The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases.

Primary and secondary cone photoreceptor death in retinal degenerative diseases, including age-related macular degeneration (AMD) and retinitis pigmentosa (RP), leads to severe visual impairment and blindness. Although the cone photoreceptor protection in retinal degenerative diseases is crucial for maintaining vision, the underlying molecular mechanisms are unclear. Here, we found that the deubiquitinase Otud7b/Cezanne is predominantly expressed in photoreceptor cells in the retina. We analyzed Otud7b-/- mice, which were subjected to light-induced damage, a dry AMD model, or were mated with an RP mouse model, and observed increased cone photoreceptor degeneration. Using RNA-sequencing and bioinformatics analysis followed by a luciferase reporter assay, we found that Otud7b downregulates NF-κB activity. Furthermore, inhibition of NF-κB attenuated cone photoreceptor degeneration in the light-exposed Otud7b-/- retina and stress-induced neuronal cell death resulting from Otud7b deficiency. Together, our findings suggest that Otud7b protects cone photoreceptors in retinal degenerative diseases by modulating NF-κB activity.

The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina.

Müller glial cells, which are the most predominant glial subtype in the retina, play multiple important roles, including the maintenance of structural integrity, homeostasis, and physiological functions of the retina. We have previously found that the Rax homeoprotein is expressed in postnatal and mature Müller glial cells in the mouse retina. However, the function of Rax in postnatal and mature Müller glial cells remains to be elucidated. In the current study, we first investigated Rax function in retinal development using retroviral lineage analysis and found that Rax controls the specification of late-born retinal cell types, including Müller glial cells in the postnatal retina. We next generated Rax tamoxifen-induced conditional KO (Rax iCKO) mice, where Rax can be depleted in mTFP-labeled Müller glial cells upon tamoxifen treatment, by crossing Raxflox/flox mice with Rlbp1-CreERT2 mice, which we have produced. Immunohistochemical analysis showed a characteristic of reactive gliosis and enhanced gliosis of Müller glial cells in Rax iCKO retinas under normal and stress conditions, respectively. We performed RNA-seq analysis on mTFP-positive cells purified from the Rax iCKO retina and found significantly reduced expression of suppressor of cytokinesignaling-3 (Socs3). Reporter gene assays showed that Rax directly transactivates the Socs3 promoter. We observed decreased expression of Socs3 in Müller glial cells of Rax iCKO retinas by immunostaining. Taken together, the present results suggest that Rax suppresses inflammation in Müller glial cells by transactivating Socs3. This study sheds light on the transcriptional regulatory mechanisms underlying retinal Müller glial cell homeostasis.

The carboxyl-terminal region of SDCCAG8 comprises a functional module essential for cilia formation as well as organ development and homeostasis.

In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8ΔC/ΔC mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8ΔC/ΔC mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8ΔC/ΔC mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein-protein interaction networks involved in cilia development.

Identification of G Protein-Coupled Receptors (GPCRs) in Primary Cilia and Their Possible Involvement in Body Weight Control.

Primary cilia are sensory organelles that harbor various receptors such as G protein-coupled receptors (GPCRs). We analyzed subcellular localization of 138 non-odorant GPCRs. We transfected GPCR expression vectors into NIH3T3 cells, induced ciliogenesis by serum starvation, and observed subcellular localization of GPCRs by immunofluorescent staining. We found that several GPCRs whose ligands are involved in feeding behavior, including prolactin-releasing hormone receptor (PRLHR), neuropeptide FF receptor 1 (NPFFR1), and neuromedin U receptor 1 (NMUR1), localized to the primary cilia. In addition, we found that a short form of dopamine receptor D2 (DRD2S) is efficiently transported to the primary cilia, while a long form of dopamine receptor D2 (DRD2L) is rarely transported to the primary cilia. Using an anti-Prlhr antibody, we found that Prlhr localized to the cilia on the surface of the third ventricle in the vicinity of the hypothalamic periventricular nucleus. We generated the Npy2r-Cre transgenic mouse line in which Cre-recombinase is expressed under the control of the promoter of Npy2r encoding a ciliary GPCR. By mating Npy2r-Cre mice with Ift80 flox mice, we generated Ift80 conditional knockout (CKO) mice in which Npy2r-positive cilia were diminished in number. We found that Ift80 CKO mice exhibited a body weight increase. Our results suggest that Npy2r-positive cilia are important for body weight control.

Presynaptic dystroglycan-pikachurin complex regulates the proper synaptic connection between retinal photoreceptor and bipolar cells.

Dystroglycan (DG) is a key component of the dystrophin-glycoprotein complex (DGC) at the neuromuscular junction postsynapse. In the mouse retina, the DGC is localized at the presynapse of photoreceptor cells, however, the function of presynaptic DGC is poorly understood. Here, we developed and analyzed retinal photoreceptor-specific DG conditional knock-out (DG CKO) mice. We found that the DG CKO retina showed a reduced amplitude and a prolonged implicit time of the ERG b-wave. Electron microscopic analysis revealed that bipolar dendrite invagination into the photoreceptor terminus is perturbed in the DG CKO retina. In the DG CKO retina, pikachurin, a DG ligand in the retina, is markedly decreased at photoreceptor synapses. Interestingly, in the Pikachurin(-/-) retina, the DG signal at the ribbon synaptic terminus was severely reduced, suggesting that pikachurin is required for the presynaptic accumulation of DG at the photoreceptor synaptic terminus, and conversely DG is required for pikachurin accumulation. Furthermore, we found that overexpression of pikachurin induces formation and clustering of a DG-pikachurin complex on the cell surface. The Laminin G repeats of pikachurin, which are critical for its oligomerization and interaction with DG, were essential for the clustering of the DG-pikachurin complex as well. These results suggest that oligomerization of pikachurin and its interaction with DG causes DG assembly on the synapse surface of the photoreceptor synaptic terminals. Our results reveal that the presynaptic interaction of pikachurin with DG at photoreceptor terminals is essential for both the formation of proper photoreceptor ribbon synaptic structures and normal retinal electrophysiology.

An essential role for RAX homeoprotein and NOTCH-HES signaling in Otx2 expression in embryonic retinal photoreceptor cell fate determination.

The molecular mechanisms underlying cell fate determination from common progenitors in the vertebrate CNS remain elusive. We previously reported that the OTX2 homeoprotein regulates retinal photoreceptor cell fate determination. While Otx2 transactivation is a pivotal process for photoreceptor cell fate determination, its transactivation mechanism in the retina is unknown. Here, we identified an evolutionarily conserved Otx2 enhancer of ∼500 bp, named embryonic enhancer locus for photoreceptor Otx2 transcription (EELPOT), which can recapitulate initial Otx2 expression in the embryonic mouse retina. We found that the RAX homeoprotein interacts with EELPOT to transactivate Otx2, mainly in the final cell cycle of retinal progenitors. Conditional inactivation of Rax results in downregulation of Otx2 expression in vivo. We also showed that NOTCH-HES signaling negatively regulates EELPOT to suppress Otx2 expression. These results suggest that the integrated activity of cell-intrinsic and -extrinsic factors on EELPOT underlies the molecular basis of photoreceptor cell fate determination in the embryonic retina.