Usher syndrome proteins ADGRV1 (USH2C) and CIB2 (USH1J) interact and share a common interactome containing TRiC/CCT-BBS chaperonins.

The human Usher syndrome (USH) is the most common form of a sensory hereditary ciliopathy characterized by progressive vision and hearing loss. Mutations in the genes ADGRV1 and CIB2 have been associated with two distinct sub-types of USH, namely, USH2C and USH1J. The proteins encoded by the two genes belong to very distinct protein families: the adhesion G protein-coupled receptor ADGRV1 also known as the very large G protein-coupled receptor 1 (VLGR1) and the Ca2+- and integrin-binding protein 2 (CIB2), respectively. In the absence of tangible knowledge of the molecular function of ADGRV1 and CIB2, pathomechanisms underlying USH2C and USH1J are still unknown. Here, we aimed to enlighten the cellular functions of CIB2 and ADGRV1 by the identification of interacting proteins, a knowledge that is commonly indicative of cellular functions. Applying affinity proteomics by tandem affinity purification in combination with mass spectrometry, we identified novel potential binding partners of the CIB2 protein and compared these with the data set we previously obtained for ADGRV1. Surprisingly, the interactomes of both USH proteins showed a high degree of overlap indicating their integration in common networks, cellular pathways and functional modules which we confirmed by GO term analysis. Validation of protein interactions revealed that ADGRV1 and CIB2 mutually interact. In addition, we showed that the USH proteins also interact with the TRiC/CCT chaperonin complex and the Bardet Biedl syndrome (BBS) chaperonin-like proteins. Immunohistochemistry on retinal sections demonstrated the co-localization of the interacting partners at the photoreceptor cilia, supporting the role of USH proteins ADGRV1 and CIB2 in primary cilia function. The interconnection of protein networks involved in the pathogenesis of both syndromic retinal dystrophies BBS and USH suggest shared pathomechanisms for both syndromes on the molecular level.

The adhesion G protein-coupled receptor VLGR1/ADGRV1 controls autophagy.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest known adhesion G protein-coupled receptor. Mutations in VLGR1/ADGRV1 cause Usher syndrome (USH), the most common form of hereditary deaf-blindness, and have been additionally linked to epilepsy. Although VLGR1/ADGRV1 is almost ubiquitously expressed, little is known about the subcellular function and signalling of the VLGR1 protein and thus about mechanisms underlying the development of diseases. Using affinity proteomics, we identified key components of autophagosomes as putative interacting proteins of VLGR1. In addition, whole transcriptome sequencing of the retinae of the Vlgr1/del7TM mouse model revealed altered expression profiles of gene-related autophagy. Monitoring autophagy by immunoblotting and immunocytochemistry of the LC3 and p62 as autophagy marker proteins revealed evoked autophagy in VLGR1-deficient hTERT-RPE1 cells and USH2C patient-derived fibroblasts. Our data demonstrate the molecular and functional interaction of VLGR1 with key components of the autophagy process and point to an essential role of VLGR1 in the regulation of autophagy at internal membranes. The close association of VLGR1 with autophagy helps to explain the pathomechanisms underlying human USH and epilepsy related to VLGR1 defects.

Monitoring paxillin in astrocytes reveals the significance of the adhesion G protein coupled receptor VLGR1/ADGRV1 for focal adhesion assembly.

VLGR1/ADGRV1 (very large G protein-coupled receptor-1) is the largest adhesion G protein-coupled receptor (aGPCR). Mutations in VLGR1/ADGRV1 are associated with human Usher syndrome, the most common form of deaf-blindness, and also with epilepsy in humans and mice. VLGR1 is expressed almost ubiquitously but is mainly found in the CNS and in the sensory cells of the eye and inner ear. Little is known about the pathogenesis of the diseases related to VLGR1. We previously identified VLGR1 as a vital component of focal adhesions (FAs) serving as a metabotropic mechanoreceptor controls cell spreading and migration. FAs are highly dynamic and turnover in response to internal and external signals. Here, we aimed to elucidate how VLGR1 participates in FA turnover. Nocodazole washouts and live cell imaging of paxillin-DsRed2 consistently showed that FA disassembly was not altered, but de novo assembly of FA was significantly delayed in Vlgr1-deficient astrocytes, indicating that VLGR1 is enrolled in FA assembly. In FRAP experiments, recovery rates were significantly reduced in Vlgr1-deficient FAs, indicating reduced turnover kinetics in VLGR1-deficient FAs. We showed that VLGR1 regulates cell migration by controlling the FA turnover during their assembly and expect novel insights into pathomechanisms related to pathogenic dysfunctions of VLGR1.

The Usher syndrome 1C protein harmonin regulates canonical Wnt signaling.

Human Usher syndrome (USH) is the most common form of hereditary combined deaf-blindness. USH is a complex genetic disorder, and the pathomechanisms underlying the disease are far from being understood, especially in the eye and retina. The USH1C gene encodes the scaffold protein harmonin which organizes protein networks due to binary interactions with other proteins, such as all USH proteins. Interestingly, only the retina and inner ear show a disease-related phenotype, although USH1C/harmonin is almost ubiquitously expressed in the human body and upregulated in colorectal cancer. We show that harmonin binds to ß-catenin, the key effector of the canonical Wnt (cWnt) signaling pathway. We also demonstrate the interaction of the scaffold protein USH1C/harmonin with the stabilized acetylated ß-catenin, especially in nuclei. In HEK293T cells, overexpression of USH1C/harmonin significantly reduced cWnt signaling, but a USH1C-R31* mutated form did not. Concordantly, we observed an increase in cWnt signaling in dermal fibroblasts derived from an USH1C R31*/R80Pfs*69 patient compared with healthy donor cells. RNAseq analysis reveals that both the expression of genes related to the cWnt signaling pathway and cWnt target genes were significantly altered in USH1C patient-derived fibroblasts compared to healthy donor cells. Finally, we show that the altered cWnt signaling was reverted in USH1C patient fibroblast cells by the application of Ataluren, a small molecule suitable to induce translational read-through of nonsense mutations, hereby restoring some USH1C expression. Our results demonstrate a cWnt signaling phenotype in USH establishing USH1C/harmonin as a suppressor of the cWnt/ß-catenin pathway.

The Adhesion GPCR VLGR1/ADGRV1 Regulates the Ca2+ Homeostasis at Mitochondria-Associated ER Membranes.

The very large G protein-coupled receptor (VLGR1, ADGRV1) is the largest member of the adhesion GPCR family. Mutations in VLGR1 have been associated with the human Usher syndrome (USH), the most common form of inherited deaf-blindness as well as childhood absence epilepsy. VLGR1 was previously found as membrane-membrane adhesion complexes and focal adhesions. Affinity proteomics revealed that in the interactome of VLGR1, molecules are enriched that are associated with both the ER and mitochondria, as well as mitochondria-associated ER membranes (MAMs), a compartment at the contact sites of both organelles. We confirmed the interaction of VLGR1 with key proteins of MAMs by pull-down assays in vitro complemented by in situ proximity ligation assays in cells. Immunocytochemistry by light and electron microscopy demonstrated the localization of VLGR1 in MAMs. The absence of VLGR1 in tissues and cells derived from VLGR1-deficient mouse models resulted in alterations in the MAM architecture and in the dysregulation of the Ca2+ transient from ER to mitochondria. Our data demonstrate the molecular and functional interaction of VLGR1 with components in MAMs and point to an essential role of VLGR1 in the regulation of Ca2+ homeostasis, one of the key functions of MAMs.

Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes.

Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.

Phylogenetic profiling and cellular analyses of ARL16 reveal roles in traffic of IFT140 and INPP5E.

The ARF family of regulatory GTPases is ancient, with 16 members predicted to have been present in the last eukaryotic common ancestor. Our phylogenetic profiling of paralogues in diverse species identified four family members whose presence correlates with that of a cilium/flagellum: ARL3, ARL6, ARL13, and ARL16. No prior evidence links ARL16 to cilia or other cell functions, despite its presence throughout eukaryotes. Deletion of ARL16 in mouse embryonic fibroblasts (MEFs) results in decreased ciliogenesis yet increased ciliary length. We also found Arl16 knockout (KO) in MEFs to alter ciliary protein content, including loss of ARL13B, ARL3, INPP5E, and the IFT-A core component IFT140. Instead, both INPP5E and IFT140 accumulate at the Golgi in Arl16 KO lines, while other intraflagellar transport (IFT) proteins do not, suggesting a specific defect in traffic from Golgi to cilia. We propose that ARL16 regulates a Golgi-cilia traffic pathway and is required specifically in the export of IFT140 and INPP5E from the Golgi.

The ARF GAPs ELMOD1 and ELMOD3 act at the Golgi and cilia to regulate ciliogenesis and ciliary protein traffic.

ELMODs are a family of three mammalian paralogues that display GTPase-activating protein (GAP) activity toward a uniquely broad array of ADP-ribosylation factor (ARF) family GTPases that includes ARF-like (ARL) proteins. ELMODs are ubiquitously expressed in mammalian tissues, highly conserved across eukaryotes, and ancient in origin, being present in the last eukaryotic common ancestor. We described functions of ELMOD2 in immortalized mouse embryonic fibroblasts (MEFs) in the regulation of cell division, microtubules, ciliogenesis, and mitochondrial fusion. Here, using similar strategies with the paralogues ELMOD1 and ELMOD3, we identify novel functions and locations of these cell regulators and compare them to those of ELMOD2, allowing the determination of functional redundancy among the family members. We found strong similarities in phenotypes resulting from deletion of either Elmod1 or Elmod3 and marked differences from those arising in Elmod2 deletion lines. Deletion of either Elmod1 or Elmod3 results in the decreased ability of cells to form primary cilia, loss of a subset of proteins from cilia, and accumulation of some ciliary proteins at the Golgi, predicted to result from compromised traffic from the Golgi to cilia. These phenotypes are reversed upon activating mutant expression of either ARL3 or ARL16, linking their roles to ELMOD1/3 actions.

Roles for ELMOD2 and Rootletin in ciliogenesis.

ELMOD2 is a GTPase-activating protein with uniquely broad specificity for ARF family GTPases. We previously showed that it acts with ARL2 in mitochondrial fusion and microtubule stability and with ARF6 during cytokinesis. Mouse embryonic fibroblasts deleted for ELMOD2 also displayed changes in cilia-related processes including increased ciliation, multiciliation, ciliary morphology, ciliary signaling, centrin accumulation inside cilia, and loss of rootlets at centrosomes with loss of centrosome cohesion. Increasing ARL2 activity or overexpressing Rootletin reversed these defects, revealing close functional links between the three proteins. This was further supported by the findings that deletion of Rootletin yielded similar phenotypes, which were rescued upon increasing ARL2 activity but not ELMOD2 overexpression. Thus, we propose that ARL2, ELMOD2, and Rootletin all act in a common pathway that suppresses spurious ciliation and maintains centrosome cohesion. Screening a number of markers of steps in the ciliation pathway supports a model in which ELMOD2, Rootletin, and ARL2 act downstream of TTBK2 and upstream of CP110 to prevent spurious release of CP110 and to regulate ciliary vesicle docking. These data thus provide evidence supporting roles for ELMOD2, Rootletin, and ARL2 in the regulation of ciliary licensing.

Ataluren for the Treatment of Usher Syndrome 2A Caused by Nonsense Mutations.

The identification of genetic defects that underlie inherited retinal diseases (IRDs) paves the way for the development of therapeutic strategies. Nonsense mutations caused approximately 12% of all IRD cases, resulting in a premature termination codon (PTC). Therefore, an approach that targets nonsense mutations could be a promising pharmacogenetic strategy for the treatment of IRDs. Small molecules (translational read-through inducing drugs; TRIDs) have the potential to mediate the read-through of nonsense mutations by inducing expression of the full-length protein. We provide novel data on the read-through efficacy of Ataluren on a nonsense mutation in the Usher syndrome gene USH2A that causes deaf-blindness in humans. We demonstrate Ataluren´s efficacy in both transiently USH2AG3142*-transfected HEK293T cells and patient-derived fibroblasts by restoring USH2A protein expression. Furthermore, we observed enhanced ciliogenesis in patient-derived fibroblasts after treatment with TRIDs, thereby restoring a phenotype that is similar to that found in healthy donors. In light of recent findings, we validated Ataluren´s efficacy to induce read-through on a nonsense mutation in USH2A-related IRD. In line with published data, our findings support the use of patient-derived fibroblasts as a platform for the validation of preclinical therapies. The excellent biocompatibility combined with sustained read-through efficacy makes Ataluren an ideal TRID for treating nonsense mutations based IRDs.