Yehong Zhuo

Caspase-8 promotes NLRP1/NLRP3 inflammasome activation and IL-1β production in acute glaucoma

Significance Retinal damage and resulting irreversible vision loss are feared complications of rapid and substantially elevated intraocular pressure (IOP) in acute glaucoma. An inflammatory response to retinal ischemia/reperfusion injury involving Toll-like receptor 4 (TLR4) and IL-1β has been implicated in disease pathogenesis; however, the underlying mechanisms remain incompletely understood. This study demonstrates the critical role of caspase-8 in IOP-induced cell death in rodent models of acute glaucoma. TLR4 signaling, mediated by caspase-8, was crucial for the activation of Nod-like receptor family, pyrin domain containing 1 (NLRP1)/NLRP3 inflammasomes and processing of IL-1β. Inhibition of either TLR4 or caspase-8 signaling significantly blocked production of IL-1β and attenuated retinal ischemic damage. These findings identify a mechanism of retinal retinal ganglion cell death and provide a previously unidentified treatment strategy to preserve vision in acute glaucoma. Acute glaucoma is a sight-threatening condition characterized by a sudden and substantial rise in intraocular pressure (IOP) and consequent retinal ganglion cell (RGC) death. Angle closure glaucoma, a common cause of glaucoma in Asia that affects tens of millions of people worldwide, often presents acutely with loss of vision, pain, and high IOP. Even when medical and surgical treatment is available, acute angle closure glaucoma can cause permanent and irreversible loss of vision. Toll-like receptor 4 (TLR4) signaling has been previously implicated in the pathogenesis of IOP-induced RGC death, although the underlying mechanisms are largely unknown. In the present study, we used an acute IOP elevation/glaucoma model to investigate the underlying mechanism of RGC death. We found that TLR4 leads to increased caspase-8 expression; this elevation increases IL-1β expression and RGC death via a caspase-1–dependent pathway involving Nod-like receptor family, pyrin domain containing 1 (NLRP1)/NLRP3 inflammasomes and a caspase-1–independent pathway. We show that inhibition of caspase-8 activation significantly attenuates RGC death by down-regulating the activation of NLRP1 and NLRP3, thus demonstrating the pivotal role of caspase-8 in the TLR4-mediated activation of inflammasomes. These findings demonstrate collectively a critical role of caspase-8 in transducing TLR4-mediated IL-1β production and RGC death and highlight signal transduction in a caspase-1–dependent NLRP1/NLRP3 inflammasome pathway and a caspase-1–independent pathway in acute glaucoma. These results provide new insight into the pathogenesis of glaucoma and point to a treatment strategy.

Generation of Retinal Ganglion–like Cells from Reprogrammed Mouse Fibroblasts

PURPOSE Somatic cells can be reprogrammed into an embryonic stem cell-like pluripotent state by Oct-3/4, Sox2, c-Myc, and Klf4. Sox2 as an essential reprogramming factor also contributes to the development of the eye and the retina. This study was conducted to determine whether induced pluripotent stem (iPS) cells express retinal progenitor cell (RPC)-related genes and whether iPS cells can directly differentiate into retinal ganglion cells (RGCs). METHODS Mouse iPS cells were induced by the ectopically expressed four factors in tail-tip fibroblasts (TTFs). The expression of RPC-related genes in iPS cells was analyzed by RT-PCR and immunofluorescence. iPS cells were induced to differentiate into RGCs by the addition of Dkk1 + Noggin (DN) + DAPT and overexpression of Math5. iPS-derived retinal ganglion (RG)-like cells were injected into the retina, and the eyes were analyzed by immunohistochemistry. RESULTS iPS cells inherently express RPC-related genes such as Pax6, Rx, Otx2, Lhx2, and Nestin. Overexpression of Math5 and addition of DN can directly differentiate iPS into retinal ganglion-like cells. These iPS-derived RG-like cells display long synapses and gene expression patterns, including Math5, Brn3b, Islet-1, and Thy1.2. Furthermore, inhibiting Hes1 by DAPT increases the expression of RGC marker genes. In addition, iPS-derived RG-like cells were able to survive but were unable to be integrated into the normal retina after transplantation. CONCLUSIONS The four factor iPS cell inherently expressed RPC-related genes, and the iPS cell could be further turned into RG-like cells by the regulation of transcription factor expression. These findings demonstrate that iPS cells are valuable for regeneration research into retinal degeneration diseases.

Serum Starvation Induced Cell Cycle Synchronization Facilitates Human Somatic Cells Reprogramming

Human induced pluripotent stem cells (iPSCs) provide a valuable model for regenerative medicine and human disease research. To date, however, the reprogramming efficiency of human adult cells is still low. Recent studies have revealed that cell cycle is a key parameter driving epigenetic reprogramming to pluripotency. As is well known, retroviruses such as the Moloney murine leukemia virus (MoMLV) require cell division to integrate into the host genome and replicate, whereas the target primary cells for reprogramming are a mixture of several cell types with different cell cycle rhythms. Whether cell cycle synchronization has potential effect on retrovirus induced reprogramming has not been detailed. In this study, utilizing transient serum starvation induced synchronization, we demonstrated that starvation generated a reversible cell cycle arrest and synchronously progressed through G2/M phase after release, substantially improving retroviral infection efficiency. Interestingly, synchronized human dermal fibroblasts (HDF) and adipose stem cells (ASC) exhibited more homogenous epithelial morphology than normal FBS control after infection, and the expression of epithelial markers such as E-cadherin and Epcam were strongly activated. Futhermore, synchronization treatment ultimately improved Nanog positive clones, achieved a 15–20 fold increase. These results suggested that cell cycle synchronization promotes the mesenchymal to epithelial transition (MET) and facilitates retrovirus mediated reprogramming. Our study, utilization of serum starvation rather than additional chemicals, provide a new insight into cell cycle regulation and induced reprogramming of human cells.

The Role of Long Noncoding RNAs in Neurodegenerative Diseases.

Long noncoding RNAs (lncRNAs) are transcripts with low protein-coding potential but occupy a large part of transcriptional output. Their roles include regulating gene expression at the epigenetic, transcriptional, and post-transcriptional level in cellular homeostasis. However, lncRNA studies are still in their infancy and the functions of the vast majority of lncRNA transcripts remain unknown. It is generally known that the function of the human nervous system largely relies on the precise regulation of gene expression. Various studies have shown that lncRNAs have a significant impact on normal neural development and on the development and progression of neurodegenerative diseases. In this review, we focused on recent studies associated with lncRNAs in neurodegenerative diseases, including Alzheimer’s disease (AD), Parkinson’s disease (PD), Huntington’s disease (HD), amyotrophic lateral sclerosis (ALS), multiple system atrophy (MSA), frontotemporal lobar degeneration (FTLD), and glaucoma. Glaucoma, caused by unexplained ganglion cell lesion and apoptosis, is now labeled as a chronic neurodegenerative disorder [1], and therefore, we discussed the association of lncRNAs with glaucoma as well. We illustrate the role of some specific lncRNAs, which may provide new insights into our understanding of the etiology and pathophysiology of the neurodegenerative diseases mentioned above.

TCF7L2 Variation and Proliferative Diabetic Retinopathy

Proliferative diabetic retinopathy (PDR) is the most severe vision-threatening complication of diabetes. For investigation of genetic association between TCF7L2 and PDR in Caucasian type 2 diabetes mellitus (T2DM) and its functional consequences, 383 T2DM patients with PDR (T2DM-PDR) and 756 T2DM patients without diabetic retinopathy (T2DM–no DR) were genotyped with rs7903146 in TCF7L2. We found that risk allele (T) frequency of rs7903146 was significantly higher in T2DM-PDR patients (allelic P = 2.52E-04). In lymphoblastoid cells induced to undergo endoplasmic reticulum (ER) stress by treatment of tunicamycin, higher fold change of TCF7L2 and VEGFA mRNA levels were observed in rs7903146-TT cells than in rs7903146-CC cells (P = 0.02 for TCF7L2; P = 0.004 for VEGFA), suggesting that ER stress plays a role in PDR pathogenesis. Silencing TCF7L2 resulted in decreased mRNA levels of both TCF7L2 and VEGFA (P < 0.001). Retinas of oxygen-induced retinopathy mice (a model for PDR) had higher TCF7L2 and VEGFA mRNA levels than those of controls (P = 2.9E-04 for TCF7L2; P = 1.9E-07 for VEGFA). Together, data from our study show that TCF7L2-rs7903146 is associated with PDR in Caucasian T2DM and suggest that TCF7L2 promotes pathological retinal neovascularization via ER stress–dependent upregulation of VEGFA.

Phacoemulsification treatment of subjects with acute primary angle closure and chronic primary angle-closure glaucoma.

PurposeTo investigate and compare the effects of phacoemulsification as an initial procedure to control intraocular pressure (IOP) in eyes with acute primary angle closure (APAC) and chronic primary angle-closure glaucoma (CPACG) with coexisting cataracts and peripheral anterior synechiae (PAS) of greater than 180 degrees. SettingProspective, cross-sectional study. MethodsThirteen patients (13 eyes) with APAC and 10 patients (10 eyes) with CPACG were enrolled in the study. IOP control in the 2 groups was compared at final follow-up. Other outcome measures included visual acuity, anterior chamber depth, gonioscopic appearance, and complications. ResultsThe postoperative IOP was reduced in both the APAC and CPACG groups. As assessed at last follow-up (median 6 mo), IOP in the APAC group was significantly lower than that in the CPACG group. The absolute success rate was 100% in the APAC group and 80% in the CPACG group. The number of hypotensive medications decreased in both APAC and CPACG group. The postoperative degree of PAS was relieved in 11 of 12 eyes with APAC and 5 of 9 eyes with CPACG. ConclusionsInitial phacoemulsification performed to provide short-term control of IOP is more efficacious in patients with APAC than in those with CPACG, especially when PAS is greater than 180 degrees.

Rapamycin Is Neuroprotective in a Rat Chronic Hypertensive Glaucoma Model

Glaucoma is a leading cause of irreversible blindness. Injury of retinal ganglion cells (RGCs) accounts for visual impairment of glaucoma. Here, we report rapamycin protects RGCs from death in experimental glaucoma model and the underlying mechanisms. Our results showed that treatment with rapamycin dramatically promote RGCs survival in a rat chronic ocular hypertension model. This protective action appears to be attributable to inhibition of neurotoxic mediators release and/or direct suppression of RGC apoptosis. In support of this mechanism, in vitro, rapamycin significantly inhibits the production of NO, TNF-α in BV2 microglials by modulating NF-κB signaling. In experimental animals, treatment with rapamycin also dramatically inhibited the activation of microglials. In primary RGCs, rapamycin was capable of direct suppression the apoptosis of primary RGCs induced by glutamate. Mechanistically, rapamycin-mediated suppression of RGCs apoptosis is by sparing phosphorylation of Akt at a site critical for maintenance of its survival-promoting activity in cell and animal model. These results demonstrate that rapamycin is neuroprotective in experimental glaucoma, possibly via decreasing neurotoxic releasing and suppressing directly apoptosis of RGCs.

Autophagy in Retinal Ganglion Cells in a Rhesus Monkey Chronic Hypertensive Glaucoma Model

Primary open angle glaucoma (POAG) is a neurodegenerative disease characterized by physiological intraocular hypertension that causes damage to the retinal ganglion cells (RGCs). In the past, RGC damage in POAG was suggested to have been attributed to RGC apoptosis. However, in the present study, we applied a model closer to human POAG through the use of a chronic hypertensive glaucoma model in rhesus monkeys to investigate whether another mode of progressive cell death, autophagy, was activated in the glaucomatous retinas. First, in the glaucomatous retinas, the levels of LC3B-II, LC3B-II/LC3B-I and Beclin 1 increased as demonstrated by Western blot analyses, whereas early or initial autophagic vacuoles (AVi) and late or degraded autophagic vacuoles (AVd) accumulated in the ganglion cell layer (GCL) and in the inner plexiform layer (IPL) as determined by transmission electron microscopy (TEM) analysis. Second, lysosome activity and autophagosome-lysosomal fusion increased in the RGCs of the glaucomatous retinas, as demonstrated by Western blotting against lysosome associated membrane protein-1 (LAMP1) and double labeling against LC3B and LAMP1. Third, apoptosis was activated in the glaucomatous eyes with increased levels of caspase-3 and cleaved caspase-3 and an increased number of TUNEL-positive RGCs. Our results suggested that autophagy was activated in RGCs in the chronic hypertensive glaucoma model of rhesus monkeys and that autophagy may have potential as a new target for intervention in glaucoma treatment.

Mobile zinc increases rapidly in the retina after optic nerve injury and regulates ganglion cell survival and optic nerve regeneration

Significance The inability of CNS pathways to regenerate after injury can lead to devastating, life-long losses in sensory, motor, and other functions. We report that after injury to the optic nerve, a widely studied CNS pathway that normally cannot regenerate, mobile zinc (Zn2+) increases rapidly in the processes of retinal interneurons (amacrine cells) and then transfers via vesicular release to retinal ganglion cells (RGCs), the injured projection neurons. Eliminating Zn2+ leads to both persistent RGC survival and substantial axon regeneration with a broad therapeutic window. These findings show that signaling between interneurons and RGCs contributes to regulating the fate of RGCs after optic nerve injury, and that Zn2+ chelation may provide a potent therapeutic approach. Retinal ganglion cells (RGCs), the projection neurons of the eye, cannot regenerate their axons once the optic nerve has been injured and soon begin to die. Whereas RGC death and regenerative failure are widely viewed as being cell-autonomous or influenced by various types of glia, we report here that the dysregulation of mobile zinc (Zn2+) in retinal interneurons is a primary factor. Within an hour after the optic nerve is injured, Zn2+ increases several-fold in retinal amacrine cell processes and continues to rise over the first day, then transfers slowly to RGCs via vesicular release. Zn2+ accumulation in amacrine cell processes involves the Zn2+ transporter protein ZnT-3, and deletion of slc30a3, the gene encoding ZnT-3, promotes RGC survival and axon regeneration. Intravitreal injection of Zn2+ chelators enables many RGCs to survive for months after nerve injury and regenerate axons, and enhances the prosurvival and regenerative effects of deleting the gene for phosphatase and tensin homolog (pten). Importantly, the therapeutic window for Zn2+ chelation extends for several days after nerve injury. These results show that retinal Zn2+ dysregulation is a major factor limiting the survival and regenerative capacity of injured RGCs, and point to Zn2+ chelation as a strategy to promote long-term RGC protection and enhance axon regeneration.

Endoplasmic reticulum stress promotes amyloid-beta peptides production in RGC-5 cells

Endoplasmic reticulum (ER) stress has been implicated in various neurodegenerative diseases, including Alzheimer’s disease. We have previously observed amyloid production in the retina of the Tg2576 transgenic mouse model of Alzheimer’s disease. In this study, we used tunicamycin-induced ER stress in RGC-5 cells, a cell line identical to the photoreceptor cell line 661W, to investigate the effect of ER stress on production of amyloid-beta (Abeta) peptides. We found that the mRNA level of amyloid-beta precursor protein (APP) remained stable, while the protein level of amyloid-beta precursor protein (APP) was decreased, the amyloid-beta precursor protein cleaving enzymes beta-site APP-cleaving enzyme 1 and presenilin 1 were upregulated, Abeta1–40 and Abeta1–42 production were increased, and reactive oxygen species production and apoptosis markers were elevated following induction of ER stress. The protein level of Abeta degradation enzymes, neprilysin, endothelin-converting enzyme 1, and endothelin-converting enzyme 2 remained unchanged during the prolonged ER stress, showing that the generation of Abeta did not result from reduction of proteolysis by these enzymes. Inclusion of group II caspase inhibitor, Z-DEVD-FMK, increased the ER stress mediated Abeta production, suggesting that they are generated by a caspase-independent mechanism. Our findings provided evidence of a role of ER stress in Abeta peptide overproduction and apoptotic pathway activation in RGC-5 cells.