Neurons derived from patients with bipolar disorder divide into intrinsically different sub-populations of neurons, predicting the patients' responsiveness to lithium.

Bipolar disorder (BD) is a progressive psychiatric disorder with more than 3% prevalence worldwide. Affected individuals experience recurrent episodes of depression and mania, disrupting normal life and increasing the risk of suicide greatly. The complexity and genetic heterogeneity of psychiatric disorders have challenged the development of animal and cellular models. We recently reported that hippocampal dentate gyrus (DG) neurons differentiated from induced pluripotent stem cell (iPSC)-derived fibroblasts of BD patients are electrophysiologically hyperexcitable. Here we used iPSCs derived from Epstein-Barr virus-immortalized B-lymphocytes to verify that the hyperexcitability of DG-like neurons is reproduced in this different cohort of patients and cells. Lymphocytes are readily available for research with a large number of banked lines with associated patient clinical description. We used whole-cell patch-clamp recordings of over 460 neurons to characterize neurons derived from control individuals and BD patients. Extensive functional analysis showed that intrinsic cell parameters are very different between the two groups of BD neurons, those derived from lithium (Li)-responsive (LR) patients and those derived from Li-non-responsive (NR) patients, which led us to partition our BD neurons into two sub-populations of cells and suggested two different subdisorders. Training a Naïve Bayes classifier with the electrophysiological features of patients whose responses to Li are known allows for accurate classification with more than 92% success rate for a new patient whose response to Li is unknown. Despite their very different functional profiles, both populations of neurons share a large, fast after-hyperpolarization (AHP). We therefore suggest that the large, fast AHP is a key feature of BD and a main contributor to the fast, sustained spiking abilities of BD neurons. Confirming our previous report with fibroblast-derived DG neurons, chronic Li treatment reduced the hyperexcitability in the lymphoblast-derived LR group but not in the NR group, strengthening the validity and utility of this new human cellular model of BD.

Boundary cap cells are highly competitive for CNS remyelination: fast migration and efficient differentiation in PNS and CNS myelin-forming cells.

During development, boundary cap cells (BC) and neural crest cell (NCC) derivatives generate Schwann cells (SC) of the spinal roots and a subpopulation of neurons and satellite cells in the dorsal root ganglia. Despite their stem-like properties, their therapeutic potential in the diseased central nervous system (CNS) was never explored. The aim of this work was to explore BC therapeutic potential for CNS remyelination. We derived BC from Krox20(Cre) x R26R(Yfp) embryos at E12.5, when Krox20 is exclusively expressed by BC. Combining microdissection and cell fate mapping, we show that acutely isolated BC are a unique population closely related but distinct from NCC and SC precursors. Moreover, when grafted in the demyelinated spinal cord, BC progeny expands in the lesion through a combination of time-regulated processes including proliferation and differentiation. Furthermore, when grafted away from the lesion, BC progeny, in contrast to committed SC, show a high migratory potential mediated through enhanced interactions with astrocytes and white matter, and possibly with polysialylated neural cell adhesion molecule expression. In response to demyelinated axons of the CNS, BC progeny generates essentially myelin-forming SC. However, in contact with axons and astrocytes, some of them generate also myelin-forming oligodendrocytes. There are two primary outcomes of this study. First, the high motility of BC and their progeny, in addition to their capacity to remyelinate CNS axons, supports the view that BC are a reservoir of interest to promote CNS remyelination. Second, from a developmental point of view, BC behavior in the demyelinated CNS raises the question of the boundary between central and peripheral myelinating cells.

Krox20 inactivation in the PNS leads to CNS/PNS boundary transgression by central glia.

CNS/PNS interfaces constitute cell boundaries, since they delimit territories with different neuronal and glial contents. Despite their potential interest in regenerative medicine, the mechanisms restricting oligodendrocytes and astrocytes to the CNS, and Schwann cells to the PNS in mammals are not known. To investigate the involvement of peripheral glia and myelin in the maintenance of the CNS/PNS boundary, we have first made use of different mouse mutants. We show that inactivation of Krox20/Egr2, a master regulatory gene for myelination in Schwann cells, results in transgression of the CNS/PNS boundary by astrocytes and oligodendrocytes and in myelination of nerve root axons by oligodendrocytes. In contrast, such migration does not occur with the Trembler(J) mutation, which prevents PNS myelination without affecting Krox20 expression. Altogether these data suggest that maintenance of the CNS/PNS boundary requires a new Krox20 function separable from myelination control. Finally, we have analyzed a human patient affected by a congenital amyelinating neuropathy, associated with the absence of the KROX20 protein in Schwann cells. In this case, the nerve roots were also invaded by oligodendrocytes and astrocytes. This indicates that transgression of the CNS/PNS boundary by central glia can occur in pathological situations in humans and suggests that the underlying mechanisms are common with the mouse.

A dual role of erbB2 in myelination and in expansion of the schwann cell precursor pool.

Neuregulin-1 provides an important axonally derived signal for the survival and growth of developing Schwann cells, which is transmitted by the ErbB2/ErbB3 receptor tyrosine kinases. Null mutations of the neuregulin-1, erbB2, or erbB3 mouse genes cause severe deficits in early Schwann cell development. Here, we employ Cre-loxP technology to introduce erbB2 mutations late in Schwann cell development, using a Krox20-cre allele. Cre-mediated erbB2 ablation occurs perinatally in peripheral nerves, but already at E11 within spinal roots. The mutant mice exhibit a widespread peripheral neuropathy characterized by abnormally thin myelin sheaths, containing fewer myelin wraps. In addition, in spinal roots the Schwann cell precursor pool is not correctly established. Thus, the Neuregulin signaling system functions during multiple stages of Schwann cell development and is essential for correct myelination. The thickness of the myelin sheath is determined by the axon diameter, and we suggest that trophic signals provided by the nerve determine the number of times a Schwann cell wraps an axon.

Transcriptional regulation of globin gene expression in the human erythroid cell line K562.

The effect of hemin on the rate of synthesis and the level of globin messenger RNA's in the human erythroid cell line K562 was examined by means of cloned hybridization probes specific for each of the human embryonic, fetal, and adult globin genes. Hemin increases both the rate of transcription and the level of accumulation of zeta-, epsilon-, gamma-, and alpha-globin messenger RNA's by a factor of 3 to 5. Thus, hemin induction of globin gene expression in K562 cells is at the level of transcription.

Biology of hepatitis B virus.

Immunochemical investigations of the viral antigens and molecular characterization of the viral DNA have elucidated the nature of the hepatitis B virus infection underlying acute, chronic, and oncogenic disorders of the liver in man. Cloning and sequencing of viral DNA have made possible studies on the structure of the genome and on certain aspects of the biology of the virus, hitherto constrained for a lack of tissue culture systems and laboratory animal models useful in its propagation.

Forward signaling mediated by ephrin-B3 prevents contralateral corticospinal axons from recrossing the spinal cord midline.

To investigate Eph-ephrin bidirectional signaling, a series of mutations were generated in the ephrin-B3 locus. The absence of both forward and reverse signaling resulted in mice with mirror movements as typified by a hopping locomotion. The corticospinal tract was defective as axons failed to respect the midline boundary of the spinal cord and bilaterally innervated both contralateral and ipsilateral motor neuron populations. A second mutation that expresses a truncated ephrin-B3 protein lacking its cytoplasmic domain did not lead to hopping, indicating that reverse signaling is not required for corticospinal innervation. Ephrin-B3 is concentrated at the spinal cord midline, while one of its receptors, EphA4, is expressed in postnatal corticospinal neurons as their fibers pathfind down the contralateral spinal cord. Our data indicate ephrin-B3 functions as a midline-anchored repellent to stimulate forward signaling in EphA4-expressing axons.

Reorganization of pontine rhythmogenic neuronal networks in Krox-20 knockout mice.

We have shown previously that the inactivation of the zinc finger gene Krox-20 affects hindbrain segmentation, resulting in the elimination of rhombomeres 3 and 5. We demonstrate here that Krox-20 homozygous mutant mice exhibit abnormally slow respiratory and jaw opening rhythms, indicating that a modification of hindbrain segmentation influences the function of neuronal networks after birth. Central neuronal networks that control respiratory frequency are made predominantly depressant by the elimination of a previously undescribed rhythm-promoting system. Recordings of rhythmic activity from the isolated hindbrain following progressive tissue transections indicate that the reorganization takes place in the caudal pontine reticular formation. The newborn (PO) Krox-20-/- mice, in which apneas are ten times longer than in wild-type animals, may be a valuable model for the study of life-threatening apneas during early infancy.

A requirement for the immediate early gene Zif268 in the expression of late LTP and long-term memories.

The induction of long-term potentiation (LTP) in the dentate gyrus of the hippocampus is associated with a rapid and robust transcription of the immediate early gene Zif268. We used a mutant mouse with a targeted disruption of Zif268 to ask whether this gene, which encodes a zinc finger transcription factor, is required for the maintenance of late LTP and for the expression of long-term memory. We show that whereas mutant mice exhibit early LTP in the dentate gyrus, late LTP is absent when measured 24 and 48 hours after tetanus in the freely moving animal. In both spatial and non-spatial learning tasks, short-term memory remained intact, whereas performance was impaired in tests requiring long-term memory. Thus, Zif268 is essential for the transition from short- to long-term synaptic plasticity and for the expression of long-term memories.

Road casualties in work-related and private contexts: occupational medical impact. Results from the ESPARR cohort.

BACKGROUND: Road accidents may impact victims' physical and/or mental health and socio-occupational life, notably including return to work. OBJECTIVES: To assess whether the occupational medical consequences sustained by subjects injured in road accidents occurring in a work-related context differ from those associated with private accidents. METHODS: 778 adults who were in work or occupational training at the time of their accident were included. Two groups were distinguished: 354 (45.5%) injured in road accidents occurring in a work-related context (commuting or on duty) and 424 (54.5%) injured in a private accident. The groups were compared on medical and occupational factors assessed on prospective follow-up at 6 months and 1 and 3 years. Multivariate analysis explored for factors associated at 6 months and 1 year with sick leave following the accident and duration of sick leave. RESULTS: There were no significant differences between groups for demographic data apart from a slightly higher injury severity in private accidents (32.5% of private accidents with MAIS3+(Maximum Abbreviated Injury Scale greater or equal to 3) vs. 23.7% for work-related accidents, p = 0.007). Victims of work-related accidents were more often on sick leave (OR = 1.8; 95% CI, 1.1-2.9). Although the length of sick leave is higher for work-related accidents that for private accidents, multivariate analysis showed that the injury severity and the post-traumatic stress disorder (PTSD) are significant factors to explain the time to return to work. There were no significant differences according to occupational impact during follow-up, notably including sick-leave duration, number of victims returning to work within 3 years and number of victims out of work due to incapacity. CONCLUSIONS: In the ESPARR (follow-up study of a road-accident population in the Rhône administrative county: Etude de Suivi d'une Population d'Accidentés de la Route dans le Rhône) cohort, the fact that a road accident occurred in a work-related context did not affect the occupational consequences.

Linker scanning mutagenesis of the 5'-flanking region of the mouse beta-major-globin gene: sequence requirements for transcription in erythroid and nonerythroid cells.

We analyzed the sequences required for transcription of the mouse beta-major-globin gene by introducing deletion and linker scanning mutations into the 5'-flanking region and then studying the effects of these mutations on beta-globin gene transcription in a HeLa cell transient expression assay or after stable introduction into mouse erythroleukemia cells. Consistent with earlier studies, we found that three distinct regions upstream from the RNA capping site are required for efficient beta-globin gene transcription in HeLa cells: the ATA box located 30 base pairs upstream from the mRNA capping site (-30), the CCAAT box located at -75, and the distal sequence element CCACACCC located at -90. In the ATA and CAAT box regions, the sequences necessary for efficient transcription extend beyond the limits of the canonical sequences. Mutations in the sequences located between the three transcriptional control elements do not significantly affect transcription in HeLa cells. Although the promoter defined in HeLa cell transfection experiments is also required for efficient transcription in mouse erythroleukemia cells, none of the mutations tested affects the regulation of beta-globin gene transcription during mouse erythroleukemia cell differentiation. Thus, DNA sequences downstream from the mRNA cap site appear to be sufficient for the regulation of beta-globin gene expression during the differentiation of mouse erythroleukemia cells in culture.

Characterization of a mouse multigene family that encodes zinc finger structures.

The Drosophila segmentation gene Krüppel encodes multiple tandemly repeated units predicted to form DNA-binding zinc fingers. We have isolated 23 bacteriophages, containing nonoverlapping inserts from a mouse genomic DNA library, on the basis of cross-hybridization under nonstringent conditions to a probe corresponding to the Krüppel finger region. Nucleotide sequence analysis of six phage DNAs indicated that they all contained regions with similarity to Krüppel and potentially encoded zinc finger domains. Within these regions, the level of similarity to Krüppel was particularly high between successive fingers. Northern (RNA) blotting analysis suggested that the mouse sequences belonged to different genes, the expression of some of which was modulated during cell differentiation and development. Hybridization experiments suggested that the similarity between some of the genes extended outside of the finger regions. In conclusion, our data suggest that the mouse genome contains a large family of evolutionarily related genes encoding possible trans-acting factors. These genes are likely to play a regulatory role at the transcriptional level.

The serum-inducible mouse gene Krox-24 encodes a sequence-specific transcriptional activator.

The mouse gene Krox-24 is transiently activated during cell cycle reentry. It encodes a protein with three zinc fingers similar to those of the transcription factor Sp1. Here we present a biochemical characterization of the gene products. Krox-24 mRNA is translated into two proteins of 82 and 88 kilodaltons, designated p82Krox-24 and p88Krox-24, respectively. p82Krox-24 is initiated at the first AUG codon of the open reading frame, whereas synthesis of p88Krox-24 starts at a non-AUG codon located upstream. Both proteins were synthesized in HeLa cells infected with recombinant vaccinia viruses expressing Krox-24 cDNAs. Under these conditions, they were found phosphorylated on serine residues and glycosylated. The availability of the proteins made possible the determination of the DNA recognition sequence. In vitro, Krox-24 bound specifically to the sequence 5'-GCG(C/G)GGGCG-3'. This sequence is similar but not identical to the Sp1 target sequence. Insertion of an oligomer for the binding site in cis, close to the herpes simplex virus thymidine kinase promoter, rendered this promoter responsive to Krox-24. Krox-24 is therefore a sequence-specific transcriptional activator. Krox-24-binding sites were found upstream of several serum-inducible genes, raising the possibility that Krox-24 is involved in the regulation of these genes.

Structure, chromosome location, and expression of the mouse zinc finger gene Krox-20: multiple gene products and coregulation with the proto-oncogene c-fos.

We have analyzed the structure and the regulation of Krox-20, a mouse zinc finger-encoding gene which is transiently activated following serum stimulation of quiescent fibroblast cells in culture. The gene is localized on chromosome 10, band B5, in the mouse, and the homologous human gene also maps to chromosome 10 (region q21.1 to q22.1). Alternative splicing of the 5'-most intron of the Krox-20 gene gives rise to mRNAs encoding putative zinc finger proteins with different N termini. The first exon contains a sequence element with strong similarity to the c-fos proto-oncogene serum response element (SRE). This element can functionally substitute for the c-fos SRE, and it binds the same nuclear protein. It is probably responsible for the serum induction of Krox-20, possibly in combination with a weaker SRE located in the 5'-flanking region of the gene. Our findings suggest that c-fos, Krox-20, and a number of immediate-early serum response genes are coregulated and that the SRE and its cognate protein are essential components of this regulatory pathway.

[Consequences for children involved in a road traffic accident: one year of observation and follow-up in the Rhone county]. / Consequénces des accidents de la circulation chez les enfants: suivi pendant un an dans le département du Rhône.

Children aged 6 to 11 who have been injured in a traffic accident were observed over a one year period, in parallel with a control group of children. More than one-third of the injured children had been hospitalized (for periods ranging anywhere between 1 and 47 days). One year later, one injured child out of ten was still suffering from pain, and/or still being treated for injuries resulting from the accident. Many other factors were linked to the initial overall level of severity of the injuries, contrary to that of pain, such as the rate and duration of hospitalization, the duration of care provided, the number of medical consultations, and absenteeism from school. Children who had been injured in a road traffic accident were found to be more anxious and nervous, in general, as well as having a high prevalence of sleeping disorders in comparison to the children in the control group.

Transactivation of Krox-20 and Krox-24 promoters by the HTLV-1 Tax protein through common regulatory elements.

The HTLV-1 Tax protein has been shown to induce the expression of host cellular genes, some of which play crucial roles in cell proliferation and differentiation. We have examined the effect of Tax on the expression of two immediate-early genes, Krox-20 and Krox-24, which encode transcription factors. Several HTLV-1-infected T-cell lines and a HeLa cell line that constitutively expresses the Tax protein have a high level of expression of the Krox-20 and Krox-24 genes. In addition, Tax transactivates the promoters of both Krox-20 and Knox-24 in a co-transfection assay. Tax-responsive elements in Krox-20 and Krox-24 include the serum response elements (SREs) and the putative cAMP-responsive element (CRE). A correlation exists between the ability of these elements to mediate Tax transactivation and their affinity for their cognate factors, the serum response factor (SRF) or the CRE-binding protein (CREB) respectively. Since Tax is also able to transactivate the human c-fos promoter through the SRE and the CRE-60, our findings support the idea that the HTLV-1 Tax protein uses common mechanisms for transactivation of these three immediate-early genes. Deregulation of their expression may contribute to malignant transformation associated with HTLV-1 infection.

An Eph-related receptor protein tyrosine kinase gene segmentally expressed in the developing mouse hindbrain.

In search of genes possibly involved in the regulation of hindbrain segmentation, we have isolated mouse cDNA clones corresponding to putative protein kinase genes by polymerase chain reaction amplification of cDNA from 9.5-day-old embryo hindbrains. In situ hybridization analysis revealed that one of these genes, Sek, was expressed in an alternating segment-restricted pattern in the developing hindbrain. Isolation and analysis of Sek cDNAs covering the entire coding sequence indicated that Sek encoded a putative receptor protein tyrosine kinase, belonging to the Eph family. These data are consistent with a role of the Sek gene product in a signal transduction process involved in pattern formation in the hindbrain.

Characterization of an Krox-24/Egr-1-responsive element in the human tumor necrosis factor promoter.

We have analyzed in various human leukemic cell lines a previously unrecognized region within the human TNF gene promoter that contains the sequence motif 5'-CCGCCCCCGCG-3'. This GC-rich sequence maps to bps -170 and -160 of the TNF gene. Electrophoretic mobility shift assays (EMSA) combined with methylation interference analysis revealed the binding of two distinct proteins with overlapping recognition sites. Supershift assays identified the constitutive transcription factor Sp1 and the immediate-early growth-response transcription factor Egr-1/Krox-24. Interestingly, this Egr-1-related factor was induced by PMA but not by TNF. The TNF gene GC-rich sequence conferred PMA responsiveness when linked to a heterologous minimal c-fos promoter. To examine the involvement of Egr-1/Krox-24 in TNF gene regulation, a Krox-24 expression vector was used, pSCTKr24. In Jurkat T cells pSCTKr24 stimulated pTNF-286CAT that contains sequences -286 to +34 of the human TNF gene fused to the chloramphenicol acetyltransferase (CAT) gene. Moreover, pSCTKr24 also stimulated the TNF gene GC-rich sequence linked to the minimal c-fos promoter. However, deletion of this site did not result in markedly reduced TNF promoter activity, suggesting that the Egr-1/Krox-24 response element may play an auxiliary role in TNF gene regulation.

Pattern of expression of the transcription factor Krox-20 in mouse hair follicle.

We examined the pattern of expression in hair follicles of the transcription factor gene Krox-20. During embryogenesis, Krox-20 is first expressed in the upper portion of the hair bud, then in the hair canal, in the sebaceous glands and in the outer root sheath. In the mature follicles, Krox-20, like Shh and TGFbetaRII, is also expressed in a sub-population of matrix keratinocytes located in a ring-like fashion in vibrissal follicles and clustered on one side of the papilla in pelage follicles. This polarized pattern rotates around the papilla as a result of sequential gene expression by individual groups of matrix cells. This peculiar pattern is not linked to follicle angling.

Krox-20 is a key regulator of rhombomere-specific gene expression in the developing hindbrain.

The morphogenesis of the vertebrate hindbrain involves a transient segmentation process leading to the formation of reiterated organisation units called rhombomeres (r). A number of regulatory genes expressed with a rhombomere-specific pattern have been identified, including the gene encoding the transcription factor Krox-20, which is restricted to r3 and r5. We have previously demonstrated that in r3 and r5 Krox-20 directly controls the transcription of Hoxa-2 and Hoxb-2. In the present study, we provide evidence that Krox-20 is required for the expression of another Hox gene, Hoxb-3, in r5 specifically. Furthermore, the regulatory role of Krox-20 is not restricted to the control of Hox gene expression, since it is also involved in the activation of a receptor tyrosine kinase gene, Sek-1, in r3 and r5 and in the repression of the follistatin gene in r3 but not in r5. In conclusion, at least five regulatory genes belonging to different families are under the direct or indirect control of Krox-20 in r3 and/or r5 and this transcription factor therefore appears as a key regulator of gene expression in the developing hindbrain.