Universiẗat, Diss., 2003--München.

Thesis--Wisconsin. / Vita. Includes bibliographical references (leaves 129-135).

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Die Maus mit progressiver motorischer Neuropathie (PMN) ist ein Modell für eine vererbte motorische Neuropathie mit progressiver Neurodegeneration. Die Degeneration der Axone geht mit homozygoten Mutationen des TBCE- Gens einher, das für das Tubulin-Chaperon-E- Protein kodiert. TBCE ist für die korrekte Dimerisierung von Alpha- und Beta-Tubulin verantwortlich. Auffallend ist, dass die PMN-Maus nach dem normalen Beginn des Hörens auch einen progressiven Hörverlust entwickelt, der durch die Degeneration des Hörnervs und den Verlust der äußeren Haarzellen (OHC) gekennzeichnet ist. Die Entwicklung dieser neuronalen und cochleären Pathologie ist Wirkung von peg-IGF-1 auf das auditorische System durch Behandlung ab dem 15. postnatalen Tag (p15). Die histologische Analyse ergab positive Auswirkungen auf die OHC-Synapsen der medialen olivocochleären (MOC) neuronalen Fasern und eine kurzfristige Abschwächung des OHC-Verlustes. Peg-IGF-1 war in der Lage, die Desorganisation der OHC-Synapsen bedingt wiederherzustellen und die Bereitstellung von cholinerger Acetyltransferase in den Präsynapsen aufrechtzuerhalten. Zur Beurteilung der auditorischen Funktion wurden frequenzspezifische Hirnstammreaktionen und otoakustische Emissionen mit Verzerrungsprodukten bei Tieren mit p21 und p28 aufgezeichnet. Trotz der positiven Auswirkungen auf die MOC-Fasern und die OHC konnte jedoch keine Wiederherstellung des Hörvermögens erreicht werden. Die vorliegende Arbeit zeigt, dass die synaptische Pathologie der efferenten MOC-Fasern in PMN-Mäusen eine besondere Form der "efferenten auditorischen Neuropathie" darstellt. Peg-IGF-1 zeigte eine otoprotektive Wirkung, indem es die Degeneration von OHCs und efferenten Synapsen verhinderte. Es sind jedoch verstärkte Anstrengungen zur Optimierung der Behandlung erforderlich, um nachweisbare Verbesserungen der Hörleistung zu erzielen. / The progressive motor neuropathy (PMN) mouse is a model of an inherited motor neuropathy disease with progressive neurodegeneration. Axon degeneration associates with homozygous mutations of the TBCE gene encoding the tubulin chaperone E protein. TBCE is responsible for the correct dimerization of alpha and beta-tubulin. Strikingly, the PMN mouse also develops a progressive hearing loss after normal hearing onset, characterized by degeneration of the auditory nerve and outer hair cell (OHC) loss. However, the development of this neuronal and cochlear pathology is not fully understood yet. Previous studies with pegylated insulin-like growth factor 1 (peg-IGF-1) treatment in this mouse model have been shown to expand lifespan, weight, muscle strength, and motor coordination. Accordingly, peg-IGF-1 was evaluated for an otoprotective effect. We investigated the effect of peg-IGF-1 on the auditory system by treatment starting at postnatal day 15 (p15). Histological analysis revealed positive effects on OHC synapses of medial olivocochlear (MOC) neuronal fibers and a short-term attenuation of OHC loss. Peg-IGF-1 was able to conditionally restore the disorganization of OHC synapses and maintain the provision of cholinergic acetyltransferase in presynapses. To assess auditory function, frequency-specific auditory brainstem responses and distortion product otoacoustic emissions were recorded in animals on p21 and p28. However, despite the positive effect on MOC fibers and OHC, no restoration of hearing could be achieved. The present work demonstrates that the synaptic pathology of efferent MOC fibers in PMN mice represents a particular form of “efferent auditory neuropathy.” Peg-IGF-1 showed an otoprotective effect by preventing the degeneration of OHCs and efferent synapses. However, enhanced efforts are needed to optimize the treatment to obtain detectable improvements in hearing performances.

[Truncated abstract] The cochlea presumably possesses a number of regulatory mechanisms to maintain cochlear sensitivity in the face of disturbances to its function. Evidence for such mechanisms can be found in the time-course of the recovery of CAP thresholds during experimental manipulations, and in observations of slow oscillations in cochlear micromechanics following exposure to low-frequency tones (the “bounce phenomenon”) and other perturbations. To increase our understanding of these oscillatory processes within the cochlea, and OHCs in particular, investigations into cochlear regulation were carried out using a combination of mathematical modelling of the ionic and mechanical interactions likely to exist within the OHCs, and electrophysiological experiments conducted in guinea pigs. The electrophysiological experiments consisted of electrocochleographic recordings and, in some cases, measurement of otoacoustic emissions, during a variety of experimental perturbations, including the application of force to the cochlear wall, exposure to very-low-frequency tones, injection of direct current into scala tympani, and intracochlear perfusions of artificial perilymph containing altered concentrations of potassium, sodium, and sucrose. To obtain a panoramic view of cochlear regulation under these conditions, software was written to enable the interleaved and near-simultaneous measurement of multiple indicators of cochlear function, including the compound action potential (CAP) threshold, amplitude and waveshape at multiple frequencies, the OHC transfer curves derived from low-frequency cochlear microphonic (CM) waveforms, distortion-product otoacoustic emissions (DPOAEs), the spectrum of the round-window neural noise (SNN), and the endocochlear potential (EP). ... The mathematical model we have developed provided a physiologically-plausible and internally-consistent explanation for the time-courses of the cochlear changes observed during a number of different perturbations. We show that much of the oscillatory behaviour within the cochlea is consistent with underlying oscillations in cytosolic calcium concentration. We conclude that a number of the discrepancies between the simulation results and the experimental data can be resolved if the cytosolic calcium functions as two distinct pools: one which controls basolateral permeability and one which controls slow motility. This two-calcium-pool model is discussed.

This thesis presents a novel biomimetic cochlea filter which closely resembles the biological cochlea behaviour. The filter is highly feasible for analogue very-large-scale integration (VLSI) circuits, which leads to a micro-watt-power and millimetre-sized hardware implementation. By virtue of such features, the presented filter contributes to a solid foundation for future biologically-inspired audio signal processors. Unlike existing works, the presented filter is developed by taking direct inspirations from the physiologically measured results of the biological cochlea. Since the biological cochlea has prominently different characteristics of frequency response from low to high frequencies, the biomimetic cochlea filter is built by cascading three sub-filters accordingly: a 2nd-order bandpass filter for the constant gentle low-frequency response, a 2nd-order tunable low-pass filter for the variable and selective centre frequency response and a 5th-order elliptic filter for the ultra-steep roll-off at stop-band. As a proof of concept, a biomimetic cochlea filter bank is built to process audio signals, which demonstrates the highly discriminative spectral decomposition and high-resolution time-frequency analysis capabilities similar to the biological cochlea. The filter has simple representation in the Laplace domain which leads to a convenient analogue circuit realisation. A floating-active-inductor circuit cell is developed to build the corresponding RLC ladder for each of the three sub-filters. The circuits are designed based on complementary metal-oxide-semiconductor (CMOS) transistors for VLSI implementation. Non-ideal factors of CMOS transistors including parasitics, noise and mismatches are extensively analysed and consciously considered in the circuit design. An analogue VLSI chip is successfully fabricated using 0.35μ m CMOS process. The chip measurements demonstrate that the centre frequency response of the filter has about 20 dB wide gain tuning range and a high quality factor reaching maximally over 19. The filter has a 20 dB/decade constant gentle low-frequency tail and an over 300 dB/decade sharp stop-band roll-off slope. The measured results agree with the filter model expectations and are comparable with the biological cochlea characteristics. Each filter channel consumes as low as 59.5 ~90μ Wpower and occupies only 0.9 mm2 area. Besides, the biomimetic cochlea filter chip is characterised from a wide range of angles and the experimental results cover not only the auditory filter specifications but also the integrated circuit design considerations. Furthermore, following the progressive development of the acoustic resonator based on microelectro- mechanical systems (MEMS) technology, a MEMS-CMOS implementation of the proposed filter becomes possible in the future. A key challenge for such implementation is the low sensing capacitance of the MEMS resonator which suffers significantly from sensitivity degradation due to the parasitic capacitance. A novel MEMS capacitive interface circuit chip is additionally developed to solve this issue. As shown in the chip results, the interface circuit is able to cancel the parasitic capacitance and increase the sensitivity of capacitive sensors by 35 dB without consuming any extra power. Besides, the chopper-stabilisation technique is employed which effectively reduces the circuit flicker noise and offsets. Due to these features, the interface circuit chip is capable of converting a 7.5 fF capacitance change of a 1-Volt-biased 0.5 pF capacitive sensor pair into a 0.745 V signal-conditioned output while consuming only 165.2μ W power.

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Outre leur polarité apico-basale, certaines cellules épithéliales développent une seconde polarité, appelée Polarité Planaire Cellulaire (PCP). L'axe de la PCP est orienté perpendiculairement à l'axe de polarité apico-basale et régit l'orientation uniforme de certaines structures, comme les poils ou cils, non seulement à l'échelle de la cellule mais également au sein du tissu. L'épithélium cochléaire est l'un des meilleurs modèles d'étude de PCP chez les mammifères. En effet, les cellules neuro-épitheliales qui le composent, soutenues par des cellules de soutien, présentent à leur apex, des touffes ciliaires dont l'orientation est parfaitement coordonnée par la voie de la polarité planaire. Les deux premiers gènes impliqués dans la PCP chez les mammifères, Vangl2 et Scrib1, ont été identifiés sur la base du phénotype de la cochlée chez les mutants. L'analyse de la localisation de Vangl2 dans l'organe de Corti a également révélé une localisation asymétrique proximo-distale et transitoire de la protéine, perpendiculaire à l'axe apico-basal classique. Cette asymétrie apparaît à la jonction entre deux types cellulaires : une cellule sensorielle ciliée et une cellule de soutien. J'ai pu montrer au cours de mes travaux de thèse que cette asymétrie était majoritairement due à une accumulation de Vangl2 du côté distal des cellules de soutien, et que dans une moindre mesure, Vangl2 pouvait ségréger du côté distal des cellules ciliées. Cette localisation subcellulaire très précise et limitée dans l'espace semble être indépendante de l'expression du gène Scrib1 dans les cellules ciliées. La délétion du gène Scrib1 dans les cellules ciliées m'a toutefois permis de mettre en évidence que ce gène avait un rôle autonome dans la régulation de la PCP, et que les cellules de soutien de l'organe de Corti pouvaient jouer un rôle prépondérant dans le contrôle de la PCP. Mes travaux ont également permis de mettre en évidence que GIPC1 avait un rôle dans la régulation de la PCP et le maintien de l'intégrité des touffes ciliaires des cellules sensorielles, et que le complexe GIPC1/Myosine VI pouvait réguler l'établissement de l'asymétrie de Vangl2 dans l'organe de Corti. / Several epithelia exhibit a second polarity perpendicular to the apico-basal axis, called planar polarity and that governs the orientation of structures such as stereocilia and hear. Our laboratory studies planar polarity, using mammalian cochlear sensory epithelium and we focus our studies on Vangl2, that we identified as the first mammalian planar polarity gene. Vangl2 encodes a four-transmembrane protein that contains a PDZ binding domain in its C-terminus tail. Vangl2 is asymmetrically located at the junction between mechanosensory hair cells and supporting cells, and this asymmetry appears important for planar cell polarity. I have shown in my thesis, using STED microscopy, that Vangl2 asymmetry is mainly due to an accumulation of Vangl2 to the distal side of supporting cells. I sought to dissect the molecular role of Vangl2 by analysing its trafficking within the cochlear epithelium. Deletion analysis shows that the last 12 amino acids, unlike its N-terminus tail are essential for Vangl2 endoplasmic reticulum sorting, its plasma membrane targeting and its function. Conditional mutant mice analysis show that Scrib1, which we have previously shown, interacts with Vangl2 through the PDZ binding domain of its C-terminal tail, is not the protein mediating this asymmetry. My work also highlight that GIPC1 had a role in the regulation of PCP and maintaining the integrity of hair bundles of sensory cells, and that the complex GIPC1/Myosin VI could regulate Vangl2 asymmetry in the organ of Corti.