Ординатура / Офтальмология / Английские материалы / Mechanisms of the Glaucomas_Shields, Tombran-Tink, Barnstable_2008

220.Mi, S., Lee, X., Shao, Z., Thill, G., Ji, B., Relton, J., Levesque, M., Allaire, N., Perrin, S., Sands, B., Crowell, T., Cate, R. L., McCoy, J. M. & Pepinsky, R. B. (2004). LINGO- 1 is a component of the Nogo-66 receptor/p75 signaling complex. Nat Neurosci 7, 221–8.

221.Park, J. B., Yiu, G., Kaneko, S., Wang, J., Chang, J., He, X. L., Garcia, K. C. & He, Z. (2005). A TNF receptor family member, TROY, is a coreceptor with Nogo receptor in mediating the inhibitory activity of myelin inhibitors. Neuron 45, 345–51.

222.Shao, Z., Browning, J. L., Lee, X., Scott, M. L., Shulga-Morskaya, S., Allaire, N., Thill, G., Levesque, M., Sah, D., McCoy, J. M., Murray, B., Jung, V., Pepinsky, R. B. & Mi, S. (2005). TAJ/TROY, an orphan TNF receptor family member, binds Nogo-66 receptor 1 and regulates axonal regeneration. Neuron 45, 353–9.

223.McKerracher, L., David, S., Jackson, D. L., Kottis, V., Dunn, R. J. & Braun, P. E. (1994). Identification of myelin-associated glycoprotein as a major myelin-derived inhibitor of neurite growth. Neuron 13, 805–11.

224.Mukhopadhyay, G., Doherty, P., Walsh, F. S., Crocker, P. R. & Filbin, M. T. (1994). A novel role for myelin-associated glycoprotein as an inhibitor of axonal regeneration. Neuron 13, 757–67.

225.Wang, K. C., Koprivica, V., Kim, J. A., Sivasankaran, R., Guo, Y., Neve, R. L. & He, Z. (2002). Oligodendrocyte-myelin glycoprotein is a Nogo receptor ligand that inhibits neurite outgrowth. Nature 417, 941–4.

226.Domeniconi, M., Cao, Z., Spencer, T., Sivasankaran, R., Wang, K., Nikulina, E., Kimura, N., Cai, H., Deng, K., Gao, Y., He, Z. & Filbin, M. (2002). Myelin-associated glycoprotein interacts with the Nogo66 receptor to inhibit neurite outgrowth. Neuron 35, 283–90.

227.Bandtlow, C. & Dechant, G. (2004). From cell death to neuronal regeneration, effects of the p75 neurotrophin receptor depend on interactions with partner subunits. Sci STKE 2004, pe24.

228.Domeniconi, M., Zampieri, N., Spencer, T., Hilaire, M., Mellado, W., Chao, M. V. & Filbin, M. T. (2005). MAG induces regulated intramembrane proteolysis of the p75 neurotrophin receptor to inhibit neurite outgrowth. Neuron 46, 849–55.

229.Brosamle, C., Huber, A. B., Fiedler, M., Skerra, A. & Schwab, M. E. (2000). Regeneration of lesioned corticospinal tract fibers in the adult rat induced by a recombinant, humanized IN-1 antibody fragment. J Neurosci 20, 8061–8.

230.Liebscher, T., Schnell, L., Schnell, D., Scholl, J., Schneider, R., Gullo, M., Fouad, K., Mir, A., Rausch, M., Kindler, D., Hamers, F. P. & Schwab, M. E. (2005). Nogo-A antibody improves regeneration and locomotion of spinal cord-injured rats. Ann Neurol 58, 706–19.

231.Koprivica, V., Cho, K. S., Park, J. B., Yiu, G., Atwal, J., Gore, B., Kim, J. A., Lin, E., Tessier-Lavigne, M., Chen, D. F. & He, Z. (2005). EGFR activation mediates inhibition of axon regeneration by myelin and chondroitin sulfate proteoglycans. Science 310, 106–10.

232.Kim, J. E., Li, S., GrandPre, T., Qiu, D. & Strittmatter, S. M. (2003). Axon regeneration in young adult mice lacking Nogo-A/B. Neuron 38, 187–99.

233.Simonen, M., Pedersen, V., Weinmann, O., Schnell, L., Buss, A., Ledermann, B., Christ, F., Sansig, G., van der Putten, H. & Schwab, M. E. (2003). Systemic deletion of the myelinassociated outgrowth inhibitor Nogo-A improves regenerative and plastic responses after spinal cord injury. Neuron 38, 201–11.

234.Zheng, B., Ho, C., Li, S., Keirstead, H., Steward, O. & Tessier-Lavigne, M. (2003). Lack of enhanced spinal regeneration in Nogo-deficient mice. Neuron 38, 213–24.

235.Zheng, B., Atwal, J., Ho, C., Case, L., He, X. L., Garcia, K. C., Steward, O. & TessierLavigne, M. (2005). Genetic deletion of the Nogo receptor does not reduce neurite

inhibition in vitro or promote corticospinal tract regeneration in vivo. Proc Natl Acad Sci USA 102, 1205–10.

236.Dimou, L., Schnell, L., Montani, L., Duncan, C., Simonen, M., Schneider, R., Liebscher, T., Gullo, M. & Schwab, M. E. (2006). Nogo-A-deficient mice reveal straindependent differences in axonal regeneration. J Neurosci 26, 5591–603.

and the molecular composition of cellular structures, such as organelles, can also be determined only at the protein level.

To identify proteins in a high-throughput fashion, proteomics utilizes various approaches, including protein electrophoresis, liquid chromatography-based peptide separation, mass spectrometry (MS), and computerized data analysis using bioinformatic tools (4). A major aspect of many proteomic strategies is the identification of proteins using an analytical fingerprint that can be used to search a sequence database. One common fingerprint is the tandem mass (MS/MS) spectrum of a peptide. Thus, an MS/MS spectrum can be algorithmically compared with predicted peptide spectra from a sequence database to identify the respective protein. Traditionally, two-dimensional (2D) gel electrophoresis has been used as a separation method to facilitate the analysis of complex protein mixtures by MS (5). More recently, gel-free strategies have been developed for comprehensive proteomic analysis using MS (6). Mass spectrometric methods are being developed that not only identify proteins in a mixture but also compare the relative level of protein expression between two different samples. Advanced strategies also enable the researchers to gain information about modifications and interactions of proteins. Emerging biochemical approaches, continuously developing software tools, and expanding databases are expected to further improve the power of proteomics for the large-scale study of gene function directly at the protein level.

Ongoing research has identified alterations in the expression level of various proteins expressed by retinal ganglion cells (RGCs) in glaucomatous eyes (7–11). It is also evident that many proteins exhibit post-translational modifications during glaucomatous neurodegeneration, including oxidation (12), phosphorylation (10,13), and glycation (14). Novel functions of proteins after such modifications have important implications in the cellular mechanisms of glaucomatous neurodegeneration. By determining protein activity and protein–protein interactions, as well as the stability, localization, and turnover of proteins, such proteome alterations are extremely important for the regulation of cell signaling and the ultimate cell fate. Proteolytic regulation of protein function is also very important for glaucomatous neurodegeneration as supported by the involvement of proteolytic caspase cascades in the final execution of RGC death (15–17). Proteomics is now being effectively used to study pathogenic processes involved in glaucomatous neurodegeneration and opens new avenues for glaucoma research aiming at the identification of biomarkers and new treatment targets.

CURRENT APPLICATIONS OF PROTEOMICS IN GLAUCOMATOUS NEURODEGENERATION

Given that the identification of alterations in the cellular protein complement can help to understand impaired cellular mechanisms, increasing number of proteomic studies over the past few years have provided important information, which significantly enhances the current understanding of glaucomatous neurodegeneration. Along with the continuous improvement and refinement of proteomic and bioinformatic tools, ongoing studies promise to obtain increasing amounts of structural and functional information, thereby providing novel treatment targets for neuroprotective interventions.

RGC Proteome Mapping

A major goal of current proteomic studies is a thorough identification of the RGC proteome to establish a specific database and a 2D proteome map (18). Identification of RGC proteins expressed under normal conditions is essential to analyze qualitative and quantitative differences in RGC protein expression under various disease conditions, including glaucoma.

A common difficulty in protein analysis is in the lysation of proteins. However, the protein lysation has not been a problem for RGCs. A main challenge of RGC proteomics is the enrichment of RGCs. It is encouraging, however, that to determine protein expression within distinct cell types, 90% purity is appropriate for sufficient sensitivity, although even a minimum amount of contamination with a few other cell types would be problematic for the gene expression studies. Current studies utilize RGCs isolated from rat retinas through the immunomagnetic selection of Thy-1.1-positive cells using a two-step process (12,13,16). RGCs isolated through this procedure have been identified based on retrograde labeling with a fluorescent tracer, cell morphology, and immunolabeling for specific markers (16). In addition, to re-examine the purity of isolated RGCs, lysated proteins have been subjected to western blot analysis using specific antibodies recognizing different retinal cell markers (18). Western blots shown in Fig. 1 confirm the expression of neurofilament protein, but the absence of other cell markers, including glial fibrillary acidic protein (an astrocyte marker); syntaxin (an amacrine cell marker); vimentin and glutamine synthetase (Müller cell markers); metabotropic glutamate receptor 6 (a bipolar cell marker); and rhodopsin (a photoreceptor marker). In contrast, western blots of protein samples obtained from unselected cells show the expression of different retinal cell markers. This enriched isolation of RGCs with quite high yields facilitates sufficient protein sampling for proteomic analysis. Despite recent advances in the sensitivity of protein detection and identification, the protein amount obtained is a unique advantage of the immunoselection technique over alternative strategies, such as the laser capture microdissection (19), which can dissect single cells within a tissue section.

Complementary approaches are currently utilized to identify the RGC proteome. One of the approaches is 2D polyacrylamide gel electrophoresis (2D-PAGE) followed by mass spectrometric analysis of the tryptic digests of excised gel spots for protein identification (see Fig. 2). This technique can be routinely applied for quantitative expression profiling of large sets of complex protein mixtures in RGC lysates. 2DPAGE enables the separation of complex mixtures of proteins according to isoelectric point (pI), molecular weight (MW), solubility, and relative abundance. Furthermore, this technique provides a map of intact proteins, which reflects changes in protein expression level, isoforms, or post-translational modifications.

To ensure high-quality datasets with minimum false-positive rates, mass spectrometric protein identification should follow common guidelines (20). For peptide mass fingerprinting, complementary and confirmatory search engines are commonly utilized in the bioinformatic analysis of mass spectrometric data obtained using matrix-assisted laser desorption ionization time-of-flight MS (MALDI-TOF/MS). Peptide masses are first subjected to a non-restricted search using the Mascot search engine. Whether the MW and pI estimated through 2D-PAGE match with the MW and pI of the identified

Fig. 1. Western blots of lysated retinal ganglion cell (RGC) proteins. For proteomic analysis, RGCs are isolated from rat retinal cell suspensions through the immunomagnetic selection of Thy-1.1-positive cells using a two-step process. Western blot analysis using specific antibodies recognizing different retinal cell markers confirm the expression of neurofilament protein (NFP), but the absence of other cell markers, including glial fibrillary acidic protein (GFAP, an astrocyte marker); syntaxin (an amacrine cell marker); vimentin and glutamine synthetase (Müller cell markers); metabotropic glutamate receptor 6 (mGluR6, a bipolar cell marker); and rhodopsin (a photoreceptor marker). In contrast, western blots of protein samples obtained from unselected cells show the expression of different retinal cell markers.

proteins through peptide mass fingerprinting is carefully evaluated. After completion of this search, the MW and pI are used to classify all of the initial significant hits into definite hits, negative hits (false positive), potential multimers or fragments, or proteins that are likely to be modified. Samples are then subjected to restricted search by the Profound search engine using predicted MW and pI ranges based on the spot positions on the gels. To provide convincing protein identity through this integrative approach between non-restricted and restricted searches, the p value for the statistical significance of the match should be <0.05, and the sequence coverage should be >10%.

Protein identification through the analysis of 2D-gels is also complemented by peptide sequences obtained using liquid chromatography coupled with tandem MS (LC-MS/MS). Multiple MS/MS spectra are acquired using multiple peptides, each satisfying the criteria for a good match in the utilized software. For protein identification, two or more peptides should show manually confirmed sequence matches. The use of tandem MS significantly increases the number of proteins that can be identified through 2D-PAGE, because it provides high-quality datasets with increased sensitivity

Fig. 2. To establish the database of normal retinal ganglion cell (RGC) proteome, RGC protein samples are subjected to 2D polyacrylamide gel electrophoresis (2D-PAGE) followed by mass spectrometric analysis of excised gel spots for protein identification. As a complementary approach to protein identification from 2D-gels, a liquid chromatography-based gel-free proteomic technique is also utilized to identify proteins directly from the tryptic digest of the protein sample.

and minimum false-positive rates. Tandem MS is also applicable to a complex mixture of proteins, in gel or in solution. This facilitates the identification of multiple proteins that may be present in one 2D-gel spot.

When required for further confirmation (sequence coverage <10%; only one peptide showing sequence match), western blot analysis is performed using specific antibodies; and immunohistochemistry is utilized to determine the cellular localization of the identified proteins. In fact, by determining the cellular localization of proteins, along with an in situ examination of tissue structures, immunohistochemistry significantly contributes to protein expression studies.

It should be recognized that there may be a number of shortcomings of the 2D-PAGE- based approach for protein identification. For example, proteins of low-abundance, integral membrane proteins, and proteins with extreme MW or pI may be difficult to identify. Fortunately, recent refinements in technique, including immobilized pH gradients, have enhanced the ability to identify these difficult proteins by 2D-gel analysis (5). Recent advances in high-resolution 2D-PAGE allow the separation of up to 10000 protein spots on a 2D-gel, and as small as 1 ng of protein per spot can be detected and quantified. Advances in gel staining also facilitate the visualization and identification of protein spots on gels. For example, Sypro Ruby, a commonly utilized fluorescent-based dye, has a greater dynamic range for protein expression and also does not impair extraction of peptides from the gel (21). As an attempt to reduce sample complexity and enrich low-abundance proteins, protein pre-fractionation techniques can be applied (22,23). For example, a multicompartment electrolyzer can successfully be utilized to separate mixtures of RGC proteins into fractions according to the pI range. The isolated fractions can then be separated on narrow pI range first dimension gel strips for subsequent 2D-gel analysis followed by MS. Current studies

support that 2D-gels obtained using pre-fractionated samples show many more spots. In addition, no protein precipitation or smears occur upon pre-fractionation, which makes loading larger sample amounts more feasible to increase the dynamic range of detection.

Gel-free approaches capable of characterizing proteins directly from entire cell lysates are also complementarily utilized to identify the RGC proteome. Digestion of proteinsample and direct analysis of the resulting peptides through microcapillary highperformance LC-MS/MS facilitates shotgun identification of complex protein mixtures without the need for prior sample fractionation (6,24). This powerful technique helps alleviate many limitations of the 2D-PAGE-based approach. Since this technique uses separation mechanisms based on charge and hydrophobicity, as opposed to pI and MW for 2D-PAGE, it can specifically facilitate the identification of low-abundance and integral membrane proteins and proteins with extreme MW or pI. In addition, a much smaller amount of protein sample is sufficient for the identification of thousands of proteins using this technique. Acquiring over 3000 MS/MS spectra using only 10 g of RGC protein sample has been achieved using a liquid chromatography-based gel-free technique during ongoing studies.

Through the complementary use of different proteomic techniques, the identified proteins include a wide variety of RGC proteins with predicted locations within the entire spectra of cellular compartments, including nuclear, mitochondrial, endoplasmic, and membrane proteins, which are involved in the cell structure, energy metabolism, signal transduction, and stress response (18). Ongoing efforts aim for a thorough identification of the RGC proteome to establish the specific database for widespread utility.

Identification of Differential Protein Expression in RGCs

Identification of time-dependent alterations in the expression and specific modifications of RGC proteins during glaucomatous neurodegeneration can provide comprehensive information about RGC response to glaucomatous injury, reveal cellular pathways associated with the neurodegenerative process of glaucoma, and identify biomarkers and treatment targets. Therefore, one of the goals of proteomic studies in the field of glaucoma is to quantitatively identify time-dependent alterations in the RGC proteome. Current studies utilize an experimental rat model of glaucoma by inducing chronic elevation of intraocular pressure (IOP) by hypertonic saline injections into episcleral veins (12,25).

To identify differentially expressed or modified RGC proteins in ocular hypertensive eyes relative to controls, RGC protein lysates can be pooled from eyes with known IOP exposure and axon loss. For 2D-PAGE-based differential display analysis, protein quantity is determined based on spot intensity in a series of 2D-gels obtained using equally loaded RGC protein samples from ocular hypertensive and control eyes, and the intensity of the matched protein spots is compared by using image analysis. Excised gel spots are then processed for mass spectrometric protein identification. Differential proteomics using this 2D-PAGE approach can reveal multiple alterations in protein expression level during glaucomatous neurodegeneration in ocular hypertensive eyes. For example, an increase or decrease in a spot intensity points to increased or decreased

protein expression. Some newly detected spots also point to the up-regulated expression of a protein which is not detectable on control gels due to a low expression level under normal conditions. Alternatively, some of the newly detected spots may result from the shift of a protein spot from its initial position due to post-translational modification or may result from proteolytic cleavage. A critical consideration for differential display analysis is the number of samples required to detect a change in protein expression. Based on statistical power calculations, 10 samples have been reported to be sufficient to detect a 30% change in intensity for 24% of the proteome, whereas the same number of samples is sensitive enough to detect a 100% intensity change for 97% of the proteins (26). Ongoing studies complement the 2D-PAGE approach of differential proteomics with gel-free quantitative analysis techniques, which facilitate high-throughput generation of quantitative protein profiles directly from protein samples (27,28). For example, relative protein abundance can be calculated using stable isotope labeling (29,30). However, it should be emphasized that there is no one best approach for quantitative proteomics, and all approaches are complementary even though technically challenging. In addition, western blot analysis should be utilized for confirmation of differential protein expression. Functional assays, as well as confirmation of the proteomic data at gene level on selected targets, should also be considered as complementary approaches when required.

The data of differentially expressed proteins in ocular hypertensive eyes are interpreted by integrating with known cellular networks. The analysis and integration of the proteomic data can also utilize complementary bioinformatic computational data mining strategies, such as clustering, functional annotation, and statistical inference (31). In addition to the identification of up-regulated or down-regulated proteins, identification of post-translational protein modifications as well as identification of interacting proteins can provide further information about specific signaling cascades involved in glaucomatous neurodegeneration.

Identification of Post-Translational Protein Modifications During

Glaucomatous Neurodegeneration

High-throughput proteomic techniques have become powerful tools for the study of protein signaling cascades and have the capability of characterizing molecular networks on a large scale. Several different strategies have been used to analyze protein modifications, and almost all are targeted to specific types of modifications. With the advent of targeted proteomic approaches to ultrasensitive MS, it is now feasible to identify post-translational modifications of proteins on a proteome-wide scale. Targeted proteomics not only identifies specific proteome changes but also characterizes biochemical consequences of these changes and discerns how these alterations govern the genesis of a pathological phenotype (32,33). Current proteomic studies to identify cellular mechanisms of glaucomatous neurodegeneration in ocular hypertensive eyes determine post-translational modifications of proteins, including oxidation, phosphorylation, and glycation. Initial findings of these studies strongly support that targeted proteomic approaches provide a very promising way to elucidate pathogenic processes involved in glaucomatous neurodegeneration at the protein level.

Protein Oxidation

On the basis of the evidence of amplified production of reactive oxygen species during glaucomatous neurodegeneration (17), proteomic analysis has recently been performed to determine whether retinal proteins are oxidatively modified during glaucomatous neurodegeneration in ocular hypertensive eyes, and if so, what the targets are for protein oxidation in these eyes (12). Retinal protein samples obtained from moderately damaged ocular hypertensive rat eyes were utilized in this study, and comparison of protein oxidation levels was obtained by identifying retinal proteins containing reactive carbonyl groups using 2D-oxyblot analysis followed by protein identification through MS (see Fig. 3). Results of this study have revealed that the oxidative protein modification occurs to a greater extent in ocular hypertensive retinas compared with the controls. Evaluation of 2D-oxyblots with Coomassie Blue-stained 2D-gels from the same samples showed that approximately 60 protein spots obtained using retinal protein lysates from ocular hypertensive retinas (out of hundreds of spots) exhibited protein carbonyl immunoreactivity, which reflects oxidatively modified proteins (see Fig. 4). Protein carbonyl immunoreactivity of individual protein spots detected on 2D-oxyblots was compared between ocular hypertensive and control eyes following normalization of spots to their protein content measured by the intensity of Coomassie Blue staining. This comparison revealed a significant increase in carbonyl immunoreactivity in individual spots obtained using retinal protein lysates from ocular hypertensive eyes compared with the controls. The proteins identified using MALDI-TOF/MS and LCMS/MS included glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a glycolytic

Fig. 3. To identify oxidatively modified retinal proteins during glaucomatous neurodegeneration, retinas are dissected from rat eyes. An aliquot of the retinal protein sample is used to detect protein carbonyls through 2D-oxyblot analysis, during which protein carbonyls react with 2,4-dihydrophenylhydrazine (DNPH) forming a hydrazone that is detected by immunolabeling of membranes with a specific anti-DNP antibody. Another aliquot of the same protein sample is used in 2D polyacrylamide gel electrophoresis (2D-PAGE). After matching the corresponding spots on 2D-oxyblots and 2D-gel images, protein spots exhibiting oxidative modification are excised from 2D-gels, and peptide masses are analyzed by using mass spectrometry. Bioinformatic databases are then searched for protein identification from the mass spectral data.