Mass spectrometry-based retina proteomics.

A subfield of neuroproteomics, retina proteomics has experienced a transformative growth since its inception due to methodological advances in enabling chemical, biochemical, and molecular biology techniques. This review focuses on mass spectrometry's contributions to facilitate mammalian and avian retina proteomics to catalog and quantify retinal protein expressions, determine their posttranslational modifications, as well as its applications to study the proteome of the retina in the context of biology, health and diseases, and therapy developments.

Proteomics-Based Identification of Retinal Protein Networks Impacted by Elevated Intraocular Pressure in the Hypertonic Saline Injection Model of Experimental Glaucoma.

Elevated intraocular pressure is considered a major cause of glaucomatous retinal neurodegeneration. To facilitate a better understanding of the underlying molecular processes and mechanisms, we report a study focusing on alterations of the retina proteome by induced ocular hypertension in a rat model of the disease. Glaucomatous processes were modeled through sclerosing the aqueous outflow routes of the eyes by hypertonic saline injections into an episcleral vein. Mass spectrometry-based quantitative retina proteomics using a label-free shotgun methodology identified over 200 proteins significantly affected by ocular hypertension. Various facets of glaucomatous pathophysiology were revealed through the organization of the findings into protein interaction networks and by pathway analyses. Concentrating on retinal neurodegeneration as a characteristic process of the disease, elevated intraocular pressure-induced alterations in the expression of selected proteins were verified by targeted proteomics based on nanoflow liquid chromatography coupled with nano-electrospray ionization tandem mass spectrometry using the parallel reaction monitoring method of data acquisition. Acquired raw data are shared through deposition to the ProteomeXchange Consortium (PXD042729), making a retina proteomics dataset on the selected animal model of glaucoma available for the first time.

[ß-Glu2]TRH Is a Functional Antagonist of Thyrotropin-Releasing Hormone (TRH) in the Rodent Brain.

Selective antagonists of thyrotropin-releasing hormone (TRH; pGlu-His-Pro-NH2), in order to enable a better understanding of this peptide's central functions, have not been identified. Using pGlu-Glu-Pro-NH2 ([Glu2]TRH) as a lead peptide and with modification at its central residue, our studies focused on some of its analogues synthesized as potential functional antagonists of TRH in the rodent brain. Among the peptides studied, the novel isomeric analogue [ß-Glu2]TRH was found to suppress the analeptic and antidepressant-like pharmacological activities of TRH without eliciting intrinsic effects in these paradigms. [ß-Glu2]TRH also completely reversed TRH's stimulation of acetylcholine turnover in the rat hippocampus without a cholinergic activity of its own, which was demonstrated through in vivo microdialysis experiments. Altogether, [ß-Glu2]TRH emerged as the first selective functional antagonist of TRH's prominent cholinergic actions, by which this endogenous peptide elicits a vast array of central effects.

Proteomics Complementation of the Rat Uterotrophic Assay for Estrogenic Endocrine Disruptors: A Roadmap of Advancing High Resolution Mass Spectrometry-Based Shotgun Survey to Targeted Biomarker Quantifications.

The widely used rat uterotrophic assay to assess known and potential estrogenic compounds only considers uterine weight gain as endpoint measurement. To complement this method with an advanced technology that reveals molecular targets, we analyzed changes in protein expression using label-free quantitative proteomics by nanoflow liquid chromatography coupled with high-resolution mass spectrometry and tandem mass spectrometry from uterine protein extracts of ovariectomized rats after daily 17ß-estradiol exposure for five days in comparison with those of vehicle-treated control animals. Our discovery-driven study revealed 165 uterine proteins significantly regulated by estrogen treatment and mapped by pathway analyses. Estrogen-regulated proteins represented cell death, survival and development, cellular growth and proliferation, and protein synthesis as top molecular and cellular functions, and a network found with the presence of nuclear estrogen receptor(s) as a prominent molecular node confirmed the relevance of our findings to hormone-associated events. An exploratory application of targeted proteomics to bisphenol A as a well-known example of an estrogenic endocrine disruptor is also presented. Overall, the results of this study have demonstrated the power of combining untargeted and targeted quantitative proteomic strategies to identify and verify candidate molecular markers for the evaluation of endocrine-disrupting chemicals to complement a conventional bioassay.

The Antagonist pGlu-ßGlu-Pro-NH2 Binds to an Allosteric Site of the Thyrotropin-Releasing Hormone Receptor.

After we identified pGlu-ßGlu-Pro-NH2 as the first functional antagonist of the cholinergic central actions of the thyrotropin-releasing hormone (TRH, pGlu-His-Pro-NH2), we became interested in finding the receptor-associated mechanism responsible for this antagonism. By utilizing a human TRH receptor (hTRH-R) homology model, we first refined the active binding site within the transmembrane bundle of this receptor to enhance TRH's binding affinity. However, this binding site did not accommodate the TRH antagonist. This directed us to consider a potential allosteric binding site in the extracellular domain (ECD). Searches for ECD binding pockets prompted the remodeling of the extracellular loops and the N-terminus. We found that different trajectories of ECDs produced novel binding cavities that were then systematically probed with TRH, as well as its antagonist. This led us to establish not only a surface-recognition binding site for TRH, but also an allosteric site that exhibited a selective and high-affinity binding for pGlu-ßGlu-Pro-NH2. The allosteric binding of this TRH antagonist is more robust than TRH's binding to its own active site. The findings reported here may shed light on the mechanisms and the multimodal roles by which the ECD of a TRH receptor is involved in agonist and/or antagonist actions.

Comparative Proteomics Analysis Reveals Unique Early Signaling Response of Saccharomyces cerevisiae to Oxidants with Different Mechanism of Action.

The early signaling events involved in oxidant recognition and triggering of oxidant-specific defense mechanisms to counteract oxidative stress still remain largely elusive. Our discovery driven comparative proteomics analysis revealed unique early signaling response of the yeast Saccharomyces cerevisiae on the proteome level to oxidants with a different mechanism of action as early as 3 min after treatment with four oxidants, namely H2O2, cumene hydroperoxide (CHP), and menadione and diamide, when protein abundances were compared using label-free quantification relying on a high-resolution mass analyzer (Orbitrap). We identified significant regulation of 196 proteins in response to H2O2, 569 proteins in response to CHP, 369 proteins in response to menadione and 207 proteins in response to diamide. Only 17 proteins were common across all treatments, but several more proteins were shared between two or three oxidants. Pathway analyses revealed that each oxidant triggered a unique signaling mechanism associated with cell survival and repair. Signaling pathways mostly regulated by oxidants were Ran, TOR, Rho, and eIF2. Furthermore, each oxidant regulated these pathways in a unique way indicating specificity of response to oxidants having different modes of action. We hypothesize that interplay of these signaling pathways may be important in recognizing different oxidants to trigger different downstream MAPK signaling cascades and to induce specific responses.

A Novel Prodrug Approach for Central Nervous System-Selective Estrogen Therapy.

Beneficial effects of estrogens in the central nervous system (CNS) results from the synergistic combination of their well-orchestrated genomic and non-genomic actions, making them potential broad-spectrum neurotherapeutic agents. However, owing to unwanted peripheral hormonal burdens by any currently known non-invasive drug administrations, the development of estrogens as safe pharmacotherapeutic modalities cannot be realized until they are confined specifically and selectively to the site of action. We have developed small-molecule bioprecursor prodrugs carrying the para-quinol scaffold on the steroidal A-ring that are preferentially metabolized in the CNS to the corresponding estrogens. Here, we give an overview of our discovery of these prodrugs. Selected examples are shown to illustrate that, independently of the route of administrations and duration of treatments, these agents produce high concentration of estrogens only in the CNS without peripheral hormonal liability. 10ß,17ß-Dihydroxyestra-1,4-dien-3-one (DHED) has been the best-studied representative of this novel type of prodrugs for brain and retina health. Specific applications in preclinical animal models of centrally-regulated and estrogen-responsive human diseases, including neurodegeneration, menopausal symptoms, cognitive decline and depression, are discussed to demonstrate the translational potential of our prodrug approach for CNS-selective and gender-independent estrogen therapy with inherent therapeutic safety.

An exploratory investigation of brain-selective estrogen treatment in males using a mouse model of Alzheimer's disease.

Estrogens are neuroprotective, and studies suggest that they may mitigate the pathology and symptoms of Alzheimer's disease (AD) in female models. However, central estrogen effects have not been examined in males in the context of AD. The purpose of this follow-up study was to assess the benefits of a brain-selective 17ß-estradiol estrogen prodrug, 10ß,17ß-hydroxyestra-1,4-dien-3-one (DHED), also in the male APPswe/PS1dE9 double-transgenic mouse model of the disease. After continuously exposing 6-month old animals to DHED for two months, their brains showed decreased amyloid precursor and amyloid-ß protein levels. The DHED-treated APPswe/PS1dE9 double transgenic subjects also exhibited enhanced performance in a cognitive task, while 17ß-estradiol treatment did not reach statistical significance. Taken together, data presented here suggest that DHED may also have therapeutic benefit in males and warrant further investigations to fully elucidate the potential of targeted estrogen therapy for a gender-independent treatment of early-stage AD.

Cholinergic Neurons in the Basal Forebrain Promote Wakefulness by Actions on Neighboring Non-Cholinergic Neurons: An Opto-Dialysis Study.

Understanding the control of sleep-wake states by the basal forebrain (BF) poses a challenge due to the intermingled presence of cholinergic, GABAergic, and glutamatergic neurons. All three BF neuronal subtypes project to the cortex and are implicated in cortical arousal and sleep-wake control. Thus, nonspecific stimulation or inhibition studies do not reveal the roles of these different neuronal types. Recent studies using optogenetics have shown that "selective" stimulation of BF cholinergic neurons increases transitions between NREM sleep and wakefulness, implicating cholinergic projections to cortex in wake promotion. However, the interpretation of these optogenetic experiments is complicated by interactions that may occur within the BF. For instance, a recent in vitro study from our group found that cholinergic neurons strongly excite neighboring GABAergic neurons, including the subset of cortically projecting neurons, which contain the calcium-binding protein, parvalbumin (PV) (Yang et al., 2014). Thus, the wake-promoting effect of "selective" optogenetic stimulation of BF cholinergic neurons could be mediated by local excitation of GABA/PV or other non-cholinergic BF neurons. In this study, using a newly designed opto-dialysis probe to couple selective optical stimulation with simultaneous in vivo microdialysis, we demonstrated that optical stimulation of cholinergic neurons locally increased acetylcholine levels and increased wakefulness in mice. Surprisingly, the enhanced wakefulness caused by cholinergic stimulation was abolished by simultaneous reverse microdialysis of cholinergic receptor antagonists into BF. Thus, our data suggest that the wake-promoting effect of cholinergic stimulation requires local release of acetylcholine in the basal forebrain and activation of cortically projecting, non-cholinergic neurons, including the GABAergic/PV neurons. SIGNIFICANCE STATEMENT: Optogenetics is a revolutionary tool to assess the roles of particular groups of neurons in behavioral functions, such as control of sleep and wakefulness. However, the interpretation of optogenetic experiments requires knowledge of the effects of stimulation on local neurotransmitter levels and effects on neighboring neurons. Here, using a novel "opto-dialysis" probe to couple optogenetics and in vivo microdialysis, we report that optical stimulation of basal forebrain (BF) cholinergic neurons in mice increases local acetylcholine levels and wakefulness. Reverse microdialysis of cholinergic antagonists within BF prevents the wake-promoting effect. This important result challenges the prevailing dictum that BF cholinergic projections to cortex directly control wakefulness and illustrates the utility of "opto-dialysis" for dissecting the complex brain circuitry underlying behavior.

Targeting Alpha Toxin To Mitigate Its Lethal Toxicity in Ferret and Rabbit Models of Staphylococcus aureus Necrotizing Pneumonia.

The role broad-spectrum antibiotics play in the spread of antimicrobial resistance, coupled with their effect on the healthy microbiome, has led to advances in pathogen-specific approaches for the prevention or treatment of serious bacterial infections. One approach in clinical testing is passive immunization with a monoclonal antibody (MAb) targeting alpha toxin for the prevention or treatment of Staphylococcus aureus pneumonia. Passive immunization with the human anti-alpha toxin MAb, MEDI4893*, has been shown to improve disease outcome in murine S. aureus pneumonia models. The species specificity of some S. aureus toxins necessitates testing anti-S. aureus therapeutics in alternate species. We developed a necrotizing pneumonia model in ferrets and utilized an existing rabbit pneumonia model to characterize MEDI4893* protective activity in species other than mice. MEDI4893* prophylaxis reduced disease severity in ferret and rabbit pneumonia models against both community-associated methicillin-resistant S. aureus (MRSA) and hospital-associated MRSA strains. In addition, adjunctive treatment of MEDI4893* with either vancomycin or linezolid provided enhanced protection in rabbits relative to the antibiotics alone. These results confirm that MEDI4893 is a promising candidate for immunotherapy against S. aureus pneumonia.

Mannich Ketones as Possible Antimycobacterial Agents.

Twenty-three known unsaturated and fused Mannich ketones and their reduced derivatives (amino alcohols) were selected for an antituberculotic study. They were screened against several mycobacterial strains including Mycobacterium tuberculosis, M. xenopi, and M. gordonae, and minimum inhibitory concentration values were also determined using the standard antituberculotic drug isoniazid (INH) as a reference. Structure-activity relationships were also studied. The mode of action of the test compounds was investigated using transmission electron microscopy, high-performance liquid chromatography, and matrix-assisted desorption/ionization mass spectrometry. Several test substances proved to be as potent as INH, but their antimycobacterial spectra were broader than that of INH. Our findings suggest that their mode of action is probably through the inhibition of mycobacterial cell wall biosynthesis.

Critical Role of Alpha-Toxin and Protective Effects of Its Neutralization by a Human Antibody in Acute Bacterial Skin and Skin Structure Infections.

Methicillin-resistant Staphylococcus aureus (MRSA) causes large-scale epidemics of acute bacterial skin and skin structure infections (ABSSSI) within communities across the United States. Animal models that reproduce ABSSSI as they occur in humans are urgently needed to test new therapeutic strategies. Alpha-toxin plays a critical role in a variety of staphylococcal infection models in mice, but its role in the pathogenesis of ABSSSI remains to be elucidated in rabbits, which are similar to humans in their susceptibility to S. aureus superantigens and certain bicomponent pore-forming leukocidins. We report here a new rabbit model of ABSSSI and show that those infected with a mutant deficient in expression of alpha-toxin (Δhla) developed a small dermonecrotic lesion, whereas those infected with isogenic USA300 MRSA wild-type or complemented Δhla strains developed ABSSSI that mimic the severe infections that occur in humans, including the large central dermonecrotic core surrounded by erythema, induration, and marked subcutaneous hemorrhage. More importantly, immunoprophylaxis with MEDI4893*, an anti-alpha-toxin human monoclonal antibody, significantly reduced the severity of disease caused by a USA300 wild-type strain to that caused by the Δhla mutant, indicating that this toxin could be completely neutralized during infection. Thus, this study illustrates a potential high standard for the development of new immunotherapeutic agents in which a toxin-neutralizing antibody provides protection to the same degree achieved with a toxin gene knockout. When MEDI4893* was administered as adjunctive therapy with a subtherapeutic dose of linezolid, the combination was significantly more efficacious than either agent alone in reducing the severity of ABSSSI.

A comparative evaluation of treatments with 17ß-estradiol and its brain-selective prodrug in a double-transgenic mouse model of Alzheimer's disease.

Estrogens are neuroprotective and, thus, potentially useful for the therapy of Alzheimer's disease; however, clinical use of hormone therapy remains controversial due to adverse peripheral effects. The goal of this study was to investigate the benefits of treatment with 10ß,17ß-dihydroxyestra-1,4-dien-3-one (DHED), a brain-selective prodrug of 17ß-estradiol, in comparison with the parent hormone using APPswe/PS1dE9 double transgenic mice to model the pathology of the disease. Ovariectomized and intact females were continuously treated with vehicle, 17ß-estradiol, or DHED via subcutaneous osmotic pumps from 6 to 8months of age. We confirmed that this prolonged treatment with DHED did not stimulate uterine tissue, whereas 17ß-estradiol treatment increased uterine weight. Amyloid precursor protein decreased in both treatment groups of intact, but not in ovariectomized double transgenic females in which ovariectomy already decreased the expression of this protein significantly. However, reduced brain amyloid-ß peptide levels could be observed for both treatments. Consequently, double-transgenic ovariectomized and intact mice had higher cognitive performance compared to untreated control animals in response to both estradiol and DHED administrations. Overall, the tested brain-selective 17ß-estradiol prodrug proved to be an effective early-stage intervention in an Alzheimer's disease-relevant mouse model without showing systemic impact and, thus, warrants further evaluation as a potential therapeutic candidate.

Direct Analysis in Real Time (DART) of an Organothiophosphate at Ultrahigh Resolution by Fourier Transform Ion Cyclotron Resonance Mass Spectrometry and Tandem Mass Spectrometry.

Direct analysis in real time (DART) is a recently developed ambient ionization technique for mass spectrometry to enable rapid and sensitive analyses with little or no sample preparation. After swab-based field sampling, the organothiophosphate malathion was analyzed using DART-Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometry (MS) and tandem mass spectrometry (MS/MS). Mass resolution was documented to be over 800,000 in full-scan MS mode and over 1,000,000 for an MS/MS product ion produced by collision-induced dissociation of the protonated analyte. Mass measurement accuracy below 1 ppm was obtained for all DART-generated ions that belonged to the test compound in the mass spectra acquired using only external mass calibration. This high mass measurement accuracy, achievable at present only through FTMS, was required for unequivocal identification of the corresponding molecular formulae.

Anti-alpha-toxin monoclonal antibody and antibiotic combination therapy improves disease outcome and accelerates healing in a Staphylococcus aureus dermonecrosis model.

Alpha-toxin (AT) is a major virulence determinant in Staphylococcus aureus skin and soft tissue infection models. We previously demonstrated that prophylactic administration of 2A3, an AT-neutralizing monoclonal antibody (MAb), prevents S. aureus disease in a mouse dermonecrosis model by neutralizing AT-mediated tissue necrosis and immune evasion. In the present study, MEDI4893*, an affinity-optimized version of 2A3, was characterized for therapeutic activity in the dermonecrosis model as a single agent and in combination with two frontline antibiotics, vancomycin and linezolid. MEDI4893* postinfection therapy was found to exhibit a therapeutic treatment window similar to that for linezolid but longer than that for vancomycin. Additionally, when combined with either vancomycin or linezolid, MEDI4893* resulted in reduced tissue damage, increased neutrophil and macrophage infiltration and abscess formation, and accelerated healing relative to those with the antibiotic monotherapies. These data suggest that AT neutralization with a potent MAb holds promise for both prophylaxis and adjunctive therapy with antibiotics and may be a valuable addition to currently available options for the treatment of S. aureus skin and soft tissue infections.

Label-free LC-MS/MS identification of phosphatidylglycerol-regulated proteins in Synechocystis sp. PCC6803.

We present a proteomics dataset combining SDS-PAGE prefractionation and data-dependent LC-MS/MS that enables the identification of phosphatidylglycerol-regulated proteins in the pgsA(-) mutant of Synechocystis sp. PCC6803, a cyanobacterium strain that grows with this indispensable phospholipid added exogenously. We searched the acquired raw data against a composite protein sequence database of Synechocystis using MASCOT, and employed Progenesis LC-MS software for label-free quantification based on extracted peptide intensities to detect changes in protein abundances upon phospholipid withdrawal. Protein identifications were validated using rigorous criteria, and our analysis of the dataset revealed 80 phosphatidylglycerol-regulated proteins involved in various cellular processes including photosynthesis, respiration, metabolism, transport, transcription, and translation. The data have been deposited to the ProteomeXchange with identifier PXD000363 (http://proteomecentral.proteomexchange.org/dataset/PXD000363).

Application of screening experimental designs to assess chromatographic isotope effect upon isotope-coded derivatization for quantitative liquid chromatography-mass spectrometry.

Isotope effect may cause partial chromatographic separation of labeled (heavy) and unlabeled (light) isotopologue pairs. Together with a simultaneous matrix effect, this could lead to unacceptable accuracy in quantitative liquid chromatography-mass spectrometry assays, especially when electrospray ionization is used. Four biologically relevant reactive aldehydes (acrolein, malondialdehyde, 4-hydroxy-2-nonenal, and 4-oxo-2-nonenal) were derivatized with light or heavy (d3-, (13)C6-, (15)N2-, or (15)N4-labeled) 2,4-dinitrophenylhydrazine and used as model compounds to evaluate chromatographic isotope effects. For comprehensive assessment of retention time differences between light/heavy pairs under various gradient reversed-phase liquid chromatography conditions, major chromatographic parameters (stationary phase, mobile phase pH, temperature, organic solvent, and gradient slope) and different isotope labelings were addressed by multiple-factor screening using experimental designs that included both asymmetrical (Addelman) and Plackett-Burman schemes followed by statistical evaluations. Results confirmed that the most effective approach to avoid chromatographic isotope effect is the use of (15)N or (13)C labeling instead of deuterium labeling, while chromatographic parameters had no general influence. Comparison of the alternate isotope-coded derivatization assay (AIDA) using deuterium versus (15)N labeling gave unacceptable differences (>15%) upon quantifying some of the model aldehydes from biological matrixes. On the basis of our results, we recommend the modification of the AIDA protocol by replacing d3-2,4-dinitrophenylhydrazine with (15)N- or (13)C-labeled derivatizing reagent to avoid possible unfavorable consequences of chromatographic isotope effects.

Direct sub-atmospheric pressure ionization mass spectrometry: Evaporation/sublimation-driven ionization is amazing, fundamentally, and practically.

This paper covers direct sub-atmospheric pressure ionization mass spectrometry (MS). The discovery, applications, and mechanistic aspects of novel ionization processes for use in MS that are not based on the high-energy input from voltage, laser, and/or high temperature but on sublimation/evaporation within a region linking a higher to lower pressure and modulated by heat and collisions, are discussed, including how this new reality has guided a series of discoveries, instrument developments, and commercialization. A research focus, inter alia, is on how best to understand, improve, and use these novel ionization processes, which convert volatile and nonvolatile compounds from solids (sublimation) or liquids (evaporation) into gas-phase ions for analysis by MS providing reproducible, accurate, sensitive, and prompt results. Our perception on how these unprecedented versus traditional ionization processes/methods relate to each other, how they can be made to coexist on the same mass spectrometer, and an outlook on new and expanded applications (e.g., clinical, portable, fast, safe, and autonomous) is presented, and is based on ST's Opening lecture presentation at the Nordic Mass spectrometry Conference, Geilo, Norway, January 2023. Focus will be on matrix-assisted ionization (MAI) and solvent-assisted ionization (SAI) MS covering the period from 2010 to 2023; a potential paradigm shift in the making.

Tissue penetration and antimicrobial activity of standard- and high-dose trimethoprim/sulfamethoxazole and linezolid in patients with diabetic foot infection.

OBJECTIVES: The purpose of this study was to conduct a pharmacokinetic and pharmacodynamic evaluation of high (320/1600 mg) and standard (160/800 mg) doses of trimethoprim/sulfamethoxazole and linezolid in outpatients with mild diabetic foot infections (DFIs). METHODS: Both viable skin/soft tissue from the infection site and serum were obtained at various times after antibiotic administration from 18 patients (6 per study group) being treated with linezolid, standard doses of trimethoprim/sulfamethoxazole or high doses of trimethoprim/sulfamethoxazole during a follow-up clinic visit. These samples were assayed for drug concentrations by liquid chromatography in tandem with mass spectrometry. Patient sera were also utilized in time-kill assays against two strains of Staphylococcus aureus and three strains of ß-haemolytic streptococci. RESULTS: The mean tissue/serum ratio for linezolid was 0.46 (range, 0.18-0.71). The mean tissue/serum ratio for trimethoprim was 1.2 (range, 0.3-4.5) for both standard and high doses, and 0.23 (range, 0.1-0.46) and 0.36 (range, 0.14-1.28) for standard and high doses of sulfamethoxazole, respectively. Linezolid exhibited inhibitory activity in time-kill assays against strains of S. aureus (0.45 ± 0.5 log10 cfu/mL) and ß-haemolytic streptococci (2.2 ± 0.6 log10 cfu/mL), while trimethoprim/sulfamethoxazole exhibited bactericidal (>3 log kill) activity against all of these isolates. These findings were consistent for each sampling time and for high as well as standard doses of trimethoprim/sulfamethoxazole. CONCLUSIONS: This pharmacokinetic/pharmacodynamic study found that trimethoprim/sulfamethoxazole exhibits good skin/soft tissue penetration in patients with DFIs as well as bactericidal activity in serum against strains of S. aureus and ß-haemolytic streptococci.

17ß-estradiol eye drops protect the retinal ganglion cell layer and preserve visual function in an in vivo model of glaucoma.

Neuroprotection in glaucoma as a curative strategy complementary to current therapies to lower intraocular pressure (IOP) is highly desirable. This study was designed to investigate neuroprotection by 17ß-estradiol (E2) to prevent retinal ganglion cell (RGC) death in a glaucoma model of surgically elevated IOP in rats. We found that daily treatment with E2-containing eye drops resulted in significant E2 concentration in the retina with concomitant profound neuroprotective therapeutic benefits, even in the presence of continually elevated IOP. The number of apoptotic cells in the RGC layer was significantly decreased in the E2-treated group, when compared to the vehicle-treated controls. Deterioration in visual acuity in these animals was also markedly prevented. Using mass spectrometry-based proteomics, beneficial changes in the expression of several proteins implicated in the maintenance of retinal health were also found in the retina of E2-treated animals. On the other hand, systemic side effects could not be avoided with the eye drops, as confirmed by the measured high circulating estrogen levels and through the assessment of the uterus representing a typical hormone-sensitive peripheral organ. Collectively, the demonstrated significant neuroprotective effect of topical E2 in the selected animal model of glaucoma provides a clear rationale for further studies aiming at targeting E2 into the eye while avoiding systemic E2 exposure to diminish undesirable off-target side effects.