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1.
Adv Exp Med Biol ; 1415: 319-325, 2023.
Article in English | MEDLINE | ID: mdl-37440051

ABSTRACT

Transcription factor EB (TFEB) plays a very important role in the maintenance of cellular homeostasis. TFEB is a transcription factor that regulates the expression of several genes in the Coordinated Lysosomal Expression and Regulation (CLEAR) network. The CLEAR network genes are known to regulate many processes associated with the autophagy pathway and lysosome biogenesis. Lysosomes, which are degradative organelles in the cell, are associated with several cellular mechanisms, such as autophagy and phagocytosis. Recent studies have shown that TFEB dysregulation and lysosomal dysfunction are associated with several degenerative diseases. Thus, enhancing TFEB activity and accompanied induction of lysosomal function and autophagy can have tremendous therapeutic potential for the treatment of several degenerative diseases including age-related macular degeneration (AMD). In this chapter, we briefly illustrate the expression and regulation of TFEB in response to several cellular stressors and discuss the effects of TFEB overexpression to induce cellular clearance functions.


Subject(s)
Gene Expression Regulation , Lysosomes , Lysosomes/metabolism , Transcription Factors/metabolism , Autophagy/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/pharmacology
2.
Nat Commun ; 13(1): 6045, 2022 10 13.
Article in English | MEDLINE | ID: mdl-36229454

ABSTRACT

The retinal pigment epithelium (RPE) plays an important role in the development of diabetic retinopathy (DR), a leading cause of blindness worldwide. Here we set out to explore the role of Akt2 signaling-integral to both RPE homeostasis and glucose metabolism-to DR. Using human tissue and genetically manipulated mice (including RPE-specific conditional knockout (cKO) and knock-in (KI) mice), we investigate whether Akts in the RPE influences DR in models of diabetic eye disease. We found that Akt1 and Akt2 activities were reciprocally regulated in the RPE of DR donor tissue and diabetic mice. Akt2 cKO attenuated diabetes-induced retinal abnormalities through a compensatory upregulation of phospho-Akt1 leading to an inhibition of vascular injury, inflammatory cytokine release, and infiltration of immune cells mediated by the GSK3ß/NF-κB signaling pathway; overexpression of Akt2 has no effect. We propose that targeting Akt1 activity in the RPE may be a novel therapy for treating DR.


Subject(s)
Diabetes Mellitus, Experimental , Diabetic Retinopathy , Proto-Oncogene Proteins c-akt/metabolism , Animals , Cytokines/metabolism , Diabetes Mellitus, Experimental/complications , Diabetes Mellitus, Experimental/genetics , Diabetes Mellitus, Experimental/metabolism , Diabetic Retinopathy/etiology , Epithelial Cells/metabolism , Glucose/metabolism , Glycogen Synthase Kinase 3 beta/genetics , Glycogen Synthase Kinase 3 beta/metabolism , Humans , Mice , NF-kappa B/metabolism , Retinal Pigment Epithelium/metabolism , Retinal Pigments/metabolism
3.
Int J Mol Sci ; 23(3)2022 Feb 01.
Article in English | MEDLINE | ID: mdl-35163631

ABSTRACT

Autophagy is a vital cellular mechanism that benefits cellular maintenance and survival during cell stress. It can eliminate damaged or long-lived organelles and improperly folded proteins to maintain cellular homeostasis, development, and differentiation. Impaired autophagy is associated with several diseases such as cancer, neurodegenerative diseases, and age-related macular degeneration (AMD). Several signaling pathways are associated with the regulation of the autophagy pathway. The glycogen synthase kinase-3 signaling pathway was reported to regulate the autophagy pathway. In this review, we will discuss the mechanisms by which the GSK-3 signaling pathway regulates autophagy. Autophagy and lysosomal function are regulated by transcription factor EB (TFEB). GSK-3 was shown to be involved in the regulation of TFEB nuclear expression in an mTORC1-dependent manner. In addition to mTORC1, GSK-3ß also regulates TFEB via the protein kinase C (PKC) and the eukaryotic translation initiation factor 4A-3 (eIF4A3) signaling pathways. In addition to TFEB, we will also discuss the mechanisms by which the GSK-3 signaling pathway regulates autophagy by modulating other signaling molecules and autophagy inducers including, mTORC1, AKT and ULK1. In summary, this review provides a comprehensive understanding of the role of the GSK-3 signaling pathway in the regulation of autophagy.


Subject(s)
Autophagy , Glycogen Synthase Kinase 3/metabolism , Signal Transduction , Animals , Glycogen Synthase Kinase 3/physiology , Humans , Lysosomes , Mechanistic Target of Rapamycin Complex 1/metabolism
4.
Cells ; 10(10)2021 09 22.
Article in English | MEDLINE | ID: mdl-34685484

ABSTRACT

Lysosomes are important for proper functioning of the retinal pigment epithelial (RPE) cells. RPE cells have a daily burden of phagocytosis of photoreceptor outer segments (POS) and also degrade cellular waste by autophagy. Here, we identified the role of Zinc-finger protein with KRAB and SCAN domains 3 (ZKSCAN3) in co-ordinate regulation of lysosomal function and autophagy in the RPE. Our studies show that in the RPE, ZKSCAN3 is predominantly nuclear in healthy cells and its nuclear expression is reduced upon nutrient deprivation. siRNA-mediated knockdown of ZKSCAN3 results in de-repression of some of the ZKSCAN3 target genes. Knockdown of ZKSCAN3 also resulted in an induction in autophagy flux, increase in the number of functional lysosomes and accompanied activation of lysosomal cathepsin B activity in ARPE-19 cells. We also demonstrated that inhibition of P38 mitogen-activated protein kinase (MAPK) retains ZKSCAN3 in the nucleus in nutrient-deprived cells. In summary, our studies elucidated the role of ZKSCAN3 as a transcriptional repressor of autophagy and lysosomal function in the RPE.


Subject(s)
Epithelial Cells/metabolism , Repressor Proteins/metabolism , Retinal Pigment Epithelium/metabolism , Transcription Factors/metabolism , Autophagy/physiology , Humans , Lysosomes/metabolism , Phagocytosis/physiology , Retinal Pigments/metabolism
5.
Int J Mol Sci ; 22(16)2021 Aug 12.
Article in English | MEDLINE | ID: mdl-34445390

ABSTRACT

Nuclear factor of activated T cells (NFAT) family of transcription factors are substrates of calcineurin and play an important role in integrating Ca2+ signaling with a variety of cellular functions. Of the five NFAT proteins (NFAT1-5), NFAT1-4 are subject to dephosphorylation and activation by calcineurin, a Ca2+-calmodulin-dependent phosphatase. Increased levels of intracellular Ca2+ activates calcineurin, which in turn dephosphorylates and promotes nuclear translocation of NFAT. We investigated the functions of NFAT proteins in the retinal pigment epithelial cells (RPE). Our results show that NFAT-mediated luciferase activity was induced upon treatment with the bacterial endotoxin, lipopolysaccharide (LPS) and treatment with the NFAT peptide inhibitor, MAGPHPVIVITGPHEE (VIVIT) decreased LPS-induced NFAT luciferase activity. LPS-induced activation of NFAT-regulated cytokines (IL-6 and IL-8) is inhibited by treatment of cells with VIVIT. We also investigated the effects of NFAT signaling on the autophagy pathway. Our results show that inhibition of NFAT with VIVIT in cells deprived of nutrients resulted in cytosolic retention of transcription Factor EB (TFEB), decreased expression of TFEB-regulated coordinated Lysosomal Expression and Regulation CLEAR network genes and decreased starvation-induced autophagy flux in the RPE cells. In summary, these studies suggest that the NFAT pathway plays an important role in the regulation of autophagy and inflammation in the RPE.


Subject(s)
Lipopolysaccharides/adverse effects , NFATC Transcription Factors/metabolism , Oligopeptides/adverse effects , Retinal Pigment Epithelium/cytology , Autophagy/drug effects , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/genetics , Cell Line , Epithelial Cells/cytology , Epithelial Cells/drug effects , Epithelial Cells/metabolism , Gene Expression Regulation/drug effects , Humans , Interleukin-6/genetics , Interleukin-8/genetics , Retinal Pigment Epithelium/drug effects , Retinal Pigment Epithelium/metabolism , Signal Transduction/drug effects
6.
Nanomaterials (Basel) ; 11(1)2021 Jan 12.
Article in English | MEDLINE | ID: mdl-33445545

ABSTRACT

Ocular diseases can deteriorate vision to the point of blindness and thus can have a major impact on the daily life of an individual. Conventional therapies are unable to provide absolute therapy for all ocular diseases due to the several limitations during drug delivery across the blood-retinal barrier, making it a major clinical challenge. With recent developments, the vast number of publications undergird the need for nanotechnology-based drug delivery systems in treating ocular diseases. The tool of nanotechnology provides several essential advantages, including sustained drug release and specific tissue targeting. Additionally, comprehensive in vitro and in vivo studies have suggested a better uptake of nanoparticles across ocular barriers. Nanoparticles can overcome the blood-retinal barrier and consequently increase ocular penetration and improve the bioavailability of the drug. In this review, we aim to summarize the development of organic and inorganic nanoparticles for ophthalmic applications. We highlight the potential nanoformulations in clinical trials as well as the products that have become a commercial reality.

7.
Cell Mol Life Sci ; 77(5): 835-851, 2020 Mar.
Article in English | MEDLINE | ID: mdl-31901947

ABSTRACT

Lipocalins are a family of secreted adipokines which play important roles in various biological processes. Lipocalin-2 (LCN-2) has been shown to be involved in acute and chronic inflammation. This particular protein is critical in the pathogenesis of several diseases including cancer, diabetes, obesity, and multiple sclerosis. Herein, we discuss the general molecular basis for the involvement of LCN-2 in acute infections and chronic disease progression and also ascertain the probable role of LCN-2 in ocular diseases, particularly in age-related macular degeneration (AMD). We elaborate on the signaling cascades which trigger LCN-2 upregulation in AMD and suggest therapeutic strategies for targeting such pathways.


Subject(s)
Lipocalin-2/genetics , Lipocalin-2/metabolism , Macular Degeneration/genetics , Macular Degeneration/pathology , Vision Disorders/genetics , Animals , Disease Models, Animal , Humans , Inflammation/pathology , Mice , Retina/pathology , Retinal Pigment Epithelium/pathology , Signal Transduction , Vision Disorders/pathology
8.
Cell Mol Biol Lett ; 24: 33, 2019.
Article in English | MEDLINE | ID: mdl-31160892

ABSTRACT

BACKGROUND: Induction of lysosomal function and autophagy is regarded as an adaptive mechanism in response to cellular stress. The transcription factor EB (TFEB) has been identified as a master regulator of lysosomal function and autophagy. TFEB is a member of the microphthalmia family of bHLH-LZ transcription factors that includes other members such as micropthalmia-associated transcription factor (MITF), TFE3, and TFEC. TFEB controls lysosome biogenesis and autophagy by upregulation of a family of genes belonging to the Coordinated Lysosomal Expression and Regulation (CLEAR) network. Here, we investigated the expression of TFEB in cells subjected to nutrient deprivation and lysosomal stress. We studied transcriptional induction of TFEB-regulated genes in response to nutrient deprivation and lysosomal stress in retinal pigment epithelial (RPE) cells. Furthermore, we also investigated the induction of autophagy and lysosomal genes upon overexpression of constitutively active form of TFEB. METHODS: Expression of TFEB and MITF protein levels were evaluated in cells subjected to prolonged periods of nutrient deprivation. mRNA levels of the CLEAR network genes was measured by quantitative real time PCR (qRT-PCR) analysis in cells deprived of nutrients, treated with ammonium chloride and upon overexpression of constitutively active TFEB. Immunostaining with LC3 antibody was used to measure autophagy flux. Labeling with lysoTracker dye was used to assess lysosomes. RESULTS: Our results show that nutrient deprivation increases protein levels of TFEB and MITF in ARPE-19 cells. Nutrient stress induces the expression of lysosomal (LAMP1, CTSD MCOLN1, SGSH) and autophagy (BECN1) genes. Lysosomal stress also increases the expression of lysosomal (ATP6V0A1 and LAMP1) and autophagy (p62 and BECN1) genes. Our results show that overexpression of constitutively active TFEB also induces the expression of CLEAR network genes. CONCLUSIONS: Collectively, these observations suggest that nutrient stress induces the protein expression of both MITF and TFEB in ARPE-19 cells. TFEB-regulated transcriptional program plays an important role in adaptive response of cells during both nutrient and lysosomal stress.


Subject(s)
Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Epithelial Cells/metabolism , Lysosomes/metabolism , Retinal Pigment Epithelium/pathology , Stress, Physiological , Adult , Ammonium Chloride/pharmacology , Animals , Cell Line , Epithelial Cells/drug effects , Gene Regulatory Networks/drug effects , Humans , Lysosomes/drug effects , Mice, Inbred C57BL , Stress, Physiological/drug effects , Transcription, Genetic/drug effects
9.
Aging Cell ; 16(2): 349-359, 2017 04.
Article in English | MEDLINE | ID: mdl-28083894

ABSTRACT

The dry (nonneovascular) form of age-related macular degeneration (AMD), a leading cause of blindness in the elderly, has few, if any, treatment options at present. It is characterized by early accumulation of cellular waste products in the retinal pigmented epithelium (RPE); rejuvenating impaired lysosome function in RPE is a well-justified target for treatment. It is now clear that amino acids and vacuolar-type H+ -ATPase (V-ATPase) regulate the mechanistic target of rapamycin, complex 1 (mTORC1) signaling in lysosomes. Here, we provide evidence for the first time that the amino acid transporter SLC36A4/proton-dependent amino acid transporter (PAT4) regulates the amino acid pool in the lysosomes of RPE. In Cryba1 (gene encoding ßA3/A1-crystallin) KO (knockout) mice, where PAT4 and amino acid levels are increased in the RPE, the transcription factors EB (TFEB) and E3 (TFE3) are retained in the cytoplasm, even after 24 h of fasting. Consequently, genes in the coordinated lysosomal expression and regulation (CLEAR) network are not activated, and lysosomal function remains low. As these mice age, expression of RPE65 and lecithin retinol acyltransferase (LRAT), two vital visual cycle proteins, decreases in the RPE. A defective visual cycle would possibly slow down the regeneration of new photoreceptor outer segments (POS). Further, photoreceptor degeneration also becomes obvious during aging, reminiscent of human dry AMD disease. Electron microscopy shows basal laminar deposits in Bruch's membrane, a hallmark of development of AMD. For dry AMD patients, targeting PAT4/V-ATPase in the lysosomes of RPE cells may be an effective means of preventing or delaying disease progression.


Subject(s)
Amino Acid Transport Systems/metabolism , Amino Acids/metabolism , Epithelial Cells/metabolism , Multiprotein Complexes/metabolism , Retinal Pigment Epithelium/cytology , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Aging/metabolism , Animals , Basic Helix-Loop-Helix Leucine Zipper Transcription Factors/metabolism , Crystallins/metabolism , Cytosol/metabolism , Epithelial Cells/ultrastructure , Gene Regulatory Networks , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1 , Mice, Knockout , Phosphorylation , Protein Binding , Retinal Photoreceptor Cell Outer Segment/metabolism , Retinal Pigment Epithelium/metabolism , Retinal Pigment Epithelium/ultrastructure , Visual Pathways/metabolism , beta-Crystallin A Chain
10.
Adv Exp Med Biol ; 854: 779-84, 2016.
Article in English | MEDLINE | ID: mdl-26427489

ABSTRACT

We have previously demonstrated that ßA3/A1-crystallin, a member of the ß/γ-crystallin superfamily, is expressed in the astrocytes and retinal pigment epithelial (RPE) cells of the eye. In order to understand the physiological functions of ßA3/A1-crystallin in RPE cells, we generated conditional knockout (cKO) mice where Cryba1, the gene encoding ßA3/A1-crystallin, is deleted specifically from the RPE using the Cre-loxP system. By utilizing the cKO model, we have shown that this protein is required by RPE cells for proper lysosomal degradation of photoreceptor outer segments (OS) that have been internalized in phagosomes and also for the proper functioning of the autophagy process. We also reported that ßA3/A1-crystallin is trafficked to lysosomes, where it regulates endolysosomal acidification by modulating the activity of the lysosomal V-ATPase complex. Our results show that the V-ATPase activity in cKO RPE is significantly lower than WT RPE. Since, V-ATPase is important for regulating lysosomal pH, we noticed that endolysosomal pH was higher in the cKO cells compared to the WT cells. Increased lysosomal pH in cKO RPE is also associated with reduced Cathepsin D activity. Cathepsin D is a major lysosomal aspartic protease involved in the degradation of the OS and hence we believe that reduced proteolytic activity contributes to impaired degradation of OS in the cKO RPE. Reduced lysosomal activity in the cKO RPE also contributes to the incomplete degradation of the autophagosomes. Our results also suggest that ßA3/A1-crystallin regulates V-ATPase activity by binding to the V0 subunit of the V-ATPase complex. Taken together, these results suggest a novel mechanism by which ßA3/A1-crystallin regulates lysosomal function by modulating the activity of V-ATPase.


Subject(s)
Crystallins/metabolism , Epithelial Cells/metabolism , Retinal Pigment Epithelium/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Animals , Cathepsin D/metabolism , Crystallins/genetics , Hydrogen-Ion Concentration , Immunoblotting , Lysosomes/metabolism , Mice, Knockout , Retinal Photoreceptor Cell Outer Segment/metabolism , Retinal Pigment Epithelium/cytology , beta-Crystallin A Chain
11.
Exp Eye Res ; 144: 46-53, 2016 Mar.
Article in English | MEDLINE | ID: mdl-26321509

ABSTRACT

The retinal pigmented epithelium (RPE) is critically important to retinal homeostasis, in part due to its very active processes of phagocytosis and autophagy. Both of these processes depend upon the normal functioning of lysosomes, organelles which must fuse with (auto)phagosomes to deliver the hydrolases that effect degradation of cargo. It has become clear that signaling through mTOR complex 1 (mTORC1), is very important in the regulation of lysosomal function. This signaling pathway is becoming a target for therapeutic intervention in diseases, including age-related macular degeneration (AMD), where lysosomal function is defective. In addition, our laboratory has been studying animal models in which the gene (Cryba1) for ßA3/A1-crystallin is deficient. These animals exhibit impaired lysosomal clearance in the RPE and pathological signs that are similar to some of those seen in AMD patients. The data demonstrate that ßA3/A1-crystallin localizes to lysosomes in the RPE and that it is a binding partner of V-ATPase, the proton pump that acidifies the lysosomal lumen. This suggests that ßA3/A1-crystallin may also be a potential target for therapeutic intervention in AMD. In this review, we focus on effector molecules that impact the lysosomal-autophagic pathway in RPE cells.


Subject(s)
Autophagy/physiology , Lysosomes/physiology , Retinal Pigment Epithelium/metabolism , Animals , Humans , Mechanistic Target of Rapamycin Complex 1 , Multiprotein Complexes/physiology , Organelle Biogenesis , TOR Serine-Threonine Kinases/physiology
12.
Biochim Biophys Acta ; 1860(1 Pt B): 287-98, 2016 Jan.
Article in English | MEDLINE | ID: mdl-26022148

ABSTRACT

BACKGROUND: Persistent fetal vasculature (PFV) is a human disease in which the fetal vasculature of the eye fails to regress normally. The fetal, or hyaloid, vasculature nourishes the lens and retina during ocular development, subsequently regressing after formation of the retinal vessels. PFV causes serious congenital pathologies and is responsible for as much as 5% of blindness in the United States. SCOPE OF REVIEW: The causes of PFV are poorly understood, however there are a number of animal models in which aspects of the disease are present. One such model results from mutation or elimination of the gene (Cryba1) encoding ßA3/A1-crystallin. In this review we focus on the possible mechanisms whereby loss of functional ßA3/A1-crystallin might lead to PFV. MAJOR CONCLUSIONS: Cryba1 is abundantly expressed in the lens, but is also expressed in certain other ocular cells, including astrocytes. In animal models lacking ßA3/A1-crystallin, astrocyte numbers are increased and they migrate abnormally from the retina to ensheath the persistent hyaloid artery. Evidence is presented that the absence of functional ßA3/A1-crystallin causes failure of the normal acidification of endolysosomal compartments in the astrocytes, leading to impairment of certain critical signaling pathways, including mTOR and Notch/STAT3. GENERAL SIGNIFICANCE: The findings suggest that impaired endolysosomal signaling in ocular astrocytes can cause PFV disease, by adversely affecting the vascular remodeling processes essential to ocular development, including regression of the fetal vasculature. This article is part of a Special Issue entitled Crystallin Biochemistry in Health and Disease.


Subject(s)
Eye Proteins/metabolism , Persistent Hyperplastic Primary Vitreous/embryology , Persistent Hyperplastic Primary Vitreous/metabolism , Retinal Vessels/abnormalities , Retinal Vessels/metabolism , beta-Crystallin A Chain/metabolism , Animals , Chronic Disease , Humans , Models, Biological
13.
Sci Rep ; 5: 8755, 2015 Mar 04.
Article in English | MEDLINE | ID: mdl-25736717

ABSTRACT

We have previously reported that in the Nuc1 rat, which has a spontaneous mutation in Cryba1 (the gene encoding ßA3/A1-crystallin), astrocytes exhibit decreased Notch signaling, leading to reduced promoter activity for glial fibrillary acidic protein (GFAP). Interestingly, in both Nuc1 astrocytes and in wild type astrocytes following knockdown of Cryba1, vascular endothelial growth factor (VEGF) secretion is decreased. This has led us to explore signaling mediators that could be regulated by ßA3/A1-crystallin to modulate both GFAP and VEGF. Several studies have shown that the signal transducer and activator of transcription 3 (STAT3) is involved in the co-regulation of GFAP and VEGF. We show that STAT3 and ßA3/A1-crystallin may co-regulate each other in astrocytes. Such co-regulation would create a positive feedback circuit; i.e., in the cytosol of astrocytes, ßA3/A1-crystallin is necessary for the phosphorylation of STAT3, which then dimerizes and translocates to the nucleus to form DNA-binding complexes, activating transcription of Cryba1. This stoichiometric co-regulation of STAT3 and Cryba1 could potentiate expression of GFAP and secretion of VEGF, both of which are essential for maintaining astrocyte and blood vessel homeostasis in the retina. Consistent with this idea, Cryba1 knockout mice exhibit an abnormal astrocyte pattern and defective remodeling of retinal vessels.


Subject(s)
Astrocytes/metabolism , Crystallins/metabolism , Optic Nerve/metabolism , STAT3 Transcription Factor/metabolism , Signal Transduction , Animals , Base Sequence , Binding Sites/genetics , Crystallins/genetics , Gene Expression , Immunoblotting , Mice, Knockout , Microscopy, Confocal , Molecular Sequence Data , Mutation , Optic Nerve/cytology , Phosphorylation , Promoter Regions, Genetic/genetics , Rats , Receptors, Notch/metabolism , Reverse Transcriptase Polymerase Chain Reaction , beta-Crystallin A Chain
14.
Aging Cell ; 13(6): 1091-4, 2014 Dec.
Article in English | MEDLINE | ID: mdl-25257511

ABSTRACT

Although chronic inflammation is believed to contribute to the pathology of age-related macular degeneration (AMD), knowledge regarding the events that elicit the change from para-inflammation to chronic inflammation in the pathogenesis of AMD is lacking. We propose here that lipocalin-2 (LCN2), a mammalian innate immunity protein that is trafficked to the lysosomes, may contribute to this process. It accumulates significantly with age in retinal pigment epithelial (RPE) cells of Cryba1 conditional knockout (cKO) mice, but not in control mice. We have recently shown that these mice, which lack ßA3/A1-crystallin specifically in RPE, have defective lysosomal clearance. The age-related increase in LCN2 in the cKO mice is accompanied by increases in chemokine (C-C motif) ligand 2 (CCL2), reactive gliosis, and immune cell infiltration. LCN2 may contribute to induction of a chronic inflammatory response in this mouse model with AMD-like pathology.


Subject(s)
Acute-Phase Proteins/metabolism , Crystallins/metabolism , Lipocalins/metabolism , Macular Degeneration/metabolism , Oncogene Proteins/metabolism , Retinal Pigment Epithelium/metabolism , Age Factors , Animals , Chronic Disease , Crystallins/genetics , Disease Models, Animal , Inflammation/genetics , Inflammation/metabolism , Inflammation/pathology , Lipocalin-2 , Macular Degeneration/genetics , Macular Degeneration/pathology , Mice , Retinal Pigment Epithelium/pathology , beta-Crystallin A Chain
15.
J Biol Chem ; 289(23): 15915-26, 2014 Jun 06.
Article in English | MEDLINE | ID: mdl-24742684

ABSTRACT

Glutamate-induced elevation in intracellular Ca(2+) has been implicated in excitotoxic cell death. Neurons respond to increased glutamate levels by activating an extracellular proteolytic cascade involving the components of the plasmin-plasminogen system. AnxA2 is a Ca(2+)-dependent phospholipid binding protein and serves as an extracellular proteolytic center by recruiting the tissue plasminogen activator and plasminogen and mediating the localized generation of plasmin. Ratiometric Ca(2+) imaging and time-lapse confocal microscopy demonstrated glutamate-induced Ca(2+) influx. We showed that glutamate translocated both endogenous and AnxA2-GFP to the cell surface in a process dependent on the activity of the NMDA receptor. Glutamate-induced translocation of AnxA2 is dependent on the phosphorylation of tyrosine 23 at the N terminus, and mutation of tyrosine 23 to a non-phosphomimetic variant inhibits the translocation process. The cell surface-translocated AnxA2 forms an active plasmin-generating complex, and this activity can be neutralized by a hexapeptide directed against the N terminus. These results suggest an involvement of AnxA2 in potentiating glutamate-induced cell death processes.


Subject(s)
Annexin A2/metabolism , Glutamic Acid/physiology , Animals , Annexin A2/chemistry , Annexin A2/genetics , Calcium/metabolism , Cell Line, Transformed , Extracellular Space , Fibrinolysin/metabolism , Green Fluorescent Proteins/genetics , Mice , Phosphorylation , Protein Transport , Proteolysis , Tyrosine/metabolism
16.
Autophagy ; 10(3): 480-96, 2014 Mar.
Article in English | MEDLINE | ID: mdl-24468901

ABSTRACT

In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/ßA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V0-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the ßA3- and ßA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.


Subject(s)
Autophagy/physiology , Crystallins/metabolism , Lysosomes/metabolism , Multiprotein Complexes/metabolism , Retinal Pigment Epithelium/metabolism , Signal Transduction , TOR Serine-Threonine Kinases/metabolism , Vacuolar Proton-Translocating ATPases/metabolism , Animals , Autophagy/genetics , Crystallins/genetics , Mechanistic Target of Rapamycin Complex 1 , Mice , Mice, Knockout , Multiprotein Complexes/genetics , Phagocytosis/physiology , Phagosomes/metabolism , Rats , Retinal Pigment Epithelium/cytology , Signal Transduction/physiology , TOR Serine-Threonine Kinases/genetics
17.
Nat Commun ; 4: 1629, 2013.
Article in English | MEDLINE | ID: mdl-23535650

ABSTRACT

Astrocytes migrate from the optic nerve into the inner retina, forming a template upon which retinal vessels develop. In the Nuc1 rat, mutation in the gene encoding ßA3/A1-crystallin disrupts both Notch signalling in astrocytes and formation of the astrocyte template. Here we show that loss of ßA3/A1-crystallin in astrocytes does not impede Notch ligand binding or extracellular cleavages. However, it affects vacuolar-type proton ATPase (V-ATPase) activity, thereby compromising acidification of the endolysosomal compartments, leading to reduced γ-secretase-mediated processing and release of the Notch intracellular domain (NICD). Lysosomal-mediated degradation of Notch is also impaired. These defects decrease the level of NICD in the nucleus, inhibiting the expression of Notch target genes. Overexpression of ßA3/A1-crystallin in those same astrocytes restored V-ATPase activity and normal endolysosomal acidification, thereby increasing the levels of γ-secretase to facilitate optimal Notch signalling. We postulate that ßA3/A1-crystallin is essential for normal endolysosomal acidification, and thereby, normal activation of Notch signalling in astrocytes.


Subject(s)
Astrocytes/metabolism , Endosomes/metabolism , Lysosomes/metabolism , Optic Nerve/metabolism , Receptor, Notch1/metabolism , Signal Transduction , Animals , Calcium-Binding Proteins/metabolism , Crystallins/metabolism , Intercellular Signaling Peptides and Proteins/metabolism , Membrane Proteins/metabolism , Mice , Optic Nerve/cytology , Serrate-Jagged Proteins
18.
Transgenic Res ; 21(5): 1033-42, 2012 Oct.
Article in English | MEDLINE | ID: mdl-22427112

ABSTRACT

Nuc1 is a spontaneous rat mutant resulting from a mutation in the Cryba1 gene, coding for ßA3/A1-crystallin. Our earlier studies with Nuc1 provided novel evidence that astrocytes, which express ßA3/A1-crystallin, have a pivotal role in retinal remodeling. The role of astrocytes in the retina is only beginning to be explored. One of the limitations in the field is the lack of appropriate animal models to better investigate the function of astrocytes in retinal health and disease. We have now established transgenic mice that overexpress the Nuc1 mutant form of Cryba1, specifically in astrocytes. Astrocytes in wild type mice show normal compact stellate structure, producing a honeycomb-like network. In contrast, in transgenics over-expressing the mutant (Nuc1) Cryba1 in astrocytes, bundle-like structures with abnormal patterns and morphology were observed. In the nerve fiber layer of the transgenic mice, an additional layer of astrocytes adjacent to the vitreous is evident. This abnormal organization of astrocytes affects both the superficial and deep retinal vascular density and remodeling. Fluorescein angiography showed increased venous dilation and tortuosity of branches in the transgenic retina, as compared to wild type. Moreover, there appear to be fewer interactions between astrocytes and endothelial cells in the transgenic retina than in normal mouse retina. Further, astrocytes overexpressing the mutant ßA3/A1-crystallin migrate into the vitreous, and ensheath the hyaloid artery, in a manner similar to that seen in the Nuc1 rat. Together, these data demonstrate that developmental abnormalities of astrocytes can affect the normal remodeling process of both fetal and retinal vessels of the eye and that ßA3/A1-crystallin is essential for normal astrocyte function in the retina.


Subject(s)
Astrocytes/physiology , Crystallins/metabolism , Retina/growth & development , Retinal Vessels/growth & development , Animals , Astrocytes/pathology , Blotting, Western , Cell Movement , Cell Shape , Crystallins/genetics , Fluorescein Angiography , Fluorescent Antibody Technique , Gene Expression Regulation , Humans , Mice , Mice, Inbred C57BL , Mice, Transgenic , Microscopy, Confocal , Mutant Proteins/genetics , Mutant Proteins/metabolism , Oocytes/cytology , Oocytes/metabolism , Promoter Regions, Genetic , Rats , Retina/pathology , Retinal Vessels/pathology , Transgenes
19.
J Biol Chem ; 286(35): 30911-30925, 2011 Sep 02.
Article in English | MEDLINE | ID: mdl-21737841

ABSTRACT

Annexin A2 (AnxA2), a Ca(2+)-dependent phospholipid-binding protein, is known to associate with the plasma membrane and the endosomal system. Within the plasma membrane, AnxA2 associates in a Ca(2+) dependent manner with cholesterol-rich lipid raft microdomains. Here, we show that the association of AnxA2 with the lipid rafts is influenced not only by intracellular levels of Ca(2+) but also by N-terminal phosphorylation at tyrosine 23. Binding of AnxA2 to the lipid rafts is followed by the transport along the endocytic pathway to be associated with the intralumenal vesicles of the multivesicular endosomes. AnxA2-containing multivesicular endosomes fuse directly with the plasma membrane resulting in the release of the intralumenal vesicles into the extracellular environment, which facilitates the exogenous transfer of AnxA2 from one cell to another. Treatment with Ca(2+) ionophore triggers the association of AnxA2 with the specialized microdomains in the exosomal membrane that possess raft-like characteristics. Phosphorylation at Tyr-23 is also important for the localization of AnxA2 to the exosomal membranes. These results suggest that AnxA2 is trafficked from the plasma membrane rafts and is selectively incorporated into the lumenal membranes of the endosomes to escape the endosomal degradation pathway. The Ca(2+)-dependent exosomal transport constitutes a novel pathway of extracellular transport of AnxA2.


Subject(s)
Annexin A2/chemistry , Endocytosis , Exosomes/metabolism , Membrane Microdomains/chemistry , Animals , Biological Transport , Biotinylation , Calcium/chemistry , Calcium-Binding Proteins/chemistry , Cell Line, Tumor , Cell Membrane/metabolism , Centrifugation, Density Gradient , Extracellular Matrix/metabolism , Ionophores/pharmacology , Mice , Microscopy, Confocal/methods , NIH 3T3 Cells , Phosphorylation , Sucrose/pharmacology , Tyrosine/chemistry
20.
J Cell Sci ; 124(Pt 9): 1453-64, 2011 May 01.
Article in English | MEDLINE | ID: mdl-21486955

ABSTRACT

Extracellular proteolysis is an indispensable requirement for the formation of new blood vessels during neovascularization and is implicated in the generation of several angiogenic regulatory molecules. Anti-proteolytic agents have become attractive therapeutic strategies in diseases associated with excessive neovascularization. Annexin A2 (AnxA2) is an endothelial cell-surface receptor for the generation of active proteolytic factors, such as plasmin. Here, we show that AnxA2 is abundantly expressed in the neovascular tufts in a murine model of neovascularization. Exposure to hypoxic conditions results in elevation of AnxA2 and tissue plasminogen activator (tPA) in human retinal microvascular endothelial cells (RMVECs). We show that the hexapeptide competitive inhibitor LCKLSL, which targets the N-terminal tPA-binding site of AnxA2, binds efficiently to cell-surface AnxA2 compared with binding of the control peptide LGKLSL. Treatment with the competitive peptide inhibits the generation of plasmin and suppresses the VEGF-induced activity of tPA under hypoxic conditions. Application of the competitive peptide in two in vivo models of angiogenesis demonstrated suppression of the angiogenic responses, which was also associated with significant changes in the vascular sprouting. These results suggest that AnxA2-mediated plasmin generation is an important event in angiogenesis and is inhibited by a specific competitive peptide that inhibits the binding of tPA to AnxA2.


Subject(s)
Annexin A2/antagonists & inhibitors , Cell Hypoxia/physiology , Neovascularization, Pathologic/drug therapy , Oligopeptides/therapeutic use , Animals , Cells, Cultured , Chick Embryo , Endothelial Cells/cytology , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Enzyme-Linked Immunosorbent Assay , Female , Humans , Mice , Microscopy, Confocal , Neovascularization, Pathologic/chemically induced , Protein Binding/drug effects , Retina/cytology , Retina/drug effects , Retina/pathology , Tissue Plasminogen Activator/metabolism
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