A new mouse-fixation device for IOP measurement in awake mice.

Glaucoma is an irreversible blinding eye disease. The mechanisms underlying glaucoma are complex. Up to now, no successful remedy has been found to completely cure the condition. High intraocular pressure (IOP) is an established risk factor for glaucoma and the only known modifiable factor for glaucoma treatment. Mice have been widely used to study glaucoma pathogenesis. IOP measurement is an important tool for monitoring the potential development of glaucomatous phenotypes in glaucoma mouse models. Currently, there are two methods of IOP measurement in mice: invasive and non-invasive. As the invasive method can cause corneal damage and inflammation, and most of the noninvasive method involves the use of anesthetics. In the course of our research, we designed a mouse fixation device to facilitate non-invasive measurements of mouse IOPs. Using this device, mouse IOPs can be accurately measured in awake mice. This device will help researchers to accurately assess mouse IOP without the use of anesthetics.

Association of variants in GJA8 with familial acorea-microphthalmia-cataract syndrome.

Congenital acorea is a rare disease with the absence of a pupil in the eye. To date, only one family and two isolated cases with congenital acorea have been reported. The gene associated with acorea has not been identified. In this study, we recruited a Chinese family acorea-microphthalmia-cataract syndrome. By analyzing the whole-exome sequencing (WES) data of this Chinese family, we revealed the association of a novel heterozygous variant, NM_005267.5:c.137G>A (p.G46E) in the gap junction protein alpha 8 (GJA8) gene encoding connexin 50 or CX50, with familial acorea-microphthalmia-cataract syndrome. Additionally, another variant, NM_005267.5:c.151G>A (p.D51N) in GJA8, was identified to co-segregate with this syndrome in an unrelated Japanese family. Ectopic expression of p.G46E and p.D51N mutant GJA8 genes in cultured cells caused protein mislocalization, suggesting that the p.G46E and p.D51N mutations in GJA8 impaired the function of the gap junction channels. These results established GJA8 as the first gene associated with familial acorea-microphthalmia-cataract syndrome.

Leukemia Inhibitory Factor Protects against Degeneration of Cone Photoreceptors Caused by RPE65 Deficiency.

BACKGROUND: Retinal pigment epithelium (RPE) 65 is a key enzyme in the visual cycle involved in the regeneration of 11-cis-retinal. Mutations in the human RPE65 gene cause Leber's congenital amaurosis (LCA), a severe form of an inherited retinal disorder. Animal models carrying Rpe65 mutations develop early-onset retinal degeneration. In particular, the cones degenerate faster than the rods. To date, gene therapy has been used successfully to treat RPE65-associated retinal disorders. However, gene therapy does not completely prevent progressive retinal degeneration in patients, possibly due to the vulnerability of cones in these patients. In the present study, we tested whether leukemia inhibitory factor (LIF), a trophic factor, protects cones in rd12 mice harboring a nonsense mutation in Rpe65. METHODS: LIF was administrated to rd12 mice by intravitreal microinjection. Apoptosis of retinal cells was analyzed by TUNEL assay. The degeneration of cone cells was evaluated by immunostaining of retinal sections and retinal flat-mounts. Signaling proteins regulated by LIF in the retinal and cultured cells were determined by immunoblotting. RESULTS: Intravitreal administration of LIF activated the STAT3 signaling pathway, thereby inhibiting photoreceptor apoptosis and preserving cones in rd12 mice. Niclosamide (NCL), an inhibitor of STAT3 signaling, effectively blocked STAT3 signaling and autophagy in cultured 661W cells treated with LIF. Co-administration of LIF with NCL to rd12 mice abolished the protective effect of LIF, suggesting that STAT3 signaling and autophagy mediate the protection. CONCLUSION: LIF is a potent factor that protects cones in rd12 mice. This finding implies that LIF can be used in combination with gene therapy to achieve better therapeutic outcomes for patients with RPE65-associated LCA.

Rhodopsin-associated retinal dystrophy: Disease mechanisms and therapeutic strategies.

Rhodopsin is a light-sensitive G protein-coupled receptor that initiates the phototransduction cascade in rod photoreceptors. Mutations in the rhodopsin-encoding gene RHO are the leading cause of autosomal dominant retinitis pigmentosa (ADRP). To date, more than 200 mutations have been identified in RHO. The high allelic heterogeneity of RHO mutations suggests complicated pathogenic mechanisms. Here, we discuss representative RHO mutations as examples to briefly summarize the mechanisms underlying rhodopsin-related retinal dystrophy, which include but are not limited to endoplasmic reticulum stress and calcium ion dysregulation resulting from protein misfolding, mistrafficking, and malfunction. Based on recent advances in our understanding of disease mechanisms, various treatment methods, including adaptation, whole-eye electrical stimulation, and small molecular compounds, have been developed. Additionally, innovative therapeutic treatment strategies, such as antisense oligonucleotide therapy, gene therapy, optogenetic therapy, and stem cell therapy, have achieved promising outcomes in preclinical disease models of rhodopsin mutations. Successful translation of these treatment strategies may effectively ameliorate, prevent or rescue vision loss related to rhodopsin mutations.

LncRNA MBNL1-AS1 knockdown increases the sensitivity of hepatocellular carcinoma to tripterine by regulating miR-708-5p-mediated glycolysis.

Hepatocellular carcinoma (HCC) is identified as a common cancer type across the world and needs novel and efficient treatment. Tripterine, a well-known compound, exerts suppressive role in HCC development. However, the related molecular mechanism of tripterine in HCC remains unclear. The expression of MBNL1-AS1in HCC tissues and cells was measured via qRT-PCR assay. MTT assay was employed to estimate cell viability. Besides, cell migration as well as invasion was determined through transwell assay. Additionally, the binding ability of miR-708-5p and MBNL1-AS1or HK2 was proved by starBase database and luciferase reporter gene assay. Moreover, the HK2 level was detected by immunoblotting. MBNL1-AS1 was reduced in HCC tissues and cells. Overexpression of MBNL1-AS1 decreased the sensitivity of HCC cells to tripterine while MBNL1-AS1 silence played opposite effect. In addition, miR-708-5p was the target of MBNL1-AS1 and was down-regulated through MBNL1-AS1 in HCC cells. Moreover, miR-708-5p suppressed glycolysis rate and reduced the expression of vital glycolytic enzyme (HK2, LDHA and PKM2) in HCC cells. Furthermore, miR-708-5p reduced HK2 expression by binding to it directly. In this investigation, we proved that LncRNA MBNL1-AS1 increased the tripterine resistance of HCC cells at least partly by mediating miR-708-5p-related glycolysis. These findings revealed a potent therapeutic target for the treatment of HCC.

LncRNA SLC9A3-AS1 knockdown increases the sensitivity of liver cancer cell to triptolide by regulating miR-449b-5p-mediated glycolysis.

Triptolide (TP) is involved in the progression of liver cancer. However, the detailed molecular network regulated through TP is still unclear. Long non-coding RNA (LncRNA) SLC9A3 exerts roles in various pathological progresses. Nevertheless, whether SLC9A3 affects the sensitivity of liver cancer cells to TP have not been uncovered. The content of SLC9A3-AS1 and miR-449b-5p was estimated by utilizing quantitative real-time polymerase-chain reaction (qRT-PCR). Cell counting kit 8 (CCK-8) assay was introduced to assess cell viability. Additionally, cell viability as well as invasion was tested via transwell assay. The direct binding between miR-449b-5p and SLC9A3-AS1 or LDHA was confirmed through luciferase reporter gene assay. Moreover, glycolysis rate was tested by calculating the uptake of glucose in addition to the production of lactate in Huh7 cells. LncRNA SLC9A3-AS1 was up-regulated in liver cancer tissue samples and cells. Knockdown of SLC9A3-AS1 notably further inhibited viability, migration as well as invasion in Huh7 cells. MiR-449b-5p was the direct downstream miRNA of SLC9A3-AS1 and was down-regulated by SLC9A3-AS1 in Huh7 cells. In addition, miR-449b-5p was reduced in liver cancer tissues and cells. Overexpressed miR-449b-5p increased the sensitivity of Huh7 cells to TP remarkably. Moreover, miR-449b-5p negatively regulated LDHA expression in Huh7 cells. This work proved that SLC9A3-AS1 increased the sensitivity of liver cancer cells to TP by regulating glycolysis rate mediated via miR-449b-5p/LDHA axis. These findings implied that TP is likely to be a potent agent for treating patients diagnosed with liver cancer.

Deep Sc-RNA sequencing decoding the molecular dynamic architecture of the human retina.

The human retina serves as a light detector and signals transmission tissue. Advanced insights into retinal disease mechanisms and therapeutic strategies require a deep understanding of healthy retina molecular events. Here, we sequenced the mRNA of over 0.6 million single cells from human retinas across six regions at nine different ages. Sixty cell sub-types have been identified from the human mature retinas with unique markers. We revealed regional and age differences of gene expression profiles within the human retina. Cell-cell interaction analysis indicated a rich synaptic connection within the retinal cells. Gene expression regulon analysis revealed the specific expression of transcription factors and their regulated genes in human retina cell types. Some of the gene's expression, such as DKK3, are elevated in aged retinas. A further functional investigation suggested that over expression of DKK3 could impact mitochondrial stability. Overall, decoding the molecular dynamic architecture of the human retina improves our understanding of the vision system.

Cholesterol homeostasis regulated by ABCA1 is critical for retinal ganglion cell survival.

Genome-wide association studies have suggested a link between primary open-angle glaucoma and the function of ABCA1. ABCA1 is a key regulator of cholesterol efflux and the biogenesis of high-density lipoprotein (HDL) particles. Here, we showed that the POAG risk allele near ABCA1 attenuated ABCA1 expression in cultured cells. Consistently, POAG patients exhibited lower ABCA1 expression, reduced HDL, and higher cholesterol in white blood cells. Ablation of Abca1 in mice failed to form HDL, leading to elevated cholesterol levels in the retina. Counting retinal ganglion cells (RGCs) by using an artificial intelligence (AI) program revealed that Abca1-deficient mice progressively lost RGCs with age. Single-cell RNA sequencing (scRNA-seq) revealed aberrant oxidative phosphorylation in the Abca1-/- retina, as well as activation of the mTORC1 signaling pathway and suppression of autophagy. Treatment of Abca1-/- mice using atorvastatin reduced the cholesterol level in the retina, thereby improving metabolism and protecting RGCs from death. Collectively, we show that lower ABCA1 expression and lower HDL are risk factors for POAG. Accumulated cholesterol in the Abca1-/- retina causes profound aberrant metabolism, leading to a POAG-like phenotype that can be prevented by atorvastatin. Our findings establish statin use as a preventive treatment for POAG associated with lower ABCA1 expression.

Automatic counting of retinal ganglion cells in the entire mouse retina based on improved YOLOv5.

Glaucoma is characterized by the progressive loss of retinal ganglion cells (RGCs), although the pathogenic mechanism remains largely unknown. To study the mechanism and assess RGC degradation, mouse models are often used to simulate human glaucoma and specific markers are used to label and quantify RGCs. However, manually counting RGCs is time-consuming and prone to distortion due to subjective bias. Furthermore, semi-automated counting methods can produce significant differences due to different parameters, thereby failing objective evaluation. Here, to improve counting accuracy and efficiency, we developed an automated algorithm based on the improved YOLOv5 model, which uses five channels instead of one, with a squeeze-and-excitation block added. The complete number of RGCs in an intact mouse retina was obtained by dividing the retina into small overlapping areas and counting, and then merging the divided areas using a non-maximum suppression algorithm. The automated quantification results showed very strong correlation (mean Pearson correlation coefficient of 0.993) with manual counting. Importantly, the model achieved an average precision of 0.981. Furthermore, the graphics processing unit (GPU) calculation time for each retina was less than 1 min. The developed software has been uploaded online as a free and convenient tool for studies using mouse models of glaucoma, which should help elucidate disease pathogenesis and potential therapeutics.

Characterization of two novel knock-in mouse models of syndromic retinal ciliopathy carrying hypomorphic Sdccag8 mutations.

Mutations in serologically defined colon cancer autoantigen protein 8 ( SDCCAG8) were first identified in retinal ciliopathy families a decade ago with unknown function. To investigate the pathogenesis of SDCCAG8-associated retinal ciliopathies in vivo, we employed CRISPR/Cas9-mediated homology-directed recombination (HDR) to generate two knock-in mouse models, Sdccag8Y236X/Y236X and Sdccag8E451GfsX467/E451GfsX467 , which carry truncating mutations of the mouse Sdccag8, corresponding to mutations that cause Bardet-Biedl syndrome (BBS) and Senior-Løken syndrome (SLS) (c.696T>G p.Y232X and c.1339-1340insG p.E447GfsX463) in humans, respectively. The two mutant Sdccag8 knock-in mice faithfully recapitulated human SDCCAG8-associated BBS phenotypes such as rod-cone dystrophy, cystic renal disorder, polydactyly, infertility, and growth retardation, with varied age of onset and severity depending on the hypomorphic strength of the Sdccag8 mutations. To the best of our knowledge, these knock-in mouse lines are the first BBS mouse models to present with the polydactyly phenotype. Major phototransduction protein mislocalization was also observed outside the outer segment after initiation of photoreceptor degeneration. Impaired cilia were observed in the mutant photoreceptors, renal epithelial cells, and mouse embryonic fibroblasts derived from the knock-in mouse embryos, suggesting that SDCCAG8 plays an essential role in ciliogenesis, and cilium defects are a primary driving force of SDCCAG8-associated retinal ciliopathies.

Identification of a novel compound heterozygous CYP4V2 variant in a patient with autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) belongs to a family of retinal disorders that is characterized by the progressive degeneration of rod and cone photoreceptors. The aim of the present study was to screen for possible disease-causing genetic variants in a non-consanguineous Chinese family with non-syndromic autosomal recessive RP. Whole-exome sequencing (WES) was performed in samples from the affected individual (the proband) and those from the two children of the proband. A novel compound heterozygous variant of c.C958T (p.R320X) and c.G1355A (p.R452H) in the Cytochrome P450 family 4 subfamily V member 2 (CYP4V2) gene was identified through WES. Subsequently, this variant was validated by direct Sanger sequencing. This compound heterozygous variant was found to be absent from other unaffected family members and 400 ethnically-matched healthy control individuals. In addition, this compound variant was co-segregated with the RP phenotype in an autosomal recessive manner. In silico analysis revealed that both c.C958T (p.R320X) and c.G1355A (p.R452H) could compromise the protein function of CYP4V2. These results strongly suggest this compound variant to be a disease-causing variant, which expands upon the spectrum of currently known CYP4V2 genetic variants associated with retinal diseases.

Exogenous PDE5 Expression Rescues Photoreceptors in RD1 Mice.

BACKGROUND: Catalytic hydrolysis of cyclic guanosine monophosphate (cGMP) by phosphodiesterase 6 (PDE6) is critical in phototransduction signalling in photoreceptors. Mutations in the genes encoding any of the three PDE6 subunits are associated with retinitis pigmentosa, the most common form of inherited retinal diseases. The RD1 mouse carries a naturally occurring nonsense mutation in the Pde6b gene. The RD1 mouse retina rapidly degenerates and fails to form rod photoreceptor outer segments due to the elevated cGMP level and subsequent excessive Ca2+ influx. In this study, we aim to test whether the PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, rescues photoreceptors in the RD1 retina. METHODS: Electroporation used the PDE5 expression plasmid to transfect neonatal RD1 mice. The mouse retina degeneration was assessed by retinal sections' stains with DAPI. The expression and localization of phototransduction proteins in photoreceptors were analysed by immunostaining. The expression of proteins in cultured cells was analysed by immunoblotting. RESULTS: The exogenous PDE5 expression, a non-photoreceptor-specific member of the PDE superfamily, prevents photoreceptor degeneration in RD1 mice. Unlike endogenous photoreceptor-specific PDE6 localised in the outer segments of photoreceptors, ectopically- expressed PDE5 was distributed in inner segments and synaptic terminals. PDE5 also promoted the development of the outer segments in RD1 mice. PDE5 co-expression with rhodopsin in cultured cells showed enhanced rhodopsin expression. CONCLUSION: Lowering the cGMP level in photoreceptors by PDE5 is sufficient to rescue photoreceptors in RD1 retinas. cGMP may also play a role in rhodopsin expression regulation in photoreceptors.

A quick protocol for the preparation of mouse retinal cryosections for immunohistochemistry.

Immunohistochemistry (IHC) using mouse retinal cryosections is widely used to study the expression and intracellular localization of proteins in mouse retinas. Conventionally, the preparation of retinal cryosections from mice involves tissue fixation, cryoprotection, the removal of the cornea and lens, embedding and sectioning. The procedure takes 1-2 days to complete. Recently, we developed a new technique for the preparation of murine retinal cryosections by coating the sclera with a layer of Super Glue. This enables us to remove the cornea and extract the lens from the unfixed murine eye without causing the eyecup to collapse. In the present study, based on this new technique, we move a step forward to modify the conventional protocol. Unlike in the conventional protocol, in this method, we first coat the unfixed mouse eyeball on the sclera with Super Glue and then remove the cornea and lens. The eyecup is then fixed, cryoprotected and sectioned. This new protocol for the preparation of retinal cryosections reduces the time for the procedure to as little as 2 h. Importantly, the new protocol consistently improves the morphology of retinal sections as well as the image quality of IHC. Thus, this new quick protocol will be greatly beneficial to the community of visual sciences by expediting research progress and improving the results of IHC.

Inhibition of mTOR signaling by rapamycin protects photoreceptors from degeneration in rd1 mice.

Retinitis pigmentosa (RP) is an inherited retinal degenerative disease that begins with defective rod photoreceptor function, followed by impaired cone function, and complete blindness in its late stage. To date, however, there is no effective treatment for RP. By carrying a nonsense mutation in the Pde6b gene, rd1 mice display elevated cGMP in conjunction with higher intracellular Ca 2+ in their rod photoreceptors, resulting in fast retinal degeneration. Ca 2+ has been linked to activation of the mammalian target of rapamycin (mTOR) pathway. The mTOR pathway integrates extracellular and intracellular signals to sense the supply of nutrients and plays a central role in regulating protein and lipid synthesis as well as apoptosis and autophagy. In the present study, we showed that mTOR and phosphorylated mTOR (p-mTOR, activated form of mTOR) are up-regulated in rd1 photoreceptors at postnatal day 10 (P10), a pre-degenerative stage. Moreover, the downstream effectors of mTOR, such as pS6K and S6K, are also increased, suggesting activation of the mTOR signaling pathway. Intravitreal administration of rapamycin, a negative regulator of mTOR, inhibits the mTOR pathway in rd1 photoreceptors. Consequently, the progression of retinal degeneration is slower and retinal function is enhanced, possibly mediated by activation of autophagy in the photoreceptors. Taken together, these results highlight rapamycin as a potential therapeutic avenue for retinal degeneration.

A novel missense variant in cathepsin C gene leads to PLS in a Chinese patient: A case report and literature review.

BACKGROUND: Papilon-Lefevre syndrome (PLS; OMIM 245000) is a rare autosomal recessive disease characterized by aggressive periodontitis and palmoplantar keratoderma. The prevalence of PLS in the general population is one to four cases per million. Although the etiology and pathogenic mechanisms underlying PLS remain largely unclear, existing evidence shows loss-of-function mutations of the cathepsin C gene (CTSC; OMIM 602365) could cause PLS. Here we found a novel variant of the CTSC gene in a Chinese PLS family and predicted the effect of the variant on the physic-chemical characters and tertiary structure of the protein. METHODS: The 1-7 coding exons and exon-intron boundaries of CTSC gene of the proband and her family were amplified and sequenced directly, and Chromas was used to read sequencing files. Furthermore, the PolyPhen-2, PROVEAN, and Mutation Taster were utilized to predict the pathogenicity of the variant. Besides, the physic-chemical and structural characters of the protein were analyzed by ProtParam, ProtScale, and SWISS-MODEL. RESULTS: Our study identified a novel homozygous variant c.763T>C (p.Cys255Arg) in exon 6 of the CTSC gene, and it was a likely pathogenic variant as predicted by PolyPhen-2, PROVEAN, and Mutation Taster. Moreover, ProtParam and Protscale revealed the variant increased the isoelectric point and hydrophilicity of the protein, and the SWISS-MODEL analysis suggested the variant was located in a critical domain for protein activity. CONCLUSION: Our study analyzed a Chinese family with PLS and identified a novel missense variant in the CTSC gene. Besides, this study retrospectively summarized 113 variants of CTSC in the world and highlighted the features of 27 CTSC variants in Chinese PLS patients. In addition, this study paid much particular attention to the relationship between CTSC variants and different phenotypes.

An improved method for preparation of mouse retinal cryosections.

Immunohistochemistry using mouse retinal cryosections is a routine assay used in vision research. However, retinal tissues are fragile, and it is difficult to obtain an ideal retinal cryosection. Here, we developed a modified method for preparing retinal cryosection. Super Glue was applied on the surface of the sclera before the cornea and the lens are removed from either the unfixed or PFA-fixed mouse eyeballs. The new methods largely prevented retinal detachment in mouse retinal cryosections. Immunostaining of retinal cryosections derived from PFA-fixed mouse eyes using rod and cone markers yielded high-quality immunofluorescent images. Immunolabeling of retinal cryosections obtained from unfixed mouse eyes using a cilium-specific marker had improved orientations of photoreceptor connecting cilia. This new method substantially improves the morphology and immunostaining results of fixed and unfixed mouse eyes.

Identification of a novel homozygous variant in the CNGA1 gene in a Chinese family with autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a complex group of hereditary retinal dystrophies. Although >60 genes have been identified to be associated with non­syndromic RP, the exact genetic variant remains elusive in numerous cases of RP. In the present study, a Chinese pedigree affected by RP with autosomal recessive inheritance, including a total of seven members with one affected patient and six unaffected individuals, was recruited. Comprehensive ophthalmic examinations were performed on the proband and the proband's unaffected daughter. Genomic DNA was extracted from peripheral blood. Whole­exome sequencing (WES) was performed for the affected individual. The candidate pathogenic variant was verified by direct Sanger sequencing. The affected individual presented with classical clinical symptoms of RP. A novel homozygous variant, c.265delC (p.L89Ffs*3) in the cyclic nucleotide­gated channel subunit α 1 gene was identified in the affected patient. This homozygous variant was absent in other unaffected family members and 600 ethnicity­matched healthy controls. The variant was co­segregated with the disease phenotype in an autosomal recessive manner.

Identification of CRB1 mutations in two Chinese consanguineous families exhibiting autosomal recessive retinitis pigmentosa.

Retinitis pigmentosa (RP) is a leading cause of inherited blindness characterized by progressive loss of retinal photoreceptor cells. The present study aimed to identify the causative gene mutations in two Chinese families with autosomal recessive retinitis pigmentosa (arRP). Two Chinese consanguineous arRP families (RP­2284 and RP­2360) were recruited in this study, involving totally three affected and 25 unaffected members. All the affected members underwent a complete ophthalmic examination, including fundus photography, multifocal electroretinography (ERG) and full field ERG. Exome sequencing was performed on the three RP patients in the two families, followed by direct Sanger sequencing in all the family members and in 1,260 unrelated controls for validation of the mutations identified. Two homozygous missense mutations in the crumbs homolog 1 (CRB1) gene, which is known to cause severe retinal dystrophies, were found to be related to the phenotype of the two arRP families. The homozygous missense mutation c.1997 T>A in CRB1 was detected in two patients in the RP­2284 family. The proband in the RP­2360 family was the only RP patient and was found to carry the novel homozygous missense mutation c.2426 A>C in CRB1. The two mutations were heterozygous or absent in the other healthy family members, and they were absent in the 1,260 controls. The amino acid changes in the CRB1 protein affected by the two mutations were predicted to be damaging by Polyohen­2. Our study reported two CRB1 mutations causing arRP in two Chinese families, which expands the CRB1 mutation spectrum of RP in the Chinese population and emphasizes the causative role of CRB1 in RP.