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Neurotrophin-3 growth factor can improve cochlear neuron survival, and localized delivery of this protein to the round window membrane in the middle ear may be able to reverse sensorineural hearing loss. Thus, the goal of this work was to develop an injectable hydrogel delivery system that can allow localized release of neurotrophin-3 in a controlled and sustained manner. We identified a PEG hydrogel formulation that uses thiol-vinyl sulfone Michael addition for crosslinking. This injectable formulation provides elastic hydrogels with higher mechanical rigidity, better bio-adhesion and longer residence time than Poloxamer hydrogels currently being investigated clinically for hearing loss. In vivo, PEG hydrogels induce local immune responses comparable to biocompatible Poloxamer hydrogels, yet they released payloads at a ~5-fold slower rate in the subcutaneous area. Based on this injectable hydrogel formulation, we designed an affinity-based protein release system by modifying PEG hydrogels with affinity peptides specific to neurotrophin-3 proteins. We verified the sustained release of neurotrophin-3 from peptide-conjugated PEG hydrogels resulting from the reversible interaction between peptides and proteins. The rate of affinity-controlled release depends on the polymer concentrations, the affinity of peptides and the peptide-to-protein ratios. Collectively, we developed an injectable hydrogel formulation for localized delivery of neurotrophin-3, which provides affinity-controlled release and longer delivery time compared to Poloxamer hydrogels.

SUMMARY Dentate gyrus (DG) development requires specification of granule cell (GC) progenitors in the hippocampal neuroepithelium, as well as their proliferation and migration into the primordial DG. We identify the Plexin family members Plxna2 and Plxna4 as important regulators of DG development. Distribution of immature GCs is regulated by Sema5A signaling through PlxnA2 and requires a functional PlxnA2 GTPase-activating protein (GAP) domain and Rap1 small GTPases. In adult Plxna2−/− but not Plxna2-GAP-deficient mice, the dentate GC layer is severely malformed, neurogenesis is compromised, and mossy fibers form aberrant synaptic boutons within CA3. Behavioral studies with Plxna2−/− mice revealed deficits in associative learning, sociability, and sensorimotor gating—traits commonly observed in neuropsychiatric disorder. Remarkably, while morphological defects are minimal in Plxna2-GAP-deficient brains, defects in fear memory and sensorimotor gating persist. Since allelic variants of human PLXNA2 and RAP1 associate with schizophrenia, our studies identify a biochemical pathway important for brain development and mental health., In Brief Zhao et al. find that Sema5A-PlexinA2 forward signaling through Rap1 GTPases is required for progenitor distribution in the developing mouse dentate gyrus. Adult Plxna2−/−, but not Plxna2-GAP-deficient, mice show defects in dentate morphology, neurogenesis, and mossy fiber connectivity. Plxna2−/− and Plxna2-GAP mice exhibit behavioral defects suggestive of neuropsychiatric illness.

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.

In the enteric nervous system (ENS), glia outnumber neurons by 4-fold and form an extensive network throughout the gastrointestinal tract. Growing evidence for the essential role of enteric glia in bowel function makes it imperative to understand better their molecular marker expression and how they relate to glia in the rest of the nervous system. We analyzed expression of markers of astrocytes and oligodendrocytes in the ENS and found, unexpectedly, that proteolipid protein 1 (PLP1) is specifically expressed by glia in adult mouse intestine. PLP1 and S100β are the markers most widely expressed by enteric glia, while glial fibrillary acidic protein expression is more restricted. Marker expression in addition to cellular location and morphology distinguishes a specific subpopulation of intramuscular enteric glia, suggesting that a combinatorial code of molecular markers can be used to identify distinct subtypes. To assess the similarity between enteric and extraenteric glia, we performed RNA sequencing analysis on PLP1-expressing cells in the mouse intestine and compared their gene expression pattern to that of other types of glia. This analysis shows that enteric glia are transcriptionally unique and distinct from other cell types in the nervous system. Enteric glia express many genes characteristic of the myelinating glia, Schwann cells and oligodendrocytes, although there is no evidence of myelination in the murine ENS. GLIA 2015;63:2040-2057.

Small-fiber neuropathy (SFN) is a disorder of peripheral nerves commonly found in patients with diabetes mellitus, HIV infection, and those receiving chemotherapy. The complexity of disease etiology has led to a scarcity of effective treatments. Using two models of progressive SFN, we show that overexpression of glial cell line-derived neurotrophic factor (GDNF) in skin keratinocytes or topical application of XIB4035, a reported nonpeptidyl agonist of GDNF receptor α1 (GFRα1), are effective treatments for SFN. We also demonstrate that XIB4035 is not a GFRα1 agonist, but rather it enhances GFRα family receptor signaling in conjunction with ligand stimulation. Taken together, our results indicate that topical application of GFRα/RET receptor signaling modulators may be a unique therapy for SFN, and we have identified XIB4035 as a candidate therapeutic agent.

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.

In acquired sensorineural hearing loss, such as that produced by noise or aging, there can be massive loss of the synaptic connections between cochlear sensory cells and primary sensory neurons, without loss of the sensory cells themselves. Because the cell bodies and central projections of these cochlear neurons survive for months to years, there is a long therapeutic window in which to re-establish functional connections and improve hearing ability. Here we show in noise-exposed mice that local delivery of neurotrophin-3 (NT-3) to the round window niche, 24 hours after an exposure that causes an immediate loss of up to 50% loss of synapses in the cochlear basal region, can regenerate pre- and post-synaptic elements at the hair cell / cochlear nerve interface. This synaptic regeneration, as documented by confocal microscopy of immunostained cochlear sensory epithelia, was coupled with a corresponding functional recovery, as seen in the suprathreshold amplitude of auditory brainstem response Wave 1. Cochlear delivery of neurotrophins in humans is likely achievable as an office procedure via transtympanic injection, making our results highly significant in a translational context.

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.

Recent studies indicate that molecules released by glia can induce synapse formation. However, what induces glia to produce such signals, their identity, and their in vivo relevance remain poorly understood. Here we demonstrate that supporting cells of the vestibular organ—cells that have many characteristics of glia—promote synapse formation only when induced by neuron-derived signals. Furthermore, we identify BDNF as the synaptogenic signal produced by these nonneuronal cells. Mice in which erbB signaling has been eliminated in supporting cells have vestibular dysfunction caused by failure of synapse formation between hair cells and sensory neurons. This phenotype correlates with reduced BDNF expression in supporting cells and is rescued by reexpression of BDNF in these cells. Furthermore, knockdown of BDNF expression in supporting cells postnatally phenocopies the loss of erbB signaling. These results indicate that vestibular supporting cells contribute in vivo to vestibular synapse formation and that this is mediated by reciprocal signals between sensory neurons and supporting cells involving erbB receptors and BDNF.

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.

Several psychiatric disorders are associated with white matter defects, suggesting that oligodendrocyte (OL) abnormalities underlie some aspects of these diseases. Neuregulin 1 (NRG1) and its receptor, erbB4, are genetically linked with susceptibility to schizophrenia and bipolar disorder. In vitro studies suggest that NRG1-erbB signaling is important for OL development. To test whether erbB signaling contributes to psychiatric disorders by regulating the structure or function of OLs, we analyzed transgenic mice in which erbB signaling is blocked in OLs in vivo . Here we show that loss of erbB signaling leads to changes in OL number and morphology, reduced myelin thickness, and slower conduction velocity in CNS axons. Furthermore, these transgenic mice have increased levels of dopamine receptors and transporters and behavioral alterations consistent with neuropsychiatric disorders. These results indicate that defects in white matter can cause alterations in dopaminergic function and behavior relevant to neuropsychiatric disorders.

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.

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, heregulin, neu differentiation factor, and glial growth factor are members of a new family of growth and differentiation factors whose effects have been assayed on Schwann cells, skeletal muscle cells, and mammary tumor cell lines. To gain insight into their roles in the CNS, we studied the expression of ARIA in the rat brain. We found ARIA mRNA in all cholinergic neurons throughoutthe CNS, including motor neurons and cells of the medial septal nucleus and the nucleus basalis of Meynert. We also found that ARIA induces tyrosine phosphorylation of a 185 kDa protein in central and peripheral targets of these cholinergic neurons. ARIA mRNA, however, is not restricted to cholinergic neurons, suggesting that it may also play a role at other types of synapses. Its distribution in germinal layers of the telencephalon and cerebellum suggests that it may also play a role in the proliferation and/or migration of neuronal and glial precursor cells.

Studies of sensorineural hearing loss have long suggested that survival of spiral ganglion neurons (SGNs) depends on trophic support provided by their peripheral targets, the inner hair cells (IHCs): following ototoxic drugs or acoustic overexposure, IHC death is rapid whereas SGN degeneration is always delayed. However, recent noise-trauma studies show that SGNs can die even when hair cells survive, and transgenic mouse models show that supporting cell dysfunction can cause SGN degeneration in the absence of IHC pathology. To reexamine this issue, we studied a model of IHC loss that does not involve noise or ototoxic drugs. Mice lacking the gene for the high-affinity thiamine transporter (Slc19a2) have normal cochlear structure and function when fed a regular (thiamine-rich) diet. However, dietary thiamine restriction causes widespread, rapid (within 10 d) loss of IHCs. Using this model, we show that SGNs can survive for months after IHC loss, indicating that (1) IHCs are not necessary for neuronal survival, (2) neuronal loss in the other hearing loss models is likely due to effects of the trauma on the sensory neurons or other inner ear cells, and (3) that other cells, most likely supporting cells of the organ of Corti, are the main source of SGN survival factors. These results overturn a long-standing dogma in the study of sensorineural hearing loss and highlight the importance of cochlear supporting cells in neuronal survival in the adult inner ear.

ARIA acetylcholine receptor-inducing activity protein, is a member of a family of ligands that includes the Neu differentiation factor, heregulin, and glial growth factor. These ligands all act through one or more receptor tyrosine kinases of approximately 185 kDa. In some conditions these ligands promote proliferation, whereas in others they induce differentiation. ARIA was originally isolated from chick brain on the basis of its ability to induce synthesis of nicotinic acetylcholine receptors in skeletal muscle. In this paper we show that ARIA is expressed in the subventricular zone of the rat brain and that it enhances the development of oligodendrocytes from bipotential (O2A) glial progenitor cells. We have also found that ARIA induces tyrosine phosphorylation of a 185-kDa protein in O2A progenitor cells. ARIA does not increase bromodeoxyuridine incorporation by oligodendrocytes but is mitogenic when added to Schwann cells in vitro. Thus, ARIA accelerates the formation of oligodendrocytes in vitro and is expressed where it could exercise the same influence in vivo.

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.

Neuregulin and the neuregulin receptor ERBB4 have been genetically and functionally implicated in schizophrenia. In this study, we used the yeast two-hybrid system to identify proteins that interact with ERBB4, to identify genes and pathways that might contribute to schizophrenia susceptibility. We identified the MAGI scaffolding proteins as ERBB4-binding proteins. After validating the interaction of MAGI proteins with ERBB4 in mammalian cells, we demonstrated that ERBB4 expression, alone or in combination with ERBB2 or ERBB3, led to the tyrosine phosphorylation of MAGI proteins, and that this could be further enhanced with receptor activation by neuregulin. As MAGI proteins were previously shown to interact with receptor phosphotyrosine phosphatase beta/zeta (RPTPbeta), we postulated that simultaneous binding of MAGI proteins to RPTPbeta and ERBB4 forms a phosphotyrosine kinase/phosphotyrosine phosphatase complex. Studies in cultured cells confirmed both a spatial and functional association between ERBB4, MAGI and RPTPbeta. Given the evidence for this functional association, we examined the genes coding for MAGI and RPTPbeta for genetic association with schizophrenia in a Caucasian United Kingdom case-control cohort (n= approximately 1400). PTPRZ1, which codes for RPTPbeta, showed significant, gene-wide and hypothesis-wide association with schizophrenia in our study (best individual single-nucleotide polymorphism allelic P=0.0003; gene-wide P=0.0064; hypothesis-wide P=0.026). The data provide evidence for a role of PTPRZ1, and for RPTPbeta signaling abnormalities, in the etiology of schizophrenia. Furthermore, the data indicate a role for RPTPbeta in the modulation of ERBB4 signaling that may in turn provide further support for an important role of neuregulin/ERBB4 signaling in the molecular basis of schizophrenia.

Recent studies indicate that neurotrophin 3 (NT3) may be important for the maintenance and function of the adult inner ear, but the pattern of postnatal NT3 expression in this organ has not been characterized. We used a reporter mouse in which cells expressing NT3 also express beta-galactosidase, allowing for their histochemical visualization, to determine the pattern of NT3 expression in cochlear and vestibular organs. We analyzed animals from birth (P0) to adult (P135). At P0, NT3 was strongly expressed in supporting cells and hair cells of all vestibular and cochlear sense organs, Reissner's membrane, saccular membrane, and the dark cells adjacent to canal organs. With increasing age, staining disappeared in most cell types but remained relatively high in inner hair cells (IHCs) and to a lesser extent in IHC supporting cells. In the cochlea, by P0 there is a longitudinal gradient (apex > base) that persists into adulthood. In vestibular maculae, staining gradients are: striolar > extrastriolar regions and supporting cells > hair cells. By P135, cochlear staining is restricted to IHCs and their supporting cells, with stronger expression in the apex than the base. By the same age, in the vestibular organs, NT3 expression is weak and restricted to saccular and utricular supporting cells. These results suggest that NT3 might play a long-term role in the maintenance and functioning of the adult auditory and vestibular systems and that supporting cells are the main source of this factor in the adult.

Hearing performance with modern cochlear implants is dependent on remaining sensory and neural elements. CNS plasticity and individual capacity for outstanding performance with meager peripheral neural survival provide some exceptions; however, both human and animal studies support a direct relationship of performance with structural integrity/function of remaining sensory and neural elements in the implanted ear. This dependency will increase with next generation implants. This has driven interventions based on increased understanding of molecular mechanisms underlying cell death and survival to enhance sensory and neural survival. It has driven tissue engineering of remaining elements with neurotrophic and other factors to induce regeneration, neurite outgrowth, and stem cell replacements of lost elements. Delivery to the cochlea is being accomplished via middle ear or transtympanic application, direct delivery in bolus or continuous administration via osmotic pumps, as coatings or channels in implants, gene transfer via viral vectors, factors incorporated in nano- and micro-particles, and via systemic administration. Current results provide a base for optimism for a future integrated system of cochlear implant-drug/gene delivery system. Each agent and delivery has risks and benefits. Challenges to select the best strategy for translation and application in humans will be discussed. [This work was s supported by NIH/NIDCD grants R01 DC010412, DC 004820, DC011294, P30 DC005188, and DOD W81XWH-12-10492.]

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.

Degeneration of cochlear sensory neurons is an important cause of hearing loss, but the mechanisms that maintain the survival of adult cochlear sensory neurons are not clearly defined. We now provide evidence implicating the neuregulin (NRG)-erbB receptor signaling pathway in this process. We found that NRG1 is expressed by spiral ganglion neurons (SGNs), whereas erbB2 and erbB3 are expressed by supporting cells of the organ of Corti, suggesting that these molecules mediate interactions between these cells. Transgenic mice in which erbB signaling in adult supporting cells is disrupted by expression of a dominant-negative erbB receptor show severe hearing loss and 80% postnatal loss of type-I SGNs without concomitant loss of the sensory cells that they contact. Quantitative RT-PCR analysis of neurotrophic factor expression shows a specific downregulation in expression of neurotrophin-3 (NT3) in the transgenic cochleas before the onset of neuronal death. Because NT3 is critical for survival of type I SGNs during development, these results suggest that it plays similar roles in the adult. Together, the data indicate that adult cochlear supporting cells provide critical trophic support to the neurons, that survival of postnatal cochlear sensory neurons depends on reciprocal interactions between neurons and supporting cells, and that these interactions are mediated by NRG and neurotrophins.

Schizophrenia is a devastating psychiatric disease that affects 0.5-1% of the world's adult population. The hypothesis that this disease is a developmental disorder of the nervous system with late onset of its characteristic symptoms has been gaining acceptance in past years. However, the anatomical, cellular and molecular bases of schizophrenia remain unclear. Numerous studies point to alterations in different aspects of brain development as possible causes of schizophrenia, including defects in neuronal migration, neurotransmitter receptor expression and myelination. Recently, the gene that encodes neuregulin-1 (NRG1) has been identified as a potential susceptibility gene for schizophrenia, and defects in the expression of erbB3, one of the NRG1 receptors, have been shown to occur in the prefrontal cortex of schizophrenic patients, suggesting that NRG1-erbB signaling is involved in the pathogenesis of schizophrenia. These findings open new approaches to defining the molecular and cellular basis of schizophrenia in more mechanistic terms.

Insulin resistance and diabetes might promote neurodegenerative disease, but a molecular link between these disorders is unknown. Many factors are responsible for brain growth, patterning, and survival, including the insulin-insulin-like growth factor (IGF)-signaling cascades that are mediated by tyrosine phosphorylation of insulin receptor substrate (IRS) proteins. Irs2 signaling mediates peripheral insulin action and pancreatic beta-cell function, and its failure causes diabetes in mice. In this study, we reveal two important roles for Irs2 signaling in the mouse brain. First, disruption of the Irs2 gene reduced neuronal proliferation during development by 50%, which dissociated brain growth from Irs1-dependent body growth. Second, neurofibrillary tangles containing phosphorylated tau accumulated in the hippocampus of old Irs2 knock-out mice, suggesting that Irs2 signaling is neuroprotective. Thus, dysregulation of the Irs2 branch of the insulin-Igf-signaling cascade reveals a molecular link between diabetes and neurodegenerative disease.

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.

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.

Motor neurons stimulate their postsynaptic muscle targets to synthesize neurotransmitter receptors. Polypeptide signaling molecules may mediate this inductive interaction. Here we report the purification of ARIA, a protein that stimulates the synthesis of muscle acetylcholine receptors, and the isolation of ARIA cDNA. Recombinant ARIA increases acetylcholine receptor synthesis greater than 3-fold, and it induces tyrosine phosphorylation of a 185 kd muscle protein. The ARIA cDNA hybridizes with mRNAs that are expressed in the spinal cord from E4, a time prior to the onset of neuromuscular synapse formation, through adulthood. By E7, hybridizing mRNAs are concentrated in motor neurons. Chicken ARIA is homologous to the rat Neu differentiation factor and human heregulin, ligands for the receptor tyrosine kinase encoded by the neu (c- erbB2, HER2 ) proto-oncogene. Our data suggest that members of the ARIA protein family promote the formation and maintenance of chemical synapses and, furthermore, that receptor tyrosine kinases play important roles in this process.

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.

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.