Neuroretinal Cell Culture Model as a Tool for the Development of New Therapeutic Approaches for Oxidative Stress-Induced Ocular Diseases, with a Focus on Glaucoma.

Glaucoma is a heterogeneous group of optic neuropathies characterized by a progressive degeneration of the retinal ganglion cells (RGCs), leading to irreversible vision loss. Nowadays, the traditional therapeutic approach to glaucoma consists of lowering the intraocular pressure (IOP), which does not address the neurodegenerative features of the disease. Besides animal models of glaucoma, there is a considerable need for in vitro experimental models to propose new therapeutic strategies for this ocular disease. In this study, we elucidated the pathological mechanisms leading to neuroretinal R28 cell death after exposure to glutamate and hydrogen peroxide (H2O2) in order to develop new therapeutic approaches for oxidative stress-induced retinal diseases, including glaucoma. We were able to show that glutamate and H2O2 can induce a decrease in R28 cell viability in a concentration-dependent manner. A cell viability of about 42% was found after exposure to 3 mM of glutamate and about 56% after exposure to 100 µM of H2O2 (n = 4). Label-free quantitative mass spectrometry analysis revealed differential alterations of 193 and 311 proteins in R28 cells exposed to 3 mM of glutamate and 100 µM of H2O2, respectively (FDR < 1%; p < 0.05). Bioinformatics analysis indicated that the protein changes were associated with the dysregulation of signaling pathways, which was similar to those observed in glaucoma. Thus, the proteomic alteration induced by glutamate was associated with the inhibition of the PI3K/AKT signaling pathway. On the other hand, H2O2-induced toxicity in R28 cells was linked to the activation of apoptosis signaling and the inhibition of the mTOR and ERK/MAPK signaling pathways. Furthermore, the data show a similarity in the inhibition of the EIF2 and AMPK signaling pathways and the activation of the sumoylation and WNT/ß-catenin signaling pathways in both groups. Our findings suggest that the exposure of R28 cells to glutamate and H2O2 could induce glaucoma-like neurodegenerative features and potentially provide a suitable tool for the development of new therapeutic strategies for retinal diseases.

Towards optimized tissue regeneration: a new 3D printable bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate.

Introduction: Autologous platelet concentrate (APC) are pro-angiogenic and can promote wound healing and tissue repair, also in combination with other biomaterials. However, challenging defect situations remain demanding. 3D bioprinting of an APC based bioink encapsulated in a hydrogel could overcome this limitation with enhanced physio-mechanical interface, growth factor retention/secretion and defect-personalized shape to ultimately enhance regeneration. Methods: This study used extrusion-based bioprinting to create a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate. Chemico-physical testing exhibited an amorphous structure characterized by high shape fidelity. Cytotoxicity assay and incubation of human osteogenic sarcoma cells (SaOs2) exposed excellent biocompatibility. enzyme-linked immunosorbent assay analysis confirmed pro-angiogenic growth factor release of the printed constructs, and co-incubation with HUVECS displayed proper cell viability and proliferation. Chorioallantoic membrane (CAM) assay explored the pro-angiogenic potential of the prints in vivo. Detailed proteome and secretome analysis revealed a substantial amount and homologous presence of pro-angiogenic proteins in the 3D construct. Results: This study demonstrated a 3D bioprinting approach to fabricate a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate with high shape fidelity, biocompatibility, and substantial pro-angiogenic properties. Conclusion: This approach may be suitable for challenging physiological and anatomical defect situations when translated into clinical use.

Proteome landscape and interactome of voltage-gated potassium channel 1.6 (Kv1.6) of the murine ophthalmic artery and neuroretina.

The voltage-gated potassium channel 1.6 (Kv1.6) plays a vital role in ocular neurovascular beds and exerts its modulatory functions via interaction with other proteins. However, the interactome and their potential roles remain unknown. Here, the global proteome landscape of the ophthalmic artery (OA) and neuroretina was mapped, followed by the determination of Kv1.6 interactome and validation of its functionality and cellular localization. Microfluorimetric analysis of intracellular [K+] and Western blot validated the native functionality and cellular expression of the recombinant Kv1.6 channel protein. A total of 54, 9 and 28 Kv1.6-interacting proteins were identified in the mouse OA and, retina of mouse and rat, respectively. The Kv1.6-protein partners in the OA, namely actin cytoplasmic 2, alpha-2-macroglobulin and apolipoprotein A-I, were implicated in the maintenance of blood vessel integrity by regulating integrin-mediated adhesion to extracellular matrix and Ca2+ flux. Many retinal protein interactors, particularly the ADP/ATP translocase 2 and cytoskeleton protein tubulin, were involved in endoplasmic reticulum stress response and cell viability. Three common interactors were found in all samples comprising heat shock cognate 71 kDa protein, Ig heavy constant gamma 1 and Kv1.6 channel. This foremost in-depth investigation enriched and identified the elusive Kv1.6 channel and, elucidated its complex interactome.

Ocular surface changes in mice with streptozotocin-induced diabetes and diabetic polyneuropathy.

PURPOSE: Diabetes mellitus (DM) is a leading risk factor for corneal neuropathy and dry eye disease (DED). Another common consequence of DM is diabetic peripheral polyneuropathy (DPN). Both complications affect around 50 % of the DM patients but the relationship between DM, DED and DPN remains unclear. METHODS: In this study, we examined mice with early onset of DM and PN after streptozotocin (STZ)-induced diabetes (DPN). We compared the early morphological changes of the sciatic nerve, dorsal root and trigeminal ganglia with the changes in the ocular surface, including tear proteomic and we also investigated respective changes in the gene expressions and morphological alterations in the eye tissues involved in tear production. RESULTS: The lacrimal gland, conjunctival goblet cells and cornea showed morphological changes along with alterations in tear proteins without any obvious signs of ocular surface inflammation. The gene expression for respectively altered tear proteins i.e., of Clusterin in cornea, Car6, Adh3a1, and Eef1a1 in eyelids, and Pigr in the lacrimal gland also showed significant changes compared to control mice. In the trigeminal ganglia like in the dorsal root ganglia neuronal cells showed swollen mitochondria and, in the latter, there was a significant increase of NADPH oxidases and MMP9 suggestive of oxidative and neuronal stress. In the dorsal root ganglia and the sciatic nerve, there was an upregulation of a number of pro-inflammatory cytokines and pain-mediating chemokines. CONCLUSION: The early ocular changes in DM Mice only affect the lacrimal gland. Which, is reflected in the tear film composition of DPN mice. Due to the high protein concentration in tear fluid in humans, proteomic analysis in addition to noninvasive investigation of goblet cells and cornea can serve as a tools for the early diagnosis of DPN, DED in clinical practice. Early treatment could delay or even prevent the ocular complications of DM such as DED and PN.

Adaptive responses of neuronal cells to chronic endoplasmic reticulum (ER) stress.

Accumulation of misfolded proteins or perturbation of calcium homeostasis leads to endoplasmic reticulum (ER) stress and is linked to the pathogenesis of neurodegenerative diseases. Hence, understanding the ability of neuronal cells to cope with chronic ER stress is of fundamental interest. Interestingly, several brain areas uphold functions that enable them to resist challenges associated with neurodegeneration. Here, we established novel clonal mouse hippocampal (HT22) cell lines that are resistant to prolonged (chronic) ER stress induced by thapsigargin (TgR) or tunicamycin (TmR) as in vitro models to study the adaption to ER stress. Morphologically, we observed a significant increase in vesicular und autophagosomal structures in both resistant lines and 'giant lysosomes', especially striking in TgR cells. While autophagic activity increased under ER stress, lysosomal function appeared slightly impaired; in both cell lines, we observed enhanced ER-phagy. However, proteomic analyses revealed that various protein clusters and signaling pathways were differentially regulated in TgR versus TmR cells in response to chronic ER stress. Additionally, bioenergetic analyses in both resistant cell lines showed a shift toward aerobic glycolysis ('Warburg effect') and a defective complex I of the oxidative phosphorylation (OXPHOS) machinery. Furthermore, ER stress-resistant cells differentially activated the unfolded protein response (UPR) comprising IRE1α and ATF6 pathways. These findings display the wide portfolio of adaptive responses of neuronal cells to chronic ER stress. ER stress-resistant neuronal cells could be the basis to uncover molecular modulators of adaptation, resistance, and neuroprotection as potential pharmacological targets for preventing neurodegeneration.

Glaucoma-Associated CDR1 Peptide Promotes RGC Survival in Retinal Explants through Molecular Interaction with Acidic Leucine Rich Nuclear Phosphoprotein 32A (ANP32A).

Glaucoma is a complex, multifactorial optic neuropathy mainly characterized by the progressive loss of retinal ganglion cells (RGCs) and their axons, resulting in a decline of visual function. The pathogenic molecular mechanism of glaucoma is still not well understood, and therapeutic strategies specifically addressing the neurodegenerative component of this ocular disease are urgently needed. Novel immunotherapeutics might overcome this problem by targeting specific molecular structures in the retina and providing direct neuroprotection via different modes of action. Within the scope of this research, the present study showed for the first time beneficial effects of the synthetic CDR1 peptide SCTGTSSDVGGYNYVSWYQ on the viability of RGCs ex vivo in a concentration-dependent manner compared to untreated control explants (CTRL, 50 µg/mL: p < 0.05 and 100 µg/mL: p < 0.001). Thereby, this specific peptide was identified first as a potential biomarker candidate in the serum of glaucoma patients and was significantly lower expressed in systemic IgG molecules compared to healthy control subjects. Furthermore, MS-based co-immunoprecipitation experiments confirmed the specific interaction of synthetic CDR1 with retinal acidic leucine-rich nuclear phosphoprotein 32A (ANP32A; p < 0.001 and log2 fold change > 3), which is a highly expressed protein in neurological tissues with multifactorial biological functions. In silico binding prediction analysis revealed the N-terminal leucine-rich repeat (LRR) domain of ANP32A as a significant binding site for synthetic CDR1, which was previously reported as an important docking site for protein-protein interactions (PPI). In accordance with these findings, quantitative proteomic analysis of the retinae ± CDR1 treatment resulted in the identification of 25 protein markers, which were significantly differentially distributed between both experimental groups (CTRL and CDR1, p < 0.05). Particularly, acetyl-CoA biosynthesis I-related enzymes (e.g., DLAT and PDHA1), as well as cytoskeleton-regulating proteins (e.g., MSN), were highly expressed by synthetic CDR1 treatment in the retina; on the contrary, direct ANP32A-interacting proteins (e.g., NME1 and PPP2R4), as well as neurodegenerative-related markers (e.g., CEND1), were identified with significant lower abundancy in the CDR1-treated retinae compared to CTRL. Furthermore, retinal protein phosphorylation and histone acetylation were also affected by synthetic CDR1, which are both partially controlled by ANP32A. In conclusion, the synthetic CDR1 peptide provides a great translational potential for the treatment of glaucoma in the future by eliciting its neuroprotective mechanism via specific interaction with ANP32A's N terminal LRR domain.

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.

Short-Term Omega-3 Supplementation Modulates Novel Neurovascular and Fatty Acid Metabolic Proteome Changes in the Retina and Ophthalmic Artery of Mice with Targeted Cyp2c44 Gene Deletion.

Cytochrome P450 (CYP) gene mutations are a common predisposition associated with glaucoma. Although the molecular mechanisms are largely unknown, omega-3 polyunsaturated fatty acids (ω-3 PUFA) and their CYP-derived bioactive mediators play crucial roles in the ocular system. Here, we elucidated the proteome and cell-signalling alterations attributed to the main human CYP2C gene deficiency using a homologous murine model (Cyp2c44-/-), and unravelled the effects of acute ω-3 PUFA supplementation in two ocular vascular beds comprising the retrobulbar ophthalmic artery (OA) and retina (R). Male Cyp2c44-/- mice (KO) and their floxed littermates (WT) were gavaged daily for 7 days with 0.01 mL/g of ω-3 PUFA composed of menhaden fish oil. Another group in respective strains served as vehicle-treated controls. OA and R were isolated at day 8 post-treatment (n = 9/group) and subjected to mass spectrometry (MS)-based proteomics and in silico bioinformatics analyses. Cyp2c44-/- resulted in significant detrimental proteome changes associated with compromised vascular integrity and degeneration in the OA and R, respectively. However, notable changes in the OA after ω-3 PUFA intake were associated with the maintenance of intercellular junctional and endothelial cell functions, as well as activation of the fatty acid metabolic pathway in the KO mice. Conversely, ω-3 PUFA supplementation profoundly influenced the regulation of a large majority of retinal proteins involved in the preservation of neuronal and phototransduction activities in WT mice, namely synaptophysin, phosducin and guanylate cyclase-1, while significantly abrogating degenerative processes in the KO mice via the regulation of, namely, synaptotagmin-1 and beta-crystallin B2. In gist, this study demonstrated that dietary supplementation with ω-3 PUFA for a short period of seven days regulated specific neuro-vasculoprotective mechanisms to preserve the functionality of the OA and R in the absence of Cyp2c44. The potential adjunct use of ω-3 PUFA for glaucoma therapy needs further investigation.

Synthetic antibody-derived immunopeptide provides neuroprotection in glaucoma through molecular interaction with retinal protein histone H3.1.

Glaucoma is a group of optic neuropathies characterized by the progressive degeneration of retinal ganglion cells (RGCs) as well as their axons leading to irreversible loss of sight. Medical management of the intraocular pressure (IOP) still represents the gold standard in glaucoma therapy, which only manages a single risk factor and does not directly address the neurodegenerative component of this eye disease. Recently, our group showed that antibody-derived immunopeptides (encoding complementarity-determining regions, CDRs) provide attractive glaucoma medication candidates and directly interfere its pathogenic mechanisms by different modes of action. In accordance with these findings, the present study showed the synthetic complementary-determining region 2 (CDR2) peptide (INSDGSSTSYADSVK) significantly increased RGC viability in vitro in a concentration-dependent manner (p < 0.05 using a CDR2 concentration of 50 µg/mL). Employing state-of the-art immunoprecipitation experiments, we confirmed that synthetic CDR2 exhibited a high affinity toward the retinal target protein histone H3.1 (HIST1H3A) (p < 0.001 and log2-fold change > 3). Furthermore, molecular dynamics (MD) simulations along with virtual docking analyses predicted potential CDR2-specific binding regions of HIST1H3A, which might represent essential post-translational modification (PTM) sites for epigenetic regulations. Quantitative mass spectrometry (MS) analysis of retinas demonstrated 39 proteins significantly affected by CDR2 treatment (p < 0.05). An up-regulation of proteins involved in the energy production (e.g., ATP5F1B and MT-CO2) as well as the regulatory ubiquitin proteasome system (e.g., PSMC5) was induced by the synthetic CDR2 peptide. On the other hand, CDR2 reduced metabolic key enzymes (e.g., DDAH1 and MAOB) as well as ER stress-related proteins (e.g., SEC22B and VCP) and these data were partially confirmed by microarray technology. Our outcome measurements indicate that specific protein-peptide interactions influence the regulatory epigenetic function of HIST1H3A promoting the neuroprotective mechanism on RGCs in vitro. In addition to IOP management, such synthetic peptides as CDR2 might serve as a synergistic immunotherapy for glaucoma in the future.

Longitudinal CSF proteome profiling in mice to uncover the acute and sustained mechanisms of action of rapid acting antidepressant (2R,6R)-hydroxynorketamine (HNK).

Delayed onset of antidepressant action is a shortcoming in depression treatment. Ketamine and its metabolite (2R,6R)-hydroxynorketamine (HNK) have emerged as promising rapid-acting antidepressants. However, their mechanism of action remains unknown. In this study, we first described the anxious and depression-prone inbred mouse strain, DBA/2J, as an animal model to assess the antidepressant-like effects of ketamine and HNK in vivo. To decode the molecular mechanisms mediating HNK's rapid antidepressant effects, a longitudinal cerebrospinal fluid (CSF) proteome profiling of its acute and sustained effects was conducted using an unbiased, hypothesis-free mass spectrometry-based proteomics approach. A total of 387 proteins were identified, with a major implication of significantly differentially expressed proteins in the glucocorticoid receptor (GR) signaling pathway, providing evidence for a link between HNK and regulation of the stress hormone system. Mechanistically, we identified HNK to repress GR-mediated transcription and reduce hormonal sensitivity of GR in vitro. In addition, mammalian target of rapamycin (mTOR) and brain-derived neurotrophic factor (BDNF) were predicted to be important upstream regulators of HNK treatment. Our results contribute to precise understanding of the temporal dynamics and molecular targets underlying HNK's rapid antidepressant-like effects, which can be used as a benchmark for improved treatment strategies for depression in future.

Cyp2c44 epoxygenase-derived epoxyeicosatrienoic acids in vascular smooth muscle cells elicit vasoconstriction of the murine ophthalmic artery.

Cytochrome P450 (CYP) signalling pathway has been shown to play a vital role in the vasoreactivity of wild type mouse ophthalmic artery. In this study, we determined the expression, vascular responses and potential mechanisms of the CYP-derived arachidonic acid metabolites. The expression of murine CYP (Cyp2c44) and soluble epoxide hydrolase (sEH) in the wild type ophthalmic artery was determined with immunofluorescence, which showed predominant expression of Cyp2c44 in the vascular smooth muscle cells (VSMC), while sEH was found mainly in the endothelium of the wild type ophthalmic artery. Artery of Cyp2c44-/- and sEH-/- mice were used as negative controls. Targeted mass spectrometry-based lipidomics analysis of endogenous epoxide and diols of the wild type artery detected only 14, 15-EET. Vasorelaxant responses of isolated vessels in response to selective pharmacological blockers and agonist were analysed ex vivo. Direct antagonism of epoxyeicosatrienoic acids (EETs) with a selective inhibitor caused partial vasodilation, suggesting that EETs may behave as vasoconstrictors. Exogenous administration of synthetic EET regioisomers significantly constricted the vessels in a concentration-dependent manner, with the strongest responses elicited by 11, 12- and 14, 15-EETs. Our results provide the first experimental evidence that Cyp2c44-derived EETs in the VSMC mediate vasoconstriction of the ophthalmic artery.

Regulation of the HTRA2 Protease Activity by an Inhibitory Antibody-Derived Peptide Ligand and the Influence on HTRA2-Specific Protein Interaction Networks in Retinal Tissues.

The mitochondrial serine protease HTRA2 has many versatile biological functions ranging from being an important regulator of apoptosis to being an essential component for neuronal cell survival and mitochondrial homeostasis. Loss of HTRA2 protease function is known to cause neurodegeneration, whereas overactivation of its proteolytic function is associated with cell death and inflammation. In accordance with this, our group verified in a recent study that the synthetic peptide ASGYTFTNYGLSWVR, encoding the hypervariable sequence part of an antibody, showed a high affinity for the target protein HTRA2 and triggered neuroprotection in an in vitro organ culture model for glaucoma. To unravel this neuroprotective mechanism, the present study showed for the first time that the synthetic CDR1 peptide significantly (p < 0.01) inhibited the proteolytic activity of HTRA2 up to 50% using a specific protease function assay. Furthermore, using state-of-the-art co-immunoprecipitation technologies in combination with high-resolution MS, we identified 50 significant protein interaction partners of HTRA2 in the retina of house swine (p < 0.01; log2 fold change > 1.5). Interestingly, 72% of the HTRA2-specific interactions (23 of 31 binders) were inhibited by additional treatment with UCF-101 (HTRA2 protease inhibitor) or the synthetic CDR peptide. On the other hand, the remaining 19 binders of HTRA2 were exclusively identified in the UCF101 and/or CDR group. However, many of the interactors were involved in the ER to Golgi anterograde transport (e.g., AP3D1), aggrephagy (e.g., PSMC1), and the pyruvate metabolism/citric acid cycle (e.g., SHMT2), and illustrated the complex protein interaction networks of HTRA2 in neurological tissues. In conclusion, the present study provides, for the first time, a comprehensive protein catalogue of HTRA2-specific interaction partners in the retina, and will serve as reference map in the future for studies focusing on HTRA2-mediated neurodegeneration.

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.

Proteomics Reveals the Potential Protective Mechanism of Hydrogen Sulfide on Retinal Ganglion Cells in an Ischemia/Reperfusion Injury Animal Model.

Glaucoma is the leading cause of irreversible blindness and is characterized by progressive retinal ganglion cell (RGC) degeneration. Hydrogen sulfide (H2S) is a potent neurotransmitter and has been proven to protect RGCs against glaucomatous injury in vitro and in vivo. This study is to provide an overall insight of H2S's role in glaucoma pathophysiology. Ischemia-reperfusion injury (I/R) was induced in Sprague-Dawley rats (n = 12) by elevating intraocular pressure to 55 mmHg for 60 min. Six of the animals received intravitreal injection of H2S precursor prior to the procedure and the retina was harvested 24 h later. Contralateral eyes were assigned as control. RGCs were quantified and compared within the groups. Retinal proteins were analyzed via label-free mass spectrometry based quantitative proteomics approach. The pathways of the differentially expressed proteins were identified by ingenuity pathway analysis (IPA). H2S significantly improved RGC survival against I/R in vivo (p < 0.001). In total 1115 proteins were identified, 18 key proteins were significantly differentially expressed due to I/R and restored by H2S. Another 11 proteins were differentially expressed following H2S. IPA revealed a significant H2S-mediated activation of pathways related to mitochondrial function, iron homeostasis and vasodilation. This study provides first evidence of the complex role that H2S plays in protecting RGC against I/R.

Bioenergetic shift and actin cytoskeleton remodelling as acute vascular adaptive mechanisms to angiotensin II in murine retina and ophthalmic artery.

Ocular vascular dysfunction is a major contributing factor to the pathogenesis of glaucoma. In recent years, there has been a renewed interest in the role of angiotensin II (Ang II) in mediating the disease progression. Despite its (patho)physiological importance, the molecular mechanisms underlying Ang II-mediated oxidative stress remain largely unexplored in the ocular vasculature. Here, we provide the first direct evidence of the alterations of proteome and signalling pathways underlying Ang II-elicited oxidative insult independent of arterial pressure changes in the ophthalmic artery (OA) and retina (R) employing an in vitro experimental model. Both R and OA were isolated from male C57Bl/6J mice (n = 15/group; n = 5/biological replicate) and incubated overnight in medium containing either vehicle or Ang II (0.1 µM) at physiological conditions. Label-free quantitative mass spectrometry (MS)-based proteomics analysis identified a differential expression of 107 and 34 proteins in the R and OA, respectively. Statistical and bioinformatics analyses revealed that protein clusters involved in actin cytoskeleton and integrin-linked kinase signalling were significantly activated in the OA. Conversely, a large majority of differentially expressed retinal proteins were involved in dysregulation of numerous energy-producing and metabolic signalling pathways, hinting to a possible shift in retinal cell bioenergetics. Particularly, Ang II-mediated downregulation of septin-7 (Sept7; p < 0.01) and superoxide dismutase [Cu-Zn] (Sod1; p < 0.05), and upregulation of troponin T, fast skeletal muscle (Tnnt3; p < 0.05) and tropomyosin alpha-3 chain (Tpm3; p < 0.01) in the OA, and significant decreased expressions of two crystallin proteins (Cryab; p < 0.05 and Crybb2; p < 0.0001) in the R were verified at the mRNA level, corroborating our proteomics findings. In summary, these results demonstrated that exogenous application of Ang II over an acute time period caused impairment of retinal bioenergetics and cellular demise, and actin cytoskeleton-mediated vascular remodelling in the OA.

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.

Synthetic Polyclonal-Derived CDR Peptides as an Innovative Strategy in Glaucoma Therapy.

The pathogenesis of glaucoma is strongly associated with the occurrence of autoimmune-mediated loss of retinal ganglion cells (RGCs) and additionally, recent evidence shows that specific antibody-derived signature peptides are significantly differentially expressed in sera of primary-open angle glaucoma patients (POAG) compared to healthy controls. Synthetically antibody-derived peptides can modulate various effector functions of the immune system and act as antimicrobial or antiviral molecules. In an ex vivo adolescent glaucoma model, this study, for the first time, demonstrates that polyclonal-derived complementarity-determining regions (CDRs) can significantly increase the survival rate of RGCs (p = 0.013). We subsequently performed affinity capture experiments that verified the mitochondrial serine protease HTRA2 (gene name: HTRA2) as a high-affinity retinal epitope target of CDR1 sequence motif ASGYTFTNYGLSWVR. Quantitative proteomic analysis of the CDR-treated retinal explants revealed increased expression of various anti-apoptotic and anti-oxidative proteins (e.g., VDAC2 and TXN) compared to untreated controls (p < 0.05) as well as decreased expression levels of cellular stress response markers (e.g., HSPE1 and HSP90AA1). Mitochondrial dysfunction, the protein ubiquitination pathway and oxidative phosphorylation were annotated as the most significantly affected signaling pathways and possibly can be traced back to the CDR-induced inhibition or modulation of the master regulator HTRA2. These findings emphasize the great potential of synthetic polyclonal-derived CDR peptides as therapeutic agents in future glaucoma therapy and provide an excellent basis for affinity-based biomarker discovery purposes.

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.

Enhanced autophagic-lysosomal activity and increased BAG3-mediated selective macroautophagy as adaptive response of neuronal cells to chronic oxidative stress.

Oxidative stress and a disturbed cellular protein homeostasis (proteostasis) belong to the most important hallmarks of aging and of neurodegenerative disorders. The proteasomal and autophagic-lysosomal degradation pathways are key measures to maintain proteostasis. Here, we report that hippocampal cells selected for full adaptation and resistance to oxidative stress induced by hydrogen peroxide (oxidative stress-resistant cells, OxSR cells) showed a massive increase in the expression of components of the cellular autophagic-lysosomal network and a significantly higher overall autophagic activity. A comparative expression analysis revealed that distinct key regulators of autophagy are upregulated in OxSR cells. The observed adaptive autophagic response was found to be independent of the upstream autophagy regulator mTOR but is accompanied by a significant upregulation of further downstream components of the canonical autophagy network such as Beclin1, WIPI1 and the transmembrane ATG9 proteins. Interestingly, the expression of the HSP70 co-chaperone BAG3, mediator of BAG3-mediated selective macroautophagy and highly relevant for the clearance of aggregated proteins in cells, was found to be increased in OxSR cells that were consequently able to effectively overcome proteotoxic stress. Overexpression of BAG3 in oxidative stress-sensitive HT22 wildtype cells partly established the vesicular phenotype and the enhanced autophagic flux seen in OxSR cells suggesting that BAG3 takes over an important part in the adaptation process. A full proteome analysis demonstrated additional changes in the expression of mitochondrial proteins, metabolic enzymes and different pathway regulators in OxSR cells as consequence of the adaptation to oxidative stress in addition to autophagy-related proteins. Taken together, this analysis revealed a wide variety of pathways and players that act as adaptive response to chronic redox stress in neuronal cells.

Sample Preparation for Mass-spectrometry-based Proteomics Analysis of Ocular Microvessels.

The use of isolated ocular blood vessels in vitro to decipher the pathophysiological state of the eye using advanced technological approaches has greatly expanded our understanding of certain diseases. Mass spectrometry (MS)-based proteomics has emerged as a powerful tool to unravel alterations in the molecular mechanisms and protein signaling pathways in the vascular beds in health and disease. However, sample preparation steps prior to MS analyses are crucial to obtain reproducible results and in-depth elucidation of the complex proteome. This is particularly important for preparation of ocular microvessels, where the amount of sample available for analyses is often limited and thus, poses a challenge for optimum protein extraction. This article endeavors to provide an efficient, rapid and robust protocol for sample preparation from an exemplary retrobulbar ocular vascular bed employing the porcine short posterior ciliary arteries. The present method focuses on protein extraction procedures from both the supernatant and pellet of the sample following homogenization, sample cleaning with centrifugal filter devices prior to one-dimensional gel electrophoresis and peptide purification steps for label-free quantification in a liquid chromatography-electrospray ionization-linear ion trap-Orbitrap MS system. Although this method has been developed specifically for proteomics analyses of ocular microvessels, we have also provided convincing evidence that it can also be readily employed for other tissue-based samples.