Saeed Akhtar

Autophagy Activation Clears ELAVL1/HuR-Mediated Accumulation of SQSTM1/p62 during Proteasomal Inhibition in Human Retinal Pigment Epithelial Cells

Age-related macular degeneration (AMD) is the most common reason of visual impairment in the elderly in the Western countries. The degeneration of retinal pigment epithelial cells (RPE) causes secondarily adverse effects on neural retina leading to visual loss. The aging characteristics of the RPE involve lysosomal accumulation of lipofuscin and extracellular protein aggregates called “drusen”. Molecular mechanisms behind protein aggregations are weakly understood. There is intriguing evidence suggesting that protein SQSTM1/p62, together with autophagy, has a role in the pathology of different degenerative diseases. It appears that SQSTM1/p62 is a connecting link between autophagy and proteasome mediated proteolysis, and expressed strongly under the exposure to various oxidative stimuli and proteasomal inhibition. ELAVL1/HuR protein is a post-transcriptional factor, which acts mainly as a positive regulator of gene expression by binding to specific mRNAs whose corresponding proteins are fundamental for key cellular functions. We here show that, under proteasomal inhibitor MG-132, ELAVL1/HuR is up-regulated at both mRNA and protein levels, and that this protein binds and post-transcriptionally regulates SQSTM1/p62 mRNA in ARPE-19 cell line. Furthermore, we observed that proteasomal inhibition caused accumulation of SQSTM1/p62 bound irreversibly to perinuclear protein aggregates. The addition of the AMPK activator AICAR was pro-survival and promoted cleansing by autophagy of the former complex, but not of the ELAVL1/HuR accumulation, indeed suggesting that SQSTM1/p62 is decreased through autophagy-mediated degradation, while ELAVL1/HuR through the proteasomal pathway. Interestingly, when compared to human controls, AMD donor samples show strong SQSTM1/p62 rather than ELAVL1/HuR accumulation in the drusen rich macular area suggesting impaired autophagy in the pathology of AMD.

Clearance of autophagy-associated dying retinal pigment epithelial cells – a possible source for inflammation in age-related macular degeneration

Retinal pigment epithelial (RPE) cells can undergo different forms of cell death, including autophagy-associated cell death during age-related macular degeneration (AMD). Failure of macrophages or dendritic cells (DCs) to engulf the different dying cells in the retina may result in the accumulation of debris and progression of AMD. ARPE-19 and primary human RPE cells undergo autophagy-associated cell death upon serum depletion and oxidative stress induced by hydrogen peroxide (H2O2). Autophagy was revealed by elevated light-chain-3 II (LC3-II) expression and electron microscopy, while autophagic flux was confirmed by blocking the autophago-lysosomal fusion using chloroquine (CQ) in these cells. The autophagy-associated dying RPE cells were engulfed by human macrophages, DCs and living RPE cells in an increasing and time-dependent manner. Inhibition of autophagy by 3-methyladenine (3-MA) decreased the engulfment of the autophagy-associated dying cells by macrophages, whereas sorting out the GFP-LC3-positive/autophagic cell population or treatment by the glucocorticoid triamcinolone (TC) enhanced it. Increased amounts of IL-6 and IL-8 were released when autophagy-associated dying RPEs were engulfed by macrophages. Our data suggest that cells undergoing autophagy-associated cell death engage in clearance mechanisms guided by professional and non-professional phagocytes, which is accompanied by inflammation as part of an in vitro modeling of AMD pathogenesis.

Zebrafish Model for the Genetic Basis of X-Linked Retinitis Pigmentosa

Retinitis pigmentosa (RP) affects 1/4000 individuals in most populations, and X-linked RP (XLRP) is one of the most severe forms of human retinal degeneration. Mutations in both the retinitis pigmentosa GTPase regulator (RPGR) gene and retinitis pigmentosa 2 (RP2) gene account for almost all cases of XLRP. The functional roles of both RPGR and RP2 in the pathogenesis of XLRP are unclear. Due to the surprisingly high degree of functional conservation between human genes and their zebrafish orthologues, the zebrafish has become an important model for human retinal disorders. In this brief review, we summarize the functional characterization of XLRP-causing genes, RPGR and RP2, in zebrafish, and highlight recent studies that provide insight into the cellular functions of both genes. This will not only shed light on disease mechanisms in XLRP but will also provide a solid platform to test RP-causing mutants before proposing XLRP gene therapy trials.

Role of epidermal growth factor receptor (EGFR) in corneal remodelling in diabetes

Purpose:u2002 This study examined the role of epidermal growth factor receptor (EGFR) signalling on the organization and remodelling of collagen fibrils (CFs) and proteoglycans (PGs) in the stroma of diabetic rat cornea.

Comparison of upstream regulators in human ex vivo cultured cornea limbal epithelial stem cells and differentiated corneal epithelial cells.

BackgroundThe surface of the human eye is covered by corneal epithelial cells (CECs) which regenerate from a small population of limbal epithelial stem cells (LESCs). Cell therapy with LESCs is a non-penetrating treatment for preventing blindness due to LESC deficiency or dysfunction. Our aim was to identify new putative molecular markers and upstream regulators in the LESCs and associated molecular pathways.ResultsGenome-wide microarray transcriptional profiling was used to compare LESCs to differentiated human CECs. Ingenuity-based pathway analysis was applied to identify upstream regulators and pathways specific to LESCs. ELISA and flow cytometry were used to measure secreted and surface expressed proteins, respectively. More than 2 fold increase and decrease in expression could be found in 1830 genes between the two cell types. A number of molecules functioning in cellular movement (381), proliferation (567), development (552), death and survival (520), and cell-to-cell signaling (290) were detected having top biological functions in LESCs and several of these were confirmed by flow cytometric surface protein analysis. Custom-selected gene groups related to stemness, differentiation, cell adhesion, cytokines and growth factors as well as angiogenesis could be analyzed. The results show that LESCs play a key role not only in epithelial differentiation and tissue repair, but also in controlling angiogenesis and extracellular matrix integrity. Some pro-inflammatory cytokines were found to be important in stemness-, differentiation- and angiogenesis-related biological functions: IL-6 and IL-8 participated in most of these biological pathways as validated by their secretion from LESC cultures.ConclusionsThe gene and molecular pathways may provide a more specific understanding of the signaling molecules associated with LESCs, therefore, help better identify and use these cells in the treatment of ocular surface diseases.

Ultrastructure features of camel cornea – collagen fibril and proteoglycans

INTRODUCTIONnu2002 The uniform distribution of collagen fibrils and proteoglycans maintain the transparency of normal cornea. We describe the ultrastructural features of camel cornea including collagen fibrils and proteoglycans (PGs).nnnMETHODSnu2002 Camel corneas (of 6-, 8-, and 10-month-old animals) were fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and processed for electron microscopy. The AnalySIS LS Professional program was used to analyze the collagen fibril diameter.nnnRESULTSnu2002 The camel cornea consists of four layers: the epithelium (227u2003μm), stroma (388u2003μm), Descemets membrane (DM), and endothelium. The epithelium constituted 36% of the camel cornea, whereas corneal stroma constituted 62% of the corneal thickness (629u2003μm). The PGs in the posterior stroma were significantly larger in number and size compared with the anterior and middle stroma. The collagen fibril diameter was 25u2003nm and interfibrillar spacing 40u2003nm. Fibrillar structures are present throughout the DM.nnnCONCLUSIONnu2002 The structure of the camel cornea is very different from human and other animals. The unique structure of the cornea might be an adaptation to help the camel to survive in a hot and dry climate. The camel cornea may also be a good model to study the effect of hot and dry climates on the cornea.

Ultrastructural and three‐dimensional study of post‐LASIK ectasia cornea

INTRODUCTION: Post‐laser in situ keratomileusis (LASIK) corneal ectasia is a serious late postoperative complication. Here, we report the ultrastructural features of the post‐LASIK cornea of two patients. METHODS: Two normal corneas (age 24 and 37 years old) and two post‐LASIK ectaic corneas from two patients (A and B) were studied. The “patient A” (age 27 years) underwent penetrating keratoplasty and “patient B” (age 31 years) underwent deep‐anterior lamellar keratoplasty. The excised corneas were processed for light and electron microscopy. A total of 120 images for three‐dimensional (3D) reconstruction were taken by using the software “Recorder” and using a bottom mounted camera “Quemesa” attached to a JOEL 1400 transmission electron microscope. The 3D images were constructed using “Visual Kai” software. RESULTS: In the post‐LASIK cornea, the hemidesmosomes, the basement membrane, and Bowman”s layer were abnormal. The stromal lamellae were thin and disorganized. The collagen fibrils (CFs) diameter and interfibrillar spacing had decreased. Aggregated microfibrils were present in the Bowmans layer and all parts of the stroma. A large number of microfilaments were present at the detachment end of the flap and residual stroma. The 3D images showed the presence of collagen microfibrils and proteoglycans (PGs) within the CF of the normal and post‐LASIK cornea. The collagen microfibrils and PGs within the CFs had degenerated in the post‐LASIK cornea. CONCLUSION: Collagen microfibrils and PGs within the CFs were degenerated, leading to the degeneration of CFs, followed by the disorganization of lamellae in post‐LASIK cornea. The CFs diameter and interfibrillar spacing decreased. Microsc. Res. Tech. 77:91–98, 2014.

Collagen Fibrils and Proteoglycans of Macular Dystrophy Cornea: Ultrastructure and 3D Transmission Electron Tomography.

We report the ultrastructure and 3D transmission electron tomography of collagen fibrils (CFs), proteoglycans (PGs), and microfibrils within the CF of corneas of patients with macular corneal dystrophy (MCD). Three normal corneas and three MCD corneas from three Saudi patients (aged 25, 31, and 49 years, respectively) were used for this study. The corneas were processed for light and electron microscopy studies. 3D images were composed from a set of 120 ultrastructural images using the program Composer and visualized using the program Visuliser Kai. 3D image analysis of MCD cornea showed a clear organization of PGs around the CF at very high magnification and degeneration of the microfibrils within the CF. Within the MCD cornea, the PG area in the anterior stroma was significantly larger than in the middle and posterior stroma. The PG area in the MCD cornea was significantly larger compared with the PG area in the normal cornea. The CF diameter and inter-fibrillar spacing of the MCD cornea were significantly smaller compared with those of the normal cornea. Ultrastructural 3D imaging showed that the production of unsulfated keratin sulfate (KS) may lead to the degeneration of micro-CFs within the CFs. The effect of the unsulfated KS was higher in the anterior stroma compared with the posterior stroma.

Effect of processing methods for transmission electron microscopy on corneal collagen fibrils diameter and spacing

Introduction: The corneal tissue was processed in fixatives and embedded in resin for transmission electron microscopy to observe the ultrastructure of the collagen fibrils (CFs). The effect of these processing methods on the CF diameter and the interfibrillar spacing was studied. Methods: Four normal human corneal buttons were used for this study. A part of each cornea was fixed in 2.5% glutaraldehyde containing cuprolinic blue in sodium acetate buffer and embedded in spurrs resin (SpurrCB). A second part of each cornea was fixed in 2.5% glutaraldehyde + osmium tetroxide and embedded spurrs resin (SpurrOsm). The third part of each cornea was fixed in paraformaldehyde (4%) and embedded in LR White at 4°C (LRWhite). Ultrathin sections were stained with uranyl acetate and lead citrate. Results: In the tissue, fixed in SpurrCB, the diameter was 38.4 ± 5.9 nm and spacing between CF was 52.5 ± 5.3 nm. In the tissue fixed in SpurrOsm, the diameter was 28.37 ± 5.84 nm and spacing between CF was 45 ± 4.57 nm. In the tissue fixed in LR White, the CF diameter was 24 ± 2.3 nm and spacing between CF was 39.0 ± 4.2 nm. The diameters and interfibrillar spacing of the tissue processed by SpurrCB, SpurrOsm, and LRWhite were significantly different (P < 0.001) from one another. Conclusion: Our study shows that there is a variation in the CF diameter and spacing depending on the method of fixation and embedding resins used. This needs to be considered when comparative studies using different methods are done. Microsc. Res. Tech. 2012.

Histological Characterization of the Dicer1 Mutant Zebrafish Retina

DICER1, a multidomain RNase III endoribonuclease, plays a critical role in microRNA (miRNA) and RNA-interference (RNAi) functional pathways. Loss of Dicer1 affects different developmental processes. Dicer1 is essential for retinal development and maintenance. DICER1 was recently shown to have another function of silencing the toxicity of Alu RNAs in retinal pigment epithelium (RPE) cells, which are involved in the pathogenesis of age related macular degeneration. In this study, we characterized a Dicer1 mutant fish line, which carries a nonsense mutation (W1457Ter) induced by N-ethyl-N-nitrosourea mutagenesis. Zebrafish DICER1 protein is highly conserved in the evolution. Zebrafish Dicer1 is expressed at the earliest stages of zebrafish development and persists into late developmental stages; it is widely expressed in adult tissues. Homozygous Dicer1 mutant fish (DICER1W1457Ter/W1457Ter) have an arrest in early growth with significantly smaller eyes and are dead at 14–18u2009dpf. Heterozygous Dicer1 mutant fish have similar retinal structure to that of control fish; the retinal pigment epithelium (RPE) cells are normal with no sign of degeneration at the age of 20 months.