A pathogenic role for JNK signaling in experimental anti-GBM glomerulonephritis.

Activation of the c-Jun NH2-terminal kinase (JNK) signaling pathway is involved in the immune response; however, little is known of its role in immune-induced renal injury. In this study, we examine JNK signaling in the rat anti-glomerular basement membrane (GBM) disease model using CC-401, a specific JNK inhibitor. Animals were given CC-401, vehicle alone or no treatment starting before anti-GBM serum injection and continued treatment until killing. In acute disease, CC-401 blocked JNK signaling and reduced proteinuria in the first 24 h. The transient neutrophil influx seen at 3 h of disease was not affected, however. Continued CC-401 treatment suppressed glomerular and tubulointerstitial damage usually seen at 14 days. The protective effect may be due to modulation of macrophage activation, as CC-401 had no effect upon glomerular macrophage infiltration at day 14 despite the suppression of glomerular lesions and a marked reduction in renal tumor necrosis factor-alpha and inducible nitric oxide synthase messenger RNA levels. Treatment with CC-401 had no apparent effect on T cell or humoral immune responses. These studies suggest that JNK signaling promotes renal injury in acute and progressive rat anti-GBM disease. JNK inhibitors may be a novel therapeutic approach for the treatment of human glomerulonephritis.

Induction of tissue plasminogen activator in differentiated NG108-15 cells.

Plasminogen activators may facilitate neurite outgrowth and neuronal migration in the developing nervous system. The expression of tissue plasminogen activator by NG108-15 neuroblastoma grown under a variety of conditions has been explored. High tissue plasminogen mRNA expression correlates with growth conditions which induce morphological differentiation and neurite outgrowth; however, NG108-15 cells grown in suspension with dibutyryl-cAMP also show a high level of tissue plasminogen activator expression.

Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

Ephrin-A5 (AL-1/RAGS) is essential for proper retinal axon guidance and topographic mapping in the mammalian visual system.

Ephrin-A5 (AL-1/RAGS), a ligand for Eph receptor tyrosine kinases, repels retinal axons in vitro and has a graded expression in the superior colliculus (SC), the major midbrain target of retinal ganglion cells. These properties implicate ephrin-A5 in the formation of topographic maps, a fundamental organizational feature of the nervous system. To test this hypothesis, we generated mice lacking ephrin-A5. The majority of ephrin-A5-/- mice develop to adulthood, are morphologically intact, and have normal anterior-posterior patterning of the midbrain. However, within the SC, retinal axons establish and maintain dense arborizations at topographically incorrect sites that correlate with locations of low expression of the related ligand ephrin-A2. In addition, retinal axons transiently overshoot the SC and extend aberrantly into the inferior colliculus (IC). This defect is consistent with the high level of ephrin-A5 expression in the IC and the finding that retinal axon growth on membranes from wild-type IC is inhibited relative to that on membranes from ephrin-A5-/- IC. These findings show that ephrin-A5 is required for the proper guidance and mapping of retinal axons in the mammalian midbrain.

Graded and lamina-specific distributions of ligands of EphB receptor tyrosine kinases in the developing retinotectal system.

Molecular gradients have been postulated to control the topographic mapping of retinal axons in their central targets. Based initially on their expression patterns, and more recently on functional studies, members of the EphA subfamily of receptor tyrosine kinases and their ephrin-A ligands have been implicated in the guidance of retinal axons along the anterior-posterior axis of the chick optic tectum. The report that a receptor of the EphB subfamily, EphB2/Cek5/Nuk/Sek3, is expressed in a high ventral to low dorsal gradient in the developing chick retina and is present on ganglion cell axons suggests that it may be involved in the mapping of retinal axons along the corresponding dorsal-ventral axis of the tectum. To address this issue, we have determined the expression and distribution of ephrin-B1/LERK-2/Cek5-L and ephrin-B2/LERK-5/Htk-L/ELF-2, ligands for EphB2, in the developing chick retinotectal system using riboprobes, immunocytochemistry, and receptor affinity probes. Both ephrin-B1 and ephrin-B2 transcripts are expressed in a high dorsal to low ventral gradient in the developing retina, complementary to the distribution of EphB2. Ephrin-B1 and ephrin-B2 proteins are predominantly found in the developing plexiform layers, suggesting a role in the development of intraretinal connections. Neither protein is detected on ganglion cell axons. In tectum, ephrin-B1 transcripts are expressed in a high dorsal to low ventral gradient in the neuroepithelium and the protein is present along the processes of radial glia and is concentrated at their endfeet in the stratum opticum, at the time retinal axons are growing through it. This distribution of ephrin-B1 suggests that it influences retinal axon mapping along the dorsal-ventral tectal axis and may also be involved in intratectal development. In contrast, ephrin-B2 transcripts and protein are localized to the deeper retinorecipient laminae in the tectum at the time retinal axons begin to arborize in them, suggesting that this ligand may influence the laminar patterning of retinal axon terminations.

Topographically specific effects of ELF-1 on retinal axon guidance in vitro and retinal axon mapping in vivo.

Topographic maps, which maintain the spatial order of neurons in the order of their axonal connections, are found throughout the nervous system. In the visual retinotectal projection, ELF-1, a ligand in the tectum, and its receptors in the retina show complementary gradients in expression and binding, indicating they may be positional labels for map development. Here we show that ELF-1 acts as a repellent axon guidance factor in vitro. In vivo, when the tectal ELF-1 pattern is modified by retroviral overexpression, retinal axons avoid ectopic ELF-1 patches and map to abnormally anterior positions. All these effects were seen on axons from temporal but not nasal retina, indicating that ELF-1 could determine nasal versus temporal retinotectal specificity, and providing a direct demonstration of a cell recognition molecule with topographically specific effects on neural map development.

Retroviral misexpression of engrailed genes in the chick optic tectum perturbs the topographic targeting of retinal axons.

We have investigated the role of the homeodomain transcription factor genes En-1 and En-2, homologs of the Drosophila segment polarity gene engrailed, in regulating the development of the retinotopic map in the chick optic tectum. The En proteins are distributed in a gradient along the rostral-caudal axis of the developing tectum, with highest amounts found caudally. Previous evidence suggests that En-1 and En-2 may regulate the polarity of the rostral-caudal axis of the tectum and the subsequent topographic mapping of retinal axons. We have tested this hypothesis by using a recombinant replication-competent retrovirus to overexpress the En-1 or En-2 genes in the developing tectum. Anterograde labeling with the axon tracer Dil was used to analyze the topographic mapping of retinal axons after the time that the retinotectal projection is normally topographically organized. Overexpression of either En-1 or En-2 perturbed the topographic targeting of retinal axons. In En-infected tecta, nasal retinal axons form an abnormally diffuse projection with numerous aberrant axons, branches, and arbors found at topographically incorrect locations, colocalized with domains of viral infection. In contrast, temporal axons did not form a diffuse projection or discrete aberrant arbors; however, many temporal axons were stunted and ended aberrantly rostral to their appropriate TZ, or in other cases either did not enter the tectum or formed a dense termination at its extreme rostral edge. These findings indicate that En-1 and En-2 are involved in regulating the development of the retinotopic map in the tectum. Furthermore, they support the hypothesis that En genes regulate the polarity of the rostral-caudal axis of the tectum, most likely by controlling the expression of retinal axon guidance molecules.

Eph receptor tyrosine kinases and their ligands in neural development.

Receptor tyrosine kinases play important roles in many developmental phenomena, including the regulation of cell proliferation, differentiation, and survival. The largest subfamily of receptor tyrosine kinases are the Eph receptors, which are widely expressed in the nervous system. With the recent identification of several Eph ligands, it has become evident that Eph receptors and their ligands are involved in the guidance of retinal axons and in the process of hindbrain segmentation.

Tissue plasminogen activator mRNA expression in granule neurons coincides with their migration in the developing cerebellum.

Tissue plasminogen activator activity in the developing cerebellum, as quantified by zymography of cerebellar homogenates from embryonic day (E) 17 to adult mice, shows a peak of activity at postnatal day (P) 7, followed by a steady 75% decrease into adulthood. Northern blot analysis reveals a similar pattern for tissue plasminogen activator mRNA levels, which are low at E17 but increase dramatically, reaching their highest levels of specific mRNA/micrograms RNA in P1-P7 mice and declining about threefold in the adult mouse. In situ hybridization of whole mouse brain sections with a tissue plasminogen activator antisense cRNA probe shows pronounce reactivity in the cerebellum. Although some binding is associated with the cerebellar meninges, the external granule layer is devoid of tissue plasminogen activator mRNA at all ages. However, highly labeled elongated cells, which also bind antibody to neuronal nuclear antigen and are adjacent to Bergmann glial fibers (i.e., migrating granule neurons), are readily visible throughout the molecular and Purkinje layers at P7 and P14. In the adult mouse cerebellum, tissue plasminogen activator mRNA labeling is restricted to cells in the Purkinje/internal granule layers. Thus, tissue plasminogen activator gene expression is induced as granule neurons leave the external granule layer and begin their inward migration.

Mechanisms and molecules controlling the development of retinal maps.

All mature vertebrates exhibit precise topographic mapping from the retina to the tectum, or its mammalian homologue, the superior colliculus (SC). In frogs and fish the development of this projection is precise from the outset; in avians retinal axon targeting is more diffuse but respects a coarse topographic matching; and in rodents early projections show no topographic specificity. Topography in avians and rodents emerges from a process of branch extension, arborization, and elimination of aberrant axonal projections. Despite these differences, the basic mechanisms controlling the development of this retinotopy are conserved. It has been hypothesized that molecules distributed in a position-dependent manner in the retina and the tectum or SC control the development of these maps. A number of candidate molecules have been identified on the basis of their distribution, or their ability to influence axonal growth in vitro. In addition, transcription factors and signaling molecules are expressed in a position-dependent manner and may regulate the expression of molecules involved in retinotopic map formation.

Tissue plasminogen activator expression in the embryonic nervous system.

Tissue plasminogen activator (tPA) expression during embryogenesis was determined by in situ hybridization of whole mouse embryos from E10.5 through E17.5. The strongest expression occurs in the basal midbrain and hindbrain, and continues posteriorly into the neural canal. This expression coincides with extensive cell migration and proliferation, and tissue remodelling of this region. tPA mRNA is also associated with cells that appear to be invading the cerebellar anlage. Presumptive proliferating and migrating cells in the olfactory neuroepithelium also express tPA. These results indicate that tPA is expressed by a number of different cell types in the developing nervous system and suggest a role for tPA in cell migration and tissue remodelling of the developing CNS.

Allergic fungal sinusitis. Report of three cases associated with dematiaceous fungi.

Most reported cases of allergic sinusitis have been attributed to Aspergillus, based on the morphologic features of the organisms in tissue sections. However, in most cases, cultures have not been done. This is a report of three cases of non-Aspergillus allergic fungal sinusitis. The patients' ages were 11, 16, and 43; two were male and one was female. Histopathologic study disclosed fungal organisms resembling Aspergillus. However, cultures of these patients' nasal secretions grew Drechslera, Exserohilum, and Bipolaris fungal organisms. The non-Aspergillus nature of these infections was further supported by positive Fontana-Masson melanin staining. The authors conclude that allergic fungal sinusitis most likely results from non- Aspergillus organisms. For definitive fungal identification, tissue culture is mandatory. When tissue is not cultured or no organisms grow, a Fontana-Masson stain can be a useful adjunct in fungal identification.