The IgSF Cell Adhesion Protein CLMP and Congenital Short Bowel Syndrome (CSBS).

The immunoglobulin-like cell adhesion molecule CLMP is a member of the CAR family of cell adhesion proteins and is implicated in human congenital short-bowel syndrome (CSBS). CSBS is a rare but very severe disease for which no cure is currently available. In this review, we compare data from human CSBS patients and a mouse knockout model. These data indicate that CSBS is characterized by a defect in intestinal elongation during embryonic development and impaired peristalsis. The latter is driven by uncoordinated calcium signaling via gap junctions, which is linked to a reduction in connexin43 and 45 levels in the circumferential smooth muscle layer of the intestine. Furthermore, we discuss how mutations in the CLMP gene affect other organs and tissues, including the ureter. Here, the absence of CLMP produces a severe bilateral hydronephrosis-also caused by a reduced level of connexin43 and associated uncoordinated calcium signaling via gap junctions.

Neuregulin 3 promotes excitatory synapse formation on hippocampal interneurons.

Hippocampal GABAergic interneurons are crucial for cortical network function and have been implicated in psychiatric disorders. We show here that Neuregulin 3 (Nrg3), a relatively little investigated low-affinity ligand, is a functionally dominant interaction partner of ErbB4 in parvalbumin-positive (PV) interneurons. Nrg3 and ErbB4 are located pre- and postsynaptically, respectively, in excitatory synapses on PV interneurons in vivo Additionally, we show that ablation of Nrg3 results in a similar phenotype as the one described for ErbB4 ablation, including reduced excitatory synapse numbers on PV interneurons, altered short-term plasticity, and disinhibition of the hippocampal network. In culture, presynaptic Nrg3 increases excitatory synapse numbers on ErbB4+ interneurons and affects short-term plasticity. Nrg3 mutant neurons are poor donors of presynaptic terminals in the presence of competing neurons that produce recombinant Nrg3, and this bias requires postsynaptic ErbB4 but not ErbB4 kinase activity. Furthermore, when presented by non-neuronal cells, Nrg3 induces postsynaptic membrane specialization. Our data indicate that Nrg3 provides adhesive cues that facilitate excitatory neurons to synapse onto ErbB4+ interneurons.

The CAR group of Ig cell adhesion proteins-Regulators of gap junctions?

Members of the CAR group of Ig-like type I transmembrane proteins mediate homotypic cell adhesion, share a common overall extracellular domain structure and are closely related at the amino acid sequence level. CAR proteins are often found at tight junctions and interact with intracellular scaffolding proteins, suggesting that they might modulate tight junction assembly or function. However, impairment of tight junction integrity has not been reported in mouse knockout models or zebrafish mutants of CAR members. In contrast, in the same knockout models deficits in gap junction communication were detected in several organ systems, including the atrioventricular node of the heart, smooth muscle cells of the intestine and the ureter and in Sertoli cells of the testes. Possible interactions between BT-IgSF and connexin41.8 on the disturbed pattern of pigment stripes found in zebrafish mutants and between ESAM and connexin43 during hematopoiesis in the mouse are also discussed. On the basis of the combined data and phenotypic similarities between CAR member mutants and connexin mutants I hypothesize that they primarily play a role in the organization of gap junction communication. Also see the video abstract here: https://youtu.be/i0yq2KhuDAE.

The role of agrin, Lrp4 and MuSK during dendritic arborization and synaptogenesis in cultured embryonic CNS neurons.

The role of agrin, Lrp4 and MuSK, key organizers of neuromuscular synaptogenesis, in the developing CNS is only poorly understood. We investigated the role of these proteins in cultured mouse embryonic cortical neurons from wildtype and from Lrp4- and MuSK-deficient mice. Neurons from Lrp4-deficient mice had fewer but longer primary dendrites and a decreased density of puncta containing excitatory and inhibitory synapse-associated proteins. Neurons from MuSK-deficient mice had an altered dendritic branching pattern but no change in the density of puncta stained by antibodies against synapse-associated proteins. Transfection of TM-agrin compensated the dendritic branching deficits in Lrp4-deficient but not in MuSK-deficient neurons. TM-agrin transfection increased the density of excitatory synaptic puncta in MuSK-deficient but not in Lrp4-deficient mice and reduced the number of inhibitory synaptic puncta irrespective of MuSK and Lrp4 expression. Addition of purified soluble agrin to microisland cultures of cortical neurons revealed an Lrp4-dependent increase in the size and density of glutamatergic synaptic puncta and in mEPSC but not in mIPSC frequency and amplitude. Thus, agrin induced an Lrp4-independent increase in dendritic branch complexity, an Lrp4-dependent increase of excitatory synaptic puncta and an Lrp4- and MuSK-independent decrease in the density of puncta containing inhibitory synapse-associated proteins. These results establish selective roles for agrin, Lrp4 and MuSK during dendritogenesis and synaptogenesis in cultured CNS neurons.

Regulation of the Natriuretic Peptide Receptor 2 (Npr2) by Phosphorylation of Juxtamembrane Serine and Threonine Residues Is Essential for Bifurcation of Sensory Axons.

cGMP signaling elicited by activation of the transmembrane receptor guanylyl cyclase Npr2 (also known as guanylyl cyclase B) by the ligand CNP controls sensory axon bifurcation of DRG and cranial sensory ganglion (CSG) neurons entering the spinal cord or hindbrain, respectively. Previous studies have shown that Npr2 is phosphorylated on serine and threonine residues in its kinase homology domain (KHD). However, it is unknown whether phosphorylation of Npr2 is essential for axon bifurcation. Here, we generated a knock-in mouse line in which the seven regulatory serine and threonine residues in the KHD of Npr2 were substituted by alanine (Npr2-7A), resulting in a nonphosphorylatable enzyme. Real-time imaging of cGMP in DRG neurons with a genetically encoded fluorescent cGMP sensor or biochemical analysis of guanylyl cyclase activity in brain or lung tissue revealed the absence of CNP-induced cGMP generation in the Npr27A/7A mutant. Consequently, bifurcation of axons, but not collateral formation, from DRG or CSG in this mouse mutant was perturbed at embryonic and mature stages. In contrast, axon branching was normal in a mouse mutant in which constitutive phosphorylation of Npr2 is mimicked by a replacement of all of the seven serine and threonine sites by glutamic acid (Npr2-7E). Furthermore, we demonstrate that the Npr27A/7A mutation causes dwarfism as described for global Npr2 mutants. In conclusion, our in vivo studies provide strong evidence that phosphorylation of the seven serine and threonine residues in the KHD of Npr2 is an important regulatory element of Npr2-mediated cGMP signaling which affects physiological processes, such as axon bifurcation and bone growth.SIGNIFICANCE STATEMENT The branching of axons is a morphological hallmark of virtually all neurons. It allows an individual neuron to innervate different targets and to communicate with neurons located in different regions of the nervous system. The natriuretic peptide receptor 2 (Npr2), a transmembrane guanylyl cyclase, is essential for the initiation of bifurcation of sensory axons when entering the spinal cord or the hindbrain. By using two genetically engineered mouse lines, we show that phosphorylation of specific serine and threonine residues in juxtamembrane regions of Npr2 are required for its enzymatic activity and for axon bifurcation. These investigations might help to understand the regulation of Npr2 and its integration in intracellular signaling systems.

The cell adhesion molecule BT-IgSF is essential for a functional blood-testis barrier and male fertility in mice.

The Ig-like cell adhesion molecule (IgCAM) BT-IgSF (brain- and testis-specific Ig superfamily protein) plays a major role in male fertility in mice. However, the molecular mechanism by which BT-IgSF supports fertility is unclear. Here, we found that it is localized in Sertoli cells at the blood-testis barrier (BTB) and at the apical ectoplasmic specialization. The absence of BT-IgSF in Sertoli cells in both global and conditional mouse mutants (i.e. AMHCre and Rosa26CreERT2 lines) resulted in male infertility, atrophic testes with vacuolation, azoospermia, and spermatogenesis arrest. Although transcripts of junctional proteins such as connexin43, ZO-1, occludin, and claudin11 were up-regulated in the absence of BT-IgSF, the functional integrity of the BTB was impaired, as revealed by injection of a BTB-impermeable component into the testes under in vivo conditions. Disruption of the BTB coincided with mislocalization of connexin43, which was present throughout the seminiferous epithelium and not restricted to the BTB as in wild-type tissues, suggesting impaired cell-cell communication in the BT-IgSF-KO mice. Because EM images revealed a normal BTB structure between Sertoli cells in the BT-IgSF-KO mice, we conclude that infertility in these mice is most likely caused by a functionally impaired BTB. In summary, our results indicate that BT-IgSF is expressed at the BTB and is required for male fertility by supporting the functional integrity of the BTB.

Cell-cell communication mediated by the CAR subgroup of immunoglobulin cell adhesion molecules in health and disease.

The immunoglobulin superfamily represents a diverse set of cell-cell contact proteins and includes well-studied members such as NCAM1, DSCAM, L1 or the contactins which are strongly expressed in the nervous system. In this review we put our focus on the biological function of a less understood subgroup of Ig-like proteins composed of CAR (coxsackievirus and adenovirus receptor), CLMP (CAR-like membrane protein) and BT-IgSF (brain and testis specific immunoglobulin superfamily). The CAR-related proteins are type I transmembrane proteins containing an N-terminal variable (V-type) and a membrane proximal constant (C2-type) Ig domain in their extracellular region which are implicated in homotypic adhesion. They are highly expressed during embryonic development in a variety of tissues including the nervous system whereby in adult stages the protein level of CAR and CLMP decreases, only BT-IgSF expression increases within age. CAR-related proteins are concentrated at specialized cell-cell communication sites such as gap or tight junctions and are present at the plasma membrane in larger protein complexes. Considerable progress has been made on the molecular structure and interactions of CAR while research on CLMP and BT-IgSF is at an early stage. Studies on mouse mutants revealed biological functions of CAR in the heart and for CLMP in the gastrointestinal and urogenital systems. Furthermore, CAR and BT-IgSF appear to regulate synaptic function in the hippocampus.

Molecular Analysis of Sensory Axon Branching Unraveled a cGMP-Dependent Signaling Cascade.

Axonal branching is a key process in the establishment of circuit connectivity within the nervous system. Molecular-genetic studies have shown that a specific form of axonal branching—the bifurcation of sensory neurons at the transition zone between the peripheral and the central nervous system—is regulated by a cyclic guanosine monophosphate (cGMP)-dependent signaling cascade which is composed of C-type natriuretic peptide (CNP), the receptor guanylyl cyclase Npr2, and cGMP-dependent protein kinase Iα (cGKIα). In the absence of any one of these components, neurons in dorsal root ganglia (DRG) and cranial sensory ganglia no longer bifurcate, and instead turn in either an ascending or a descending direction. In contrast, collateral axonal branch formation which represents a second type of axonal branch formation is not affected by inactivation of CNP, Npr2, or cGKI. Whereas axon bifurcation was lost in mouse mutants deficient for components of CNP-induced cGMP formation; the absence of the cGMP-degrading enzyme phosphodiesterase 2A had no effect on axon bifurcation. Adult mice that lack sensory axon bifurcation due to the conditional inactivation of Npr2-mediated cGMP signaling in DRG neurons demonstrated an altered shape of sensory axon terminal fields in the spinal cord, indicating that elaborate compensatory mechanisms reorganize neuronal circuits in the absence of bifurcation. On a functional level, these mice showed impaired heat sensation and nociception induced by chemical irritants, whereas responses to cold sensation, mechanical stimulation, and motor coordination are normal. These data point to a critical role of axon bifurcation for the processing of acute pain perception.

Dorsal root ganglion axon bifurcation tolerates increased cyclic GMP levels: the role of phosphodiesterase 2A and scavenger receptor Npr3.

A cyclic GMP (cGMP) signaling pathway, comprising C-type natriuretic peptide (CNP), its guanylate cyclase receptor Npr2, and cGMP-dependent protein kinase I, is critical for the bifurcation of dorsal root ganglion (DRG) and cranial sensory ganglion axons when entering the mouse spinal cord and the hindbrain respectively. However, the identity and functional relevance of phosphodiesterases (PDEs) that degrade cGMP in DRG neurons are not completely understood. Here, we asked whether regulation of the intracellular cGMP concentration by PDEs modulates the branching of sensory axons. Real-time imaging of cGMP with a genetically encoded fluorescent cGMP sensor, RT-PCR screens, in situ hybridization, and immunohistology combined with the analysis of mutant mice identified PDE2A as the major enzyme for the degradation of CNP-induced cGMP in embryonic DRG neurons. Tracking of PDE2A-deficient DRG sensory axons in conjunction with cGMP measurements indicated that axon bifurcation tolerates increased cGMP concentrations. As we found that the natriuretic peptide scavenger receptor Npr3 is expressed by cells associated with dorsal roots but not in DRG neurons itself at early developmental stages, we analyzed axonal branching in the absence of Npr3. In Npr3-deficient mice, the majority of sensory axons showed normal bifurcation, but a small population of axons (13%) was unable to form T-like branches and generated turns in rostral or caudal directions only. Taken together, this study shows that sensory axon bifurcation is insensitive to increases of CNP-induced cGMP levels and Npr3 does not have an important scavenging function in this axonal system.

Bifurcation of axons from cranial sensory neurons is disabled in the absence of Npr2-induced cGMP signaling.

Axonal branching is a prerequisite for the establishment of complex neuronal circuits and their capacity for parallel information processing. Previously, we have identified a cGMP signaling pathway composed of the ligand C-type natriuretic peptide (CNP), its receptor, the guanylyl cyclase natriuretic peptide receptor 2 (Npr2), and the cGMP-dependent kinase Iα (cGKIα) that regulates axon bifurcation of dorsal root ganglion (DRG) neurons in the spinal cord. Now we asked whether this cascade also controls axon bifurcation elsewhere in the nervous system. An Npr2-lacZ reporter mouse line was generated to clarify the pattern of the CNP receptor expression. It was found that during the period of axonal outgrowth, Npr2 and cGKIα were strongly labeled in neurons of all cranial sensory ganglia (gV, gVII, gVIII, gIX, and gX). In addition, strong complementary expression of CNP was detected in the hindbrain at the entry zones of sensory afferents. To analyze axon branching in individual Npr2-positive neurons, we generated a mouse mutant expressing a tamoxifen-inducible variant of Cre recombinase expressed under control of the Npr2-promoter (Npr2-CreER(T2)). After crossing this strain with conditional reporter mouse lines, we revealed that the complete absence of Npr2 activity indeed prohibited the bifurcation of cranial sensory axons in their entrance region. Consequently, axons only turned in either an ascending or descending direction, while collateral formation and growth of the peripheral arm was not affected. These findings indicate that in neurons of the cranial sensory ganglia, as in DRG neurons, cGMP signals are necessary for the execution of an axonal bifurcation program.

The IgCAM BT-IgSF (IgSF11) is essential for connexin43-mediated astrocyte-astrocyte coupling in mice.

The type I transmembrane protein BT-IgSF is predominantly localized in the brain and testes. It belongs to the CAR subgroup of Ig cell adhesion proteins, that are hypothesized to regulate connexin expression or localization. Here, we studied the putative link between BT-IgSF and connexins in astrocytes, ependymal cells and neurons of the mouse. Global knockout of BT-IgSF caused an increase in the clustering of connexin43 (Gja1), but not of connexin30 (Gjb6), on astrocytes and ependymal cells. Additionally, knockout animals displayed reduced expression levels of connexin43 protein in the cortex and hippocampus. Importantly, analysis of biocytin spread in hippocampal or cortical slices from mature mice of either sex revealed a decrease in astrocytic cell-cell coupling in the absence of BT-IgSF. Blocking either protein biosynthesis or proteolysis showed that the lysosomal pathway increased connexin43 degradation in astrocytes. Localization of connexin43 in subcellular compartments was not impaired in astrocytes of BT-IgSF mutants. In contrast to connexin43 the localization and expression of connexin36 (Gjd2) on neurons was not affected by the absence of BT-IgSF. Overall, our data indicate that the IgCAM BT-IgSF is essential for correct gap junction-mediated astrocyte-to-astrocyte cell communication.Significance Statement Astrocytes regulate a variety of physiological processes in the developing and adult brain that are essential for proper brain function. Astrocytes form extensive networks in the brain and communicate via gap junctions. Disruptions of gap junction coupling are found in several diseases such as neurodegeneration or epilepsy. Here, we demonstrate that the cell adhesion protein BT-IgSF is essential for gap junction mediated coupling between astrocytes in the cortex and hippocampus.

Impaired presynaptic function and elimination of synapses at premature stages during postnatal development of the cerebellum in the absence of CALEB (CSPG5/neuroglycan C).

Chicken acidic leucine-rich EGF-like domain-containing brain protein (CALEB), also known as chondroitin sulfate proteoglycan (CSPG)5 or neuroglycan C, is a neural chondroitin sulfate-containing and epidermal growth factor (EGF)-domain-containing transmembrane protein that is implicated in synaptic maturation. Here, we studied the role of CALEB within the developing cerebellum. Adult CALEB-deficient mice displayed impaired motor coordination in Rota-Rod experiments. Analysis of the neuronal connectivity of Purkinje cells by patch-clamp recordings demonstrated impairments of presynaptic maturation of inhibitory synapses. GABAergic synapses on Purkinje cells revealed decreased evoked amplitudes, altered paired-pulse facilitation and reduced depression after repetitive stimulation at early postnatal but not at mature stages. Furthermore, the elimination of supernumerary climbing fiber synapses on Purkinje cells was found to occur at earlier developmental stages in the absence of CALEB. For example, at postnatal day 8 in wild-type mice, 54% of Purkinje cells had three or more climbing fiber synapses in contrast to mutants where this number was decreased to less than 25%. The basic properties of the climbing fiber Purkinje cell synapse remained unaffected. Using Sholl analysis of dye-injected Purkinje cells we revealed that the branching pattern of the dendritic tree of Purkinje cells was not impaired in CALEB-deficient mice. The alterations observed by patch-clamp recordings correlated with a specific pattern and timing of expression of CALEB in Purkinje cells, i.e. it is dynamically regulated during development from a high chondroitin sulfate-containing form to a non-chondroitin sulfate-containing form. Thus, our results demonstrated an involvement of CALEB in the presynaptic differentiation of cerebellar GABAergic synapses and revealed a new role for CALEB in synapse elimination in Purkinje cells.

Retinal pigment epithelium protein of 65 kDA gene-linked retinal degeneration is not modulated by chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C/ chondroitin sulfate proteoglycan 5.

PURPOSE: To analyze in vivo the function of chicken acidic leucine-rich epidermal growth factor-like domain containing brain protein/Neuroglycan C (gene symbol: Cspg5) during retinal degeneration in the Rpe65⁻/⁻ mouse model of Leber congenital amaurosis. METHODS: We resorted to mice with targeted deletions in the Cspg5 and retinal pigment epithelium protein of 65 kDa (Rpe65) genes (Cspg5⁻/⁻/Rpe65⁻/⁻). Cone degeneration was assessed with cone-specific peanut agglutinin staining. Transcriptional expression of rhodopsin (Rho), S-opsin (Opn1sw), M-opsin (Opn1mw), rod transducin α subunit (Gnat1), and cone transducin α subunit (Gnat2) genes was assessed with quantitative PCR from 2 weeks to 12 months. The retinal pigment epithelium (RPE) was analyzed at P14 with immunodetection of the retinol-binding protein membrane receptor Stra6. RESULTS: No differences in the progression of retinal degeneration were observed between the Rpe65⁻/⁻ and Cspg5⁻/⁻/Rpe65⁻/⁻ mice. No retinal phenotype was detected in the late postnatal and adult Cspg5⁻/⁻ mice, when compared to the wild-type mice. CONCLUSIONS: Despite the previously reported upregulation of Cspg5 during retinal degeneration in Rpe65⁻/⁻ mice, no protective effect or any involvement of Cspg5 in disease progression was identified.

Regulation of adhesion by flexible ectodomains of IgCAMs.

To perform their diverse biological functions the adhesion activities of the cell adhesion molecules of the immunoglobulin superfamily (IgCAMs) might be regulated by local clustering, proteolytical shedding of their ectodomains or rapid recycling to and from the plasma membrane. Another form of regulation of adhesion might be obtained through flexible ectodomains of IgCAMs which adopt distinct conformations and which in turn modulate their adhesion activity. Here, we discuss variations in the conformation of the extracellular domains of CEACAM1 and CAR that might influence their binding and signaling activities. Furthermore, we concentrate on alternative splicing of single domains and short segments in the extracellular regions of L1 subfamily members that might affect the organization of the N-terminal located Ig-like domains. In particular, we discuss variations of the linker sequence between Ig-like domains 2 and 3 (D2 and D3) that is required for the horseshoe conformation.

Lack of the Ig cell adhesion molecule BT-IgSF (IgSF11) induced behavioral changes in the open maze, water maze and resident intruder test.

The brain- and testis-specific Ig superfamily protein (BT-IgSF, also termed IgSF11) is a homotypic cell adhesion protein. In the nervous system, BT-IgSF regulates the stability of AMPA receptors in the membrane of cultured hippocampal neurons, modulates the connectivity of chandelier cells and controls gap junction-mediated astrocyte-astrocyte communication. Here, we performed behavioral tests in BT-IgSF-deficient mice. BT-IgSF-deficient mice were similar to control littermates with respect to their reflexes, motor coordination and gating, and associative learning. However, BT-IgSF-deficient mice displayed an increased tendency to stay in the central illuminated areas in the open field and O-Maze paradigms suggesting reduced anxiety or increased scotophobia (fear of darkness). Although BT-IgSF-deficient mice initially found the platform in the water maze their behavior was compromised when the platform was moved, indicating reduced behavioral flexibility. This deficit was overcome by longer training to improve their spatial memory. Furthermore, male BT-IgSF-deficient mice displayed increased aggression towards an intruder. Our results show that specific behaviors are modified by the lack of BT-IgSF and demonstrate a contribution of BT-IgSF to network functions.

GIPC1 regulates MACC1-driven metastasis.

Background: Identification of cancer metastasis-relevant molecular networks is desired to provide the basis for understanding and developing intervention strategies. Here we address the role of GIPC1 in the process of MACC1-driven metastasis. MACC1 is a prognostic indicator for patient metastasis formation and metastasis-free survival. MACC1 controls gene transcription, promotes motility, invasion and proliferation of colon cancer cells in vitro, and causes tumor growth and metastasis in mice. Methods: By using yeast-two-hybrid assay, mass spectrometry, co-immunoprecipitation and peptide array we analyzed GIPC1 protein binding partners, by using the MACC1 gene promoter and chromatin immunoprecipitation and electrophoretic mobility shift assay we probed for GIPC1 as transcription factor. We employed GIPC1/MACC1-manipulated cell lines for in vitro and in vivo analyses, and we probed the GIPC1/MACC1 impact using human primary colorectal cancer (CRC) tissue. Results: We identified MACC1 and its paralogue SH3BP4 as protein binding partners of the protein GIPC1, and we also demonstrated the binding of GIPC1 as transcription factor to the MACC1 promoter (TSS to -60 bp). GIPC1 knockdown reduced endogenous, but not CMV promoter-driven MACC1 expression, and diminished MACC1-induced cell migration and invasion. GIPC1 suppression reduced tumor growth and metastasis in mice intrasplenically transplanted with MACC1-overexpressing CRC cells. In human primary CRC specimens, GIPC1 correlates with MACC1 expression and is of prognostic value for metastasis formation and metastasis-free survival. Combination of MACC1 and GIPC1 expression improved patient survival prognosis, whereas SH3BP4 expression did not show any prognostic value. Conclusions: We identified an important, dual function of GIPC1 - as protein interaction partner and as transcription factor of MACC1 - for tumor progression and cancer metastasis.

The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord.

Sensory axonal projections into the spinal cord display a highly stereotyped pattern of T- or Y-shaped axon bifurcation at the dorsal root entry zone (DREZ). Here, we provide evidence that embryonic mice with an inactive receptor guanylyl cyclase Npr2 or deficient for cyclic guanosine monophosphate-dependent protein kinase I (cGKI) lack the bifurcation of sensory axons at the DREZ, i.e., the ingrowing axon either turns rostrally or caudally. This bifurcation error is maintained to mature stages. In contrast, interstitial branching of collaterals from primary stem axons remains unaffected, indicating that bifurcation and interstitial branching are processes regulated by a distinct molecular mechanism. At a functional level, the distorted axonal branching at the DREZ is accompanied by reduced synaptic input, as revealed by patch clamp recordings of neurons in the superficial layers of the spinal cord. Hence, our data demonstrate that Npr2 and cGKI are essential constituents of the signaling pathway underlying axonal bifurcation at the DREZ and neuronal connectivity in the dorsal spinal cord.

Signalling mechanisms regulating axonal branching in vivo.

Identification of the molecular mechanisms underlying axonal branching in vivo has begun in several neuronal systems, notably the projections formed by dorsal root ganglion (DRG) neurons or retinal ganglion cells (RGC). cGMP signalling is essential for sensory axon bifurcation at the spinal cord, whereas brain-derived neurotrophic factor (BDNF) and ephrinA signalling establish position-dependent branching of RGC axons. In the latter system, the degradation of specific signalling components, via the ubiquitin-proteasome system, may provide an additional mechanism involved in axon branching of RGC. The process of arborisation is essential for neurons to innervate multiple targets and to build topographic maps. The various forms of branching found in different types of neurons are regulated by distinct signalling pathways activated by multiple extracellular cues in addition to axonal guidance factors. These signalling cascades, together with transcriptional programs, most likely interact and trigger the polymerisation or depolymerisation of the actin and tubulin cytoskeleton to regulate branching.

C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons.

Neuronal circuits are shaped during development by the coordinated action of guidance factors and signals that regulate axonal branching. Unlike guidance cues, the molecules and signaling cascades that underlie axonal branching remain to be resolved. Here we show that the secreted molecule C-type natriuretic peptide (CNP) induces a cGMP signaling cascade via its receptor particulate guanylyl cyclase Npr2 which is essential for sensory axon bifurcation at the dorsal root entry zone (DREZ) of the spinal cord. In contrast, another form of sensory axon branching-collateral formation-is not affected by this pathway. We also demonstrate that cGMP signaling via the nitric oxide-stimulated soluble guanylyl cyclase system (NO-GC) is dispensable for sensory axon branching. Functionally, the bifurcation error in CNP mutant mice is maintained at mature stages and results in a reduced input on secondary neurons as detected by patch-clamp recordings.

The IgCAM CAR Regulates Gap Junction-Mediated Coupling on Embryonic Cardiomyocytes and Affects Their Beating Frequency.

The IgCAM coxsackie-adenovirus receptor (CAR) is essential for embryonic heart development and electrical conduction in the mature heart. However, it is not well-understood how CAR exerts these effects at the cellular level. To address this question, we analyzed the spontaneous beating of cultured embryonic hearts and cardiomyocytes from wild type and CAR knockout (KO) embryos. Surprisingly, in the absence of the CAR, cultured cardiomyocytes showed increased frequencies of beating and calcium cycling. Increased beatings of heart organ cultures were also induced by the application of reagents that bind to the extracellular region of the CAR, such as the adenovirus fiber knob. However, the calcium cycling machinery, including calcium extrusion via SERCA2 and NCX, was not disrupted in CAR KO cells. In contrast, CAR KO cardiomyocytes displayed size increases but decreased in the total numbers of membrane-localized Cx43 clusters. This was accompanied by improved cell-cell coupling between CAR KO cells, as demonstrated by increased intercellular dye diffusion. Our data indicate that the CAR may modulate the localization and oligomerization of Cx43 at the plasma membrane, which could in turn influence electrical propagation between cardiomyocytes via gap junctions.