Expression of neurotrophins and Trk receptors in the developing,adult, and regenerating avian cochlea

We studied the expression of neurotrophins and their Trk receptors in the chicken cochlea. Based on in situ hybridization, brain-derived neurotrophic factor (BDNF) is the major neurotrophin there, in contrast to the mammalian cochlea, where neurotrophin-3 (NT-3) predominates. NT-3 mRNA labeling was weak and found only during a short time period in the early cochles. During embryogenesis, BDNF mRNA was first seen in early differentiating hair cells. Afferent cochlear neurons expressed trkB mRNA from the early stages of gangliogenesis onward. In accordance, in vitro, BDNF promoted survival of dissociated neurons and stimulated neuritogenesis from ganglionic explants. High levels of BDNF mRNA in hair cells and trkB mRNA in cochlear neurons persisted in the mature cochlea. In addition, mRNA for the truncated TrkB receptor was expressed in nonneuronal cells, specifically in supporting cells, located adjacent to the site of BDNF synthesis and nerve endings. Following acoustic trauma, regenerated hair cells acquired BDNF mRNA expression at early stages of differentiation. Truncated trkB mRNA was lost from supporting cells that regenerated into hair cells. High levels of BDNF mRNA persisted in surviving hair cells and trkB mRNA in cochlear neurons after noise exposure. These results suggest that in the avian cochlea, peripheral target-derived BDNF contributes to the onset and maintenance of hearing function by supporting neuronal survival and regulating the (re)innervation process. Truncated TrkB receptors may regulate the BDNF concentration available to neurites, and they might have an important role during reinnervation. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 1019–1033, 1997     

NTPDase1 and NTPDase2 immunolocalization in mouse cochlea: implications for regulation of p2 receptor signaling.

Cellular, molecular, and physiological studies have demonstrated an important signaling role for ATP and related nucleotides acting via P2 receptors in the cochlea of the inner ear. Signal modulation is facilitated by ectonucleotidases, a heterologous family of surface-located enzymes involved in extracellular nucleotide hydrolysis. Our previous studies have implicated CD39/NTPDase1 and CD39L1/NTPDase2, members of the ectonucleoside triphosphate diphosphohydrolase (E-NTPDase) family, as major ATP-hydrolyzing enzymes in the tissues lining the cochlear endolymphatic and perilymphatic compartments. NTPDase1 hydrolyzes both nucleoside triphosphates and diphosphates. In contrast, NTPDase2 is a preferential nucleoside triphosphatase. This study characterizes expression of these E-NTPDases in the mouse cochlea by immunohistochemistry. NTPDase1 can be immunolocalized to the cochlear vasculature and neural tissues (primary auditory neurons in the spiral ganglion). In contrast, NTPDase2 immunolabeling was principally localized to synaptic regions of the sensory inner and outer hair cells, stereocilia and cuticular plates of the outer hair cells, supporting cells of the organ of Corti (Deiters' cells and inner border cells), efferent nerve fibers located in the intraganglionic spiral bundle, and in the outer sulcus and root region of the spiral ligament. This differential expression of NTPDase1 and 2 in the cochlea suggests spatial regulation of P2 receptor signaling, potentially involving different nucleotide species and hydrolysis kinetics.     

Unaltered D1, D2, D4, and D5 dopamine receptor mRNA expression and distribution in the spinal cord of the D3 receptor knockout mouse

Dopamine (DA) acts through five receptor subtypes (D1–D5). We compared expression levels and distribution patterns of all DA mRNA receptors in the spinal cord of wild-type (WT) and loss of function D3 receptor knockout (D3KO) animals. D3 mRNA expression was increased in D3KO, but no D3 receptor protein was associated with cell membranes, supporting the previously reported lack of function. In contrast, mRNA expression levels and distribution patterns of D1, D2, D4, and D5 receptors were similar between WT and D3KO animals. We conclude that D3KO spinal neurons do not compensate for the loss of function of the D3 receptor with changes in the other DA receptor subtypes. This supports use of D3KO animals as a model to provide insight into D3 receptor dysfunction in the spinal cord.     

Developmentally regulated expression of ectonucleotidases NTPDase5 and NTPDase6 and UDP-responsive P2Y receptors in the rat cochlea

Ectonucleoside triphosphate diphosphohydrolases (E-NTPDases) regulate complex extracellular P2 receptor signalling pathways in mammalian tissues by hydrolysing extracellular nucleotides to the respective nucleosides. All enzymes from this family (NTPDase1-8) are expressed in the adult rat cochlea. This study reports the changes in expression of NTPDase5 and NTPDase6 in the developing rat cochlea. These two intracellular members of the E-NTPDase family can be released in a soluble form and show preference for nucleoside 5′-diphosphates, such as UDP and GDP. Here, we demonstrate differential spatial and temporal patterns for NTPDase5 and NTPDase6 expression during cochlear development, which are indicative of both cytosolic and extracellular action via pyrimidines. NTPDase5 is noted during the early postnatal period in developing sensory hair cells and supporting Deiters’ cells of the organ of Corti, and primary auditory neurons located in the spiral ganglion. In contrast, NTPDase6 is confined to the embryonic and early postnatal hair cell bundles. NTPDase6 immunolocalisation in the developing cochlea underpins its putative role in hair cell bundle development, probably via cytosolic action, whilst NTPDase5 may have a broader extracellular role in the development of sensory and neural tissues in the rat cochlea. Both NTPDase5 and NTPDase6 colocalize with UDP-preferring P2Y₄, P2Y₆ and P2Y₁₄ receptors during cochlear development, but this strong association was lost in the adult cochlea. Spatiotemporal topographic expression of NTPDase5 and NTPDase6 and P2Y receptors in adult and developing cochlear tissues provide strong support for the role of pyrimidinergic signalling in cochlear development.     

Immunological Detection of Isoforms of the Somatostatin Receptor Subtype, SSTR2

Abstract: Somatostatin (SRIF) induces its diverse physiological actions through interactions with different receptor subtypes. Multiple SRIF receptor subtypes have recently been cloned. To analyze the physical properties of receptor subtype SSTR2, two different peptide-directed antibodies were generated against SSTR2. Antibody “2e3,” directed against the peptide SSCTINWPGESGAWYT (residues 191–206), corresponding to a region in the predicted third extracellular domain of mouse SSTR2, and antibody “2i4,” directed against the peptide SGTEDGERSDS (residues 333–343) from the predicted cytoplasmic tail of mouse SSTR2, were developed. In Chinese hamster ovary (CHO) cells stably expressing the mouse SSTR2 gene (CHOB), the antibody 2e3 recognized specifically a protein of 93-kDa protein by immunoblotting. No specific immunoreactivity was detected by 2e3 in nontransfected CHO cells or CHO cells stably expressing vector alone or human SSTR1 or mouse SSTR3 genes. The antibody 2i4 specifically immunoprecipitated SSTR2 solubilized from CHOB cells that could be labeled with the SSTR2-specific ligand ¹²⁵I-MK-678. Furthermore, both 2e3 and 2i4 specifically immunoprecipitated 93-kDa [³⁵S]methionine-labeled proteins from CHOB cells, indicating that they recognize the same proteins. In contrast to studies in CHOB cells, immunoblotting studies showed that 2e3 detected specifically a single 148-kDa protein from different regions of the rat brain that have previously been shown to express high levels of SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. In contrast, no immunoreactivity was detected in rat kidney, liver, or lung, which do not express SSTR2. No 93-kDa protein was detected specifically in the rat brain. The 148-kDa protein detected by 2e3 is an SRIF receptor because 2e3 and 2i4 specifically immunoprecipitated solubilized rat brain SRIF receptors that could be reversibly labeled with ¹²⁵I-MK-678. As in rat brain, 2e3 interacted specifically with a single 148-kDa protein in rat pituitary, in the rat pancreatic cell line AR42J, and in the HEK 293 cell line derived from human kidney, all of which express SSTR2 mRNA and SRIF receptors with high affinity for ¹²⁵I-MK-678. These findings indicate that rat brain and pituitary, as well as a pancreatic and a kidney cell line, express primarily a form of SSTR2 different from CHOB cells. The multiple forms of SSTR2 may result from differential post-translational processing of SSTR2 because 2e3 immunoprecipitated 41-kDa in vitro translation products generated from mRNA extracted from CHOB and AR42J cells. This 41-kDa protein has the predicted size of unprocessed SSTR2. These results demonstrate that 2e3 and 2i4 antibodies interact specifically with SSTR2. Detection of two different size proteins by the SSTR2 peptide-directed antibodies suggests the existence of multiple forms of SSTR2.     

Up-regulation of Thrombospondin-2 in Akt1-null Mice Contributes to Compromised Tissue Repair Due to Abnormalities in Fibroblast Function

Vascular remodeling is essential for tissue repair and is regulated by multiple factors, including thrombospondin-2 (TSP2) and hypoxia/VEGF-induced activation of Akt. In contrast to TSP2 knock-out (KO) mice, Akt1 KO mice have elevated TSP2 expression and delayed tissue repair. To investigate the contribution of increased TSP2 to Akt1 KO mice phenotypes, we generated Akt1/TSP2 double KO (DKO) mice. Full-thickness excisional wounds in DKO mice healed at an accelerated rate when compared with Akt1 KO mice. Isolated dermal Akt1 KO fibroblasts expressed increased TSP2 and displayed altered morphology and defects in migration and adhesion. These defects were rescued in DKO fibroblasts or after TSP2 knockdown. Conversely, the addition of exogenous TSP2 to WT cells induced cell morphology and migration rates that were similar to those of Akt1 KO cells. Akt1 KO fibroblasts displayed reduced adhesion to fibronectin with manganese stimulation when compared with WT and DKO cells, revealing an Akt1-dependent role for TSP2 in regulating integrin-mediated adhesions; however, this effect was not due to changes in β1 integrin surface expression or activation. Consistent with these results, Akt1 KO fibroblasts displayed reduced Rac1 activation that was dependent upon expression of TSP2 and could be rescued by a constitutively active Rac mutant. Our observations show that repression of TSP2 expression is a critical aspect of Akt1 function in tissue repair.     

Notch Signaling Limits Supporting Cell Plasticity in the Hair Cell-Damaged Early Postnatal Murine Cochlea

In mammals, auditory hair cells are generated only during embryonic development and loss or damage to hair cells is permanent. However, in non-mammalian vertebrate species, such as birds, neighboring glia-like supporting cells regenerate auditory hair cells by both mitotic and non-mitotic mechanisms. Based on work in intact cochlear tissue, it is thought that Notch signaling might restrict supporting cell plasticity in the mammalian cochlea. However, it is unresolved how Notch signaling functions in the hair cell-damaged cochlea and the molecular and cellular changes induced in supporting cells in response to hair cell trauma are poorly understood. Here we show that gentamicin-induced hair cell loss in early postnatal mouse cochlear tissue induces rapid morphological changes in supporting cells, which facilitate the sealing of gaps left by dying hair cells. Moreover, we provide evidence that Notch signaling is active in the hair cell damaged cochlea and identify Hes1, Hey1, Hey2, HeyL, and Sox2 as targets and potential Notch effectors of this hair cell-independent mechanism of Notch signaling. Using Cre/loxP based labeling system we demonstrate that inhibition of Notch signaling with a γ- secretase inhibitor (GSI) results in the trans-differentiation of supporting cells into hair cell-like cells. Moreover, we show that these hair cell-like cells, generated by supporting cells have molecular, cellular, and basic electrophysiological properties similar to immature hair cells rather than supporting cells. Lastly, we show that the vast majority of these newly generated hair cell-like cells express the outer hair cell specific motor protein prestin.     

The zinc finger transcription factor Gfi1, implicated in lymphomagenesis,is required for inner ear hair cell differentiation and survival

Gfi1 was first identified as causing interleukin 2-independent growth in T cells and lymphomagenesis in mice. Much work has shown that Gfi1 and Gfi1b, a second mouse homolog, play pivotal roles in blood cell lineage differentiation. However, neither Gfi1 nor Gfi1b has been implicated in nervous system development, even though their invertebrate homologues, senseless in Drosophila and pag-3 in C. elegans are expressed and required in the nervous system. We show that Gfi1 mRNA is expressed in many areas that give rise to neuronal cells during embryonic development in mouse, and that Gfi1 protein has a more restricted expression pattern. By E12.5 Gfi1 mRNA is expressed in both the CNS and PNS as well as in many sensory epithelia including the developing inner ear epithelia. At later developmental stages, Gfi1 expression in the ear is refined to the hair cells and neurons throughout the inner ear. Gfi1 protein is expressed in a more restricted pattern in specialized sensory cells of the PNS, including the eye, presumptive Merkel cells, the lung and hair cells of the inner ear. Gfi1 mutant mice display behavioral defects that are consistent with inner ear anomalies, as they are ataxic, circle, display head tilting behavior and do not respond to noise. They have a unique inner ear phenotype in that the vestibular and cochlear hair cells are differentially affected. Although Gfi1-deficient mice initially specify inner ear hair cells, these hair cells are disorganized in both the vestibule and cochlea. The outer hair cells of the cochlea are improperly innervated and express neuronal markers that are not normally expressed in these cells. Furthermore, Gfi1 mutant mice lose all cochlear hair cells just prior to and soon after birth through apoptosis. Finally, by five months of age there is also a dramatic reduction in the number of cochlear neurons. Hence, Gfi1 is expressed in the developing nervous system, is required for inner ear hair cell differentiation, and its loss causes programmed cell death.     

Coexpression of human somatostatin receptor-2 (SSTR2) and SSTR3 modulates antiproliferative signaling and apoptosis

Background

Somatostatin (SST) via five G_i coupled receptors namely SSTR1-5 is known to inhibit cell proliferation by cytostatic and cytotoxic mechanisms. Heterodimerization plays a crucial role in modulating the signal transduction pathways of SSTR subtypes. In the present study, we investigated human SSTR2/SSTR3 heterodimerization, internalization, MAPK signaling, cell proliferation and apoptosis in HEK-293 cells in response to SST and specific agonists for SSTR2 and SSTR3.

Results

Although in basal conditions, SSTR2 and SSTR3 colocalize at the plasma membrane and exhibit heterodimerization, the cell surface distribution of both receptors decreased upon agonist activation and was accompanied by a parallel increase in intracellular colocalization. Receptors activation by SST and specific agonists significantly decreased cAMP levels in cotransfected cells in comparison to control. Agonist-mediated modulation of pERK1/2 was time and concentration-dependent, and pronounced in serum-deprived conditions. pERK1/2 was inhibited in response to SST; conversely receptor-specific agonist treatment caused inhibition at lower concentration and activation at higher concentration. Strikingly, ERK1/2 phosphorylation was sustained upon prolonged treatment with SST but not with receptor-specific agonists. On the other hand, SST and receptor-specific agonists modulated p38 phosphorylation time-dependently. The receptor activation in cotransfected cells exhibits G_i-dependent inhibition of cell proliferation attributed to increased PARP-1 expression and TUNEL staining, whereas induction of p21 and p27^Kip1 suggests a cytostatic effect.

Conclusion

Our study provides new insights in SSTR2/SSTR3 mediated signaling which might help in better understanding of the molecular interactions involving SSTRs in tumor biology.     

Aggression and arginine vasopressin immunoreactivity regulation by androgen receptor and estrogen receptor alpha

In the following study, we asked which steroid receptors regulate aggression and arginine vasopressin (AVP) immunoreactivity (– ir) in several limbic regions. Using spontaneous mutant and knockout mice, we generated a novel cross of mice whose offspring lacked estrogen receptor α (ERα), androgen receptor (AR) or both ERα and AR. The wild-type (WT) males and females were compared with ERα knockout (ERαKO) male, mutated AR (Tfm) male and ERαKO/Tfm (double knockout; DKO) male littermates. Animals were gonadectomized and treated with 17β-estradiol (E2) prior to resident-intruder aggression tests. WT and Tfm males showed aggression whereas WT females, ERαKO and DKO males did not. In the lateral septum, WT and Tfm male brains had significantly denser AVP-ir as compared with WT females and DKO males. ERαKO male brains were intermediate in the amount of AVP-ir present. In the medial amygdala, brains from all genotypes had equivalent AVP-ir, except DKO males, which had significantly less AVP-ir. Overall, the expression of aggressive behavior coincided with AVP-ir in WT, Tfm and DKO males. However, in ERαKO males and WT females, the amount of AVP-ir was not associated with resident-intruder aggression. In sum we have shown that E2 acts via ERα to regulate aggression in male mice. In contrast both ERα and AR contribute to AVP-ir in limbic brain regions.     

Identification of distinct signalling pathways for somatostatin receptors SSTR1 and SSTR2 as revealed by microphysiometry.

Somatostatin receptors (SSTRs) are known to mediate diverse cellular responses. Most target cell express more than one SSTR isoform, making it difficult to define the signalling pathway used by individual receptor subtypes. Thus, we have expressed SSTR1 or SSTR2 in rat pituitary F4C1 cells which lack endogenous SSTRs. Using a silicon-based biosensor system, the Cytosensor microphysiometer, which measures the extracellular acidification rate (ECAR) in real time, we have studied the responses to SS mediated by either SSTR1 or SSTR2. In control F4C1 cells, SS had no effect on the basal ECAR. In transfected cells expressing only SSTR1, SS caused a unique decrease in ECAR in a concentration-dependent manner. Receptor-mediated decreases in ECAR have not been reported previously. In F4C1 cells expressing only SSTR2, SS induced a bidirectional ECAR response, a rapid increase followed by a decrease below basal. Two SS analogues, MK678 and CH275, induced characteristic ECAR responses with the expected receptor selectivities for SSTR1 or SSTR2. Pretreatment of F4C1 cells with pertussis toxin abolished the decreases in ECAR mediated by both SSTR1 and SSTR2, but only partially reduced the increase in ECAR mediated by SSTR2. The decrease in ECAR did not depend on a decrease in intracellular cAMP. The ECAR responses to SS were modestly attenuated by methylisobutylamiloride (MIA), an inhibitor of the ubiquitous Na(+)-H+ exchanger NHE1. Removal of extracellular Na+ greatly inhibited the ECAR responses to SS, demonstrating a role for both amiloride-sensitive and -insensitive Na(+)-dependent acid transport mechanisms in SS-induced extracellular acidification. In conclusion, we have identified and characterized different signalling pathways for SSTR1 and SSTR2 in pituitary cells as measured by microphysiometry.     

<Emphasis Type="Italic">Lmx1a</Emphasis> is required for segregation of sensory epithelia and normal ear histogenesis and morphogenesis

At embryonic day 8.5, the LIM-homeodomain factor Lmx1a is expressed throughout the otic placode but becomes developmentally restricted to non-sensory epithelia of the ear (endolymphatic duct, ductus reuniens, cochlea lateral wall). We confirm here that the ears of newborn dreher (Lmx1a ^dr) mutants are dysmorphic. Hair cell markers such as Atoh1 and Myo7 reveal, for the first time, that newborn Lmx1a mutants have only three sensory epithelia: two enlarged canal cristae and one fused epithelium comprising an amalgamation of the cochlea, saccule, and utricle (a “cochlear-gravistatic” endorgan). The enlarged anterior canal crista develops by fusion of horizontal and anterior crista, whereas the posterior crista fuses with an enlarged papilla neglecta that may extend into the cochlear lateral wall. In the fused endorgan, the cochlear region is distinguished from the vestibular region by markers such as Gata3, the presence of a tectorial membrane, and cochlea-specific innervation. The cochlea-like apex displays minor disorganization of the hair and supporting cells. This contrasts with the basal half of the cochlear region, which shows a vestibular epithelium-like organization of hair cells and supporting cells. The dismorphic features of the cochlea are also reflected in altered gene expression patterns. Fgf8 expression expands from inner hair cells in the apex to most hair cells in the base. Two supporting cell marker proteins, Sox2 and Prox1, also differ in their cellular distribution between the base and the apex. Sox2 expression expands in mutant canal cristae prior to their enlargement and fusion and displays a more diffuse and widespread expression in the base of the cochlear region, whereas Prox1 is not detected in the base. These changes in Sox2 and Prox1 expression suggest that Lmx1a expression restricts and sharpens Sox2 expression, thereby defining non-sensory and sensory epithelium. The adult Lmx1a mutant organ of Corti shows a loss of cochlear hair cells, suggesting that the long-term maintenance of hair cells is also disrupted in these mutants. This work was supported by grants from the NCRR/COBRE (P20 RR 018788; D.H.N.) and NIH (RO1 DC 005590; B.F.). Parts of this investigation were conducted in a facility constructed with support of a Research Facilities Improvement Program Grant from the National Center for Research Resources, National Institutes of Health. We acknowledge the use of the confocal microscope facility of the NCCB, supported by EPSCoR EPS-0346476 (CFD 47.076), and of the University of Nebraska microarray facility, supported by NCRR/COBRE.     

Notch signaling regulates the pattern of auditory hair cell differentiation in mammals

The development of the mammalian cochlea is an example of patterning in the peripheral nervous system. Sensory hair cells and supporting cells in the cochlea differentiate via regional and cell fate specification. The Notch signaling components shows both distinct and overlapping expression patterns of Notch1 receptor and its ligands Jagged1 (Jag1) and Jagged2 (Jag2) in the developing auditory epithelium of the rat. On embryonic day 16 (E16), many precursor cells within the K?lliker's organ immunostained for the presence of both Notch1 and Jag1, while the area of hair cell precursors did not express either Notch1 and Jag1. During initial events of hair cell differentiation between E18 and birth, Notch1 and Jag1 expression predominated in supporting cells and Jag2 in nascent hair cells. Early after birth, Jag2 expression decreased in hair cells while the pattern of Notch1 expression now included both supporting cells and hair cells. We show that the normal pattern of hair cell differentiation is disrupted by alteration of Notch signaling. A decrease of either Notch1 or Jag1 expression by antisense oligonucleotides in cultures of the developing sensory epithelium resulted in an increase in the number of hair cells. Our data suggest that the Notch1 signaling pathway is involved in a complex interplay between the consequences of different ligand-Notch1 combinations during cochlear morphogenesis and the phases of hair cell differentiation.     

Cell cycle regulation in the inner ear sensory epithelia: Role of cyclin D1 and cyclin-dependent kinase inhibitors

Sensory hair cells and supporting cells of the mammalian cochlea and vestibular (balance) organs exit the cell cycle during embryogenesis and do not proliferate thereafter. Here, we have studied the mechanisms underlying the maintenance of the postmitotic state and the proliferative capacity of these cells. We provide the first evidence of the role of cyclin D1 in cell cycle regulation in these cells. Cyclin D1 expression disappeared from embryonic hair cells as differentiation started. The expression was transiently upregulated in cochlear hair cells early postnatally, paralleling the spatiotemporal pattern of unscheduled cell cycle re-entry of cochlear hair cells from the p19^Ink4d/p21^Cip1 compound mutant mice. Cyclin D1 misexpression in vitro in neonatal vestibular HCs from these mutant mice triggered S-phase re-entry. Thus, cyclin D1 suppression is important for hair cell's quiescence, together with the maintained expression of cyclin-dependent kinase inhibitors. In contrast to hair cells, cyclin D1 expression was maintained in supporting cells when differentiation started. The expression continued during the neonatal period when supporting cells have been shown to re-enter the cell cycle upon stimulation with exogenous mitogens. Thereafter, the steep decline in supporting cell's proliferative activity paralleled with cyclin D1 downregulation. Thus, cyclin D1 critically contributes to the proliferative plasticity of supporting cells. These data suggest that targeted cyclin D1 induction in supporting cells might be an avenue for proliferative regeneration in the inner ear.     

Cloning of a novel somatostatin receptor, SSTR3, coupled to adenylylcyclase.

The gene encoding a novel mouse somatostatin receptor termed mSSTR3 was isolated and characterized. The sequence of mSSTR3 shows 46 and 47% identity with mSSTR1 and mSSTR2, respectively. mSSTR3 binds somatostatin-14 and somatostatin-28 with high affinity, but shows very low affinity for the somatostatin analogs MK-678 and SMS-201-995. In addition, mSSTR3 is coupled to pertussis toxin-sensitive G proteins and mediates somatostatin inhibition of forskolin-stimulated and dopamine D1 receptor-stimulated cAMP formation, indicating that it is coupled to adenylylcyclase. The pharmacological properties of mSSTR3 and its ability to couple with adenylylcyclase distinguish SSTR3 from the other cloned somatostatin receptors and indicates that it mediates biological functions different from SSTR1 or SSTR2. In situ hybridization indicates that SSTR3 mRNA is widely distributed in the mouse brain, and its expression in the nucleus of the lateral olfactory tract and in the piriform cortex, the primary olfactory cortex in the rodent brain, suggests that SSTR3 may participate in the processing and modulation of primary sensory information.