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1.
Aging Dis ; 2024 04 22.
Artículo en Inglés | MEDLINE | ID: mdl-38739943

RESUMEN

Prior studies have emphasized a bioenergetic crisis in the retinal pigment epithelium (RPE) as a critical factor in the development of age-related macular degeneration (AMD). The isoforms Fructose-1,6-bisphosphate aldolase C (ALDOC) and pyruvate kinase M2 (PKM2) have been proposed to play a role in AMD pathogenesis. While PKM2 and ALDOC are crucial for aerobic glycolysis in the neural retina, they are not as essential for the RPE. In this study, we examined the expression and activity of PKM2 and ALDOC in both young and aged RPE cells, as well as in the retina and RPE tissue of mice, including an experimentally induced AMD mouse model. Our findings reveal an upregulation in PKM2 and ALDOC expression, accompanied by increased pyruvate kinase activity, in the aged and AMD mouse RPE. Conversely, there is a decrease in ALDOC expression but an increase in PKM2 expression and pyruvate kinase activity in the aged and AMD retina. Overall, our study indicates that aged and AMD RPE cells tend to favor aerobic glycolysis, while this tendency is diminished in the aged and AMD retina. These results underscore the significance of targeting PKM2 and ALDOC in the RPE as a promising therapeutic approach to address the bioenergetic crisis and prevent vision loss in AMD.

2.
Aging Dis ; 2024 Jan 30.
Artículo en Inglés | MEDLINE | ID: mdl-38300646

RESUMEN

Insulin-like growth factor-1 (IGF-1) plays a diverse role in the retina, exerting its effects in both normal and diseased conditions. Deficiency of IGF-1 in humans leads to issues such as microcephaly, mental retardation, deafness, and postnatal growth failure. IGF-1 is produced in the retinal pigment epithelium (RPE) and activates the IGF-1 receptor (IGF-1R) in photoreceptor cells. When IGF-1R is absent in rod cells, it results in the degeneration of photoreceptors, emphasizing the neuroprotective function of IGF signaling in these cells. Contrastingly, in neovascular age-related macular degeneration (AMD), there is an overexpression of both IGF-1 and IGF-1R in RPE. The mechanisms behind this altered regulation of IGF-1 in diseased states are currently unknown. This comprehensive review provides recent insights into the role of IGF-1 in the health and disease of the retina, raising several unanswered questions that still need further investigation.

3.
PNAS Nexus ; 2(3): pgad063, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-37007713

RESUMEN

Phosphoinositides (PIPs) are a family of minor acidic phospholipids in the cell membrane. Phosphoinositide (PI) kinases and phosphatases can rapidly convert one PIP product into another resulting in the generation of seven distinct PIPs. The retina is a heterogeneous tissue composed of several cell types. In the mammalian genome, around 50 genes encode PI kinases and PI phosphatases; however, there are no studies describing the distribution of these enzymes in the various retinal cell types. Using translating ribosome affinity purification, we have identified the in vivo distribution of PI-converting enzymes from the rod, cone, retinal pigment epithelium (RPE), Müller glia, and retinal ganglion cells, generating a physiological atlas for PI-converting enzyme expression in the retina. The retinal neurons, rods, cones, and RGCs, are characterized by the enrichment of PI-converting enzymes, whereas the Müller glia and RPE are characterized by the depletion of these enzymes. We also found distinct differences between the expression of PI kinases and PI phosphatases in each retinal cell type. Since mutations in PI-converting enzymes are linked to human diseases including retinal diseases, the results of this study will provide a guide for what cell types are likely to be affected by retinal degenerative diseases brought on by changes in PI metabolism.

4.
PNAS Nexus ; 2(3): pgad038, 2023 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-36896135

RESUMEN

The postmitotic retina is highly metabolic and the photoreceptors depend on aerobic glycolysis for an energy source and cellular anabolic activities. Lactate dehydrogenase A (LDHA) is a key enzyme in aerobic glycolysis, which converts pyruvate to lactate. Here we show that cell-type-specific actively translating mRNA purification by translating ribosome affinity purification shows a predominant expression of LDHA in rods and cones and LDHB in the retinal pigment epithelium and Müller cells. We show that genetic ablation of LDHA in the retina resulted in diminished visual function, loss of structure, and a loss of dorsal-ventral patterning of the cone-opsin gradient. Loss of LDHA in the retina resulted in increased glucose availability, promoted oxidative phosphorylation, and upregulated the expression of glutamine synthetase (GS), a neuron survival factor. However, lacking LDHA in Müller cells does not affect visual function in mice. Glucose shortage is associated with retinal diseases, such as age-related macular degeneration (AMD), and regulating the levels of LDHA may have therapeutic relevance. These data demonstrate the unique and unexplored roles of LDHA in the maintenance of a healthy retina.

5.
Cell Death Dis ; 13(7): 613, 2022 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-35840554

RESUMEN

Insulin-like growth factor I (IGF-1) is a neurotrophic factor and is the ligand for insulin-like growth factor 1 receptor (IGF-1R). Reduced expression of IGF-1 has been reported to cause deafness, mental retardation, postnatal growth failure, and microcephaly. IGF-1R is expressed in the retina and photoreceptor neurons; however, its functional role is not known. Global IGF-1 KO mice have age-related vision loss. We determined that conditional deletion of IGF-1R in photoreceptors and pan-retinal cells produces age-related visual function loss and retinal degeneration. Retinal pigment epithelial cell-secreted IGF-1 may be a source for IGF-1R activation in the retina. Altered retinal, fatty acid, and phosphoinositide metabolism are observed in photoreceptor and retinal cells lacking IGF-1R. Our results suggest that the IGF-1R pathway is indispensable for photoreceptor survival, and activation of IGF-1R may be an essential element of photoreceptor and retinal neuroprotection.


Asunto(s)
Factor I del Crecimiento Similar a la Insulina , Células Fotorreceptoras de Vertebrados , Degeneración Retiniana , Animales , Factor I del Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/metabolismo , Ratones , Neuronas/metabolismo , Neuroprotección/genética , Neuroprotección/fisiología , Células Fotorreceptoras de Vertebrados/metabolismo , Receptor IGF Tipo 1/genética , Receptor IGF Tipo 1/metabolismo , Retina/metabolismo , Degeneración Retiniana/genética , Degeneración Retiniana/metabolismo
6.
J Biol Chem ; 298(6): 101944, 2022 06.
Artículo en Inglés | MEDLINE | ID: mdl-35447116

RESUMEN

Mechanistic target of rapamycin (mTOR) and mTOR complex 1 (mTORC1), linchpins of the nutrient sensing and protein synthesis pathways, are present at relatively high levels in the ganglion cell layer (GCL) and retinal ganglion cells (RGCs) of rodent and human retinas. However, the role of mTORCs in the control of protein synthesis in RGC is unknown. Here, we applied the SUrface SEnsing of Translation (SUnSET) method of nascent protein labeling to localize and quantify protein synthesis in the retinas of adult mice. We also used intravitreal injection of an adeno-associated virus 2 vector encoding Cre recombinase in the eyes of mtor- or rptor-floxed mice to conditionally knockout either both mTORCs or only mTORC1, respectively, in cells within the GCL. A novel vector encoding an inactive Cre mutant (CreΔC) served as control. We found that retinal protein synthesis was highest in the GCL, particularly in RGC. Negation of both complexes or only mTORC1 significantly reduced protein synthesis in RGC. In addition, loss of mTORC1 function caused a significant reduction in the pan-RGC marker, RNA-binding protein with multiple splicing, with little decrease of the total number of cells in the RGC layer, even at 25 weeks after adeno-associated virus-Cre injection. These findings reveal that mTORC1 signaling is necessary for maintaining the high rate of protein synthesis in RGCs of adult rodents, but it may not be essential to maintain RGC viability. These findings may also be relevant to understanding the pathophysiology of RGC disorders, including glaucoma, diabetic retinopathy, and optic neuropathies.


Asunto(s)
Glaucoma , Células Ganglionares de la Retina , Animales , Glaucoma/metabolismo , Diana Mecanicista del Complejo 1 de la Rapamicina/metabolismo , Ratones , Retina/metabolismo , Células Ganglionares de la Retina/metabolismo
7.
Artículo en Inglés | MEDLINE | ID: mdl-35427794

RESUMEN

Reversible phosphorylation of phosphatidylinositol by phosphoinositide (PI) kinases and phosphatases generates seven distinct phosphoinositide phosphates, called phosphoinositides or PIPs. All seven PIPs are formed in the retina and photoreceptor cells. Around 50 genes in the mammalian genome encode PI kinases and PI phosphatases. There are no studies available on the distribution of these enzymes in the retina and photoreceptors. AIM: To employ Ribosomal Targeting Strategy and Nuclear Labeling to Analyze Phosphoinositide Signatures in rod-photoreceptor cells. METHODS: HA-tagging of ribosomal protein Rpl22 was induced with Cre-recombinase under the control of the rhodopsin promoter. Actively translating mRNAs associated with polyribosomes were isolated by immunoprecipitation with HA antibody, followed by RNA isolation and gene identification. We also isolated biotinylated-rod nuclei from NuTRAP mice under the control of the rhodopsin-Cre promoter and analyzed nuclear phosphoinositides. RESULTS: Our results indicate that the expression of class I and class III PI 3-kinase, PI4K IIIß, PI 5-kinase, PIKfyve, PI3-phosphatases, MTMR2, 4, 6, 7, 14, PI4-phosphatase, TMEM55A, PI 5-phosphatases, SYNJI, INPP5B, INPP5E, INPP5F, SKIP and other phosphatases with dual substrate specificity, PTPMT1, SCAM1, and FIG4 are highly enriched in rod photoreceptor cells compared with the retina and cone-like retina. Our analysis identified the presence of PI(4)P, PI(3,4)P2, PI(3,5)P2, and PI(4,5)P2 in the rod nuclei. CONCLUSIONS: Our studies for the first time demonstrate the expression of PI kinases, PI phosphatases, and nuclear PIPs in rod photoreceptor cells. The NuTRAP mice may be useful not only for epigenetic and transcriptomic studies but also for in vivo cell-specific lipidomics research.


Asunto(s)
Monoéster Fosfórico Hidrolasas , Células Fotorreceptoras , Ribosomas , 1-Fosfatidilinositol 4-Quinasa , Animales , Flavoproteínas , Ratones , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfoinosítido Fosfatasas , Monoéster Fosfórico Hidrolasas/genética , Monoéster Fosfórico Hidrolasas/metabolismo , Células Fotorreceptoras/metabolismo , Proteínas Tirosina Fosfatasas no Receptoras , Rodopsina
8.
Commun Biol ; 4(1): 850, 2021 07 08.
Artículo en Inglés | MEDLINE | ID: mdl-34239035

RESUMEN

The retinal pigmented epithelium (RPE) is a monolayer of multifunctional cells located at the back of the eye. High membrane turnover and polarization, including formation of actin-based apical microvilli, are essential for RPE function and retinal health. Herein, we demonstrate an important role for ßA3/A1-crystallin in RPE. ßA3/A1-crystallin deficiency leads to clathrin-mediated epidermal growth factor receptor (EGFR) endocytosis abnormalities and actin network disruption at the apical side that result in RPE polarity disruption and degeneration. We found that ßA3/A1-crystallin binds to phosphatidylinositol transfer protein (PITPß) and that ßA3/A1-crystallin deficiency diminishes phosphatidylinositol 4,5-biphosphate (PI(4,5)P2), thus probably decreasing ezrin phosphorylation, EGFR activation, internalization, and degradation. We propose that ßA3/A1-crystallin acquired its RPE function before evolving as a structural element in the lens, and that in the RPE, it modulates the PI(4,5)P2 pool through PITPß/PLC signaling axis, coordinates EGFR activation, regulates ezrin phosphorylation and ultimately the cell polarity.


Asunto(s)
Polaridad Celular/fisiología , Endocitosis , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Cadena A de beta-Cristalina/metabolismo , Animales , Polaridad Celular/genética , Proteínas del Citoesqueleto/metabolismo , Células Epiteliales/ultraestructura , Humanos , Ratones Noqueados , Fosfatidilinositol 4,5-Difosfato/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Fosforilación , Unión Proteica , Epitelio Pigmentado de la Retina/citología , Cadena A de beta-Cristalina/genética
9.
Biomolecules ; 11(4)2021 04 19.
Artículo en Inglés | MEDLINE | ID: mdl-33921658

RESUMEN

Protein tyrosine kinases and protein phosphatases play a critical role in cellular regulation. The length of a cellular response depends on the interplay between activating protein kinases and deactivating protein phosphatases. Protein tyrosine phosphatase 1B (PTP1B) and growth factor receptor-bound protein 14 (Grb14) are negative regulators of receptor tyrosine kinases. However, in the retina, we have previously shown that PTP1B inactivates insulin receptor signaling, whereas phosphorylated Grb14 inhibits PTP1B activity. In silico docking of phosphorylated Grb14 and PTP1B indicate critical residues in PTP1B that may mediate the interaction. Phosphoinositides (PIPs) are acidic lipids and minor constituents in the cell that play an important role in cellular processes. Their levels are regulated by growth factor signaling. Using phosphoinositide binding protein probes, we observed increased levels of PI(3)P, PI(4)P, PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 in PTP1B knockout mouse retina and decreased levels of these PIPs in Grb14 knockout mouse retina. These observations suggest that the interplay between PTP1B and Grb14 can regulate PIP metabolism.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Fosfatidilinositoles/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 1/metabolismo , Retina/metabolismo , Proteínas Adaptadoras Transductoras de Señales/química , Animales , Sitios de Unión , Ratones , Unión Proteica , Proteína Tirosina Fosfatasa no Receptora Tipo 1/química
10.
J Lipid Res ; 62: 100041, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-32540927

RESUMEN

The field of phosphoinositide signaling has expanded significantly in recent years. Phosphoinositides (also known as phosphatidylinositol phosphates or PIPs) are universal signaling molecules that directly interact with membrane proteins or with cytosolic proteins containing domains that directly bind phosphoinositides and are recruited to cell membranes. Through the activities of phosphoinositide kinases and phosphoinositide phosphatases, seven distinct phosphoinositide lipid molecules are formed from the parent molecule, phosphatidylinositol. PIP signals regulate a wide range of cellular functions, including cytoskeletal assembly, membrane budding and fusion, ciliogenesis, vesicular transport, and signal transduction. Given the many excellent reviews on phosphoinositide kinases, phosphoinositide phosphatases, and PIPs in general, in this review, we discuss recent studies and advances in PIP lipid signaling in the retina. We specifically focus on PIP lipids from vertebrate (e.g., bovine, rat, mouse, toad, and zebrafish) and invertebrate (e.g., Drosophila, horseshoe crab, and squid) retinas. We also discuss the importance of PIPs revealed from animal models and human diseases, and methods to study PIP levels both in vitro and in vivo. We propose that future studies should investigate the function and mechanism of activation of PIP-modifying enzymes/phosphatases and further unravel PIP regulation and function in the different cell types of the retina.


Asunto(s)
Fosfatidilinositoles
11.
Biology (Basel) ; 9(11)2020 Nov 07.
Artículo en Inglés | MEDLINE | ID: mdl-33171845

RESUMEN

The major pathway for the production of the low-abundance membrane lipid phosphatidylinositol 3-phosphate (PI(3)P) synthesis is catalyzed by class III phosphoinositide 3-kinase (PI3K) Vps34. The absence of Vps34 was previously found to disrupt autophagy and other membrane-trafficking pathways in some sensory neurons, but the roles of phosphatidylinositol 3-phosphate and Vps34 in cone photoreceptor cells have not previously been explored. We found that the deletion of Vps34 in neighboring rods in mouse retina did not disrupt cone function up to 8 weeks after birth, despite diminished rod function. Immunoblotting and lipid analysis of cones isolated from the cone-dominant retinas of the neural retina leucine zipper gene knockout mice revealed that both PI(3)P and Vps34 protein are present in mouse cones. To determine whether Vps34 and PI(3)P are important for cone function, we conditionally deleted Vps34 in cone photoreceptor cells of the mouse retina. Overall retinal morphology and rod function appeared to be unaffected. However, the loss of Vps34 in cones resulted in the loss of structure and function. There was a substantial reduction throughout the retina in the number of cones staining for M-opsin, S-opsin, cone arrestin, and peanut agglutinin, revealing degeneration of cones. These studies indicate that class III PI3K, and presumably PI(3)P, play essential roles in cone photoreceptor cell function and survival.

12.
Exp Eye Res ; 197: 108131, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32622801

RESUMEN

The retina is one of the most metabolically active tissues, yet the processes that control retinal metabolism remains poorly understood. The mTOR complex (mTORC) that drives protein and lipid biogenesis and autophagy has been studied extensively in regards to retinal development and responses to optic nerve injury but the processes that regulate homeostasis in the adult retina have not been determined. We previously demonstrated that normal adult retina has high rates of protein synthesis compared to skeletal muscle, associated with high levels of mechanistic target of rapamycin (mTOR), a kinase that forms multi-subunit complexes that sense and integrate diverse environmental cues to control cell and tissue physiology. This study was undertaken to: 1) quantify expression of mTOR complex 1 (mTORC1)- and mTORC2-specific partner proteins in normal adult rat retina, brain and liver; and 2) to localize these components in normal human, rat, and mouse retinas. Immunoblotting and immunoprecipitation studies revealed greater expression of raptor (exclusive to mTORC1) and rictor (exclusive for mTORC2) in normal rat retina relative to liver or brain, as well as the activating mTORC components, pSIN1 and pPRAS40. By contrast, liver exhibits greater amounts of the mTORC inhibitor, DEPTOR. Immunolocalization studies for all three species showed that mTOR, raptor, and rictor, as well as most other known components of mTORC1 and mTORC2, were primarily localized in the inner retina with mTORC1 primarily in retinal ganglion cells (RGCs) and mTORC2 primarily in glial cells. In addition, phosphorylated ribosomal protein S6, a direct target of the mTORC1 substrate ribosomal protein S6 kinase beta-1 (S6K1), was readily detectable in RGCs, indicating active mTORC1 signaling, and was preserved in human donor eyes. Collectively, this study demonstrates that the inner retina expresses high levels of mTORC1 and mTORC2 and possesses active mTORC1 signaling that may provide cell- and tissue-specific regulation of homeostatic activity. These findings help to define the physiology of the inner retina, which is key for understanding the pathophysiology of optic neuropathies, glaucoma and diabetic retinopathy.


Asunto(s)
Regulación de la Expresión Génica , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Diana Mecanicista del Complejo 2 de la Rapamicina/genética , ARN/genética , Enfermedades de la Retina/genética , Células Ganglionares de la Retina/metabolismo , Animales , Modelos Animales de Enfermedad , Humanos , Immunoblotting , Masculino , Diana Mecanicista del Complejo 1 de la Rapamicina/biosíntesis , Diana Mecanicista del Complejo 2 de la Rapamicina/biosíntesis , Ratones , Ratones Endogámicos C57BL , ARN/metabolismo , Ratas , Ratas Sprague-Dawley , Enfermedades de la Retina/metabolismo , Enfermedades de la Retina/patología , Células Ganglionares de la Retina/patología , Transducción de Señal
13.
Biology (Basel) ; 9(6)2020 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-32545642

RESUMEN

Inositol phospholipids play an important role in cell physiology. The inositol head groups are reversibly phosphorylated to produce seven distinct phosphorylated inositides, commonly referred to as phosphoinositides (PIs). These seven PIs are dynamically interconverted from one PI to another by the action of PI kinases and PI phosphatases. The PI signals regulate a wide variety of cellular functions, including organelle distinction, vesicular transport, cytoskeletal organization, nuclear events, regulation of ion channels, cell signaling, and host-pathogen interactions. Most of the studies of PIs in ocular tissues are based on the PI enzymes and PI phosphatases. In this study, we examined the PI levels in the cornea, retinal pigment epithelium (RPE), and retina using PI-binding protein as probes. We have examined the lipids PI(3)P, PI(4)P, PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3, and each is present in the cornea, RPE, and retina. Alterations in the levels of these PIs in mouse models of retinal disease and corneal infections have been reported, and the results of our study will help in the management of anomalous phosphoinositide metabolism in ocular tissues.

14.
Front Cell Dev Biol ; 8: 266, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-32426353

RESUMEN

One hundred years ago, Otto Heinrich Warburg observed that postmitotic retinal cells are the highest oxygen-consuming cells in the body. He compared these cells to actively growing mitotic tumor cells since both cells reprogram glucose for anabolic processes, which include lipid, protein, and RNA/DNA synthesis, and for antioxidant metabolism. To achieve this metabolic reprogramming, cancer cells preferentially express a less active dimeric form, the M2 isoform of pyruvate kinase (PKM2), which shuttles glucose toward the accumulation of glycolytic intermediates that redirect cell activities into anabolic processes. Similar to cancer cells, retinal photoreceptors predominantly express the M2 isoform of PKM2. This isoform performs both metabolic and non-metabolic functions in photoreceptor cells. This review focuses on the metabolic and non-metabolic roles of pyruvate kinases in photoreceptor cell functions.

15.
Sci Rep ; 10(1): 7456, 2020 05 04.
Artículo en Inglés | MEDLINE | ID: mdl-32366925

RESUMEN

The main therapeutic goal for diabetic retinopathy (DR) is to prevent vision loss in patients with diabetes mellitus. Identifying the visual complications at a preclinical juncture will offer an early therapeutic window for diagnosis and intervention. Very recently, we found that pyruvate kinase M2 isoform (PKM2) regulates visual function through regulation of a key enzyme, phosphodiesterase 6ß (Pde6ß), involved in modulating photoreceptor functions. A recent study showed that the activation of PKM2 protects mitochondrial integrity in diabetic nephropathy. In the present study, we examined the role of PKM2 in DR in a mouse model that has both phenotypes of obesity and type II diabetes. In DR, we found decreased expression of PKM2 and Pde6ß expression, but not PKM1. Consistent with decreased Pde6ß expression, the db/db mice had reduced rod photoreceptor function. We found increased pyruvate kinase activity and a decreased ratio of reduced/oxidized redox in db/db mouse retina compared with control retinas. There was no significant difference in the levels of lactate between db/db and control mouse retina. Our findings suggest that reduced expression of PKM2 with unchanged PKM1 expression might be responsible for higher pyruvate kinase activity in db/db mouse retina. Our studies suggest that PKM2 has a role in DR. The results support that PKM2 may serve as a therapeutic target in the treatment of DR.


Asunto(s)
Retinopatía Diabética/enzimología , Piruvato Quinasa/metabolismo , Animales , Fosfodiesterasas de Nucleótidos Cíclicos Tipo 6/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Modelos Animales de Enfermedad , Electrorretinografía , Regulación Enzimológica de la Expresión Génica , Glicerol/metabolismo , Glucólisis , Isoenzimas , Ácido Láctico/metabolismo , Ratones , Ratones Endogámicos C57BL , Oxidación-Reducción , Fenotipo , Retina/metabolismo , Epitelio Pigmentado de la Retina/metabolismo
16.
Cell Biol Int ; 44(4): 1020-1027, 2020 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-31889373

RESUMEN

We previously reported a ligand-independent and rhodopsin-dependent insulin receptor (IR) neuroprotective signaling pathway in both rod and cone photoreceptor cells, which is activated through protein-protein interaction. Our previous studies were performed with either retina or isolated rod or cone outer segment preparations and the expression of IR signaling proteins were examined. The isolation of outer segments with large portions of the attached inner segments is a technical challenge. Optiprep™ density gradient medium has been used to isolate the cells and subcellular organelles, Optiprep™ is a non-ionic iodixanol-based medium with a density of 1.320 g/mL. We employed this method to examine the expression of IR and its signaling proteins, and activation of one of the downstream effectors of the IR in isolated photoreceptor cells. Identification of the signaling complexes will be helpful for therapeutic targeting in disease conditions.


Asunto(s)
Receptor de Insulina/metabolismo , Segmento Interno de las Células Fotorreceptoras Retinianas/metabolismo , Segmento Externo de las Células Fotorreceptoras Retinianas/metabolismo , Rodopsina/metabolismo , Animales , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Segmento Interno de las Células Fotorreceptoras Retinianas/ultraestructura , Segmento Externo de las Células Fotorreceptoras Retinianas/ultraestructura , Transducción de Señal
17.
J Mol Biol Ther ; 1: 44-55, 2019.
Artículo en Inglés | MEDLINE | ID: mdl-34528026

RESUMEN

Several nanotechnology podiums have gained remarkable attention in the area of medical sciences, including diagnostics and treatment. In the past decade, engineered multifunctional nanoparticles have served as drug and gene carriers. The most important aspect of translating nanoparticles from the bench to bedside is safety. These nanoparticles should not elicit any immune response and should not be toxic to humans or the environment. Lipid-based nanoparticles have been shown to be the least toxic for in vivo applications, and significant progress has been made in gene and drug delivery employing lipid-based nanoassemblies. Several excellent reviews and reports discuss the general use and application of lipid-based nanoparticles; our review focuses on the application of lipid-based nanoparticles for the treatment of ocular diseases, and recent advances in and updates on their use.

18.
Cell Death Dis ; 9(7): 737, 2018 07 03.
Artículo en Inglés | MEDLINE | ID: mdl-29970877

RESUMEN

The tumor form of pyruvate kinase M2 has been suggested to promote cellular anabolism by redirecting the metabolism to cause accumulation of glycolytic intermediates and increasing flux through the pentose phosphate pathway, which is a metabolic pathway parallel to glycolysis. Both rod and cone photoreceptors express the tumor form of pyruvate kinase M2. Recent studies from our laboratory show that PKM2 is functionally important for rod photoreceptor structure, function, and viability. However, the functional role of PKM2 in cones is not known. In this study, we conditionally deleted PKM2 in cones (cone-cre PKM2-KO) and found that loss of PKM2 results in the upregulation of PKM1 and a significant loss of cone function and cone degeneration in an age-dependent manner. Gene expression studies on cone-cre PKM2-KO show decreased expression of genes regulating glycolysis, PPP shunt, and fatty acid biosynthesis. Consistent with these observations, cones lacking PKM2 have significantly shorter cone outer segments than cones with PKM2. Our studies clearly suggest that PKM2 is essential for the anabolic process in cones to keep them alive for normal functioning and to support cone structure.


Asunto(s)
Piruvato Quinasa/metabolismo , Animales , Electrorretinografía , Glucólisis/genética , Glucólisis/fisiología , Immunoblotting , Ratones , Ratones Noqueados , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Piruvato Quinasa/genética , Retina/metabolismo , Retina/patología , Células Fotorreceptoras Retinianas Conos/metabolismo , Células Fotorreceptoras Retinianas Conos/patología
19.
Adv Exp Med Biol ; 1074: 117-123, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29721935

RESUMEN

Over the last few years, huge progress has been made in the understanding of molecular mechanisms underlying the pathogenesis of retinal degenerative diseases. Such knowledge has led to the development of gene therapy approaches to treat these devastating disorders. Non-viral gene delivery has been recognized as a prospective treatment for retinal degenerative diseases. In this review, we will summarize the constituent characteristics and recent applications of three representative nanoparticles (NPs) in ocular therapy.


Asunto(s)
Técnicas de Transferencia de Gen , Terapia Genética/métodos , Vectores Genéticos/administración & dosificación , Nanopartículas/administración & dosificación , Degeneración Retiniana/terapia , Animales , Humanos , Liposomas/administración & dosificación , Nanopartículas del Metal/administración & dosificación , Ratones Noqueados , Polietilenglicoles/administración & dosificación , Polímeros , cis-trans-Isomerasas/deficiencia , cis-trans-Isomerasas/genética
20.
Adv Exp Med Biol ; 1074: 491-497, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29721981

RESUMEN

Studies form our laboratory and others show that the oncogenic tyrosine kinase and serine threonine kinase signaling pathways are essential for cone photoreceptor survival. These pathways are downregulated in mouse models of retinal degenerative diseases. In the present study, we found that activation mutants of mTOR delayed the death of cones in a mouse model of retinal degeneration. These studies suggest that oncogenic protein kinases may be useful as therapeutic agents to treat retinal degenerations that affect cones.


Asunto(s)
Células Fotorreceptoras Retinianas Conos/efectos de los fármacos , Retinitis Pigmentosa/terapia , Serina-Treonina Quinasas TOR/uso terapéutico , Animales , Factores de Transcripción con Cremalleras de Leucina de Carácter Básico/deficiencia , Supervivencia Celular , Modelos Animales de Enfermedad , Proteínas del Ojo , Genes Sintéticos , Inyecciones Intraoculares , Ratones , Ratones Endogámicos BALB C , Ratones Noqueados , Ratones Mutantes , Regiones Promotoras Genéticas , Receptor de Insulina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/uso terapéutico , Serina-Treonina Quinasas TOR/genética
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