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

Regulation of the renal 11ß-hydroxysteroid dehydrogenase activity by stress

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

Clarin-1 acts as a modulator of mechanotransduction activity and presynaptic ribbon assembly

Clarin-1 is a four-transmembrane protein expressed by hair cells and photoreceptors. Mutations in its corresponding gene are associated with Usher syndrome type 3, characterized by late-onset and progressive hearing and vision loss in humans. Mice carrying mutations in the clarin-1 gene have hair bundle dysmorphology and a delay in synapse maturation. In this paper, we examined the expression and function of clarin-1 in zebrafish hair cells. We observed protein expression as early as 1 d postfertilization. Knockdown of clarin-1 resulted in inhibition of FM1-43 incorporation, shortening of the kinocilia, and mislocalization of ribeye b clusters. These phenotypes were fully prevented by co-injection with clarin-1 transcript, requiring its C-terminal tail. We also observed an in vivo interaction between clarin-1 and Pcdh15a. Altogether, our results suggest that clarin-1 is functionally important for mechanotransduction channel activity and for proper localization of synaptic components, establishing a critical role for clarin-1 at the apical and basal poles of hair cells.</jats:p

Coordinate roles for Itgα8 and Pcdh15 in the regulation of cilia biogenesis in sensory cells

The organism's perception of its surroundings depends on sensory systems and the highly specialized cilia present in the neurosensory cells. Here, we described the existence of an integrin alpha 8 (Itga8)/protocadherin-15a (Pcdh15a) ciliary complex in neuromast hair cells. Depletion of the complex via down-regulation or loss of function mutations leads to a dysregulation of cilia biogenesis and endocytosis. At the molecular level, removal of the complex blocks the access of Rab8a into the cilia as well as normal recruitment of ciliary cargo by centriolar satellites. These defects can be reversed by the introduction of a constitutively active form of Rhoa, suggesting that Itga8/Pcdh15a complex mediates its effect through the activation of this small-GTPase and probably by the regulation of actin cytoskeleton dynamics. Our data points to a novel mechanism involved in the regulation of sensory cilia development, with the corresponding implications for normal sensory function.</jats:p

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

α1β1 Integrin/Rac1-Dependent Mesangial Invasion of Glomerular Capillaries in Alport Syndrome

Alport syndrome, hereditary glomerulonephritis with hearing loss, results from mutations in type IV collagen COL4A3, COL4A4, or COL4A5 genes. The mechanism for delayed glomerular disease onset is unknown. Comparative analysis of Alport mice and CD151 knockout mice revealed progressive accumulation of laminin 211 in the glomerular basement membrane. We show mesangial processes invading the capillary loops of both models as well as in human Alport glomeruli, as the likely source of this laminin. l-NAME salt–induced hypertension accelerated mesangial cell process invasion. Cultured mesangial cells showed reduced migratory potential when treated with either integrin-linked kinase inhibitor or Rac1 inhibitor, or by deletion of integrin α1. Treatment of Alport mice with Rac1 inhibitor or deletion of integrin α1 reduced mesangial cell process invasion of the glomerular capillary tuft. Laminin α2–deficient Alport mice show reduced mesangial process invasion, and cultured laminin α2–null cells showed reduced migratory potential, indicating a functional role for mesangial laminins in progression of Alport glomerular pathogenesis. Collectively, these findings predict a role for biomechanical insult in the induction of integrin α1β1–dependent Rac1-mediated mesangial cell process invasion of the glomerular capillary tuft as an initiation mechanism of Alport glomerular pathology

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