ABSTRACT: 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.
Project description:Corneal endothelium (CE) is among the most metabolically active tissues in the body. This elevated metabolic rate helps the CE maintain corneal transparency by its ion and fluid transport properties, which when disrupted, leads to visual impairment. Here we demonstrate that glutamine catabolism (glutaminolysis) through TCA cycle generates a large fraction of the ATP needed to maintain CE function, and this glutaminolysis is severely disrupted in cells deficient in NH<sub>3</sub>:H<sup>+</sup> cotransporter Solute Carrier Family 4 Member 11 (SLC4A11). Considering SLC4A11 mutations leads to corneal endothelial dystrophy and sensorineural deafness, our results indicate that SLC4A11-associated developmental and degenerative disorders result from altered glutamine catabolism. Overall, our results describe an important metabolic mechanism that provides CE cells with the energy required to maintain high level transport activity, reveal a direct link between glutamine metabolism and developmental and degenerative neuronal diseases, and suggest an approach for protecting the CE during ophthalmic surgeries.
Project description:SLC4A11, a member of the SLC4 family of bicarbonate transporters, is a widely expressed integral membrane protein, abundant in kidney and cornea. Mutations of SLC4A11 cause some cases of the blinding corneal dystrophies, congenital hereditary endothelial dystrophy, and Fuchs endothelial corneal dystrophy. These diseases are marked by fluid accumulation in the corneal stroma, secondary to defective fluid reabsorption by the corneal endothelium. The role of SLC4A11 in these corneal dystrophies is not firmly established, as SLC4A11 function remains unclear. To clarify the normal function(s) of SLC4A11, we characterized the protein following expression in the simple, low-background expression system Xenopus laevis oocytes. Since plant and fungal SLC4A11 orthologs transport borate, we measured cell swelling associated with accumulation of solute borate. The plant water/borate transporter NIP5;1 manifested borate transport, whereas human SLC4A11 did not. SLC4A11 supported osmotically driven water accumulation that was electroneutral and Na+ independent. Studies in oocytes and HEK293 cells could not detect Na+-coupled HCO3- transport or Cl-/HCO3- exchange by SLC4A11. SLC4A11 mediated electroneutral NH3 transport in oocytes. Voltage-dependent OH- or H+ movement was not measurable in SLC4A11-expressing oocytes, but SLC4A11-expressing HEK293 cells manifested low-level cytosolic acidification at baseline. In mammalian cells, but not oocytes, OH-/H+ conductance may arise when SLC4A11 activates another protein or itself is activated by another protein. These data argue against a role of human SLC4A11 in bicarbonate or borate transport. This work provides additional support for water and ammonia transport by SLC4A11. When expressed in oocytes, SLC4A11 transported NH3, not NH3/H.
Project description: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.
Project description:Vps35 (vacuolar protein sorting 35) is a major component of retromer that selectively promotes endosome-to-Golgi retrieval of transmembrane proteins. Dysfunction of retromer is a risk factor for the pathogenesis of Parkinson's disease (PD) and Alzheimer's disease (AD). However, Vps35/retromer's function in the eye or the contribution of Vps35-deficiency to eye degenerative disorders remains to be explored. Here we provide evidence for a critical role of Vps35 in mouse corneal dystrophy. Vps35 is expressed in mouse and human cornea. Mouse cornea from Vps35 heterozygotes (Vps35+/-) show features of dystrophy, such as loss of both endothelial and epithelial cell densities, disorganizations of endothelial, stroma, and epithelial cells, excrescences in the Descemet membrane, and corneal edema. Additionally, corneal epithelial cell proliferation was reduced in Vps35-deficient mice. Intriguingly, cell surface targeting of SLC4A11, a membrane transport protein (OH- /H+ /NH3 /H2O) of corneal endothelium, whose mutations have been identified in patients with corneal dystrophy, was impaired in Vps35-deficient cells and cornea. Taken together, these results suggest that SLC4A11 appears to be a Vps35/retromer cargo, and Vps35-regulation of SLC4A11 trafficking may underlie Vps35/retromer regulation of corneal dystrophy.
Project description:The corneal endothelium (CE) dysfunction impairs optical transparency and leads to corneal allograft failure. Morphologically, CE cells are characterized by premature senescence at the late stage of corneal graft. However, the detailed molecular mechanisms are largely unknown. Here we found that transforming growth factor-? (TGF-?) is elevated in the CE of late graft failure. In addition, senescence-associated gene p21 and p16 are increased as well, which is consistent with their elevation upon TGF-? treatment in human corneal endothelial cell B4G12. Furthermore, TGF-? treatment leads to high positive ratio of SA-?-gal, indicating B4G12 cells undergo cellular senescence. Mechanistically, we demonstrated that TGF-? could induce mitochondrial ROS (mtROS) production and mtROS scavenger could rescue CE cell senescence upon TGF-? treatment. Our study provides new evidence that elevated TGF-? plays a crucial role in the CE cell senescence and loss in chronic corneal graft failure, which could be potential targets for clinical treatment.
Project description:The SLC4A11 gene encodes the bicarbonate-transporter-related protein BTR1, which is mutated in syndromes characterized by vision and hearing loss. Signs of these diseases [congenital hereditary endothelial dystrophy (CHED) and Harboyan syndrome] are evident in mouse models of Slc4a11 disruption. However, the intrinsic activity of Slc4a11 remains controversial, complicating assignment of its (patho)physiological role. Most studies concur that Slc4a11 transports H+ (or the thermodynamically equivalent species OH-) rather than HCO3-, but disparities have arisen as to whether the transport is coupled to another species such as Na+ or NH3/NH4+ Here for the first time, we examine the action of mouse Slc4a11 in Xenopus oocytes. We simultaneously monitor changes in intracellular pH, membrane potential, and conductance as we alter extracellular pH, revealing the electrical and chemical driving forces that underlie the observed ion fluxes. We find that mSlc4a11 is an ideally selective H+/OH- conductive pathway, the action of which is uncoupled from the cotransport of any other ion. We also find that the activity of mSlc4a11 is independently enhanced by both extracellular and intracellular alkalinization, suggesting OH- as the most likely substrate and providing a novel explanation for the apparent NH3-dependence of Slc4a11-mediated currents reported by others. We suggest that the unique properties of Slc4a11 action underlie its value as a pH regulator in corneal endothelial cells.
Project description:Purpose:To report molecular genetic findings in six probands with congenital hereditary endothelial dystrophy (CHED) variably associated with hearing loss (also known as Harboyan syndrome). Furthermore, we developed a cellular model to determine if disease-associated variants induce aberrant SLC4A11 pre-mRNA splicing. Methods:Direct sequencing of the entire SLC4A11 coding region was performed in five probands. In one individual, whole genome sequencing was undertaken. The effect of c.2240+5G>A on pre-mRNA splicing was evaluated in a corneal endothelial-like (CE-like) cell model expressing SLC4A11. CE-like cells were derived from autologous induced pluripotent stem cells (iPSCs) via neural crest cells exposed to B27, PDGF-BB, and DKK-2. Total RNA was extracted, and RT-PCR was performed followed by Sanger and a targeted next generation sequencing (NGS) approach to identify and quantify the relative abundance of alternatively spliced transcripts. Results:In total, 11 different mutations in SLC4A11 evaluated as pathogenic were identified; of these, c.1237G>A, c.2003T>C, c.1216+1G>A, and c.2240+5G>A were novel. The c.2240+5G>A variant was demonstrated to result in aberrant pre-mRNA splicing. A targeted NGS approach confirmed that the variant introduces a leaky cryptic splice donor site leading to the production of a transcript containing an insertion of six base pairs with the subsequent introduction of a premature stop codon (p.Thr747*). Furthermore, a subset of transcripts comprising full retention of intron 16 also were observed, leading to the same functionally null allele. Conclusions:This proof-of-concept study highlights the potential of using CE-like cells to investigate the pathogenic consequences of SLC4A11 disease-associated variants.
Project description:Corneal endothelial dystrophy is a progressive disease with gradual loss of vision and characterized by degeneration and dysfunction of corneal endothelial cells. Mutations in SLC4A11, a Na+ dependent OH- transporter, cause congenital hereditary endothelial dystrophy (CHED) and Fuchs' endothelial corneal dystrophy (FECD), the two most common forms of endothelial degeneration. Along with genetic factors, oxidative stress plays a role in pathogenesis of several corneal diseases. In this study we looked into the role of SLC4A11 in antioxidant stress response in human corneal endothelial cells (HCEnC). We found increased expression of SLC4A11 in presence of oxidative stress. Depletion of SLC4A11 using targeted siRNA, caused an increase in reactive oxygen species, cytochrome c, lowered mitochondrial membrane potential, and reduced cell viability during oxidative stress. Moreover, SLC4A11 was found to be necessary for NRF2 mediated antioxidant gene expression in HCEnC. On the other hand, over expression of SLC4A11 reduces reactive oxygen species levels and increases cell viability. Lastly, CHED tissue specimens show evidence of oxidative stress and reduced expression of NRF2. In conclusion, our data suggests a possible role of SLC4A11 in regulating oxidative stress, and might be responsible for both the etiology and treatment of corneal endothelial dystrophy.
Project description:Fuchs endothelial corneal dystrophy (FECD) is a leading cause of corneal endothelial (CE) degeneration resulting in impaired visual acuity. It is a genetically complex and age-related disorder, with higher incidence in females. In this study, we established a nongenetic FECD animal model based on the physiologic outcome of CE susceptibility to oxidative stress by demonstrating that corneal exposure to ultraviolet A (UVA) recapitulates the morphological and molecular changes of FECD. Targeted irradiation of mouse corneas with UVA induced reactive oxygen species (ROS) production in the aqueous humor, and caused greater CE cell loss, including loss of ZO-1 junctional contacts and corneal edema, in female than male mice, characteristic of late-onset FECD. UVA irradiation caused greater mitochondrial DNA (mtDNA) and nuclear DNA (nDNA) damage in female mice, indicative of the sex-driven differential response of the CE to UVA, thus accounting for more severe phenotype in females. The sex-dependent effect of UVA was driven by the activation of estrogen-metabolizing enzyme CYP1B1 and formation of reactive estrogen metabolites and estrogen-DNA adducts in female but not male mice. Supplementation of N-acetylcysteine (NAC), a scavenger of reactive oxygen species (ROS), diminished the morphological and molecular changes induced by UVA in vivo. This study investigates the molecular mechanisms of environmental factors in FECD pathogenesis and demonstrates a strong link between UVA-induced estrogen metabolism and increased susceptibility of females for FECD development.
Project description:Fuchs' endothelial corneal dystrophy (FECD), which affects approximately 5% of the population over 40 in the U.S.A., is a major cause of corneal transplantation. FECD is associated with mutations of a variety of unrelated genes: SLC4A11, COL8A2, TCF8, and LOXHD1. The current pathological description of the dystrophy includes deficiency of corneal endothelium (CE) pump function and induction of the unfolded protein response (UPR). This study aims to determine the contribution of the two mechanisms by assessing the expression levels of (1) seven endothelial ion transporters known to regulate stromal hydration and (2) UPR related genes in a set of six CE samples obtained from FECD patients compared to that of normal controls.CE samples collected during FECD keratoplasty or from an eye bank (normal control) were transferred into an RNA stabilizing agent and refrigerated. Total RNA from each CE specimen was individually extracted. The expression levels of ion transporters and UPR genes were tested using quantitative real-time (RT) PCR and a UPR specific PCR array, respectively.In normal CE, the comparative expression levels of ion transporters in decreasing order were SLC4A11, Na(+)/K(+) ATPase, pNBCe1, and NHE1, followed by the isoforms of monocarboxylate transporters (MCTs). In FECD samples, Na(+)/K(+) ATPase and MCTs 1 and 4 were significantly downregulated compared to normal controls (p<0.05). The PCR array tested 84 UPR related genes. Data analysis showed upregulation of 39 genes and downregulation of three genes, i.e., approximately 51% of the tested genes had their expression altered in FECD samples with a difference greater than ± twofold regulation. Thirteen of the altered genes showed significant changes (p<0.05). The PCR array results were validated by quantitative RT-PCR.FECD samples had evident UPR with significant changes in the expression of the protein processing pathway genes. The significant downregulation of ion transporters indicates simultaneous compromised CE pump function in Fuchs' dystrophy.