The E50K optineurin mutation impacts autophagy-mediated degradation of TDP-43 and leads to RGC apoptosis in vivo and in vitro.

The glaucoma-associated E50K mutation in optineurin (OPTN) is known to affect autophagy and cause the apoptosis of retinal ganglion cells (RGCs), but the pathogenic mechanism remains unclear. In this study, we investigated whether the OPTN (E50K) mutation caused TDP-43 aggregation by disrupting autophagy in vivo and in vitro. OPTN (E50K) mutant mice were generated and analysed for genotype and phenotype. Adeno-associated virus type 2 vectors containing either GFP only, GFP-tagged wild-type OPTN or GFP-tagged E50K-mutated OPTN were used to transfect R28 cells. Loss of RGCs decreased retinal thickness and visual impairment were observed in OPTN (E50K) mice compared with WT mice. Moreover, overexpression of E50K OPTN induced R28 cell apoptosis. Increased p62/SQSTM1 and LC3-II levels indicated that autophagic flux was inhibited and contributed to TDP-43 aggregation in vivo and in vitro. We found that rapamycin effectively reduced the aggregation of TDP-43 in OPTN (E50K) mice and decreased the protein levels of p62/SQSTM1 and the autophagic marker LC3-II. Moreover, rapamycin increased the RGC number and visual function of E50K mice. In addition, we also observed increased cytoplasmic TDP-43 in the spinal cord and motor dysfunction in 24-month-old OPTN (E50K) mice, indicating that TDP-43 accumulation may be the common pathological mechanism of glaucoma and amyotrophic lateral sclerosis (ALS). In conclusion, the disruption of autophagy by OPTN (E50K) affected the degradation of TDP-43 and may play an important role in OPTN (E50K)-mediated glaucomatous retinal neurodegeneration.

Synergistic neuroprotective effect of rasagiline and idebenone against retinal ischemia-reperfusion injury via the Lin28-let-7-Dicer pathway.

Retinal ischemia-reperfusion (RIR) injury causes neuronal degeneration and initiates various optic nerve diseases. This study aimed to investigate the synergistic neuroprotective effect of rasagiline and idebenone against RIR injury. A combination of rasagiline and idebenone was administered intraperitoneally immediately after establishment of the RIR model. Treatment with the combination of the two drugs resulted in a significant restoration of retinal thickness and retinal ganglion cells. Apoptosis of cells in ganglion cell layers was also ameliorated, suggesting that the effect of the two drugs was synergistic and the expression of brain-derived neurotrophic factor increased. Furthermore, idebenone and rasagiline induced the expression of Lin28A and Lin28B, respectively, which resulted in a reduced expression of microRNAs in the let-7 family and an increased protein output of Dicer. The data obtained from gene overexpression and knockdown experiments indicated that let-7 and Dicer were necessary for the synergistic neuroprotective effect of the two drugs. Our findings suggested that combination therapy with rasagiline and idebenone produced a synergistic effect that ameliorated RIR injury and restored visual function. In addition, the combined treatment provided neuroprotection via enhancement of the selective regulation of let-7 by Lin28A/B. These findings implied that a treatment with the combination of rasagiline and idebenone is a feasible treatment option for optic nerve diseases.

Decreased expression of miR­9 due to E50K OPTN mutation causes disruption of the expression of BDNF leading to RGC­5 cell apoptosis.

The aims of the present study were to investigate the effect of E50K optineurin (OPTN) mutation on RGC­5 cells and to define the role of microRNA­9 (miR­9) in this system. Transfected RGC­5 cells were used to evaluate the effects of E50K OPTN on the expression of miR­9 and subsequent disruption of RGC­5 cell apoptosis was analyzed using western blotting. The results showed that the expression of E50K OPTN was associated with a marked reduction in the levels of miR­9 in the E50K OPTN­transfected RGC­5 cells. The E50K OPTN­dependent reductions in miR­9 led to increased expression of the transcriptional repressor, RE1­silencing transcription factor and decreased the expression of brain­derived neurotrophic factor. Thus, E50K OPTN may disrupt the expression of miR­9, suggesting a potential mechanism by which E50K OPTN mutation may lead to RGC­5 cell apoptosis.

Lentiviral Delivery of Small Hairpin RNA Targeting Connective Tissue Growth Factor Blocks Profibrotic Signaling in Tenon's Capsule Fibroblasts.

PURPOSES: Trabeculectomy is a surgical procedure for lowering intraocular pressure in glaucoma patients, in which excessive scarring leading to failure of the filtering bleb adversely affects the surgical outcome. Heightened Tenon's capsule fibroblast (TCF) proliferation and extracellular matrix (ECM) deposition are implicated in this process but endogenous factors that regulate TCF functions remain largely elusive. This study sought to elucidate the role of connective tissue growth factor (CTGF) in the regulation of TCF phenotypes and signaling. METHODS: Expression of CTGF in scarring and nonscarring Tenon's capsules was measured by real-time PCR and immunofluorescence. Knockdown of CTGC was achieved by lentivirus delivery of small-hairpin RNA. Cell proliferation was measured by CCK8, cell cycle progression, and apoptosis by flow cytometry, adhesion, migration, and invasion of TCF by functional assays in vitro. Proteins and cytokines related to fibrosis were measured by Western blot and ELISA, respectively. RESULTS: Expression of CTGF was significantly upregulated in scarring Tenon's capsules and their isolated fibroblasts when compared with the nonfibrotic counterparts. Functionally, targeting CTGF with lentivirus-delivered small-hairpin RNA inhibited the proliferation, adhesion, migration, and invasion of TCFs, accompanied by downregulation of p38 and nuclear factor-κB as well as matrix metalloproteinase-2, cyclin D1, and collagen I. In addition, lentiviral targeting of CTGF reduced the release of fibrosis-related cytokines from TCFs and inhibited TCF-conditioned, medium-induced macrophage chemotaxis. CONCLUSIONS: Our study supports a crucial role of CTGF in the regulation of TCF proliferation and ECM deposition. Targeting CTGF using lentiviral vector may be a promising approach for preventing excessive scarring after trabeculectomy.

Expression profiling of microRNAs in optineurin (E50K) mutant transgenic mice.

An E50K substitution in the transcription factor optineurin (OPTN) induces primary open-angle glaucoma (POAG). To explore the potential role of microRNAs (miRNAs) in E50K OPTN-induced POAG, miRNA expression profiling was performed on retinal samples from OPTN (E50K) transgenic and wild-type mice. The retinas were collected from 30 transgenic and 30 wild-type mice, and miRNA expression was evaluated using a genome-wide miRNA microarray. miRNAs that were differentially expressed in retinal samples from OPTN (E50K) transgenic mice were identified and validated by reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additional gene ontology and signaling pathway analyses were performed using bioinformatics tools. A total of 48 miRNAs exhibited increased or decreased expression in the retinas from OPTN (E50K) transgenic mice when compared with the expression in the retinas from wild-type mice. A total of 5 miRNAs with increased expression in OPTN (E50K) transgenic mice could be grouped into one cluster as they belong to the miR-8 family and may act as regulators in the development of POAG in OPTN (E50K) transgenic mice. RT-qPCR results confirmed significantly increased expression of miR-141 in the retinas of OPTN (E50K) transgenic mice as compared to wild-type mice. In conclusion, these results show that certain miRNAs are differentially expressed in the retinas of OPTN (E50K) transgenic mice and may play roles in the pathogenesis of POAG induced by OPTN (E50K).

[Protective effects of metallothionein induced by dexamethasone against ischemia/reperfusion injury of myocardium of isolated rat heart].

OBJECTIVE: To investigate the protective effects of metallothionein (MT) induced by dexamethasone (DEX) against ischemia/reperfusion (I/R) injury of myocardium of isolated rat heart. METHODS: Thirty-two Sprague-Dawley (SD) rats were divided randomly into the DEX and control groups. In the former group, the rats were pretreated with DEX, and in latter group distilled water was given before their hearts were isolated for Langendorff perfusion and I/R. MT was assessed by Western blotting. The left ventricular developed pressure (LVDP), maximal change rate of intraventricular pressure rise/down (+/-dp/dt max), coronary artery flow (CF) and reperfusion arrhythmias were observed dynamically before ischemia and during 60-minute reperfusion following 30-minute ischemia, The hearts were perfused with triphenyltetrazolium (TTC) after 60-minute reperfusion. The myocardial infarct size was measured with Adobe Photoshop. The levels of MB isoenzyme of creatine kinase (CK-MB), malonaldehyde (MDA), total superoxide dismutase (T-SOD), CuZn-SOD, catalase (CAT), glutathione peroxidase (GSH-Px) and the activities of Na+-K+-ATPase, Ca2+-Mg2+-ATPase were detected. RESULTS: Compared with control group, the expression of MT was significantly increased (3.085+/-1.065 vs. 1.028+/-0.016, P<0.05), the LVDP, +/-dp/dt max and CF were greatly improved (all P<0.05), the accumulated point of ventricular arrhythmia and the infarct size were significantly reduced in DEX group [(2.00+/-1.41) scores vs. (6.63+/-4.24) scores and (28.38+/-11.22)% vs. (47.39+/-8.30)%, respectively, both P<0.01]. The level of CK-MB was significantly lowered in the DEX group compared with control group [(8.69+/-4.16)U/g vs. (18.15+/-5.59) U/g, P<0.01], and myocardium MDA content was decreased (P<0.05). Moreover, the levels of T-SOD, CuZn-SOD, CAT, GSH-Px, and the activities of Na+-K+-ATPase and Ca2+-Mg2+-ATPase were significantly increased (P<0.05 or/and P<0.01) in DEX group. CONCLUSION: DEX induces upregulation of MT, which attenuates I/R injury in rat heart.

Genomic analysis of the rat lung following elemental mercury vapor exposure.

Elemental mercury (Hg0) is a highly toxic chemical with increasing public health concern. Although the lung receives the highest exposure to Hg0 vapor, it is resistant to Hg0 toxicity relative to the kidney and brain. In an earlier study, exposure of rats to 4 mg Hg0 vapor/m3, 2 h per day for 10 days, did not produce pathological alterations in the lung but increased metallothionein and glutathione S-transferase in the kidney. This study was undertaken to examine pulmonary gene expression associated with Hg0 vapor inhalation. Total RNA was extracted from lung tissues of rats, previously exposed to air or Hg0 vapor, and subjected to microarray analysis. Hg0 vapor exposure increased the expression of genes encoding inflammatory responses, such as chemokines, tumor necrosis factor-alpha (TNFalpha), TNF-receptor-1, interleukin-2 (IL-2), IL-7, prostaglandin E2 receptor, and heat-shock proteins. As adaptive responses, glutathione S-transferases (GST-pi, mGST1), metallothionein, and thioredoxin peroxidase were all increased in response to Hg exposure. Some transporters, such as multidrug resistance-associated protein (MRP), P-glycoprotein, and zinc transporter ZnT1, were also increased in an attempt to reduce pulmonary Hg load. The expression of transcription factor c-jun/AP-1 and PI3-kinases was suppressed, while the expression of protein kinase-C was increased. Expression of epidermal fatty acid-binding protein was also enhanced. Real-time RT-PCR and Western blot analyses confirmed the microarray results. In summary, genomic analysis revealed an array of gene alterations in response to Hg0 vapor exposure, which could be important for the development of pulmonary adaptation to Hg during Hg0 vapor inhalation.