Role for a Novel Usher Protein Complex in Hair Cell Synaptic Maturation

The molecular mechanisms underlying hair cell synaptic maturation are not well understood. Cadherin-23 (CDH23), protocadherin-15 (PCDH15) and the very large G-protein coupled receptor 1 (VLGR1) have been implicated in the development of cochlear hair cell stereocilia, while clarin-1 has been suggested to also play a role in synaptogenesis. Mutations in CDH23, PCDH15, VLGR1 and clarin-1 cause Usher syndrome, characterized by congenital deafness, vestibular dysfunction and retinitis pigmentosa. Here we show developmental expression of these Usher proteins in afferent spiral ganglion neurons and hair cell synapses. We identify a novel synaptic Usher complex comprised of clarin-1 and specific isoforms of CDH23, PCDH15 and VLGR1. To establish the in vivo relevance of this complex, we performed morphological and quantitative analysis of the neuronal fibers and their synapses in the Clrn1−/− mouse, which was generated by incomplete deletion of the gene. These mice showed a delay in neuronal/synaptic maturation by both immunostaining and electron microscopy. Analysis of the ribbon synapses in Ames waltzerav3J mice also suggests a delay in hair cell synaptogenesis. Collectively, these results show that, in addition to the well documented role for Usher proteins in stereocilia development, Usher protein complexes comprised of specific protein isoforms likely function in synaptic maturation as well

Influencia del estrés sobre la regulación de la actividad 11ß-hidroxiesteroide deshidrogenasa renal

Este trabajo pretende dilucidar algunos de los mecanismos mediados por el estrés y por los glucocorticoides, sobre la actividad de la llβ-hidroxiesteroide deshidrogenasa 2 (11βHSD2). Con este fin se obtuvieron los siguientes aportes: La presencia de la glándula adrenal intacta permite que el animal responda, ante una situación de estrés, con un aumento en la actividad y abundancia de la 11βHSD2 renal; produciéndose un aumento en el catabolismo de los glucocorticoides circulantes. En ausencia de la glándula adrenal, la respuesta del organismo difiere dependiendo de la situación de estrés ensayada. Para el caso de las ratas sometidas a una sobrecarga simulada, existe un incremento de la actividad y abundancia de la 11βHSD2 renal, mientras que, los animales sometidos a una acidosis metabólica, sólo evidencian un incremento en la actividad enzimática con la administración de glucocorticoides. Este incremento se produce sin un aumento en la expresión de la enzima. La administración aguda de corticosterona a ratas adrenalectomizadas, produjo un aumento progresivo de la actividad y abundancia enzimática, hasta la dosis fisiológica de 68 μg de corticosterona/ 100 g de peso corporal. La administración crónica de glucocorticoides, sólo produjo un incremento en la actividad y abundancia de la 11βHSD2, con la dosis suprafisiológica de 308 μg de corticosterona/100 g peso corporal. Existe un incremento progresivo de la actividad enzimática a medida que aumentan los niveles de glucocorticoides circulantes, hasta una cierta concentración (concentración umbral) a partir de la cual la actividad enzimática se vuelve independiente de los niveles de corticosterona. Esta falta de correlación entre los niveles de corticosterona y la actividad enzimática, junto con el aumento de actividad observado en animales adrenalectomizados estresados llevó a plantear la posibilidad de la existencia de algún factor extra-adrenal involucrado en la regulación de la actividad de la 11βHSD2. La linea celular MDCK, derivada de riñón distal de perro posee actividad 11β-deshidrogenasa. La serotonina, posible factor involucrado en la respuesta de la 11βHSD2 frente al estrés, estimula la actividad 11βHSD2 en esta línea celular. Esta estimulación depende de la concentración de serotonina utilizada y de la forma de administración. Además, serotonina muestra un efecto multifásico sobre la estimulación de la actividad enzimática. La acción de serotonina sobre las células MDCK, estaría mediada por receptores 5HT1Dα

Adrenal gland involvement in the regulation of renal 11beta-hydroxysteroid dehydrogenase 2

Renal 11beta-hydroxysteroid dehydrogenase 2 (HSD2) catalyzes the conversion of active glucocorticoids to inert 11beta-keto compounds, thereby preventing the illicit binding of these hormones to mineralocorticoid receptors (MRs) and, thus, conferring aldosterone specificity. Absence or inhibition of HSD2 activity, originates a hypertensive syndrome with sodium retention and increased potassium elimination. Recent studies from our laboratory reported an increment of HSD2 activity in intact-stressed rats. To evaluate the adrenal involvement in this increase, we analyzed HSD2 activity and protein abundance in Intact, Sham-operated, and adrenalectomized rats under stress situations (gavage with an overload of 200 mM HCl (10 ml) and simulated gavage) or with corticosterone replacement. HSD2 activity was assessed in renal microsomal preparations obtained from different groups of animals. HSD2 protein abundance was measured by Western-blot. Circulating corticosterone was determined by radioimmunoassay. Sham-operated animals showed an increase in HSD2 activity and abundance compared to Intact and adrenalectomized rats suggesting the involvement of stress-related adrenal factors in HSD2 regulation. In the case of acidotic adrenalectomized animals, there was an increase in renal HSD2 activity when, along with the HCl overload, the rats were injected with corticosterone. This increment occurred without an increase in enzyme abundance. These results suggest the importance of circulating levels of glucocorticoids to respond to a metabolic acidosis, through regulation of HSD2 stimulation. The group subjected to a simulated gavage showed an increase in enzyme activity and protein abundance, thus demonstrating the need for both adrenal and extra-factors in the modulation of renal HSD2. The adrenalectomized animals injected with different doses of corticosterone, produced a progressive increase in enzyme activity and abundance, being significant for the dose of 68 microg corticosterone/100 g body weight. The highest dose (308 microg/100 g body weight) did not show any variation in activity and abundance compared to the control group. This biphasic effect of glucocorticoids could be explained taking into account their permissive and suppressive actions, depending on their blood levels. Knowing that stress induces multifactorial responses, it should not be surprising to observe a differential regulation in renal HSD2, confirming that different stressors act through different factors of both, adrenal and extra-adrenal origin.Fil: Zallocchi, Marisa Laura. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Prog.de Regulación Hormonal y Metabolica(p); ArgentinaFil: Matkovic, Laura Beatriz. Universidad Catolica de Córdoba. Facultad de Medicina. Departamento de Química Biologica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Prog.de Regulación Hormonal y Metabolica(p); ArgentinaFil: Calvo, Juan Carlos. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Biología y Medicina Experimental. Fundación de Instituto de Biología y Medicina Experimental. Instituto de Biología y Medicina Experimental; ArgentinaFil: Damasco, Maria Cristina. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Prog.de Regulación Hormonal y Metabolica(p); Argentin

Moderate Light-Induced Degeneration of Rod Photoreceptors with Delayed Transducin Translocation in shaker1 Mice

Rod photoreceptors in shaker1 mice, a USH1B mouse model, show that delayed transducin translocation and moderate light exposure can induce their degeneration. These findings reveal that, contrary to earlier studies, shaker1 mice possess a robust retinal phenotype that may link to defective rod protein translocation and suggest that USH1B animal models are therefore likely vulnerable to light-induced photoreceptor damage, even under moderate light

Characterization of quinoxaline derivatives for protection against iatrogenically induced hearing loss

Hair cell loss is the leading cause of hearing and balance disorders in humans. It can be caused by many factors, including noise, aging, and therapeutic agents. Previous studies have shown the therapeutic potential of quinoxaline against drug-induced ototoxicity. Here, we screened a library of 68 quinoxaline derivatives for protection against aminoglycoside-induced damage of hair cells from the zebrafish lateral line. We identified quinoxaline-5-carboxylic acid (Qx28) as the best quinoxaline derivative that provides robust protection against both aminoglycosides and cisplatin in zebrafish and mouse cochlear explants. FM1-43 and aminoglycoside uptake, as well as antibiotic efficacy studies, revealed that Qx28 is neither blocking the mechanotransduction channels nor interfering with aminoglycoside antibacterial activity, suggesting that it may be protecting the hair cells by directly counteracting the ototoxin’s mechanism of action. Only when animals were incubated with higher doses of Qx28 did we observe a partial blockage of the mechanotransduction channels. Finally, we assessed the regulation of the NF-κB pathway in vitro in mouse embryonic fibroblasts and in vivo in zebrafish larvae. Those studies showed that Qx28 protects hair cells by blocking NF-κB canonical pathway activation. Thus, Qx28 is a promising and versatile otoprotectant that can act across different species and toxins

Light-induced translocation of RGS9-1 and Gβ5L in mouse rod photoreceptors.

The transducin GTPase-accelerating protein complex, which determines the photoresponse duration of photoreceptors, is composed of RGS9-1, Gβ5L and R9AP. Here we report that RGS9-1 and Gβ5L change their distribution in rods during light/dark adaptation. Upon prolonged dark adaptation, RGS9-1 and Gβ5L are primarily located in rod inner segments. But very dim-light exposure quickly translocates them to the outer segments. In contrast, their anchor protein R9AP remains in the outer segment at all times. In the dark, Gβ5L's interaction with R9AP decreases significantly and RGS9-1 is phosphorylated at S(475) to a significant degree. Dim light exposure leads to quick de-phosphorylation of RGS9-1. Furthermore, after prolonged dark adaptation, RGS9-1 and transducin Gα are located in different cellular compartments. These results suggest a previously unappreciated mechanism by which prolonged dark adaptation leads to increased light sensitivity in rods by dissociating RGS9-1 from R9AP and redistributing it to rod inner segments

Usher proteins are present at the apical and basal aspects of P3 hair cells.

<p>Isolated hair cells were immunostained for the Usher proteins (green) and myosin7A (magenta) and counter-stained with phalloidin (red). Arrowheads: Usher staining at the base of the hair cells. Asterisks: apical staining and co-localization with phalloidin. <b>A a′, a″</b>: CDH23. <b>B, b′, b″:</b> VLGR1, <b>C, c′, c″:</b> PCDH15, <b>D, d′, d″:</b> clarin-1. Scale bar: <b>A–D:</b> 2 µm; <b>a′–d″:</b> 4 µm.</p

Clrn1−/− mouse has immature synaptic contacts.

<p>P3 (<b>A–D</b>), P9 (<b>E–H</b>) and P14 (<b>I–L</b>) IHC ribbon synapses immunostained for the pre-synaptic marker RIBEYE (red) and the post-synaptic marker GluR2/3 (green). Bar graph: Quantitative analysis of the ribbons in WT and <i>Clrn1−/−</i> IHCs. Student's t test shows significant differences at P3 only (asterisk). <b>M–P:</b> Ultrastructural analysis of WT (<b>M, O</b>) and <i>Clrn1−/−</i> (<b>N, P</b>) P9 synapses at the base of OHCs (<b>M–N</b>) and IHCs (<b>O–P</b>). Small arrows denote synaptic contacts between hair cells and neuronal fibers. Insets: magnification of pointed area. N: nucleus. nf: neuronal fiber.. Scale bars. <b>A–L:</b> 2 µm, <b>N–Q</b>: 500 nm.</p