Altered neuronal network and rescue in a human MECP2 duplication model.

Increased dosage of methyl-CpG-binding protein-2 (MeCP2) results in a dramatic neurodevelopmental phenotype with onset at birth. We generated induced pluripotent stem cells (iPSCs) from patients with the MECP2 duplication syndrome (MECP2dup), carrying different duplication sizes, to study the impact of increased MeCP2 dosage in human neurons. We show that cortical neurons derived from these different MECP2dup iPSC lines have increased synaptogenesis and dendritic complexity. In addition, using multi-electrodes arrays, we show that neuronal network synchronization was altered in MECP2dup-derived neurons. Given MeCP2 functions at the epigenetic level, we tested whether these alterations were reversible using a library of compounds with defined activity on epigenetic pathways. One histone deacetylase inhibitor, NCH-51, was validated as a potential clinical candidate. Interestingly, this compound has never been considered before as a therapeutic alternative for neurological disorders. Our model recapitulates early stages of the human MECP2 duplication syndrome and represents a promising cellular tool to facilitate therapeutic drug screening for severe neurodevelopmental disorders.

Human cystic fibrosis embryonic stem cell lines derived on placental mesenchymal stromal cells.

This study describes the production of two new human embryonic stem cell (hESC) lines affected by cystic fibrosis. These cell lines are heterozygous compounds, each a carrier of the DF508 mutations associated either with E585X or with 3849+10 kb C-->T. The derivation process was performed on irradiated human placental mesenchymal stromal cells and designed to minimize contact with xeno-components. This new source of feeder cells is easy to obtain and devoid of ethical concerns. The cells have a great capacity to proliferate which reduces the need for continuous preparation of new feeder cell lines. In addition, three normal hESC lines were obtained in the same conditions. The five stem cell lines retained hESC-specific features, including an unlimited and undifferentiated proliferation capacity, marker expression and the maintenance of stable karyotype. They also demonstrated pluripotency in vitro, forming cell lineages of the three germ layers, as indicated by immunolocalization of beta-tubulin, alpha-fetoprotein and actin. These new genetic cell lines represent an important in-vitro tool to study the physiological processes underlying this genetic disease, drug screening, and tissue engineering.

[Molecular mechanisms of neuronal connective tissue genesis during the course of cerebral cortex development]. / Mécanismes moléculaires de la genèse de la connectivité neuronale au cours du développement du cortex cérébral.

The cerebral cortex constitutes one of the most complex structures in our brain. In correlation with its elaborate functions, it is characterized by the great complexity of its neuronal connections, but the mechanisms responsible for the generation of these connections remain poorly known. We have recently initiated the characterization of a new multigenic family of axon guidance factors, the ephrin/Eph gene family, during the development of neuronal connections in the mouse cortex. Combining expression studies, in vitro guidance essays, and in vivo analysis of mutant mice, enabled us to demonstrate the critical role of ephrin/Eph genes in the development of cortical networks. Mutant mice for ephrin/Eph genes display a topographic distortion of their cortical somatosensory map, as well as ectopic projections from the motor thalamus to the somatosensory cortex. The identification of factors like ephrins, capable to (re)specify the pattern of neuronal connections, has implications for our understanding of pathological brain development (epilepsy, abnormal movements, psychiatric diseases), and in the perspective of the rational design of cell therapies of neurodegenerative diseases.

Enhanced plasticity of retinothalamic projections in an ephrin-A2/A5 double mutant.

Ascending sensory information reaches primary sensory cortical areas via thalamic relay neurons that are organized into modality-specific compartments or nuclei. Although the sensory relay nuclei of the thalamus show consistent modality-specific segregation of afferents, we now show in a wild-type mouse strain that the visual pathway can be surgically "rewired" so as to induce permanent retinal innervation of auditory thalamic cell groups. Applying the same rewiring paradigm to a transgenic mouse lacking the EphA receptor family ligands ephrin-A2 and ephrin-A5 results in more extensive rewiring than in the wild-type strain. We also show for the first time that ephrin-A2 and ephrin-A5 define a distinct border between visual and auditory thalamus. In the absence of this ephrin-A2/A5 border and after rewiring surgery, retinal afferents are better able to invade and innervate the deafferented auditory thalamus. These data suggest that signals that induce retinal axons to innervate the denervated auditory thalamus may compete with barriers, such as the ephrins, that serve to contain them within the normal target. The present findings thus show that the targeting of retinothalamic projections can be surgically manipulated in the mouse and that such plasticity can be controlled by proteins known to regulate topographic mapping.

Malformation of the functional organization of somatosensory cortex in adult ephrin-A5 knock-out mice revealed by in vivo functional imaging.

The molecular mechanisms that coordinate the functional organization of the mammalian neocortex are largely unknown. We tested the involvement of a putative guidance label, ephrin-A5, in the functional organization of the somatosensory cortex by quantifying the functional representations of individual whiskers in vivo in adult ephrin-A5 knock-out mice, using intrinsic signal optical imaging. In wild-type mice ephrin-A5 is expressed in a gradient in the somatosensory cortex during development. In adult ephrin-A5 knock-out mice, we found a spatial gradient of change in the amount of cortical territory shared by individual whisker functional representations across the somatosensory cortex, as well as a gradient of change in the distance between the functional representations. Both gradients of change were in correspondence with the developmental expression gradient of ephrin-A5 in wild-type mice. These changes involved malformations of the cortical spacing of the thalamocortical components, without concurrent malformations of the intracortical components of individual whisker functional representations. Overall, these results suggest that a developmental guidance label, such as ephrin-A5, is involved in establishing certain spatial relationships of the functional organization of the adult neocortex, and they underscore the advantage of investigating gene manipulation using in vivo functional imaging.

A mapping label required for normal scale of body representation in the cortex.

The neocortical primary somatosensory area (S1) consists of a map of the body surface. The cortical area devoted to different regions, such as parts of the face or hands, reflects their functional importance. Here we investigated the role of genetically determined positional labels in neocortical mapping. Ephrin-A5 was expressed in a medial > lateral gradient across S1, whereas its receptor EphA4 was in a matching gradient across the thalamic ventrobasal (VB) complex, which provides S1 input. Ephrin-A5 had topographically specific effects on VB axon guidance in vitro. Ephrin-A5 gene disruption caused graded, topographically specific distortion in the S1 body map, with medial regions contracted and lateral regions expanded, changing relative areas up to 50% in developing and adult mice. These results provide evidence for within-area thalamocortical mapping labels and show that a genetic difference can cause a lasting change in relative scale of different regions within a topographic map.

The ephrins and Eph receptors in neural development.

The Eph receptors are the largest known family of receptor tyrosine kinases. Initially all of them were identified as orphan receptors without known ligands, and their specific functions were not well understood. During the past few years, a corresponding family of ligands has been identified, called the ephrins, and specific functions have now been identified in neural development. The ephrins and Eph receptors are implicated as positional labels that may guide the development of neural topographic maps. They have also been implicated in pathway selection by axons, the guidance of cell migration, and the establishment of regional pattern in the nervous system. The ligands are anchored to cell surfaces, and most of the functions so far identified can be interpreted as precise guidance of cell or axon movement. This large family of ligands and receptors may make a major contribution to the accurate spatial patterning of connections and cell position in the nervous system.

Topographic guidance labels in a sensory projection to the forebrain.

Visual connections to the mammalian forebrain are known to be patterned by neural activity, but it remains unknown whether the map topography of such higher sensory projections depends on axon guidance labels. Here, we show complementary expression and binding for the receptor EphA5 in mouse retina and its ligands ephrin-A2 and ephrin-A5 in multiple retinal targets, including the major forebrain target, the dorsal lateral geniculate nucleus (dLGN). These ligands can act in vitro as topographically specific repellents for mammalian retinal axons and are necessary for normal dLGN mapping in vivo. The results suggest a general and economic modular mechanism for brain mapping whereby a projecting field is mapped onto multiple targets by repeated use of the same labels. They also indicate the nature of a coordinate system for the mapping of sensory connections to the forebrain.

Molecular cloning and chromosomal mapping of olfactory receptor genes expressed in the male germ line: evidence for their wide distribution in the human genome.

Olfactory receptor genes constitute the largest family of G protein-coupled receptors. We have previously shown that members of this family are expressed during spermatogenesis, and that the corresponding proteins are displayed on mature sperm cells. In each mammalian species, a restricted subset of olfactory receptors is expressed in male germ cells and displays a pattern of expression suggestive of their potential implication in the control of sperm physiology. In addition to the cDNA fragments available previously, we now report the molecular cloning of two olfactory receptor cDNAs from a human testis library. Five olfactory receptor genes expressed in germ cells were localized in the human genome by radiation hybrid mapping. Three of the genes map to the short arm of chromosome 19 (19p13.1-19p31.3), one to chromosome 11 (11q22.1-22.3), and one to chromosome 17 (17q21-22). The former two localizations fall within clusters previously identified for members of the putative olfactory receptor gene family expressed in olfactory mucosa. Similarly, sequence analysis has revealed that these testicular genes share no distinctive structural features from the other, non-testicular, members of the family. The expression of a subset of olfactory receptor genes in the male germ line is therefore not correlated to their belonging to a specific structural subgroup, or to a specific gene cluster or chromosomal segment.

Specific repertoire of olfactory receptor genes in the male germ cells of several mammalian species.

Olfactory receptors constitute the largest family among G protein-coupled receptors, with up to 1000 members expected. We have previously shown that genes belonging to this family were expressed in the male germ line from both dog and human. We have subsequently demonstrated the presence of one of the corresponding olfactory receptor proteins during dog spermatogenesis and in mature sperm cells. In this study, we investigated whether the unexpected pattern of expression of olfactory receptors in the male germ line was conserved in other mammalian species. Using reverse transcription-PCR with primers specific for the olfactory receptor gene family, about 20 olfactory receptor cDNA fragments were cloned from the testis of each mammalian species tested. As a whole, they displayed no sequence specificity compared to other olfactory receptors, but highly homologous, possibly orthologous, genes were amplified from different species. Finally, their pattern of expression, as determined by RNase protection assay, revealed that many but not all of these receptors were expressed predominantly in testis. The male germ line from each mammalian species tested ins thus characterized by a specific repertoire of olfactory receptors, which display a pattern of expression suggestive of their potential implication in the control of sperm maturation, migration, or fertilization.

Olfactory receptors are displayed on dog mature sperm cells.

Olfactory receptors constitute a huge family of structurally related G protein-coupled receptors, with up to a thousand members expected. We have shown previously that genes belonging to this family were expressed in the male germ line from both dog and human. The functional significance of this unexpected site of expression was further investigated in the present study. We demonstrate that a few dog genes representative of various subfamilies of olfactory receptors are expressed essentially in testis, with little or no expression in olfactory mucosa. Other randomly selected members of the family show the expected site of expression, restricted to the olfactory system. Antibodies were generated against the deduced amino acid sequence of the most abundantly expressed olfactory receptor gene in dog testis. The purified serum was able to detect the gene product (DTMT receptor) in late round and elongated spermatids, as well as in the cytoplasmic droplet that characterizes the maturation of dog sperm cells, and on the tail midpiece of mature spermatozoa. Western blotting further confirmed the presence of a 40-kD immunoreactive protein in the membrane of mature sperm cells. Altogether , these results demonstrate that the main expression site of a subset of the large olfactory receptor gene family is not olfactory mucosa but testis. This expression correlates with the presence of the corresponding protein during sperm cell maturation, and on mature sperm cells. The pattern of expression is consistent with a role as sensor for unidentified chemicals possibly involved in the control of mammalian sperm maturation, migration, and/or fertilization.

Soluble and particulate Ins(1,4,5)P3/Ins(1,3,4,5)P4 5-phosphatase in bovine brain.

Ins(1,4,5)P3 5-phosphatase catalyses the dephosphorylation of Ins(1,4,5)P3 in the 5 position. At 1 microM Ins(1,4,5)P3, 10-15% of total activity of a bovine brain homogenate was measured in the soluble fraction, whereas 85-90% was in the particulate fraction. Particulate activity could be solubilized by cholate or, to a lower extent, by 2 M KCl. Two soluble enzymes (type I and type II) could be fractionated by DEAE-Sephacel chromatography. Soluble activities have been further purified by blue-Sepharose, Sephacryl S-200 and phosphocellulose chromatography. Specific activities reached 10-30 mumol.min-1 mg protein-1 for type I and were 10-20 times lower for type II. Type I and type II Ins(1,4,5)P3 5-phosphatase displayed different Km values and molecular masses, as estimated by gel filtration. Type I dephosphorylated both Ins(1,4,5)P3 and Ins(1,3,4,5)P4; in contrast, type II specifically dephosphorylated Ins(1,4,5)P3 but not Ins(1,3,4,5)P4. Type I Ins(1,4,5)P3 5-phosphatase eluted as a single peak of activity with an apparent molecular mass of 51 kDa when gel filtration was performed in the presence of cholate. This molecular mass is identical to the molecular mass estimated for the particulate Ins(1,4,5)P3 5-phosphatase that was solubilized by cholate. Km values for Ins(1,4,5)P3 and Ins(1,3,4,5)P4 obtained with type I Ins(1,4,5)P3 5-phosphatase were 11 microM and 1 microM, respectively. Similar values were obtained with particulate Ins(1,4,5)P3 5-phosphatase. In conclusion, the catalytic domains of type I and particulate Ins(1,4,5)P3 5-phosphatase activity may be very similar, if not identical, but different from type II phosphatase.