Analysis of Sphingosine and Sphinganine from the Aqueous Humor for Signaling Studies Using Ultrahigh-Performance Liquid Chromatography-Mass Spectrometry.

Sphingolipids, including sphingosine and sphinganine, are one of the major classes of lipids. They serve as constituents of cell membranes and lipid rafts and aid in the performance of cell-cell communication and adhesion. Abnormal levels of sphingolipids in the aqueous humor can indicate impaired sphingolipid metabolism and associated ocular pathologies. Sphingolipids can be extracted from the aqueous humor by the methyl-tert-butyl ether (MTBE) lipid extraction method and subsequently analyzed by liquid chromatography-mass spectrometry (LC-MS). This chapter describes a modified protocol for an MTBE lipid extraction from the aqueous humor, followed by analysis with ultrahigh-performance liquid chromatography-mass spectrometry (UHPLC-MS).

Optimized Lipidomics Extraction of Sphingosine and Sphinganine from Optic Nerve for Signaling Studies.

Interconvertible sphingolipid metabolites represent germane constituents of eukaryotic membranes and are vital in the regulation of cellular homeostasis, proliferation, survival, and induction of autophagy. This protocol describes a step-by-step method for extractions of sphingosine and sphinganine from mammalian tissue samples, particularly from the murine optic nerve. These lipids are partitioned into a binary mixture of chloroform and methanol in a modified Bligh and Dyer method. This is followed with reverse phase ultrahigh-performance liquid chromatography fractionation with a C18+ column and subsequent tandem mass spectrometry (UHPLC-MS-MS) analysis of the biological abundance. These free sphingoid bases dissociate to form structurally distinctive carbocation product ions that can be confirmed with annotations of lipidomic databases or in-house fragmentation software.

Diacylglycerol Signaling in Retinal Ganglion Cells.

With impaired retinal ganglion cell (RGC) function and eventual RGC death, there is a heightened risk of experiencing glaucoma-induced blindness or other optic neuropathies. Poor RGC efficiency leads to limited transmission of visual signals between the retina and the brain by RGC axons. Increased focus on studying lipid messengers found in neurons such as endocannabinoids (eCBs) has importance due to their potential axonal pathway regenerative properties. 2-Arachidonoylglycerol (2-AG), a common eCB, is synthesized from an sn-1 hydrolysis reaction between diacylglycerol (DAG) and diacylglycerol lipase (DAGL). Examination of DAG production allows for future downstream analysis in relation to DAGL functionality. Here, we describe protocol guidelines for extracting RGCs from mouse retinas and subsequent mass spectrometry analysis of the DAG content present within the RGCs.

Downregulation of ATP8B2 to Assess Plasmalogen Distribution and Far1 Expression in Primary Trabecular Meshwork Cells.

The trabecular meshwork (TM) from primary open-angle glaucoma (POAG) cases has been found to contain decreased levels of intracellular plasmalogens. Plasmalogens are a subset of lipids involved in diverse cellular processes such as intracellular signaling, membrane asymmetry, and protein regulation. Proper plasmalogen biosynthesis is regulated by rate-limiting enzyme fatty acyl-CoA reductase (Far1). ATPase phospholipid transporting 8B2 (ATP8B2) is a type IV P-type ATPase responsible for the asymmetric distribution of plasmalogens between the intracellular and extracellular leaflets of the plasma membranes. Here we describe the methodology for extraction and culturing of TM cells from corneal tissue and subsequent downregulation of ATP8B2 using siRNA transfection. Further quantification and downstream effects of ATP8B2 gene knockdown will be analyzed utilizing immunoblotting techniques.

Quantifying ABCA1/apoA-1 Signaling Pathways with AFM Imaging and Lipidomic Analysis.

The role of lipid metabolic pathways in the pathophysiology of primary open-angle glaucoma (POAG) has been thoroughly elucidated, with pathways involved in lipid-related disorders such as hypercholesterolemia and hyperlipoprotein accumulation being of particular interest. The ABCA1/apoA-1 transduction pathway moderates reverse cholesterol transport (RCT), facilitating the transport of free cholesterol (FC) and phospholipids (PL) and preventing intracellular lipid aggregates in retinal ganglion cells (RGCs) due to excess FCs and PLs. A deficiency of ABCA1 transporters, and thus, dysregulation of the ABCA1/apoA-1 transduction pathway, may potentiate cellular lipid accumulation, which affects the structural and mechanical features of the cholesterol-rich RGC membranes. Atomic force microscopy (AFM) is a cutting-edge imaging technique suitable for imaging topographical surfaces of a biological specimen and determining its mechanical properties and structural features. The versatility and precision of this technique may prove beneficial in understanding the effects of ABCA1/apoA-1 pathway downregulation and decreased cholesterol efflux in RGCs and their membranes. In this protocol, ABCA1-/- RGC mouse models are prepared over the course of 3 days and are then compared with non-knockout ABCA1 RGC mouse models through AFM imaging of topographical surfaces to examine the difference in membrane dynamics of knockout vs. non-knockout models. Intracellular and extracellular levels of lipids are quantified through high-performance liquid chromatography with tandem mass spectrometry (HPLC-MS/MS).

Analysis of Lipoprotein Signaling in Iris Melanocytes.

Lipids are compounds involved in many biologic functions including cell structure, metabolism, energy storage and are involved in signaling. A prominent lipid in these functions is cholesterol. Cholesterol also plays a part in the signaling of melanocytes, which contain melanosomes. The maturation of these melanosomes happens during melanocyte growth. The deficit of melanogenesis or melanosome maturation is associated with ocular albinism in the eye. Aberrations of melanosome maturation are also associated with pigment dispersion syndrome. Albinism and pigment dispersion manifestations are systemic. Both melanogenesis and melanocyte maturation are affected by cholesterol metabolism. Cholesterol signaling is a part of many pathways in the body, and evaluating these signals can have implications in systemic disease processes of melanogenesis and melanosome maturation, like ocular albinism and pigment dispersion. Cholesterol is carried by lipoprotein particles. Low-density lipoprotein (LDL) is usually the transport vehicle for cholesterol to reach tissues and organelles. The LDL uptake on cells often sends out a cascade of internal signaling within the cells. We describe here LDL signaling related to lipase activity changes using enzymatic methods with a kit. We describe analyses of cholesterol esters and free cholesterol with liquid chromatography and gas chromatography with or in tandem with mass spectrometry (GC-MS and LC-MS/MS). These analyses will provide insight into melanosome maturation and melanogenesis. The methods described here are applicable to all melanocytes within the body of a model mammalian organism.

Association of Primary Open-Angle Glaucoma with Diabetic Retinopathy among Patients with Type 1 and Type 2 Diabetes: A Large Global Database Study.

PURPOSE: To assess the correlation between primary open-angle glaucoma (POAG) and the risk of developing diabetic retinopathy (DR) in patients with type 1 diabetes mellitus (T1DM) and type 2 diabetes mellitus (T2DM). DESIGN: A retrospective cohort study leveraging the global patient database of TriNetX Research Network. PARTICIPANTS: The study included 44 359 patients with diabetes mellitus (DM) with POAG and 4 393 300 patients with DM without any glaucoma ≥ 18 years of age. Propensity score matching harmonized the cohorts to 39 680 patients each, covering diagnoses from January 1, 2005, to January 1, 2023. METHODS: We analyzed data using specific International Classification of Diseases, 10th Revision (ICD-10) codes for DM and glaucoma. We matched the cohorts using propensity score matching, adjusting for age, sex, race/ethnicity, blood markers, relevant medical history, and ophthalmic service use. MAIN OUTCOME MEASURES: The primary outcome was the first-time occurrence of DR, including nonproliferative DR (NPDR) and proliferative DR (PDR), in patients with DM with and without glaucoma at 1-, 5-, and 10-year intervals from their individual index dates. RESULTS: At 10 years, patients with T1DM with POAG exhibited a heightened risk for any DR (adjusted risk ratios [RRs], 4.12; 95% confidence interval [CI], 3.05-5.57, P < 0.0001) and PDR (RR, 7.02; 95% CI, 3.62-13.61, P < 0.0001). Patients with T2DM and POAG also faced an increased 10-year risk for any DR (RR, 2.47; 95% CI, 2.28-2.68, P < 0.0001) and PDR (RR, 3.82; 95% CI, 3.09-4.70, P < 0.0001). The combined association of POAG on DR risk in those with T1DM and T2DM at 10 years was found to be significantly higher among patients with POAG (5.45%) compared with those without glaucoma (2.12%) (adjusted hazard ratio [aHR], 2.33; 95% CI, 2.14-2.53). The cumulative incidence of DR was significantly higher in the POAG group compared with nonglaucoma counterparts after a decade (log-rank P < 0.001). CONCLUSIONS: Our findings underscore a substantial association between POAG and DR development in both T1DM and T2DM patients, emphasizing the need for vigilant screening and comprehensive management in glaucomatous patients with DM to mitigate the risk of DR. Future research should delve into elucidating the causal mechanisms driving these observed associations. FINANCIAL DISCLOSURE(S): The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Appropriate patient population for future visual system axon regeneration therapies.

A number of blinding diseases caused by damage to the optic nerve result in progressive vision loss or loss of visual acuity. Secondary glaucoma results from traumatic injuries, pseudoexfoliation or pigmentary dispersion syndrome. Progressive peripheral vision loss is common to all secondary glaucoma irrespective of the initial event. Axon regeneration is a potential therapeutic avenue to restore lost vision in these patients. In contrast to the usual approach of having the worst possible patient population for initial therapies, axon regeneration may require consideration of appropriate patient population even for initial treatment trials. The current state of axon regeneration therapies, their potential future and suitable patient population when ready is discussed in this perspective. The selection of patients are important for adoption of axon regeneration specifically in the areas of central nervous system regenerative medicine. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology Neurological Diseases > Biomedical Engineering Metabolic Diseases > Molecular and Cellular Physiology.

Currently available prostanoids for the treatment of glaucoma and ocular hypertension: A review.

Recent advancements in prostaglandin analogs (PGAs) have reinforced their role in managing intraocular pressure (IOP). Latanoprost excels in 24-h IOP control, while various PGAs offer similar effectiveness and side effects, generic PGAs perform as well as branded ones, and a notable IOP rise observed upon PGA discontinuation. Formulations with or without preservatives show comparable IOP reduction and adherence, often surpassing benzalkonium chloride (BAK)-preserved options. Emergent PGAs, such as latanoprostene bunod, fixed-dose netarsudil combined with latanoprost, and omidenepag Isopropyl, offer enhanced or non-inferior IOP reduction. The bimatoprost implant introduces a novel administration method with effective IOP reduction. These developments underscore ongoing progress in PGA-focused ophthalmological research. This article offers a comprehensive review of available prostanoid analogs and explores new developments.

Mass spectrometry dataset of LC-MS lipidomics analysis of Xenopus laevis optic nerve.

CNS injuries of the anuran amphibian, Xenopus laevis, are uniquely suited for studying the molecular compositions of neuronal regeneration of retinal ganglion cells (RGC) due to a functional recovery of optic axons disparate to adult mammalian analogues. RGCs and their optic nerve axons undergo irreversible neurodegeneration in glaucoma and associated optic neuropathies, resulting in blindness in mammals. Conversely, Xenopus demonstrates RGC lifetime-spanning regenerative capabilities after optic nerve crush [1], inciting opportunities to compare de novo regeneration and develop efficient pharmaceutical approaches for vision restoration. Studies revealing lipidome alterations during optic nerve regeneration are sparse and could serve as a solid foundation for these underlying molecular changes. We profile the lipid changes in a transgenic line of 1 year old Xenopus laevis Tg(islet2b:gfp) frogs that were either left untreated (naïve) or had a monocular surgery of either a left optic crush injury (crush) or sham surgery (sham). Matching controls of uninjured right optic nerves were also collected (control). Tg(islet2b:gfp) frogs were allowed to recover for 7,12,18, and 27 days post optic nerve crush. Following euthanasia, the optic nerves were collected for lipidomic analysis. A modified Bligh and Dyer method [2] was used for lipid extraction, followed by untargeted mass spectrometry lipid profiling with a Q Exactive Orbitrap Mass Spectrometer coupled with a Vanquish Horizon Binary UHPLC LC-MS system (LC MS-MS). The raw scans were analyzed and quantified with LipidSearch 5.0 and the statistical analysis was conducted through Metaboanalyst 5.0. This data is available at Metabolomics Workbench, study ID [ST002414].

Metabolomics dataset of zebrafish optic nerve regeneration after injury.

Zebrafish (Danio rerio) have the capacity for successful adult optic nerve regeneration. In contrast, mammals lack this intrinsic ability and undergo irreversible neurodegeneration seen in glaucoma and other optic neuropathies. Optic nerve regeneration is often studied using optic nerve crush, a mechanical neurodegenerative model. Untargeted metabolomic studies within successful regenerative models are deficient. Evaluation of tissue metabolomic changes in active zebrafish optic nerve regeneration can elucidate prioritized metabolite pathways that can be targeted in mammalian systems for therapeutic development. Female and male (6 month to 1 year old wild type) right zebrafish optic nerves were crushed and collected three days after. Contralateral, uninjured optic nerves were collected as controls. The tissue was dissected from euthanized fish and frozen on dry ice. Samples were pooled for each category (female crush, female control, male crush, male control) and pooled at n = 31 to obtain sufficient metabolite concentrations for analysis. Optic nerve regeneration at 3 days post crush was demonstrated by microscope visualization of GFP fluorescence in Tg(gap43:GFP) transgenic fish. Metabolites were extracted using a Precellys Homogenizer and a serial extraction method: (1) 1:1 Methanol/Water and (2) 8:1:1 Acetonitrile/Methanol/Acetone. Metabolites were analyzed by untargeted liquid chromatography-mass spectrometry (LC MS-MS) profiling using a Q-Exactive Orbitrap instrument coupled with Vanquish Horizon Binary UHPLC LC-MS system. Metabolites were identified and quantified using Compound Discoverer 3.3 and isotopic internal metabolites standards.

Glaucomatous aqueous humor vesicles are smaller and differ in composition compared to controls.

Cells communicate with each other using vesicles of varying sizes, including a specific repertoire known as exosomes. We isolated aqueous humor (AH)-derived vesicles using two different methods: ultracentrifugation and an exosome isolation kit. We confirmed a unique vesicle size distribution in the AH derived from control and primary open-angle glaucoma (POAG) patients using various techniques, including Nanotracker, dynamic light scattering, atomic force imaging, and electron microscopy. Bonafide vesicle and/or exosome markers were present by dot blot in both control and POAG AH-derived vesicles. Marker levels differed between POAG and control samples, while non-vesicle negative markers were absent in both. Quantitative labeled (iTRAQ) proteomics showed a reduced presence of a specific protein, STT3B, in POAG compared to controls, which was further confirmed using dot blot, Western blot, and ELISA assays. Along the lines of previous findings with AH profiles, we found vast differences in the total phospholipid composition of AH vesicles in POAG compared to controls. Electron microscopy further showed that the addition of mixed phospholipids alters the average size of vesicles in POAG. We found that the cumulative particle size of type I collagen decreased in the presence of Cathepsin D, which normal AH vesicles were able to protect against, but POAG AH vesicles did not. AH alone had no effect on collagen particles. We observed a protective effect on collagen particles with an increase in artificial vesicle sizes, consistent with the protective effects observed with larger control AH vesicles but not with the smaller-sized POAG AH vesicles. Our experiments suggest that AH vesicles in the control group provide greater protection for collagen beams compared to POAG, and their increased vesicle sizes are likely contributing factors to this protection.

Ocular Treatments Targeting Separate Prostaglandin Receptors in Mice Exhibit Alterations in Intraocular Pressure and Optic Nerve Lipidome.

Background: Prostaglandin (PG) receptor agonists are the first-line eyedrop medication treatment for glaucoma. The pathophysiology of this disease is not completely known, and elevated intraocular pressure (IOP) is the key risk factor. The membranes of the axons (of the retinal ganglion cells) passing through the optic nerve (ON) head experience significant damage. Lipids are an essential component of the cell's membranes, and their profile changes owing to neurodegeneration. In this investigation, three agonists for distinct PG receptors were used to lower IOP and to determine their effect on the ON lipids. We utilized DBA/2J mice as a model of progressive IOP increase and C57BL/6J mice as a model of ON crush. Methods: DBA/2J and C57BL/6J mice were treated daily for 2 weeks with Latanoprost, PF-04217329, or Rivenprost. The IOP was measured every 2 days and pattern electroretinogram was conducted for DBA/2J throughout the study. Lipidomics of ONs were performed for each model and treatment group. Results: Of the tested compounds, Latanoprost and Rivenprost were the most effective agents decreasing IOP in DBA/2J mice. Triglyceride levels increased in the ONs of DBA/2J mouse model, but phosphatidylethanolamine levels underwent highest level changes in the C57BL/6J mouse model when treated with Latanoprost. Conclusions: Topical ocular FP- and EP4-receptor agonists appreciably lowered IOP in the DBA/2J mice representing pigmentary glaucoma. The observed changes in ON lipidomics in the different models of neurodegeneration suggest possible use of such measures in the development of more effective medicines for both IOP reduction and ON protection.

Glycerophospholipid Analysis of Optic Nerve Regeneration Models Indicate Potential Membrane Order Changes Associated with the Lipidomic Shifts.

Purpose: Optic nerve (ON) injury causes irreversible degeneration, leading to vision loss that cannot be restored with available therapeutics. Current therapies slow further degeneration but do not promote regeneration. New regenerative factors have been discovered that are successful in vivo. However, the mechanisms of efficient long-distance regeneration are still unknown. Membrane expansion by lipid insertion is an essential regenerative process, so lipid profiles for regenerating axons can provide insight into growth mechanisms. This article's analysis aims to add to the increasingly available ON regeneration lipid profiles and relate it to membrane order/properties. Methods: In this study, we present an analysis of glycerophospholipids, one of the largest axonal lipid groups, from three mammalian ON regeneration lipid profiles: Wnt3a, Zymosan + CPT-cAMP, and Phosphatase/Tensin homolog knockout (PTENKO) at 7 and 14 days post crush (dpc). Significant lipid classes, species, and ontological properties were crossreferenced between treatments and analyzed using Metaboanalyst 5.0 and Lipid Ontology (LION). Membrane order changes associated with significant lipid classes were evaluated by C-Laurdan dye and exogenous lipids provided to a neuroblastoma cell line. Results and Conclusions: At 7 dpc, ONs show increased lysoglycerophospholipids and decreased phosphatidylethanolamines (PEs)/negative intrinsic curvature lipids. At 14 dpc, regenerative treatments show divergence: Wnt3a displays higher lysoglycerophospholipid content, while Zymosan and PTENKO decrease lysoglycerophospholipids and increase phosphatidylcholine (PC)-related species. Membrane order imaging indicates lysoglycerophospholipids decreases membrane order while PE and PC had no significant membrane order effects. Understanding these changes will allow therapeutic development targeting lipid metabolic pathways that can be used for vision loss treatments.

Isolation of Mitochondrial Lipids and Mass Spectrometric Analysis.

Mitochondria participate in many important metabolic processes in the body. The lipid profile of mitochondria is especially important in membrane regulation and pathway signaling. The isolation and study of these lipids can provide unparalleled information about the mechanisms behind these cellular processes. In this chapter, we describe a protocol to isolate mitochondrial lipids from homogenized murine optic nerves. The lipid extraction was performed using butanol-methanol (BUME) and subsequently analyzed using liquid chromatography-mass spectrometry. Further analysis of the raw data was conducted using LipidSearch™ and MetaboAnalyst 4.0.

Characterization of Gangliosides from Mouse Optic Nerve Samples Using Mass Spectrometry.

This chapter focuses on identifying gangliosides in the optic nerve of the mouse using mass spectrometry techniques. The described protocol will also permit the characterization of the sample's lipidome. Two deuterium-labeled ganglioside standards and a general lipid class standard will be utilized for extraction efficiency and quantification. Using reversed-phase high-performance liquid chromatography (HPLC) coupled to a Q Exactive mass spectrometer, the samples will be analyzed. The method will consist of both an untargeted approach and a targeted approach with a ganglioside-specific inclusion list.

High-Resolution Liquid Chromatography-Mass Spectrometry for Lipidomics.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is a powerful tool for identification and classification of lipids. Ultra-high performance liquid chromatography (UHPLC) allows for robust separations of complex mixtures, while high-resolution mass spectrometry (HRMS) identifies compounds with efficiency and accuracy (Zullig T and Kofeler HC, Mass Spectrom Rev 40:162-176, 2021). The high specificity and sensitivity of mass spectrometry makes it the method of choice when analyzing lipids (Kofeler HC, J Lipid Res 62:100138, 2021). Untargeted mass spectrometry identifies all lipids within a sample and is useful for identification and further discovery. This chapter describes the use of a Q Exactive mass spectrometer to perform an untargeted LC-MS/MS lipidomics analysis.

Lipidomics Analysis with Triple Quadrupole Mass Spectrometry.

Lipids serve an essential role in multiple cellular functions including signaling, metabolism, energy storage, and membrane constitution. Lipidomics, the study of lipids using analytical chemistry, allows for the study of disease states and cellular metabolism. Shotgun lipidomics is a technique that involves direct-infusion electrospray ionization (ESI) and analysis with a triple quadrupole mass spectrometer. Triple quadrupole mass spectrometry is ideally suited for lipidomics analysis because it allows for class-specific identification of lipids. Individual lipid class can be identified by the adjustment of three parameters-collision energy, ion mode, and scan type. This chapter describes the use of a triple quadrupole mass spectrometer, the TSQ Quantum Access MAX, to perform lipidomics analysis with high sensitivity, accuracy, and precision.