Chen Liang

To investigate the potential relationship between subretinal microglia and transplanted donor photoreceptors. Photoreceptor precursors were transplanted into wild-type mice and rd1 mice by trans-scleral injection. Immunohistochemistry was employed to detect microglia and macrophages. PlX5622 feed was used to achieve microglia depletion and microglia repopulation. RNA-seq and qPCR were utilized to evaluate gene expression. Confocal microscopy was used to observe the interaction between microglia and donor photoreceptors. Donor photoreceptors survived in rd1 mice but not in wild-type mice after trans-scleral injection. The microglial cells closely interacted with donor cells. While donor cells failed to survive in rd1 mice after microglia depletion, they could survive following microglia repopulation. The RNA-seq analysis showed a pro-neurodevelopmental effect of sub-retinal microglia/RPE tissue in rd1 mice. Subretinal microglia supported donor photoreceptor survival in rd1 mice.

Centrosomal protein dysfunction might cause ciliopathies. However, the role of centrosomal proteins in male infertility remains poorly defined. Here, we identified a pathogenic splicing mutation inCEP78in male infertile patients with severely reduced sperm number and motility, and the typical multiple morphological abnormalities of the sperm flagella phenotype. We further createdCep78knockout mice, which showed an extremely low sperm count, completely aberrant sperm morphology, and approximately null sperm motility. The infertility of the patients and knockout mice could not be rescued by an intracytoplasmic sperm injection treatment. Mechanistically, CEP78 might regulate USP16 expression, which further stabilizes Tektin levels via the ubiquitination pathway.Cep78knockout mice also exhibited impairments in retina and outer hair cells of the cochlea. Collectively, our findings identified nonfunctional CEP78 as an indispensable factor contributing to male infertility and revealed a role for this gene in regulating retinal and outer hair cell function in mice.

Purpose To investigate the potential genetic defects in a five-generation Chinese family with autosomal dominant congenital cataract (ADCC). Methods Whole exome sequencing was performed to search the variants in the candidate genes associated with congenital cataract. Sanger sequencing was used to validate the variants and examine their co-segregation in the patients and their relatives. The potential effect of the variants was analyzed using several bioinformatic methods and further examined through Western blotting and co-immunoprecipitation. Results A missense variant c. 71 G > T (p. Gly24Val) in the CRYBA4 gene, a known ADCC candidate gene, was identified to be heterozygously present in the patients and co-segregate with cataract in the family. The mutation was absent in all of the searched databases, including our in-house exome sequences of 10,000 Chinese. The alignments of the amino acid sequences of CRYBA4 in a variety of species revealed that the amino acid residue Gly24 was evolutionarily highly conserved, and the in silico analysis predicted that the missense mutation of Gly24Val was damaging for the protein structure and function of CRYBA4. Then, the in vitro expression analysis further revealed that the Gly24Val mutation in CRYBA4 inhibited its binding with CRYBB1. The impaired interaction of β-crystallin proteins may affect their water-solubility and contribute to the formation of precipitates in lens fiber cells. Conclusion We identified a novel missense variant in the CRYBA4 gene as a pathogenic mutation of ADCC in a Chinese family. Our finding expanded the CRYBA4 variation spectrum associated with congenital cataracts.

Photoreceptor transplantation can rescue the retinal function of late-stage rd1 mice. Many studies have used synaptic markers to suggest that there are synaptic connections after transplantation, but how donor and host cells are connected remains unknown. Many molecules are needed for triad ribbon synapse formation in wild-type mice. Among them, pikachurin is an important extracellular matrix protein that bridges the pre- and postsynaptic components. To investigate the mechanism of the synaptic connection between donor photoreceptor and host retina, we studied the expression of pikachurin in late-stage rd1 mice before and after transplantation. The results showed that the full-length form of pikachurin could still be detected in the degenerated retina. After photoreceptors were transplanted to the subretinal space of rd1 or wild-type mice, pikachurin was detected in the cytoplasm of most donor photoreceptor cells. Pikachurin puncta may represent the cleaved form of the protein and may indicate synapse generation, but it was barely observed in the donor mass of wild-type mice (3.83 ± 3.17 puncta per 100 donor cells). In contrast, pikachurin puncta could be found in the graft of the rd1 mouse retina, but the number was low (21.35 ± 9.48 puncta per 100 donor cells). In addition, 54.12 ± 8.45% of bassoon puncta were paired with pikachurin puncta and 45.5 ± 6.33% were not, indicating that there were fewer pikachurin puncta than bassoon. These results suggest that pikachurin is involved in only a portion of the synaptic connection between the donor photoreceptor and host retina.

Von Hippel–Lindau (Vhl) protein inhibits hypoxia-inducible factor (Hif), yet its deletion in murine retina does not cause the extensive angiogenesis expected with Hif induction. The mechanism is unclear. Here we show that retinoblastoma tumor suppressor (Rb1) constrains expression of Hif target genes in the Vhl ^–/– retina. Deleting Rb1 induced extensive retinal neovascularization and autophagic ablation of photoreceptors in the Vhl ^–/– retina. RNA-sequencing, ChIP, and reporter assays showed Rb1 recruitment to and repression of certain Hif target genes. Activating Rb1 by deleting cyclin D1 induced a partial defect in the retinal superficial vascular plexus. Unexpectedly, removing Vhl suppressed retinoblastoma formation in murine Rb1/Rbl1 –deficient retina but generated subretinal vascular growths resembling retinal angiomatous proliferation (RAP) and retinal capillary hemangioblastoma (RCH). Most stromal cells in the RAP/RCH–like lesions were Sox9 ⁺ , suggesting a Müller glia origin, and expressed Lgals3, a marker of human brain hemangioblastoma. Thus, the Rb family limit Hif target gene expression in the Vhl ^–/– retina, and removing this inhibitory signal generates new models for RAP and RCH.

Purpose To determine whether the GNAQ/11 mutation correlated with the outcome of patients with uveal melanoma (UM) when genetic heterogeneity was considered. Methods We performed a retrospective study of sixty-seven patients with UM. The heterogeneity of GNAQ/11 was examined by using droplet digital PCR. The correlation between metastasis and heterogeneity of the GNAQ/11 mutation was analysed. Disease free survival curves were constructed using the Kaplan-Meier method, and the Wilcoxon log-rank test was used to compare the curves. Results The GNAQ/11 mutation ratio was varied between each case. Among these patients, 28.35% of them harboured homogeneous mutation of GNAQ/11, 62.69% present heterogeneous mutation and 8.96% didn’t present either GNAQ or GNA11 mutation. The tumour with heterogeneous mutation of GNAQ/11 has a higher metastatic rate than that with homogeneous mutation (13/29 vs 1/18, p=0.027). In Kaplan-Meier analysis, metastasis-free survival was not significantly associated with either homogeneous or heterogeneous mutation of GNAQ/11. Conclusion The mutation ratio of GNAQ/11 in UM was quite variable. The tumour with heterogeneous mutation of GNAQ/11 is more likely to develop a poor prognosis than that with homogeneous mutation of GNAQ/11.