Activation of Pax3 target genes is necessary but not sufficient for neurogenesis in the ophthalmic trigeminal placode.

Vertebrate cranial neurogenic placodes are relatively simple model systems for investigating the control of sensory neurogenesis. The ophthalmic trigeminal (opV) placode, for which the earliest specific marker is the paired domain homeodomain transcription factor Pax3, forms cutaneous sensory neurons in the ophthalmic lobe of the trigeminal ganglion. We previously showed that Pax3 expression in avian opV placode cells correlates with specification and commitment to a Pax3+, cutaneous sensory neuron fate. Pax3 can act as a transcriptional activator or repressor, depending on the cellular context. We show using mouse Splotch(2H) mutants that Pax3 is necessary for the normal neuronal differentiation of opV placode cells. Using an electroporation construct encoding a Pax3-Engrailed fusion protein, which represses Pax3 target genes, we show that activation of Pax3 target genes is required cell-autonomously within chick opV placode cells for expression of the opV placode markers FGFR4 and Ngn2, maintenance of the preplacodal marker Eya2, expression of Pax3 itself (suggesting that Pax3 autoregulates), neuronal differentiation and delamination. Mis-expression of Pax3 in head ectoderm is sufficient to induce FGFR4 and Ngn2 expression, but neurons do not differentiate, suggesting that additional signals are necessary to enable Pax3+ cells to differentiate as neurons. Mis-expression of Pax3 in the Pax2+ otic and epibranchial placodes also downregulates Pax2 and disrupts otic vesicle closure, suggesting that Pax3 is sufficient to alter the identity of these cells. Overall, our results suggest that activation of Pax3 target genes is necessary but not sufficient for neurogenesis in the opV placode.

Analysis of the action of euxanthone, a plant-derived compound that stimulates neurite outgrowth.

We have investigated the neurite growth-stimulating properties of euxanthone, a xanthone derivative isolated from the Chinese medicinal plant Polygala caudata. Euxanthone was shown to exert a marked stimulatory action on neurite outgrowth from chick embryo dorsal root ganglia explanted in collagen gels, in the absence of added neurotrophins. It was also shown to promote cell survival in explanted chick embryo ganglia, and to stimulate neurite outgrowth from isolated adult rat primary sensory neurons in vitro. The further finding that euxanthone stimulates neurite outgrowth from explants of chick embryo retina and ventral spinal cord suggests an action on signaling pathways downstream of neuronal receptors for specific neurotrophic factors. Consistent with this, euxanthone did not promote neurite outgrowth from non-transfected PC12 cells, or from PC12 cells transfected with TrkB or TrkC, under conditions in which these cells extended neurites in response to, respectively, the neurotrophins nerve growth factor, brain-derived neurotrophic factor and neurotrophin 3. Western blot analysis of euxanthone-stimulated dorsal root ganglion explants showed that expression of phospho-mitogen-activated protein (MAP) kinase was up-regulated after 1 h of euxanthone-treatment. Inhibition of the MAP kinase pathway using PD98059, a specific inhibitor of MAP kinase kinase, blocked all euxanthone-stimulated neurite outgrowth. However, analysis of phospho-Akt expression indicated that the phosphatidylinositol-3 kinase-Akt pathway, another major signaling pathway engaged by neurotrophins, is not significantly activated by euxanthone. These results suggest that euxanthone promotes neurite outgrowth by selectively activating the MAP kinase pathway.

Pet dogs owned by lupus patients are at a higher risk of developing lupus.

The aim of this study is to determine whether pet dogs owned by patients with systemic lupus erythematosus (SLE) are at a higher risk of developing SLE. Diagnosis of canine SLE was mainly based on the 11 diagnostic criteria for human SLE and two marked immunological features of canine SLE. Among 59 pet dogs owned by 37 SLE patients, 11 (18.64%) were ANA positive, and three (5.08%) had SLE. In contrast, of 187 pet dogs owned by non-SLE households, nine (4.81%) were ANA positive, and none (0%) had SLE. Among 650 outpatient dogs registered in the veterinary hospital, 34 (5.23%) were ANA positive, and six (0.92%) had SLE. Frequency of ANA and SLE among pet dogs owned by SLE patients was significantly higher than in pet dogs owned by non-SLE households (P = 0.001 for ANA; P = 0.013 for SLE) and in outpatient dogs (P < 0.001 for ANA; P = 0.032 for SLE). With respect to canine SLE development, the relative risk or risk ratio (R) of human SLE contact varied from 5.5 (compared with outpatient dogs) to near the infinite (compared with dogs owned by non-SLE households). The prevalence of canine SLE among pet dogs of SLE patients was therefore estimated to be 508 per 10 000 [95% confidence interval (95% CI), 0-1068]. In conclusion, pet dogs with human SLE contact were at a higher risk of developing SLE. Our results indicate that a common environmental factor or zoonotic agent may be involved in the development of human and canine SLE.

Somite polarity and segmental patterning of the peripheral nervous system.

The analysis of the outgrowth pattern of spinal axons in the chick embryo has shown that somites are polarized into anterior and posterior halves. This polarity dictates the segmental development of the peripheral nervous system: migrating neural crest cells and outgrowing spinal axons traverse exclusively the anterior halves of the somite-derived sclerotomes, ensuring a proper register between spinal axons, their ganglia and the segmented vertebral column. Much progress has been made recently in understanding the molecular basis for somite polarization, and its linkage with Notch/Delta, Wnt and Fgf signalling. Contact-repulsive molecules expressed by posterior half-sclerotome cells provide critical guidance cues for axons and neural crest cells along the anterior-posterior axis. Diffusible repellents from surrounding tissues, particularly the dermomyotome and notochord, orient outgrowing spinal axons in the dorso-ventral axis ('surround repulsion'). Repulsive forces therefore guide axons in three dimensions. Although several molecular systems have been identified that may guide neural crest cells and axons in the sclerotome, it remains unclear whether these operate together with considerable overall redundancy, or whether any one system predominates in vivo.

Requirement of the JIP1 scaffold protein for stress-induced JNK activation.

The c-Jun N-terminal kinase (JNK) signal transduction pathway is activated in response to the exposure of cells to environmental stress. Components of the JNK signaling pathway interact with the JIP1 scaffold protein. JIP1 is located in the neurites of primary hippocampal neurons. However, in response to stress, JIP1 accumulates in the soma together with activated JNK and phosphorylated c-Jun. Disruption of the Jip1 gene in mice by homologous recombination prevented JNK activation caused by exposure to excitotoxic stress and anoxic stress in vivo and in vitro. These data show that the JIP1 scaffold protein is a critical component of a MAP-kinase signal transduction pathway.

JNK3 contributes to c-Jun activation and apoptosis but not oxidative stress in nerve growth factor-deprived sympathetic neurons.

The stress activated protein kinase pathway culminates in c-Jun phosphorylation mediated by the Jun Kinases (JNKs). The role of the JNK pathway in sympathetic neuronal death is unclear in that apoptosis is not inhibited by a dominant negative protein of one JNK kinase, SEK1, but is inhibited by CEP-1347, a compound known to inhibit this overall pathway but not JNKs per se. To evaluate directly the apoptotic role of the JNK isoform that is selectively expressed in neurons, JNK3, we isolated sympathetic neurons from JNK3-deficient mice and quantified nerve growth factor (NGF) deprivation-induced neuronal death, oxidative stress, c-Jun phosphorylation, and c-jun induction. Here, we report that oxidative stress in neurons from JNK3-deficient mice is normal after NGF deprivation. In contrast, NGF-deprivation-induced increases in the levels of phosphorylated c-Jun, c-jun, and apoptosis are each inhibited in JNK3-deficient mice. Overall, these results indicate that JNK3 plays a critical role in activation of c-Jun and apoptosis in a classic model of cell-autonomous programmed neuron death.

Programmed cell death of developing mammalian neurons after genetic deletion of caspases.

An analysis of programmed cell death of several populations of developing postmitotic neurons after genetic deletion of two key members of the caspase family of pro-apoptotic proteases, caspase-3 and caspase-9, indicates that normal neuronal loss occurs. Although the amount of cell death is not altered, the death process may be delayed, and the cells appear to use a nonapoptotic pathway of degeneration. The neuronal populations examined include spinal interneurons and motor, sensory, and autonomic neurons. When examined at both the light and electron microscopic levels, the caspase-deficient neurons exhibit a nonapoptotic morphology in which nuclear changes such as chromatin condensation are absent or reduced; in addition, this morphology is characterized by extensive cytoplasmic vacuolization that is rarely observed in degenerating control neurons. There is also reduced terminal deoxynucleotidyl transferase-mediated biotinylated UTP nick end labeling in dying caspase-deficient neurons. Despite the altered morphology and apparent temporal delay in cell death, the number of neurons that are ultimately lost is indistinguishable from that seen in control animals. In contrast to the striking perturbations in the morphology of the forebrain of caspase-deficient embryos, the spinal cord and brainstem appear normal. These results are consistent with the growing idea that the involvement of specific caspases and the occurrence of caspase-independent programmed cell death may be dependent on brain region, cell type, age, and species or may be the result of specific perturbations or pathology.

Mechanisms of programmed cell death in the developing brain.

Programmed cell death (apoptosis) is an important mechanism that determines the size and shape of the vertebrate nervous system. Recent gene-targeting studies have indicated that homologs of the cell-death pathway in the nematode Caenorhabditis elegans have analogous functions in apoptosis in the developing mammalian brain. However, epistatic genetic analysis has revealed that the apoptosis of progenitor cells during early embryonic development and apoptosis of postmitotic neurons at later stage of brain development have distinct roles and mechanisms. These results provide new insight on the significance and mechanism of neural cell death in mammalian brain development.

Caspase-3 is required for apoptosis-associated DNA fragmentation but not for cell death in neurons deprived of potassium.

Caspases are crucial effectors of the cell death pathway activated by virtually all apoptosis-inducing stimuli within neurons and nonneuronal cells. Among the caspases, caspase-3 (CPP32) appears to play a pivotal role and has been found to be necessary for developmentally regulated cell death in the brain. We have used mice lacking caspase-3 (-/-CPP32) to examine its involvement in cultured cerebellar granule neurons induced to undergo apoptosis by potassium deprivation (K+). We find that, following K+ deprivation, neurons from -/-CPP32 mice die to the same extent as those from normal (+/+) mice. Although a small delay in the induction of cell death is observed in -/-CPP32 neurons, the rate of cell death is generally comparable to that of +/+ cultures. Though not critical for neuronal death, caspase-3 is required for DNA fragmentation and chromatin condensation as judged by the absence of these apoptotic features in -/-CPP32 neurons. Boc.Asp.fmk, a pan caspase inhibitor, partially protects +/+ neurons from low-K+-mediated cell death and does so to the same extent in -/-CPP32 cultures, suggesting the involvement of a caspase other than caspase-3 in cell death. However, the protective effect of boc.Asp.fmk is not seen beyond 24 hr, suggesting that the effect of caspase inhibition is one of delaying rather than preventing apoptosis. The more selective caspase inhibitors DEVD.fmk, IETD.fmk, and VEID.fmk fail to affect cell death, indicating that members inhibited by these agents (such as caspases - 6 ,7, 8, 9 and 10) are also not involved in low-K+-mediated apoptosis.

The role of cell death in regulating the size and shape of the mammalian forebrain.

The size of the cerebral cortex is determined by the rate of production of neurons and glial cells in the proliferative ventricular and subventricular zones. Recent studies from targeted mutations of different death-effector gene families indicate that programmed cell death (PCD) plays an important role in cell production and early morphogenesis of the mammalian forebrain before the formation of neuronal connections. For example, disruption of the c/Jun N-kinase signaling pathway by double-targeted mutation of both Jnk1 and Jnk2 results in increased PCD in the forebrain leading to precocious degeneration of cerebral precursors. In contrast, disturbance of the caspase cascade by targeted disruption of either casp-9 or casp-3 leads to decreased PCD causing expansion and exencephaly of the forebrain as well as supernumerary neurons in the cerebral cortex. The supernumerary neurons in these knockout mice align radially and form an expanded cortical plate which begins to form cerebral convolutions. Thus, the precise coordination of different apoptotic signaling pathways during early stages of neurogenesis is crucial for regulation of the proper cortical size and shape.

The Jnk1 and Jnk2 protein kinases are required for regional specific apoptosis during early brain development.

The c-Jun NH2-terminal kinase (Jnk) family is implicated in apoptosis, but its function in brain development is unclear. Here, we address this issue using mutant mice lacking different members of the family (Jnk1, Jnk2, and Jnk3). Mice deficient in Jnk1, Jnk2, Jnk3, and Jnk1/Jnk3 or Jnk2/Jnk3 double mutants all survived normally. Compound mutants lacking Jnk1 and Jnk2 genes were embryonic lethal and had severe dysregulation of apoptosis in brain. Specifically, there was a reduction of cell death in the lateral edges of hindbrain prior to neural tube closure. In contrast, increased apoptosis and caspase activation were found in the mutant forebrain, leading to precocious degeneration. These results suggest that Jnk1 and Jnk2 regulate region-specific apoptosis during early brain development.

Reduced apoptosis and cytochrome c-mediated caspase activation in mice lacking caspase 9.

Caspases are essential components of the mammalian cell death machinery. Here we test the hypothesis that Caspase 9 (Casp9) is a critical upstream activator of caspases through gene targeting in mice. The majority of Casp9 knockout mice die perinatally with a markedly enlarged and malformed cerebrum caused by reduced apoptosis during brain development. Casp9 deletion prevents activation of Casp3 in embryonic brains in vivo, and Casp9-deficient thymocytes show resistance to a subset of apoptotic stimuli, including absence of Casp3-like cleavage and delayed DNA fragmentation. Moreover, the cytochrome c-mediated cleavage of Casp3 is absent in the cytosolic extracts of Casp9-deficient cells but is restored after addition of in vitro-translated Casp9. Together, these results indicate that Casp9 is a critical upstream activator of the caspase cascade in vivo.

Bioflavonoids commonly and potently induce tyrosine dephosphorylation/inactivation of oncogenic proline-directed protein kinase FA in human prostate carcinoma cells.

In this study, we investigate the effect of bioflavonoids on the activity and phosphotyrosine content of oncogenic proline-directed protein kinase FA (PDPK FA) in human prostate carcinoma cells. Chronic treatment of human prostate carcinoma cells with low concentrations of quercetin, apigenin, and kaempferol commonly and potently induced tyrosine dephosphorylation and concurrent inactivated oncogenic PDPK FA in a concentration-dependent manner. This is demonstrated by a specific assay of this kinase's activity in the immunoprecipitates from the cell extracts followed by immunoblotting and phosphotyrosine analysis. The results indicate that bioflavonoids may function as common tyrosine kinase inhibitors to inhibit PDPK FA-specific tyrosine kinase and thereby to induce tyrosine dephosphorylation/inactivation of this oncogenic kinase in human carcinoma cells. Under this condition, quercetin, apigenin, and kaempferol can also inhibit cell growth in a similar concentration-dependent manner. The results further indicate that inhibition of tyrosine phosphorylation/activation of this oncogenic PDPK represents a new mode of action mechanism for bioflavonoids during the antiproliferation process in human carcinoma cells.

The naturally occurring PKC inhibitor sphingosine and tumor promoter phorbol ester potentially induce tyrosine phosphorylation/activation of oncogenic proline-directed protein kinase FA/GSK-3alpha in a common signalling pathway.

When serum-starved A431 cells were treated with 200 nM phorbol ester TPA for 15 min, the cellular activity of protein kinase FA/glycogen synthase kinase-3alpha (kinase FA/GSK-3alpha) could be decreased to approximately 25% of control. Conversely, when treated with 1 microM TPA for 24 hr, the activity could be reversibly increased to approximately 200% of Control. The naturally occurring protein kinase C (PKC) inhibitor sphingosine at a concentration of 27 microM could also induce activation of kinase FA/GSK-3alpha to approximately 200% of control within 60 min. Further, when cells were chronically treated with 1 microM TPA for 24 hr and then with 27 microM sphingosine for 60 min, the activity of kinase FA/GSK-3alpha could only be increased to approximately 200% of control. Furthermore, when cells were pretreated with sphingosine and then acutely treated with TPA, the acute TPA effect on kinase FA/GSK-3alpha activity could be abolished by genistein or tyrosine phosphorylation, which could be blocked by genistein or tyrosine phosphatase, but could be reversed by orthovanadate. Taken together, the results demonstrate that TPA/sphingosine induce tyrosine phosphorylation and concurrent activation of kinase FA/GSK-3alpha in a common signalling pathway. Since TPA and sphingosine are potent PKC modulators, the results further suggest a potential role of PKC in modulating tyrosine phosphorylation/activation of kinase FA/GSK-3alpha. Kinetic studies on seven subtypes of PKC further demonstrate a specific involvement of PKCE in this tyrosine phosphorylation/activation process. This provides a new mode of signal transduction between these two important serine/threonine kinases in cells.

Restrictive clonal allocation in the chimeric mouse brain.

Whether, and to what extent, lineage restriction contributes to the organization of the mammalian brain remains unclear. Here we address this issue by examining the distribution of clonally related cells in chimeric mice generated by injecting genetically tagged embryonic stem (ES) cells into blastocyst embryos. Our examination of postnatal chimeric brains revealed that the vast majority of labeled ES cell descendents were confined within a different subset of brain regions in each animal. Moreover, the deployment of labeled cells in different brain regions was distinctive. The pattern of ordered and binomial colonization suggested that early diversified founder cells may constrain the fates of their descendants through a restriction of dispersion. In addition, the symmetrical distribution of ES cell descendants suggests that bilaterally corresponding structures may arise from a common set of progenitor cells. Finally, clones of cells formed a continuous band within the deep strata of the neocortex. This later finding in conjunction with the radial distribution of clones in remaining layers observed in previous studies indicates that the cerebral neocortex may derive from two groups of founder cells, which is consistent with the hypothesis of dual phylogenetic origins of the mammalian cerebral cortex.

Absence of excitotoxicity-induced apoptosis in the hippocampus of mice lacking the Jnk3 gene.

Excitatory amino acids induce both acute membrane depolarization and latent cellular toxicity, which often leads to apoptosis in many neurological disorders. Recent studies indicate that glutamate toxicity may involve the c-Jun amino-terminal kinase (JNK) group of mitogen-activated protein (MAP) kinases. One member of the JNK family, Jnk3, may be required for stress-induced neuronal apoptosis, as it is selectively expressed in the nervous system. Here we report that disruption of the gene encoding Jnk3 in mice caused the mice to be resistant to the excitotoxic glutamate-receptor agonist kainic acid: they showed a reduction in seizure activity and hippocampal neuron apoptosis was prevented. Although application of kainic acid imposed the same level of noxious stress, the phosphorylation of c-Jun and the transcriptional activity of the AP-1 transcription factor complex were markedly reduced in the mutant mice. These data indicate that the observed neuroprotection is due to the extinction of a Jnk3-mediated signalling pathway, which is an important component in the pathogenesis of glutamate neurotoxicity.

Overexpression of protein kinase FA/GSK-3 alpha (a proline-directed protein kinase) correlates with human hepatoma dedifferentiation/progression.

Computer analysis of protein phosphorylation sites sequence revealed that transcriptional factors and viral oncoproteins are prime targets for regulation of proline-directed protein phosphorylation, suggesting an association of the proline-directed protein kinase (PDPK) family with neoplastic transformation and tumorigenesis. In this report, an immunoprecipitate activity assay of protein kinase FA/glycogen synthase kinase-3 alpha (kinase F(A)/GSK-3 alpha) (a member of PDPK family) has been optimized for human hepatoma and used to demonstrate for the first time significantly increased (P < 0.01) activity in poorly differentiated SK-Hep-1 hepatoma (24.2 +/- 2.8 units/mg) and moderately differentiated Mahlavu hepatoma (14.5 +/- 2.2 units/mg) when compared to well differentiated Hep 3B hepatoma (8.0 +/- 2.4 units/mg). Immunoblotting analysis revealed that increased activity of kinase FA/GSK-3 alpha is due to overexpression of the protein. Elevated kinase FA/GSK-3 alpha expression in human hepatoma biopsies relative to normal liver tissue was found to be even more profound. This kinase appeared to be fivefold overexpressed in well differentiated hepatoma and 13-fold overexpressed in poorly differentiated hepatoma when compared to normal liver tissue. Taken together, the results provide initial evidence that overexpression of kinase FA/GSK-3 alpha is involved in human hepatoma dedifferentiation/progression. Since kinase FA/GSK-3 alpha is a PDPK, the results further support a potential role of this kinase in human liver tumorigenesis, especially in its dedifferentiation/progression.