Age-Related Macular Degeneration and Mitochondria-Associated Autoantibodies: A Review of the Specific Pathogenesis and Therapeutic Strategies.

Age-related macular degeneration (AMD) is a severe retinal disease that causes irreversible visual loss and blindness in elderly populations worldwide. The pathological mechanism of AMD is complex, involving the interactions of multiple environmental and genetic factors. A poor understanding of the disease leads to limited treatment options and few effective prevention methods. The discovery of autoantibodies in AMD patients provides an opportunity to explore the pathogenesis and treatment direction of the disease. This review focuses on the mitochondria-associated autoantibodies and summarizes the functional roles of mitochondria under physiological conditions and their alterations during the pathological states. Additionally, it discusses the crosstalk between mitochondria and other organelles, as well as the mitochondria-related therapeutic strategies in AMD.

Association of autoantibody levels with different stages of age-related macular degeneration (AMD): Results from the population-based Gutenberg Health Study (GHS).

PURPOSE: Anti-retinal autoantibodies are assumed to be associated with age-related macular degeneration (AMD). To our knowledge, this is the first evaluation of autoantibodies in human sera of participants with different stages of AMD in a large population-based, observational cohort study in Germany. METHODS: The Gutenberg Health Study (GHS) is a population-based, observational cohort study in Germany, including 15,010 participants aged between 35 and 74. Amongst others, non-mydriatic fundus photography (Visucam PRO NM™, Carl Zeiss Meditec AG, Jena, Germany) was performed. Fundus images of the first 5000 participants were graded based on the Rotterdam Eye Study classification. Sera of participants with AMD (n=541) and sera of age-matched participants without AMD (n=490) were analyzed by antigen-microarrays. Besides descriptive statistics, autoantibody-levels were compared by Mann-Whitney-U test and the associations of level of autoantibodies with AMD were calculated by logistic regression analysis. Likewise, possible associations of the autoantibodies and both clinical and laboratory parameters on AMD subjects were analyzed. RESULTS: Autoantibodies against transferrin (p<0.001) were significantly downregulated in participants with early AMD and soft, distinct drusen (≥63 µm) or pigmentary abnormalities only compared to Controls. Mitogen-activated protein kinase 3 (p=0.041), glutathione peroxidase 4 (p=0.048), clusterin (p=0.045), lysozyme (p=0.19), protein kinase C substrate 80K-H (p=0.02), heat shock 70 kDa protein 1A (p=0.04) and insulin (p=0.018) show a trend between Control and participants with early AMD and soft, distinct drusen (≥63 µm) or pigmentary abnormalities only. CONCLUSIONS: This study contributes to a growing knowledge of autoantibodies in association with different AMD stages compared to controls in the context of a large population-based study in Germany. Especially autoantibodies against inflammatory proteins were downregulated in participants with early AMD and soft, distinct drusen (≥63 µm) or pigmentary abnormalities only.

Lectin-Based Affinity Enrichment and Characterization of N-Glycoproteins from Human Tear Film by Mass Spectrometry.

The glycosylation of proteins is one of the most common post-translational modifications (PTMs) and plays important regulatory functions in diverse biological processes such as protein stability or cell signaling. Accordingly, glycoproteins are also a consistent part of the human tear film proteome, maintaining the proper function of the ocular surface and forming the first defense barrier of the ocular immune system. Irregularities in the glycoproteomic composition of tear film might promote the development of chronic eye diseases, indicating glycoproteins as a valuable source for biomarker discovery or drug target identification. Therefore, the present study aimed to develop a lectin-based affinity method for the enrichment and concentration of tear glycoproteins/glycopeptides and to characterize their specific N-glycosylation sites by high-resolution mass spectrometry (MS). For method development and evaluation, we first accumulated native glycoproteins from human tear sample pools and assessed the enrichment efficiency of different lectin column systems by 1D gel electrophoresis and specific protein stainings (Coomassie and glycoproteins). The best-performing multi-lectin column system (comprising the four lectins ConA, JAC, WGA, and UEA I, termed 4L) was applied to glycopeptide enrichment from human tear sample digests, followed by MS-based detection and localization of their specific N-glycosylation sites. As the main result, our study identified a total of 26 N glycosylation sites of 11 N-glycoproteins in the tear sample pools of healthy individuals (n = 3 biological sample pools). Amongst others, we identified tear film proteins lactotransferrin (N497 and N642, LTF), Ig heavy chain constant α-1 (N144 and 340, IGHA1), prolactin-inducible protein (N105, PIP), and extracellular lacritin (N105, LACRT) as highly reliable and significant N glycoproteins, already associated with the pathogenesis of various chronic eye diseases such as dry eye syndrome (DES). In conclusion, the results of the present study will serve as an important tear film N-glycoprotein catalog for future studies focusing on human tear film and ocular surface-related inflammatory diseases.

CKAMP44 controls synaptic function and strength of relay neurons during early development of the dorsal lateral geniculate nucleus.

Relay neurons of the dorsal lateral geniculate nucleus (dLGN) receive inputs from retinal ganglion cells via retinogeniculate synapses. These connections undergo pruning in the first 2 weeks after eye opening. The remaining connections are strengthened several-fold by the insertion of AMPA receptors (AMPARs) into weak or silent synapses. In this study, we found that the AMPAR auxiliary subunit CKAMP44 is required for receptor insertion and function of retinogeniculate synapses during development. Genetic deletion of CKAMP44 resulted in decreased synaptic strength and a higher number of silent synapses in young (P9-11) mice. Recovery from desensitisation of AMPARs was faster in CKAMP44 knockout (CKAMP44-/- ) than in wild-type mice. Moreover, loss of CKAMP44 increased the probability of inducing plateau potentials, which are known to be important for eye-specific input segregation and retinogeniculate synapse maturation. The anatomy of relay neurons in the dLGN was changed in young CKAMP44-/- mice showing a transient increase in dendritic branching that normalised during later development (P26-33). Interestingly, input segregation in young CKAMP44-/- mice was not affected when compared to wild-type mice. These results demonstrate that CKAMP44 promotes maturation and modulates function of retinogeniculate synapses during early development of the visual system without affecting input segregation. KEY POINTS: Expression of CKAMP44 starts early during development of the dorsal lateral geniculate nucleus (dLGN) and remains stable in relay neurons and interneurons. Genetic deletion of CKAMP44 decreases synaptic strength and increases silent synapse number in dLGN relay neurons; increases the rate of recovery from desensitisation of AMPA receptors in dLGN relay neurons; and reduces synaptic short-term depression in retinogeniculate synapses. The probability of inducing plateau potentials is elevated in relay neurons of CKAMP44-/- mice. Eye-specific input segregation is unaffected in the dLGN of CKAMP44-/- mice. Deletion of CKAMP44 mildly affects dendritic arborisation of relay neurons in the dLGN.

Dynamin-like Protein 1 (DNML1) as a Molecular Target for Antibody-Based Immunotherapy to Treat Glaucoma.

Slow and progressive loss of retinal ganglion cells (RGCs) is the main characteristic of glaucoma, the second leading cause of blindness worldwide. Previous studies have shown that impaired mitochondrial dynamics could facilitate retinal neurodegeneration. Mitochondrial dynamics are regulated directly (fission) or more indirectly (fusion) by dynamin-like protein 1 (DNML1). Therefore, DNM1L might be a promising target for an antibody-based approach to treat glaucoma. The consequences of targeting endogenous DNM1L by antibodies in a glaucoma animal model have not been investigated yet. Here, we show that the intravitreal application of an anti-DNM1L antibody showed protective effects regarding the survival of RGCs and their axons in the retinal nerve fiber layer (RNFL). Antibody treatment also improved retinal functionality, as observed by electroretinography (Ganzfeld ERG). Western blot analysis revealed altered DNM1L phosphorylation and altered expression of proteins related to apoptosis suggesting a decreased apoptosis rate. Mass spectrometry analysis revealed 28 up-regulated and 21 down-regulated proteins (p < 0.05) in both experimental groups. Protein pathway analysis showed that many proteins interacted directly with the target protein DNM1L and could be classified into three main protein clusters: Vesicle traffic-associated (NSF, SNCA, ARF1), mitochondrion-associated (HSP9A, SLC25A5/ANT2, GLUD1) and cytoskeleton-associated (MAP1A) signaling pathway. Our results demonstrate that DNM1L is a promising target for an antibody-based approach to glaucoma therapy.

A Monoclonal Anti-HMGB1 Antibody Attenuates Neurodegeneration in an Experimental Animal Model of Glaucoma.

Neuroinflammation is a crucial process for the loss of retinal ganglion cells (RGC), a major characteristic of glaucoma. High expression of high-mobility group box protein 1 (HMGB1) plays a detrimental role in inflammatory processes and is elevated in the retinas of glaucoma patients. Therefore, this study aimed to investigate the effects of the intravitreal injection of an anti-HMGB1 monoclonal antibody (anti-HMGB1 Ab) in an experimental animal model of glaucoma. Two groups of Spraque Dawley rats received episcleral vein occlusion to chronically elevate intraocular pressure (IOP): (1) the IgG group, intravitreal injection of an unspecific IgG as a control, n = 5, and (2) the HMGB1 group, intravitreal injection of an anti-HMGB1 Ab, n = 6. IOP, retinal nerve fiber layer thickness (RNFLT), and the retinal flash response were monitored longitudinally. Post-mortem examinations included immunohistochemistry, microarray, and mass spectrometric analysis. RNFLT was significantly increased in the HMGB1 group compared with the IgG group (p < 0.001). RGC density showed improved neuronal cell survival in the retina in HMGB1 compared with the IgG group (p < 0.01). Mass spectrometric proteomic analysis of retinal tissue showed an increased abundance of RNA metabolism-associated heterogeneous nuclear ribonucleoproteins (hnRNPs), such as hnRNP U, D, and H2, in animals injected with the anti-HMGB1 Ab, indicating that the application of the antibody may cause increased gene expression. Microarray analysis showed a significantly decreased expression of C-X-C motif chemokine ligand 8 (CXCL8, p < 0.05) and connective tissue growth factor (CTGF, p < 0.01) in the HMGB1 group. Thus, these data suggest that intravitreal injection of anti-HMGB1 Ab reduced HMGB1-dependent inflammatory signaling and mediated RGC neuroprotection.

Tear Film-specific Biomarkers in Glaucoma Patients. / Tränenfilmspezifische Biomarker bei Glaukompatienten.

Glaucoma is a group of chronic eye diseases that lead to degeneration of retinal ganglion cells (RGCs) and their axons followed by irreversible loss of vision in the patient. Glaucoma is a disease that initially evolves asymptomatically with the first symptoms appearing only at an advanced stage of this eye disease. For this reason, it is always necessary to develop state-of-the-art technologies and methods for the identification and characterization of new, specific biomarkers for the early diagnosis of glaucoma. Therefore, the analysis of biological fluids, as in this case the tear fluid of patients, represents an attractive source to identify new specific as well as sensitive biomarkers in glaucoma. These biomarkers could be involved in the pathophysiological processes of glaucoma or possibly serve for diagnostic differentiation of various types of glaucoma.

Contribution of the Commensal Microflora to the Immunological Homeostasis and the Importance of Immune-Related Drug Development for Clinical Applications.

Not long ago, self-reactive immune activity was considered as pathological trait. A paradigm shift has now led to the recognition of autoimmune processes as part of natural maintenance of molecular homeostasis. The immune system is assigned further roles beneath the defense against pathogenic organisms. Regarding the humoral immune system, the investigation of natural autoantibodies that are frequently found in healthy individuals has led to further hypotheses involving natural autoimmunity in other processes as the clearing of cellular debris or decrease in inflammatory processes. However, their role and origin have not been entirely clarified, but accumulating evidence links their formation to immune reactions against the gut microbiome. Antibodies targeting highly conserved proteins of the commensal microflora are suggested to show self-reactive properties, following the paradigm of the molecular mimicry. Here, we discuss recent findings, which demonstrate potential links of the commensal microflora to the immunological homeostasis and highlight the possible implications for various diseases. Furthermore, specific components of the immune system, especially antibodies, have become a focus of attention for the medical management of various diseases and provide attractive treatment options in the future. Nevertheless, the development and optimization of such macromolecules still represents a very time-consuming task, shifting the need to more medical agents with simple structural properties and low manufacturing costs. Synthesizing only the biologically active sites of antibodies has become of great interest for the pharmaceutical industry and offers a wide range of therapeutic application areas as it will be discussed in the present review article.

Minocycline reduces inflammatory response and cell death in a S100B retina degeneration model.

BACKGROUND: Previous studies noted that intravitreal injection of S100B triggered a glaucoma-like degeneration of retina and optic nerve as well as microglia activation after 14 days. The precise role of microglia in our intravitreal S100B model is still unclear. Hence, microglia were inhibited through minocycline. The aim is to investigate whether microglia have a significant influence on the degeneration process or whether they are only a side effect in the model studied here. METHODS: Minocycline was applied daily in rats by intraperitoneal injection using two different concentrations (13.5 mg/kg body weight, 25 mg/kg body weight). One day after treatment start, S100B or PBS was intravitreally injected in one eye per rat. The naïve groups received no injections. This resulted in a total of five groups (naïve n = 14, PBS n = 14, S100B n = 13, 13.5 mg/kg mino n = 15, 25 mg/kg mino n = 15). At day 14, electroretinogram measurements were performed, followed by immunofluorescence and label-free quantitative proteomics analysis. The focus of these investigations was on the survival of RGCs as well as their axons, the response of the microglia, and the identification of further pathological modes of action of S100B. RESULTS: The best signal transmission was detected via ERG in the 13.5 mg/kg mino group. The inhibition of the microglia protected optic nerve neurofilaments and decreased the negative impact of S100B on RGCs. However, the minocycline treatment could not trigger complete protection of RGCs. Furthermore, in retina and optic nerve, the minocycline treatment reduced the number and activity of S100B-triggered microglia in a concentration-dependent manner. Proteomics analysis showed that S100B application led to numerous metabolic functions and cellular stress, mainly an increased inflammatory response, glycolysis, and mitochondrial dysfunction, which caused oxidative stress in the retina. Importantly, the protective capability of lower dose of minocycline was unraveled by suppressing the apoptotic, inflammatory, and the altered metabolic processes caused by S100B insult in the retina. CONCLUSION: Intravitreally injected S100B not only led to a pro-inflammatory microglial reaction, but also a mitochondrial and metabolic dysfunction. Also, these results suggest that an excessive microglial response may be a significant degenerative factor, but not the only trigger for increased cell death.

Peptides of the variable IgG domain as potential biomarker candidates in primary open-angle glaucoma (POAG).

Autoantibody profiling has gained increasing interest in the research field of glaucoma promising the detection of highly specific and sensitive marker candidates for future diagnostic purposes. Recent studies demonstrated that immune responses are characterized by the expression of congruent or similar complementarity determining regions (CDR) in different individuals and could be used as molecular targets in biomarker discovery. Main objective of this study was to characterize glaucoma-specific peptides from the variable region of sera-derived immunoglobulins using liquid chromatography--mass spectrometry (LC-MS)-based quantitative proteomics. IgG was purified from sera of 13 primary open-angle glaucoma patients (POAG) and 15 controls (CTRL) and subsequently digested into Fab and Fc by papain. Fab was further purified, tryptic digested and measured by LC-MS/MS. Discovery proteomics revealed in total 75 peptides of the variable IgG domain showing significant glaucoma-related level changes (P < 0.05; log2 fold change ≥ 0.5): 6 peptides were high abundant in POAG sera, whereas 69 peptides were low abundant in comparison to CTRL group. Via accurate inclusion mass screening strategy 28 IgG V domain peptides were further validated showing significantly decreased expression levels in POAG sera. Amongst others 5 CDR1, 2 CDR2 and 1 CDR3 sequences. In addition, we observed significant shifts in the variable heavy chain family distribution and disturbed κ/λ ratios in POAG patients in contrast to CTRL. These findings strongly indicate that glaucoma is accompanied by systemic effects on antibody production and B cell maturation possibly offering new prospects for future diagnostic or therapy purposes.

Comparative Quantitative Analysis of Porcine Optic Nerve Head and Retina Subproteomes.

Optic nerve head (ONH) and retina (RET) are the main sites of damage in neurodegenerative optic neuropathies including glaucoma. Up to date, little is known about the molecular interplay between these two adjoining ocular components in terms of proteomics. To close this gap, we investigated ONH and RET protein extracts derived from porcine eyes (n = 12) (Sus scrofa domestica Linnaeus 1758) using semi-quantitative mass spectrometry (MS)-based proteomics comprising bottom-up LC-ESI MS/MS and targeted SPE-MALDI-TOF MS analysis. In summary, more than 1600 proteins could be identified from the ONH/RET tissue complex. Moreover, ONH and RET displayed tissue-specific characteristics regarding their qualitative and semi-quantitative protein compositions. Gene ontology (GO)-based functional and protein-protein interaction analyses supported a close functional connection between the metabolic-related RET and the structural-associated ONH subproteomes, which could be affected under disease conditions. Inferred from the MS findings, stress-associated proteins including clusterin, ceruloplasmin, and endoplasmin can be proposed as extracellular mediators of the ONH/ RET proteome interface. In conclusion, ONH and RET show obvious proteomic differences reflecting characteristic functional features which have to be considered for future protein biomarker profiling studies.

An In-Depth View of the Porcine Trabecular Meshwork Proteome.

The house swine (Sus scrofa domestica Linnaeus 1758) is an important model organism regarding the study of neurodegenerative diseases, especially ocular neuropathies such as glaucoma. This is due to the high comparability of the porcine and human eye regarding anatomy and molecular features. In the pathogenesis of glaucoma, the trabecular meshwork (TM) forms a key ocular component in terms of intraocular pressure (IOP) elevation. Thereby, functional TM abnormalities are correlated with distinct proteomic alterations. However, a detailed analysis of the TM proteome has not been realized so far. Since the porcine eye has high potential as a model system to study ocular diseases such as glaucoma, the present study focuses on the in-depth analysis of the porcine TM proteome. By use of a bottom-up (BU) mass spectrometric (MS) platform utilizing electrospray ionization liquid chromatography tandem MS (LC-ESI-MS/MS) considering database-dependent and peptide de novo sequencing, more than 3000 TM proteins were documented with high confidence (FDR < 1%). A distinct number of proteins with neuronal association were revealed. To the best to our knowledge, many of these protein species have not been reported for TM tissue before such as reelin, centlein and high abundant neuroblast differentiation-associated protein AHNAK (AHNAK). Thereby, AHNAK might play a superordinate role in the TM regarding proposed tissue involvement in barrier function. Also, a high number of secretory proteins could be identified. The generated TM proteomic landscape underlines a multifunctional character of the TM beyond representing a simple drainage system. Finally, the protein catalogue of the porcine TM provides an in-depth view of the TM molecular landscape and will serve as an important reference map in terms of glaucoma research utilizing porcine animal models, porcine TM tissues and/or cultured TM cells.

[Endothelial Cell Reaction to Elevated Hydrostatic Pressure and Oxidative Stress in Vitro]. / Reaktion der Endothelzellen auf kurzzeitig physiologisch erhöhte hydrostatische Drücke und oxidativen Stress in vitro.

INTRODUCTION: Endothelial dysfunction has become a strongly discussed factor regarding glaucoma pathogenesis. In addition to peripapillary bleedings as signs of vascular damage, there is a definite correlation between glaucoma and vascular dysregulation syndrome. The aim of this study was to evaluate endothelial cell reaction to moderately elevated hydrostatic pressure and oxidative stress in vitro. METHODS: In vitro, primarily dissociated brain microvascular endothelial cells (BMECs) were exposed to moderately elevated hydrostatic pressure (60 and 120 mmHg) in a special pressure chamber. Additionally, cells primarily exposed to pressure, and cells not exposed to pressure, were incubated with low amounts of H2O2. A live/dead assay was performed to evaluate cell viability. Immunohistochemical staining against actin was used for morphological evaluation. RESULTS: Neither 60 nor 120 mmHg of elevated pressure had a viability changing effect on primary endothelial cells. Secondary, no big morphological changes could be discovered. However, against a low concentration of oxidative stress, BMECs showed high vulnerability. A difference in reaction to cells stressed with high pressure before could not be shown. CONCLUSION: Direct effects, in terms of higher vulnerability or morphological changes of moderately elevated high pressure on endothelial cells, could not be shown. However, the reaction to low amounts of oxidative stress indicates the involvement of endothelial cells in the pathogenesis of glaucoma and the special role of oxidative stress when referring to endothelial dysfunction in glaucomatous disease.

[Morphological and Quantitative Changes in Retinal and Optic Nerve Vessels in Experimental Glaucoma Model with Elevated IOP for 7 Weeks]. / Erhöhter Augeninnendruck für 7 Wochen induziert lokale Gefäßveränderungen im experimentellen Glaukommodell in vivo.

INTRODUCTION: Glaucoma is characterised by progressive loss of retinal ganglion cells and axons. Experimental research has concentrated on understanding the pathophysiological mechanisms involved in glaucomatous damage. It is still a matter of debate whether neurons or capillaries are primarily damaged by elevated intraocular pressure (IOP). The aim of this study was to detect IOP-induced vascular changes in the vessels of the optic nerve head and the main vessels of the retina in vivo. METHODS: Experimental glaucoma was induced in adult Sprague Dawley rats by cauterisation of three episcleral veins of the left eye (n = 3). In vivo, retinal vessel calibre was measured manually using a peripapillary scan with SD-OCT (Heidelberg Engineering) at baseline and after seven weeks of IOP elevation. The animals were then sacrificed and the optic nerve was fixed with 30% glutaraldehyde and cross-sections stained with paraphenylene diamine to mark the vessels. Contralateral eyes served as controls. Pictures were taken and number of vessels, vessel calibre and area were calculated and compared. RESULTS: IOP was significantly elevated (p < 0.001). In optic nerve cross sections, the number of capillaries did not differ significantly between animals with elevated IOP and controls. However, vessel calibre and area were significantly reduced (p < 0.001) in glaucomatous optic nerves. The calibre of the retinal vessels was significantly lowered - by 9.22% (p = 0.021). CONCLUSION: Retinal arterioles and optic nerve capillaries respond sensitively to abnormal pressure elevation in vivo, showing high and early vulnerability. The vascular responses may influence secondary neuronal responses, which culminate in the death of ganglion cells and blindness, as occurs in clinical glaucoma.

Relaxin 2 fails to lower intraocular pressure and to dilate retinal vessels in rats.

PURPOSE: Recently, the vasodilator relaxin 2 has been introduced as a treatment for acute heart failure. However, its role on vessels of the eye and intraocular pressure (IOP) remains unclear though it has been hypothesized to induce a decrease IOP after intramuscular injection in humans. We aimed to test whether the hormone relaxin 2 lowers IOP and dilates retinal vessels in animals. METHODS: The IOP of female Sprague-Dawley rats before and after application of relaxin 2 was measured using an Icare Tonolab device calibrated for rats. Recombinant human relaxin 2 in phosphate-buffered saline with 0.1% bovine serum albumin was either applied as eye drops (1000, 2000 or 3000 ng/ml), injected intravitreally (500 ng/ml) or intravenously (13.3 µg/kg body weight). Retinal vessel thickness was monitored using infrared fundus images compiled with optical coherence tomography (Heidelberg Engineering) before and several time points after application of relaxin 2. RESULTS: Neither topical nor intravitreous or intravenous application of relaxin 2 lowered the IOP or changed the arterial or venous vessel diameter after 1 or 3 h after application. DISCUSSION: Now that relaxin 2 is more easily available, the hormone came again into focus as a potential glaucoma therapeutic. However, our study in rats could not support the hypothesis that relaxin 2 lowers IOP or dilates retinal vessels.

DNA mismatch repair activity of MutLα is regulated by CK2-dependent phosphorylation of MLH1 (S477).

MutLα, a heterodimer consisting of MLH1 and PMS2, is a key player of DNA mismatch repair (MMR), yet little is known about its regulation. In this study, we used mass spectrometry to identify phosphorylated residues within MLH1 and PMS2. The most frequently detected phosphorylated amino acid was serine 477 of MLH1. Pharmacological treatment indicates that Casein kinase II (CK2) could be responsible for the phosphorylation of MLH1 at serine 477 in vivo. In vitro kinase assay verified MLH1 as a substrate of CK2. Most importantly, using in vitro MMR assay we could demonstrate that p-MLH1S477 lost MMR activity. Moreover, we found that levels of p-MLH1S477 varied during the cell cycle. In summary, we identified that phosphorylation of MLH1 by CK2 at amino acid position 477 can switch off MMR activity in vitro. Since CK2 is overexpressed in many tumors and is able to inactivate MMR, the new mechanism here described could have an important impact on tumors overactive in CK2.

Comparison of Two Solid-Phase Extraction (SPE) Methods for the Identification and Quantification of Porcine Retinal Protein Markers by LC-MS/MS.

Proper sample preparation protocols represent a critical step for liquid chromatography-mass spectrometry (LC-MS)-based proteomic study designs and influence the speed, performance and automation of high-throughput data acquisition. The main objective of this study was to compare two commercial solid-phase extraction (SPE)-based sample preparation protocols (comprising SOLAµTM HRP SPE spin plates from Thermo Fisher Scientific and ZIPTIP® C18 pipette tips from Merck Millipore) for analytical performance, reproducibility, and analysis speed. The house swine represents a promising animal model for studying human eye diseases including glaucoma and provides excellent requirements for the qualitative and quantitative MS-based comparison in terms of ocular proteomics. In total six technical replicates of two protein fractions [extracted with 0.1% dodecyl-ß-maltoside (DDM) or 1% trifluoroacetic acid (TFA)] of porcine retinal tissues were subjected to in-gel trypsin digestion and purified with both SPE-based workflows (N = 3) prior to LC-MS analysis. On average, 550 ± 70 proteins (1512 ± 199 peptides) and 305 ± 48 proteins (806 ± 144 peptides) were identified from DDM and TFA protein fractions, respectively, after ZIPTIP® C18 purification, and SOLAµTM workflow resulted in the detection of 513 ± 55 proteins (1347 ± 180 peptides) and 300 ± 33 proteins (722 ± 87 peptides), respectively (FDR < 1%). Venn diagram analysis revealed an average overlap of 65 ± 2% (DDM fraction) and 69 ± 4% (TFA fraction) in protein identifications between both SPE-based methods. Quantitative analysis of 25 glaucoma-related protein markers also showed no significant differences (P > 0.05) regarding protein recovery between both SPE methods. However, only glaucoma-associated marker MECP2 showed a significant (P = 0.02) higher abundance in ZIPTIP®-purified replicates in comparison to SOLAµTM-treated study samples. Nevertheless, this result was not confirmed in the verification experiment using in-gel trypsin digestion of recombinant MECP2 (P = 0.24). In conclusion, both SPE-based purification methods worked equally well in terms of analytical performance and reproducibility, whereas the analysis speed and the semi-automation of the SOLAµTM spin plates workflow is much more convenient in comparison to the ZIPTIP® C18 method.

The potential impact of recent insights into proteomic changes associated with glaucoma.

INTRODUCTION: Glaucoma, a major ocular neuropathy, is still far from being understood on a molecular scale. Proteomic workflows revealed glaucoma associated alterations in different eye components. By using state-of-the-art mass spectrometric (MS) based discovery approaches large proteome datasets providing important information about glaucoma related proteins and pathways could be generated. Corresponding proteomic information could be retrieved from various ocular sample species derived from glaucoma experimental models or from original human material (e.g. optic nerve head or aqueous humor). However, particular eye tissues with the potential for understanding the disease's molecular pathomechanism remains underrepresented. Areas covered: The present review provides an overview of the analysis depth achieved for the glaucomatous eye proteome. With respect to different eye regions and biofluids, proteomics related literature was found using PubMed, Scholar and UniProtKB. Thereby, the review explores the potential of clinical proteomics for glaucoma research. Expert commentary: Proteomics will provide important contributions to understanding the molecular processes associated with glaucoma. Sensitive discovery and targeted MS approaches will assist understanding of the molecular interplay of different eye components and biofluids in glaucoma. Proteomic results will drive the comprehension of glaucoma, allowing a more stringent disease hypothesis within the coming years.

Proteomic profiling reveals crucial retinal protein alterations in the early phase of an experimental glaucoma model.

PURPOSE: Clinical glaucoma is difficult to assess in terms of molecular pathophysiology, prompting studies in experimental models of glaucoma. The purpose of this study was to investigate quantitative changes in retinal protein expression at the onset of experimental glaucoma in rats. Analyzing the proteome provides a suitable tool to decipher the pathophysiological processes in glaucomatous degeneration. METHODS: Thermic cauterization of episcleral veins was utilized to elevate the intraocular pressure in Sprague Dawley rats. Morphological changes were surveyed on a cellular level with a staining of Brn3a-positive cells. The retinal nerve fiber layer was investigated using optical coherence tomography (OCT, Heidelberg Engineering) and the optic nerve was analyzed by an axonal grading system. Mass spectrometry-featured quantitative proteomics and immunohistochemical staining was used to identify specifically altered proteins in the course of intraocular pressure elevation and initial neurodegeneration. Proteomic data were further analyzed with Ingenuity Pathway Analysis and Cytoscape to analyze further molecular associations. RESULTS: The intraocular pressure rose significantly (p < 0.001) for the follow-up period of 3 weeks after which animals were sacrificed. Eyes exposed to an elevated intraocular pressure showed an initial decrease of retinal ganglion cells, retinal nerve fiber layer (p < 0.05) and an impairment of the optic nerve (p < 0.01). Mass spectrometry led to the identification and quantification of 931 retinal proteins, whereas 32 were considerably altered. Bioinformatics-assisted clustering revealed that a majority of these proteins are functionally associated with cell differentiation, apoptosis and stress response. The creation of an interactive protein network showed that numerous altered proteins are connected regarding their cellular function. Protein kinase b, mitogen-activated protein kinase 1 and the NF-κB complex seem to be essential molecules in this context. CONCLUSIONS: In conclusion, these results provide further lines of evidence that substantial molecular changes occur at the onset of the disease, identifying potential key players, which might be useful as biomarkers for diagnostics and development of medical treatment in the future.

Course of serum autoantibodies in patients after acute angle-closure glaucoma attack.

BACKGROUND: The aim of our investigation was to analyze the autoantibody -reactivities of patients after acute angle-closure glaucoma (AACG) by means of a protein microarray approach to identify intraocular pressure(IOP)-dependent antibodies. METHODS: Collected sera from different study time points (AACG n = 6, 0, 2, 4 and 12 weeks) and control group (CTRL n = 11, 0 and 12 weeks) were analyzed. Protein-microarrays were incubated with sera, and occurring immunoreactivities were visualized with fluorescence labeled secondary antibodies. To detect changes, spot intensities were digitized and compared with statistical techniques. RESULTS: Three autoantibodies with significant level-alteration in the time course of the survey could be identified. Immunoreactivities to heat shock 27-kDa protein (HSP27), tubulin-tyrosine ligase-like protein 12 (TTLL12), and neuron-specific enolase (NSE) show an increasing linear trend from week 0 up to week 12 with a positive correlation coefficient (P ≤ 0.05, r ≥ 0.4). In the CTRL- group, no significant alterations could be detected in corresponding autoantibody-level. Analysis of variance revealed significant changes of antibody-level between certain time points (anti-HSP27 antibody [week 0 vs. 2], anti-TTLL12 antibody [week 0 vs. 12], and anti-NSE antibody [week 4 vs. 12] [P ≤ 0.05, respectively]) in AACG group. CONCLUSIONS: With this autoantibodies profiling approach, we were able to detect autoimmune reactivities in sera of patients without former indication for glaucomatous damage after rise of IOP due to AACG attack. After further validation in subsequent studies, this autoantibodies could give further insights into the pathogenesis of glaucoma and could possibly help to understand the effect of IOP on glaucomatous optic neuropathy.