Astroglial Connexin 43 Regulates Synaptic Vesicle Release at Hippocampal Synapses.

Connexin 43, an astroglial gap junction protein, is enriched in perisynaptic astroglial processes and plays major roles in synaptic transmission. We have previously found that astroglial Cx43 controls synaptic glutamate levels and allows for activity-dependent glutamine release to sustain physiological synaptic transmissions and cognitiogns. However, whether Cx43 is important for the release of synaptic vesicles, which is a critical component of synaptic efficacy, remains unanswered. Here, using transgenic mice with a glial conditional knockout of Cx43 (Cx43-/-), we investigate whether and how astrocytes regulate the release of synaptic vesicles from hippocampal synapses. We report that CA1 pyramidal neurons and their synapses develop normally in the absence of astroglial Cx43. However, a significant impairment in synaptic vesicle distribution and release dynamics were observed. In particular, the FM1-43 assays performed using two-photon live imaging and combined with multi-electrode array stimulation in acute hippocampal slices, revealed a slower rate of synaptic vesicle release in Cx43-/- mice. Furthermore, paired-pulse recordings showed that synaptic vesicle release probability was also reduced and is dependent on glutamine supply via Cx43 hemichannel (HC). Taken together, we have uncovered a role for Cx43 in regulating presynaptic functions by controlling the rate and probability of synaptic vesicle release. Our findings further highlight the significance of astroglial Cx43 in synaptic transmission and efficacy.

The SMC-like RecN protein is at the crossroads of several genotoxic stress responses in Escherichia coli.

Introduction: DNA damage repair (DDR) is an essential process for living organisms and contributes to genome maintenance and evolution. DDR involves different pathways including Homologous recombination (HR), Nucleotide Excision Repair (NER) and Base excision repair (BER) for example. The activity of each pathway is revealed with particular drug inducing lesions, but the repair of most DNA lesions depends on concomitant or subsequent action of the multiple pathways. Methods: In the present study, we used two genotoxic antibiotics, mitomycin C (MMC) and Bleomycin (BLM), to decipher the interplays between these different pathways in E. coli. We combined genomic methods (TIS and Hi-SC2) and imaging assays with genetic dissections. Results: We demonstrate that only a small set of DDR proteins are common to the repair of the lesions induced by these two drugs. Among them, RecN, an SMC-like protein, plays an important role by controlling sister chromatids dynamics and genome morphology at different steps of the repair processes. We further demonstrate that RecN influence on sister chromatids dynamics is not equivalent during the processing of the lesions induced by the two drugs. We observed that RecN activity and stability requires a pre-processing of the MMC-induced lesions by the NER but not for BLM-induced lesions. Discussion: Those results show that RecN plays a major role in rescuing toxic intermediates generated by the BER pathway in addition to its well-known importance to the repair of double strand breaks by HR.

Shigella IpaA mediates actin bundling through diffusible vinculin oligomers with activation imprint.

Upon activation, vinculin reinforces cytoskeletal anchorage during cell adhesion. Activating ligands classically disrupt intramolecular interactions between the vinculin head and tail domains that bind to actin filaments. Here, we show that Shigella IpaA triggers major allosteric changes in the head domain, leading to vinculin homo-oligomerization. Through the cooperative binding of its three vinculin-binding sites (VBSs), IpaA induces a striking reorientation of the D1 and D2 head subdomains associated with vinculin oligomerization. IpaA thus acts as a catalyst producing vinculin clusters that bundle actin at a distance from the activation site and trigger the formation of highly stable adhesions resisting the action of actin relaxing drugs. Unlike canonical activation, vinculin homo-oligomers induced by IpaA appear to keep a persistent imprint of the activated state in addition to their bundling activity, accounting for stable cell adhesion independent of force transduction and relevant to bacterial invasion.

A conserved molecular template underlies color pattern diversity in estrildid finches.

The color patterns that adorn animals' coats not only exhibit extensive diversity linked to various ecological functions but also display recurrences in geometry, orientation, or body location. How processes of pattern formation shape such phenotypic trends remains a mystery. Here, we surveyed plumage color patterns in passerine finches displaying extreme apparent variation and identified a conserved set of color domains. We linked these domains to putative embryonic skin regions instructed by early developmental tissues and outlined by the combinatory expression of few genetic markers. We found that this embryonic prepattern is largely conserved in birds displaying drastic color differences in the adult, interspecies variation resulting from the masking or display of each domain depending on their coloration. This work showed that a simple molecular landscape serves as common spatial template to extensive color pattern variation in finches, revealing that early conserved landmarks and molecular pathways are a major cause of phenotypic trends.

Extended sister-chromosome catenation leads to massive reorganization of the E. coli genome.

In bacteria, chromosome segregation occurs progressively from the origin to terminus within minutes of replication of each locus. Between replication and segregation, sister loci are held in an apparent cohesive state by topological links. The decatenation activity of topoisomerase IV (Topo IV) is required for segregation of replicated loci, yet little is known about the structuring of the chromosome maintained in a cohesive state. In this work, we investigated chromosome folding in cells with altered decatenation activities. Within minutes after Topo IV inactivation, massive chromosome reorganization occurs, associated with increased in contacts between nearby loci, likely trans-contacts between sister chromatids, and in long-range contacts between the terminus and distant loci. We deciphered the respective roles of Topo III, MatP and MukB when TopoIV activity becomes limiting. Topo III reduces short-range inter-sister contacts suggesting its activity near replication forks. MatP, the terminus macrodomain organizing system, and MukB, the Escherichia coli SMC, promote long-range contacts with the terminus. We propose that the large-scale conformational changes observed under these conditions reveal defective decatenation attempts involving the terminus area. Our results support a model of spatial and temporal partitioning of the tasks required for sister chromosome segregation.

OTX2 stimulates adult retinal ganglion cell regeneration.

Retinal ganglion cell (RGC) axons provide the only link between the light sensitive and photon transducing neural retina and visual centers of the brain. RGC axon degeneration occurs in a number of blinding diseases and the ability to stimulate axon regeneration from surviving ganglion cells could provide the anatomic substrate for restoration of vision. OTX2 is a homeoprotein transcription factor expressed in the retina and previous studies showed that, in response to stress, exogenous OTX2 increases the in vitro and in vivo survival of RGCs. Here we examined and quantified the effects of OTX2 on adult RGC axon regeneration in vitro and in vivo. The results show that exogenous OTX2 stimulates the regrowth of axons from RGCs in cultures of dissociated adult retinal cells and from explants of adult retinal tissue and that RGCs respond directly to OTX2 as regrowth is observed in cultures of purified adult rat RGCs. Importantly, after nerve crush in vivo, we observed a positive effect of OTX2 on the number of regenerating axons up to the optic chiasm within 14 days post crush and a very modest level of acuity absent in control mice. The effect of OTX2 on RGC survival and regeneration is of potential interest for degenerative diseases affecting this cell type. All animal procedures were approved by the local "Comié d'éιthique en expérimentation animale n°59" and authorization n° 00702.01 delivered March 28, 2014 by the French "Ministére de l'enseignement supérieur et de la recherche".

The Crohn's disease-related bacterial strain LF82 assembles biofilm-like communities to protect itself from phagolysosomal attack.

Patients with Crohn's disease exhibit abnormal colonization of the intestine by adherent invasive E. coli (AIEC). They adhere to epithelial cells, colonize them and survive inside macrophages. It appeared recently that AIEC LF82 adaptation to phagolysosomal stress involves a long lag phase in which many LF82 cells become antibiotic tolerant. Later during infection, they proliferate in vacuoles and form colonies harboring dozens of LF82 bacteria. In the present work, we investigated the mechanism sustaining this phase of growth. We found that intracellular LF82 produced an extrabacterial matrix that acts as a biofilm and controls the formation of LF82 intracellular bacterial communities (IBCs) for several days post infection. We revealed the crucial role played by the pathogenicity island encoding the yersiniabactin iron capture system to form IBCs and for optimal LF82 survival. These results illustrate that AIECs use original strategies to establish their replicative niche within macrophages.

Shigella IpaA Binding to Talin Stimulates Filopodial Capture and Cell Adhesion.

The Shigella type III effector IpaA contains three binding sites for the focal adhesion protein vinculin (VBSs), which are involved in bacterial invasion of host cells. Here, we report that IpaA VBS3 unexpectedly binds to talin. The 2.5 Å resolution crystal structure of IpaA VBS3 in complex with the talin H1-H4 helices shows a tightly folded α-helical bundle, which is in contrast to the bundle unraveling upon vinculin interaction. High-affinity binding to talin H1-H4 requires a core of hydrophobic residues and electrostatic interactions conserved in talin VBS H46. Remarkably, IpaA VBS3 localizes to filopodial distal adhesions enriched in talin, but not vinculin. In addition, IpaA VBS3 binding to talin was required for filopodial adhesions and efficient capture of Shigella. These results point to the functional diversity of VBSs and support a specific role for talin binding by a subset of VBSs in the formation of filopodial adhesions.

Hydrogen peroxide (H2O2) controls axon pathfinding during zebrafish development.

It is now becoming evident that hydrogen peroxide (H2O2), which is constantly produced by nearly all cells, contributes to bona fide physiological processes. However, little is known regarding the distribution and functions of H2O2 during embryonic development. To address this question, we used a dedicated genetic sensor and revealed a highly dynamic spatio-temporal pattern of H2O2 levels during zebrafish morphogenesis. The highest H2O2 levels are observed during somitogenesis and organogenesis, and these levels gradually decrease in the mature tissues. Biochemical and pharmacological approaches revealed that H2O2 distribution is mainly controlled by its enzymatic degradation. Here we show that H2O2 is enriched in different regions of the developing brain and demonstrate that it participates to axonal guidance. Retinal ganglion cell axonal projections are impaired upon H2O2 depletion and this defect is rescued by H2O2 or ectopic activation of the Hedgehog pathway. We further show that ex vivo, H2O2 directly modifies Hedgehog secretion. We propose that physiological levels of H2O2 regulate RGCs axonal growth through the modulation of Hedgehog pathway.

Absence of cell surface expression of human ACE leads to perinatal death.

Renal tubular dysgenesis (RTD) is a recessive autosomal disease characterized most often by perinatal death. It is due to the inactivation of any of the major genes of the renin-angiotensin system (RAS), one of which is the angiotensin I-converting enzyme (ACE). ACE is present as a tissue-bound enzyme and circulates in plasma after its solubilization. In this report, we present the effect of different ACE mutations associated with RTD on ACE intracellular trafficking, secretion and enzymatic activity. One truncated mutant, R762X, responsible for neonatal death was found to be an enzymatically active, secreted form, not inserted in the plasma membrane. In contrast, another mutant, R1180P, was compatible with life after transient neonatal renal insufficiency. This mutant was located at the plasma membrane and rapidly secreted. These results highlight the importance of tissue-bound ACE versus circulating ACE and show that the total absence of cell surface expression of ACE is incompatible with life. In addition, two missense mutants (W594R and R828H) and two truncated mutants (Q1136X and G1145AX) were also studied. These mutants were neither inserted in the plasma membrane nor secreted. Finally, the structural implications of these ACE mutations were examined by molecular modelling, which suggested some important structural alterations such as disruption of intra-molecular non-covalent interactions (e.g. salt bridges).

Deletion of astroglial connexins weakens the blood-brain barrier.

Astrocytes, the most prominent glial cell type in the brain, send specialized processes named endfeet, which enwrap blood vessels and express a large molecular repertoire dedicated to the physiology of the vascular system. One of the most striking properties of astrocyte endfeet is their enrichment in gap junction protein connexins 43 and 30 (Cx43 and Cx30) allowing for direct intercellular trafficking of ions and small signaling molecules through perivascular astroglial networks. The contribution of astroglial connexins to the physiology of the brain vascular system has never been addressed. Here, we show that Cx43 and Cx30 expression at the level of perivascular endfeet starts from postnatal days 2 and 12 and is fully mature at postnatal days 15 and 20, respectively, indicating that astroglial perivascular connectivity occurs and develops during postnatal blood-brain barrier (BBB) maturation. We demonstrate that mice lacking Cx30 and Cx43 in GFAP (glial fibrillary acidic protein)-positive cells display astrocyte endfeet edema and a partial loss of the astroglial water channel aquaporin-4 and ß-dystroglycan, a transmembrane receptor anchoring astrocyte endfeet to the perivascular basal lamina. Furthermore, the absence of astroglial connexins weakens the BBB, which opens upon increased hydrostatic vascular pressure and shear stress. These results demonstrate that astroglial connexins are necessary to maintain BBB integrity.

Asymmetrical interhemispheric connections develop in cat visual cortex after early unilateral convergent strabismus: anatomy, physiology, and mechanisms.

In the mammalian primary visual cortex, the corpus callosum contributes to the unification of the visual hemifields that project to the two hemispheres. Its development depends on visual experience. When this is abnormal, callosal connections must undergo dramatic anatomical and physiological changes. However, data concerning these changes are sparse and incomplete. Thus, little is known about the impact of abnormal postnatal visual experience on the development of callosal connections and their role in unifying representation of the two hemifields. Here, the effects of early unilateral convergent strabismus (a model of abnormal visual experience) were fully characterized with respect to the development of the callosal connections in cat visual cortex, an experimental model for humans. Electrophysiological responses and 3D reconstruction of single callosal axons show that abnormally asymmetrical callosal connections develop after unilateral convergent strabismus, resulting from an extension of axonal branches of specific orders in the hemisphere ipsilateral to the deviated eye and a decreased number of nodes and terminals in the other (ipsilateral to the non-deviated eye). Furthermore this asymmetrical organization prevents the establishment of a unifying representation of the two visual hemifields. As a general rule, we suggest that crossed and uncrossed retino-geniculo-cortical pathways contribute successively to the development of the callosal maps in visual cortex.

Direct projections of omnipause neurons to reticulospinal neurons: a double-labeling light microscopic study in the cat.

Omnipause neurons (OPNs) are inhibitory neurons located in the midline region of the caudal pons. Their role in gating the discharges of saccade-related burst neurons is well known, but there is no agreement concerning their influence on brainstem neurons that control other muscle groups participating in rapid gaze shifts. In the present study, we inquired whether OPNs project directly to pontobulbar reticulospinal neurons (RSNs) in the cat. Retrograde transport of horseradish peroxidase from the cervical spinal cord was used to label RSNs and an anterograde tracer (biocytin) was iontophoresed at sites of extracellular recording of the OPN activity. Somadendritic characteristics of biocytin-labeled OPNs were largely similar to those obtained previously with intracellular labeling. Three-dimensional reconstruction of axonal trajectories and collaterals revealed that projections of OPNs, regarded as a population, are bilateral. Their terminals were restricted to the reticular formation and midline structures throughout the rostral bulbar and pontine tegmentum. Appositions of synaptic boutons originating from five fully stained OPNs were detected on 38 retrogradely labeled RSNs, each of the OPNs contacting 3-13 cells. The numbers of boutons (1-46; mean 11.8) on the RSN somata and proximal dendrites indicate that the anatomical strength of paired OPN-RSN connections is comparable to that of other similarly studied inhibitory neurons in the cat. The existence of connections with RSNs supports the hypothesis of a generalized influence of OPNs on several effectors participating in orienting gaze shifts as opposed to the idea of their strict specialization for the control of eye saccades.