image-guided-optical-coherence-tomography-to-assess-structural

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Ocular diseases, such as age-related macular degeneration, glaucoma, retinitis pigmentosa, and uveitis, are always accompanied by retinal structural ...

Wenzhou Medical University

Precursor messenger RNA (Pre-mRNA) splicing is an essential biological process in eukaryotic cells. Genetic mutations in many spliceosome genes confer human eye diseases. Mutations in the pre-mRNA splicing factor, RP9 (also known as PAP1), predispose autosomal dominant retinitis pigmentosa (adRP) with an early onset and severe vision loss. However, underlying molecular mechanisms of the RP9 mutation causing photoreceptor degeneration remains fully unknown. Here, we utilize the CRISPR/Cas9 system to generate both the Rp9 gene knockout (KO) and point mutation knock in (KI) (Rp9, c.A386T, P.H129L) which is analogous to the reported one in the retinitis pigmentosa patients (RP9, c.A410T, P.H137L) in 661 W retinal photoreceptor cells in vitro. We found that proliferation and migration were significantly decreased in the mutated cells. Gene expression profiling by RNA-Seq demonstrated that RP associated genes, Fscn2 and Bbs2, were down-regulated in the mutated cells. Furthermore, pre-mRNA splicing of the Fscn2 gene was markedly affected. Our findings reveal a functional relationship between the ubiquitously expressing RP9 and the disease-specific gene, thereafter provide a new insight of disease mechanism in RP9-related retinitis pigmentosa.

Targeted RP9 ablation and mutagenesis in mouse photoreceptor cells by CRISPR-Cas9.

The etiology of the highly myopic condition has been unclear for decades. We investigated the genetic contributions to early-onset high myopia (EOHM), which is defined as having a refraction of less than or equal to -6 diopters before the age of 6, when children are less likely to be exposed to high educational pressures. Trios (two nonmyopic parents and one child) were examined to uncover pathogenic mutations using whole-exome sequencing. We identified parent-transmitted biallelic mutations or de novo mutations in as-yet-unknown or reported genes in 16 probands. Interestingly, an increased rate of de novo mutations was identified in the EOHM patients. Among the newly identified candidate genes, a  mutation was identified in one EOHM proband. Expanded screening of 1,040 patients found an additional four mutations in the same gene. Then, we generated  mutant mice to further elucidate the functional impact of this gene and observed typical myopic phenotypes, including an elongated axial length. Using a trio-based exonic screening study in EOHM, we deciphered a prominent role for de novo mutations in EOHM patients without myopic parents. The discovery of a disease gene, , provides insights into myopic development and its etiology, which expands our current understanding of high myopia and might be useful for future treatment and prevention.

Trio-based exome sequencing arrests de novo mutations in early-onset high myopia.

MicroRNAs (miRNAs) are known to be essential for retinal maturation and functionality; however, the role of the most abundant miRNAs, the miR-183/96/182 cluster (miR-183 cluster), in photoreceptor cells remains unclear. Here we demonstrate that ablation of two components of the miR-183 cluster, miR-183 and miR-96, significantly affects photoreceptor maturation and maintenance in mice. Morphologically, early-onset dislocated cone nuclei, shortened outer segments and thinned outer nuclear layers are observed in the miR-183/96 double-knockout (DKO) mice. Abnormal photoreceptor responses, including abolished photopic electroretinography (ERG) responses and compromised scotopic ERG responses, reflect the functional changes in the degenerated retina. We further identify Slc6a6 as the cotarget of miR-183 and miR-96. The expression level of Slc6a6 is significantly higher in the DKO mice than in the wild-type mice. In contrast, Slc6a6 is down-regulated by adeno-associated virus-mediated overexpression of either miR-183 or miR-96 in wild-type mice. Remarkably, both silencing and overexpression of Slc6a6 in the retina are detrimental to the electrophysiological activity of the photoreceptors in response to dim light stimuli. We demonstrate that miR-183/96-mediated fine-tuning of Slc6a6 expression is indispensable for photoreceptor maturation and maintenance, thereby providing insight into the epigenetic regulation of photoreceptors in mice.

miR-183/96 plays a pivotal regulatory role in mouse photoreceptor maturation and maintenance.

Circular RNAs (circRNAs) belong to an endogenous class of RNA molecules with both ends covalently linked in a circle. Although their expression pattern in the mammalian brain has been well studied, the characteristics and functions of circRNAs in retinas remain unknown. To reveal the whole expression profiles of circRNAs in the neural retina, we investigated retinal RNAs of human, monkey, mouse, pig, zebrafish and tree shrew and detected thousands of circRNAs showing conservation and variation in the retinas across different vertebrate species. We further investigated one of the abundant circRNAs, circPDE4B, identified in human retina. Silencing of circPDE4B significantly inhibited the proliferation of human A549 cells. Functional assays demonstrated that circPDE4B could sponge miR-181C, thereby altering the cell phenotype. We have explored the retinal circRNA repertoires across human and different vertebrates, which provide new insights into the important role of circRNAs in the vertebrate retinas, as well as in related human diseases.

Circular RNAs in human and vertebrate neural retinas.

MicroRNA-182 (miR-182) is abundantly expressed in mammalian retinas; however, the association between miR-182 and retinal function remains unclear. In this study, we explored whether miR-182 contributes to functional decline in retinas using a miR-182 depleted mouse.

Deletion of miR-182 Leads to Retinal Dysfunction in Mice.

Retinitis pigmentosa (RP) is the most common manifestation of inherited retinal diseases with high degree of genetic, allelic, and phenotypic heterogeneity. CEP250 encodes the C-Nap1 protein and has been associated with various retinal phenotypes. Here, we report the identification of a mutation (c.562C>T, p.R188*) in the CEP250 in a consanguineous family with nonsyndromic RP. To gain insights into the molecular pathomechanism underlying CEP250 defects and the functional relevance of CEP250 variants in humans, we conducted a functional characterization of CEP250 variant using a novel Cep250 knockin mouse line. Remarkably, the disruption of Cep250 resulted in severe impairment of retinal function and significant retinal morphological alterations. The homozygous knockin mice showed significantly reduced retinal thickness and ERG responses. This study not only broadens the spectrum of phenotypes associated with CEP250 mutations, but also, for the first time, elucidates the function of CEP250 in photoreceptors using a newly established animal model.

Functional characterization of CEP250 variant identified in nonsyndromic retinitis pigmentosa.

Circular RNAs (circRNAs) represent a class of noncoding RNAs with a wide expression pattern, and they constitute an important layer of the genome regulatory network. To date, the expression pattern and regulatory potency of circRNAs in the retina, a key part of the central nervous system, are not yet well understood. In this study, RNAs from five stages (E18.5, P1, P7, P14, and P30) of mouse retinal development were sequenced. A total of 9,029 circRNAs were identified. Most circRNAs were expressed in different stages with a specific signature, and their expression patterns were different from those of their host linear transcripts. Some circRNAs could act as sponges for several retinal microRNAs (miRNAs). Furthermore, circTulp4 could function as a competitive endogenous RNA (ceRNA) to regulate target genes. Remarkably, silencing circTulp4 in vivo led to mice having a thin outer nuclear layer (ONL) and defective retinal function. In addition, we found that circRNAs were dysregulated at a much earlier time point than that of disease onset in a retinal degeneration model (rd8 mice). In summary, we provide the first circRNA expression atlas during retinal development and highlight a key biological role for circRNAs in retinal development and degeneration.

The Circular RNome of Developmental Retina in Mice.

Microbial ecosystems interact with the human body and affect human health. The microbial community on the ocular surface remains an underexplored territory despite its importance as the first line of defense barrier that protects the eye and ultimately sight. We investigated how age and sex affected human ocular surface microbiome, and in the present study wanted to understand how geographic difference shaped the microbiome in the ocular surface.

Geographic Difference Shaped Human Ocular Surface Metagenome of Young Han Chinese From Beijing, Wenzhou, and Guangzhou Cities.

Non-coding RNAs (ncRNAs) are key players in variety of biogenesis and biological functions. Their aberrant expression has been implicated in disease progression. NcRNAs can be divided into short ncRNAs whose subtypes are mainly microRNA (miRNA), long non-coding RNA (lncRNA) and circular RNA (circRNA). They are involved in cellular processes, including gene regulation, development and disease. The retina is a remarkably sophisticated instrument with interconnected cell types and is the primary target of many genetic diseases. In addition, in terms of retinal dyshomeostasis and inflammation, ncRNAs seems to play critical roles in many retinal diseases. Here, we provide an overview of ncRNAs in developing retina. We also review how does these ncRNAs function in various retinal diseases including animal and human models. These data indicate that ncRNAs regulate cellular processes including cell proliferation, differentiation, apoptosis and contribute to initiation and progression of retinal diseases.

Emerging roles of non-coding RNAs in retinal diseases: A review.

Cdr1as is the abundant circular RNA (circRNA) in human and vertebrate retinas. However, the role of Cdr1as in the retina remains unknown. In this study, we aimed to generate a Cdr1as knockout (KO) mouse model and investigate the retinal consequences of Cdr1as loss of function. Through  hybridization (ISH), we demonstrated that Cdr1as is mainly expressed in the inner retina. Using CRISPR/Cas9 targeting Cdr1as, we successfully generated KO mice. We carried out ocular examinations in the KO mice until postnatal day 500. Compared with the age-matched wild-type (WT) siblings, the KO mice displayed increased b-wave amplitude of photopic electrophysiological response and reduced vision contrast sensitivity. Through small RNA profiling of the retinas, we determined that miR-7 was downregulated, while its target genes were upregulated. Taken together, our results demonstrated for the first time that Cdr1as ablation led to a mild retinal consequence in mice, indicating that Cdr1as abundance is not indispensable for retinal development and maintenance.

Abundant Neural circRNA Cdr1as Is Not Indispensable for Retina Maintenance.

Potocki-Shaffer syndrome (PSS) is a rare contiguous gene deletion syndrome marked by haploinsufficiency of genes in chromosomal region 11p11.2p12. Approximately 50 cases of PSS have been reported; however, a syndrome with a PSS-like clinical phenotype caused by 11p11.12p12 duplication has not yet been reported.

11p11.12p12 duplication in a family with intellectual disability and craniofacial anomalies.

Argonaute proteins are key players in small RNA-guided gene silencing processes. Ago2 is the member of the Argonaute subfamily with slicer endonuclease activity and is critical for microRNA homeostasis and indispensable for biological development. However, the impact of Ago2 dysregulation in the retina remains to be fully explored. In this study, we studied the role of Ago2 in mouse retina.

Retinal Degeneration Caused by Ago2 Disruption.

Abundant retinal microRNA-183 cluster (miR-183C) has been reported to be a key player in photoreceptor development and functionality in mice. However, whether there is a protagonist in this cluster remains unclear. Here, we used a mutant mouse model to study the role of miR-96, a member of miR-183C, in photoreceptor development and functionality.

Depletion of miR-96 Delays, But Does Not Arrest, Photoreceptor Development in Mice.

To investigate the levels of cytokines in the plasma of patients with multiple myeloma(MM), and explore its clinical significance.

Xuejiao Chen

State Key Laboratory of Ophthalmology,
Optometry,
and Vision Science,
Eye Hospital,
State Key Laboratory of Ophthalmology, Optometry, and Vision Science, Eye Hospital

Wenzhou Medical University

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

Xuejiao Chen

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Wenzhou Medical University

Image-Guided Optical Coherence Tomography to Assess Structural Changes in Rodent Retinas

State Key Laboratory of Ophthalmology,
Optometry,
and Vision Science,
Eye Hospital,
State Key Laboratory of Ophthalmology, Optometry, and Vision Science, Eye Hospital