Crx, a Novel otx-like Homeobox Gene, Shows Photoreceptor-Specific Expression and Regulates Photoreceptor Differentiation

AbstractWe have isolated a novel otx-like homeobox gene, Crx, from the mouse retina. Crx expression is restricted to developing and mature photoreceptor cells. CRX bound and transactivated the sequence TAATCC/A, which is found upstream of several photoreceptor-specific genes, including the opsin genes from many species. Overexpression of Crx using a retroviral vector increased the frequency of clones containing exclusively rod photoreceptors and reduced the frequency of clones containing amacrine interneurons and Müller glial cells. In addition, presumptive photoreceptor cells expressing a dominant-negative form of CRX failed to form proper photoreceptor outer segments and terminals. Crx is a novel photoreceptor-specific transcription factor and plays a crucial role in the differentiation of photoreceptor cells

Cloning and characterization of mr-s, a novel SAM domain protein, predominantly expressed in retinal photoreceptor cells

BACKGROUND: Sterile alpha motif (SAM) domains are ~70 residues long and have been reported as common protein-protein interaction modules. This domain is found in a large number of proteins, including Polycomb group (PcG) proteins and ETS family transcription factors. In this work, we report the cloning and functional characterization of a novel SAM domain-containing protein, which is predominantly expressed in retinal photoreceptors and the pineal gland and is designated mouse mr-s (major retinal SAM domain protein). RESULTS: mr-s is evolutionarily conserved from zebrafish through human, organisms through which the mechanism of photoreceptor development is also highly conserved. Phylogenetic analysis suggests that the SAM domain of mr-s is most closely related to a mouse polyhomeotic (ph) ortholog, Mph1/Rae28, which is known as an epigenetic molecule involved in chromatin modifications. These findings provide the possibility that mr-s may play a critical role by regulating gene expression in photoreceptor development. mr-s is preferentially expressed in the photoreceptors at postnatal day 3–6 (P3-6), when photoreceptors undergo terminal differentiation, and in the adult pineal gland. Transcription of mr-s is directly regulated by the cone-rod homeodomain protein Crx. Immunoprecipitation assay showed that the mr-s protein self-associates mainly through the SAM domain-containing region as well as ph. The mr-s protein localizes mainly in the nucleus, when mr-s is overexpressed in HEK293T cells. Moreover, in the luciferase assays, we found that mr-s protein fused to GAL4 DNA-binding domain functions as a transcriptional repressor. We revealed that the repression activity of mr-s is not due to a homophilic interaction through its SAM domain but to the C-terminal region. CONCLUSION: We identified a novel gene, mr-s, which is predominantly expressed in retinal photoreceptors and pineal gland. Based on its expression pattern and biochemical analysis, we predict that mr-s may function as a transcriptional repressor in photoreceptor cells and in pinealocytes of the pineal gland

Synaptogenesis and outer segment formation are perturbed in the neural retina of Crx mutant mice

BACKGROUND: In Leber's congenital amaurosis (LCA), affected individuals are blind, or nearly so, from birth. This early onset suggests abnormal development of the neural retina. Mutations in genes that affect the development and/or function of photoreceptor cells have been found to be responsible in some families. These examples include mutations in the photoreceptor transcription factor, Crx. RESULTS: A Crx mutant strain of mice was created to serve as a model for LCA and to provide more insight into Crx's function. In this study, an ultrastructural analysis of the developing retina in Crx mutant mice was performed. Outer segment morphogenesis was found to be blocked at the elongation stage, leading to a failure in production of the phototransduction apparatus. Further, Crx-/- photoreceptors demonstrated severely abnormal synaptic endings in the outer plexiform layer. CONCLUSIONS: This is the first report of a synaptogenesis defect in an animal model for LCA. These data confirm the essential role this gene plays in multiple aspects of photoreceptor development and extend our understanding of the basic pathology of LCA

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 cytokine signaling-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.Yoshimoto T., Chaya T., Varner L.R., et al. The Rax homeoprotein in Müller glial cells is required for homeostasis maintenance of the postnatal mouse retina. Journal of Biological Chemistry 299, 105461 (2023); https://doi.org/10.1016/j.jbc.2023.105461

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

Varner L.R., Chaya T., Maeda Y., et al. The deubiquitinase Otud7b suppresses cone photoreceptor degeneration in mouse models of retinal degenerative diseases. iScience 27, 109380 (2024); https://doi.org/10.1016/j.isci.2024.109380.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

Cleavage of amyloid-β precursor protein (APP) by membrane-type matrix metalloproteinases

金沢大学がん研究所がん病態制御Amyloid-β precursor protein (APP) was identified on expression cloning from a human placenta cDNA library as a gene product that modulates the activity of membrane-type matrix metalloproteinase-1 (MT1-MMP). Co-expression of MT1-MMP with APP in HEK293T cells induced cleavage and shedding of the APP ectodomain when co-expressed with APP adaptor protein Fe65. Among the MT-MMPs tested, MT3-MMP and MT5-MMP also caused efficient APP shedding. The recombinant APP protein was cleaved by MT3-MMP in vitro at the A463-M 464, N579-M580, H622-S 623, and H685-Q686 peptide bonds, which included a cleavage site within the amyloid β peptide region known to produce a C-terminal fragment. The Swedish-type mutant of APP, which produces a high level of amyloid β peptide, was more effectively cleaved by MT3-MMP than wild-type APP in both the presence and absence of Fe65; however, amyloid β peptide production was not affected by MT3-MMP expression. Expression of MT3-MMP enhanced Fe65-dependent transactivation by APP fused to the Gal4 DNA-binding and transactivation domains. These results suggest that MT1-MMP, MT3-MMP and MT5-MMP should play an important role in the regulation of APP functions in tissues including the central nervous system. © 2006 The Japanese Biochemical Society

Cleavage of Amyloid-β Precursor Protein (APP) by Membrane-Type Matrix Metalloproteinases

Division of Molecular Viology and Oncolog

Identification of Autoantibodies against TRPM1 in Patients with Paraneoplastic Retinopathy Associated with ON Bipolar Cell Dysfunction

Background: Paraneoplastic retinopathy (PR), including cancer-associated retinopathy (CAR) and melanoma-associated retinopathy (MAR), is a progressive retinal disease caused by antibodies generated against neoplasms not associated with the eye. While several autoantibodies against retinal antigens have been identified, there has been no known autoantibody reacting specifically against bipolar cell antigens in the sera of patients with PR. We previously reported that the transient receptor potential cation channel, subfamily M, member 1 (TRPM1) is specifically expressed in retinal ON bipolar cells and functions as a component of ON bipolar cell transduction channels. In addition, this and other groups have reported that human TRPM1 mutations are associated with the complete form of congenital stationary night blindness. The purpose of the current study is to investigate whether there are autoantibodies against TRPM1 in the sera of PR patients exhibiting ON bipolar cell dysfunction. Methodology/Principal Findings: We performed Western blot analysis to identify an autoantibody against TRPM1 in the serum of a patient with lung CAR. The electroretinograms of this patient showed a severely reduced ON response wit