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International audience; Heterozygous mutations in JAGGED1, encoding a single-pass transmembrane ligand for the Notch receptors, cause Alagille syndrome (AGS), a polymalformative disorder affecting the liver, heart, eyes and skeleton and characterized by a peculiar facies. Most of the JAGGED1 mutations generate premature termination codons, and as a result, two pathogenic mechanisms causing AGS have been proposed: haploinsufficiency or a dominant-negative effect of putative truncated proteins. To determine whether missense or protein-truncating mutations in JAGGED1 can lead to the synthesis and function of abnormal proteins, we performed cell culture experiments. We showed that human JAGGED1 undergoes a metalloprotease-dependent cleavage resulting in the shedding of its extracellular domain and that this domain seems able to fulfill a biological function in vitro, probably by antagonizing Notch signaling. Moreover, the soluble form of JAGGED1 was able to compete with the transmembrane ligand. Mutant proteins with missense or nonsense mutations were synthesized and gave rise to a chord-like phenotype and a migration defect when expressed by stably transfected cells. These chord-like structures were similar to the phenotype exhibited by fibroblasts isolated from a fetus with a protein-truncating mutation. Results obtained from Notch signaling inhibition and Notch reporter assays showed that this chord-like phenotype, exhibited by mutant JAGGED1 transfectants, may result from an inhibitory effect on Notch signaling. Altogether, our results favor a dominant-negative mechanism of some JAGGED1 mutations in AGS.

Neurogenesis persists throughout life in the rodent subventricular zone (SVZ) and subgranular zone (SGZ) and increases in the adult after brain injury. In this study, postnatal day 7 rats underwent middle cerebral artery electrocoagulation and transient homolateral common carotid artery occlusion, a lesioning protocol that resulted in ipsilateral (IL) forebrain ischemic injury, leading to a cortical cavity 3 weeks later. The effects of neonatal ischemia on hemispheric damage, cell death, cell proliferation, and neurogenesis were examined 4 hours to 6 weeks later by the terminal deoxynucleotidyl transferase dUTP nick-end labeling assay and immunohistochemistry of Ki-67 in proliferating cells and of doublecortin, a microtubule-associated protein expressed only by immature neurons. Neonatal ischemic injury resulted in persistent reduced IL and transient reduced contralateral (CL) hemispheric areas, a consequence of sustained and transient cell death in the IL and CL areas, respectively. Ki-67 immunostaining revealed 3 peaks of newly generated cells in the dorsal SVZ and SGZ in the IL side and also in the CL side at 48 hours and 7 and 28 days after ischemia. Double immunofluorescence revealed that most of the Ki-67-positive cells were astrocytes at 48 hours. Ischemic injury also stimulated SVZ neurogenesis, based on increased doublecortin immunostaining in both SVZs at 7 to 14 days after injury. Doublecortin-positive neurons remained visible around the lesion at 21 days but displayed an immature shape in discrete chains or clusters. Although unilateral ischemic damage was produced, results indicate successful regenerative changes in the CL hemisphere, allowing anatomical recovery.

This paper examines the molecular machinery involved in membrane exchange within the plant endomembrane system. A study has been undertaken on beta-COP-like proteins in plant cells using M3A5, an antibody raised against the conserved sequence of mammalian beta-COP proteins. In mammalian cells, beta-COP proteins are part of a complex named the coatomer, which probably recruits some specific areas of the endomembrane system. Immunofluorescence analyses by confocal laser scanning microscopy showed that beta-COP-like proteins marked predominantly the plant Golgi apparatus. Other proteins known to be part of a potential machinery for COPI vesicle formation (gamma-COP, beta'-COP and Arf1 proteins) were immunolocalized on the same membraneous structures as beta-COP. Moreover, beta-COP and other COPI antibodies stained the cell plate in dividing cells. It is further shown that, in maize root cells, and in contrast to observations upon mammalian cells, the drug Brefeldin A (BFA) does not induce the release of beta-COP and Arf1 proteins from the Golgi membrane into the cytosol. These data clearly demonstrate that the antibody M3A5 is a valuable marker for studies on trafficking events in plant cells. They also report for the first time the location of COP components in plant tissue at the light level, especially on a model well known for secretion, i.e. the maize root cells. They also suggest that the membrane recruitment machinery may function in a plant-specific way.