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Joseph R Casey - One of the best experts on this subject based on the ideXlab platform.
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defective cell adhesion function of solute transporter SLC4A11 in endothelial corneal dystrophies
Human Molecular Genetics, 2019Co-Authors: Darpan Malhotra, Martin Jung, Claudia Fechertrost, Matthew Lovatt, Gary S L Peh, Sergei Y Noskov, Jodhbir S Mehta, Richard Zimmermann, Joseph R CaseyAbstract:Corneal endothelial cell (CEnC) loss is often associated with blinding endothelial corneal dystrophies: dominantly inherited, common (5%) Fuchs endothelial corneal dystrophy (FECD) and recessive, rare congenital hereditary endothelial dystrophy (CHED). Mutations of SLC4A11, an abundant corneal solute transporter, cause CHED and some cases of FECD. The link between defective SLC4A11 solute transport function and CEnC loss is, however, unclear. Cell adhesion assays using SLC4A11-transfected HEK293 cells and primary human CEnC revealed that SLC4A11 promotes adhesion to components of Descemet's membrane (DM), the basement membrane layer to which CEnC bind. An antibody against SLC4A11 extracellular loop 3 (EL3) suppressed cell adhesion, identifying EL3 as the DM-binding site. Earlier studies showed that some SLC4A11 mutations cause FECD and CHED by impairing solute transport activity or cell surface trafficking. Without affecting these functions, FECD-causing mutations in SLC4A11-EL3 compromised cell adhesion capacity. In an energy-minimized SLC4A11-EL3 three-dimensional model, these mutations cluster and are buried within the EL3 structure. A GST fusion protein of SLC4A11-EL3 interacts with principal DM protein, COL8A2, as identified by mass spectrometry. Engineered SLC4A11-EL3-containing protein, STIC (SLC4A11-EL3 Transmembrane-GPA Integrated Chimera), promotes cell adhesion in transfected HEK293 cells and primary human CEnC, confirming the cell adhesion role of EL3. Taken together, the data suggest that SLC4A11 directly binds DM to serve as a cell adhesion molecule (CAM). These data further suggest that cell adhesion defects contribute to FECD and CHED pathology. Observations with STIC point toward a new therapeutic direction in these diseases: replacement of lost cell adhesion capacity.
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Human Corneal Expression of SLC4A11, a Gene Mutated in Endothelial Corneal Dystrophies
Scientific Reports, 2019Co-Authors: Darpan Malhotra, Sampath K Loganathan, Anthony M Chiu, Chris M. Lukowski, Joseph R CaseyAbstract:Two blinding corneal dystrophies, pediatric-onset congenital hereditary endothelial dystrophy (CHED) and some cases of late-onset Fuchs endothelial corneal dystrophy (FECD), are caused by SLC4A11 mutations. Three N-terminal SLC4A11 variants: v1, v2 and v3 are expressed in humans. We set out to determine which of these transcripts and what translated products, are present in corneal endothelium as these would be most relevant for CHED and FECD studies. Reverse transcription PCR (RT-PCR) and quantitative RT-PCR revealed only v2 and v3 mRNA in human cornea, but v2 was most abundant. Immunoblots probed with variant-specific antibodies revealed that v2 protein is about four times more abundant than v3 in human corneal endothelium. Bioinformatics and protein analysis using variant-specific antibodies revealed that second methionine in the open reading frame (M36) acts as translation initiation site on SLC4A11 v2 in human cornea. The v2 variants starting at M1 (v2-M1) and M36 (v2-M36) were indistinguishable in their cell surface trafficking and transport function (water flux). Structural homology models of v2-M36 and v3 suggest structural differences but their significance remains unclear. A combination of bioinformatics, RNA quantification and isoform-specific antibodies allows us to conclude that SLC4A11 variant 2 with start site M36 is predominant in corneal endothelium.
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ophthalmic nonsteroidal anti inflammatory drugs as a therapy for corneal dystrophies caused by SLC4A11 mutation
Investigative Ophthalmology & Visual Science, 2018Co-Authors: Kumari Alka, Joseph R CaseyAbstract:Purpose SLC4A11 is a plasma membrane protein of corneal endothelial cells. Some mutations of the SLC4A11 gene result in SLC4A11 protein misfolding and failure to mature to the plasma membrane. This gives rise to some cases of Fuchs' endothelial corneal dystrophy (FECD) and congenital hereditary endothelial dystrophy (CHED). We screened ophthalmic nonsteroidal anti-inflammatory drugs (NSAIDs) for their ability to correct SLC4A11 folding defects. Methods Five ophthalmic NSAIDs were tested for their therapeutic potential in some genetic corneal dystrophy patients. HEK293 cells expressing CHED and FECD-causing SLC4A11 mutants were grown on 96-well dishes in the absence or presence of NSAIDs. Ability of NSAIDs to correct mutant SLC4A11 cell-surface trafficking was assessed with a bioluminescence resonance energy transfer (BRET) assay and by confocal microscopy. The ability of mutant SLC4A11-expressing cells to mediate water flux (SLC4A11 mediates water flux across the corneal endothelial cell basolateral membrane as part of the endothelial water pump) was measured upon treatment with ophthalmic NSAIDs. Results BRET-assays revealed significant rescue of SLC4A11 mutants to the cell surface by 4 of 5 NSAIDs tested. The NSAIDs, diclofenac and nepafenac, were effective in moving endoplasmic reticulum-retained missense mutant SLC4A11 to the cell surface, as measured by confocal immunofluorescence. Among intracellular-retained SLC4A11 mutants, 20 of 30 had significant restoration of cell surface abundance upon treatment with diclofenac. Diclofenac restored mutant SLC4A11 water flux activity to the level of wild-type SLC4A11 in some cases. Conclusions These results encourage testing diclofenac eye drops as a treatment for corneal dystrophy in patients whose disease is caused by some SLC4A11 missense mutations.
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molecular phenotype of SLC4A11 missense mutants setting the stage for personalized medicine in corneal dystrophies
Human Mutation, 2018Co-Authors: Kumari Alka, Joseph R CaseyAbstract:SLC4A11 mutations cause cases of congenital hereditary endothelial dystrophy (CHED), Harboyan syndrome (HS), and Fuchs endothelial corneal dystrophy (FECD). Defective water reabsorption from corneal stroma by corneal endothelial cells (CECs) leads to these corneal dystrophies. SLC4A11, in the CEC basolateral membrane, facilitates transmembrane movement of H2 O, NH3 , and H+ -equivalents. Some SLC4A11 disease mutants have impaired folding, leading to a failure to move to the cell surface, which in some cases can be corrected by the drug, glafenine. To identify SLC4A11 mutants that are targets for folding-correction therapy, we examined 54 SLC4A11 missense mutants. Cell-surface trafficking was assessed on immunoblots, by the level of mature, high molecular weight, cell surface-associated form, and using a bioluminescence resonance energy transfer assay. Low level of cell surface trafficking was found in four out of 18 (20%) of FECD mutants, 19/ out of 31 (61%) of CHED mutants, and three out of five (60%) of HS mutants. Amongst ER-retained mutants, 16 showed increased plasma membrane trafficking when grown at 30°C, suggesting that their defect has potential for rescue. CHED-causing point mutations mostly resulted in folding defects, whereas the majority of FECD missense mutations did not affect trafficking, implying functional impairment. We identified mutations that make patients candidates for folding correction of their corneal dystrophy.
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SLC4A11 three dimensional homology model rationalizes corneal dystrophy causing mutations
Human Mutation, 2017Co-Authors: Katherine E Badior, Kumari Alka, Joseph R CaseyAbstract:We studied the structural effects of point mutations of a membrane protein that cause genetic disease. SLC4A11 is a membrane transport protein (OH- /H+ /NH3 /H2 O) of basolateral corneal endothelium, whose mutations cause some cases of congenital hereditary endothelial dystrophy and Fuchs endothelial corneal dystrophy. We created a three-dimensional homology model of SLC4A11 membrane domain, using Band 3 (SLC4A1) crystal structure as template. The homology model was assessed in silico and by analysis of mutants designed on the basis of the model. Catalytic pathway mutants p.Glu675Gln, p.His724Arg, and p.His724Ala impaired SLC4A11 transport. p.Ala720Leu, in a region of extended structure of the proposed translocation pore, failed to mature to the cell surface. p.Gly509Lys, located in an open region at the core domain/gate domain interface, had wild-type level of transport function. The molecular phenotype of 37 corneal dystrophy-causing point mutants was rationalized, based on their location in the homology model. Four map to the substrate translocation pathway, 25 to regions of close transmembrane helix packing, three to the dimeric interface, and five lie in extramembraneous loops. The model provides a view of the spectrum of effects of disease mutations on membrane protein structure and provides a tool to analyze pathogenicity of additional newly discovered SLC4A11 mutants.
Eranga N Vithana - One of the best experts on this subject based on the ideXlab platform.
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transmembrane water flux through SLC4A11 a route defective in genetic corneal diseases
Human Molecular Genetics, 2013Co-Authors: Gonzalo L Vilas, Sampath K Loganathan, Eranga N Vithana, Jun Liu, Jodhbir S Mehta, Andri K Riau, James D Young, Joseph R CaseyAbstract:Three genetic corneal dystrophies [congenital hereditary endothelial dystrophy type 2 (CHED2), Harboyan syndrome and Fuchs endothelial corneal dystrophy] arise from mutations of the SLC4A11 gene, which cause blindness from fluid accumulation in the corneal stroma. Selective transmembrane water conductance controls cell size, renal fluid reabsorption and cell division. All known water-channelling proteins belong to the major intrinsic protein family, exemplified by aquaporins (AQPs). Here we identified SLC4A11, a member of the solute carrier family 4 of bicarbonate transporters, as an unexpected addition to known transmembrane water movement facilitators. The rate of osmotic-gradient driven cell-swelling was monitored in Xenopus laevis oocytes and HEK293 cells, expressing human AQP1, NIP5;1 (a water channel protein from plant), hCNT3 (a human nucleoside transporter) and human SLC4A11. hCNT3-expressing cells swelled no faster than control cells, whereas SLC4A11-mediated water permeation at a rate about half that of some AQP proteins. SLC4A11-mediated water movement was: (i) similar to some AQPs in rate; (ii) uncoupled from solute-flux; (iii) inhibited by stilbene disulfonates (classical SLC4 inhibitors); (iv) inactivated in one CHED2 mutant (R125H). Localization of AQP1 and SLC4A11 in human and murine corneal (apical and basolateral, respectively) suggests a cooperative role in mediating trans-endothelial water reabsorption. SLC4A11(-/-) mice manifest corneal oedema and distorted endothelial cells, consistent with loss of a water-flux. Observed water-flux through SLC4A11 extends the repertoire of known water movement pathways and call for a re-examination of explanations for water movement in human tissues.
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SLC4A11 is an eipa sensitive na permeable phi regulator
American Journal of Physiology-cell Physiology, 2013Co-Authors: Diego G. Ogando, Eranga N Vithana, Wenlin Zhang, Supriya S Jalimarada, Joseph A. BonannoAbstract:SLC4A11, a member of the solute linked cotransporter 4 family that is comprised predominantly of bicarbonate transporters, was described as an electrogenic 2Na+-B(OH)4− (borate) cotransporter and a...
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mice with a targeted disruption of SLC4A11 model the progressive corneal changes of congenital hereditary endothelial dystrophy
Investigative Ophthalmology & Visual Science, 2013Co-Authors: Shyam S Chaurasia, Eranga N Vithana, Jodhbir S MehtaAbstract:Purpose To establish an animal model of congenital hereditary endothelial dystrophy (CHED) using SLC4A11 knockout (KO) mice and evaluate the abnormalities in the cornea and kidney. Methods The SLC4A11 KO mouse model was generated by gene deletion. Corneal abnormalities were evaluated using slit-lamp photography, anterior segment optical coherence tomography (AS-OCT), immunohistochemistry, RT-PCR, corneal endothelial cell staining, and electron microscopy. The temporal corneal changes were also monitored. Histological and functional changes of the kidney were also evaluated. Results Successful knockout of the SLC4A11 gene was confirmed by immunohistochemistry and RT-PCR. Slit-lamp photography and AS-OCT showed progressive corneal edema. Increased corneal endothelial cell size with decreased corneal endothelial cell density was observed with increased age. Scanning electron microscopy also revealed progressive cell swelling and distortion of the hexagonal cell morphology with time. Transmission electron microscopy showed characteristic ultrastructural findings of CHED, including endothelial vacuolization, thickening of the Descemet membrane, disorganization of collagen fibril, deposition of amorphous material, and progression of these changes with age. Decreased urine osmolarity and electrolyte concentrations suggesting abnormality in water resorption were also detected. Conclusions Our SLC4A11 KO mouse model successfully represents clinical manifestations of human CHED. We were able to show chronological corneal progression for the first time in a knockout mouse model as well as renal abnormalities.
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ion transport function of SLC4A11 in corneal endothelium
Investigative Ophthalmology & Visual Science, 2013Co-Authors: Supriya S Jalimarada, Eranga N Vithana, Diego G. Ogando, Joseph A. BonannoAbstract:PURPOSE Mutations in SLC4A11, a member of the SLC4 superfamily of bicarbonate transporters, give rise to corneal endothelial cell dystrophies. SLC4A11 is a putative Na⁺ borate and Na⁺:OH⁻ transporter. Therefore we ask whether SLC4A11 in corneal endothelium transports borate (B[OH]₄⁻), bicarbonate (HCO3⁻), or hydroxyl (OH⁻) anions coupled to Na⁺. METHODS SLC4A11 expression in cultured primary bovine corneal endothelial cells (BCECs) was determined by semiquantitative PCR, SDS-PAGE/Western blotting, and immunofluorescence staining. Ion transport function was examined by measuring intracellular pH (pHi) or Na⁺ ([Na⁺](i)) in response to Ringer solutions with/without B(OH)₄⁻ or HCO₃⁻ after overexpressing or small interfering RNA (siRNA) silencing of SLC4A11. RESULTS SLC4A11 is localized to the basolateral membrane in BCEC. B(OH)₄⁻ (2.5-10 mM) in bicarbonate-free Ringer induced a rapid small acidification (0.01 pH unit) followed by alkalinization (0.05-0.1 pH unit), consistent with diffusion of boric acid into the cell followed by B(OH)₄⁻. However, the rate of B(OH)₄⁻-induced pHi change was unaffected by overexpression of SLC4A11. B(OH)₄⁻ did not induce significant changes in resting [Na⁺(i)] or the amplitude and rate of acidification caused by Na⁺ removal. siRNA-mediated knockdown of SLC4A11 (∼70%) did not alter pHi responses to CO₂/HCO₃⁻-rich Ringer, Na⁺-free induced acidification, or the rate of Na⁺ influx in the presence of bicarbonate. However, in the absence of bicarbonate, siSLC4A11 knockdown significantly decreased the rate (43%) and amplitude (48%) of acidification due to Na⁺ removal and recovery (53%) upon add-back. Additionally, the rate of acid recovery following NH₄⁺ prepulse was decreased significantly (27%) by SLC4A11 silencing. CONCLUSIONS In corneal endothelium, SLC4A11 displays robust Na⁺-coupled OH⁻ transport, but does not transport B(OH)₄⁻ or HCO₃⁻.
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Differential expression of the Slc4 bicarbonate transporter family in murine corneal endothelium and cell culture.
Molecular vision, 2013Co-Authors: William Shei, Tin Aung, Jun Liu, Hla Myint Htoon, Eranga N VithanaAbstract:Purpose To characterize the relative expression levels of all the solute carrier 4 (Slc4) transporter family members (Slc4a1–SLC4A11) in murine corneal endothelium using real-time quantitative (qPCR), to identify further important members besides SLC4A11 and Slc4a4, and to explore how close to the baseline levels the gene expressions remain after cells have been subjected to expansion and culture.
Joseph A. Bonanno - One of the best experts on this subject based on the ideXlab platform.
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mitoros due to loss of SLC4A11 in corneal endothelial cells induces er stress lysosomal dysfunction and impairs autophagy
bioRxiv, 2020Co-Authors: Rajalekshmy Shyam, Diego G. Ogando, Moonjung Choi, Joseph A. BonannoAbstract:Recent studies from SLC4A11 KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in the SLC4A11 KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treated SLC4A11 KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections of SLC4A11 KO mice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy in SLC4A11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.
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ammonia sensitive SLC4A11 mitochondrial uncoupling reduces glutamine induced oxidative stress
Redox biology, 2019Co-Authors: Diego G. Ogando, Moonjung Choi, Rajalekshmy Shyam, Joseph A. BonannoAbstract:SLC4A11 is a NH3 sensitive membrane transporter with H+ channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown. Here we demonstrate that SLC4A11 is localized to the inner mitochondrial membrane of CE and SLC4A11 transfected PS120 fibroblasts, where it acts as an NH3-sensitive mitochondrial uncoupler that enhances glutamine-dependent oxygen consumption, electron transport chain activity, and ATP levels by suppressing damaging Reactive Oxygen Species (ROS) production. In the presence of glutamine, SLC4A11-/- (KO) mouse CE generate significantly greater mitochondrial superoxide, a greater proportion of damaged depolarized mitochondria, and more apoptotic cells than WT. KO CE can be rescued by MitoQ, reducing NH3 production by GLS1 inhibition or dimethyl αKetoglutarate supplementation, or by BAM15 mitochondrial uncoupling. SLC4A11 KO mouse corneal edema can be partially reversed by αKetoglutarate eye drops. Moreover, we demonstrate that this role for SLC4A11 is not specific to CE cells, as SLC4A11 knockdown in glutamine-addicted colon carcinoma cells reduced glutamine catabolism, increased ROS production, and inhibited cell proliferation. Overall, our studies reveal a unique metabolic mechanism that reduces mitochondrial oxidative stress while promoting glutamine catabolism.
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3071 Cell Survival in Corneal Endothelial Dystrophies
Journal of Clinical and Translational Science, 2019Co-Authors: Rajalekshmy Shyam, Diego G. Ogando, Moonjung Choi, Joseph A. BonannoAbstract:OBJECTIVES/SPECIFIC AIMS: Purpose - The goal of this study is to understand how loss of the membrane protein SLC4A11 alters endothelial cell metabolism thereby producing Corneal Endothelial Dystrophy. Studies from our lab indicated that glutamine-dependent mitochondrial dysfunction is one of the outcomes of SLC4A11 loss. In the current study, we ask if autophagy and mitophagy pathways and the signaling pathways that regulate these processes are altered in SLC4A11 KO cells. METHODS/STUDY POPULATION: Methods – Immortalized mouse WT and SLC4A11 KO cell lines were incubated in DMEM with and without 0.5mM glutamine for 6 hours. In order to assess mitophagy, cells were stained using Lysotracker Red and Mitotracker Green. Colocalization co-efficients of red and green channels were obtained for at least 35 cells using Zeiss-Zen Pro software. Student’s t-test was used to determine statistical significance. For Western Blots, antibodies against LC3b, AMPK, pAMPK, and b-actin were used to examine autophagy flux and potential signaling pathways that regulate autophagy. RESULTS/ANTICIPATED RESULTS: Results – In the presence of glutamine, the colocalization co-efficient of Lysotracker Red and Mitotracker Green channels was significantly increased in KO cells (0.74 ±0.18) relative to WT (0.58±0.20) with a p-value ≤0.0024. In the absence of glutamine, the colocalization co-efficient was reversed, for KO cells 0.54 ±0.14 and for WT cells 0.77±0.0.16 with a p-value ≤0.0001, suggesting increased mitophagy by glutamine in KO cells. Western Blots indicated that glutamine increased autophagy flux, as indicated by increased levels of LC3b following bafilomycin A treatment in KO cells. Concomitantly, there was an increase in pAMPK/AMPK levels suggesting a potential mechanism for increased mitophagy. DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusion and Future studies –Our data indicates enhanced mitophagy as well as autophagy in SLC4A11 KO cells. Future studies will determine whether these processes regulate cell survival in mouse models of corneal endothelial dystrophies.
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Conditionally Immortal SLC4A11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in SLC4A11-/- Mouse Model.
Investigative ophthalmology & visual science, 2017Co-Authors: Wenlin Zhang, Diego G. Ogando, Edward Kim, Moonjung Choi, Jason M. Tenessen, Joseph A. BonannoAbstract:Purpose To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched SLC4A11+/+ and SLC4A11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy. Methods We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with SLC4A11+/+ and SLC4A11-/- C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and SLC4A11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS). Results The immortalized SLC4A11+/+ and SLC4A11-/- mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. SLC4A11-/- MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the SLC4A11-/- mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with SLC4A11+/+ MCECs. Conclusions This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in SLC4A11-/- MCECs. Furthermore, SLC4A11-/- MCECs recapitulate the glutaminolysis defects observed in SLC4A11-/- mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.
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conditionally immortal SLC4A11 mouse corneal endothelial cell line recapitulates disrupted glutaminolysis seen in SLC4A11 mouse model
Investigative Ophthalmology & Visual Science, 2017Co-Authors: Wenlin Zhang, Diego G. Ogando, Edward Kim, Moonjung Choi, Jason M. Tenessen, Joseph A. BonannoAbstract:Purpose To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched SLC4A11+/+ and SLC4A11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy. Methods We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with SLC4A11+/+ and SLC4A11-/- C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and SLC4A11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS). Results The immortalized SLC4A11+/+ and SLC4A11-/- mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. SLC4A11-/- MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the SLC4A11-/- mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with SLC4A11+/+ MCECs. Conclusions This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in SLC4A11-/- MCECs. Furthermore, SLC4A11-/- MCECs recapitulate the glutaminolysis defects observed in SLC4A11-/- mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.
Diego G. Ogando - One of the best experts on this subject based on the ideXlab platform.
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mitoros due to loss of SLC4A11 in corneal endothelial cells induces er stress lysosomal dysfunction and impairs autophagy
bioRxiv, 2020Co-Authors: Rajalekshmy Shyam, Diego G. Ogando, Moonjung Choi, Joseph A. BonannoAbstract:Recent studies from SLC4A11 KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in the SLC4A11 KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treated SLC4A11 KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections of SLC4A11 KO mice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy in SLC4A11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.
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ammonia sensitive SLC4A11 mitochondrial uncoupling reduces glutamine induced oxidative stress
Redox biology, 2019Co-Authors: Diego G. Ogando, Moonjung Choi, Rajalekshmy Shyam, Joseph A. BonannoAbstract:SLC4A11 is a NH3 sensitive membrane transporter with H+ channel-like properties that facilitates Glutamine catabolism in Human and Mouse corneal endothelium (CE). Loss of SLC4A11 activity induces oxidative stress and cell death, resulting in Congenital Hereditary Endothelial Dystrophy (CHED) with corneal edema and vision loss. However, the mechanism by which SLC4A11 prevents ROS production and protects CE is unknown. Here we demonstrate that SLC4A11 is localized to the inner mitochondrial membrane of CE and SLC4A11 transfected PS120 fibroblasts, where it acts as an NH3-sensitive mitochondrial uncoupler that enhances glutamine-dependent oxygen consumption, electron transport chain activity, and ATP levels by suppressing damaging Reactive Oxygen Species (ROS) production. In the presence of glutamine, SLC4A11-/- (KO) mouse CE generate significantly greater mitochondrial superoxide, a greater proportion of damaged depolarized mitochondria, and more apoptotic cells than WT. KO CE can be rescued by MitoQ, reducing NH3 production by GLS1 inhibition or dimethyl αKetoglutarate supplementation, or by BAM15 mitochondrial uncoupling. SLC4A11 KO mouse corneal edema can be partially reversed by αKetoglutarate eye drops. Moreover, we demonstrate that this role for SLC4A11 is not specific to CE cells, as SLC4A11 knockdown in glutamine-addicted colon carcinoma cells reduced glutamine catabolism, increased ROS production, and inhibited cell proliferation. Overall, our studies reveal a unique metabolic mechanism that reduces mitochondrial oxidative stress while promoting glutamine catabolism.
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3071 Cell Survival in Corneal Endothelial Dystrophies
Journal of Clinical and Translational Science, 2019Co-Authors: Rajalekshmy Shyam, Diego G. Ogando, Moonjung Choi, Joseph A. BonannoAbstract:OBJECTIVES/SPECIFIC AIMS: Purpose - The goal of this study is to understand how loss of the membrane protein SLC4A11 alters endothelial cell metabolism thereby producing Corneal Endothelial Dystrophy. Studies from our lab indicated that glutamine-dependent mitochondrial dysfunction is one of the outcomes of SLC4A11 loss. In the current study, we ask if autophagy and mitophagy pathways and the signaling pathways that regulate these processes are altered in SLC4A11 KO cells. METHODS/STUDY POPULATION: Methods – Immortalized mouse WT and SLC4A11 KO cell lines were incubated in DMEM with and without 0.5mM glutamine for 6 hours. In order to assess mitophagy, cells were stained using Lysotracker Red and Mitotracker Green. Colocalization co-efficients of red and green channels were obtained for at least 35 cells using Zeiss-Zen Pro software. Student’s t-test was used to determine statistical significance. For Western Blots, antibodies against LC3b, AMPK, pAMPK, and b-actin were used to examine autophagy flux and potential signaling pathways that regulate autophagy. RESULTS/ANTICIPATED RESULTS: Results – In the presence of glutamine, the colocalization co-efficient of Lysotracker Red and Mitotracker Green channels was significantly increased in KO cells (0.74 ±0.18) relative to WT (0.58±0.20) with a p-value ≤0.0024. In the absence of glutamine, the colocalization co-efficient was reversed, for KO cells 0.54 ±0.14 and for WT cells 0.77±0.0.16 with a p-value ≤0.0001, suggesting increased mitophagy by glutamine in KO cells. Western Blots indicated that glutamine increased autophagy flux, as indicated by increased levels of LC3b following bafilomycin A treatment in KO cells. Concomitantly, there was an increase in pAMPK/AMPK levels suggesting a potential mechanism for increased mitophagy. DISCUSSION/SIGNIFICANCE OF IMPACT: Conclusion and Future studies –Our data indicates enhanced mitophagy as well as autophagy in SLC4A11 KO cells. Future studies will determine whether these processes regulate cell survival in mouse models of corneal endothelial dystrophies.
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Conditionally Immortal SLC4A11-/- Mouse Corneal Endothelial Cell Line Recapitulates Disrupted Glutaminolysis Seen in SLC4A11-/- Mouse Model.
Investigative ophthalmology & visual science, 2017Co-Authors: Wenlin Zhang, Diego G. Ogando, Edward Kim, Moonjung Choi, Jason M. Tenessen, Joseph A. BonannoAbstract:Purpose To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched SLC4A11+/+ and SLC4A11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy. Methods We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with SLC4A11+/+ and SLC4A11-/- C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and SLC4A11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS). Results The immortalized SLC4A11+/+ and SLC4A11-/- mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. SLC4A11-/- MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the SLC4A11-/- mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with SLC4A11+/+ MCECs. Conclusions This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in SLC4A11-/- MCECs. Furthermore, SLC4A11-/- MCECs recapitulate the glutaminolysis defects observed in SLC4A11-/- mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.
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conditionally immortal SLC4A11 mouse corneal endothelial cell line recapitulates disrupted glutaminolysis seen in SLC4A11 mouse model
Investigative Ophthalmology & Visual Science, 2017Co-Authors: Wenlin Zhang, Diego G. Ogando, Edward Kim, Moonjung Choi, Jason M. Tenessen, Joseph A. BonannoAbstract:Purpose To establish conditionally immortal mouse corneal endothelial cell lines with genetically matched SLC4A11+/+ and SLC4A11-/- mice as a model for investigating pathology and therapies for SLC4A11 associated congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy. Methods We intercrossed H-2Kb-tsA58 mice (Immortomouse) expressing an IFN-γ dependent and temperature-sensitive mutant of the SV40 large T antigen (tsTAg) with SLC4A11+/+ and SLC4A11-/- C57BL/6 mice. The growth characteristics of the cell lines was assessed by doubling time. Ion transport activities (Na+/H+ exchange, bicarbonate, lactate, and SLC4A11 ammonia transport) were analyzed by intracellular pH measurement. The metabolic status of the cell lines was assessed by analyzing TCA cycle intermediates via gas chromatography mass spectrometry (GC-MS). Results The immortalized SLC4A11+/+ and SLC4A11-/- mouse corneal endothelial cells (MCECs) remained proliferative through passage 49 and maintained similar active ion transport activity. As expected, proliferation was temperature sensitive and IFN-γ dependent. SLC4A11-/- MCECs exhibited decreased proliferative capacity, reduced NH3:H+ transport, altered expression of glutaminolysis enzymes similar to the SLC4A11-/- mouse, and reduced proportion of TCA cycle intermediates derived from glutamine with compensatory increases in glucose flux compared with SLC4A11+/+ MCECs. Conclusions This is the first report of the immortalization of MCECs. Ion transport of the immortalized endothelial cells remains active, except for NH3:H+ transporter activity in SLC4A11-/- MCECs. Furthermore, SLC4A11-/- MCECs recapitulate the glutaminolysis defects observed in SLC4A11-/- mouse corneal endothelium, providing an excellent tool to study the pathogenesis of SLC4A11 mutations associated with corneal endothelial dystrophies and to screen potential therapeutic agents.
Ira Kurtz - One of the best experts on this subject based on the ideXlab platform.
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Energy Shortage in Human and Mouse Models of SLC4A11-Associated Corneal Endothelial Dystrophies.
Investigative ophthalmology & visual science, 2020Co-Authors: Wenlin Zhang, Ira Kurtz, Ricardo Frausto, Doug D Chung, Christopher G Griffis, Liyo Kao, Angela Chen, Rustam Azimov, Alapakkam P Sampath, Anthony J. AldaveAbstract:Purpose To elucidate the molecular events in solute carrier family 4 member 11 (SLC4A11)-deficient corneal endothelium that lead to the endothelial dysfunction that characterizes the dystrophies associated with SLC4A11 mutations, congenital hereditary endothelial dystrophy (CHED) and Fuchs endothelial corneal dystrophy 4. Methods Comparative transcriptomic analysis (CTA) was performed in primary human corneal endothelial cells (pHCEnC) and murine corneal endothelial cells (MCEnC) with normal and reduced levels of SLC4A11 (SLC4A11 KD pHCEnC) and SLC4A11 (SLC4A11-/- MCEnC), respectively. Validation of differentially expressed genes was performed using immunofluorescence staining of CHED corneal endothelium, as well as western blot and quantitative PCR analysis of SLC4A11 KD pHCEnC and SLC4A11-/- MCEnC. Functional analyses were performed to investigate potential functional changes associated with the observed transcriptomic alterations. Results CTA revealed inhibition of cell metabolism and ion transport function as well as mitochondrial dysfunction, leading to reduced adenosine triphosphate (ATP) production, in SLC4A11 KD pHCEnC and SLC4A11-/- MCEnC. Co-localization of SNARE protein STX17 with mitochondria marker COX4 was observed in CHED corneal endothelium, as was activation of AMPK-p53/ULK1 in both SLC4A11 KD pHCEnC and SLC4A11-/- MCEnC, providing additional evidence of mitochondrial dysfunction and mitophagy. Reduced Na+-dependent HCO3- transport activity and altered NH4Cl-induced membrane potential changes were observed in SLC4A11-/- MCEnC. Conclusions Reduced steady-state ATP levels and subsequent activation of the AMPK-p53 pathway provide a link between the metabolic functional deficit and transcriptome alterations, as well as evidence of insufficient ATP to maintain the Na+/K+-ATPase corneal endothelial pump as the cause of the edema that characterizes SLC4A11-associated corneal endothelial dystrophies.
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SLC4A11 function evidence for h oh and nh3 h transport
American Journal of Physiology-cell Physiology, 2020Co-Authors: Liyo Kao, Rustam Azimov, Sergei Y Noskov, Natalia Abuladze, Debra K Newman, Xuesi M Shao, Hristina R Zhekova, Alexander Pushkin, Ira KurtzAbstract:Whether SLC4A11 transports ammonia and its potential mode of ammonia transport (NH4+, NH3, or NH3-2H+ transport have been proposed) are controversial. In the absence of ammonia, whether SLC4A11 med...
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energy crisis in human and mouse models of SLC4A11 associated corneal endothelial dystrophies
bioRxiv, 2019Co-Authors: Wenlin Zhang, Ira Kurtz, Ricardo Frausto, Doug D Chung, Christopher G Griffis, Liyo Kao, Angela Chen, Rustam Azimov, Alapakkam P SampathAbstract:Abstract Mutations in the solute-linked carrier family 4 member 11 (SLC4A11) gene are associated with congenital hereditary endothelial dystrophy (CHED), Fuchs endothelial corneal dystrophy and Harboyan syndrome, in all of which visually significant cornea edema may require corneal transplantation. However, the pathogenesis of SLC4A11-associated corneal endothelial dystrophies remains to be elucidated. Recent evidence suggested cellular respiration reprogramming and mitochondrial oxidative stress in SLC4A11-deficient corneal endothelium. Given the complexity of cellular metabolic regulation and its cell type specific impact on cellular physiology, we systemically analyzed the transcriptome of SLC4A11 knock-down primary human corneal endothelium (SLC4A11 KD pHCEnC) and corneal endothelial cells derived from SLC4A11-/- mice (SLC4A11-/- MCEnC) to provide a comprehensive characterization of the transcriptome profile changes resulting from loss of SLC4A11. To identify the conserved molecular mechanisms that lead to cornea endothelial dysfunction in both the human and murine models, we performed comparative transcriptomic analysis. Our analysis identified inhibition of cell metabolism and ion transport function as well as mitochondria dysfunction as shared between SLC4A11 KD pHCEnC and SLC4A11-/- MCEnC. Functional analysis confirmed the absence of SLC4A11-mediated NH4Cl-induced membrane depolarization in SLC4A11-/- MCEnC. Transcriptome of SLC4A11 KD pHCEnC and SLC4A11-/- MCEnC identified downregulation of Na+-HCO3- transporter (NBCe1, SLC4A4), a key player in corneal endothelial ‘pump’ function, and upregulation of Syntaxin 17 (STX17), an initiator of mitophagy. NBCe1 and STX17 were further analyzed in SLC4A11-/- MCEnC for functional impact and in SLC4A11 KD pHCEnC and corneal endothelium from individuals with CHED for protein expression, all showed consistent changes with transcriptome. CHED corneal endothelium also showed decreased immunostaining intensity for mitochondria markers suggesting decreased mitochondira density. In SLC4A11 KD pHCEnC and SLC4A11-/- MCEnC, steady state ATP depletion and ATP sensing AMPK-p53 pathway activation were observed, consistent with the prediction using transcriptome data that transcriptional factor p53 were responsible for the transcriptomic changes. These findings suggest that insufficient energy fueling the corneal endothelial ‘pump’, as a result of metabolic inhibition and failing mitochondria, is the direct cause of clinical phenotype of corneal edema in SLC4A11-associated corneal endothelial dystrophies.
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SLC4A11 disruption causes duct cell loss and impairs nacl reabsorption in female mouse submandibular glands
Physiological Reports, 2019Co-Authors: Ningyan Yang, Taro Mukaibo, Xin Gao, Ira Kurtz, James E MelvinAbstract:SLC4A11, a member of the Slc4 HCO3 - transporter family, has a wide tissue distribution. In mouse salivary glands, the expression of SLC4A11 mRNA was more than eightfold greater than the other nine members of the Slc4 gene family. The SLC4A11 protein displayed a diffuse subcellular distribution in both the acinar and duct cells of mouse submandibular glands (SMG). SLC4A11 disruption induced a significant increase in the Na+ and Cl- concentrations of stimulated SMG saliva, whereas it did not affect the fluid secretion rate in response to either β-adrenergic or cholinergic receptor stimulation. Heterologous expressed mouse SLC4A11 acted as a H+ /OH- transporter that was uncoupled of Na+ or Cl- movement, and this activity was blocked by ethyl-isopropyl amiloride (EIPA) but not 4,4'-Diisothiocyanato-2,2'-stilbenedisulfonic acid (DIDS). SLC4A11 disruption revealed that SLC4A11 does not play a major role in intracellular pH regulation in mouse salivary gland cells. In contrast, NaCl reabsorption was impaired in the SMG saliva of female compared to male SLC4A11 null mice, which correlated with the loss of duct cells and a decrease in expression of the duct-cell-specific transcription factor Ascl3. Together, our results suggest that SLC4A11 expression regulates the number of ducts cells in the mouse SMG and consequently NaCl reabsorption.
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Expression of SLC4A11 protein in mouse and rat medulla: a candidate transporter involved in outer medullary ammonia recycling
Wiley, 2019Co-Authors: Michael T. Gee, Ira Kurtz, Thomas L. PannabeckerAbstract:Abstract SLC4A11 is a multifunctional membrane transporter involved with H+ transport, NH3 and alkaline pH stimulated H+ transport, and water transport. The role of SLC4A11 in the kidney is not well understood. A prior study has shown that in murine kidney, SLC4A11/LacZ staining is primarily in the long‐looped descending thin limb (DTL) as determined by colocalization with aquaporin 1 (AQP1), a protein that is expressed in some, but not all, descending thin limb segments. Using a previously characterized polyclonal antibody, we demonstrate the selective expression of SLC4A11 in the upper DTLs (which are AQP1‐positive) in the outer medulla and inner medulla with little or no expression in the lower DTLs (which are AQP‐1‐null). SLC4A11 also colocalized with AQP1 and the urea transporter UT‐B in the mouse descending vasa recta, but was absent in mouse and rat ascending vasa recta. Mouse, but not rat, outer medullary collecting duct cells also labeled for SLC4A11. Our results are compatible with the hypothesis that in the inner stripe of the outer medulla, SLC4A11 plays a role in the countercurrent transport of ammonia absorbed from the outer medullary thick ascending limb and secreted into the long‐looped DTLs. SLC4A11 can potentially modulate the rate of ammonia transport in the mouse outer medullary collecting duct. Our data suggest functionally unique SLC4A11 pathways in mouse and rat and complement previous studies of DTL Na+, urea and water permeability indicating that the upper and lower DTLs of long‐looped nephrons are functionally distinct