Leanne J. Cooper

α-Synuclein implicated in Parkinson’s disease is present in extracellular biological fluids, including human plasma

Parkinsons disease (PD) and other related disorders are characterized by the accumulation of fibrillar aggregates of α‐synuclein protein (α‐syn) inside brain cells. It is likely that the formation of α‐syn aggregates plays a seminal role in the pathogenesis of at least some of these diseases, because two different mutations in the gene encoding α‐syn have been found in inherited forms of PD. α‐Syn is mainly expressed by neuronal cells and is generally considered to exist as a cytoplasmic protein. Here, we report the unexpected identification of α‐syn in conditioned culture media from untransfected and α‐syn‐transfected human neuroblastoma cells, as well as in human cerebrospinal fluid and blood plasma. The method used was immunocapture by using anti‐α‐syn antibodies coupled to magnetic beads, followed by detection on Western blots. In all cases, α‐syn was identified as a single 15 kDa band, which co‐migrated with a recombinant form of the protein and reacted with five different antibodies to α‐syn. Our findings suggest that cells normally secrete α‐syn into their surrounding media, both in vitro and in vivo. The detection of extracellular α‐syn and/or its modified forms in body fluids, particularly in human plasma, offers new opportunities for the development of diagnostic tests for PD and related diseases.

Tight junction-related protein expression and distribution in human corneal epithelium.

PURPOSE To investigate the expression and cellular distribution of the tight junction-related proteins occludin, claudin and ZO-1 in human corneal epithelium. METHODS Light and electron immunohistochemistry was used to determine tissue distribution of occludin, claudin-1 and ZO-1 in the human corneal epithelium. Reverse transcription-polymerase chain reaction was used to reveal claudin mRNA expression in human corneal epithelium. RESULTS In transverse sections, occludin and ZO-1 were localized at the apical cell-cell junctions between superficial cells in stratified corneal epithelium. Both basal and basolateral membranes of superficial cells were stained by the claudin-1 antibody, but no apical membrane staining was observed. In en face sections, claudin-1 and ZO-1 antibodies showed as bands that corresponded to the junctional complex. Claudin-1 staining of superficial cell cytoplasm was also observed. Occludin staining was seen at the junctional complex, where it was not continuous, but dotted along the cell junctions. The transcripts for claudin-1 and several other claudin isotypes, such as -2, -3, -4, -7, -9 and -14 were identified. CONCLUSION Not only occludin, but also some claudins act as integral transmembrane proteins in the corneal epithelium. ZO-1 is a component of the corneal epithelial tight junction, as it is in most epithelial cells.

Comparison of ultrastructure, tight junction-related protein expression and barrier function of human corneal epithelial cells cultivated on amniotic membrane with and without air-lifting

PURPOSE To evaluate the usefulness of the air-lifting technique for culturing corneal limbal epithelial cells on amniotic membrane (AM) for use in ocular surface reconstruction. A cultured sheet that has a good barrier function should be better for this purpose. In corneal epithelium, tight junctions (TJ) play a vital role in the barrier function. The TJ complex includes the integral transmembrane proteins occludin and the claudins, and some membrane-associated proteins such as ZO-1. In this paper, we investigated the barrier function and the expression of TJ related proteins. METHODS Corneal limbal epithelium obtained from donor corneas and cultivated on acellular AM was divided into two groups. These were the non-air-lifting (Non-AL) group, which was continuously submerged in medium, and the air-lifting (AL) group, which was submerged in medium for 3 weeks, then exposed to air by lowering the medium level. Morphology and the permeability to horseradish peroxidase (HRP) were determined by electron microscopy. Tight junction (TJ)-related protein and mRNA expression changes were assessed by immunoblotting and reverse transcription-polymerase chain reaction. RESULTS The cultures of both groups formed 4-5-layer-thick, well-stratified epithelium. The AL cultures had tightly packed epithelial cells with all the HRP/diaminobenzidine (DAB) reaction product accumulated on the apical surface of the superficial cells. The Non-AL culture, by contrast, had more loosely packed epithelial cells with larger intercellular spaces. The HRP/DAB reaction product penetrated the intercellular space to a depth of 3-4 cell layers. Statistically, there was a significant difference in intercellular spaces and desmosome count in the superficial cells between the groups. With AL, TJ-related proteins localized at the apical portion of the lateral membrane. TJ-related protein and mRNA amounts were not changed by AL while claudin subtype expression became more consistent and closer to that of in vivo corneal epithelium. CONCLUSIONS The AL technique reduces intercellular spaces in the superficial cells and promotes the formation of the barrier function. It is useful in culturing corneal epithelial cells for use in ocular surface reconstruction.

Cultivated corneal endothelial transplantation in a primate: possible future clinical application in corneal endothelial regenerative medicine.

Purpose: To review our attempt to devise a method of cultivated corneal endothelial transplantation using primates in which corneal endothelium, like that of humans, has low proliferative ability. Methods: Monkey corneal endothelial cells (MCECs) were cultivated, with subcultures grown on collagen type I carriers. The corneal endothelia of 6 eyes of 6 monkeys were scraped intensively, after which cultivated MCEC sheets were inserted into the anterior chamber of 4 eyes. As controls, a collagen sheet without MCECs was transplanted in 1 eye of a monkey, and a suspension of cultivated MCECs was injected into the anterior chamber of 1 eye of another monkey. Results: MCECs produced a confluent monolayer of closely attached hexagonal cells, which expressed both ZO-1 and Na+-K+ adenosine triphosphatase. Early postoperative period MCEC sheets were attached to Descemet membrane, and corneal clarity was recovered. Six months after transplantation, MCEC-transplanted corneas remained clear, and closely attached hexagonal cells were observed. In 1 animal with longer observation, polygonal cells were observed by in vivo specular microscopy at a density of >2000 cells/mm2 and remained >1600 cells/mm2 for ≤2 years. Control eyes showed irreversible bullous keratopathy throughout the observation period. Conclusions: Cultivated MCECs become attached to the transplanted eye and maintain a clear cornea ≤2 years postoperatively, suggesting that corneal endothelial cells of primates might have proliferative ability in vivo once they have been cultured and proliferated in vitro. Our monkey model constitutes an important step forward for regenerative medicine with possible future application in patients with corneal endothelial dysfunction.

Galantamine inhibits β-amyloid aggregation and cytotoxicity

The ability of galantamine (Reminyl) to inhibit the aggregation and toxicity of the beta-amyloid peptide (Abeta) was investigated. Galantamine showed concentration-dependent inhibition of aggregation of both Abeta 1-40 and Abeta 1-42, as determined by an ELISA method. Electron microscope studies of Abeta 1-40 incubated in the presence of galantamine revealed fibrils that were disordered and clumped in appearance. MTT and lactate dehydrogenase assays, employing SH-SY5Y human neuroblastoma cells, showed that galantamine reduced the cytotoxicity induced by Abeta 1-40. Galantamine also dramatically reduced Abeta 1-40-induced cellular apoptosis in these cells. There is some evidence that galantamine may not be acting purely as a symptomatic treatment. Disease-modifying effects of the drug could be due to an additional effect on Abeta aggregation and/or toxicity.

Copper-mediated formation of hydrogen peroxide from the amylin peptide:a novel mechanism for degeneration of islet cells in type-2 diabetes mellitus?

Amyloid deposits derived from the amylin peptide accumulate within pancreatic islet β‐cells in most cases of type‐2 diabetes mellitus (T2Dm). Human amylin ‘oligomers’ are toxic to these cells. Using two different experimental techniques, we found that H2O2 was generated during the aggregation of human amylin into amyloid fibrils. This process was greatly stimulated by Cu(II) ions, and human amylin was retained on a copper affinity column. In contrast, rodent amylin, which is not toxic, failed to generate any H2O2 and did not interact with copper. We conclude that the formation of H2O2 from amylin could contribute to the progressive degeneration of islet cells in T2Dm.

AN INVESTIGATION INTO THE COMPOSITION OF AMNIOTIC MEMBRANE USED FOR OCULAR SURFACE RECONSTRUCTION

Purpose: Cultivated limbal epithelial transplantation using an amniotic membrane (AM) carrier is now widely used for ocular surface reconstruction. The reasons for the exceptional success of AM as a carrier are not fully understood but are believed to be related to its unique composition. In this project we characterize, at the ultrastructural level, the extracellular matrix (ECM) components present in AM. We also compare the distribution of ECM components of cellular AM with that of denuded AM. Methods: Scanning, transmission, and atomic force microscopy was used to examine the structure of cellular and denuded amniotic membranes. Immunogold labeling with a panel of antibodies against ECM molecules was carried out on cellular and denuded AM. Results: Heparan sulfate, fibronectin, and laminin were present at high concentration in the lamina densa, Collagen IV was the major component of the basal lamina. Type I collagen was confined to the stroma along with significant amounts of keratan and chondroitin sulfate. Both cellular and denuded AMs had similar distributions of the ECM components. Conclusions: We were able to determine the distribution of ECM molecules in the lamina densa, basal lamina, and stroma of AM at the ultrastructural level. The removal of amniotic epithelial cells using our protocol does not appear to have any significant effects on the structure of the basal lamina or the distribution of ECM components.

An investigation of removed cultivated epithelial transplants in patients after allocultivated corneal epithelial transplantation.

Objective: To investigate the ultrastructural changes of removed cultivated corneal epithelial transplants using scanning and transmission electron microscopy. Methods: Allocultivated corneal epithelial transplantation, using an amniotic membrane carrier, was carried out on 3 patients. The primary diagnoses consisted of 1 with acute-phase chemical burn, one with drug-induced pseudopemphigoid, and 1 with Stevens-Johnson syndrome. After a period of several months the transplants were removed from these patients because of graft opacities. The removed transplants were then prepared for examination by scanning and transmission electron microscopy. Results: In all 3 cases there was a similar pattern of findings: the amniotic membrane remained intact, although it had become partially vascularized and invaded by keratocytes. Inflammatory cells were present in the epithelial layer and within the amniotic membrane. Most of the amniotic membrane was covered by conjunctival epithelial cells and goblet cells. Only a few areas of normal cultivated corneal epithelial cells were found. Conclusions: We suggest that the process of allograft rejection is responsible for the corneal epithelial loss and that this is followed by conjunctival invasion onto the amniotic membrane.

An ultrastructural investigation of dermatopontin-knockout mouse corneas

Introduction  The corneal stroma is composed of a network of heterotypic collagen fibrils, proteoglycans and matrix proteins. Transparency of the tissue principally requires the uniformity of fibril diameters and interfibrillar distances and the presence of a quasi‐hexagonal lattice arrangement of parallel fibrils. Keratan sulfate proteoglycans (KSPGs) have a crucial role and the KS chains are clearly required for the maintenance of transparency. Undersulfation of corneal KS results in tissue opacity and the lumican (a KSPG) knockout mouse shows corneal opacity and the disruption of collagen fibril diameters and interfibrillar distances ( Chakravarti et al. 1998 ). Biochemical analysis has shown that dermatopontin is an abundant component of the extracellular matrix and that it interacts with KSPGs via the KS chains. This study aims to determine whether dermatopontin has a direct role in corneal matrix organization by investigating the corneal ultrastructure of dermatopontin‐null (dpt–/–) mouse corneas.