Leptin Elongates Hypothalamic Neuronal Cilia via Transcriptional Regulation and Actin Destabilization.

Terminally differentiated neurons have a single, primary cilium. The primary cilia of hypothalamic neurons play a critical role in sensing metabolic signals. We recently showed that mice with leptin deficiency or resistance have shorter cilia in the hypothalamic neurons, and leptin treatment elongates cilia in hypothalamic neurons. Here, we investigated the molecular mechanisms by which leptin controls ciliary length in hypothalamic neurons. In N1 hypothalamic neuronal cells, leptin treatment increased the expression of intraflagellar transport proteins. These effects occurred via phosphatase and tensin homolog/glycogen synthase kinase-3ß-mediated inhibition of the transcriptional factor RFX1. Actin filament dynamics were also involved in leptin-promoted ciliary elongation. Both leptin and cytochalasin-D treatment induced F-actin disruption and cilium elongation in hypothalamic neurons that was completely abrogated by co-treatment with the F-actin polymerizer phalloidin. Our findings suggest that leptin elongates hypothalamic neuronal cilia by stimulating the production of intraflagellar transport proteins and destabilizing actin filaments.

Fetus sound stimulation: cilia memristor effect of signal transduction.

BACKGROUND: This experimental study evaluates fetal middle cerebral artery (MCA) circulation after the defined prenatal acoustical stimulation (PAS) and the role of cilia in hearing and memory and could explain signal transduction and memory according to cilia optical-acoustical properties. METHODS: PAS was performed twice on 119 no-risk term pregnancies. We analyzed fetal MCA circulation before, after first and second PAS. RESULTS: Analysis of the Pulsatility index basic (PIB) and before PAS and Pulsatility index reactive after the first PAS (PIR 1) shows high statistical difference, representing high influence on the brain circulation. Analysis of PIB and Pulsatility index reactive after the second PAS (PIR 2) shows no statistical difference. Cilia as nanoscale structure possess magnetic flux linkage that depends on the amount of charge that has passed between two-terminal variable resistors of cilia. Microtubule resistance, as a function of the current through and voltage across the structure, leads to appearance of cilia memory with the "memristor" property. CONCLUSION: Acoustical and optical cilia properties play crucial role in hearing and memory processes. We suggest that fetuses are getting used to sound, developing a kind of memory patterns, considering acoustical and electromagnetically waves and involving cilia and microtubules and try to explain signal transduction.

Pathological features in the LmnaDhe/+ mutant mouse provide a novel model of human otitis media and laminopathies.

Genetic predisposition is recognized as an important pathogenetic factor in otitis media (OM) and associated diseases. Mutant Lmna mice heterozygous for the disheveled hair and ears allele (Lmna(Dhe/+)) exhibit early-onset, profound hearing deficits and other pathological features mimicking human laminopathy associated with the LMNA mutation. We assessed the effects of the Lmna(Dhe/+) mutation on development of OM and pathological abnormalities characteristic of laminopathy. Malformation and abnormal positioning of the eustachian tube, accompanied by OM, were observed in all of the Lmna(Dhe/+) mice (100% penetrance) as early as postnatal day P12. Scanning electronic microscopy revealed ultrastructural damage to the cilia in middle ears that exhibited OM. Hearing assessment revealed significant hearing loss, paralleling that in human OM. Expression of NF-κB, TNF-α, and TGF-ß, which correlated with inflammation and/or bony development, was up-regulated in the ears or in the peritoneal macrophages of Lmna(Dhe/+) mice. Rugous, disintegrative, and enlarged nuclear morphology of peritoneal macrophages and hyperphosphatemia were found in Lmna(Dhe/+) mutant mice. Taken together, these features resemble the pathology of human laminopathies, possibly revealing some profound pathology, beyond OM, associated with the mutation. The Lmna(Dhe/+) mutant mouse provides a novel model of human OM and laminopathy.

Toward understanding the genetic basis of neural tube defects.

Neural tube defects (NTDs) represent a common group of severe congenital malformations that result from failure of neural tube closure during early development. Their etiology is quite complex involving environmental and genetic factors and their underlying molecular and cellular pathogenic mechanisms remain poorly understood. Animal studies have recently demonstrated an essential role for the planar cell polarity pathway (PCP) in mediating a morphogenetic process called convergent extension during neural tube formation. Alterations in members of this pathway lead to NTDs in vertebrate models, representing novel and exciting candidates for human NTDs. Genetic studies in NTDs have focused mainly on folate-related genes based on the finding that perinatal folic acid supplementation reduces the risk of NTDs by 60-70%. A few variants in these genes have been found to be significantly associated with an increased risk for NTDs. The candidate gene approach investigating genes involved in neurulation has failed to identify major causative genes in the etiology of NTDs. Despite this history of generally negative findings, we are achieving a rapid and impressive progress in understanding the genetic basis of NTDs, based mainly on the powerful tool of animal models.

Intraflagellar transport particles participate directly in cilium-generated signaling in Chlamydomonas.

Primary cilia are widely used for signal transduction during development and in homeostasis and are assembled and maintained by intraflagellar transport (IFT). Here, we have dissected the role of IFT in signaling within the flagella (structural and functional counterparts of cilia) of the biflagellated green alga Chlamydomonas. Using a conditional IFT mutant enables us to deplete the IFT machinery from intact, existing flagella. We identify a cGMP-dependent protein kinase (CrPKG) within flagella as the substrate of a protein tyrosine kinase activated by flagellar adhesion during fertilization. We demonstrate that flagellar adhesion stimulates association of CrPKG with a new flagellar compartment. Moreover, formation of the compartment requires IFT, and IFT particles themselves are part of the compartment. Our results lead to a model in which the IFT machinery is required not only for assembling cilia and flagella but also for organizing a signaling pathway within the organelles during cilium-generated signaling.

Ophiopogon root (Radix Ophiopogonis) prevents ultra-structural damage by SO2 in an epithelial injury model for studies of mucociliary transport.

We studied the action of the herb, Ophiopogon root (OR) in a epithelial injury model, hypothesizing that it may have beneficial effects on mucociliary transport following injury to the palate induced by sodium metabisulphite (MB) which releases SO(2) on contact with water. OR (extract from 1g of root/ml)-incubated palates and non-incubated palates were compared to assess the effect of MB on mucociliary clearance on the bull frog palate. MB 10(-1) M, acutely increased mucociliary clearance time (MCT) by 254.5 +/- 57.3% in untreated and 243.3 +/- 98.5% in OR-incubated palates, (over all significance assessed by one-way ANOVA, F = 12.82, p < 0.001, df = 8,54 for MB and F = 10.56, p < 0.001, df = 8,54 for OR). MCT returned to normal during recovery in OR-treated palates following MB. In untreated palates, MCT did not return to control values during a similar recovery period. ANOVA comparing MCTs in the recovery period in untreated vs OR-treated palates was significantly different (F = 2.92, p < 0.03, df = 5,36). SEM images of epithelial tissue, analyzed by morphometry, showed a 25 +/- 12% loss of ciliated cells in untreated palates and little or no damage to cilia in OR-treated palates. Intact groups of ciliated cells were found in SEM micrographs of mucus from MB-treated palates. We conclude that the loss of cilia or ciliated cells prevented full recovery of MCT after MB in untreated palates. In OR-incubated palates, mucociliary transport was completely restored within 20 min after topical application of MB, possibly through a protective action on the extra-cellular matrix.

Cultured peritoneal mesothelial cells exhibit apical primary cilia.

This study examined primary cilia on cultured human and rabbit peritoneal mesothelial cells (PMC) and investigated factors that influence ciliary expression. Primary cilia were examined with indirect immunocytochemistry, laser scanning confocal microscopy and scanning electron microscopy. Ciliary expression was evaluated in cultures with or without l-cysteine (0.25 mM) or exposure to Ca(2+)-free Krebs-Ringer solution supplemented with EGTA, 0.5 mM. This treatment disrupted cell monolayer integrity. Cilia were counted and normalized to total cell counts using NIH image. Primary cilia were identified on both human and rabbit PMC. Cells treated with l-cysteine expressed significantly more cilia compared with monolayers deprived of l-cysteine. Exposure to Ca(2+)-free Krebs-Ringer solution significantly reduced cilia (5.9+/-1.0%, n=7). Although ciliary expression could be augmented with l-cysteine, approximately 60% of human PMC and 84% of rabbit PMC did not exhibit cilia. Together, these data show that monolayers of PMC express apical cilia that can be augmented with l-cysteine, independently of increased cell density.

Drosophila KAP interacts with the kinesin II motor subunit KLP64D to assemble chordotonal sensory cilia, but not sperm tails.

BACKGROUND: Kinesin II-mediated anterograde intraflagellar transport (IFT) is essential for the assembly and maintenance of flagella and cilia in various cell types. Kinesin associated protein (KAP) is identified as the non-motor accessory subunit of Kinesin II, but its role in the corresponding motor function is not understood. RESULTS: We show that mutations in the Drosophila KAP (DmKap) gene could eliminate the sensory cilia as well as the sound-evoked potentials of Johnston's organ (JO) neurons. Ultrastructure analysis of these mutants revealed that the ciliary axonemes are absent. Mutations in Klp64D, which codes for a Kinesin II motor subunit in Drosophila, show similar ciliary defects. All these defects are rescued by exclusive expression of DmKAP and KLP64D/KIF3A in the JO neurons of respective mutants. Furthermore, reduced copy number of the DmKap gene was found to enhance the defects of hypomorphic Klp64D alleles. Unexpectedly, however, both the DmKap and the Klp64D mutant adults produce vigorously motile sperm with normal axonemes. CONCLUSIONS: KAP plays an essential role in Kinesin II function, which is required for the axoneme growth and maintenance of the cilia in Drosophila type I sensory neurons. However, the flagellar assembly in Drosophila spermatids does not require Kinesin II and is independent of IFT.

Tip link loss and recovery on chick short hair cells following intense exposure to sound.

Stereocilia tip links on chick short hair cells (SHCs) were counted in the 'patch' lesion produced by acoustic overstimulation. Tip links were also counted on tall hair cells (THCs) immediately superior to the lesion. Eight groups were studied with three exposed to intense sound for differing durations. Three other groups were allowed to recover from the longest exposure for different time periods. Tip link counts from non-exposed control hair cells came from two other groups. Chicks exposed for 4, 24 or 48 h to a 120-dB SPL 0.9-kHz pure tone showed SHC tip link loss of 30.3, 40.6, and 35.5%, respectively. Chicks exposed for 48 h were allowed to recover for 24, 96 or 288 h, and showed systematic tip link recovery to control levels. Tip link loss and recovery in THCs adjacent to the patch lesion were identical to that seen in SHCs. After 288 h of recovery, surviving SHCs were distinguished from newly regenerated SHCs in the patch lesion. A comparison of tip link presence in the surviving (74%) and regenerated (84%) SHCs revealed a significant difference. These results suggest that the process of tip link destruction and recovery following acoustic overstimulation is the same for THCs and SHCs. This observation is surprising based on differences in the degree of acoustic injury to THC and SHC regions of the papillae, and the difference between THC and SHC sensory hair bundle stimulation.

Plasma membrane Ca2+-ATPase isoform 2a is the PMCA of hair bundles.

Mechanoelectrical transduction channels of hair cells allow for the entry of appreciable amounts of Ca(2+), which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca(2+) that enters transduction channels is extruded by the plasma membrane Ca(2+)-ATPase (PMCA), a Ca(2+) pump that is highly concentrated in hair bundles and may be essential for normal hair cell function. Because PMCA isozymes and splice forms are regulated differentially and have distinct biochemical properties, we determined the identity of hair bundle PMCA in frog and rat hair cells. By screening a bullfrog saccular cDNA library, we identified abundant PMCA1b and PMCA2a clones as well as rare PMCA2b and PMCA2c clones. Using immunocytochemistry and immunoprecipitation experiments, we showed in bullfrog sacculus that PMCA1b is the major isozyme of hair cell and supporting cell basolateral membranes and that PMCA2a is the only PMCA present in hair bundles. This complete segregation of PMCA1 and PMCA2 isozymes holds for rat auditory and vestibular hair cells; PMCA2a is the only PMCA isoform in hair bundles of outer hair cells and vestibular hair cells and is the predominant PMCA of hair bundles of inner hair cells. Our data suggest that hair cells control plasma membrane Ca(2+)-pumping activity by targeting specific PMCA isozymes to distinct subcellular locations. Because PMCA2a is the only Ca(2+) pump present at appreciable levels in hair bundles, the biochemical properties of this pump must account fully for the physiological features of transmembrane Ca(2+) pumping in bundles.

Confocal analysis of primary cilia structure and colocalization with the Golgi apparatus in chondrocytes and aortic smooth muscle cells.

Detyrosinated and acetylated alpha-tubulins represent a stable pool of tubulin typically associated with microtubules of the centrosome and primary cilium of eukaryotic cells. Although primary cilium-centrosome and centrosome-Golgi relationships have been identified independently, the precise structural relationship between the primary cilium and Golgi has yet to be specifically defined. Confocal immunohistochemistry was used to localize detyrosinated (ID5) and acetylated (6-11B-1) tubulin antibodies in primary cilia of chondrocytes and smooth muscle cells, and to demonstrate their relationship to the Golgi complex identified by complementary lectin staining with wheat germ agglutinin. The results demonstrate the distribution and inherent structural variation of primary cilia tubulins, and the anatomical interrelationship between the primary cilium, the Golgi apparatus and the nucleus. We suggest that these interrelationships may form part of a functional feedback mechanism which could facilitate the directed secretion of newly synthesized connective tissue macromolecules.

Interaction of Haemophilus parasuis with nasal and tracheal mucosa following intranasal inoculation of cesarean derived colostrum deprived (CDCD) swine.

Twenty-three cesarean derived, colostrum deprived pigs were obtained at 5 wk of age and inoculated intranasally with either 1.4 x 10(8) colony forming units of Haemophilus parasuis or sterile phosphate buffered saline. Pigs were euthanized at 4, 8, 12, 18, 26, or 36 h post-inoculation and tissues from the oropharynx and respiratory tract were obtained for qualitative bacterial culture, immunohistochemistry for H. parasuis antigens, and light and transmission electron microscopy. Haemophilus parasuis was consistently isolated from the nasal cavity (17/17, 100%) and trachea (13/17, 76%) and rarely isolated from the lung (3/17, 18%) and blood stream (1/17, 6%) of infected pigs. Antigens of H. parasuis were sporadically detected on the nasal mucosa (6/17, 35%) and trachea (8/17, 47%). Light microscopic lesions included submucosal and intraepithelial infiltrates of neutrophils and infrequent, patchy loss of cilia. Ultrastructural changes in nasal mucosal epithelial cells included cell protrusion, loss of cilia, and dilation of the cytocavitary network. Bacteria were infrequently identified and were either within an amorphous material at the apical surface of the cilia or were between individual cilia. These results suggest H. parasuis associates with the nasal mucosa and can induce a suppurative rhinitis with nasal mucosal epithelial cell degeneration. This process may represent an initial event in the pathogenesis of H. parasuis infection of swine.

Changes in distortion product otoacoustic emissions and outer hair cells following interrupted noise exposures.

Changes in distortion product otoacoustic emissions (DPOAEs) were examined during and after interrupted noise exposures and compared to the condition of the outer hair cells (OHCs) and inner hair cells (IHCs) as assessed by scanning electron microscopy (SEM). Binaural, adult chinchillas were exposed to a 95 dB SPL, octave band noise centered at 0.5 kHz for 15 days using a 3 h on/9 h off schedule. DPOAEs were measured before, during and after the exposures. DPOAE amplitudes decreased significantly during the first few days of the interrupted noise exposures and then began to recover. At most frequencies, the emission amplitudes recovered completely to pre-exposure baseline values by five days after the last exposure. The results of the present study indicate that the changes in DPOAE amplitude paralleled the recovery in the amplitude and threshold of the compound action potentials as reported previously (Boettcher et al., 1992). Although the DPOAEs completely recovered, considerable OHC loss and stereocilia disarray was evident even four weeks after exposure.

Intercellular contacts between chick stereocilia after acoustic overstimulation.

The goal of this study was to analyze the distribution of actin and the shape of stereocilia of chick hair cells that survive acoustic trauma. Chicks were exposed to intense octave band noise for 4 h. They were killed either immediately after the exposure, after 6 or after 72 h. The basilar papillae were examined using scanning electron microscopy and fluorescence microscopy, with phalloidin as an actin-specific probe. Injured hair cells which survived the trauma displayed disorganized stereocilia bundles, elongated stereocilia, and supernumerary stereocilia bundles. Tips of stereocilia in the damaged region of the basilar papilla appeared to be in contact with tips of stereocilia of neighboring hair cells. These contacts may represent 'stress links' which appear in traumatized hair cells. These results show that substantial changes in stereocilia occur within hours of exposure to intense noise. We speculate that surviving hair cells may play a role in the process of repair of the basilar papilla after noise trauma and that the changes in stereocilia structure described here are related to this role.

Quantitative histochemistry of phosphorus in the vestibular gelatinous membrane: an electron probe X-ray microanalytical study.

Electron probe X-ray microanalysis was used to study the phosphorus concentration in the otolithic gelatinous membrane of the saccule and the utricle with scanning electron microscopy. The otolithic membranes were plunge-frozen in liquid N2 and freeze-dried. Quantitative analysis was carried out with an energy dispersive detector using the peak-to-background ratio method and different concentrations of KH2PO4 salts dissolved in dextran solutions. The otolithic gelatinous membrane consists of a 25-30 microns-thick layer overlying the cilia of the hair cells. Elements detected in the gelatinous membrane are: Na, P, S, Cl, K and Ca. Although Student's t-test did not show significant differences between saccular and utricular concentrations of phosphorus, the distribution of this element in the two organs was different. Regression analysis established that the concentrations of phosphorus in the saccular and utricular gelatinous membrane were dependent. The regression equation was: y = 18.02x2 + 133.9 (r = 0.83, P < 0.05) where y is the concentration of phosphorus in the utricle, and x2 the concentration of phosphorus in the saccule. The findings obtained in the present study could be related to structural differences in organic phosphate residues of the phosphoproteins associated to collagen, or to different polyphosphoinositide turnover rates in the cell membrane.

[Normal structure of stereocilia and recovery from ciliary damage in the organ of Corti after acoustic overstimulation].

The normal structure of outer hair cell (OHC) stereocilia in the organ of Corti, as well as damage and the recovery of OHC stereocilia after acoustic overstimulation, were demonstrated using scanning electron microscopy and tannic acid-osmium staining techniques. The highest row of OHC stereocilia is known to show an orderly gradation in height along the length of the cochlea. The present study demonstrated that the middle and lower rows of stereocilia possess a similar height gradation pattern. These findings suggest that the orderly gradation of stereocilia may play an important role in the tuning capability of the organ of Corti sensory cell. To investigate the mechanisms of recovery from ciliary acoustic damage, guinea pigs were exposed to a 4.00 kHz pure tone at an intensity of 120 dB for 120 minutes. All animals showed temporarily elevated hearing thresholds, which had returned to normal one week later. The first detectable change after acoustic overstimulation was a derangement of the cilia with a loss of ciliary interconnections. The tip links connecting the tips of the stereocilia to their taller neighbours were also affected showing elongation or disappearance. In comparing ciliary damage immediately after and one week after acoustic overstimulation, no signs of recovery in hair cell cilia which had been already lost, could be detected, while stereocilia with slight damage seemed to recover in the early post-sound exposure stage. Some lost tip links also seem to reappear in surviving cilia. In addition side links have the possibility of recovery.

Changes in the stereocilia and non-monotonic pattern of threshold shift after exposure to impulse noise.

The recovery pattern of threshold shift (TS) and 'dynamic' changes in the stereocilia after exposure to high-level impulse noise in guinea pigs were investigated. 33 albino guinea pigs were exposed to 10 impulse noises at the rate of 1/min. The noise had peak level of 166 dB SPL and a duration of 0.1 ms. Thirteen of the exposed animals were used to systematically measure threshold shifts at regular intervals from 0.5 h to 30 days post-exposure by click auditory cortex evoked response (AC-ER). Twelve of the animals who had typical TS at the same intervals were killed for scanning electron microscopic examination. The recovery pattern of threshold shifts was non-monotonic, which was different from that seen with continuous noise. There was an increase in TS after the exposure and the maximum level of TS was found at 8 h post-exposure. Morphological analysis also showed delayed changes in the stereocilia after exposure. The severity of changes in the stereocilia reached a peak at 8 h, at which time complete fusion of hair bundles took place. The tectorial material found on the tips of the stereocilia may be responsible for the sequence of the changes.

Rhopalium development in Aurelia aurita ephyrae.

Rhopalia of developing ephyrae were examined using the SEM and TEM at 24 h intervals following strobilation induction. Kinocilia are shorter in the ephyra stage than in polyps. A few ephyra-type kinocilia are found in rhopalia as early as 24 h after induction, before a distinct rhopalium is seen. By 72 h, the shorter kinocilia predominate and are almost as numerous as in ephyrae (120 h). Many of the kinocilia are associated with mechanoreceptor cells (MR) found in the rhopalia. These MR cells are compared to those reported for medusae. Although newly released ephyrae lack a touch plate, the MR cells in their rhopalia along with the statocyst and neuromuscular system apparently enable these organisms to detect and respond to gravity.

Electron microscopic observation of communication between inner ear stereocilia under normal and noise stimulated conditions.

The ultrastructure of communication between inner ear stereocilia under normal and noise stimulated conditions was studied in the guinea pig. With ruthenium red added to the pre- and post fixation solutions, the connections between the stereocilia could be observed in more detail than with ordinary fixation. After exposure to 1,000 Hz 110 dBSPL sound for 3 h, parts of the connections between the stereocilia had disappeared, and adherence between the adjoining stereocilia was observed. The results of our study lend support to the fact that the congregation of stereocilia after noise stimulation is due to the sticky adjoining stereocilia membrane.

Effect of acoustic overstimulation on the glycocalyx and the ciliary interconnections in the organ of Corti: high resolution scanning electron microscopic investigation.

Changes in ciliary interconnections in the organ of Corti are described after acoustic overstimulation using a special high resolution scanning electron microscope and tannic acid-osmium staining technique, giving an almost three dimensional view. Guinea pigs were exposed to a 3.85 kHz pure tone at an intensity of 120 dB for 22.5 minutes. The first detectable change was a disarrangement of the cilia with a loosening of the interconnections. The ciliary plasma membrane presented with an abnormally smooth appearance. The tip links connecting the tips of the stereocilia to their taller neighbours were also affected showing elongation or even disappearance. The fine granules which cover the tips of the tallest stereocilia of the outer hair cells were decreased. These findings suggest that acoustic overstimulation may affect the carbohydrate metabolism exceeding to degeneration of ciliary interconnections resulting in a disarrangement and detachment of cilia. The tip links, which may participate in sensory cell transduction, seem also to be affected by acoustic overstimulation.