Search

Age-related hearing loss (ARHL) is the most prevalent sensory deficit in the elderly. This progressive pathology often has psychological and medical comorbidities, including social isolation, depression, and cognitive decline. Despite ARHL's enormous societal and economic impact, no therapies to prevent or slow its progression exist. Loss of synapses between inner hair cells (IHCs) and spiral ganglion neurons (SGNs), a.k.a. IHC synaptopathy, is an early event in cochlear aging, preceding neuronal and hair cell loss. To determine if age-related IHC synaptopathy can be prevented, and if this impacts the time-course of ARHL, we tested the effects of cochlear overexpression of neurotrophin-3 (Ntf3) starting at middle age. We chose Ntf3 because this neurotrophin regulates the formation of IHC-SGN synapses in the neonatal period. We now show that triggering Ntf3 overexpression by IHC supporting cells starting in middle age rapidly increases the amplitude of sound-evoked neural potentials compared with age-matched controls, indicating that Ntf3 produces a positive effect on cochlear function when the pathology is minimal. Furthermore, near the end of their lifespan, Ntf3-overexpressing mice have milder ARHL, with larger sound-evoked potentials along the ascending auditory pathway and reduced IHC synaptopathy compared with age-matched controls. Our results also provide evidence that an age-related decrease in cochlear Ntf3 expression contributes to ARHL and that Ntf3 supplementation could serve as a therapeutic for this prevalent disorder. Furthermore, these findings suggest that factors that regulate synaptogenesis during development could prevent age-related synaptopathy in the brain, a process involved in several central nervous system degenerative disorders.

Noise exposures causing only transient threshold shifts can destroy auditory-nerve synapses without damaging hair cells. Here, we asked whether virally mediated neurotrophin3 (NT3) overexpression can repair this damage. CBA/CaJ mice at 6 wks were injected unilaterally with adeno-associated virus (AAV) containing either NT3 or GFP genes, via the posterior semicircular canal, 3 wks prior to, or 5 hrs after, noise exposure. Controls included exposed animals receiving vehicle only, and unexposed animals receiving virus. Thresholds were measured 2 wks post-exposure, just before cochleas were harvested for histological analysis. In separate virus-injected animals, unexposed cochleas were extracted for qRT-PCR. The GFP reporter showed that inner hair cells (IHCs) were transfected throughout the cochlea, and outer hair cells mainly in the apex. qRT-PCR showed 4- to 10-fold overexpression of NT3 from 1–21 days post-injection, and 1.7-fold overexpression at 40 days. AAV-NT3 delivered prior to noise exposure produced a dose-dependent reduction of synaptopathy, with nearly complete rescue at some cochlear locations. In unexposed ears, NT3 overexpression did not affect thresholds, however GFP overexpression caused IHC loss. In exposed ears, NT3 overexpression increased permanent threshold shifts. Thus, although NT3 overexpression can minimize noise-induced synaptic damage, the forced overexpression may be harmful to hair cells themselves during cochlear overstimulation.

Producing hair cells of the inner ear is the major goal of ongoing research that combines advances in developmental and stem cell biology. The recent advent of an inner ear organoid protocol-resulting in three-dimensional stem cell-derived tissues resembling vestibular sensory epithelia-has sparked interest in applications such as regeneration, drug discovery, and disease modeling. In this study, we adapted this protocol for a novel mouse embryonic stem cell line with a fluorescent reporter for Pax2 expression. We used Pax2EGFP/+ organoid formation to model otic induction, the pivotal developmental event when preplacodal tissue adopts otic fate. We found upregulation of Pax2 and activation of ERK downstream of fibroblast growth factor signaling in organoid formation as in embryonic inner ear development. Pax2 expression was evident from the EGFP reporter beginning at the vesicle formation stage and persisting through generation of the sensory epithelium. The native ventralizing signal sonic hedgehog was largely absent from the cell aggregates as otic vesicles began to form, confirming the dorsal vestibular organoid fate. Nonetheless, cochlear- or vestibular-like neurons appeared to delaminate from the derived otic vesicles and formed synaptic contacts with hair cells in the organoids. Cell lines with transcriptional reporters such as Pax2EGFP/+ facilitate direct evaluation of morphological changes during organoid production, a major asset when establishing and validating the culture protocol.

Sensory epithelia of the inner ear contain two major cell types: hair cells and supporting cells. It has been clear for a long time that hair cells play critical roles in mechanoreception and synaptic transmission. In contrast, until recently the more abundant supporting cells were viewed primarily as serving primarily structural and homeostatic functions. In this review we discuss the growing information about the roles that supporting cells play in the development, function and maintenance of the inner ear, their activities in pathological states, their potential for hair cell regeneration, and the mechanisms underlying these processes.

Upon peripheral nerve injury, specific molecular events, including increases in the expression of selected neurotrophic factors, are initiated to prepare the tissue for regeneration. However, the mechanisms underlying these events and the nature of the cells involved are poorly understood. We used the injury-induced upregulation of glial cell-derived neurotrophic factor (GDNF) expression as a tool to gain insights into these processes. We found that both myelinating and nonmyelinating Schwann cells are responsible for the dramatic increase in GDNF expression after injury. We also demonstrate that the GDNF upregulation is mediated by a signaling cascade involving activation of Schwann cell purinergic receptors, followed by protein kinase C signaling which activates protein kinase D (PKD), which leads to increased GDNF transcription. Given the potent effects of GDNF on survival and repair of injured peripheral neurons, we propose that targeting these pathways may yield therapeutic tools to treat peripheral nerve injury and neuropathies.

Transient receptor potential (TRP) channels have been implicated as sensors of diverse stimuli in mature neurons. However, developmental roles for TRP channels in the establishment of neuronal connectivity remain largely unexplored. Here, we identify an essential function for TRPC5, a member of the canonical TRP subfamily, in the regulation of dendrite patterning in the mammalian brain. Strikingly, TRPC5 knockout mice harbor long, highly branched granule neuron dendrites with impaired dendritic claw differentiation in the cerebellar cortex. In vivo RNAi analyses suggest that TRPC5 regulates dendrite morphogenesis in the cerebellar cortex in a cell-autonomous manner. Correlating with impaired dendrite patterning in the cerebellar cortex, behavioral analyses reveal that TRPC5 knockout mice have deficits in gait and motor coordination. Finally, we uncover the molecular basis of TRPC5's function in dendrite patterning. We identify the major protein kinase calcium/calmodulin-dependent kinase II β (CaMKIIβ) as a critical effector of TRPC5 function in neurons. Remarkably, TRPC5 forms a complex specifically with CaMKIIβ, but not the closely related kinase CaMKIIα, and thereby induces the CaMKIIβ-dependent phosphorylation of the ubiquitin ligase Cdc20-APC at the centrosome. Accordingly, centrosomal CaMKIIβ signaling mediates the ability of TRPC5 to regulate dendrite morphogenesis in neurons. Our findings define a novel function for TRPC5 that couples calcium signaling to a ubiquitin ligase pathway at the centrosome and thereby orchestrates dendrite patterning and connectivity in the brain.

Neuregulin-1 (NRG1) signaling has multiple functions in neurons and glia. The data in this study show that NRG1 may also possess significant signaling and cytoprotective properties in human brain microvascular endothelial cells (BMECs). Neuregulin-1 mRNA and protein expression are present in these cells, and NRG1 receptors erbB2 and erbB3 are phosphorylated in response to NRG1. Neuregulin-1 triggers clear biologic responses in BMECs—elevated phospho-Akt levels, increased ring formation in a Matrigel assay, and decreased cell death after oxidative injury with H2O2. These data suggest that NRG1 signaling is functional and cytoprotective in BMECs.

The formation of morphologically and functionally mature dendrites is a key event in neuronal maturation and the establishment of functional neuronal networks, but the signals that regulate mammalian dendritic development remain poorly understood. Here we show that the erbB receptor signalling pathway, which modulates expression of several neurotransmitter receptors, also regulates dendritic development of cerebellar granule cells in the intact cerebellum. These results suggest that neuregulin-erbB signalling may control a program of postsynaptic development, from initiating dendrite morphogenesis to the formation and maturation of the postsynaptic apparatus.

Signaling by the Notch1 receptor is critical for the formation of radial glia in the developing nervous system. We have shown previously that Notch1 regulates the molecular and morphological differentiation of radial glia through the transcriptional activation of at least two genes, brain lipid binding protein (BLBP) and the erbB2 receptor tyrosine kinase. However, the mechanisms by which this occurs remained undefined. Here we demonstrate that Notch1 effects on radial glia gene expression are mediated by two downstream mechanisms, one that the depends on Suppressor of Hairless [Su(H)] and the other on Deltex1 (DTX1). These two Notch1-binding proteins contribute to the regulation of BLBP and erbB2 expression, respectively. Importantly, our results suggest that, although these events can occur simultaneously, a hierarchical relationship might exist between DTX1 and Su(H), because overexpression of DTX1 or a dominant-negative form of this protein inhibits Su(H)-mediated events but not vice versa. In contrast to the effects of DTX1 overexpression, interference RNA-mediated knock-down of DTX1 blocks Notch1-induced erbB2 promoter activation and radial glia formation selectively, without affecting Su(H)-dependent pathways, indicating that loss of DTX1 expression and expression of dominant-negative DTX1 result in different alterations in cell differentiation and gene expression. Together, these results show that Notch1 regulates radial glia formation through two distinct transcriptional mechanisms and that the outcomes of Notch1 signaling may depend on the relative expression levels of its coregulators.

Supporting cells in the cochlea play critical roles in the development, maintenance, and function of sensory hair cells and auditory neurons. Although the loss of hair cells or auditory neurons results in sensorineural hearing loss, the consequence of supporting cell loss on auditory function is largely unknown. In this study, we specifically ablated inner border cells (IBCs) and inner phalangeal cells (IPhCs), the two types of supporting cells surrounding inner hair cells (IHCs) in mice in vivo. We demonstrate that the organ of Corti has the intrinsic capacity to replenish IBCs/IPhCs effectively during early postnatal development. Repopulation depends on the presence of hair cells and cells within the greater epithelial ridge and is independent of cell proliferation. This plastic response in the neonatal cochlea preserves neuronal survival, afferent innervation, and hearing sensitivity in adult mice. In contrast, the capacity for IBC/IPhC regeneration is lost in the mature organ of Corti, and consequently IHC survival and hearing sensitivity are impaired significantly, demonstrating that there is a critical period for the regeneration of cochlear supporting cells. Our findings indicate that the quiescent neonatal organ of Corti can replenish specific supporting cells completely after loss in vivo to guarantee mature hearing function.

During development of the nervous system, neural progenitors arise in proliferative zones, then exit the cell cycle and migrate away from these zones. Here we show that migration of cerebellar granule cells out of their proliferative zone, the external granule cell layer (EGL), is impaired in Bdnf–/– mice. The reason for impaired migration is that BDNF directly and acutely stimulates granule cell migration. Purified Bdnf–/– granule cells show defects in initiation of migration along glial fibers and in Boyden chamber assays. This phenotype can be rescued by exogenous BDNF. Using time-lapse video microscopy we find that BDNF is acutely motogenic as it stimulates migration of individual granule cells immediately after addition. The stimulation of migration reflects both a chemokinetic and chemotactic effect of BDNF. Collectively, these data demonstrate that BDNF is directly motogenic for granule cells and provides a directional cue promoting migration from the EGL to the internal granule cell layer (IGL).Movies available on-line

Transcription of the brain-derived neurotrophic factor (BDNF) gene is regulated in a calcium- and neuron-selective manner; however, the mechanisms that underlie this selectivity are not known. We have characterized a new calcium-response element, CaRE1, that is required for activity-dependent transcription of BDNF exon III and have cloned a transcription factor, CaRF, that activates transcription from BDNF promoter III in a CaRE1-dependent manner. The transcriptional activity of CaRF is regulated in a calcium- and neuron-selective manner, suggesting that CaRF may confer selectivity upon the activity-dependent induction of BDNF exon III expression.

Background & Aims When the glial fibrillary acidic protein (GFAP) promoter is used to express cellular toxins that eliminate glia in mice, intestinal epithelial permeability and proliferation increase; this led to the concept that glia are required for maintenance of the gastrointestinal epithelium. Many enteric glia, however, particularly in the mucosa, do not express GFAP. In contrast, virtually all enteric glia express proteolipid protein 1 (PLP1). We investigated whether elimination of PLP1-expressing cells compromises epithelial maintenance or gastrointestinal motility. Methods We generated mice that express tamoxifen-inducible Cre recombinase under control of the Plp1 promoter and carry the diptheria toxin subunit A (DTA) transgene in the Rosa26 locus (Plp1 CreER ;Rosa26 DTA mice). In these mice, PLP1-expressing glia are selectively eliminated without affecting neighboring cells. We measured epithelial barrier function and gastrointestinal motility in these mice and littermate controls, and analyzed epithelial cell proliferation and ultrastructure from their intestinal tissues. To compare our findings with those from previous studies, we also eliminated glia with ganciclovir in Gfap HSV-TK mice. Results Expression of DTA in PLP1-expressing cells selectively eliminated enteric glia from the small and large intestines, but caused no defects in epithelial proliferation, barrier integrity, or ultrastructure. In contrast, administration of ganciclovir to Gfap HSV-TK mice eliminated fewer glia but caused considerable non-glial toxicity and epithelial cell death. Elimination of PLP1-expressing cells did not reduce survival of neurons in the intestine, but altered gastrointestinal motility in female, but not male, mice. Conclusions Using the Plp1 promoter to selectively eliminate glia in mice, we found that enteric glia are not required for maintenance of the intestinal epithelium, but are required for regulation of intestinal motility in females. Previous observations supporting the concept that maintenance of the intestinal epithelium requires enteric glia can be attributed to non-glial toxicity in Gfap HSV-TK mice and epithelial-cell expression of GFAP. Contrary to widespread notions, enteric glia are therefore not required for epithelial homeostasis. However, they regulate intestinal motility in a sex-dependent manner.

Neurotrophin-3 (Ntf3) and brain derived neurotrophic factor (Bdnf) are critical for sensory neuron survival and establishment of neuronal projections to sensory epithelia in the embryonic inner ear, but their postnatal functions remain poorly understood. Using cell-specific inducible gene recombination in mice we found that, in the postnatal inner ear, Bbnf and Ntf3 are required for the formation and maintenance of hair cell ribbon synapses in the vestibular and cochlear epithelia, respectively. We also show that supporting cells in these epithelia are the key endogenous source of the neurotrophins. Using a new hair cell CreERT line with mosaic expression, we also found that Ntf3's effect on cochlear synaptogenesis is highly localized. Moreover, supporting cell-derived Ntf3, but not Bbnf, promoted recovery of cochlear function and ribbon synapse regeneration after acoustic trauma. These results indicate that glial-derived neurotrophins play critical roles in inner ear synapse density and synaptic regeneration after injury. DOI: http://dx.doi.org/10.7554/eLife.03564.001, eLife digest Noise-induced hearing loss is common, and can result from prolonged exposure to moderate levels of noise that are not perceived as painful or even unpleasant. Some hearing loss can be attributed to the death of hair cells in a part of the inner ear called the cochlea. When sound waves hit the cochlea, they cause the fluid inside it to vibrate: the hair cells detect these vibrations and convert them into electrical signals that are sent along neurons to the brain. However, vibrations that are too strong can destroy hair cells. Increasing evidence suggests that hearing loss also results from damage to the synapses that connect the hair cells and the neurons in the cochlea. During development of the inner ear, molecules called growth factors are needed to ensure the survival of these neurons. Wan et al. predicted that these growth factors might also have a role in adult animals, and that producing more of them might help to safeguard hearing from the damaging effects of noise. Consistent with this, mice that were genetically modified to lack a growth factor called neurotrophin-3 had cochleae that did not work properly and had fewer synapses between hair cells and neurons compared to control mice. Conversely, mice that produced too much neurotrophin-3 had more synapses than controls and also recovered more quickly from the effects of 2 hr exposure to 100 dB noise (roughly the volume of a pneumatic drill). Studies of the cochlea revealed that the extra neurotrophin-3 had boosted the regeneration of synapses damaged by the noise. The beneficial effects of neurotrophin-3 were still seen when overproduction was started shortly after noise exposure, suggesting that it could have therapeutic potential. This is particularly significant in the light of recent evidence that the loss of synapses often comes before the death of hair cells in both age-related hearing loss and noise-induced hearing loss. DOI: http://dx.doi.org/10.7554/eLife.03564.002

Eph receptors transduce short-range repulsive signals for axon guidance by modulating actin dynamics within growth cones. We report the cloning and characterization of ephexin, a novel Eph receptor-interacting protein that is a member of the Dbl family of guanine nucleotide exchange factors (GEFs) for Rho GTPases. Ephrin-A stimulation of EphA receptors modulates the activity of ephexin leading to RhoA activation, Cdc42 and Rac1 inhibition, and cell morphology changes. In addition, expression of a mutant form of ephexin in primary neurons interferes with ephrin-A-induced growth cone collapse. The association of ephexin with Eph receptors constitutes a molecular link between Eph receptors and the actin cytoskeleton and provides a novel mechanism for achieving highly localized regulation of growth cone motility.

The innervation-induced down-regulation of fetal-type acetylcholine receptor (AChR) expression in developing muscle fibers has largely been attributed to nerve-evoked muscle activity; however, there is increasing evidence that a neural trophic factor also contributes to this receptor down-regulation. Previous studies from this laboratory have shown that neural extracts contain a factor which decreases fetal-type AChR expression in skeletal muscle cell lines and therefore may account for the proposed inhibitory neurotrophic influence. The current study investigated possible intracellular signaling molecules involved in this receptor down-regulation and demonstrated that activation of protein kinase C and p70S6k appeared to be important in receptor down-regulation. Decreases in AChR density were independent of myogenin. In addition, the receptor down-regulation was independent of neuregulin, which also induces p70S6k activity. These studies demonstrate that neural extracts contain an inhibitory factor which can down-regulate fetal-type AChR expression independently of nerve-evoked muscle activity through intracellular signaling molecules which are known to regulate AChR expression. © 2000 John Wiley & Sons, Inc. J Neurobiol 42: 190–201, 2000

Human epidermal growth factor receptor 4 (HER4) is a member of the epidermal growth factor (EGF) receptor subfamily of receptor tyrosine kinases that is activated by neuregulins (NRG), betacellulin (BTC), and heparin-binding EGF-like growth factor. Sequencing of full-length human HER4 cDNAs revealed the existence of two HER4 isoforms that differed by insertion of either 23 or 13 alternative amino acids in the extracellular juxtamembrane (JM) region. The 23-amino acid form (HER4 JM-a) and the 13-amino acid form (HER4 JM-b) were expressed in a tissue-specific manner, as demonstrated by reverse transcriptase-polymerase chain reaction analysis of mouse and human tissues. Both isoforms were expressed in neural tissues such as cerebellum, whereas kidney expressed HER4 JM-a only and heart HER4 JM-b only. In situ hybridization using specific oligonucleotides demonstrated transcription of both JM-a and JM-b isoforms in the mouse cerebellum. Tyrosine phosphorylation analysis indicated that both receptor isoforms were activated to the same extent by NRG-β1 and BTC, and to a lesser extent by NRG-α1 and heparin-binding EGF-like growth factor. A functional difference was found, however, in response to phorbol ester treatment. Stimulation of cells with phorbol ester resulted in a loss of 125I-NRG-β1 binding and in a reduction of total cell-associated HER4 protein in HER4 JM-a transfectants but not in HER4 JM-b transfectants. It was concluded that novel alternatively spliced isoforms of HER4 exist, that they are distributed differentially in vivo in mouse and human tissues, that they are both activated by HER4 ligands, and that they may represent cleavable and noncleavable forms of HER4.

ARIA is a member of a family of polypeptide growth and differentiation factors that also includes glial growth factor (GGF), neu differentiation factor, and heregulin. ARIA mRNA is expressed in all cholinergic neurons of the central nervous systems of rats and chicks, including spinal cord motor neurons. In vitro, ARIA elevates the rate of acetylcholine receptor incorporation into the plasma membrane of primary cultures of chick myotubes. To study whether ARIA may regulate the synthesis of junctional synaptic acetylcholine receptors in chick embryos, we have developed riboprobes and polyclonal antibody reagents that recognize isoforms of ARIA that include an amino-terminal immunoglobulin C2 domain and examined the expression and distribution of ARIA in motor neurons and at the neuromuscular junction. We detected significant ARIA mRNA expression in motor neurons as early as embryonic day 5, around the time that motor axons are making initial synaptic contacts with their target muscle cells. In older embryos and postnatal animals, we found ARIA protein concentrated in the synaptic cleft at neuromuscular junctions, consistent with transport down motor axons and release at nerve terminals. At high resolution using immunoelectron microscopy, we detected ARIA immunoreactivity exclusively in the synaptic basal lamina in a pattern consistent with binding to synapse specific components on the presynaptic side of the basal lamina. These results support a role for ARIA as a trophic factor released by motor neuron terminals that may regulate the formation of mature neuromuscular synapses.

Determining the molecular mechanisms behind synaptogenesis (synapse formation and maintenance) is of great importance for understanding higher brain function as well as disease states such as Alzheimer's and muscular dystrophy. Neuregulin-1 (NRG-1), a nerve derived protein, was isolated based on its ability to stimulate new acetylcholine receptor (AChR) formation on muscle. This molecule has been hypothesized to enforce the high density of AChRs on the post-synaptic membrane in neuromuscular synapses, however, its role in vivo has been difficult to study due to the early death of NRG and ErbB mutants. Therefore, we have developed a microfluidic system, mimicking a synapse by focally delivering nerve derived proteins to a cell chamber containing myotubes, to study synaptogenesis. Also, the device's focal delivery capacity coupled with patterning of the culture chamber surface allows us to ask questions with spatial variables. As a platform for our AChR kinetics studies, we have examined complex, aneural AChR clusters found on muscle and first reported by Kummer et al. in 2004. These features are good models of in vivo post-synaptic areas, because they have similar topologies, protein expression and developmental patterns. After staining with fluorescent bungarotoxin, we have found that neuregulin decreases the half-life of receptors in pretzels by 21.4%. We have also confirmed that NRG-1 increases receptor insertion into pretzels. Afterwards, we examined the extent NRG-1 activation travels in the long, multi-nucleated muscle cell using mRNA in situ hybridization.

Several mutations in Drosophila impair learning and the cAMP cascade. We report here that the fine morphology of an identified mechanosensory neuron is abnormal in two of these mutants, dunce (dnc) and rutabaga (rut). The neuron innervating the antero-notopleural bristle was filled with horseradish peroxidase and studied at the light- and electron-microscopy level. In the mutants dnc and rut, this neuron has an abnormally large number of side branches and varicosities in a defined segment of the axon. In wild-type flies, age tends to decrease the number of side branches and variacosities in the same axonal segment that is affected by the mutations. Ultrastructural studies are compatible with the interpretation that the varicosities are potential synaptic sites. The results suggest that the cAMP cascade plays a role in shaping neuronal connectivity.

In the enteric nervous system (ENS), glia outnumber neurons by 4 to 6-fold and form an extensive network throughout the gastrointestinal tract. Enteric glia are essential for normal gastrointestinal function and play roles in regulating epithelial barrier integrity, epithelial cell proliferation and neuronal support. While glial subtypes can be clearly distinguished in the central and peripheral nervous systems (CNS and PNS), it remains unknown whether similar glial diversity exists in the ENS. Because of their morphology and expression of Glial Fibrillary Acidic Protein (GFAP), until recently, enteric glia were thought to resemble astrocytes. We tested the hypothesis that enteric glia instead constitute a unique and heterogeneous group of glial cells. To define the level of heterogeneity, we first analyzed expression of the markers S100β, Sox10, GFAP, and proteolipid protein 1 (PLP1) in the small and large intestine of adult mice. Sox10 and S100β are widely expressed by enteric glia throughout the intestine. GFAP expression, however, is more restricted. Marker expression in combination with cellular location reproducibly distinguished subpopulations of enteric glia, suggesting that functional subtypes are likely to exist. Unexpectedly, we found that PLP1 is widely expressed by enteric glia, although they do not myelinate axons. We then performed RNA sequencing analysis (RNA-Seq) on PLP1-expressing cells in the mouse intestine and compared their gene expression to that of other types of glia in the CNS and PNS. This showed that enteric glia are transcriptionally distinct from other glial classes, and share the greatest similarity to myelinating glia. The gene expression database generated by this study will facilitate future studies of glial function in gastrointestinal physiology.

To investigate the role of erbB signaling in the interactions between peripheral axons and myelinating Schwann cells, we generated transgenic mice expressing a dominant-negative erbB receptor in these glial cells. Mutant mice have delayed onset of myelination, thinner myelin, shorter internodal length, and smaller axonal caliber in adulthood. Consistent with the morphological defects, transgenic mice also have slower nerve conduction velocity and defects in their responses to mechanical stimulation. Molecular analysis indicates that erbB signaling may contribute to myelin formation by regulating transcription of myelin genes. Analysis of sciatic nerves showed a reduction in the levels of expression of myelin genes in mutant mice. In vitro assays revealed that neuregulin-1 (NRG1) induces expression of myelin protein zero (P0). Furthermore, we found that the effects of NRG1 on P0 expression depend on the NRG1 isoform used. When NRG1 is presented to Schwann cells in the context of cell-cell contact, type III but not type I NRG1 regulates P0 gene expression. These results suggest that disruption of the NRG1-erbB signaling pathway could contribute to the pathogenesis of peripheral neuropathies with hypomyelination and neuropathic pain. Fil: Chen, Suzhen. Harvard Medical School; Estados Unidos Fil: Velardez, Miguel Omar. Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Departamento de Biología Celular e Histología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay; Argentina Fil: Warot, Xavier. Boston University; Estados Unidos Fil: Yu, Zhao-Xue. Boston University; Estados Unidos Fil: Miller, Shyra J.. University of Cincinnati; Estados Unidos Fil: Cros, Didier. Harvard Medical School; Estados Unidos Fil: Corfas, Gabriel. Harvard Medical School; Estados Unidos

After cell birth, almost all neurons in the mammalian central nervous system migrate. It is unclear whether and how cell migration is coupled with neurogenesis. Here we report that proneural basic helix-loop-helix (bHLH) transcription factors not only initiate neuronal differentiation but also potentiate cell migration. Mechanistically, proneural bHLH factors regulate the expression of genes critically involved in migration, including down-regulation of RhoA small GTPase and up-regulation of doublecortin and p35, which, in turn, modulate the actin and microtubule cytoskeleton assembly and enable newly generated neurons to migrate. In addition, we report that several DNA-binding-deficient proneural genes that fail to initiate neuronal differentiation still activate migration, whereas a different mutation of a proneural gene that causes a failure in initiating cell migration still leads to robust neuronal differentiation. Collectively, these data suggest that transcription programs for neurogenesis and migration are regulated by bHLH factors through partially distinct mechanisms.

Radial glia cells both generate neurons and physically guide nascent neurons to their target destination in the cortex, and as such they are essential for CNS development. It has been proposed that in the developing cerebellum, neuronal contact induces radial glia formation, however, the mechanisms involved in this process are not well understood. Here we demonstrate that neuronal induction of radial glia formation is the result of sequential signaling through Notch1 and erbB receptors. First, Notch1 activation by neuronal contact induces the glial expression of the brain lipid binding protein (BLBP) and erbB2 genes. Interestingly, two different signaling pathways mediate these effects of Notch1 on transcription, BLBP expression being dependent on Su(H), whereas erbB2 is regulated by a yet unidentified Notch1 pathway. The subsequent increase in erbB2 receptor expression makes the glia more responsive to neuronal NRG, which then induces the morphological transformation into radial glia. Thus, these results unveil some of the mechanisms underlying radial glia formation.

The intermediate filament glial fibrillary acidic protein (GFAP) is a classic marker for several types of glial cells, including astrocytes and nonmyelinating Schwann cells. The pattern of expression of GFAP in the postnatal murine inner ear, from postnatal day 3 (P3) to P38, was studied by anti-GFAP immunostaining in wild-type mice as well as in two lines of transgenic mice expressing either beta-galactosidase (LacZ) or green fluorescent protein (GFP) under the control of the GFAP promoter. Analysis of protein and promoter activity shows that several classes of supporting cells in the sensory epithelia, as well as Schwann cells and satellite cells express GFAP. Early after birth, all cochlear supporting cells express GFAP, in a gradient decreasing in intensity from base to apex. After P15, GFAP expression in the organ of Corti is mostly restricted to supporting cells of the inner hair cell area (i.e., inner border and inner phalangeal cells) and outer hair cell area (i.e., Deiters' cells). A small population of limbic cells also showed expression in a base-to-apex gradient. In the vestibular organs, high expression was detected in supporting cells in extrastriolar regions of the utricular macula and in the canal ampullae, with weaker staining in the saccular macula. These results suggest that supporting cells of the inner ear have important similarities to glial cells and may play roles similar to those of astrocytes or Schwann cells in supporting the normal development and maintenance of neurons and sensory cells of the inner ear.

HER4 is a member of the epidermal growth factor receptor family and has an essential function in heart and neural development. Identification of two HER4 isoforms, HER4 JM-a and JM-b, which differ in their extracellular juxtamembrane region and in their susceptibility to cleavage after phorbol ester stimulation, showed that the juxtamembrane region of the receptor is critical for proteolysis. We now demonstrate that phorbol ester and pervanadate are effective stimuli for HER4 JM-a processing and that the HER4 JM-b isoform does not undergo cleavage in response to any of the stimuli studied. We also show that HER4 JM-a is not cleaved in cells lacking the metalloprotease tumor necrosis factor-alpha-converting enzyme (TACE) and that reexpression of TACE in these cells restores constitutive and regulated processing of HER4 JM-a. Moreover, we show that the sequence specific to the HER4 JM-a juxtamembrane region is sufficient to confer susceptibility to phorbol 12-myristate 13-acetate-induced cleavage of the HER2 receptor. In conclusion, we provide evidence that TACE is essential for the regulated shedding of the HER4 JM-a receptor.

Neuregulin (NRG), a growth and differentiation factor that signals via erbB receptor tyrosine kinases, has been shown to have biological effects in both the CNS and the peripheral nervous system. We report here that erbB4 is expressed in mature cerebellar granule cells, where it appears to be concentrated at the granule cell postsynaptic terminals. We also show that one form of NRG, Ig-NRG, plays a crucial role in aspects of cerebellar granule cell developmentin vitro. First, Ig-NRG treatment of granule cells in culture selectively induces the expression of the GABAAreceptor β2 subunit. This increase in subunit expression is paralleled by an increase in functional GABAAreceptors. In contrast to its effects on GABAAreceptor subunit expression, Ig-NRG does not upregulate NMDA receptor N2B and N2C subunit expression. Second, we demonstrate that Ig-NRG also enhances neurite outgrowth from cultured granule cells. Ig-NRG does not, however, act as a survival factor for the granule cells. We have compared the effect of Ig-NRG with the effects of brain-derived neurotrophic factor (BDNF), a neurotrophin that exerts specific effects on granule cells in culture, and found that BDNF does not mimic the effects of Ig-NRG on GABAAreceptor subunit expression. Our results show that Ig-NRG has specific effects on granule cell development and maturation and may regulate these processesin vivo.

Receptor tyrosine kinases (RTKs) were believed until recently to act at the cell membrane in a singular fashion (i.e., binding of ligands on the extracellular domain would activate the intrinsic tyrosine kinase activity in the intracellular domain), which would then start a cascade involving other intracellular signaling molecules that would act as effectors. However, new evidence indicates that some RTKs can signal through a different modality; they can move into the nucleus where they directly exert their actions. Although some studies have showed that the proteolytically released intracellular domain of several RTKs can move to the nucleus where they influence gene expression and cell function, others suggest that RTKs can also move to the nucleus as holoproteins. The identification of this novel signaling mechanism calls for a critical reevaluation of the mechanisms of action of RTKs and their biological roles.

ARIA is a glycoprotein purified from chick brain on the basis of its ACh receptor-inducing activity (ARIA). In this study we present evidence that ARIA increases the number of voltage-gated sodium channels in chick muscle as well as the number of ACh receptors (AChRs). Exposure of chick myotubes to ARIA increased by twofold the number of 3H-saxitoxin binding, an effect that is comparable to the increase of AChRs assayed by 125I-alpha-bungarotoxin (125I-alpha-BTX) binding. We also documented effects of ARIA on myoblasts: the number of 125I-alpha-BTX binding sites in the mononucleated muscle cells was increased by 1.5-fold, and the peak TTX-sensitive inward currents increased by the same amount. No change was detected in the voltage dependence of channel activation, in mean channel current, or in mean channel open time. Thus, the Na+ channel is the first molecule, other than AChR subunits, whose expression has been shown to be induced by ARIA. Since sodium channels are concentrated at motor end plates, our results provide circumstantial evidence that ARIA may regulate several genes expressed at developing neuromuscular junctions. Moreover, the finding that ARIA's effects extend to mononucleated myoblasts suggests that this protein may be important during the earliest stages of muscle formation and innervation.

Motoneurons promote the accumulation of acetylcholine receptors (AChRs) at developing neuromuscular synapses. The AChR-inducing activity protein ARIA, which is purified from chicken brain and increases the synthesis of AChRs in chicken myotubes, may play a crucial role in this process. Here we show that ARIA induces the rapid tyrosine phosphorylation of a M(r) 185,000 protein (p185) in muscle cells. Phosphorylation of p185 correlates with AChR induction at each stage of ARIA purification. Moreover, medium conditioned by spinal cord motoneurons stimulates AChR synthesis and p185 phosphorylation. Studies with membrane-impermeant reagents and 125I-labeled ARIA indicate that p185 is a transmembrane ARIA-receptor tyrosine kinase. Our data suggests that muscle AChR synthesis can be regulated through tyrosine phosphorylation.

Bergmann glial cells play critical roles in the structure and function of the cerebellum. During development, their radial processes serve as guides for migrating granule neurons and their terminal endfeet tile to form the glia limitans. As the cerebellum matures, Bergmann glia perform important roles in synaptic transmission and synapse maintenance, while continuing to serve as essential structural elements. Despite growing evidence of the diverse functions of Bergmann glia, the molecular mechanisms that mediate these functions have remained largely unknown. As a step toward identifying the molecular repertoire underlying Bergmann glial function, here we examine global gene expression in individual Bergmann glia from developing (P6) and mature (P30) mouse cerebellum. When we select for developmentally regulated genes, we find that transcription factors and ribosomal genes are particularly enriched at P6 relative to P30; whereas synapse associated molecules are enriched at P30 relative to P6. We also analyze genes expressed at high levels at both ages. In all these categories, we find genes that were not previously known to be expressed in glial cells, and discuss novel functions some of these genes may potentially play in Bergmann glia. We also show that Bergmann glia, even in the adult, express a large set of genes thought to be specific to stem cells, suggesting that Bergmann glia may retain neural precursor potential as has been proposed. Finally, we highlight several genes that in the cerebellum are expressed in Bergmann glia but not astrocytes, and may therefore serve as new, specific markers for Bergmann glia.

We have studied the effect of formamidines onDrosophila melanogaster. Low concentrations of formamidines are toxic to adultDrosophila. A mutant with reduced cAMP synthesis displays increased resistance to the toxin. Formamidines also reduce viability ofDrosophila eggs and retard imago eclosion. At sublethal concentrations, formamidines markedly affect the flies' behavior. Upon injection, the compounds increase muscle activity. Upon feeding, formamidines induce motor excitation, reduce phototaxis and impair olfactory learning without affecting the ability to recognize an olfactory cue. In vitro, two formamidines were found to inhibit octopamine-stimulated adenylate cyclase without affecting the basal activity of the enzyme, while a third one was found to stimulate adenylate cyclase; this stimulation was blocked by phentolamine and to a lesser degree by propranolol, thus resembling the effect of octopamine. The binding of [3H]octopamine toDrosophila head membranes was also inhibited. Taken together, our results indicate that formamidines interact with octopaminergic systems inDrosophila, exert both peripheral and central effects in the fly, and could be used to dissect the roles of octopamine in development and behavior, including behavioral plasticity. The results also suggest that formamidines could be used to select mutants in aminergic transmission and in the cAMP cascade.

Summary Ephs regulate growth cone repulsion, a process controlled by the actin cytoskeleton. The guanine nucleotide exchange factor (GEF) ephexin1 interacts with EphA4 and has been suggested to mediate the effect of EphA on the activity of Rho GTPases, key regulators of the cytoskeleton and axon guidance. Using cultured ephexin1 −/− mouse neurons and RNA interference in the chick, we report that ephexin1 is required for normal axon outgrowth and ephrin-dependent axon repulsion. Ephexin1 becomes tyrosine phosphorylated in response to EphA signaling in neurons, and this phosphorylation event is required for growth cone collapse. Tyrosine phosphorylation of ephexin1 enhances ephexin1's GEF activity toward RhoA while not altering its activity toward Rac1 or Cdc42, thus changing the balance of GTPase activities. These findings reveal that ephexin1 plays a role in axon guidance and is regulated by a switch mechanism that is specifically tailored to control Eph-mediated growth cone collapse.