Carbamazepine reduces disease severity in a mouse model of metaphyseal chondrodysplasia type Schmid caused by a premature stop codon (Y632X) in the Col10a1 gene.

Mutations, mostly in the region of the COL10A1 gene encoding the C-terminal non-collagenous domain, cause the dwarfism metaphyseal chondrodysplasia type Schmid (MCDS). In most cases, the disease mechanism involves the misfolding of the mutant protein causing increased endoplasmic reticulum (ER) stress and an unfolded protein response (UPR). However, in an iliac crest biopsy, the COL10A1 p.Y632X mutation was found to produce instability of the mutant mRNA such that little mutant protein may be produced. To investigate the disease mechanism further, a gene-targeted mouse model of the Col10a1 p.Y632X mutation was generated. In this model, the mutant mRNA showed no instability, and in mice heterozygous for the mutation, mutant and wild-type mRNAs were present at equal concentrations. The protein was translated from the mutant allele and retained within the cell, triggering increased ER stress and a UPR. The mutation produced a relatively severe form of MCDS. Nevertheless, treatment of the mice with carbamazepine (CBZ), a drug which stimulates intracellular proteolysis and alleviates ER stress, effectively reduced the disease severity in this model of MCDS caused by a premature stop codon in the Col10a1 gene. Specifically, the drug reduced ER stress in the growth plate, restored growth plate architecture toward the wild-type state, significantly increased bone growth and within 2 weeks of treatment corrected the MCDS-induced hip distortion. These results indicate that CBZ is likely to be effective in ongoing clinical trials against all forms of MCDS whether caused by premature stop codons or substitutions.

Ablation of retinal horizontal cells from adult mice leads to rod degeneration and remodeling in the outer retina.

In the brain, including the retina, interneurons show an enormous structural and functional diversity. Retinal horizontal cells represent a class of interneurons that form triad synapses with photoreceptors and ON bipolar cells. At this first retinal synapse, horizontal cells modulate signal transmission from photoreceptors to bipolar cells by feedback and feedforward inhibition. To test how the fully developed retina reacts to the specific loss of horizontal cells, these interneurons were specifically ablated from adult mice using the diphtheria toxin (DT)/DT-receptor system and the connexin57 promoter. Following ablation, the retinal network responded with extensive remodeling: rods retracted their axons from the outer plexiform layer and partially degenerated, whereas cones survived. Cone pedicles remained in the outer plexiform layer and preserved synaptic contacts with OFF but not with ON bipolar cells. Consistently, the retinal ON pathway was impaired, leading to reduced amplitudes and prolonged latencies in electroretinograms. However, ganglion cell responses showed only slight changes in time course, presumably because ON bipolar cells formed multiple ectopic synapses with photoreceptors, and visual performance, assessed with an optomotor system, was only mildly affected. Thus, the loss of an entire interneuron class can be largely compensated even by the adult retinal network.

Epigenetic regulation of promiscuous gene expression in thymic medullary epithelial cells.

Thymic central tolerance comprehensively imprints the T-cell receptor repertoire before T cells seed the periphery. Medullary thymic epithelial cells (mTECs) play a pivotal role in this process by virtue of promiscuous expression of tissue-restricted autoantigens. The molecular regulation of this unusual gene expression, in particular the involvement of epigenetic mechanisms is only poorly understood. By studying promiscuous expression of the mouse casein locus, we report that transcription of this locus proceeds from a delimited region ("entry site") to increasingly complex patterns along with mTEC maturation. Transcription of this region is preceded by promoter demethylation in immature mTECs followed upon mTEC maturation by acquisition of active histone marks and local locus decontraction. Moreover, analysis of two additional gene loci showed that promiscuous expression is transient in single mTECs. Transient gene expression could conceivably add to the local diversity of self-antigen display thus enhancing the efficacy of central tolerance.

CaMKII inhibition due to TRIC-B loss-of-function dysregulates SMAD signaling in osteogenesis imperfecta.

Ca2+ is a second messenger that regulates a variety of cellular responses in bone, including osteoblast differentiation. Mutations in trimeric intracellular cation channel B (TRIC-B), an endoplasmic reticulum channel specific for K+, a counter ion for Ca2+flux, affect bone and cause a recessive form of osteogenesis imperfecta (OI) with a still puzzling mechanism. Using a conditional Tmem38b knock out mouse, we demonstrated that lack of TRIC-B in osteoblasts strongly impairs skeleton growth and structure, leading to bone fractures. At the cellular level, delayed osteoblast differentiation and decreased collagen synthesis were found consequent to the Ca2+ imbalance and associated with reduced collagen incorporation in the extracellular matrix and poor mineralization. The impaired SMAD signaling detected in mutant mice, and validated in OI patient osteoblasts, explained the osteoblast malfunction. The reduced SMAD phosphorylation and nuclear translocation were mainly caused by alteration in Ca2+ calmodulin kinase II (CaMKII)-mediated signaling and to a less extend by a lower TGF-ß reservoir. SMAD signaling, osteoblast differentiation and matrix mineralization were only partially rescued by TGF-ß treatment, strengthening the impact of CaMKII-SMAD axes on osteoblast function. Our data established the TRIC-B role in osteoblasts and deepened the contribution of the CaMKII-SMAD signaling in bone.

The human deafness-associated connexin 30 T5M mutation causes mild hearing loss and reduces biochemical coupling among cochlear non-sensory cells in knock-in mice.

Mutations in the GJB2 and GJB6 genes, respectively, coding for connexin26 (Cx26) and connexin30 (Cx30) proteins, are the most common cause for prelingual non-syndromic deafness in humans. In the inner ear, Cx26 and Cx30 are expressed in different non-sensory cell types, where they largely co-localize and may form heteromeric gap junction channels. Here, we describe the generation and characterization of a mouse model for human bilateral middle/high-frequency hearing loss based on the substitution of an evolutionarily conserved threonine by a methionine residue at position 5 near the N-terminus of Cx30 (Cx30T5M). The mutation was inserted in the mouse genome by homologous recombination in mouse embryonic stem cells. Expression of the mutated Cx30T5M protein in these transgenic mice is under the control of the endogenous Cx30 promoter and was analysed via activation of the lacZ reporter gene. When probed by auditory brainstem recordings, Cx30(T5M/T5M) mice exhibited a mild, but significant increase in their hearing thresholds of about 15 dB at all frequencies. Immunolabelling with antibodies to Cx26 or Cx30 suggested normal location of these proteins in the adult inner ear, but western blot analysis showed significantly down-regulated the expression levels of Cx26 and Cx30. In the developing cochlea, electrical coupling, probed by dual patch-clamp recordings, was normal. However, transfer of the fluorescent tracer calcein between cochlear non-sensory cells was reduced, as was intercellular Ca(2+) signalling due to spontaneous ATP release from connexin hemichannels. Our findings link hearing loss to decreased biochemical coupling due to the point-mutated Cx30 in mice.

The neuronal connexin36 interacts with and is phosphorylated by CaMKII in a way similar to CaMKII interaction with glutamate receptors.

Electrical synapses can undergo activity-dependent plasticity. The calcium/calmodulin-dependent kinase II (CaMKII) appears to play a critical role in this phenomenon, but the underlying mechanisms of how CaMKII affects the neuronal gap junction protein connexin36 (Cx36) are unknown. Here we demonstrate effective binding of (35)S-labeled CaMKII to 2 juxtamembrane cytoplasmic domains of Cx36 and in vitro phosphorylation of this protein by the kinase. Both domains reveal striking similarities with segments of the regulatory subunit of CaMKII, which include the pseudosubstrate and pseudotarget sites of the kinase. Similar to the NR2B subunit of the NMDA receptor both Cx36 binding sites exhibit phosphorylation-dependent interaction and autonomous activation of CaMKII. CaMKII and Cx36 were shown to be significantly colocalized in the inferior olive, a brainstem nucleus highly enriched in electrical synapses, indicating physical proximity of these proteins. In analogy to the current notion of NR2B interaction with CaMKII, we propose a model that provides a mechanistic framework for CaMKII and Cx36 interaction at electrical synapses.

The WNT1G177C mutation specifically affects skeletal integrity in a mouse model of osteogenesis imperfecta type XV.

The recent identification of homozygous WNT1 mutations in individuals with osteogenesis imperfecta type XV (OI-XV) has suggested that WNT1 is a key ligand promoting the differentiation and function of bone-forming osteoblasts. Although such an influence was supported by subsequent studies, a mouse model of OI-XV remained to be established. Therefore, we introduced a previously identified disease-causing mutation (G177C) into the murine Wnt1 gene. Homozygous Wnt1G177C/G177C mice were viable and did not display defects in brain development, but the majority of 24-week-old Wnt1G177C/G177C mice had skeletal fractures. This increased bone fragility was not fully explained by reduced bone mass but also by impaired bone matrix quality. Importantly, the homozygous presence of the G177C mutation did not interfere with the osteoanabolic influence of either parathyroid hormone injection or activating mutation of LRP5, the latter mimicking the effect of sclerostin neutralization. Finally, transcriptomic analyses revealed that short-term administration of WNT1 to osteogenic cells induced not only the expression of canonical WNT signaling targets but also the expression of genes encoding extracellular matrix modifiers. Taken together, our data demonstrate that regulating bone matrix quality is a primary function of WNT1. They further suggest that individuals with WNT1 mutations should profit from existing osteoanabolic therapies.

The TSG101 protein binds to connexins and is involved in connexin degradation.

Gap junctions mediate electrical and metabolic communication between cells in almost all tissues and are proposed to play important roles in cellular growth control, differentiation and embryonic development. Gap junctional communication and channel assembly were suggested to be regulated by interaction of connexins with different proteins including kinases and phosphatases. Here, we identified the tumor susceptibility gene 101 (TSG101) protein to bind to the carboxyterminal tail of connexin45 in a yeast two-hybrid protein interaction screen. Glutathione S-transferase pull down experiments and immunoprecipitation revealed that not only connexin45 but also connexin30.2, -36, and -43 carboxyterminal regions were associated with TSG101 protein in pull down analyses and that connexin31, -43 and -45 co-precipitate with endogenous TSG101 protein in lysates from HM1 embryonic stem cells. TSG101 has been shown to be involved in cell cycle control, transcriptional regulation and turnover of endocytosed proteins. Thus, we decided to study the functional role of this interaction. SiRNA mediated knock down of TSG101 in HM1 embryonic stem cells led to increased levels of connexin43 and -45, prolonged half life of these connexins and increased transfer of microinjected Lucifer yellow. Our results suggest that TSG101 is involved in the degradation of connexins via interaction with connexin proteins.

Gnathodiaphyseal dysplasia is not recapitulated in a respective mouse model carrying a mutation of the Ano5 gene.

Mutations in the gene ANO5, encoding for the transmembrane protein Anoctamin 5 (Ano5), have been identified to cause gnathodiaphyseal dysplasia (GDD) in humans, a skeletal disorder characterized by sclerosis of tubular bones, increased fracture risk and fibro-osseous lesions of the jawbones. To better understand the pathomechanism of GDD we have generated via Crispr/CAS9 gene editing a mouse model harboring the murine equivalent (Ano5 p.T491F) of a GDD-causing ANO5 mutation identified in a previously reported patient. Skeletal phenotyping by contact radiography, µCT and undecalcified histomorphometry was performed in male mice, heterozygous and homozygous for the mutation, at the ages of 12 and 24 weeks. These mice did not display alterations of skeletal microarchitecture or mandible morphology. The results were confirmed in female mice and animals derived from a second, independent clone. Finally, no skeletal phenotype was observed in mice lacking ~40% of their Ano5 gene due to a frameshift mutation. Therefore, our results indicate that Ano5 is dispensable for bone homeostasis in mice, at least under unchallenged conditions, and that these animals may not present the most adequate model to study the physiological role of Anoctamin 5.

Gating, permselectivity and pH-dependent modulation of channels formed by connexin57, a major connexin of horizontal cells in the mouse retina.

Mouse connexin57 (Cx57) is expressed most abundantly in horizontal cells of the retina, and forms gap junction (GJ) channels, which constitute a structural basis for electrical and metabolic intercellular communication, and unapposed hemichannels (UHCs) that are involved in an exchange of ions and metabolites between the cytoplasm and extracellular milieu. By combining fluorescence imaging and dual whole-cell voltage clamp methods, we showed that HeLa cells expressing Cx57 and C-terminally fused with enhanced green fluorescent protein (Cx57-EGFP) form junctional plaques (JPs) and that only cell pairs exhibiting at least one JP demonstrate cell-to-cell electrical coupling and transfer of negatively and positively charged dyes with molecular mass up to approximately 400 Da. The permeability of the single Cx57 GJ channel to Alexa fluor-350 is approximately 90-fold smaller than the permeability of Cx43, while its single channel conductance (57 pS) is only 2-fold smaller than Cx43 (110 pS). Gating of Cx57-EGFP/Cx45 heterotypic GJ channels reveal that Cx57 exhibit a negative gating polarity, i.e. channels tend to close at negativity on the cytoplasmic side of Cx57. Alkalization of pH(i) from 7.2 to 7.8 increased gap junctional conductance (g(j)) of approximately 100-fold with pK(a) = 7.41. We show that this g(j) increase was caused by an increase of both the open channel probability and the number of functional channels. Function of Cx57 UHCs was evaluated based on the uptake of fluorescent dyes. We found that under control conditions, Cx57 UHCs are closed and open at [Ca(2+)](o) = approximately 0.3 mm or below, demonstrating that a moderate reduction of [Ca(2+)](o) can facilitate the opening of Cx57 UHCs. This was potentiated with intracellular alkalization. In summary, our data show that the open channel probability of Cx57 GJs can be modulated by pH(i) with very high efficiency in the physiologically relevant range and may explain pH-dependent regulation of cell-cell coupling in horizontal cell in the retina.

A Distinct Pool of Nav1.5 Channels at the Lateral Membrane of Murine Ventricular Cardiomyocytes.

Background: In cardiac ventricular muscle cells, the presence of voltage-gated sodium channels Nav1.5 at the lateral membrane depends in part on the interaction between the dystrophin-syntrophin complex and the Nav1.5 C-terminal PDZ-domain-binding sequence Ser-Ile-Val (SIV motif). α1-Syntrophin, a PDZ-domain adaptor protein, mediates the interaction between Nav1.5 and dystrophin at the lateral membrane of cardiac cells. Using the cell-attached patch-clamp approach on cardiomyocytes expressing Nav1.5 in which the SIV motif is deleted (ΔSIV), sodium current (INa) recordings from the lateral membrane revealed a SIV-motif-independent INa. Since immunostaining has suggested that Nav1.5 is expressed in transverse (T-) tubules, this remaining INa might be carried by channels in the T-tubules. Of note, a recent study using heterologous expression systems showed that α1-syntrophin also interacts with the Nav1.5 N-terminus, which may explain the SIV-motif independent INa at the lateral membrane of cardiomyocytes. Aim: To address the role of α1-syntrophin in regulating the INa at the lateral membrane of cardiac cells. Methods and Results: Patch-clamp experiments in cell-attached configuration were performed on the lateral membranes of wild-type, α1-syntrophin knockdown, and ΔSIV ventricular mouse cardiomyocytes. Compared to wild-type, a reduction of the lateral INa was observed in myocytes from α1-syntrophin knockdown hearts. Similar to ΔSIV myocytes, a remaining INa was still recorded. In addition, cell-attached INa recordings from lateral membrane did not differ significantly between non-detubulated and detubulated ΔSIV cardiomyocytes. Lastly, we obtained evidence suggesting that cell-attached patch-clamp experiments on the lateral membrane cannot record currents carried by channels in T-tubules such as calcium channels. Conclusion: Altogether, these results suggest the presence of a sub-pool of sodium channels at the lateral membrane of cardiomyocytes that is independent of α1-syntrophin and the PDZ-binding motif of Nav1.5, located in membrane domains outside of T-tubules. The question of a T-tubular pool of Nav1.5 channels, however, remains open.

Testing the Cre-mediated genetic switch for the generation of conditional knock-in mice.

The Cre-mediated genetic switch combines the ability of Cre recombinase to stably invert or excise a DNA fragment depending upon the orientation of flanking mutant loxP sites. In this work, we have tested this strategy in vivo with the aim to generate two conditional knock-in mice for missense mutations in the Impad1 and Clcn7 genes causing two different skeletal dysplasias. Targeting constructs were generated in which the Impad1 exon 2 and an inverted exon 2* and the Clcn7 exon 7 and an inverted exon 7* containing the point mutations were flanked by mutant loxP sites in a head-to-head orientation. When the Cre recombinase is present, the DNA flanked by the mutant loxP sites is expected to be stably inverted leading to the activation of the mutated exon. The targeting vectors were used to generate heterozygous floxed mice in which inversion of the wild-type with the mutant exon has not occurred yet. To generate knock-in mice, floxed animals were mated to a global Cre-deleter mouse strain for stable inversion and activation of the mutation. Unexpectedly the phenotype of homozygous Impad1 knock-in animals overlaps with the lethal phenotype described previously in Impad1 knock-out mice. Similarly, the phenotype of homozygous Clcn7 floxed mice overlaps with Clcn7 knock-out mice. Expression studies by qPCR and RT-PCR demonstrated that mutant mRNA underwent abnormal splicing leading to the synthesis of non-functional proteins. Thus, the skeletal phenotypes in both murine strains were not caused by the missense mutations, but by aberrant splicing. Our data demonstrate that the Cre mediated genetic switch strategy should be considered cautiously for the generation of conditional knock-in mice.

SLC10A7 mutations cause a skeletal dysplasia with amelogenesis imperfecta mediated by GAG biosynthesis defects.

Skeletal dysplasia with multiple dislocations are severe disorders characterized by dislocations of large joints and short stature. The majority of them have been linked to pathogenic variants in genes encoding glycosyltransferases, sulfotransferases or epimerases required for glycosaminoglycan synthesis. Using exome sequencing, we identify homozygous mutations in SLC10A7 in six individuals with skeletal dysplasia with multiple dislocations and amelogenesis imperfecta. SLC10A7 encodes a 10-transmembrane-domain transporter located at the plasma membrane. Functional studies in vitro demonstrate that SLC10A7 mutations reduce SLC10A7 protein expression. We generate a Slc10a7-/- mouse model, which displays shortened long bones, growth plate disorganization and tooth enamel anomalies, recapitulating the human phenotype. Furthermore, we identify decreased heparan sulfate levels in Slc10a7-/- mouse cartilage and patient fibroblasts. Finally, we find an abnormal N-glycoprotein electrophoretic profile in patient blood samples. Together, our findings support the involvement of SLC10A7 in glycosaminoglycan synthesis and specifically in skeletal development.

The Lrp4R1170Q Homozygous Knock-In Mouse Recapitulates the Bone Phenotype of Sclerosteosis in Humans.

Sclerosteosis is a rare autosomal recessive bone disorder marked by hyperostosis of the skull and tubular bones. Initially, we and others reported that sclerosteosis was caused by loss-of-function mutations in SOST, encoding sclerostin. More recently, we identified disease-causing mutations in LRP4, a binding partner of sclerostin, in three sclerosteosis patients. Upon binding to sclerostin, LRP4 can inhibit the canonical WNT signaling that is known to be an important pathway in the regulation of bone formation. To further investigate the role of LRP4 in the bone formation process, we generated an Lrp4 mutated sclerosteosis mouse model by introducing the p.Arg1170Gln mutation in the mouse genome. Extensive analysis of the bone phenotype of the Lrp4R1170Q/R1170Q knock-in (KI) mouse showed the presence of increased trabecular and cortical bone mass as a consequence of increased bone formation by the osteoblasts. In addition, three-point bending analysis also showed that the increased bone mass results in increased bone strength. In contrast to the human sclerosteosis phenotype, we could not observe syndactyly in the forelimbs or hindlimbs of the Lrp4 KI animals. Finally, we could not detect any significant changes in the bone formation and resorption markers in the serum of the mutant mice. However, the serum sclerostin levels were strongly increased and the level of sclerostin in the tibia was decreased in Lrp4R1170Q/R1170Q mice, confirming the role of LRP4 as an anchor for sclerostin in bone. In conclusion, the Lrp4R1170Q/R1170Q mouse is a good model for the human sclerosteosis phenotype caused by mutations in LRP4 and can be used in the future for further investigation of the mechanism whereby LRP4 regulates bone formation. © 2017 American Society for Bone and Mineral Research.

Impaired calcium homeostasis is associated with sudden cardiac death and arrhythmias in a genetic equivalent mouse model of the human HRC-Ser96Ala variant.

AIMS: The histidine-rich calcium-binding protein (HRC) Ser96Ala variant has previously been identified as a potential biomarker for ventricular arrhythmias and sudden cardiac death in patients with idiopathic dilated cardiomyopathy. Herein, the role of this variant in cardiac pathophysiology is delineated through a novel mouse model, carrying the human mutation in the homologous mouse position. METHODS AND RESULTS: The mouse HRC serine 81, homologous to human HRC serine 96, was mutated to alanine, using knock-in gene targeting. The HRC-Ser81Ala mice presented increased mortality in the absence of structural or histological abnormalities, indicating that early death may be arrhythmia-related. Indeed, under stress-but not baseline-conditions, the HRC-Ser81Ala mice developed ventricular arrhythmias, whilst at the cardiomyocyte level they exhibited increased occurrence of triggered activity. Cardiac contraction was decreased in vivo, ex vivo, and in vitro. Additionally, Ca2+ transients and SR Ca2+ load were both reduced suggesting that cytosolic Ca2+ overload is not the underlying proarrhythmic mechanism. Interestingly, total SR Ca2+ leak was increased in HRC-Ser81Ala cardiomyocytes, without an increase in Ca2+ spark and wave frequency. However, Ca2+ wave propagation was significantly slower and the duration of the associated Na/Ca exchange current was increased. Moreover, action potential duration was also increased. Notably, Ca2+/Calmodulin kinase II (CaMKII) phosphorylation of the ryanodine receptor was increased, whilst KN-93, an inhibitor of CaMKII, reduced the occurrence of arrhythmias. CONCLUSIONS: The homologous mutation Ser81Ala in HRC in mice, corresponding to Ser96Ala in humans, is associated with sudden death and depressed cardiac function. Ventricular arrhythmias are related to abnormal Ca2+ cycling across the SR. The data further support a role for CaMKII with the perspective to treat arrhythmias through CaMKII inhibition.

Cardiac-specific ablation of synapse-associated protein SAP97 in mice decreases potassium currents but not sodium current.

BACKGROUND: Membrane-associated guanylate kinase (MAGUK) proteins are important determinants of ion channel organization in the plasma membrane. In the heart, the MAGUK protein SAP97, encoded by the DLG1 gene, interacts with several ion channels via their PDZ domain-binding motif and regulates their function and localization. OBJECTIVE: The purpose of this study was to assess in vivo the role of SAP97 in the heart by generating a genetically modified mouse model in which SAP97 is suppressed exclusively in cardiomyocytes. METHODS: SAP97(fl/fl) mice were generated by inserting loxP sequences flanking exons 1-3 of the SAP97 gene. SAP97(fl/fl) mice were crossed with αMHC-Cre mice to generate αMHC-Cre/SAP97(fl/fl) mice, thus resulting in a cardiomyocyte-specific deletion of SAP97. Quantitative reverse transcriptase-polymerase chain reaction, western blots, and immunostaining were performed to measure mRNA and protein expression levels, and ion channel localization. The patch-clamp technique was used to record ion currents and action potentials. Echocardiography and surface ECGs were performed on anesthetized mice. RESULTS: Action potential duration was greatly prolonged in αMHC-Cre/SAP97(fl/fl) cardiomyocytes compared to SAP97(fl/fl) controls, but maximal upstroke velocity was unchanged. This was consistent with the decreases observed in IK1, Ito, and IKur potassium currents and the absence of effect on the sodium current INa. Surface ECG revealed an increased corrected QT interval in αMHC-Cre/SAP97(fl/fl) mice. CONCLUSION: These data suggest that ablation of SAP97 in the mouse heart mainly alters potassium channel function. Based on the important role of SAP97 in regulating the QT interval, DLG1 may be a susceptibility gene to be investigated in patients with congenital long QT syndrome.

Cell-specific cre recombinase expression allows selective ablation of glutamate receptors from mouse horizontal cells.

In the mouse retina, horizontal cells form an electrically coupled network and provide feedback signals to photoreceptors and feedforward signals to bipolar cells. Thereby, horizontal cells contribute to gain control at the first visual synapse and to the antagonistic organization of bipolar and ganglion cell receptive fields. However, the nature of horizontal cell output remains a matter of debate, just as the exact contribution of horizontal cells to center-surround antagonism. To facilitate studying horizontal cell function, we developed a knockin mouse line which allows ablating genes exclusively in horizontal cells. This knockin line expresses a Cre recombinase under the promoter of connexin57 (Cx57), a gap junction protein only expressed in horizontal cells. Consistently, in Cx57+/Cre mice, Cre recombinase is expressed in almost all horizontal cells (>99%) and no other retinal neurons. To test Cre activity, we crossbred Cx57+/Cre mice with a mouse line in which exon 11 of the coding sequence for the ionotropic glutamate receptor subunit GluA4 was flanked by two loxP sites (GluA4fl/fl). In GluA4fl/fl:Cx57+/Cre mice, GluA4 immunoreactivity was significantly reduced (∼ 50%) in the outer retina where horizontal cells receive photoreceptor inputs, confirming the functionality of the Cre/loxP system. Whole-cell patch-clamp recordings from isolated horizontal cell somata showed a reduction of glutamate-induced inward currents by ∼ 75%, suggesting that the GluA4 subunit plays a major role in mediating photoreceptor inputs. The persistent current in GluA4-deficient cells is mostly driven by AMPA and to a very small extent by kainate receptors as revealed by application of the AMPA receptor antagonist GYKI52466 and concanavalin A, a potentiator of kainate receptor-mediated currents. In summary, the Cx57+/Cre mouse line provides a versatile tool for studying horizontal cell function. GluA4fl/fl:Cx57+/Cre mice, in which horizontal cells receive less excitatory input, can thus be used to analyze the contribution of horizontal cells to retinal processing.

Gap junctions between neuronal inputs but not gonadotropin-releasing hormone neurons control estrous cycles in the mouse.

The role of gap junctions in the neural control of fertility remains poorly understood. Using acute brain slices from adult GnRH-green fluorescent protein transgenic mice, individual GnRH neurons were filled with a mixture of fluorescent dextran and neurobiotin. No dye transfer was found between any GnRH neurons, although approximately 30% of GnRH neurons exchanged neurobiotin with closely apposed cells. Dual electrophysiological recordings from pairs of GnRH neurons revealed an absence of electrical coupling. Using adult connexin 36 (Cx36)-cyan fluorescent protein transgenic mice, Cx36 was identified in cells within several hypothalamic brain regions, including 64% of preoptic area kisspeptin neurons but not in GnRH neurons. To assess the potential role of Cx36 in non-GnRH neurons within the GnRH neuronal network (i.e. neurons providing afferent inputs to GnRH neurons), a calmodulin kinase IIα-Cre (CKC)-LoxP strategy was used to generate mice with a neuron-specific deletion of Cx36 beginning in the first postnatal week. Mutant female mice exhibited normal puberty onset but disordered estrous cyclicity, although their fecundity was normal as was their estrogen-negative and -positive feedback mechanisms. The effects of adult deletion of Cx36 from neurons were assessed using a tamoxifen-dependent inducible CKC-Cx36 transgenic strategy. Mutant mice exhibited the same reproductive phenotype as the CKC-Cx36 animals. Together these observations demonstrate that there is no gap junctional coupling between GnRH neurons. However, it is apparent that other neurons within the GnRH neuronal network, potentially the preoptic kisspeptin neurons, are dependent on Cx36 gap junctions and that this is critical for normal estrous cyclicity.

Mouse lens connexin23 (Gje1) does not form functional gap junction channels but causes enhanced ATP release from HeLa cells.

In the mouse genome, 20 connexin genes have been detected that code for proteins of high sequence identity in the two extracellular loops, especially six conserved cysteine residues. The mouse connexin23 (Cx23) gene (Gje1) differs from all other connexin genes in vertebrates, since it codes for a protein that contains only 4 instead of 6 cysteine residues in the extracellular loops. Recently, two zebrafish connexin genes (Cx23a and Cx23b) have been identified, and a mouse mutant in the Gje1 gene has been described that exhibits a developmental defect in the lens. Here, we have compared the Cx23 gene in different mammalian species and found no transcripts in cDNA libraries of primates. Furthermore, all primate genomes analyzed contain stop codons in the Cx23 sequence, indicating inactivation of the orthologous primate GJE1 gene. No Cx23 mRNA was found in human eye. In order to analyze the properties of mouse Cx23 channels, we isolated HeLa cell clones stably expressing wild-type mCx23 or mCx23 fused to eGFP. Cells expressing Cx23-eGFP demonstrated its insertion in the plasma membrane but no punctate staining in contacting membranes characteristic for junctional plaques. In addition, we tested whether Cx23 forms functional gap junction channels electrophysiologically in cell pairs as well as by microinjection of neurobiotin and found that mouse Cx23 did not form gap junction channels in HeLa cells. However, there was a significant release of ATP from different Cx23 HeLa cell clones, even in the presence of normal culture medium with high calcium ion concentration, suggesting a hemichannel-based function of Cx23. Therefore, Cx23 seems to share functional properties with pannexin (hemi) channels rather than gap junction channels of other connexins.

Connexin57 is expressed in dendro-dendritic and axo-axonal gap junctions of mouse horizontal cells and its distribution is modulated by light.

Mouse horizontal cells are coupled by gap junctions composed of connexin57. These gap junctions are regulated by ambient light via multiple neuromodulators including dopamine. In order to analyze the distribution and structure of horizontal cell gap junctions in the mouse retina, and examine the effects of light adaptation on gap junction density, we developed antibodies that detect mouse retinal connexin57. Using immunohistochemistry in retinal slices, flat-mounted retinas, and dissociated retinal cells, we showed that connexin57 is expressed in the dendrites and axon terminal processes of mouse horizontal cells. No staining was found in retinas of connexin57-deficient mice. Significantly more connexin57-positive puncta were found in the distal than in the proximal outer plexiform layer, indicating a higher level of expression in axon terminal processes than in the dendrites. We also examined the gap junctions using immunoelectron microscopy and showed that connexin57 does not form hemichannels in the horizontal cell dendritic tips. Light adaptation resulted in a significant increase in the number of connexin57-immunoreactive plaques in the outer plexiform layer, consistent with previously reported effects of light adaptation on connexin57 expression in the mouse retina. This study shows for the first time the detailed location of connexin57 expression within mouse horizontal cells, and provides the first ultrastructural data on mouse horizontal cell gap junctions.