Notch signaling in the development of the inner ear: Lessons from Drosophila

The sensory patches in the ear of a vertebrate can be compared with the mechanosensory bristles of a fly. This comparison has led to the discovery that lateral inhibition mediated by the Notch cell–cell signaling pathway, first characterized in Drosophila and crucial for bristle development, also has a key role in controlling the pattern of sensory hair cells and supporting cells in the ear. We review the arguments for considering the sensory patches of the vertebrate ear and bristles of the insect to be homologous structures, evolved from a common ancestral mechanosensory organ, and we examine more closely the role of Notch signaling in each system. Using viral vectors to misexpress components of the Notch pathway in the chick ear, we show that a simple lateral-inhibition model based on feedback regulation of the Notch ligand Delta is inadequate for the ear just as it is for the fly bristle. The Notch ligand Serrate1, expressed in supporting cells in the ear, is regulated by lateral induction, not lateral inhibition; commitment to become a hair cell is not simply controlled by levels of expression of the Notch ligands Delta1, Serrate1, and Serrate2 in the neighbors of the nascent hair cell; and at least one factor, Numb, capable of blocking reception of lateral inhibition is concentrated in hair cells. These findings reinforce the parallels between the vertebrate ear and the fly bristle and show how study of the insect system can help us understand the vertebrate.

Cochlear mechanisms from a phylogenetic viewpoint

The hearing organ of the inner ear was the last of the paired sense organs of amniotes to undergo formative evolution. As a mechanical sensory organ, the inner-ear hearing organ's function depends highly on its physical structure. Comparative studies suggest that the hearing organ of the earliest amniote vertebrates was small and simple, but possessed hair cells with a cochlear amplifier mechanism, electrical frequency tuning, and incipient micromechanical tuning. The separation of the different groups of amniotes from the stem reptiles occurred relatively early, with the ancestors of the mammals branching off first, approximately 320 million years ago. The evolution of the hearing organ in the three major lines of the descendents of the stem reptiles (e.g., mammals, birds-crocodiles, and lizards-snakes) thus occurred independently over long periods of time. Dramatic and parallel improvements in the middle ear initiated papillar elongation in all lineages, accompanied by increased numbers of sensory cells with enhanced micromechanical tuning and group-specific hair-cell specializations that resulted in unique morphological configurations. This review aims not only to compare structure and function across classification boundaries (the comparative approach), but also to assess how and to what extent fundamental mechanisms were influenced by selection pressures in times past (the phylogenetic viewpoint).

Mechanical bases of frequency tuning and neural excitation at the base of the cochlea: Comparison of basilar-membrane vibrations and auditory-nerve-fiber responses in chinchilla

We review the mechanical origin of auditory-nerve excitation, focusing on comparisons of the magnitudes and phases of basilar-membrane (BM) vibrations and auditory-nerve fiber responses to tones at a basal site of the chinchilla cochlea with characteristic frequency ≈ 9 kHz located 3.5 mm from the oval window. At this location, characteristic frequency thresholds of fibers with high spontaneous activity correspond to magnitudes of BM displacement or velocity in the order of 1 nm or 50 μm/s. Over a wide range of stimulus frequencies, neural thresholds are not determined solely by BM displacement but rather by a function of both displacement and velocity. Near-threshold, auditory-nerve responses to low-frequency tones are synchronous with peak BM velocity toward scala tympani but at 80–90 dB sound pressure level (in decibels relative to 20 microPascals) and at 100–110 dB sound pressure level responses undergo two large phase shifts approaching 180°. These drastic phase changes have no counterparts in BM vibrations. Thus, although at threshold levels the encoding of BM vibrations into spike trains appears to involve only relatively minor signal transformations, the polarity of auditory-nerve responses does not conform with traditional views of how BM vibrations are transmitted to the inner hair cells. The response polarity at threshold levels, as well as the intensity-dependent phase changes, apparently reflect micromechanical interactions between the organ of Corti, the tectorial membrane and the subtectorial fluid, and/or electrical and synaptic processes at the inner hair cells.

Rearrangement of Actin Microfilaments in Plant Root Hairs Responding to Rhizobium etli Nodulation Signals1

The response of the actin cytoskeleton to nodulation (Nod) factors secreted by Rhizobium etli has been studied in living root hairs of bean (Phaseolus vulgaris) that were microinjected with fluorescein isothiocyanate-phalloidin. In untreated control cells or cells treated with the inactive chitin oligomer, the actin cytoskeleton was organized into long bundles that were oriented parallel to the long axis of the root hair and extended into the apical zone. Upon exposure to R. etli Nod factors, the filamentous actin became fragmented, as indicated by the appearance of prominent masses of diffuse fluorescence in the apical region of the root hair. These changes in the actin cytoskeleton were rapid, observed as soon as 5 to 10 min after application of the Nod factors. It was interesting that the filamentous actin partially recovered in the continued presence of the Nod factor: by 1 h, long bundles had reformed. However, these cells still contained a significant amount of diffuse fluorescence in the apical zone and in the nuclear area, presumably indicating the presence of short actin filaments. These results indicate that Nod factors alter the organization of actin microfilaments in root hair cells, and this could be a prelude for the formation of infection threads.

Mutation in mitochondrial DNA as a cause of presbyacusis

Much of the hearing loss that occurs in old age is likely to be due to the long-term deterioration of the mitochondria in the different structures of the cochlea. The current review surveys some of the basic information on mitochondria and mitochondrial DNA, as a background to their possible involvement in presbyacusis. It is likely that oxygen radicals damage mitochondrial DNA and other components of the mitochondria, such as their proteins and lipids. This further compromises both oxidative phosphorylation and the repair processes in mitochondria, setting up a vicious cycle of degradation. Evidence is presented from inherited point mutations on the possibly most critical sites for mutations in mitochondrial DNA associated with hearing loss. It is suggested that random sorting and clonal expansion of mutations both maintain the integrity of the pool of mitochondrial DNA molecules and give rise to the apoptosis that leads to loss of vulnerable cells, and hence to deafness. It is moreover suggested that apoptosis of the vulnerable cells of the inner ear may to some extent be preventable, or at least delayed. Copyright (C) 2004 S. Karger AG, Basel.

Tertiapin-Q blocks recombinant and native large conductance K+ channels in a use-dependent manner

Tertiapin, a short peptide from honey bee venom, has been reported to specifically block the inwardly rectifying K+ (Kir) channels, including G protein-coupled inwardly rectifying potassium channel (GIRK) 1 + GIRK4 heteromultimers and ROMK1 homomultimers. In the present study, the effects of a stable and functionally similar derivative of tertiapin, tertiapin-Q, were examined on recombinant human voltage-dependent Ca2+-activated large conductance K+ channel (BK or MaxiK; alpha-subunit or hSlo1 homomultimers) and mouse inwardly rectifying GIRK1 + GIRK2 (i.e., Kir3.1 and Kir3.2) heteromultimeric K+ channels expressed in Xenopus oocytes and in cultured newborn mouse dorsal root ganglion (DRG) neurons. In two-electrode voltage-clamped oocytes, tertiapin-Q (1-100 nM) inhibited BK-type K+ channels in a use- and concentration-dependent manner. We also confirmed the inhibition of recombinant GIRK1 + GIRK2 heteromultimers by tertiapin-Q, which had no effect on endogenous depolarization- and hyperpolarization-activated currents sensitive to extracellular divalent cations (Ca2+, Mg2+, Zn2+, and Ba2+) in defolliculated oocytes. In voltage-clamped DRG neurons, tertiapin-Q voltage- and use-dependently inhibited outwardly rectifying K+ currents, but Cs+-blocked hyperpolarization-activated inward currents including I-H were insensitive to tertiapin-Q, baclofen, barium, and zinc, suggesting absence of functional GIRK channels in the newborn. Under current-clamp conditions, tertiapin-Q blocked the action potential after hyperpolarization (AHP) and increased action potential duration in DRG neurons. Taken together, these results demonstrate that the blocking actions of tertiapin-Q are not specific to Kir channels and that the blockade of recombinant BK channels and native neuronal AHP currents is use-dependent. Inhibition of specific types of Kir and voltage-dependent Ca2+-activated K+ channels by tertiapin-Q at nanomolar range via different mechanisms may have implications in pain physiology and therapy.

SK channels regulate excitatory synaptic transmission and plasticity in the lateral amygdala

At glutamatergic synapses, calcium influx through NMDA receptors (NMDARs) is required for long-term potentiation (LTP); this is a proposed cellular mechanism underlying memory and learning. Here we show that in lateral amygdala pyramidal neurons, SK channels are also activated by calcium influx through synaptically activated NMDARs, resulting in depression of the synaptic potential. Thus, blockade of SK channels by apamin potentiates fast glutamatergic synaptic potentials. This potentiation is blocked by the NMDAR antagonist AP5 (D(-)-2-amino-5-phosphono-valeric acid) or by buffering cytosolic calcium with BAPTA. Blockade of SK channels greatly enhances LTP of cortical inputs to lateral amygdala pyramidal neurons. These results show that NMDARs and SK channels are colocalized at glutamatergic synapses in the lateral amygdala. Calcium influx through NMDARs activates SK channels and shunts the resultant excitatory postsynaptic potential. These results demonstrate a new role for SK channels as postsynaptic regulators of synaptic efficacy.

Enhanced information transmission with signal-dependent noise in an array of nonlinear elements

We have investigated information transmission in an array of threshold units that have signal-dependent noise and a common input signal. We demonstrate a phenomenon similar to stochastic resonance and suprathreshold stochastic resonance with additive noise and show that information transmission can be enhanced by a nonzero level of noise. By comparing system performance to one with additive noise we also demonstrate that the information transmission of weak signals is significantly better with signal-dependent noise. Indeed, information rates are not compromised even for arbitrary small input signals. Furthermore, by an appropriate selection of parameters, we observe that the information can be made to be (almost) independent of the level of the noise, thus providing a robust method of transmitting information in the presence of noise. These result could imply that the ability of hair cells to code and transmit sensory information in biological sensory systems is not limited by the level of signal-dependent noise. © 2007 The American Physical Society.

Association of SNPs in LCP1 and CTIF with hearing in 11 year old children:findings from the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort and the G-EAR consortium

BACKGROUND: The genetic basis of hearing loss in humans is relatively poorly understood. In recent years, experimental approaches including laboratory studies of early onset hearing loss in inbred mouse strains, or proteomic analyses of hair cells or hair bundles, have suggested new candidate molecules involved in hearing function. However, the relevance of these genes/gene products to hearing function in humans remains unknown. We investigated whether single nucleotide polymorphisms (SNPs) in the human orthologues of genes of interest arising from the above-mentioned studies correlate with hearing function in children. METHODS: 577 SNPs from 13 genes were each analysed by linear regression against averaged high (3, 4 and 8 kHz) or low frequency (0.5, 1 and 2 kHz) audiometry data from 4970 children in the Avon Longitudinal Study of Parents and Children (ALSPAC) birth-cohort at age eleven years. Genes found to contain SNPs with low p-values were then investigated in 3417 adults in the G-EAR study of hearing. RESULTS: Genotypic data were available in ALSPAC for a total of 577 SNPs from 13 genes of interest. Two SNPs approached sample-wide significance (pre-specified at p = 0.00014): rs12959910 in CBP80/20-dependent translation initiation factor (CTIF) for averaged high frequency hearing (p = 0.00079, β = 0.61 dB per minor allele); and rs10492452 in L-plastin (LCP1) for averaged low frequency hearing (p = 0.00056, β = 0.45 dB). For low frequencies, rs9567638 in LCP1 also enhanced hearing in females (p = 0.0011, β = -1.76 dB; males p = 0.23, β = 0.61 dB, likelihood-ratio test p = 0.006). SNPs in LCP1 and CTIF were then examined against low and high frequency hearing data for adults in G-EAR. Although the ALSPAC results were not replicated, a SNP in LCP1, rs17601960, is in strong LD with rs9967638, and was associated with enhanced low frequency hearing in adult females in G-EAR (p = 0.00084). CONCLUSIONS: There was evidence to suggest that multiple SNPs in CTIF may contribute a small detrimental effect to hearing, and that a sex-specific locus in LCP1 is protective of hearing. No individual SNPs reached sample-wide significance in both ALSPAC and G-EAR. This is the first report of a possible association between LCP1 and hearing function.

Control of thrombin signaling through PI3K is a mechanism underlying plasticity between hair follicle dermal sheath and papilla cells.

In hair follicles, dermal papilla (DP) and dermal sheath (DS) cells exhibit striking levels of plasticity, as each can regenerate both cell types. Here, we show that thrombin induces a phosphoinositide 3-kinase (PI3K)-Akt pathway-dependent acquisition of DS-like properties by DP cells in vitro, involving increased proliferation rate, acquisition of ;myofibroblastic' contractile properties and a decreased capacity to sustain growth and survival of keratinocytes. The thrombin inhibitor protease nexin 1 [PN-1, also known as SERPINE2) regulates all those effects in vitro. Accordingly, the PI3K-Akt pathway is constitutively activated and expression of myofibroblastic marker smooth-muscle actin is enhanced in vivo in hair follicle dermal cells from PN-1(-/-) mice. Furthermore, physiological PN-1 disappearance and upregulation of the thrombin receptor PAR-1 (also known as F2R) during follicular regression in wild-type mice also correlate with such changes in DP cell characteristics. Our results indicate that control of thrombin signaling interferes with hair follicle dermal cells plasticity to regulate their function.

Amino acid substitutions in σ1 and μ1 outer capsid proteins are selected during mammalian reovirus adaptation to Vero cells

Establishment of viral persistence in cell culture has previously led to the selection of mammalian reovirus mutants, although very few of those have been characterized in details. In the present study, reovirus was adapted to Vero cells that, in contrast to classically-used L929 cells, are inefficient in supporting the early steps of reovirus uncoating and are also unable to produce interferon as an antiviral response once infection occurs. The Vero cell-adapted reovirus exhibits amino acids substitutions in both the σ1 and μ1 proteins. This contrasts with uncoating mutants from persistently-infected L929 cells, and various other cell types, that generally harbor amino acids substitutions in the σ3 outer capsid protein. The Vero cell-adapted virus remained sensitive to an inhibitor of lysosomal proteases; furthermore, in the absence of selective pressure for its maintenance, t he virus has partially lost its ability to resist interferon. The positions of the amino acids substitutions on the known protein structures suggest an effect on binding of the viral σ1 protein to the cell surface and on μ1 disassembly from the outer capsid.

Putative outer membrane proteins of Leptospira interrogans stimulate human umbilical vein endothelial cells (HUVECS) and express during infection

Cell adhesion molecules (CAMs) are surface receptors present in eukaryotic cells that mediate cell-cell or cell-extracellular matrix interactions. Vascular endothelium stimulation in vitro that lead to the upregulation of CAMs was reported for the pathogenic spirochaetes, including rLIC10365 of Leptospira interrogans. In this study, we report the cloning of LIC10507, LIC10508, LIC10509 genes of L interrogans using Escherichia coli as a host system. The rational for selecting these sequences is due to their location in L. interrogans serovar Copenhageni genome that has a potential involvement in pathogenesis. The genes encode for predicted lipoproteins with no assigned functions. The purified recombinant proteins were capable to promote the upregulation of intercellular adhesion molecule 1 (ICAM-1) and E-selectin on monolayers of human umbilical vein endothelial cells (HUVECS). In addition, the coding sequences are expressed in the renal tubules of animal during bacterial experimental infection. The proteins are probably located at the outer membrane of the bacteria since they are detected in detergent-phase of L interrogans Triton X-114 extract. Altogether our data suggest a possible involvement of these proteins during bacterial infection and provide new insights into the role of this region in the pathogenesis of Leptospira. (C) 2008 Elsevier Ltd. All rights reserved.

Detection of cytokeratins in ghost cells of calcifying cystic odontogenic tumor indicates an altered keratinization and hair follicle differentiation for their development

Calcifying cystic odontogenic tumors (CCOTs) are benign cystic lesions of odontogenic origin characterized by an ameloblastoma-like epithelium and the presence of a group of cells named ghost cells. The pattern of cytokeratin (Ck) expression on these lesions remains unclear and needs to be clarified. To this end, the expression of Ck6, Ck13, Ck14, Ck18, and Ck19 in the epithelium lining of 7 cases of CCOTs was evaluated by immunohistochemistry. For this, the epithelium lining was divided into 3 distinct regions: basal layer, suprabasal layer, and the compartment composed of ghost cells. In this study, 6 cases (85.7%) were classified as type 1 and 1 (14.3%) as type 4. All cases were negative for Ck13 and Ck18, despite the epithelial layer, as well as in the ghost cells. Ck6 was only positive in the ghost cells. Positivity for Ck14 and Ck19 was found in the basal and suprabasal layers, including the ghost cells. The results showing positivity for Ck14 and Ck19 in all of the analyzed cases reinforce CCOT as being of odontogenic origin, and the restricted expression of Ck6 in the ghost cells may be indicative that these cells suffer an altered differentiation into hair follicles in CCOTs. © 2013 Elsevier Inc. All rights reserved.

Mitochondrial Swelling and Incipient Outer Membrane Rupture in Preapoptotic and Apoptotic Cells

Outer mitochondrial membrane (OMM) rupture was first noted in isolated mitochondria in which the inner mitochondrial membrane (IMM) had lost its selective permeability. This phenomenon referred to as mitochondrial permeability transition (MPT) refers to a permeabilized inner membrane that originates a large swelling in the mitochondrial matrix, which distends the outer membrane until it ruptures. Here, we have expanded previous electron microscopic observations that in apoptotic cells, OMM rupture is not caused by a membrane stretching promoted by a markedly swollen matrix. It is shown that the widths of the ruptured regions of the OMM vary from 6 to 250 nm. Independent of the perforation size, herniation of the mitochondrial matrix appeared to have resulted in pushing the IMM through the perforation. A large, long focal herniation of the mitochondrial matrix, covered with the IMM, was associated with a rupture of the OMM that was as small as 6 nm. Contextually, the collapse of the selective permeability of the IMM may precede or follow the release of the mitochondrial proteins of the intermembrane space into the cytoplasm. When the MPT is a late event, exit of the intermembrane space proteins to the cytoplasm is unimpeded and occurs through channels that transverse the outer membrane, because so far, the inner membrane is impermeable. No channel within the outer membrane can expose to the cytoplasm a permeable inner membrane, because it would serve as a conduit for local herniation of the mitochondrial matrix. Anat Rec, 2012. (c) 2012 Wiley Periodicals, Inc.