The molecular mechanism of human stem cell-derived extracellular vesicles in retinal repair and regeneration.

Extracellular vesicles (EVs), including microvesicles (MVs) and exosomes, play a critical role in metabolic regulation and intracellular communication. Stem cell-derived EVs are considered to have the potential for regeneration, like stem cells, while simultaneously avoiding the risk of immune rejection or tumour formation. The therapeutic effect of stem cell-derived EVs has been proven in many diseases. However, the molecular mechanism of stem cell-derived EVs in retinal repair and regeneration has not been fully clarified. In this review, we described the biological characteristics of stem cell-derived EVs, summarized the current research on stem cell-derived EV treatment in retinal repair and regeneration, and discussed the potential and challenges of stem cell-derived EVs in translational medicine.

Complement C3 deficiency alleviates alkylation-induced retinal degeneration in mice.

BACKGROUND: It has been found that the extensive use of anticancer drugs containing DNA-alkylating agents not only target cancer cells but also cause retinal inflammation through toxic intermediates. Complement C3 (C3) is a core component of the complement activation pathway, and dysregulation of the complement pathway is involved in several retinal degenerative diseases. However, whether C3 plays a critical role in alkylation-induced retinal degeneration is unclear. METHODS: Following treatment with the alkylating agent methyl methane sulfonate (MMS), the C3 mRNA and protein level was measured, DNA damage and photoreceptor cell death were assessed in both wild-type (WT) C57BL/6J and C3 knockout (KO) mice. RESULTS: We determined that complement pathway is activated following MMS treatment, and C3 knockout (KO) increased the rate of photoreceptor cell survival and preserved visual function. The mRNA levels of nuclear erythroid-related factor 2 (Nrf2) and related genes were higher after MMS application in C3 KO mice. CONCLUSION: In summary, our study found that C3 KO promotes photoreceptor cell survival and activates the Nrf2 signaling pathway in the context of alkylation-induced retinal degeneration.

Experimental Study of the Biological Properties of Human Embryonic Stem Cell-Derived Retinal Progenitor Cells.

Retinal degenerative diseases are among the leading causes of blindness worldwide, and cell replacement is considered as a promising therapeutic. However, the resources of seed cells are scarce. To further explore this type of therapy, we adopted a culture system that could harvest a substantial quantity of retinal progenitor cells (RPCs) from human embryonic stem cells (hESCs) within a relatively short period of time. Furthermore, we transplanted these RPCs into the subretinal spaces of Royal College of Surgeons (RCS) rats. We quantified the thickness of the treated rats' outer nuclear layers (ONLs) and explored the visual function via electroretinography (ERG). It was found that the differentiated cells expressed RPC markers and photoreceptor progenitor markers. The transplanted RPCs survived for at least 12 weeks, resulting in beneficial effects on the morphology of the host retina, and led to a significant improvement in the visual function of the treated animals. These therapeutic effects suggest that the hESCs-derived RPCs could delay degeneration of the retina and partially restore visual function.

c-Kit⁺ cells isolated from human fetal retinas represent a new population of retinal progenitor cells.

Definitive surface markers for retinal progenitor cells (RPCs) are still lacking. Therefore, we sorted c-Kit(+) and stage-specific embryonic antigen-4(-) (SSEA4(-)) retinal cells for further biological characterization. RPCs were isolated from human fetal retinas (gestational age of 12-14 weeks). c-Kit(+)/SSEA4(-) RPCs were sorted by fluorescence-activated cell sorting, and their proliferation and differentiation capabilities were evaluated by using immunocytochemistry and flow cytometry. The effectiveness and safety were assessed following injection of c-Kit(+)/SSEA4(-) cells into the subretina of Royal College of Surgeons (RCS) rats. c-Kit(+) cells were found in the inner part of the fetal retina. Sorted c-Kit(+)/SSEA4(-) cells expressed retinal stem cell markers. Our results clearly demonstrate the proliferative potential of these cells. Moreover, c-Kit(+)/SSEA4(-) cells differentiated into retinal cells that expressed markers of photoreceptor cells, ganglion cells and glial cells. These cells survived for at least 3 months after transplantation into the host subretinal space. Teratomas were not observed in the c-Kit(+)/SSEA4(-)-cell group. Thus, c-Kit can be used as a surface marker for RPCs, and c-Kit(+)/SSEA4(-) RPCs exhibit the ability to self-renew and differentiate into retinal cells.

[Mitochondrial 12S rRNA variants studies in 456 subjects with hearing loss in seven schools for deaf and mutes in Zhejiang province].

OBJECTIVE: To investigate mutational spectrum and frequency of the mitochondrial 12S rRNA gene in Chinese subjects with aminoglycoside-induced and non-syndromic hearing loss. METHODS: Total of 456 subjects with non-syndromic hearing loss were recruited from seven schools for deaf-mutes in Zhejiang province. Genomic DNA was extracted from the whole blood, and then the DNA fragment was amplified spanning the 12S rRNA gene, followed by sequencing and analyzed. RESULTS: Thirty-one variants were identified by mutation analysis of 12S rRNA gene in these subjects. The frequency of the known 1555A > G mutation was 4.4% (20/456). Prevalence of other putative deafness-associated mutation at positions 961 and 1095 were 2.0% (9/456) and 0.7% (3/456) respectively. Furthermore, the 1027A > G, 1109T > C and 1431G > A variants conferred increased sensitivity to ototoxic drugs or non-syndromic deafness as they were absent in 449 Chinese controls and localized at highly conserved nucleotides of this 12S rRNA gene. Moreover, clinical data showed a wide range of age-of-onset, variety of severity and various audiometric configurations in subjects carrying the 1555A > G mutation. CONCLUSIONS: Our data demonstrated that the mitochondrial 12S rRNA gene is the hot spot for mutations associated with aminoglycoside ototoxicity and non-syndromic hearing loss. Nuclear modifier genes, mitochondrial haplotypes and environmental factors might play a role in the phenotypic manifestation of these mutations.

The orphan nuclear hormone receptor ERRbeta controls rod photoreceptor survival.

Mutation of rod photoreceptor-enriched transcription factors is a major cause of inherited blindness. We identified the orphan nuclear hormone receptor estrogen-related receptor beta (ERRbeta) as selectively expressed in rod photoreceptors. Overexpression of ERRbeta induces expression of rod-specific genes in retinas of wild-type as well as Nrl(-/-) mice, which lack rod photoreceptors. Mutation of ERRbeta results in dysfunction and degeneration of rods, whereas inverse agonists of ERRbeta trigger rapid rod degeneration, which is rescued by constitutively active mutants of ERRbeta. ERRbeta coordinates expression of multiple genes that are rate-limiting regulators of ATP generation and consumption in photoreceptors. Furthermore, enhancing ERRbeta activity rescues photoreceptor defects that result from loss of the photoreceptor-specific transcription factor Crx. Our findings demonstrate that ERRbeta is a critical regulator of rod photoreceptor function and survival, and suggest that ERRbeta agonists may be useful in the treatment of certain retinal dystrophies.

Determination of catechin in lotus rhizomes by high-performance liquid chromatography.

A novel method was developed to analyze lotus rhizome polyphenolic catechin using high-performance liquid chromatography (HPLC). The retain time of catechin was 14.72 min under the optimized condition. Mass spectrometry was further employed to qualify and quantify the purity of the catechin peak. Good linearity (R=0.9997) was obtained within the range of 50-1,000 ng. The coefficient of variance was determined as 5.2%, with a recovery rate of 97%. The detection and quantification limitations of catechin were 23 ng and 50 ng, respectively. The catechin level was 0.0025% in the lotus rhizome, and 0.011% in the knot of the lotus rhizome (Nelumbo nucifera cv. 'damao jie'). The optimized conditions of HPLC for catechin detection in the lotus rhizome matrix were as follows: the SuperlcosIL™ LC-18 analytical column (150 mm×4.6 mm, 5 µm), methanol-water-acetic acid (10:90:1, volume ratio) as the mobile phase, an UV detector at 280 nm, a flow rate of 0.8 ml/min, column temperature at 30°C, and an injection volume of 10 µl.

Crx activates opsin transcription by recruiting HAT-containing co-activators and promoting histone acetylation.

The homeodomain transcription factor Crx is required for expression of many photoreceptor genes in the mammalian retina. The mechanism by which Crx activates transcription remains to be determined. Using protein-protein interaction assays, Crx was found to interact with three co-activator proteins (complexes): STAGA, Cbp and p300, all of which possess histone acetyl-transferase (HAT) activity. To determine the role of Crx-HAT interactions in target gene chromatin modification and transcriptional activation, quantitative RT-PCR and chromatin immunoprecipitation were performed on Crx target genes, rod and cone opsins, in developing mouse retina. Although cone opsins are transcribed earlier than rhodopsin during development, the transcription of each gene is preceded by the same sequence of events in their promoter and enhancer regions: (i) binding of Crx, followed by (ii) binding of HATs, (iii) the acetylation of histone H3, then (iv) binding of other photoreceptor transcription factors (Nrl and Nr2e3) and RNA polymerase II. In Crx knockout mice (Crx(-/-)), the association of HATs and AcH3 with target promoter/enhancer regions was significantly decreased, which correlates with aberrant opsin transcription and photoreceptor dysfunction in these mice. Similar changes to the opsin chromatin were seen in Y79 retinoblastoma cells, where opsin genes are barely transcribed. These defects in Y79 cells can be reversed by expressing a recombinant Crx or applying histone deacetylase inhibitors. Altogether, these results suggest that one mechanism for Crx-mediated transcriptional activation is to recruit HATs to photoreceptor gene chromatin for histone acetylation, thereby inducing and maintaining appropriate chromatin configurations for transcription.

Polyglutamine-expanded ataxin-7 inhibits STAGA histone acetyltransferase activity to produce retinal degeneration.

Spinocerebellar ataxia type 7 (SCA7) is characterized by cone-rod dystrophy retinal degeneration and is caused by a polyglutamine [poly(Q)] expansion within ataxin-7, a protein of previously unknown function. Here, we report that ataxin-7 is an integral component of the mammalian STAGA (SPT3-TAF9-ADA-GCN5 acetyltransferase) transcription coactivator complex, interacts directly with the GCN5 histone acetyltransferase component of STAGA, and mediates a direct interaction of STAGA with the CRX (cone-rod homeobox) transactivator of photoreceptor genes. Consistent with these results, chromatin immunoprecipitation assays document retinal-specific association of CRX, GCN5, and acetylated histone H3 with CRX target genes. RNA interference studies also implicate ataxin-7 and GCN5 in CRX-dependent gene activation, and histone deacetylase inhibitors restore the compromised expression of a CRX target gene in an ataxin-7-deficient background. Significantly, in relation to SCA7, poly(Q)-expanded ataxin-7 gets incorporated into STAGA and, in a dominant-negative manner, inhibits the nucleosomal histone acetylation function of STAGA GCN5 both in vitro and, based on chromatin immunoprecipitation assays, in SCA7 transgenic mice. These results suggest that the normal function of a poly(Q) disease protein may intersect with its pathogenic mechanism, an observation with significant implications for the molecular basis of all poly(Q) disorders and ultimately for their treatment.

Sp4 is expressed in retinal neurons, activates transcription of photoreceptor-specific genes, and synergizes with Crx.

To investigate the molecular mechanisms of photoreceptor-specific gene transcription, we examined the role of the neuronal-enriched Sp4 nuclear protein in transcription from the rod-specific beta-PDE and rod opsin gene promoters and compared it to the ubiquitous members of the Sp family, Sp1 and Sp3. Sp4 activates both the rod opsin and beta-PDE promoters, whereas Sp1 activates only the rod opsin promoter and Sp3 activates neither promoter. Interestingly, Sp1 and Sp3 competitively repress Sp4-mediated activation of the beta-PDE promoter. In addition, Sp4, Sp1, and Sp3 each show functional synergy with the photoreceptor-enriched Crx transcriptional regulator on the rod opsin promoter but not the beta-PDE promoter, although Sp4-mediated activation was the most significant. Sp4, Sp1, and Sp3 bind Crx in co-immunoprecipitation experiments, and their zinc finger domains as well as the Crx homedomain are necessary and sufficient for these interactions. Chromatin immunoprecipitation showed that the rod opsin and beta-PDE promoters are targets of both Sp4 and Crx, which further supports Sp4-Crx interactions in vivo in the context of retinal chromatin environment. In situ hybridization and immunohistochemistry demonstrated that Sp4 is abundantly expressed in various neurons of all retinal layers, and thus co-localizes or overlaps with multiple retina-restricted and -enriched genes, its putative targets. Our results indicate that photoreceptor-specific gene transcription is controlled by the combinatorial action of Sp4 and Crx. The other Sp family members may be involved in photoreceptor-specific transcription directly or through their competition with Sp4. These data suggest the potential importance of Sp4 in retinal neurobiology and pathology.