Nigel J. Fullwood

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.

Using Fourier transform IR spectroscopy to analyze biological materials

IR spectroscopy is an excellent method for biological analyses. It enables the nonperturbative, label-free extraction of biochemical information and images toward diagnosis and the assessment of cell functionality. Although not strictly microscopy in the conventional sense, it allows the construction of images of tissue or cell architecture by the passing of spectral data through a variety of computational algorithms. Because such images are constructed from fingerprint spectra, the notion is that they can be an objective reflection of the underlying health status of the analyzed sample. One of the major difficulties in the field has been determining a consensus on spectral pre-processing and data analysis. This manuscript brings together as coauthors some of the leaders in this field to allow the standardization of methods and procedures for adapting a multistage approach to a methodology that can be applied to a variety of cell biological questions or used within a clinical setting for disease screening or diagnosis. We describe a protocol for collecting IR spectra and images from biological samples (e.g., fixed cytology and tissue sections, live cells or biofluids) that assesses the instrumental options available, appropriate sample preparation, different sampling modes as well as important advances in spectral data acquisition. After acquisition, data processing consists of a sequence of steps including quality control, spectral pre-processing, feature extraction and classification of the supervised or unsupervised type. A typical experiment can be completed and analyzed within hours. Example results are presented on the use of IR spectra combined with multivariate data processing.

Amniotic Membrane as a Substrate for Cultivating Limbal Corneal Epithelial Cells for Autologous Transplantation in Rabbits.

Purpose. To examine the viability of using human amniotic membrane as substrate for culturing corneal epithelial cells and transplanting them onto severely injured rabbit eyes. Methods. An ocular-surface injury was created in the right eye of eight rabbits by a lamellar keratectomy extending 5 mm outside the limbus. Next, from the limbal region of the uninjured left eyes of five of these animals, a small biopsy of corneal epithelial cells was taken and cultured on acellular human amniotic membrane. One month later, the invading conjunctiva that covered the corneal surface of all eight injured eyes was surgically removed. Five of the eyes then received grafts of amniotic membrane containing autologous cultured epithelial cells, whereas the other three received grafts of acellular amniotic membrane alone. Results. A confluent primary culture of limbal corneal epithelial cells was established on acellular human amniotic membrane after 14 days. Cells were partially stratified and fairly well attached to the underlying amniotic membrane, although a fully formed basement membrane was not evident. The three rabbits that received amniotic membrane transplantation alone all had total epithelial defects on the graft in the early postoperative period. Eyes that were grafted with amniotic membrane that contained cultivated epithelial cells, however, were all successfully epithelialized up to 5 days after surgery. Conclusion. Autologous transplantation of cultivated corneal epithelium is feasible by using acellular amniotic membrane as a carrier.

Distinguishing cell types or populations based on the computational analysis of their infrared spectra.

Infrared (IR) spectroscopy of intact cells results in a fingerprint of their biochemistry in the form of an IR spectrum; this has given rise to the new field of biospectroscopy. This protocol describes sample preparation (a tissue section or cytology specimen), the application of IR spectroscopy tools, and computational analysis. Experimental considerations include optimization of specimen preparation, objective acquisition of a sufficient number of spectra, linking of the derived spectra with tissue architecture or cell type, and computational analysis. The preparation of multiple specimens (up to 50) takes 8 h; the interrogation of a tissue section can take up to 6 h (∼100 spectra); and cytology analysis (n = 50, 10 spectra per specimen) takes 14 h. IR spectroscopy generates complex data sets and analyses are best when initially based on a multivariate approach (principal component analysis with or without linear discriminant analysis). This results in the identification of class clustering as well as class-specific chemical entities.

Corneal and scleral collagens—a microscopist’s perspective

This paper reviews our existing understanding of the distribution and organisation of collagen types within the corneal and scleral stroma from a microscopical perspective. The contribution of various types of light microscopy, electron microscopy and atomic force microscopy to this field are separately discussed. Light microscopy was used in the earliest studies of the cornea and lead to the first description of the lamellar structure of the stroma. More recently polarised light microscopy has been used to obtain specific information on fibril orientation within individual lamellae. Light microscope immunolabelling techniques have been utilised to determine the distribution of several collagen types within the cornea and sclera, while recent developments in confocal microscopy have allowed detailed observations to be made on live cornea. Scanning electron microscopy has proved useful in determining the 3D organisation of lamellae within both corneal and scleral stroma. The transmission electron microscope was responsible for first revealing the regular diameter and high degree of order of the collagen fibrils present in the corneal stroma and contrasting this with the irregular diameter of fibrils present in sclera. This finding lead directly to the formulation of a theory of corneal transparency based on the uniformity of fibril diameter and packing. The use of specialised stains such as cuprolinic blue allowed direct observation of the glycosaminoglycan chains on proteoglycan molecules in cornea and sclera. These images allowed the binding sites of the proteoglycans along the collagen fibrils to be identified and provided convincing evidence for the importance of the proteoglycan molecules in collagen fibril organisation. Immunogold labelling has been used to map the distribution of several collagen types within the corneal and scleral stroma at the ultrastructural level and provided critical evidence for the role of type V collagen in the regulation of fibril diameter within the cornea. Specialised freezing-etching techniques have revealed the surface features of the collagen fibrils in corneal stroma, indicating clearly the presence of crossbridge structures between fibrils. The technique of rotary shadowing has been used to determine the conformation of several collagen types. In more recent years atomic force microscopy has been applied to the study of the corneal stroma. It has largely confirmed the observations made by the transmission electron microscope and provided independent evidence of crossbridge structures between the collagen fibres in cornea and sclera. The full potential of this technique has yet to be realised.

Using Raman spectroscopy to characterize biological materials.

Raman spectroscopy can be used to measure the chemical composition of a sample, which can in turn be used to extract biological information. Many materials have characteristic Raman spectra, which means that Raman spectroscopy has proven to be an effective analytical approach in geology, semiconductor, materials and polymer science fields. The application of Raman spectroscopy and microscopy within biology is rapidly increasing because it can provide chemical and compositional information, but it does not typically suffer from interference from water molecules. Analysis does not conventionally require extensive sample preparation; biochemical and structural information can usually be obtained without labeling. In this protocol, we aim to standardize and bring together multiple experimental approaches from key leaders in the field for obtaining Raman spectra using a microspectrometer. As examples of the range of biological samples that can be analyzed, we provide instructions for acquiring Raman spectra, maps and images for fresh plant tissue, formalin-fixed and fresh frozen mammalian tissue, fixed cells and biofluids. We explore a robust approach for sample preparation, instrumentation, acquisition parameters and data processing. By using this approach, we expect that a typical Raman experiment can be performed by a nonspecialist user to generate high-quality data for biological materials analysis.

A strategy for designing inhibitors of α-synuclein aggregation and toxicity as a novel treatment for Parkinson's disease and related disorders

Convergent biochemical and genetic evidence suggests that the formation of α‐synuclein (α‐syn) protein deposits is an important and, probably, seminal step in the development of Parkinsons disease (PD), dementia with Lewy bodies (DLB) and multiple system atrophy (MSA). It has been reported that transgenic animals overexpressing human α‐syn develop lesions similar to those found in the brain in PD, together with a progressive loss of dopaminergic cells and associated abnormalities of motor function. Inhibiting and/or reversing α‐syn self‐aggregation could, therefore, provide a novel approach to treating the underlying cause of these diseases. We synthesized a library of overlapping 7‐mer peptides spanning the entire α‐syn sequence, and identified amino acid residues 64‒100 of α‐syn as the binding region responsible for its self‐ association. Modified short peptides containing α‐syn amino acid sequences from part of this binding region (residues 69‒72), named α‐syn inhibitors (ASI), were found to interact with full‐ length α‐syn and block its assembly into both early oligomers and mature amyloid‐like fibrils. We also developed a cell‐permeable inhibitor of α‐syn aggregation (ASID), using the polyarginine peptide delivery system. This ASID peptide was able to inhibit the DNA damage induced by Fe(II) in neuronal cells transfected with α‐syn(A53T), a familial PD‐associated mutation. ASI peptides without this delivery system did not reverse levels of Fe(II)‐induced DNA damage. Furthermore, the ASID peptide increased (P<0.0005) the number of cells stained positive for Bcl‐2, while significantly (P<0.05) decreasing the percentage of cells stained positive for BAX. These short peptides could serve as lead compounds for the design of peptidomimetic drugs to treat PD and related disorders.

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.

Synchrotron x-ray diffraction studies of the cornea, with implications for stromal hydration

The intermolecular and interfibrillar spacings of collagen in bovine corneal stroma have been measured as a function of tissue hydration. Data were recorded from low- and high-angle x-ray diffraction patterns obtained using a high intensity synchrotron source. The most frequently occurring interfibrillar spacing varied from 34 nm in dry corneas to 76 nm at H = 5 (the hydration, H, is defined as the ratio of the weight of water to the dry weight). The most frequently occurring intermolecular Bragg spacing increased from 1.15 nm (dry) to approximately 1.60 nm at normal hydration (H approximately 3.2) and continued to increase only slowly above normal hydration. Most of the increase in the intermolecular spacing occurred between H = O and H = 1. Over this hydration range the interfibrillar and intermolecular spacings moved in tandem, which suggests that the initial water goes equally within and between the fibrils. Above H = 1 water goes preferentially between the fibrils. The results suggest that, even at normal hydration, water does not fill the interfibrillar space uniformly, and a proportion is located in another space or compartment. In dried-then-rehydrated corneas, a larger proportion of the water goes into this other compartment. In both cases, it is possible to postulate a second set or population of fibrils that are more widely and irregularly separated and therefore do not contribute significantly to the diffraction pattern.