Therapeutic effect of NLRP3 inhibition on hearing loss induced by systemic inflammation in a CAPS-associated mouse model.

BACKGROUND: Cryopyrin-associated periodic syndrome (CAPS) is an inherited autoinflammatory disease caused by a gain-of-function mutation in NLRP3. Although CAPS patients frequently suffer from sensorineural hearing loss, it remains unclear whether CAPS-associated mutation in NLRP3 is associated with the progression of hearing loss. METHODS: We generated a mice with conditional expression of CAPS-associated NLRP3 mutant (D301N) in cochlea-resident CX3CR1 macrophages and examined the susceptibility of CAPS mice to inflammation-mediated hearing loss in a local and systemic inflammation context. FINDINGS: Upon lipopolysaccharide (LPS) injection into middle ear cavity, NLRP3 mutant mice exhibited severe cochlear inflammation, inflammasome activation and hearing loss. However, this middle ear injection model induced a considerable hearing loss in control mice and inevitably caused an inflammation-independent hearing loss possibly due to ear tissue damages by injection procedure. Subsequently, we optimized a systemic LPS injection model, which induced a significant hearing loss in NLRP3 mutant mice but not in control mice. Peripheral inflammation induced by a repetitive low dose of LPS injection caused a blood-labyrinth barrier disruption, macrophage infiltration into cochlea and cochlear inflammasome activation in an NLRP3-dependent manner. Interestingly, both cochlea-infiltrating and -resident macrophages contribute to peripheral inflammation-mediated hearing loss of CAPS mice. Furthermore, NLRP3-specific inhibitor, MCC950, as well as an interleukin-1 receptor antagonist significantly alleviated systemic LPS-induced hearing loss and inflammatory phenotypes in NLRP3 mutant mice. INTERPRETATION: Our findings reveal that CAPS-associated NLRP3 mutation is critical for peripheral inflammation-induced hearing loss in our CAPS mice model, and an NLRP3-specific inhibitor can be used to treat inflammation-mediated sensorineural hearing loss. FUNDING: National Research Foundation of Korea Grant funded by the Korean Government and the Team Science Award of Yonsei University College of Medicine.

CXCL12 is required for stirrup-shaped stapes formation during mammalian middle ear development.

BACKGROUND: The mammalian middle ear comprises a chain of three ossicles-the malleus, incus, and stapes-each of which has a unique morphology for efficiently transmitting sound information. In particular, the stapes, which is attached to the inner ear, is stirrup-shaped with a head and base connected by two crural arches, forming the stapedial foramen, through which the stapedial artery passes. However, how the stapes acquires this critical stirrup shape for association with the stapedial artery during development is not clear. RESULTS: C-X-C motif chemokine ligand 12 (CXCL12) is a chemoattractant essential for cellular movement and angiogenesis. In Cxcl12 -/- embryos, migration of neural crest cells into the prospective middle ear regions and their mesenchymal condensation to form the three ossicles proceed normally in correct alignment with each other and the inner ear. However, in the absence of CXCL12, the stapes loses its stirrup shape and instead exhibits a columnar shape lacking the crural arches and central hole. In addition, although the stapedial artery initially forms during early mesenchymal condensation of the stapes, it degenerates without CXCL12 function. CONCLUSION: CXCL12 plays an essential role in establishing the stirrup-shaped architecture of the stapes, possibly by maintaining the stapedial foramen and stapedial artery throughout development.

A pathogen-derived metabolite induces microglial activation via odorant receptors.

Microglia (MG), the principal neuroimmune sentinels in the brain, continuously sense changes in their environment and respond to invading pathogens, toxins, and cellular debris, thereby affecting neuroinflammation. Microbial pathogens produce small metabolites that influence neuroinflammation, but the molecular mechanisms that determine whether pathogen-derived small metabolites affect microglial activation of neuroinflammation remain to be elucidated. We hypothesized that odorant receptors (ORs), the largest subfamily of G protein-coupled receptors, are involved in microglial activation by pathogen-derived small metabolites. We found that MG express high levels of two mouse ORs, Olfr110 and Olfr111, which recognize a pathogenic metabolite, 2-pentylfuran, secreted by Streptococcus pneumoniae. These interactions activate MG to engage in chemotaxis, cytokine production, phagocytosis, and reactive oxygen species generation. These effects were mediated through the Gαs -cyclic adenosine monophosphate-protein kinase A-extracellular signal-regulated kinase and Gßγ -phospholipase C-Ca2+ pathways. Taken together, our results reveal a novel interplay between the pathogen-derived metabolite and ORs, which has major implications for our understanding of microglial activation by pathogen recognition. DATABASE: Model data are available in the PMDB database under the accession number PM0082389.

Gene therapy for hereditary hearing loss by SLC26A4 mutations in mice reveals distinct functional roles of pendrin in normal hearing.

Rationale: Mutations of SLC26A4 that abrogate pendrin, expressed in endolymphatic sac, cochlea and vestibule, are known to cause autosomal recessive sensorineural hearing loss with enlargement of the membranous labyrinth. This is the first study to demonstrate the feasibility of gene therapy for pendrin-related hearing loss. Methods: We used a recombinant viral vector to transfect Slc26a4 cDNA into embryonic day 12.5 otocysts of pendrin-deficient knock-out (Slc26a4∆/∆ ) and pendrin-deficient knock-in (Slc26a4tm1Dontuh/tm1Dontuh ) mice. Results: Local gene-delivery resulted in spatially and temporally limited pendrin expression, prevented enlargement, failed to restore vestibular function, but succeeded in the restoration of hearing. Restored hearing phenotypes included normal hearing as well as sudden, fluctuating, and progressive hearing loss. Conclusion: Our study illustrates the feasibility of gene therapy for pendrin-related hearing loss, suggests differences in the requirement of pendrin between the cochlea and the vestibular labyrinth, and documents that insufficient pendrin expression during late embryonal and early postnatal development of the inner ear can cause sudden, fluctuating and progressive hearing loss without obligatory enlargement of the membranous labyrinth.

Targeted Gene Delivery into the Mammalian Inner Ear Using Synthetic Serotypes of Adeno-Associated Virus Vectors.

Targeting specific cell types in the mammalian inner ear is important for treating genetic hearing loss due to the different cell type-specific functions. Adeno-associated virus (AAV) is an efficient in vivo gene transfer vector, and it has demonstrated promise for treating genetic hearing loss. Although more than 100 AAV serotypes have been identified, few studies have investigated whether AAV can be distributed to specific inner ear cell types. Here we screened three EGFP-AAV reporter constructs (serotypes DJ, DJ8, and PHP.B) in the neonatal mammalian inner ear by injection via the round window membrane to determine the cellular specificity of the AAV vectors. Sensory hair cells, supporting cells, cells in Reissner's membrane, interdental cells, and root cells were successfully transduced. Hair cells in the cochlear sensory epithelial region were the most frequently transduced cell type by all tested AAV serotypes. The recombinant DJ serotype most effectively transduced a range of cell types at a high rate. Our findings provide a basis for improving treatment of hereditary hearing loss using targeted AAV-mediated gene therapy.

Impact of miR-192 and miR-194 on cyst enlargement through EMT in autosomal dominant polycystic kidney disease.

Altered miRNA (miR) expression occurs in various diseases. However, the therapeutic effect of miRNAs in autosomal dominant polycystic kidney disease (ADPKD) is unclear. Genome-wide analyses of miRNA expression and DNA methylation status were conducted to identify crucial miRNAs in end-stage ADPKD. miR-192 and -194 levels were down-regulated with hypermethylation at these loci, mainly in the intermediate and late stages, not in the early stage, of cystogenesis, suggesting their potential impact on cyst expansion. Cyst expansion has been strongly associated with endothelial-mesenchymal transition (EMT). Zinc finger E-box-binding homeobox-2 and cadherin-2, which are involved in EMT, were directly regulated by miR-192 and -194. The therapeutic effect of miR-192 and -194 in vivo and in vitro were assessed. Restoring these miRs by injection of precursors influenced the reduced size of cysts in Pkd1 conditional knockout mice. miR-192 and -194 may act as potential therapeutic targets to control the expansion and progression of cysts in patients with ADPKD.-Kim, D. Y., Woo, Y. M., Lee, S., Oh, S., Shin, Y., Shin, J.-O., Park, E. Y., Ko, J. Y., Lee, E. J., Bok, J., Yoo, K. H., Park, J. H. Impact of miR-192 and miR-194 on cyst enlargement through EMT in autosomal dominant polycystic kidney disease.

Inhibition of the Zeb family prevents murine palatogenesis through regulation of apoptosis and the cell cycle.

Mammalian palate separates the oral and nasal cavities for normal feeding, breathing and speech. The palatal shelves are a pair of maxillary prominences that consist of the neural crest-derived mesenchyme and surrounding epithelium. Palatogenesis is completed by the fusion of the midline epithelial seam (MES) after the medial edge epithelium (MEE) cells make contact between the palatal shelves. Various cellular and molecular events, such as apoptosis, cell proliferation, cell migration, and epithelial-mesenchymal transition (EMT), are involved in palatogenesis. The Zeb family of transcription factors is an essential player during normal embryonic development. The distinct role of the Zeb family has not been thoroughly elucidated to date. In mouse palate, the Zeb family factors are expressed in the palatal mesenchyme until MEE contact. Interestingly, the expression of the Zeb family has also been observed in MES, which is already fused with the mesenchymal region. The regulatory roles of the Zeb family in palatogenesis have not been elucidated to date. The purpose of this study is to determine the Zeb family effects on the cellular events. To investigate the functions of the Zeb family, siRNA targeting Zeb family was used to treat in vitro organ culture for temporary inhibition of the Zeb family during palatogenesis. In the cultured palate containing siRNA, MES was clearly observed, and E-cadherin, an epithelial marker, was still expressed. Inhibition of the Zeb family results in the suppression of apoptosis, increased cell proliferation, and defective cell migration in the developing palate. Our data suggest that the Zeb family plays multiple roles in the stimulation and inhibition of apoptosis and cell proliferation and efficient mesenchymal cell migration during palatogenesis.

CTCF Regulates Otic Neurogenesis via Histone Modification in the Neurog1 Locus.

The inner ear is a complex sensory organ responsible for hearing and balance. Formation of the inner ear is dependent on tight regulation of spatial and temporal expression of genes that direct a series of developmental processes. Recently, epigenetic regulation has emerged as a crucial regulator of the development of various organs. However, what roles higher-order chromatin organization and its regulator molecules play in inner ear development are unclear. CCCTC-binding factor (CTCF) is a highly conserved 11-zinc finger protein that regulates the three-dimensional architecture of chromatin, and is involved in various gene regulation processes. To delineate the role of CTCF in inner ear development, the present study investigated inner ear-specific Ctcf knockout mouse embryos (Pax2-Cre; Ctcffl/fl ). The loss of Ctcf resulted in multiple defects of inner ear development and severely compromised otic neurogenesis, which was partly due to a loss of Neurog1 expression. Furthermore, reduced Neurog1 gene expression by CTCF knockdown was found to be associated with changes in histone modification at the gene's promoter, as well as its upstream enhancer. The results of the present study demonstrate that CTCF plays an essential role in otic neurogenesis by modulating histone modification in the Neurog1 locus.

Exocyst Complex Member EXOC5 Is Required for Survival of Hair Cells and Spiral Ganglion Neurons and Maintenance of Hearing.

The exocyst, an octameric protein complex consisting of Exoc1 through Exoc8, was first determined to regulate exocytosis by targeting vesicles to the plasma membrane in yeast to mice. In addition to this fundamental role, the exocyst complex has been implicated in other cellular processes. In this study, we investigated the role of the exocyst in cochlear development and hearing by targeting EXOC5, a central exocyst component. Deleting Exoc5 in the otic epithelium with widely used Cre lines resulted in early lethality. Thus, we generated two different inner ear-specific Exoc5 knockout models by crossing Gfi1Cre mice with Exoc5f/f mice for hair cell-specific deletion (Gfi1Cre/+;Exoc5f/f) and by in utero delivery of rAAV-iCre into the otocyst of embryonic day 12.5 for deletion throughout the otic epithelium (rAAV2/1-iCre;Exoc5f/f). Gfi1Cre/+;Exoc5f/f mice showed relatively normal hair cell morphology until postnatal day 20, after which hair cells underwent apoptosis accompanied by disorganization of stereociliary bundles, resulting in progressive hearing loss. rAAV2/1-iCre;Exoc5f/f mice exhibited abnormal neurite morphology, followed by apoptotic degeneration of spiral ganglion neurons (SGNs) and hair cells, which led to profound and early-onset hearing loss. These results demonstrate that Exoc5 is essential for the normal development and survival of cochlear hair cells and SGNs, as well as the functional maintenance of hearing.

Temporal and spatial expression patterns of Hedgehog receptors in the developing inner and middle ear.

The mammalian inner ear is a complex organ responsible for balance and hearing. Sonic hedgehog (Shh), a member of the Hedgehog (Hh) family of secreted proteins, has been shown to play important roles in several aspects of inner ear development, including dorsoventral axial specification, cochlear elongation, tonotopic patterning, and hair cell differentiation. Hh proteins initiate a downstream signaling cascade by binding to the Patched 1 (Ptch1) receptor. Recent studies have revealed that other types of co-receptors can also mediate Hh signaling, including growth arrest-specific 1 (Gas1), cell-adhesion molecules-related/down-regulated by oncogenes (Cdon), and biregional Cdon binding protein (Boc). However, little is known about the role of these Hh co-receptors in inner ear development. In this study, we examined the expression patterns of Gas1, Cdon, and Boc, as well as that of Ptch1, in the developing mouse inner ear from otocyst (embryonic day (E) 9.5) until birth and in the developing middle ear at E15.5. Ptch1, a readout of Hh signaling, was expressed in a graded pattern in response to Shh signaling throughout development. Expression patterns of Gas1, Cdon, and Boc differed from that of Ptch1, and each Hh co-receptor was expressed in specific cells and domains in the developing inner and middle ear. These unique and differential expression patterns of Hh co-receptors suggest their roles in mediating various time- and space-specific functions of Shh during ear development.

Profiling of miRNAs and target genes related to cystogenesis in ADPKD mouse models.

Autosomal polycystic kidney disease (ADPKD) is a common inherited renal disease characterized by the development of numerous fluid-filled cysts in both kidneys. We investigated miRNA-mediated regulatory systems and networks that play an important role during cystogenesis through integrative analysis of miRNA- and RNA-seq using two ADPKD mouse models (conditional Pkd1- or Pkd2-deficient mice), at three different time points (P1, P3, and P7). At each time point, we identified 13 differentially expressed miRNAs (DEmiRs) and their potential targets in agreement with cyst progression in both mouse models. These targets were involved in well-known signaling pathways linked to cystogenesis. More specifically, we found that the actin cytoskeleton pathway was highly enriched and connected with other well-known pathways of ADPKD. We verified that miR-182-5p regulates actin cytoskeleton rearrangement and promotes ADPKD cystogenesis by repressing its target genes-Wasf2, Dock1, and Itga4-in vitro and in vivo. Our data suggest that actin cytoskeleton may play an important role in renal cystogenesis, and miR-182-5p is a novel regulator of actin cytoskeleton and cyst progression. Furthermore, this study provides a systemic network of both key miRNAs and their targets associated with cyst growth in ADPKD.

Spatiotemporal expression patterns of clusterin in the mouse inner ear.

Clusterin (CLU) is an extracellular chaperone protein that is implicated in diverse physiological and pathophysiological cellular processes. CLU expression is upregulated in response to cellular stress and under certain conditions, such as neurodegenerative disease and cancer. CLU primarily functions as a chaperone that exerts cytoprotective effects by removing cellular debris and misfolded proteins and also acts as a signaling molecule that regulates pro-survival pathways. Deafness is caused by genetic factors and various extrinsic insults, including ototoxic drugs, exposure to loud sounds and aging. Considering its cytoprotectivity, CLU may also mediate cellular defense mechanisms against hearing loss due to cellular stresses. To understand the function of CLU in the inner ear, we analyze CLU expression patterns in the mouse inner ear during development and in the adult stage. Results of quantitative real-time polymerase chain reaction analysis showed that Clu mRNA levels in the inner ear were increased during embryogenesis and were constantly expressed in the adult. Detailed spatial expression patterns of Clu both in the mRNA and protein levels were analyzed throughout various developmental stages via in situ hybridization and immunofluorescence staining. Clu expression was found in specific domains of developing inner ear starting from the otocyst stage, mainly adjacent to the prosensory domain of the cochlear epithelium. In the mature inner ear, Clu expression was observed in Deiter's cells and pillar cells of the organ of Corti, outer sulcus and in basal cells of the stria vascularis in the cochlea. These specific spatiotemporal expression patterns suggest the possible roles of CLU in inner ear development and in maintaining proper hearing function.

Pannexin 3 is required for normal progression of skeletal development in vertebrates.

The vertebrate skeletal system has various functions, including support, movement, protection, and the production of blood cells. The development of cartilage and bones, the core components of the skeletal system, is mediated by systematic inter- and intracellular communication among multiple signaling pathways in differentiating progenitors and the surrounding tissues. Recently, Pannexin (Panx) 3 has been shown to play important roles in bone development in vitro by mediating multiple signaling pathways, although its roles in vivo have not been explored. In this study, we generated and analyzed Panx3 knockout mice and examined the skeletal phenotypes of panx3 morphant zebrafish. Panx3(-/-) embryos exhibited delays in hypertrophic chondrocyte differentiation and osteoblast differentiation as well as the initiation of mineralization, resulting in shortened long bones in adulthood. The abnormal progression of hypertrophic chondrogenesis appeared to be associated with the sustained proliferation of chondrocytes, which resulted from increased intracellular cAMP levels. Similarly, osteoblast differentiation and mineralization were delayed in panx3 morphant zebrafish. Taken together, our results provide evidence of the crucial roles of Panx3 in vertebrate skeletal development in vivo.

The effect of novel mutations on the structure and enzymatic activity of unconventional myosins associated with autosomal dominant non-syndromic hearing loss.

Mutations in five unconventional myosin genes have been associated with genetic hearing loss (HL). These genes encode the motor proteins myosin IA, IIIA, VI, VIIA and XVA. To date, most mutations in myosin genes have been found in the Caucasian population. In addition, only a few functional studies have been performed on the previously reported myosin mutations. We performed screening and functional studies for mutations in the MYO1A and MYO6 genes in Korean cases of autosomal dominant non-syndromic HL. We identified four novel heterozygous mutations in MYO6. Three mutations (p.R825X, p.R991X and Q918fsX941) produce a premature truncation of the myosin VI protein. Another mutation, p.R205Q, was associated with diminished actin-activated ATPase activity and actin gliding velocity of myosin VI in an in vitro analysis. This finding is consistent with the results of protein modelling studies and corroborates the pathogenicity of this mutation in the MYO6 gene. One missense variant, p.R544W, was found in the MYO1A gene, and in silico analysis suggested that this variant has deleterious effects on protein function. This finding is consistent with the results of protein modelling studies and corroborates the pathogenic effect of this mutation in the MYO6 gene.

Conditional deletion of pten leads to defects in nerve innervation and neuronal survival in inner ear development.

All cellular phenomena and developmental events, including inner ear development, are modulated through harmonized signaling networks. Phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor, is a major signaling component involved in cross talk with key regulators of development; i.e., Wnt, Notch, and bone morphogenetic proteins. Although Pten function has been studied in various systems, its role in inner ear development is poorly understood. Here, we used inner ear-specific Pten conditional knockout mice and examined the characteristics of the inner ear. In a detailed analysis of the phenotype, reduced cochlear turning and widened epithelia were observed. Phalloidin staining of sensory epithelium revealed that hair cell patterns were disturbed; i.e., additional rows of hair cells were discovered. The neural abnormality revealed a reduction in and disorganization of nerve fibers, including apoptosis at the neural precursor stage. Pten deficiency induced increased phosphorylation of Akt at Ser473. The elevation of inhibitory glycogen synthase kinase 3ß Ser9 phosphorylation (pGSK3ß) was sustained until the neuronal differentiation stage at embryonic day 14.5, instead of pGSK3ß downregulation. This is the first report on the influence of Pten/Akt/GSK3ß signaling on the development of spiral ganglia. These results suggest that Pten is required for the maintenance of neuroblast number, neural precursors, and differentiation in the inner ear.

Developmental gene expression profiling along the tonotopic axis of the mouse cochlea.

The mammalian cochlear duct is tonotopically organized such that the basal cochlea is tuned to high frequency sounds and the apical cochlea to low frequency sounds. In an effort to understand how this tonotopic organization is established, we searched for genes that are differentially expressed along the tonotopic axis during neonatal development. Cochlear tissues dissected from P0 and P8 mice were divided into three equal pieces, representing the base, middle and apex, and gene expression profiles were determined using the microarray technique. The gene expression profiles were grouped according to changes in expression levels along the tonotopic axis as well as changes during neonatal development. The classified groups were further analyzed by functional annotation clustering analysis to determine whether genes associated with specific biological function or processes are particularly enriched in each group. These analyses identified several candidate genes that may be involved in cochlear development and acquisition of tonotopy. We examined the expression domains for a few candidate genes in the developing mouse cochlea. Tnc (tenacin C) and Nov (nephroblastoma overexpressed gene) are expressed in the basilar membrane, with increased expression toward the apex, which may contribute to graded changes in the structure of the basilar membrane along the tonotopic axis. In addition, Fst (Follistatin), an antagonist of TGF-ß/BMP signaling, is expressed in the lesser epithelial ridge and at gradually higher levels towards the apex. The graded expression pattern of Fst is established at the time of cochlear specification and maintained throughout embryonic and postnatal development, suggesting its possible role in the organization of tonotopy. Our data will provide a good resource for investigating the developmental mechanisms of the mammalian cochlea including the acquisition of tonotopy.

Hoxc8 represses BMP-induced expression of Smad6.

Proper regulation of bone morphogenetic protein (BMP) signaling is critical for correct patterning and morphogenesis of various tissues and organs. A well known feedback mechanism is a BMP-mediated induction of Smad6, an inhibitor of BMP signaling. Hoxc8, one of the Hox family transcription factors, has also been shown to negatively regulate BMP-mediated gene expression. Here we add another level of Hoxc8 regulation on BMP signaling. Our results show that Hoxc8, when over-expressed in C3H10T1/2 or C2C12 cells, suppressed basal Smad6 promoter activity and its mRNA expression. Activation of Smad6 transcription either by BMP2 treatment or Smad1 over-expression was also abolished by Hoxc8. When chromatin was precipitated from mouse embryos with anti-Smad1 or anti-Hoxc8 antibody, Smad6 promoter sequence was enriched, suggesting that Hoxc8 proteins make complexes with Smad1 in the Smad6 promoter region. Yet, a lack of Hox binding motifs in the Smad6 promoter sequence suggests that instead of directly binding to the DNA, Hoxc8 may regulate Smad6 expression via an indirect mechanism. Our results suggest that the Smad6-mediated negative feedback mechanism on BMP signaling may be balanced by the repression of Smad6 expression by Hoxc8.

The third helix of the murine Hoxc8 homeodomain facilitates protein transduction in mammalian cells.

Previously, we have demonstrated that purified Hoxc8 homeoprotein has the ability to penetrate the cellular membrane and can be transduced efficiently into COS-7 cells. Moreover, the Hoxc8 protein is able to form a complex with DNA molecules in vitro and helps the DNA be delivered intracellularly, serving as a gene delivery vehicle. Here, we further analyzed the membrane transduction activity of Hoxc8 protein and provide the evidence that the 16 amino acid (a.a.191-206, 2.23 kDa) third helix of murine Hoxc8 protein is an efficient protein transduction domain (PTD). When the 16 amino acid peptide was fused at the carboxyl terminal of enhanced green fluorescence protein (EGFP), the fusion proteins were transduced efficiently into the primary pig fetal fibroblast cells. The transduction efficiency increased in a concentration-dependent manner up to 1 microM, and appeared to plateau above a concentration of 1 microM. When tandem multimers of PTD, EGFP-PTD(2), EGFP-PTD(3), EGFP-PTD(4), and EGFP-PTD(5), were analyzed at 500 nM of concentration, the penetrating efficiency increased in a dose-dependent manner. As the number of PTDs increased, the EGFP signal also increased, although the signal maintained plateau after EGFP-PTD(3). These results indicate that the 16 amino acid third helix is the key element responsible for the membrane transduction activity of Hoxc8 proteins, and further suggest that the small peptide could serve as a therapeutic delivery vehicle for large cargo proteins.

CaMKII and CaMKIV mediate distinct prosurvival signaling pathways in response to depolarization in neurons.

By fusing the CaMKII-inhibitory peptide AIP to GFP, we constructed a specific and effective CaMKII inhibitor, GFP-AIP. Expression of GFP-AIP and/or dominant-inhibitory CaMKIV in cultured neonatal rat spiral ganglion neurons (SGNs) shows that CaMKII and CaMKIV act additively and in parallel to mediate the prosurvival effect of depolarization. Depolarization or expression of constitutively active CaMKII functionally inactivates Bad, indicating that this is one means by which CaMKII promotes neuronal survival. CaMKIV, but not CaMKII, requires CREB to promote SGN survival, consistent with the exclusively nuclear localization of CaMKIV and indicating that the principal prosurvival function of CaMKIV is activation of CREB. Consistent with this, a constitutively active CREB construct that provides a high level of CREB activity promotes SGN survival, although low levels of CREB activity did not do so. Also, in apoptotic SGNs, activation of CREB by depolarization is disabled, presumably as part of a cellular commitment to apoptosis.

Opposing gradients of Gli repressor and activators mediate Shh signaling along the dorsoventral axis of the inner ear.

Organization of the vertebrate inner ear is mainly dependent on localized signals from surrounding tissues. Previous studies demonstrated that sonic hedgehog (Shh) secreted from the floor plate and notochord is required for specification of ventral (auditory) and dorsal (vestibular) inner ear structures, yet it was not clear how this signaling activity is propagated. To elucidate the molecular mechanisms by which Shh regulates inner ear development, we examined embryos with various combinations of mutant alleles for Shh, Gli2 and Gli3. Our study shows that Gli3 repressor (R) is required for patterning dorsal inner ear structures, whereas Gli activator (A) proteins are essential for ventral inner ear structures. A proper balance of Gli3R and Gli2/3A is required along the length of the dorsoventral axis of the inner ear to mediate graded levels of Shh signaling, emanating from ventral midline tissues. Formation of the ventral-most otic region, the distal cochlear duct, requires robust Gli2/3A function. By contrast, the formation of the proximal cochlear duct and saccule, which requires less Shh signaling, is achieved by antagonizing Gli3R. The dorsal vestibular region requires the least amount of Shh signaling in order to generate the correct dose of Gli3R required for the development of this otic region. Taken together, our data suggest that reciprocal gradients of GliA and GliR mediate the responses to Shh signaling along the dorsoventral axis of the inner ear.