilamentos/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Sinapsis/metabolismo , Actinas/metabolismo , Animales , Sitios de Unión , Cateninas/metabolismo , Células Cultivadas , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteínas de Microfilamentos/química , Proteínas de Microfilamentos/genética , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismoRESUMEN
A synapse is a cell adhesion structure that permits a neuron to pass a chemical or electrical signal to another neuron. They connect neurons and form neural networks that are essential for brain functions, such as learning and memory. At a chemical synapse, the presynapse and the postsynapse are connected by cell adhesion molecules. The presynapse contains synaptic vesicles and their release machinery, whereas the postsynapse contains postsynaptic densities and receptors for the neurotransmitters. Many proteins constituting a synapse have been identified, but their life-span expression profiles remain elusive. Here, we investigated the expression levels of representative synapse-related proteins by Western blot using the extranuclear supernatant fraction of the brains of mice at various ages. These proteins were classified into seven groups depending on their expression profiles during the embryonic stage, those from postnatal day 6 (P6) to P30, and those after P90. The expression levels of the majority of the proteins were gradually increased from the embryonic stage and then decreased at P14 or P30. After P90, the expression levels were not markedly changed or, in some proteins, increased. These results indicate that the expression levels of the synapse-related proteins are regulated orderly in an aging-dependent manner.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/metabolismo , Regulación del Desarrollo de la Expresión Génica , Sinapsis/metabolismo , Animales , Encéfalo/crecimiento & desarrollo , Cadherinas/genética , Cadherinas/metabolismo , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Homólogo 4 de la Proteína Discs Large , Guanilato-Quinasas/genética , Guanilato-Quinasas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Endogámicos C57BL , NectinasRESUMEN
A hippocampal mossy fiber synapse has a complex structure and is implicated in learning and memory. In this synapse, the mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions and wrap around a multiply-branched spine, forming synaptic junctions. We have recently shown using transmission electron microscopy, immunoelectron microscopy and serial block face-scanning electron microscopy that atypical puncta adherentia junctions are formed in the afadin-deficient mossy fiber synapse and that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities and the density of synaptic vesicles docked to active zones are decreased in the afadin-deficient synapse. We investigated here the roles of afadin in the functional differentiations of the mossy fiber synapse using the afadin-deficient mice. The electrophysiological studies showed that both the release probability of glutamate and the postsynaptic responsiveness to glutamate were markedly reduced, but not completely lost, in the afadin-deficient mossy fiber synapse, whereas neither long-term potentiation nor long-term depression was affected. These results indicate that afadin plays roles in the functional differentiations of the presynapse and the postsynapse of the hippocampal mossy fiber synapse.
Asunto(s)
Proteínas de Microfilamentos/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Animales , Células Cultivadas , Ácido Glutámico/metabolismo , Potenciación a Largo Plazo , Ratones , Ratones Endogámicos C57BL , Proteínas de Microfilamentos/genética , Fibras Musgosas del Hipocampo/fisiología , Fibras Musgosas del Hipocampo/ultraestructura , Densidad Postsináptica/metabolismo , Densidad Postsináptica/fisiología , Densidad Postsináptica/ultraestructuraRESUMEN
A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure. We have previously shown using afadin-deficient mice that afadin plays multiple roles in the structural and functional differentiations of this synapse. We investigated here using a co-culture system with cultured hippocampal neurons and non-neuronal COS-7 cells expressing synaptogenic cell adhesion molecules (CAMs) whether afadin is involved in the presynaptic differentiation of hippocampal synapses. Postsynaptic CAMs NGL-3 (alias, a Lrrc4b gene product) and neuroligin induced presynaptic differentiation by trans-interacting with their respective presynaptic binding CAMs LAR (alias, a Ptprf gene product) and neurexin. This activity of NGL-3, but not neuroligin, was dependent on afadin, but not the afadin-binding presynaptic CAM nectin-1. The afadin-binding postsynaptic CAM nectin-3 did not induce presynaptic differentiation. Immunofluorescence and immunoelectron microscopy analyses showed that afadin was localized mainly at puncta adherentia junctions, but partly at synaptic junctions, of the mossy fiber synapse. ß-Catenin and γ-catenin known to bind to LAR were co-immunoprecipitated with afadin from the lysate of mouse brain. These results suggest that afadin is involved in the NGL-3-LAR system-induced presynaptic differentiation of hippocampal neurons cooperatively with ß-catenin and γ-catenin in a nectin-1-independent manner.
Asunto(s)
Proteínas Ligadas a GPI/metabolismo , Hipocampo/metabolismo , Proteínas de Microfilamentos/metabolismo , Fibras Musgosas del Hipocampo/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neurogénesis , Neuronas/metabolismo , Animales , Células COS , Células Cultivadas , Chlorocebus aethiops , Proteínas Ligadas a GPI/genética , Hipocampo/citología , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos ICR , Proteínas de Microfilamentos/genética , Fibras Musgosas del Hipocampo/ultraestructura , Nectinas/genética , Nectinas/metabolismo , Proteínas del Tejido Nervioso/genética , Neuronas/citología , Unión Proteica , beta Catenina/metabolismo , gamma Catenina/metabolismoRESUMEN
The hippocampal formation with tightly packed neurons, mainly at the dentate gyrus, CA3, CA2, and CA1 regions, constitutes a one-way neural circuit, which is associated with learning and memory. We previously showed that the cell adhesion molecules nectins and its binding protein afadin play roles in the formation of the mossy fiber synapses which are formed between the mossy fibers of the dentate gyrus granule cells and the dendrites of the CA3 pyramidal cells. We showed here that in the afadin-deficient hippocampal formation, the dentate gyrus granules cells and the CA3, CA2, and CA1 pyramidal cells were abnormally located; the mossy fiber trajectory was abnormally elongated; the CA3 pyramidal cells were abnormally differentiated; and the densities of the presynaptic boutons on the mossy fibers and the apical dendrites of the CA3 pyramidal cells were decreased. These results indicate that afadin plays roles not only in the formation of the mossy fiber synapses but also in the formation of the cellular architecture of the hippocampus and the dentate gyrus.
Asunto(s)
Región CA3 Hipocampal/citología , Giro Dentado/citología , Proteínas de Microfilamentos/metabolismo , Células Piramidales/citología , Animales , Región CA3 Hipocampal/crecimiento & desarrollo , Región CA3 Hipocampal/metabolismo , Células Cultivadas , Giro Dentado/crecimiento & desarrollo , Giro Dentado/metabolismo , Ratones , Proteínas de Microfilamentos/genética , Fibras Musgosas del Hipocampo/metabolismo , Neurogénesis , Células Piramidales/metabolismo , Sinapsis/metabolismoRESUMEN
Mammary gland development is induced by the actions of various hormones to form a structure consisting of collecting ducts and milk-secreting alveoli, which comprise two types of epithelial cells known as luminal and basal cells. These cells adhere to each other by cell adhesion apparatuses whose roles in hormone-dependent mammary gland development remain largely unknown. Here we identified a novel cell adhesion apparatus at the boundary between the luminal and basal cells in addition to desmosomes. This apparatus was formed by the trans-interaction between the cell adhesion molecules nectin-4 and nectin-1, which were expressed in the luminal and basal cells, respectively. Nectin-4 of this apparatus further cis-interacted with the prolactin receptor in the luminal cells to enhance the prolactin-induced prolactin receptor signaling for alveolar development with lactogenic differentiation. Thus, a novel nectin-mediated cell adhesion apparatus regulates the prolactin receptor signaling for mammary gland development.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Glándulas Mamarias Animales/crecimiento & desarrollo , Receptores de Prolactina/metabolismo , Transducción de Señal , Animales , Adhesión Celular , Moléculas de Adhesión Celular/análisis , Comunicación Celular , Femenino , Células HEK293 , Humanos , Glándulas Mamarias Animales/metabolismo , Glándulas Mamarias Animales/ultraestructura , Ratones Endogámicos C57BL , Nectinas , Prolactina/metabolismoRESUMEN
Proper operation of a neural circuit relies on both excitatory and inhibitory synapses. We previously showed that cell adhesion molecules nectin-1 and nectin-3 are localized at puncta adherentia junctions of the hippocampal mossy fiber glutamatergic excitatory synapses and that they do not regulate the excitatory synaptic transmission onto the CA3 pyramidal cells. We studied here the roles of these nectins in the GABAergic inhibitory synaptic transmission onto the CA3 pyramidal cells using nectin-1-deficient and nectin-3-deficient cultured mouse hippocampal slices. In these mutant slices, the amplitudes and frequencies of miniature excitatory postsynaptic currents were indistinguishable from those in the control slices. In the nectin-1-deficient slices, but not in the nectin-3-deficient slices, however, the amplitude of miniature inhibitory postsynaptic currents (mIPSCs) was larger than that in the control slices, although the frequency of the mIPSCs was not different between these two groups of slices. In the dissociated culture of hippocampal neurons from the nectin-1-deficient mice, the amplitude and frequency of mIPSCs were indistinguishable from those in the control neurons. Nectin-1 was not localized at or near the GABAergic inhibitory synapses. These results indicate that nectin-1 regulates the neuronal activities in the CA3 region of the hippocampus by suppressing the GABAergic inhibitory synaptic transmission.
Asunto(s)
Región CA3 Hipocampal/metabolismo , Moléculas de Adhesión Celular/metabolismo , Neuronas GABAérgicas/metabolismo , Potenciales Postsinápticos Inhibidores , Transmisión Sináptica , Animales , Región CA3 Hipocampal/ultraestructura , Células Cultivadas , Potenciales Postsinápticos Excitadores , Neuronas GABAérgicas/ultraestructura , Glutamatos/metabolismo , Ratones , Nectinas , Células Piramidales/metabolismo , Sinapsis/metabolismo , Sinapsis/ultraestructuraRESUMEN
Activity-dependent synaptic plasticity is a fundamental cellular process for learning and memory. While electrophysiological plasticity has been intensively studied, morphological plasticity is less clearly understood. This study investigated the effect of presynaptic stimulation on the morphology of a giant mossy fiber-CA3 pyramidal cell synapse, and found that the mossy fiber bouton altered its morphology with an increase in the number of segments. This activity-dependent alteration in morphology required the activation of glutamate receptors and an increase in postsynaptic calcium concentration. In addition, the intercellular retrograde messengers nitric oxide and arachidonic acid were necessary. Simultaneous recordings demonstrated that the morphological complexity of the presynaptic bouton and the amplitude of excitatory postsynaptic currents were well correlated. Thus, a single mossy fiber synapse has the potential for activity-dependent morphological plasticity at the presynaptic bouton, which may be important for learning and memory.
Asunto(s)
Región CA3 Hipocampal/fisiología , Potenciales Postsinápticos Excitadores , Fibras Musgosas del Hipocampo/fisiología , Células Piramidales/citología , Animales , Ácido Araquidónico/metabolismo , Región CA3 Hipocampal/citología , Región CA3 Hipocampal/metabolismo , Calcio/metabolismo , Ratones , Ratones Endogámicos C57BL , Fibras Musgosas del Hipocampo/metabolismo , Plasticidad Neuronal , Óxido Nítrico/metabolismo , Células Piramidales/metabolismo , Células Piramidales/fisiología , Receptores de Glutamato/metabolismoRESUMEN
Olfactory mitral cells extend lateral secondary dendrites that contact the lateral secondary and apical primary dendrites of other mitral cells in the external plexiform layer (EPL) of the olfactory bulb. The lateral dendrites further contact granule cell dendrites, forming dendrodendritic reciprocal synapses in the EPL. These dendritic structures are critical for odor information processing, but it remains unknown how they are formed. We recently showed that the immunoglobulin-like cell adhesion molecule nectin-1 constitutes a novel adhesion apparatus at the contacts between mitral cell lateral dendrites, between mitral cell lateral and apical dendrites, and between mitral cell lateral dendrites and granule cell dendritic spine necks in the deep sub-lamina of the EPL of the developing mouse olfactory bulb and named them nectin-1 spots. We investigated here the role of the nectin-1 spots in the formation of dendritic structures in the EPL of the mouse olfactory bulb. We showed that in cultured nectin-1-knockout mitral cells, the number of branching points of mitral cell dendrites was reduced compared to that in the control cells. In the deep sub-lamina of the EPL in the nectin-1-knockout olfactory bulb, the number of branching points of mitral cell lateral dendrites and the number of dendrodendritic reciprocal synapses were reduced compared to those in the control olfactory bulb. These results indicate that the nectin-1 spots regulate the branching of mitral cell dendrites in the deep sub-lamina of the EPL and suggest that the nectin-1 spots are required for odor information processing in the olfactory bulb.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Dendritas/fisiología , Regulación de la Expresión Génica/genética , Neuronas/citología , Bulbo Olfatorio/citología , Actinas/genética , Actinas/metabolismo , Animales , Biotina/análogos & derivados , Moléculas de Adhesión Celular/genética , Células Cultivadas , Dextranos , Embrión de Mamíferos , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Imagenología Tridimensional , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Proteínas Asociadas a Microtúbulos/metabolismo , Nectinas , Proteínas del Tejido Nervioso/metabolismo , Ubiquitina Tiolesterasa/metabolismo , Proteína 1 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
l-Afadin was originally purified from rat brain as an actin filament (F-actin)-binding protein that was homologous to the AF-6 gene product. Concomitantly, s-afadin that did not show an F-actin-binding capability was copurified with l-afadin. Structurally, s-afadin lacks the C-terminal F-actin-binding domain but has two short sequences that were not present in l-afadin. The properties and roles of l-afadin have intensively been investigated, but those of s-afadin have poorly been understood. We show here an additional difference in their biochemical properties other than binding to F-actin between l-afadin and s-afadin. Both l-afadin and s-afadin bound to nectins, immunoglobulin-like cell adhesion molecules, whereas s-afadin more preferentially bound to nectins than l-afadin. The PDZ domain of l-afadin and s-afadin was essential for their binding to nectin-3. The dilute domain of l-afadin negatively regulated its binding to nectin-3, but the deletion of the C-terminal F-actin-binding domain of l-afadin did not increase the binding of l-afadin to nectin-3. These results indicate that the s-afadin-specific C-terminal inserts may be involved in its preference of binding to nectin-3 and raise the possibility that there are proteins other than nectins that more preferentially bind s-afadin than l-afadin.
Asunto(s)
Moléculas de Adhesión Celular/metabolismo , Proteínas de Microfilamentos/metabolismo , Neuronas/metabolismo , Actinas/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/metabolismo , Adhesión Celular , Células Cultivadas , Ratones Endogámicos C57BL , Nectinas , Unión ProteicaRESUMEN
Mammalian tissues and organs are composed of different types of cells that adhere to each other homotypically (i.e. interactions between cells of the same cell type) or heterotypically (i.e. interactions between different cell types), forming a variety of cellular patterns, including mosaic patterns. At least three types of cell-cell adhesion have been observed: symmetric homotypic, asymmetric homotypic and heterotypic cell adhesions. Cadherins and nectins, which are known cell-cell adhesion molecules, mediate these cell adhesions. Cadherins comprise a family of more than 100 members, but they are primarily involved in homophilic trans-interactions (i.e. interactions between the same cadherin members) between opposing cells. By contrast, the nectin family comprises only four members, and these proteins form both homophilic and heterophilic trans-interactions (i.e. interactions between the same and different nectin members on opposing cells). In addition, heterophilic trans-interactions between nectins are much stronger than homophilic trans-interactions. Because of these unique properties, nectins have crucial roles in asymmetric homotypic cell-cell adhesion at neuronal synapses and in various types of heterotypic cell-cell adhesions. We summarize recent progress in our understanding of the biology of nectins and discuss their roles in heterotypic cell-cell adhesions, whose formation cannot be solely explained by the action of cadherins.
Asunto(s)
Moléculas de Adhesión Celular/fisiología , Adhesión Celular/fisiología , Uniones Adherentes/fisiología , Animales , Moléculas de Adhesión Celular/genética , Moléculas de Adhesión Celular/metabolismo , Humanos , NectinasRESUMEN
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.
RESUMEN
The immunogroblin (Ig) superfamily proteins characterized by the presence of Ig-like domains are involved in various cellular functions. The properties of the Ig-like domains to form rod-like structures and to bind specifically to other proteins make them ideal for cell surface receptors and cell adhesion molecules (CAMs). Ig-CAMs, nectins in mammals and Echinoid in Drosophila, are crucial components of cadherin-based adherens junctions in the epithelium. Nectins form cell-cell adhesion by their trans-interactions and recruit cadherins to the nectin-initiated cell-cell adhesion site to establish adherens junctions. Thereafter junction adhesion molecules, occludin, and claudins, are recruited to the apical side of adherens junctions to establish tight junctions. The recruitment of these molecules by nectins is mediated both by the direct and indirect interactions of afadin with many proteins, such as catenins, and zonula occludens proteins, and by the nectin-induced reorganization of the actin cytoskeleton. Nectins contribute to the formation of both homotypic and heterotypic types of cell-cell junctions, such as synapses in the brain, contacts between pigment and non-pigment cell layers of the ciliary epithelium in the eye, Sertoli cell-spermatid junctions in the testis, and sensory cells and supporting cells in the sensory organs. In addition, cis- and trans-interactions of nectins with various cell surface proteins, such as integrins, growth factor receptors, and nectin-like molecules (Necls) play important roles in the regulation of many cellular functions, such as cell polarization, movement, proliferation, differentiation, survival, and cell sorting. Furthermore, the Ig-CAMs are implicated in many human diseases including viral infections, ectodermal dysplasia, cancers, and Alzheimer's disease.
Asunto(s)
Uniones Adherentes/metabolismo , Moléculas de Adhesión Celular/metabolismo , Adhesión Celular/fisiología , Inmunoglobulinas/metabolismo , Receptores Inmunológicos/metabolismo , Animales , Humanos , Transducción de SeñalRESUMEN
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.
Asunto(s)
Neuronas Colinérgicas/química , Dendritas/química , Habénula/química , Nectinas/análisis , Sinapsis/química , Secuencia de Aminoácidos , Animales , Animales Recién Nacidos , Neuronas Colinérgicas/metabolismo , Dendritas/genética , Dendritas/metabolismo , Habénula/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Nectinas/deficiencia , Nectinas/genética , Sinapsis/genética , Sinapsis/metabolismoRESUMEN
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.
Asunto(s)
Marcación de Gen/métodos , Integrasas/genética , Neuronas/metabolismo , Oocitos/metabolismo , Recombinación Genética/genética , Espermatozoides/metabolismo , Animales , Femenino , Genes Reporteros , Células Germinativas , Masculino , Ratones , Ratones Transgénicos , MosaicismoRESUMEN
The medial habenula (MHb), implicated in stress, depression, memory, and nicotine withdrawal syndromes, receives septal inputs and sends efferents to the interpeduncular nucleus. We previously showed that the immunoglobulin-like cell adhesion molecules (CAMs) nectin-2α and nectin-2δ are expressed in astrocytes in the brain, but their expression in neurons remains unknown. We showed here by immunofluorescence microscopy that nectin-2α, but not nectin-2δ, was prominently expressed in the cholinergic neurons in the developing and adult MHbs and localized at the boundary between the adjacent somata of the clustered cholinergic neurons where the voltage-gated A-type K+ channel Kv4.2 was localized. Analysis by immunoelectron microscopy on this boundary revealed that Kv4.2 was localized at the membrane specializations (MSs) with plasma membrane darkening in an asymmetrical manner, whereas nectin-2α was localized on the apposed plasma membranes mostly at the outside of these MSs, but occasionally localized at their edges and insides. Nectin-2α at this boundary was not colocalized with the nectin-2α-binding protein afadin, other CAMs, or their interacting peripheral membrane proteins, suggesting that nectin-2α forms a cell adhesion apparatus different from the Kv4.2-associated MSs. Genetic ablation of nectin-2 delayed the localization of Kv4.2 at the boundary between the adjacent somata of the clustered cholinergic neurons in the developing MHb. These results revealed the unique localization of nectin-2α and its regulatory role in the localization of Kv4.2 at the MSs in the MHb.
Asunto(s)
Neuronas Colinérgicas/metabolismo , Habénula/citología , Nectinas/metabolismo , Terminales Presinápticos/metabolismo , Canales de Potasio Shal/metabolismo , Fracciones Subcelulares/metabolismo , Animales , Animales Recién Nacidos , Neuronas Colinérgicas/citología , Regulación de la Expresión Génica/genética , Habénula/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Nectinas/genética , Proteínas del Tejido Nervioso/metabolismo , Fosfopiruvato Hidratasa/metabolismo , beta-Galactosidasa/genética , beta-Galactosidasa/metabolismoRESUMEN
A hippocampal mossy fiber synapse, which is implicated in learning and memory, has a complex structure in which mossy fiber boutons attach to the dendritic shaft by puncta adherentia junctions (PAJs) and wrap around a multiply-branched spine, forming synaptic junctions. Here, we electron microscopically analyzed the ultrastructure of this synapse in afadin-deficient mice. Transmission electron microscopy analysis revealed that typical PAJs with prominent symmetrical plasma membrane darkening undercoated with the thick filamentous cytoskeleton were observed in the control synapse, whereas in the afadin-deficient synapse, atypical PAJs with the symmetrical plasma membrane darkening, which was much less in thickness and darkness than those of the control typical PAJs, were observed. Immunoelectron microscopy analysis revealed that nectin-1, nectin-3, and N-cadherin were localized at the control typical PAJs, whereas nectin-1 and nectin-3 were localized at the afadin-deficient atypical PAJs to extents lower than those in the control synapse and N-cadherin was localized at their nonjunctional flanking regions. These results indicate that the atypical PAJs are formed by nectin-1 and nectin-3 independently of afadin and N-cadherin and that the typical PAJs are formed by afadin and N-cadherin cooperatively with nectin-1 and nectin-3. Serial block face-scanning electron microscopy analysis revealed that the complexity of postsynaptic spines and mossy fiber boutons, the number of spine heads, the area of postsynaptic densities, and the density of synaptic vesicles docked to active zones were decreased in the afadin-deficient synapse. These results indicate that afadin plays multiple roles in the complex ultrastructural morphogenesis of hippocampal mossy fiber synapses.
Asunto(s)
Hipocampo/citología , Proteínas de Microfilamentos/metabolismo , Morfogénesis/fisiología , Fibras Musgosas del Hipocampo/ultraestructura , Neuronas/ultraestructura , Sinapsis/metabolismo , Animales , Cadherinas/metabolismo , Adhesión Celular/fisiología , Moléculas de Adhesión Celular/metabolismo , Dendritas/metabolismo , Dendritas/ultraestructura , Regulación de la Expresión Génica/genética , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas de Microfilamentos/genética , Modelos Neurológicos , Fibras Musgosas del Hipocampo/metabolismo , Nectinas/metabolismo , Neuronas/metabolismo , Canales de Potasio/genética , Canales de Potasio/metabolismo , Canales de potasio activados por Sodio , Terminales Presinápticos/metabolismo , Terminales Presinápticos/ultraestructura , Sinapsis/ultraestructura , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Nectins are Ca2+-independent immunoglobulin-like cell-cell adhesion molecules. In the nervous system, among four members (nectin-1, -2, -3, and -4), nectin-1 and -3 are asymmetrically localized at puncta adherentia junctions formed between the mossy fiber terminals and the dendrites of CA3 pyramidal neurons in the mouse hippocampus and heterophilic trans-interactions between nectin-1 and nectin-3 are involved in the selective interaction of axons and dendrites of cultured neurons. By contrast, nectin-2, which has two splicing variants, nectin-2α and -2δ, has not been well characterized in the brain. We showed here that nectin-2α was expressed in both cultured mouse neurons and astrocytes whereas nectin-2δ was selectively expressed in the astrocytes. Nectin-2δ was localized at the adhesion sites between adjacent cultured astrocytes, but in the brain it was localized on the plasma membranes of astrocytic perivascular endfoot processes facing the basement membrane of blood vessels. Genetic ablation of nectin-2 caused degeneration of astrocytic perivascular endfoot processes and neurons in the cerebral cortex. These results uncovered for the first time the localization and critical functions of nectin-2 in the brain.