Correlation of Cytokine Levels and Microglial Cell Infiltration during Retinal Degeneration in RCS Rats

Microglial cells, which are immunocompetent cells, are involved in all diseases of the central nervous system. During their activation in various diseases, a variety of soluble factors are released. In the present study, the correlation between cytokine levels and microglial cell migration in the course of retinal degeneration of Royal College of Surgeons (RCS) rats was evaluated. MFG-E8 and CD11b were used to confirm the microglial cells. In the retina of RCS rats, the mRNA expression of seven genes (MFG-E8 and its integrins αυ and ß5, CD11b and the cytokines TNF-α, IL-1ß, and MCP-1) formed almost similar bimodal peak distributions, which were centred at P7 and P45 to P60. In contrast, in rdy rats, which comprised the control group, a unimodal peak distribution centred at P14 was observed. The gene expression accompanied the activation and migration of microglial cells from the inner to the outer layer of the retina during the process of degeneration. Principal component analysis and discriminant function analysis revealed that the expression of these seven genes, especially TNF-α and CD11b, positively correlated with retinal degeneration and microglial activity during retinal degeneration in RCS rats, but not in the control rats. Furthermore, linear regression analysis demonstrated a significant correlation between the expression of these genes and the activation of microglial cells in the dystrophic retina. Our findings suggest that the suppression of microglial cells and the blockade of their cytotoxic effects may constitute a novel therapeutic strategy for treating photoreceptor death in various retinal disorders

Essential Role of MFG-E8 for Phagocytic Properties of Microglial Cells

Milk fat globule factor-E8 (MFG-E8) has been regarded as a key factor involved in the phagocytosis of apoptotic cells. We induced a lentivirus into the microglial cells for the augmentation or abrogation of MFG-E8 expression in mouse microglial cells, and investigated phagocytosis of phosphatidylserine tagged human red blood cells (hRBCs) in co-cultures. Increased MFG-E8 levels were associated with a significant increase in phagocytic activity compared to the controls. Conversely, phagocytosis dramitically decreased due to the abrogation of MFG-E8. In addition, the expression of the inflammatory cytokines, TNF-α and IL-1β, also increased or decreased in the microglial cells with the augmentation or abrogation of MFG-E8, respectively. Our findings indicate that the enhanced expression of MFG-E8 could increase phagocytosis of apoptotic cells; conversely, the rate of phagocytosis and the expression of inflammatory cytokines decreased when MFG-E8 expression was knocked down. Our results confirm that MFG-E8 plays an important role in phagocytosis, and possibly serves as an essential signal molecule for microglial cells

High-throughput cell-based screening reveals a role for ZNF131 as a repressor of ERalpha signaling

<p>Abstract</p> <p>Background</p> <p>Estrogen receptor α (ERα) is a transcription factor whose activity is affected by multiple regulatory cofactors. In an effort to identify the human genes involved in the regulation of ERα, we constructed a high-throughput, cell-based, functional screening platform by linking a response element (ERE) with a reporter gene. This allowed the cellular activity of ERα, in cells cotransfected with the candidate gene, to be quantified in the presence or absence of its cognate ligand E2.</p> <p>Results</p> <p>From a library of 570 human cDNA clones, we identified zinc finger protein 131 (ZNF131) as a repressor of ERα mediated transactivation. ZNF131 is a typical member of the BTB/POZ family of transcription factors, and shows both ubiquitous expression and a high degree of sequence conservation. The luciferase reporter gene assay revealed that ZNF131 inhibits ligand-dependent transactivation by ERα in a dose-dependent manner. Electrophoretic mobility shift assay clearly demonstrated that the interaction between ZNF131 and ERα interrupts or prevents ERα binding to the estrogen response element (ERE). In addition, ZNF131 was able to suppress the expression of pS2, an ERα target gene.</p> <p>Conclusion</p> <p>We suggest that the functional screening platform we constructed can be applied for high-throughput genomic screening candidate ERα-related genes. This in turn may provide new insights into the underlying molecular mechanisms of ERα regulation in mammalian cells.</p

Postnatal onset of retinal degeneration by loss of embryonic Ezh2 repression of Six1

Some adult-onset disorders may be linked to dysregulated embryonic development, yet the mechanisms underlying this association remain poorly understood. Congenital retinal degenerative diseases are blinding disorders characterized by postnatal degeneration of photoreceptors, and affect nearly 2 million individuals worldwide, but ∼50% do not have a known mutation, implicating contributions of epigenetic factors. We found that embryonic deletion of the histone methyltransferase (HMT) Ezh2 from all retinal progenitors resulted in progressive photoreceptor degeneration throughout postnatal life, via derepression of fetal expression of Six1 and its targets. Forced expression of Six1 in the postnatal retina was sufficient to induce photoreceptor degeneration. Ezh2, although enriched in the embryonic retina, was not present in the mature retina; these data reveal an Ezh2-mediated feed-forward pathway that is required for maintaining photoreceptor homeostasis in the adult and suggest novel targets for retinal degeneration therapy

IGFBPL1 Regulates Axon Growth through IGF-1-mediated Signaling Cascades

Activation of axonal growth program is a critical step in successful optic nerve regeneration following injury. Yet the molecular mechanisms that orchestrate this developmental transition are not fully understood. Here we identified a novel regulator, insulin-like growth factor binding protein-like 1 (IGFBPL1), for the growth of retinal ganglion cell (RGC) axons. Expression of IGFBPL1 correlates with RGC axon growth in development, and acute knockdown of IGFBPL1 with shRNA or IGFBPL1 knockout in vivo impaired RGC axon growth. In contrast, administration of IGFBPL1 promoted axon growth. Moreover, IGFBPL1 bound to insulin-like growth factor 1 (IGF-1) and subsequently induced calcium signaling and mammalian target of rapamycin (mTOR) phosphorylation to stimulate axon elongation. Blockage of IGF-1 signaling abolished IGFBPL1-mediated axon growth, and vice versa, IGF-1 required the presence of IGFBPL1 to promote RGC axon growth. These data reveal a novel element in the control of RGC axon growth and suggest an unknown signaling loop in the regulation of the pleiotropic functions of IGF-1. They suggest new therapeutic target for promoting optic nerve and axon regeneration and repair of the central nervous system

A pathologic MYOC mutation results in a novel skeletal muscle phenotype

MYOC is the gene with mutations most strongly associated with glaucoma. In the eye, MYOC transcripts are found within the trabecular meshwork, ciliary body, and retina. Other tissues with high MYOC transcript levels are skeletal muscle and heart. Despite intensive research efforts, the function of wild-type MYOC remains unknown and how mutant MYOC causes pathology is ambiguous. We hoped that by investigating mutant MYOC in a non-ocular tissue we would obtain novel insight into mutant MYOC pathology. For this study, we utilized a transgenic mouse with CMV-driven expression of cDNA encoding for a pathologic human MYOC Y437H mutant protein and we examined its skeletal muscle. Electron micrographs showed that sarcomeres in the skeletal muscle of mutant CMV-MYOC-Y437H mice had multiple M-bands. Western blots of soluble muscle lysates showed that there was a decrease in two M-band proteins, myomesin 1 (Myom1) and muscle creatine kinase (Ckm). Immunoprecipitation experiments identified CKM as a MYOC binding partner. We believe that binding of mutant MYOC to Ckm is changing sacromere M-band ultrastructure and we suggest that this may adversely impact normal muscle function. Subsequently, we speculate that a person with both a family history of glaucoma as well as a history of muscle ailments may potentially be carrying a MYOC gene mutation and physicians should be aware of this so as to enable early identification of individuals at high-risk for glaucoma

Feasibility Study on Online Diagnosis of Aging and Deterioration of Medium Voltage (MV) Three-Core Cable Based on Impedance Spectroscopy

In order to explore the feasibility of impedance spectroscopy in the application of online diagnosis of aging and deterioration of medium voltage (MV) three-core cables, based on the theory of transmission line equation, an aging and deterioration model of the MV three-core cable has been established. The impedance spectroscopy of the cable head-end under healthy and different aging and degradation states has been simulated and analyzed. Further, the effects the extent of aging, local deterioration size and position, line length, load rate and other factors on the impedance spectroscopy have been studied. According to the influence of the various factors on the impedance spectroscopy, a set of aging and deterioration diagnosis procedures and methods are proposed. The simulation results indicate that the state of the cable can be divided into 4 categories of &#x201C;healthy,&#x201D; &#x201C;overall aging,&#x201D; &#x201C;large-size deterioration&#x201D; and &#x201C;small-size deterioration.&#x201D; The impedance spectroscopies of different states have certain characteristics. These characteristics can be determined by specific criterion indicators such as the monotonous decrease of the value of the resonance peaks and the phase amplitudes, the value of the resonance peaks, and the eigen propagation frequency, etc. Therefore, impedance spectroscopy has broad application prospects for online diagnosis of aging and deterioration of MV three-core cable

Mutant myocilin impacts sarcomere ultrastructure in mouse gastrocnemius muscle.

Myocilin (MYOC) is the gene with mutations most common in glaucoma. In the eye, MYOC is in trabecular meshwork, ciliary body, and retina. Other tissues with high MYOC transcript levels are skeletal muscle and heart. To date, the function of wild-type MYOC remains unknown and how mutant MYOC causes high intraocular pressure and glaucoma is ambiguous. By investigating mutant MYOC in a non-ocular tissue we hoped to obtain novel insight into mutant MYOC pathology. For this study, we utilized a transgenic mouse expressing human mutant MYOC Y437H protein and we examined its skeletal (gastrocnemius) muscle phenotype. Electron micrographs showed that sarcomeres in the skeletal muscle of mutant CMV-MYOC-Y437H mice had multiple M-bands. Western blots of soluble muscle lysates from transgenics indicated a decrease in two M-band proteins, myomesin 1 (MYOM1) and muscle creatine kinase (CKM). Immunoprecipitation identified CKM as a MYOC binding partner. Our results suggest that binding of mutant MYOC to CKM is changing sarcomere ultrastructure and this may adversely impact muscle function. We speculate that a person carrying the mutant MYOC mutation will likely have a glaucoma phenotype and may also have undiagnosed muscle ailments or vice versa, both of which will have to be monitored and treated

Transplantation of Human Neural Progenitor Cells Expressing IGF-1 Enhances Retinal Ganglion Cell Survival.

We have previously characterized human neuronal progenitor cells (hNP) that can adopt a retinal ganglion cell (RGC)-like morphology within the RGC and nerve fiber layers of the retina. In an effort to determine whether hNPs could be used a candidate cells for targeted delivery of neurotrophic factors (NTFs), we evaluated whether hNPs transfected with an vector that expresses IGF-1 in the form of a fusion protein with tdTomato (TD), would increase RGC survival in vitro and confer neuroprotective effects in a mouse model of glaucoma. RGCs co-cultured with hNPIGF-TD cells displayed enhanced survival, and increased neurite extension and branching as compared to hNPTD or untransfected hNP cells. Application of various IGF-1 signaling blockers or IGF-1 receptor antagonists abrogated these effects. In vivo, using a model of glaucoma we showed that IOP elevation led to reductions in retinal RGC count. In this model, evaluation of retinal flatmounts and optic nerve cross sections indicated that only hNPIGF-TD cells effectively reduced RGC death and showed a trend to improve optic nerve axonal loss. RT-PCR analysis of retina lysates over time showed that the neurotrophic effects of IGF-1 were also attributed to down-regulation of inflammatory and to some extent, angiogenic pathways. This study shows that neuronal progenitor cells that hone into the RGC and nerve fiber layers may be used as vehicles for local production and delivery of a desired NTF. Transplantation of hNPIGF-TD cells improves RGC survival in vitro and protects against RGC loss in a rodent model of glaucoma. Our findings have provided experimental evidence and form the basis for applying cell-based strategies for local delivery of NTFs into the retina. Application of cell-based delivery may be extended to other disease conditions beyond glaucoma

The detection of phosphatidylserine externalization in hRBCs labelled with Annexin V FITC fluorescence (flow cytometry) (A–F) and the phagocytosis of these cells by mouse microglial cells (G–H).

<p>(A–F) PS externalization was (35.70%) most successful with incubation for 10 min with 20 µM NEM and then incubated with 20 µM PSox for 30 min. Although, 47.50% of hRBCs showed PS externalization after being incubated for 30 min with 30 µM NEM and then 30 min with 20 µM PSox for 30 min, cells were fragile and considered unsuitable for use. (G) Prelabled normal hRBCs were very rarely recognised by microglial cells, but (H) phagocytosis was common for prelabeled PSox-hRBS cells. (I) The hRBSCs phagocytosis rate was significantly higher for PSox-treated cells. Immunoflurescent staining indicated that the cultured cells expressed CD11b and MFG-E8 (J–L). (M) The majority of cultured microglial cells can incorporate microbeads (green) suggesting that they have the ability of non-specific phagocytosis. Scale bars: 50 µm for G–H, 100 µm for J–M. ** <i>P</i><0.01. Abbreviations: Con, control; PSox: mixture of oxidized and nonoxidized phosphatidylserine; hRBCs, human red blood cells.</p