Neuroprotective effects of C3 exoenzyme in excitotoxic retinopathy.

The purpose of this study is to evaluate the neuroprotective effects of C3 exoenzyme (C3) on N-methyl-d-aspartate (NMDA)-induced retinopathy in rats. C3 was expressed in Escherichia. coli and purified by affinity chromatography. Immunofluorescence was performed in NIH 3T3 cells treated with C3 to verify the cellular uptake of the protein. NMDA was injected intravitreally into rat eyes with or without C3. At various time points after injection, eyes were enucleated. Hematoxylin/eosin staining was performed on retina cross-sections for morphological analysis. Survival and apoptosis of cells in the ganglion cell layer (GCL) were assessed by cresyl violet staining and terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) on retina flat-mounts. RhoA levels in retina cells were evaluated by Western blot to detect C3 uptake in vivo. The cellular uptake of C3 was verified by immunofluorescence. Damage including a decrease in inner plexiform layer (IPL) thickness and reduction of cell density in the GCL, corresponding to apoptosis of neurons, was induced by intravitreal injection of NMDA. Protection against this damage was observed following co-injection of C3 and NMDA. RhoA ADP-ribosylation induced by C3 was confirmed by Western blot. Our results suggest that C3 exerts neuroprotective effects against excitotoxic damage induced by NMDA.

PNAS4 knockout does not induce obviously neurocytes apoptosis and abnormal development in mice brain.

Apoptosis is one kind of programmed cell death and contributes to development of a variety of organs such as brain. PNAS4 has been reported to be a novel apoptosis-related gene. Overexpression and knocking down of PNAS4 would cause zebrafish and Xenopus lavis developmental abnormalities. But its function and apoptotic mechanism in mammals are still unknown. Here, we first reported that established PNAS4 CKO (conditional knock out) mice using recombineering technology. We prepared its polyclonal antibodies which recognized both myc-PNAS4 overexpression protein and WT and CKO mice brain tissue and MEFS cells with high titre and specificity. Further we detected that PNAS4 was highly expressed in the embryonic period. However, we observed neither neural structural abnormality nor apoptosis signal in PNAS4 CKO mice brain. Our data suggested that PNAS4 was not involved in mice brain development and apoptosis.

NGS-based spinal muscular atrophy carrier screening of 10,585 diverse couples in China: a pan-ethnic study.

In this study, we performed a spinal muscular atrophy carrier screening investigation with NGS-based method. First, the validation for NGS-based method was implemented in 2255 samples using real-time PCR. The concordance between the NGS-based method and real-time PCR for the detection of SMA carrier and patient were up to 100%. Then, we applied this NGS-based method in 10,585 self-reported normal couples (34 Chinese ethnic groups from 5 provinces in South China) for SMA carrier screening. The overall carrier frequency was 1 in 73.8 (1.4%). It varied substantially between ethnic groups, highest in Dai ethnicity (4.3%), and no significant difference was found between five provinces. One couple was detected as carriers with an elevated risk of having an SMA affected baby. The distribution of SMN1:SMN2 genotype was also revealed in this study. Among the individuals with normal phenotype, the exon 7 copy-number ratio of SMN1 to SMN2 proved the gene conversion between them. With NGS-based method, we investigated SMA carrier status in Chinese population for the first time, and our results demonstrated that it is a promising alternative for SMA carrier screening and could provide data support and reference for future clinical application.

Exome sequencing identifies compound heterozygous mutations in CYP4V2 in a pedigree with retinitis pigmentosa.

Retinitis pigmentosa (RP) is a heterogeneous group of progressive retinal degenerations characterized by pigmentation and atrophy in the mid-periphery of the retina. Twenty two subjects from a four-generation Chinese family with RP and thin cornea, congenital cataract and high myopia is reported in this study. All family members underwent complete ophthalmologic examinations. Patients of the family presented with bone spicule-shaped pigment deposits in retina, retinal vascular attenuation, retinal and choroidal dystrophy, as well as punctate opacity of the lens, reduced cornea thickness and high myopia. Peripheral venous blood was obtained from all patients and their family members for genetic analysis. After mutation analysis in a few known RP candidate genes, exome sequencing was used to analyze the exomes of 3 patients III2, III4, III6 and the unaffected mother II2. A total of 34,693 variations shared by 3 patients were subjected to several filtering steps against existing variation databases. Identified variations were verified in the rest family members by PCR and Sanger sequencing. Compound heterozygous c.802-8_810del17insGC and c.1091-2A>G mutations of the CYP4V2 gene, known as genetic defects for Bietti crystalline corneoretinal dystrophy, were identified as causative mutations for RP of this family.

Arg124Cys mutation of the TGFBI gene in a Chinese pedigree of Reis-Bücklers corneal dystrophy.

AIM: To analyze mutations in transforming growth factor beta-induced (TGFBI) gene in a Chinese pedigree with Reis-Bücklers corneal dystrophy (RBCD, also known as GCD3). METHODS: In a five-generation Chinese family, eight members were identified with RBCD and the rest were unaffected. All members of the family underwent complete ophthalmologic examinations. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: A single heterozygous C>T (R124C) point mutation was found in exon 4 of TGFBI in all the affected members of the pedigree, but not in the unaffected members. CONCLUSION: R124C which was a known mutation for lattice corneal dystrophy type I, segregated with the RBCD in this pedigree. This elucidated the correlation between genotype and phenotype in a Chinese family of RBCD.

TGFBI gene mutation analysis in a Chinese pedigree of Avellino corneal dystrophy.

AIM: To analyze phenotype and genotype of a Chinese pedigree with Avellino corneal dystrophy (ACD). METHODS: Complete ophthalmic examinations were performed on all the family members. Exons of TGFBI were amplified by polymerase chain reaction, sequenced, and compared with a reference database. RESULTS: A single heterozygous G>A (R124H) point mutation was identified in exon 4 of TGFBI in three affected members and two unaffected children who were offsprings of the affected members, but not in the other family members. CONCLUSION: Mutation R124H in TGFBI was identified in this pedigree and appeared to be the disease causing mutation. Atypical phenotype and low penetrance was observed in this pedigree.

Rheb1 is required for mTORC1 and myelination in postnatal brain development.

mTor kinase is involved in cell growth, proliferation, and differentiation. The roles of mTor activators, Rheb1 and Rheb2, have not been established in vivo. Here, we report that Rheb1, but not Rheb2, is critical for embryonic survival and mTORC1 signaling. Embryonic deletion of Rheb1 in neural progenitor cells abolishes mTORC1 signaling in developing brain and increases mTORC2 signaling. Remarkably, embryonic and early postnatal brain development appears grossly normal in these Rheb1f/f,Nes-cre mice with the notable exception of deficits of myelination. Conditional expression of Rheb1 transgene in neural progenitors increases mTORC1 activity and promotes myelination in the brain. In addition the Rheb1 transgene rescues mTORC1 signaling and hypomyelination in the Rheb1f/f,Nes-cre mice. Our study demonstrates that Rheb1 is essential for mTORC1 signaling and myelination in the brain, and suggests that mTORC1 signaling plays a role in selective cellular adaptations, rather than general cellular viability.