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

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

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

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

Clrn1−/− mouse has immature type I afferent fibers.

<p>P6 WT (<b>A, </b><b>C, E, G</b>) or <i>Clrn1−/−</i> (<b>B, D, F, H</b>) whole mount organ of Corti (<b>A–B</b>) or cochlea cross-sections (<b>C–H</b>) were labeled with the neuronal tracer (<b>A–D</b>, red) and counter-stained with phalloidin for F-actin (green, <b>C–D</b>) or with peripherin (pph, green, <b>E–H</b>). Arrowheads: specific staining at the base of the OHCs. Scale bars: <b>A–B</b>: 20 µm, <b>C–D</b>: 10 µm, <b>E–F</b>: 15 µm and <b>G–H</b>: 6 µm.</p

Isoform specificity by transient knockdown and antibody pre-adsorption.

<p><b>Panel A:</b> Western blot analysis showing different degrees of knockdown for each isoform of the three different Usher proteins. Blots were stripped and re-probed for β-actin as a loading control. Red numbers are the percentage of expression relative to the scrambled and after normalization with β-actin. Isoforms were named according to previously establish nomenclature systems. Molecular protein standards are indicated to the left of each membrane. Sc: scrambled; Sp: specific siRNA, nd: no-detectable. <b>Panel B:</b> Western blot analysis of whole P3 inner ear (EAR) and neuroretina (EYE) in the presence of the affinity purify antibody (A–P) or the pre-adsorbed antibody (F-Th) by the corresponding peptide immunogen. Membranes were stripped and immunoblotted for β-actin as loading control. For CDH23, V1 corresponds to V1a and V1b, V2 corresponds to V2a and V2b, V3 corresponds to V3a and V3b and V5 corresponds to V5a and V5b.</p

Laminin α2-Mediated Focal Adhesion Kinase Activation Triggers Alport Glomerular Pathogenesis

<div><p>It has been known for some time that laminins containing α1 and α2 chains, which are normally restricted to the mesangial matrix, accumulate in the glomerular basement membranes (GBM) of Alport mice, dogs, and humans. We show that laminins containing the α2 chain, but not those containing the α1 chain activates focal adhesion kinase (FAK) on glomerular podocytes <i>in vitro</i> and <i>in vivo</i>. CD151-null mice, which have weakened podocyte adhesion to the GBM rendering these mice more susceptible to biomechanical strain in the glomerulus, also show progressive accumulation of α2 laminins in the GBM, and podocyte FAK activation. Analysis of glomerular mRNA from both models demonstrates significant induction of MMP-9, MMP-10, MMP-12, MMPs linked to GBM destruction in Alport disease models, as well as the pro-inflammatory cytokine IL-6. SiRNA knockdown of FAK in cultured podocytes significantly reduced expression of MMP-9, MMP-10 and IL-6, but not MMP-12. Treatment of Alport mice with TAE226, a small molecule inhibitor of FAK activation, ameliorated fibrosis and glomerulosclerosis, significantly reduced proteinuria and blood urea nitrogen levels, and partially restored GBM ultrastructure. Glomerular expression of MMP-9, MMP-10 and MMP-12 mRNAs was significantly reduced in TAE226 treated animals. Collectively, this work identifies laminin α2-mediated FAK activation in podocytes as an important early event in Alport glomerular pathogenesis and suggests that FAK inhibitors, if safe formulations can be developed, might be employed as a novel therapeutic approach for treating Alport renal disease in its early stages.</p></div

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

Laminin α2, but not laminin α1 activates FAK on podocytes <i>in vivo</i> and <i>in vitro</i>.

<p>Panels A–C; 7 week old wild type glomerulus stained with antibodies specific for laminin 111 and pFAK³⁹⁷ show absence of pFAK immunostaining. Panels D–F; 7 week Alport glomerulus stained with antibodies specific for laminin α1 and pFAK³⁹⁷ pFAK immunostaining in podocytes adjacent to laminin α1-immunopositive GBM. Panels G-I show the same immunostaining as for D–F using Alport mice that do not express laminin α2 (the dy/dy muscular dystrophy mutation). Note the absence of pFAK³⁹⁷ immunostaining even though GBM is immunopositive for laminin α1. Panel J. Wild type podocytes were differentiated for 2 weeks and then plated on placental laminin, EHS laminin, or merosin for 15 hours. Extracts were prepared and analyzed by western blot for expression of pFAK³⁹⁷ and total FAK. β-actin was used as a loading control). Panel K shows quantitative analysis of pFAK397 relative to total FAK for several western blots. Panel L shows real time qRT-PCR results for transcripts endocing the indicated MMPs, demonstrating significantly elevated expression of MMP-9 and MMP-10 for cells cultured on merosin (MERO) relative to cells cultured on placental laminin (PLAM). Scale bar = 10 µm.</p

Usher protein expression by type II afferent neurons and SGN cell bodies.

<p><b>Panel A:</b> P3 cochlea cross-sections dual-immunostained for peripherin (pph, green; <b>A–H</b>) and CDH23 (<b>B</b>), PCDH15 (<b>D</b>), VLGR1 (<b>F</b>) or clarin-1 (<b>H</b>) (red). Arrowheads: co-localization at the neuronal terminals. Scale bar: 5 µm. <b>Panel B:</b> P3 SGN cross-sections dual-immunostained with peripherin (green) and CDH23 (<b>I</b>), PCDH15 (<b>J</b>); VLGR1 (<b>K</b>) or clarin-1 (<b>L</b>) (red). Scale bar: 6 µm.</p