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Venkatesan A, Ridilla M, Castro N, Wolosin JM, Henty-Ridilla JL, Knox BE, Ganapathy PS, Brown JS, DeVincentis Iii AF, Sieminski S, Bernstein AM. Mitochondrial and microtubule defects in Exfoliation Glaucoma. Free Radic Biol Med 2025; 233:226-239. [PMID: 40180018 DOI: 10.1016/j.freeradbiomed.2025.03.046] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/22/2025] [Revised: 03/20/2025] [Accepted: 03/30/2025] [Indexed: 04/05/2025]
Abstract
Exfoliation Syndrome is an age-related systemic condition characterized by large aggregated fibrillar material deposition in the anterior eye tissues. This aggregate formation and deposition on the aqueous humor outflow pathway are significant risk factors for developing Exfoliation Glaucoma (XFG). XFG is a multifactorial late-onset disease that shares common features of neurodegenerative diseases, such as increased protein aggregation, impaired protein degradation, and oxidative and cellular stress. XFG patients display decreased mitochondrial membrane potential and mitochondrial DNA deletions. Here, using Tenon Capsule Fibroblasts (TFs) from patients without glaucoma (No Glaucoma, NG) and XFG patients, we found that XFG TFs have impaired mitochondrial bioenergetics and increased reactive oxygen species accumulation. These defects are associated with mitochondrial abnormalities as XFG TFs exhibit smaller mitochondria that contain dysmorphic cristae, with increased mitochondrial localization to lysosomes and slowed mitophagic flux. Mitochondrial dysfunction in the XFG TFs was associated with hyperdynamic microtubules, decreased acetylated tubulin, and increased HDAC6 activity. Treatment of XFG TFs with a mitophagy inducer, Urolithin A (UA), and a mitochondrial biogenesis inducer, Nicotinamide Ribose (NR), improved mitochondrial bioenergetics and reduced ROS accumulation. Our results demonstrate that XFG TFs have abnormal mitochondria and suggest that mitophagy inducers may represent a potential class of therapeutics for reversing mitochondrial dysfunction in XFG patients.
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Affiliation(s)
- Arunkumar Venkatesan
- Department of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY, USA
| | | | - Nileyma Castro
- Department of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY, USA; New York VA Health Care, Syracuse VA Medical Center, Syracuse, NY, USA
| | - J Mario Wolosin
- Eye and Vision Research Institute, Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY, USA
| | - Jessica L Henty-Ridilla
- Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA
| | - Barry E Knox
- Department of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY, USA; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA
| | - Preethi S Ganapathy
- Department of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY, USA
| | - Jamin S Brown
- Retina-Vitreous Surgeons of Central New York, Syracuse, NY, USA
| | | | | | - Audrey M Bernstein
- Department of Ophthalmology and Visual Sciences, SUNY Upstate Medical University, Syracuse, NY, USA; New York VA Health Care, Syracuse VA Medical Center, Syracuse, NY, USA; Department of Biochemistry and Molecular Biology, SUNY Upstate Medical University, Syracuse, NY, USA
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Venkatesan A, Ridilla M, Castro N, Wolosin JM, Henty-Ridilla JL, Knox BE, Ganapathy PS, Brown JS, DeVincentis AF, Sieminski S, Bernstein AM. Mitochondrial and Microtubule Defects in Exfoliation Glaucoma. BIORXIV : THE PREPRINT SERVER FOR BIOLOGY 2024:2024.11.25.625249. [PMID: 39651300 PMCID: PMC11623661 DOI: 10.1101/2024.11.25.625249] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 12/11/2024]
Abstract
Exfoliation Syndrome (XFS) is an age-related systemic condition characterized by large aggregated fibrillar material deposition in the anterior eye tissues. This aggregate formation and deposition on the aqueous humor outflow pathway are significant risk factors for developing Exfoliation Glaucoma (XFG), a secondary open-angle glaucoma. XFG is a complex, multifactorial late-onset disease that shares common features of neurodegenerative diseases, such as altered cellular processes with increased protein aggregation, impaired protein degradation, and oxidative and cellular stress. XFG patients display decreased mitochondrial membrane potential and mitochondrial DNA deletions. Here, using Tenon Capsule Fibroblasts (TFs) from Normal (No Glaucoma) and XFG patients, we found that XFG TFs have impaired mitochondrial bioenergetics and increased reactive oxygen species (ROS) accumulation. These defects are associated with mitochondrial abnormalities as XFG TFs exhibit smaller mitochondria that contain dysmorphic cristae, with an increase in mitochondrial localization to lysosomes and slowed mitophagy flux. Mitochondrial dysfunction in the XFG TFs was associated with an increase in the dynamics of the microtubule cytoskeleton, decreased acetylated tubulin, and increased HDAC6 activity. Treatment of XFG TFs with a mitophagy inducer, Urolithin A, and a mitochondrial biogenesis inducer, NAD + precursor, Nicotinamide Ribose, improved mitochondrial bioenergetics and reduced ROS accumulation. Our results demonstrate abnormal mitochondria in XFG TFs and suggest that mitophagy inducers may represent a potential class of therapeutics for reversing mitochondrial dysfunction in XFG patients.
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Meyer KJ, Mercer HE, Roos BR, Fingert JH, Anderson MG. Minimal phenotypes in transgenic mice with the human LOXL1/LOXL1-AS1 locus associated with exfoliation glaucoma. Vision Res 2024; 223:108464. [PMID: 39151208 PMCID: PMC11381136 DOI: 10.1016/j.visres.2024.108464] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/17/2024] [Revised: 07/31/2024] [Accepted: 07/31/2024] [Indexed: 08/18/2024]
Abstract
Exfoliation syndrome is a leading cause of secondary glaucoma worldwide. Among the risk-factors for exfoliation syndrome and exfoliation glaucoma that have been investigated, a genetic association with 15q24.1 is among the most striking. The leading candidates for the causal gene at this locus are LOXL1 and/or LOXL1-AS1, but studies have not yet coalesced in establishing, or ruling out, either candidate. Here, we contribute to studies of the 15q24.1 locus by making a partially humanized mouse model in which 166 kb of human genomic DNA from the 15q24.1 locus was introduced into the mouse genome via BAC transgenesis (B6-Tg(RP11-71M11)Andm). Transgenic expression of human genes in the BAC was only detectable for LOXL1-AS1. One cohort of 34 mice (21 experimental hemizygotes and 13 non-carrier control littermates) was assessed by slit-lamp exams and SD-OCT imaging at early (1-2 months) and mid (4-5 months) time points; fundus exams were performed at 5 months of age. A second smaller cohort (3 hemizygotes) were aged extensively (>12 months) to screen for overt abnormalities. Across all genotypes and ages, 136 slit-lamp exams, 128 SD-OCT exams, and 42 fundus exams detected no overt indices of exfoliation syndrome. Quantitatively, small, but statistically significant, age-related declines in ganglion cell complex thickness and total retinal thickness were detected in the hemizygotes at 4 months of age. Overall, this study demonstrates complexity in gene regulation from the 15q24.1 locus and suggests that LOXL1-AS1 is unlikely to be a monogenic cause of exfoliation syndrome but may contribute to glaucomatous retinal damage.
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Affiliation(s)
- Kacie J Meyer
- Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, United States; Institute for Vision Research, University of Iowa, Iowa City, IA, United States
| | - Hannah E Mercer
- Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, United States; Institute for Vision Research, University of Iowa, Iowa City, IA, United States
| | - Ben R Roos
- Institute for Vision Research, University of Iowa, Iowa City, IA, United States; Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, United States
| | - John H Fingert
- Institute for Vision Research, University of Iowa, Iowa City, IA, United States; Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, United States
| | - Michael G Anderson
- Department of Molecular Physiology and Biophysics, University of Iowa, Iowa City, IA, United States; Institute for Vision Research, University of Iowa, Iowa City, IA, United States; Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, United States; Center for the Prevention and Treatment of Visual Loss, Iowa City VA Health Care System, Iowa City, IA, United States.
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Wu HJ, Krystofiak E, Kuchtey J, Kuchtey RW. Enhanced Optic Nerve Expansion and Altered Ultrastructure of Elastic Fibers Induced by Lysyl Oxidase Inhibition in a Mouse Model of Marfan Syndrome. THE AMERICAN JOURNAL OF PATHOLOGY 2024; 194:1317-1328. [PMID: 38548269 PMCID: PMC11317902 DOI: 10.1016/j.ajpath.2024.03.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/27/2023] [Revised: 02/27/2024] [Accepted: 03/11/2024] [Indexed: 04/09/2024]
Abstract
Two major constituents of exfoliation material, fibrillin-1 and lysyl oxidase-like 1 (encoded by FBN1 and LOXL1), are implicated in exfoliation glaucoma, yet their individual contributions to ocular phenotype are minor. To test the hypothesis that a combination of FBN1 mutation and LOXL1 deficiency exacerbates ocular phenotypes, the pan-lysyl oxidase inhibitor β-aminopropionitrile (BAPN) was used to treat adult wild-type (WT) mice and mice heterozygous for a missense mutation in Fbn1 (Fbn1C1041G/+) for 8 weeks and their eyes were examined. Although intraocular pressure did not change and exfoliation material was not detected in the eyes, BAPN treatment worsened optic nerve and axon expansion in Fbn1C1041G/+ mice, an early sign of axonal damage in rodent models of glaucoma. Disruption of elastic fibers was detected only in Fbn1C1041G/+ mice, which increased with BAPN treatment, as shown by histologic and immunohistochemical staining of the optic nerve pia mater. Transmission electron microscopy showed that Fbn1C1041G/+ mice had fewer microfibrils, smaller elastin cores, and a lower density of elastic fibers compared with WT mice in control groups. BAPN treatment led to elastin core expansion in both WT and Fbn1C1041G/+ mice, but an increase in the density of elastic fiber was confined to Fbn1C1041G/+ mice. LOX inhibition had a stronger effect on optic nerve and elastic fiber parameters in the context of Fbn1 mutation, indicating the Marfan mouse model with LOX inhibition warrants further investigation for exfoliation glaucoma pathogenesis.
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Affiliation(s)
- Hang-Jing Wu
- Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Evan Krystofiak
- Cell Imaging Shared Resource, Vanderbilt University, Nashville, Tennessee
| | - John Kuchtey
- Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee
| | - Rachel W Kuchtey
- Vanderbilt Eye Institute, Vanderbilt University Medical Center, Nashville, Tennessee; Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee.
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Mueller A, Lam I, Kishor K, Lee RK, Bhattacharya S. Secondary glaucoma: Toward interventions based on molecular underpinnings. WIREs Mech Dis 2024; 16:e1628. [PMID: 37669762 DOI: 10.1002/wsbm.1628] [Citation(s) in RCA: 4] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/07/2023] [Revised: 08/08/2023] [Accepted: 08/10/2023] [Indexed: 09/07/2023]
Abstract
Glaucoma is a heterogeneous group of progressive diseases that leads to irreversible blindness. Secondary glaucoma refers to glaucoma caused by a known underlying condition. Pseudoexfoliation and pigment dispersion syndromes are common causes of secondary glaucoma. Their respective deposits may obstruct the trabecular meshwork, leading to aqueous humor outflow resistance, ocular hypertension, and optic neuropathy. There are no disease-specific interventions available for either. Pseudoexfoliation syndrome is characterized by fibrillar deposits (pseudoexfoliative material) on anterior segment structures. Over a decade of multiomics analyses taken together with the current knowledge on pseudoexfoliative glaucoma warrant a re-think of mechanistic possibilities. We propose that the presence of nucleation centers (e.g., vitamin D binding protein), crosslinking enzymes (e.g., transglutaminase 2), aberrant extracellular matrix, flawed endocytosis, and abnormal aqueous-blood barrier contribute to the formation of proteolytically resistant pseudoexfoliative material. Pigment dispersion syndrome is characterized by abnormal iridolenticular contact that disrupts iris pigment epithelium and liberates melanin granules. Iris melanogenesis is aberrant in this condition. Cytotoxic melanogenesis intermediates leak out of melanosomes and cause iris melanocyte and pigment epithelium cell death. Targeting melanogenesis can likely decrease the risk of pigmentary glaucoma. Skin and melanoma research provides insights into potential therapeutics. We propose that specific prostanoid agonists and fenofibrates may reduce melanogenesis by inhibiting cholesterol internalization and de novo synthesis. Additionally, melatonin is a potent melanogenesis suppressor, antioxidant, and hypotensive agent, rendering it a valuable agent for pigmentary glaucoma. In pseudoexfoliative glaucoma, where environmental insults drive pseudoexfoliative material formation, melatonin's antioxidant and hypotensive properties may offer adjunct therapeutic benefits. This article is categorized under: Neurological Diseases > Molecular and Cellular Physiology.
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Affiliation(s)
- Anna Mueller
- Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA
- Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, USA
| | - Isabel Lam
- Idaho College of Osteopathic Medicine, Meridian, Idaho, USA
| | - Krishna Kishor
- Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA
- Miami Integrative Metabolomics Research Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA
| | - Richard K Lee
- Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA
- Miami Integrative Metabolomics Research Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA
| | - Sanjoy Bhattacharya
- Department of Ophthalmology, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA
- Miami Integrative Metabolomics Research Center, Bascom Palmer Eye Institute, University of Miami Miller School of Medicine, Miami, Florida, USA
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Aboobakar IF, Wiggs JL. The genetics of glaucoma: Disease associations, personalised risk assessment and therapeutic opportunities-A review. Clin Exp Ophthalmol 2022; 50:143-162. [PMID: 35037362 DOI: 10.1111/ceo.14035] [Citation(s) in RCA: 25] [Impact Index Per Article: 8.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/18/2021] [Revised: 12/14/2021] [Accepted: 12/15/2021] [Indexed: 12/23/2022]
Abstract
Glaucoma refers to a heterogenous group of disorders characterised by progressive loss of retinal ganglion cells and associated visual field loss. Both early-onset and adult-onset forms of the disease have a strong genetic component. Here, we summarise the known genetic associations for various forms of glaucoma and the possible functional roles for these genes in disease pathogenesis. We also discuss efforts to translate genetic knowledge into clinical practice, including gene-based tests for disease diagnosis and risk-stratification as well as gene-based therapies.
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Affiliation(s)
- Inas F Aboobakar
- Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA
| | - Janey L Wiggs
- Department of Ophthalmology, Massachusetts Eye and Ear, Harvard Medical School, Boston, Massachusetts, USA
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De Maria A, Zientek KD, David LL, Wilmarth PA, Bhorade AM, Harocopos GJ, Huang AJW, Hong AR, Siegfried CJ, Tsai LM, Sheybani A, Bassnett S. Compositional Analysis of Extracellular Aggregates in the Eyes of Patients With Exfoliation Syndrome and Exfoliation Glaucoma. Invest Ophthalmol Vis Sci 2021; 62:27. [PMID: 34964803 PMCID: PMC8740535 DOI: 10.1167/iovs.62.15.27] [Citation(s) in RCA: 10] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/24/2022] Open
Abstract
Purpose Exfoliation syndrome (XFS) is a condition characterized by the production of insoluble fibrillar aggregates (exfoliation material; XFM) in the eye and elsewhere. Many patients with XFS progress to exfoliation glaucoma (XFG), a significant cause of global blindness. We used quantitative mass spectrometry to analyze the composition of XFM in lens capsule specimens and in aqueous humor (AH) samples from patients with XFS, patients with XFG and unaffected individuals. Methods Pieces of lens capsule and samples of AH were obtained with consent from patients undergoing cataract surgery. Tryptic digests of capsule or AH were analyzed by high-performance liquid chromatography–mass spectrometry and relative differences between samples were quantified using the tandem mass tag technique. The distribution of XFM on the capsular surface was visualized by SEM and super-resolution light microscopy. Results A small set of proteins was consistently upregulated in capsule samples from patients with XFS and patients with XFG, including microfibril components fibrillin-1, latent transforming growth factor-β–binding protein-2 and latent transforming growth factor-β–binding protein-3. Lysyl oxidase-like 1, a cross-linking enzyme associated with XFS in genetic studies, was an abundant XFM constituent. Ligands of the transforming growth factor-β superfamily were prominent, including LEFTY2, a protein best known for its role in establishing the embryonic body axis. Elevated levels of LEFTY2 were also detected in AH from patients with XFG, a finding confirmed subsequently by ELISA. Conclusions This analysis verified the presence of suspected XFM proteins and identified novel components. Quantitative comparisons between patient samples revealed a consistent XFM proteome characterized by strong expression of fibrillin-1, lysyl oxidase-like-1, and LEFTY2. Elevated levels of LEFTY2 in the AH of patients with XFG may serve as a biomarker for the disease.
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Affiliation(s)
- Alicia De Maria
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Keith D Zientek
- Proteomics Shared Resource, Oregon Health and Science University, Portland, Oregon, United States
| | - Larry L David
- Department of Chemical Physiology & Biochemistry, Oregon Health and Science University, Portland, Oregon, United States
| | - Phillip A Wilmarth
- Proteomics Shared Resource, Oregon Health and Science University, Portland, Oregon, United States
| | - Anjali M Bhorade
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States
| | - George J Harocopos
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States.,Department of Pathology & Immunology, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Andrew J W Huang
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Augustine R Hong
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Carla J Siegfried
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Linda M Tsai
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Arsham Sheybani
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States
| | - Steven Bassnett
- Department of Ophthalmology & Visual Sciences, Washington University School of Medicine, St. Louis, Missouri, United States
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9
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Proteomics of pseudoexfoliation materials in the anterior eye segment. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2021; 127:271-290. [PMID: 34340770 DOI: 10.1016/bs.apcsb.2021.03.004] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/21/2022]
Abstract
Pseudoexfoliation syndrome (PEX) is characterized by the production of white extracellular fluffy clumps of microfibrillar material that aggregates in various organs throughout the body but is known to cause disease in the eye. The accumulation of PEX material (PEXM) in the anterior segment ocular structures is believed to cause an increase in intraocular pressure (IOP) resulting in pseudoexfoliation glaucoma (PEXG). The onset of PEXG is often bilateral but asymmetric-one eye often presents with glaucoma prior to the other eye. Proteomics has been used to identify key proteins involved in PEXM formation with the end goal of developing effective treatments for PEX and PEXG which may act through inhibiting the formation of the PEX aggregates. To date, a variety of proteins with various molecular functions have been identified from extracted anterior segment structures and fluids, such as aqueous humor (AH) and blood serum of patients affected by PEX. From past studies, some proteins identified in AH, lens capsule epithelium, iris tissue, and blood serum samples include vitamin D binding protein (GC), apolipoprotein A4 (APOA4), lysyl oxidase like-1 (LOXL1), complement C3, beta-crystalline B1, and B2, and antithrombin-III (SERPINC1). Each of these proteins have been observed in eyes with PEX at varying levels within the different eye structures. In this review, we further examine the anterior segment ocular proteomics of PEXM from past studies to better understand the mechanism of PEX and PEXG development. Both genetic and environmental risk factors have been implicated to be involved in the development of PEX and PEXG. This field is at an early stage of investigation identifying how these factors modify proteins both at the expression and functional level to cause changes leading to the pathophysiology of PEX glaucoma.
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10
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Greene AG, Eivers SB, McDonnell F, Dervan EWJ, O'Brien CJ, Wallace DM. Differential Lysyl oxidase like 1 expression in pseudoexfoliation glaucoma is orchestrated via DNA methylation. Exp Eye Res 2020; 201:108349. [PMID: 33188817 DOI: 10.1016/j.exer.2020.108349] [Citation(s) in RCA: 9] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2020] [Revised: 10/14/2020] [Accepted: 11/03/2020] [Indexed: 10/23/2022]
Abstract
Pseudoexfoliation syndrome (PXF) is the most common cause of secondary open angle glaucoma worldwide. Single nucleotide polymorphisms (SNPs) in the gene Lysyl oxidase like 1 (LOXL1) are strongly associated with the development of pseudoexfoliation glaucoma (PXFG). However, these SNPs are also present in 50-80% of the general population, suggestive of other factors being involved in the pathogenesis of PXFG. In this study, we aimed to investigate the influence of epigenetic regulation, specifically DNA methylation, on LOXL1 expression in PXFG using human tenons fibroblasts (HTFs), aqueous humour and serum samples from donors with and without PXFG. LOXL1 expression in HTFs was measured by qPCR and Western Blotting and LOXL1 concentration in aqueous humour was determined by ELISA. Global DNA methylation levels were quantified using an ELISA for 5-methylcytosine. MeDIP assays assessed the methylation status of the LOXL1 promoter region. Expression of methylation-associated enzymes (DNMT1, DNMT3a and MeCP2) were determined by qPCR and inhibited by 0.3 μM 5-azacytidine (5-aza). Results showed that LOXL1 expression was significantly decreased in PXFG HTFs compared with Control HTFs at gene (Fold change 0.37 ± 0.05, P < 0.01) level and showed a decrease, when measured at the protein level (Fold change 0.65 ± 0.42, P = 0.22), however this was not found to be significant. LOXL1 concentration was increased in the aqueous of PXFG patients compared with Controls (2.76 ± 0.78 vs. 1.79 ± 0.33 ng/ml, P < 0.01). Increased global methylation (56.07% ± 4.87% vs. 32.39% ± 4.29%, P < 0.01) was observed in PXFG HTFs compared with Control HTFs, as was expression of methylation-associated enzymes (DNMT1 1.58 ± 0.30, P < 0.05, DNMT3a 1.89 ± 0.24, P < 0.05, MeCP2 1.63 ± 0.30, P < 0.01). Methylation-associated enzymes were also increased when measured at protein level (DNMT1 5.70 ± 2.64, P = 0.04, DNMT3a 1.79 ± 1.55, P = 0.42, MeCP2 1.64 ± 1.33, P = 0.45). LOXL1 promoter methylation was increased in patients with PXFG compared to Control patients in both blood (3.98 ± 2.24, 2.10 ± 1.29, P < 0.05) and HTF cells (37.31 ± 22.0, 8.66 ± 10.40, P < 0.01). Treatment of PXFG HTFs with in 5-azacytidine increased LOXL1 expression when compared with untreated PXFG HTFs (Fold change 2.26 ± 0.67, P < 0.05). These data demonstrate that LOXL1 expression is altered in PXFG via DNA methylation and that reversal of these epigenetic changes may represent future potential therapeutic targets in the management of PXFG.
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Affiliation(s)
- Alison G Greene
- Clinical Research Centre, School of Medicine, University College Dublin, Ireland
| | - Sarah B Eivers
- Clinical Research Centre, School of Medicine, University College Dublin, Ireland
| | - Fiona McDonnell
- Clinical Research Centre, School of Medicine, University College Dublin, Ireland
| | - Edward W J Dervan
- Department of Ophthalmology, Mater Misericordiae University Hospital, Eccles Street, Dublin 7, Ireland
| | - Colm J O'Brien
- Clinical Research Centre, School of Medicine, University College Dublin, Ireland; Department of Ophthalmology, Mater Misericordiae University Hospital, Eccles Street, Dublin 7, Ireland
| | - Deborah M Wallace
- Clinical Research Centre, School of Medicine, University College Dublin, Ireland.
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11
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Boughton BA, Thomas ORB, Demarais NJ, Trede D, Swearer SE, Grey AC. Detection of small molecule concentration gradients in ocular tissues and humours. JOURNAL OF MASS SPECTROMETRY : JMS 2020; 55:e4460. [PMID: 31654531 DOI: 10.1002/jms.4460] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 07/15/2019] [Revised: 10/02/2019] [Accepted: 10/17/2019] [Indexed: 06/10/2023]
Abstract
The eye is an elegant organ consisting of a number of tissues and fluids with specialised functions that together allow it to effectively transmit and transduce light input to the brain for visual perception. One key determinant of this integrated function is the spatial relationship of ocular tissues. Biomolecular distributions within the main ocular tissues cornea, lens, and retina have been studied extensively in isolation, yet the potential for metabolic communication between ocular tissues via the ocular humours has been difficult to visualise. To address this limitation, the current study presents a method to map spatial distributions of metabolites and small molecules in whole eyes, including ocular humours. Using a tape-transfer system and freeze-drying, the spatial distribution of ocular small molecules was investigated in mouse, rat, fish (black bream), and rabbit eyes using negative ion mode MALDI imaging mass spectrometry. Full-scan imaging was used for discovery experiments, while MS/MS imaging for identification and localisation was also demonstrated. In all eyes, metabolites such as glutathione and phospholipids were localised in the main ocular tissues. In addition, in rodent eyes, major metabolites were distributed relatively uniformly in ocular humours. In contrast, both uniform and spatially defined ocular metabolite distributions were observed in the black bream eye. Tissue and ocular humour distributions were reproducible, as demonstrated by the three-dimensional analysis of a mouse eye, and able to be captured with high spatial resolution analysis. The presented method could be used to further investigate the role of inter-tissue metabolism in ocular health, and to support the development of therapeutics to treat major ocular diseases.
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Affiliation(s)
- Berin A Boughton
- Metabolomics Australia, University of Melbourne, Melbourne, Australia
| | - Oliver R B Thomas
- School of BioSciences, University of Melbourne, Melbourne, Australia
| | - Nicholas J Demarais
- School of Biological Sciences, University of Auckland, Auckland, New Zealand
| | | | - Stephen E Swearer
- School of BioSciences, University of Melbourne, Melbourne, Australia
| | - Angus C Grey
- School of Medical Sciences, University of Auckland, Auckland, New Zealand
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12
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Greene AG, Eivers SB, Dervan EWJ, O'Brien CJ, Wallace DM. Lysyl Oxidase Like 1: Biological roles and regulation. Exp Eye Res 2020; 193:107975. [PMID: 32070696 DOI: 10.1016/j.exer.2020.107975] [Citation(s) in RCA: 31] [Impact Index Per Article: 6.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/25/2019] [Revised: 01/12/2020] [Accepted: 02/13/2020] [Indexed: 12/11/2022]
Abstract
Lysyl Oxidase Like 1 (LOXL1) is a gene that encodes for the LOXL1 enzyme. This enzyme is required for elastin biogenesis and collagen cross-linking, polymerising tropoelastin monomers into elastin polymers. Its main role is in elastin homeostasis and matrix remodelling during injury, fibrosis and cancer development. Because of its vast range of biological functions, abnormalities in LOXL1 underlie many disease processes. Decreased LOXL1 expression is observed in disorders of elastin such as Cutis Laxa and increased expression is reported in fibrotic disease such as Idiopathic Pulmonary Fibrosis. LOXL1 is also downregulated in the lamina cribrosa in pseudoexfoliation glaucoma and genetic variants in the LOXL1 gene have been linked with an increased risk of developing pseudoexfoliation glaucoma and pseudoexfoliation syndrome. However the two major risk alleles are reversed in certain ethnic groups and are present in a large proportion of the normal population, implying complex genetic and environmental regulation is involved in disease pathogenesis. It also appears that the non-coding variants in intron 1 of LOXL1 may be involved in the regulation of LOXL1 expression. Gene alteration may occur via a number of epigenetic and post translational mechanisms such as DNA methylation, long non-coding RNAs and microRNAs. These may represent future therapeutic targets for disease. Environmental factors such as hypoxia, oxidative stress and ultraviolet radiation exposure alter LOXL1 expression, and it is likely a combination of these genetic and environmental factors that influence disease development and progression. In this review, we discuss LOXL1 properties, biological roles and regulation in detail with a focus on pseudoexfoliation syndrome and glaucoma.
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Affiliation(s)
- Alison G Greene
- UCD Clinical Research Centre, School of Medicine, University College Dublin, Ireland.
| | - Sarah B Eivers
- UCD Clinical Research Centre, School of Medicine, University College Dublin, Ireland
| | - Edward W J Dervan
- Dept. of Ophthalmology, Mater Misericordiae University Hospital, Eccles Street, Dublin 7, Ireland
| | - Colm J O'Brien
- UCD Clinical Research Centre, School of Medicine, University College Dublin, Ireland; Dept. of Ophthalmology, Mater Misericordiae University Hospital, Eccles Street, Dublin 7, Ireland
| | - Deborah M Wallace
- UCD Clinical Research Centre, School of Medicine, University College Dublin, Ireland
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Bernstein AM, Ritch R, Wolosin JM. LOXL1 folding in exfoliation glaucoma. ADVANCES IN PROTEIN CHEMISTRY AND STRUCTURAL BIOLOGY 2019; 118:273-288. [PMID: 31928728 PMCID: PMC7589528 DOI: 10.1016/bs.apcsb.2019.09.005] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Exfoliation syndrome (XFS) is an age-related disease defined by the deposition of aggregated fibrous material (XFM) in the peri-cellular space. Principal morbidity occurs in the eye, where XFM accumulates on the anterior ocular tissues. GWAS have found that certain genetic variants of lysyl oxidase-like 1 (LOXL1), a matrix cross-linking enzyme that is required for elastic fiber formation confer risk for the development of XFS, but are not a single causative factor as many genetically affected individuals do not develop XFS or subsequent glaucoma (XFG). We have found that XFG cells display defects in lysosomes, microtubules, autophagy, and mitochondria resembling defects found in cells from age-related syndromes, such as the main neurodegenerative diseases. In the majority of these diseases, the determining cellular factor is a protein containing intrinsically disordered regions (IDRs) and displaying a high propensity for aggregation. We have found that in XFG patient-derived cells, LOXL1 protein is actively subjected to autophagic clearance, suggesting that LOXL1 is undergoing aggregation. In silico analysis demonstrates that LOXL1's first 369 aa constitute an IDR with the highest disorder probability peak centering around the known risk positions. Experimentally, we have found over-expression of either unmodified LOXL1 or fluorescent chimeras preserving the well-structured N-terminus cause copious intracellular aggregation and that aggregation wanes when the high IDR peaks are deleted. Overall, our work suggests that XFS/G results from the aggregation of the LOXL1 protein coupled with a reduction of cellular proteostasis capabilities in aging, resulting in a chronic build-up of LOXL1-containing protein aggregates.
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Affiliation(s)
- Audrey M. Bernstein
- SUNY Upstate Medical University, Department of Ophthalmology and Visual Sciences, Syracuse, NY, United States
| | - Robert Ritch
- Einhorn Clinical Research Center, New York Eye and Ear Infirmary of Mount Sinai, New York, NY, United States
| | - J. Mario Wolosin
- Eye and Vision Research Institute, Department of Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY, United States
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Schlötzer-Schrehardt U, Zenkel M. The role of lysyl oxidase-like 1 (LOXL1) in exfoliation syndrome and glaucoma. Exp Eye Res 2019; 189:107818. [PMID: 31563608 DOI: 10.1016/j.exer.2019.107818] [Citation(s) in RCA: 27] [Impact Index Per Article: 4.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/26/2019] [Revised: 09/23/2019] [Accepted: 09/25/2019] [Indexed: 12/27/2022]
Abstract
Exfoliation syndrome (XFS) is an age-related systemic disease that affects the extracellular matrix. It increases the risk of glaucoma (exfoliation glaucoma, XFG) and susceptibility to diseases of elastin-rich connective tissues. LOXL1 (lysyl oxidase-like 1) is still recognized as the major genetic effect locus in XFS and XFG in all populations worldwide, although its genetic architecture is incompletely understood. LOXL1 is a key cross-linking enzyme in elastic fiber formation and remodeling, which is compatible with the pathogenetic concept of XFS as a specific type of elastosis. This review provides an overview on the current knowledge about the role of LOXL1 in the etiology and pathophysiology of XFS and XFG. It covers the known genetic associations at the LOXL1 locus, potential mechanisms of gene regulation, implications of LOXL1 in XFS-associated fibrosis and connective tissue homeostasis, its role in the development of glaucoma and associated systemic diseases, and the currently available LOXL1-based in vivo and in vitro models. Finally, it also identifies gaps in knowledge and suggests potential areas for future research.
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Affiliation(s)
| | - Matthias Zenkel
- Department of Ophthalmology, University of Erlangen-Nürnberg, Schwabachanlage 6, 91054, Erlangen, Germany
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Han J, Permentier H, Bischoff R, Groothuis G, Casini A, Horvatovich P. Imaging of protein distribution in tissues using mass spectrometry: An interdisciplinary challenge. Trends Analyt Chem 2019. [DOI: 10.1016/j.trac.2018.12.016] [Citation(s) in RCA: 22] [Impact Index Per Article: 3.7] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022]
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Abstract
Exfoliation syndrome (XFS) produces deleterious ocular aging and has protean systemic manifestations. Local ocular production of TGFβ1 is of central importance in XFS. TGFβ1 appears to induce the expression of LOXL1 and the production of other extracellular matrix components which are known to be present in exfoliation material. Furthermore, results from several studies find that the aqueous humor of exfoliation glaucoma patients exhibits a decreased antioxidant defense and increased oxidative stress systems. Finally, studies show that the levels of interleukin-6 and interleukin-8 in the aqueous humor of XFS patients were 3-fold higher than in controls. Overall TGFβ1, as well as a prooxidative and proinflammatory environment seems to play an important role in XFS.
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Affiliation(s)
- Teresa Borrás
- Department of Ophthalmology, University of North Carolina School of Medicine, Chapel Hill, NC
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Salamanca D, Gómez-Chaparro JL, Hidalgo A, Labella F. Differential expression of proteome in aqueous humor in patients with and without glaucoma. ARCHIVOS DE LA SOCIEDAD ESPANOLA DE OFTALMOLOGIA 2018; 93:160-168. [PMID: 29439810 DOI: 10.1016/j.oftal.2017.12.019] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/28/2017] [Revised: 11/20/2017] [Accepted: 12/06/2017] [Indexed: 06/08/2023]
Abstract
OBJECTIVE To determine quantitative and qualitative differences of aqueous humor proteome in patients with and without glaucoma. METHOD Observational, descriptive and cross-sectional study of 12 patients (8 men; 4 women) with and without glaucoma. There are 3 groups of minority proteins with serum equimolar contribution of each of the patients. Specimens were obtained during cataract surgery from patients without glaucoma (performed with retrobulbar anaesthesia [cataract retrobulbar patient -CRP-;n=4] or topical [cataract topical patient -CTP-; n=4]), or from patients with glaucoma (performed with retrobulbar anaesthesia [glaucoma retrobulbar patient -GRP-; n=4]). The humor proteome samples were frozen at -80°C until processing by trypsin digestion to obtain tryptic peptides, and then performing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) to obtain the proteome and its differential expression between groups. Statistical analysis was performed using the SPSS v.17 program. RESULTS The study included 12 patients, aged (mean±standard deviation) 74.50±9.53 years. Concentrations obtained: 0.48±0.25μg/μl for CRP, 0.28±0.04μg/μl for CTP, and 0.35±0.16μg/μl for GRP. A total of 309 proteins were identified, of which 205, 210, and 182 were in CRP, CTP, and GRP, respectively. A total of 114 proteins were common to all three groups, 50 were exclusive to CRP, 58 to CTP, and 27 to GRP. CONCLUSIONS In this pilot study, a quantitative difference was found in the protein expression of humor among patients with glaucoma, there being 27 proteins unique to patients with glaucomatous disease.
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Affiliation(s)
- D Salamanca
- Universidad de Córdoba (UCO), Córdoba, España.
| | - J L Gómez-Chaparro
- Distrito Córdoba-Guadalquivir (SAS), Córdoba, España; Instituto de Investigación Biomédica de Córdoba (IMIBIC), Córdoba, España
| | - A Hidalgo
- Departamento de Neuro-Oftalmología, Uveítis, Inmunología y Glaucoma, Hospital La Arruzafa, Córdoba, España
| | - F Labella
- Universidad de Córdoba (UCO), Córdoba, España
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Abstract
In this short report we review previous work toward the identification of the protein and cellular sources of exfoliation glaucoma and described our recent finding on dysfunction of autophagy in Tenon capsule fibroblasts obtained from exfoliation syndrome glaucoma patients at the time of surgery and discuss the potential implications of these findings for understanding the cellular sources of the disease.
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Affiliation(s)
| | - Robert Ritch
- Einhorn Clinical Research Center, New York Eye and Ear Infirmary of Mount Sinai, New York, NY
| | - Audrey M Bernstein
- Departments of Ophthalmology
- Pharmacology and Systems Therapeutics, Icahn School of Medicine at Mount Sinai
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Garweg JG, Zandi S, Gerhardt C, Pfister IB. Isoforms of TGF-β in the aqueous humor of patients with pseudoexfoliation syndrome and a possible association with the long-term stability of the capsular bag after cataract surgery. Graefes Arch Clin Exp Ophthalmol 2017; 255:1763-1769. [PMID: 28660443 DOI: 10.1007/s00417-017-3724-8] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2017] [Revised: 05/26/2017] [Accepted: 06/13/2017] [Indexed: 01/18/2023] Open
Abstract
BACKGROUND Pseudoexfoliation syndrome (PEXS) may go along with capsular bag shrinkage and luxation. In the present study, we focus on an association of isoforms of TGF-β with capsular bag luxation. METHODS Aqueous humor was collected intraoperatively from 20 healthy controls and from 73 otherwise healthy patients with PEXS [PEXS without complications (PEX, n = 33), late PEXS with glaucoma (PEXG, n = 30) and with IOL and capsular bag luxation (PEXL, n = 10)]. The concentrations of TGF-β1, TGF-β2 and TGF-β3 were compared using the Bio-Plex® multiplex beads system based on the non-parametric Kruskal-Wallis H test (p < 0.01). RESULTS Concentrations of TGF-β 1, TGF-β 2 and TGF-β 3 were higher in the sub-groups PEX and PEXG than in controls (TGF-β 1; p = 0.009 and 0.0005; TGF-β 2; p = 0.002 and 0.005 and TGF-β 3; 0.0005 and 0.0005; respectively), whereas for TGF β2, no significant difference between controls and PEXL was revealed (p = 1.0). TGF-β2 concentrations were elevated in a similar degree in early PEX and PEXG, but not in PEXL compared to controls (p = 0.002). The concentrations of of TGF-β 1 and TGF-β 3 increased in parallel with the progression of disease. The levels of TGF-β 3, however, did not attain pathophysiological levels (>100 pg/ml) in any group. CONCLUSIONS A stage-dependent increase in the concentrations of TGF-β1 and TGF-β3, but not of TGF-β2, accords to the shrinkage of the capsular bag. This could increase the tension on the zonular fibers and contribute to luxation of the capsular bag.
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Affiliation(s)
- Justus G Garweg
- Swiss Eye Institute and Berner Augenklinik am Lindenhofspital, Bremgartenstrasse 119, CH-3012, Bern, Switzerland. .,University of Bern, Bern, Switzerland.
| | - Souska Zandi
- Swiss Eye Institute and Berner Augenklinik am Lindenhofspital, Bremgartenstrasse 119, CH-3012, Bern, Switzerland
| | - Christin Gerhardt
- Swiss Eye Institute and Berner Augenklinik am Lindenhofspital, Bremgartenstrasse 119, CH-3012, Bern, Switzerland.,University of Bern, Bern, Switzerland
| | - Isabel B Pfister
- Swiss Eye Institute and Berner Augenklinik am Lindenhofspital, Bremgartenstrasse 119, CH-3012, Bern, Switzerland.,University of Bern, Bern, Switzerland
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20
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Aboobakar IF, Johnson WM, Stamer WD, Hauser MA, Allingham RR. Major review: Exfoliation syndrome; advances in disease genetics, molecular biology, and epidemiology. Exp Eye Res 2016; 154:88-103. [PMID: 27845061 DOI: 10.1016/j.exer.2016.11.011] [Citation(s) in RCA: 97] [Impact Index Per Article: 10.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/04/2016] [Revised: 10/06/2016] [Accepted: 11/10/2016] [Indexed: 12/20/2022]
Abstract
Exfoliation syndrome (XFS) is a common age-related disorder that leads to deposition of extracellular fibrillar material throughout the body. The most recognized disease manifestation is exfoliation glaucoma (XFG), which is a common cause of blindness worldwide. Recent developments in XFS genetics, cell biology and epidemiology have greatly improved our understanding of the etiology of this complex inherited disease. This review summarizes current knowledge of XFS pathogenesis, identifies gaps in knowledge, and discusses areas for future research.
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Affiliation(s)
- Inas F Aboobakar
- Department of Ophthalmology, Duke University Medical Center, Durham, NC, USA
| | - William M Johnson
- Department of Ophthalmology, Duke University Medical Center, Durham, NC, USA
| | - W Daniel Stamer
- Department of Ophthalmology, Duke University Medical Center, Durham, NC, USA
| | - Michael A Hauser
- Department of Ophthalmology, Duke University Medical Center, Durham, NC, USA
| | - R Rand Allingham
- Department of Ophthalmology, Duke University Medical Center, Durham, NC, USA.
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21
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Ghaste M, Mistrik R, Shulaev V. Applications of Fourier Transform Ion Cyclotron Resonance (FT-ICR) and Orbitrap Based High Resolution Mass Spectrometry in Metabolomics and Lipidomics. Int J Mol Sci 2016; 17:ijms17060816. [PMID: 27231903 PMCID: PMC4926350 DOI: 10.3390/ijms17060816] [Citation(s) in RCA: 92] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/01/2016] [Revised: 05/14/2016] [Accepted: 05/17/2016] [Indexed: 02/02/2023] Open
Abstract
Metabolomics, along with other "omics" approaches, is rapidly becoming one of the major approaches aimed at understanding the organization and dynamics of metabolic networks. Mass spectrometry is often a technique of choice for metabolomics studies due to its high sensitivity, reproducibility and wide dynamic range. High resolution mass spectrometry (HRMS) is a widely practiced technique in analytical and bioanalytical sciences. It offers exceptionally high resolution and the highest degree of structural confirmation. Many metabolomics studies have been conducted using HRMS over the past decade. In this review, we will explore the latest developments in Fourier transform mass spectrometry (FTMS) and Orbitrap based metabolomics technology, its advantages and drawbacks for using in metabolomics and lipidomics studies, and development of novel approaches for processing HRMS data.
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Affiliation(s)
- Manoj Ghaste
- Department of Biological Sciences, College of Arts and Sciences, University of North Texas, Denton, TX 76203, USA.
| | | | - Vladimir Shulaev
- Department of Biological Sciences, College of Arts and Sciences, University of North Texas, Denton, TX 76203, USA.
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22
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Biological effect of LOXL1 coding variants associated with pseudoexfoliation syndrome. Exp Eye Res 2016; 146:212-223. [PMID: 26997634 DOI: 10.1016/j.exer.2016.03.013] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2015] [Revised: 03/01/2016] [Accepted: 03/13/2016] [Indexed: 01/08/2023]
Abstract
Pseudoexfoliation (PEX) syndrome is a systemic disease involving the extracellular matrix. It increases the risk of glaucoma, an irreversible cause of blindness, and susceptibility to heart disease, stroke and hearing loss. Single nucleotide polymorphisms (SNPs) in the LOXL1 (Lysyl oxidase-like 1) gene are the major known genetic risk factor for PEX syndrome. Two coding SNPs, rs1048861 (G > T; Arg141Leu) and rs3825942 (G > A; Gly153Asp), in the LOXL1 gene are strongly associated with the disease risk in multiple populations worldwide. In the present study, we investigated functional effects of these SNPs on the LOXL1 protein. We show through molecular modelling that positions 141 and 153 are likely surface residues and hence possible recognition sites for protein-protein interactions; the Arg141Leu and Gly153Asp substitutions cause charge changes that would lead to local differences in protein electrostatic potential and in turn the potential to modify protein-protein interactions. In RFL-6 rat fetal lung fibroblast cells ectopically expressing the LOXL1 protein variants related to PEX (Arg141_Gly153, Arg141_Asp153 or Leu141_Gly153), immunoprecipitation of the secreted variants showed differences in their processing by endogenous proteins, possibly Bone morphogenetic protein-1 (BMP-1) that cleaves and leads to enzymatic activation of LOXL1. Immunofluorescence labelling of the ectopically expressed protein variants in RFL-6 cells showed no significant difference in their extracellular accumulation tendency. In conclusion, this is the first report of a biological effect of the coding SNPs in the LOXL1 gene associated with PEX syndrome, on the LOXL1 protein. The findings indicate that the disease associated coding variants themselves may be involved in the manifestation of PEX syndrome.
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Bowrey HE, Anderson DM, Pallitto P, Gutierrez DB, Fan J, Crouch RK, Schey KL, Ablonczy Z. Imaging mass spectrometry of the visual system: Advancing the molecular understanding of retina degenerations. Proteomics Clin Appl 2016; 10:391-402. [PMID: 26586164 DOI: 10.1002/prca.201500103] [Citation(s) in RCA: 18] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/15/2015] [Revised: 08/15/2015] [Accepted: 11/11/2015] [Indexed: 11/08/2022]
Abstract
Visual sensation is fundamental for quality of life, and loss of vision to retinal degeneration is a debilitating condition. The eye is the only part of the central nervous system that can be noninvasively observed with optical imaging. In the clinics, various spectroscopic methods provide high spatial resolution images of the fundus and the developing degenerative lesions. However, the currently utilized tools are not specific enough to establish the molecular underpinnings of retinal diseases. In contrast, mass spectrometric imaging (MSI) is a powerful tool to identify molecularly specific disease indicators and classification markers. This technique is particularly well suited to the eye, where molecular information can be correlated with clinical data collected via noninvasive diagnostic imaging modalities. Recent studies during the last few recent years have uncovered a plethora of new spatially defined molecular information on several vision-threatening diseases, including age-related macular degeneration, Stargardt disease, glaucoma, cataract, as well as lipid disorders. Even though MS inside the eye cannot be performed noninvasively, by linking diagnostic and molecular information, these studies are the first step toward the development of smart ophthalmic diagnostic and surgical tools. Here, we provide an overview of current approaches applying MSI technology to ocular pathology.
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Affiliation(s)
- Hannah E Bowrey
- Brain Health Institute, Rutgers University, New Brunswick, NJ, USA
| | - David M Anderson
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Patrick Pallitto
- Robert Wood Johnson Medical School, Rutgers University, New Brunswick, NJ, USA
| | - Danielle B Gutierrez
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Jie Fan
- Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA
| | - Rosalie K Crouch
- Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA
| | - Kevin L Schey
- Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA
| | - Zsolt Ablonczy
- Department of Ophthalmology, Medical University of South Carolina, Charleston, SC, USA
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Anderson DMG, Spraggins JM, Rose KL, Schey KL. High spatial resolution imaging mass spectrometry of human optic nerve lipids and proteins. JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY 2015; 26:940-7. [PMID: 25893273 PMCID: PMC5650057 DOI: 10.1007/s13361-015-1143-9] [Citation(s) in RCA: 27] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 02/03/2015] [Revised: 03/17/2015] [Accepted: 03/17/2015] [Indexed: 05/11/2023]
Abstract
The human optic nerve carries signals from the retina to the visual cortex of the brain. Each optic nerve is comprised of approximately one million nerve fibers that are organized into bundles of 800-1200 fibers surrounded by connective tissue and supportive glial cells. Damage to the optic nerve contributes to a number of blinding diseases including: glaucoma, neuromyelitis optica, optic neuritis, and neurofibromatosis; however, the molecular mechanisms of optic nerve damage and death are incompletely understood. Herein we present high spatial resolution MALDI imaging mass spectrometry (IMS) analysis of lipids and proteins to define the molecular anatomy of the human optic nerve. The localization of a number of lipids was observed in discrete anatomical regions corresponding to myelinated and unmyelinated nerve regions as well as to supporting connective tissue, glial cells, and blood vessels. A protein fragment from vimentin, a known intermediate filament marker for astrocytes, was observed surrounding nerved fiber bundles in the lamina cribrosa region. S100B was also found in supporting glial cell regions in the prelaminar region, and the hemoglobin alpha subunit was observed in blood vessel areas. The molecular anatomy of the optic nerve defined by MALDI IMS provides a firm foundation to study biochemical changes in blinding human diseases.
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Affiliation(s)
- David M G Anderson
- Department of Biochemistry, Mass Spectrometry Research Center, Vanderbilt University School of Medicine, Nashville, TN, 37232, USA
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Update on pseudoexfoliation syndrome pathogenesis and associations with intraocular pressure, glaucoma and systemic diseases. Curr Opin Ophthalmol 2015; 26:82-9. [PMID: 25594764 DOI: 10.1097/icu.0000000000000132] [Citation(s) in RCA: 54] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
Abstract
PURPOSE OF REVIEW Pseudoexfoliation (PEX) syndrome is a common age-related disorder affecting intraocular and extraocular tissues. This review focuses on recent publications related with the pathogenesis and associations of PEX syndrome with intraocular pressure (IOP), glaucoma and systemic diseases. RECENT FINDINGS In PEX tissues, expression of lysyl oxidase-like 1 (LOXL1) was found to be markedly dysregulated. This may adversely affect elastin metabolism and lead to elastotic alteration in tissues such as lamina cribrosa. There is increasing evidence that cellular stress conditions and low-grade chronic inflammatory processes are involved in the pathogenesis of PEX. Although there is an increased risk for glaucoma development in patients with PEX and ocular hypertension as compared with non-PEX patients with ocular hypertension, LOXL1 single nucleotide polymorphisms were not associated with intraocular pressure (IOP) differences. Lack of association of PEX with all-cause mortality or dementia has been reported recently. The association with vascular diseases is not consistent among different studies. SUMMARY Despite the high prevalence of the LOXL1 variants in the general population, a much lower proportion of the population develops PEX, suggesting that in addition to LOXL1, other genetic, epigenetic and environmental factors may contribute to the development of PEX. Also, LOXL1 cannot help to identify those with PEX at increased risk for glaucoma development. Increased risk for glaucoma development in PEX patients who present with increased IOP may be related to other factors beyond IOP, contributing to increased vulnerability of the optic nerve to glaucoma development in the presence of PEX.
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27
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Elsobky S, Crane AM, Margolis M, Carreon TA, Bhattacharya SK. Review of application of mass spectrometry for analyses of anterior eye proteome. World J Biol Chem 2014; 5:106-114. [PMID: 24921002 PMCID: PMC4050106 DOI: 10.4331/wjbc.v5.i2.106] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 11/19/2013] [Revised: 01/16/2014] [Accepted: 03/04/2014] [Indexed: 02/05/2023] Open
Abstract
Proteins have important functional roles in the body, which can be altered in disease states. The eye is a complex organ rich in proteins; in particular, the anterior eye is very sophisticated in function and is most commonly involved in ophthalmic diseases. Proteomics, the large scale study of proteins, has greatly impacted our knowledge and understanding of gene function in the post-genomic period. The most significant breakthrough in proteomics has been mass spectrometric identification of proteins, which extends analysis far beyond the mere display of proteins that classical techniques provide. Mass spectrometry functions as a “mass analyzer” which simplifies the identification and quantification of proteins extracted from biological tissue. Mass spectrometric analysis of the anterior eye proteome provides a differential display for protein comparison of normal and diseased tissue. In this article we present the key proteomic findings in the recent literature related to the cornea, aqueous humor, trabecular meshwork, iris, ciliary body and lens. Through this we identified unique proteins specific to diseases related to the anterior eye.
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28
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Cillero-Pastor B, Heeren RMA. Matrix-Assisted Laser Desorption Ionization Mass Spectrometry Imaging for Peptide and Protein Analyses: A Critical Review of On-Tissue Digestion. J Proteome Res 2013; 13:325-35. [DOI: 10.1021/pr400743a] [Citation(s) in RCA: 71] [Impact Index Per Article: 5.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/12/2022]
Affiliation(s)
- Berta Cillero-Pastor
- FOM Institute AMOLF, Biomolecular Imaging Mass Spectrometry (BIMS), AMOLF Science Park 104, 1098 XG Amsterdam, The Netherlands
| | - Ron M. A. Heeren
- FOM Institute AMOLF, Biomolecular Imaging Mass Spectrometry (BIMS), AMOLF Science Park 104, 1098 XG Amsterdam, The Netherlands
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