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1.
Int J Mol Sci ; 25(12)2024 Jun 12.
Article in English | MEDLINE | ID: mdl-38928162

ABSTRACT

Polyamine (PA) spermidine (SPD) plays a crucial role in aging. Since SPD accumulates in glial cells, particularly in Müller retinal cells (MCs), the expression of the SPD-synthesizing enzyme spermidine synthase (SpdS) in Müller glia and age-dependent SpdS activity are not known. We used immunocytochemistry, Western blot (WB), and image analysis on rat retinae at postnatal days 3, 21, and 120. The anti-glutamine synthetase (GS) antibody was used to identify glial cells. In the neonatal retina (postnatal day 3 (P3)), SpdS was expressed in almost all progenitor cells in the neuroblast. However, by day 21 (P21), the SpdS label was pronouncedly expressed in multiple neurons, while GS labels were observed only in radial Müller glial cells. During early cell adulthood, at postnatal day 120 (P120), SpdS was observed solely in ganglion cells and a few other neurons. Western blot and semi-quantitative analyses of SpdS labeling showed a dramatic decrease in SpdS at P21 and P120 compared to P3. In conclusion, the redistribution of SpdS with aging indicates that SPD is first synthesized in all progenitor cells and then later in neurons, but not in glia. However, MCs take up and accumulate SPD, regardless of the age-associated decrease in SPD synthesis in neurons.


Subject(s)
Ependymoglial Cells , Retina , Spermidine Synthase , Animals , Rats , Spermidine Synthase/metabolism , Spermidine Synthase/genetics , Retina/metabolism , Ependymoglial Cells/metabolism , Aging/metabolism , Spermidine/metabolism , Neuroglia/metabolism , Animals, Newborn
2.
Cancers (Basel) ; 14(11)2022 Jun 05.
Article in English | MEDLINE | ID: mdl-35681787

ABSTRACT

Triple-negative breast cancer (TNBC) is the most aggressive, metastatic, and lethal breast cancer subtype. To improve the survival of TNBC patients, it is essential to explore new signaling pathways for the further development of effective drugs. This study aims to investigate the role of the secretory carrier membrane protein 3 (SCAMP3) in TNBC and its association with the epidermal growth factor receptor (EGFR). Through an internalization assay, we demonstrated that SCAMP3 colocalizes and redistributes EGFR from the cytoplasm to the perinucleus. Furthermore, SCAMP3 knockout decreased proliferation, colony and tumorsphere formation, cell migration, and invasion of TNBC cells. Immunoblots and degradation assays showed that SCAMP3 regulates EGFR through its degradation. In addition, SCAMP3 modulates AKT, ERK, and STAT3 signaling pathways. TNBC xenograft models showed that SCAMP3 depletion delayed tumor cell proliferation at the beginning of tumor development and modulated the expression of genes from the PDGF pathway. Additionally, analysis of TCGA data revealed elevated SCAMP3 expression in breast cancer tumors. Finally, patients with TNBC with high expression of SCAMP3 showed decreased RFS and DMFS. Our findings indicate that SCAMP3 could contribute to TNBC development through the regulation of multiple pathways and has the potential to be a target for breast cancer therapy.

3.
J Biophotonics ; 15(6): e202200002, 2022 06.
Article in English | MEDLINE | ID: mdl-35243792

ABSTRACT

Eye shine in the dark has attracted many researchers to the field of eye optics, but the initial studies of subwavelength arrangements in tapetum began only with the development of electronic microscopy at the end of the 20th century. As a result of a number of studies, it was shown that the reflective properties of the tapetum are due to their specialized cellular subwavelength microstructure (photonic crystals). These properties, together with the mutual orientation of the crystals, lead to a significant increase in reflection, which, in turn, enhances the sensitivity of the eye. In addition, research confirmed that optical mechanisms of reflection in the tapetum are very similar even for widely separated species. Due to progress in the field of nano-optics, researchers now have a better understanding of the main principles of this phenomenon. In this review, we summarize electron microscopic and functional studies of tapetal structures in the main vertebrate classes. This allows data on the microstructure of the tapetum to be used to improve our understanding of the visual system.


Subject(s)
Choroid , Vertebrates , Animals , Choroid/ultrastructure , Microscopy, Electron
4.
FEBS Open Bio ; 12(1): 95-105, 2022 01.
Article in English | MEDLINE | ID: mdl-34592066

ABSTRACT

Accumulation of amyloid in breast cancer is a well-known phenomenon, but only immunoglobulin light-chain amyloidosis (AL) or transthyretin (TTR) amyloid had been detected in human breast tumor samples previously. We recently reported that another amyloidogenic peptide, amyloid beta (Aß), is present in an aggregated form in animal and human high-grade gliomas and suggested that it originates systemically from the blood, possibly generated by platelets. To study whether breast cancers are also associated with these Aß peptides and in what form, we used a nude mouse model inoculated with triple-negative inflammatory breast cancer cell (SUM-149) xenografts, which develop noticeable tumors. Immunostaining with two types of specific antibodies for Aß identified the clear presence of Aß peptides associated with (a) carcinoma cells and (b) extracellular aggregated amyloid (also revealed by Congo red and thioflavin S staining). Aß peptides, in both cells and in aggregated amyloid, were distributed in clear gradients, with maximum levels near blood vessels. We detected significant presence of amyloid precursor protein (APP) in the walls of blood vessels of tumor samples, as well as in carcinoma cells. Finally, we used ELISA to confirm the presence of elevated levels of mouse-generated Aß40 in tumors. We conclude that Aß in inflammatory breast cancer tumors, at least in a mouse model, is always present and is concentrated near blood vessels. We also discuss here the possible pathways of Aß accumulation in tumors and whether this phenomenon could represent the specific signature of high-grade cancers.


Subject(s)
Alzheimer Disease , Inflammatory Breast Neoplasms , Alzheimer Disease/metabolism , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Animals , Heterografts , Humans , Mice , Mice, Transgenic
5.
Front Immunol ; 11: 571083, 2020.
Article in English | MEDLINE | ID: mdl-33123145

ABSTRACT

As do many other immunity-related blood cells, platelets release antimicrobial peptides that kill bacteria, fungi, and even certain viruses. Here we review the literature suggesting that there is a similarity between the antimicrobials released by other blood cells and the amyloid-related Aß peptide released by platelets. Analyzing the literature, we also propose that platelet-generated Aß amyloidosis may be more common than currently recognized. This systemic Aß from a platelet source may participate in various forms of amyloidosis in pathologies ranging from brain cancer, glaucoma, skin Aß accumulation, and preeclampsia to Alzheimer's disease and late-stage Parkinson's disease. We also discuss the advantages and disadvantages of specific animal models for studying platelet-related Aß. This field is undergoing rapid change, as it evaluates competing ideas in the light of new experimental observations. We summarized both in order to clarify the role of platelet-generated Aß peptides in amyloidosis-related health disorders, which may be helpful to researchers interested in this growing area of investigation.


Subject(s)
Alzheimer Disease/immunology , Amyloid beta-Peptides/metabolism , Amyloidosis/immunology , Blood Platelets/immunology , Brain/immunology , Parkinson Disease/immunology , Animals , Autoantibodies/metabolism , Disease Models, Animal , Humans
6.
Biomolecules ; 9(8)2019 08 08.
Article in English | MEDLINE | ID: mdl-31398804

ABSTRACT

It is well known that amyloid beta (Aß) peptides are generated in blood vessels, released into the brain during thrombosis, and temporarily accumulate in this organ after injury. Here we demonstrate that 24 h after transient middle cerebral artery occlusion (tMCAO), one of the standard models of focal ischemic stroke, Aß peptide accumulates in the brain, concentrating on the blood vessel walls. Because Aß oligomers are known to induce significant damage to brain cells, they act as an additional damaging factor during ischemic stroke. Considering that they have been shown to form ion channels in cells, affecting osmotic balance, we used an Aß peptide channel blocker, tromethamine (2-amino-2-(hydroxymethyl) propane-1,3-diol), to prevent this additional injury. Tromethamine injected 0.1 g/100 g body weight intraperitoneally at 5 min before tMCAO decreased water content in the damaged hemisphere, as measured by dry brain weight. Congo red staining, which binds only to Aß oligomer plaques (amyloid), showed that there was no significant presence of plaques. Therefore, we suggest that Aß peptide oligomers are responsible for some of the brain damage during stroke and that blockage of the ion channels that they form could be beneficial in treating this complex neurological syndrome.


Subject(s)
Amyloid beta-Peptides/metabolism , Blood Vessels/chemistry , Brain/metabolism , Infarction, Middle Cerebral Artery/metabolism , Amyloid beta-Peptides/antagonists & inhibitors , Animals , Blood Vessels/drug effects , Blood Vessels/metabolism , Brain/drug effects , Brain/pathology , Female , Infarction, Middle Cerebral Artery/drug therapy , Infarction, Middle Cerebral Artery/pathology , Inflammation/drug therapy , Inflammation/metabolism , Inflammation/pathology , Male , Rats , Rats, Sprague-Dawley , Tromethamine/pharmacology
7.
Molecules ; 24(13)2019 Jun 28.
Article in English | MEDLINE | ID: mdl-31261621

ABSTRACT

In vivo tissue transparency in the visible light spectrum is beneficial for many research applications that use optical methods, whether it involves in vivo optical imaging of cells or their activity, or optical intervention to affect cells or their activity deep inside tissues, such as brain tissue. The classical view is that a tissue is transparent if it neither absorbs nor scatters light, and thus absorption and scattering are the key elements to be controlled to reach the necessary transparency. This review focuses on the latest genetic and chemical approaches for the decoloration of tissue pigments to reduce visible light absorption and the methods to reduce scattering in live tissues. We also discuss the possible molecules involved in transparency.


Subject(s)
Optical Imaging/methods , Optogenetics/methods , Animals , Humans , Light , Scattering, Radiation
8.
Nutrients ; 11(5)2019 May 19.
Article in English | MEDLINE | ID: mdl-31109134

ABSTRACT

Breast cancer (BC) is the second leading cause of cancer death among women worldwide. The main cause of BC morbidity and mortality is the invasiveness capacity of cancer cells that may lead to metastasis. Here, we aimed to investigate the therapeutic efficacy of Ganoderma lucidum extract (GLE)-a medicinal mushroom with anticancer properties-on BC motility via the Rac/Lamellipodin pathway. GLE treatment effects were tested on MDA-MB-231 breast cancer cells. The effects were tested on cell viability, migration and invasion. Pulldowns, immunoblotting, and immunofluorescence were used to measure Rac activity and the expression of proteins involved in cell migration and in lamellipodia formation, respectively. As a result, GLE suppressed BC cell viability, migration, and invasion capacity. GLE impaired Rac activity, as well as downregulated Lamellipodin, ENA/VASP, p-FAK (Tyr925), Cdc42, and c-Myc expression. Lamellipodia formation was significantly reduced by GLE. In conclusion, we demonstrate that GLE reduces Rac activity and downregulates signaling molecules involved in lamellipodia formation. These novel findings serve as basis for further studies to elucidate the potential of GLE as a therapeutic agent regulating the Rac/Lamellipodin pathway in BC metastasis.


Subject(s)
Antineoplastic Agents/therapeutic use , Biological Products/therapeutic use , Breast Neoplasms/therapy , Carrier Proteins/metabolism , Cell Movement/drug effects , Membrane Proteins/metabolism , Reishi , rac GTP-Binding Proteins/metabolism , Antineoplastic Agents/pharmacology , Biological Products/pharmacology , Breast Neoplasms/metabolism , Cell Line, Tumor , Cell Survival/drug effects , DNA-Binding Proteins/metabolism , Down-Regulation , Female , Humans , Neoplasm Invasiveness/prevention & control , Proto-Oncogene Proteins c-myc/metabolism , Pseudopodia/drug effects , Signal Transduction
9.
Int J Mol Sci ; 20(10)2019 May 20.
Article in English | MEDLINE | ID: mdl-31137462

ABSTRACT

Immunostaining with specific antibodies has shown that innate amyloid beta (Aß) is accumulated naturally in glioma tumors and nearby blood vessels in a mouse model of glioma. In immunofluorescence images, Aß peptide coincides with glioma cells, and enzyme-linked immunosorbent assay (ELISA) have shown that Aß peptide is enriched in the membrane protein fraction of tumor cells. ELISAs have also confirmed that the Aß(1-40) peptide is enriched in glioma tumor areas relative to healthy brain areas. Thioflavin staining revealed that at least some amyloid is present in glioma tumors in aggregated forms. We may suggest that the presence of aggregated amyloid in glioma tumors together with the presence of Aß immunofluorescence coinciding with glioma cells and the nearby vasculature imply that the source of Aß peptides in glioma can be systemic Aß from blood vessels, but this question remains unresolved and needs additional studies.


Subject(s)
Amyloid beta-Peptides/metabolism , Brain Neoplasms/metabolism , Glioblastoma/metabolism , Peptide Fragments/metabolism , Animals , Cell Line, Tumor , Mice , Mice, Inbred C57BL
10.
Histol Histopathol ; 34(8): 843-856, 2019 Aug.
Article in English | MEDLINE | ID: mdl-30945258

ABSTRACT

Amyloid beta (Aß) peptides have been implicated in both Alzheimer's disease (AD) and glaucoma and have been shown to be the key etiological factor in these dangerous health complications. On the other hand, it is well known that Aß peptide can be generated from its precursor protein and massively released from the blood to nearby tissue upon the activation of platelets due to their involvement in innate immunity and inflammation processes. Here we review evidence about the development of AD and glaucoma neuronal damage showing their dependence on platelet count and activation. The correlation between the effect on platelet count and the effectiveness of anti-AD and anti-glaucoma therapies suggest that platelets may be an important player in these diseases.


Subject(s)
Alzheimer Disease/blood , Amyloid beta-Peptides/metabolism , Amyloid beta-Protein Precursor/metabolism , Blood Platelets/metabolism , Glaucoma/blood , Alzheimer Disease/etiology , Glaucoma/etiology , Humans
11.
Microsc Microanal ; 24(5): 545-552, 2018 10.
Article in English | MEDLINE | ID: mdl-30253817

ABSTRACT

Transparent cells in the vertebrate optical tract, such as lens fiber cells and corneal epithelium cells, have specialized proteins that somehow permit only a low level of light scattering in their cytoplasm. It has been shown that both cell types contain (1) beaded intermediate filaments as well as (2) α-crystallin globulins. It is known that genetic and chemical alterations to these specialized proteins induce cytoplasmic opaqueness and visual complications. Crystallins were described previously in the retinal Müller cells of frogs. In the present work, using immunocytochemistry, fluorescence confocal imaging, and immuno-electron microscopy, we found that αA-crystallins are present in the cytoplasm of retinal Müller cells and in the photoreceptors of rats. Given that Müller glial cells were recently described as "living light guides" as were photoreceptors previously, we suggest that αA-crystallins, as in other highly transparent cells, allow Müller cells and photoreceptors to minimize intraretinal scattering during retinal light transmission.


Subject(s)
Ependymoglial Cells/metabolism , Lens, Crystalline/metabolism , Neuroglia/metabolism , Photoreceptor Cells/metabolism , alpha-Crystallins/metabolism , Animals , Cytoplasm/metabolism , Ependymoglial Cells/cytology , Eye/pathology , Immunohistochemistry , Lens, Crystalline/chemistry , Light , Microscopy, Immunoelectron , Optical Imaging , Photoreceptor Cells/cytology , Rats , Rats, Sprague-Dawley , Retina/cytology , Retina/metabolism , Retinal Rod Photoreceptor Cells/cytology , Retinal Rod Photoreceptor Cells/metabolism , alpha-Crystallin A Chain/chemistry , alpha-Crystallin A Chain/metabolism , alpha-Crystallins/chemistry
12.
Neuroscience ; 384: 54-63, 2018 08 01.
Article in English | MEDLINE | ID: mdl-29800717

ABSTRACT

A-kinase-anchoring proteins, AKAPs, are scaffolding proteins that associate with kinases and phosphatases, and direct them to a specific submembrane site to coordinate signaling events. AKAP150, a rodent ortholog of human AKAP79, has been extensively studied in neurons, but very little is known about the localization and function of AKAP150 in astrocytes, the major cell type in brain. Thus, in this study, we assessed the localization of AKAP150 in astrocytes and elucidated its role during physiological and ischemic conditions. Herein, we demonstrate that AKAP150 is localized in astrocytes and is up-regulated during ischemia both in vitro and in vivo. Knock-down of AKAP150 by RNAi depolarizes the astrocytic membrane potential and substantially reduces by 80% the ability of astrocytes to take up extracellular potassium during ischemic conditions. Therefore, upregulation of AKAP150 during ischemia preserves potassium conductance and the associated hyperpolarized membrane potential of astrocytes; properties of astrocytes needed to maintain extracellular brain homeostasis. Taken together, these data suggest that AKAP150 may play a pivotal role in the neuroprotective mechanism of astrocytes during pathological conditions.


Subject(s)
A Kinase Anchor Proteins/metabolism , Astrocytes/metabolism , Brain Ischemia/metabolism , Stroke/metabolism , Up-Regulation , Animals , Male , Neurons/metabolism , Rats , Rats, Sprague-Dawley
13.
Exp Eye Res ; 173: 160-178, 2018 08.
Article in English | MEDLINE | ID: mdl-29753728

ABSTRACT

It has been shown that mammalian retinal glial (Müller) cells act as living optical fibers that guide the light through the retinal tissue to the photoreceptor cells (Agte et al., 2011; Franze et al., 2007). However, for nonmammalian species it is unclear whether Müller cells also improve the transretinal light transmission. Furthermore, for nonmammalian species there is a lack of ultrastructural data of the retinal cells, which, in general, delivers fundamental information of the retinal function, i.e. the vision of the species. A detailed study of the cellular ultrastructure provides a basic approach of the research. Thus, the aim of the present study was to investigate the retina of the spectacled caimans at electron and light microscopical levels to describe the structural features. For electron microscopy, we used a superfast microwave fixation procedure in order to achieve more precise ultrastructural information than common fixation techniques. As result, our detailed ultrastructural study of all retinal parts shows structural features which strongly indicate that the caiman retina is adapted to dim light and night vision. Various structural characteristics of Müller cells suppose that the Müller cell may increase the light intensity along the path of light through the neuroretina and, thus, increase the sensitivity of the scotopic vision of spectacled caimans. Müller cells traverse the whole thickness of the neuroretina and thus may guide the light from the inner retinal surface to the photoreceptor cell perikarya and the Müller cell microvilli between the photoreceptor segments. Thick Müller cell trunks/processes traverse the layers which contain light-scattering structures, i.e., nerve fibers and synapses. Large Müller cell somata run through the inner nuclear layer and contain flattened, elongated Müller cell nuclei which are arranged along the light path and, thus, may reduce the loss of the light intensity along the retinal light path. The oblique arrangement of many Müller cell trunks/processes in the inner plexiform layer and the large Müller cell somata in the inner nuclear layer may suggest that light guidance through Müller cells increases the visual sensitivity. Furthermore, an adaptation of the caiman retina to low light levels is strongly supported by detailed ultrastructural data of other retinal parts, e.g. by (i) the presence of a guanine-based retinal tapetum, (ii) the rod dominance of the retina, (iii) the presence of photoreceptor cell nuclei, which penetrate the outer limiting membrane, (iv) the relatively low densities of photoreceptor and neuronal cells which is compensated by (v) the presence of rods with long and thick outer segments, that may increase the probability of photon absorption. According to a cell number analysis, the central and temporal areas of the dorsal tapetal retina, which supports downward prey detection in darker water, are the sites of the highest diurnal contrast/color vision, i.e. cone vision and of the highest retinal light sensitivity, i.e. rod vision.


Subject(s)
Adaptation, Ocular/physiology , Alligators and Crocodiles , Night Vision/physiology , Retina/ultrastructure , Animals , Cell Count , Female , Male , Microscopy, Electron , Photoreceptor Cells, Vertebrate/ultrastructure , Retina/physiology , Retinal Pigment Epithelium/ultrastructure
14.
Exp Eye Res ; 173: 91-108, 2018 08.
Article in English | MEDLINE | ID: mdl-29763583

ABSTRACT

In this study, we show the capability of Müller glial cells to transport light through the inverted retina of reptiles, specifically the retina of the spectacled caimans. Thus, confirming that Müller cells of lower vertebrates also improve retinal light transmission. Confocal imaging of freshly isolated retinal wholemounts, that preserved the refractive index landscape of the tissue, indicated that the retina of the spectacled caiman is adapted for vision under dim light conditions. For light transmission experiments, we used a setup with two axially aligned objectives imaging the retina from both sides to project the light onto the inner (vitreal) surface and to detect the transmitted light behind the retina at the receptor layer. Simultaneously, a confocal microscope obtained images of the Müller cells embedded within the vital tissue. Projections of light onto several representative Müller cell trunks within the inner plexiform layer, i.e. (i) trunks with a straight orientation, (ii) trunks which are formed by the inner processes and (iii) trunks which get split into inner processes, were associated with increases in the intensity of the transmitted light. Projections of light onto the periphery of the Müller cell endfeet resulted in a lower intensity of transmitted light. In this way, retinal glial (Müller) cells support dim light vision by improving the signal-to-noise ratio which increases the sensitivity to light. The field of illuminated photoreceptors mainly include rods reflecting the rod dominance of the of tissue. A subpopulation of Müller cells with downstreaming cone cells led to a high-intensity illumination of the cones, while the surrounding rods were illuminated by light of lower intensity. Therefore, Müller cells that lie in front of cones may adapt the intensity of the transmitted light to the different sensitivities of cones and rods, presumably allowing a simultaneous vision with both receptor types under dim light conditions.


Subject(s)
Alligators and Crocodiles/physiology , Ependymoglial Cells/physiology , Light , Night Vision/physiology , Retina/physiology , Vision, Ocular/physiology , Animals , Eye Proteins/metabolism , Female , Male , Microscopy, Confocal , Retinal Cone Photoreceptor Cells/physiology , Retinal Rod Photoreceptor Cells/physiology
15.
Invest Ophthalmol Vis Sci ; 58(7): 3046-3054, 2017 06 01.
Article in English | MEDLINE | ID: mdl-28622396

ABSTRACT

Purpose: The mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch's membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1-/- mice. Methods: Proteoglycans in mouse Bruch's membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch's membrane/choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography. Results: HSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch's membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch's membrane was impaired. In contrast, the proteoglycan amount in Efemp1-/- Bruch's membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch's membrane. Diffusion across Efemp1-/- Bruch's membrane was enhanced. Conclusions: Mutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch's membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch's membrane.


Subject(s)
Bruch Membrane/metabolism , DNA/genetics , Extracellular Matrix Proteins/genetics , Macular Degeneration/genetics , Mutation , Proteoglycans/metabolism , Aging/genetics , Animals , Bruch Membrane/pathology , DNA Mutational Analysis , Disease Models, Animal , Extracellular Matrix Proteins/metabolism , Macular Degeneration/diagnosis , Macular Degeneration/metabolism , Mice , Mice, Mutant Strains
16.
J Biol Chem ; 291(14): 7716-26, 2016 Apr 01.
Article in English | MEDLINE | ID: mdl-26867573

ABSTRACT

TheKCNJ10gene encoding Kir4.1 contains numerous SNPs whose molecular effects remain unknown. We investigated the functional consequences of uncharacterized SNPs (Q212R, L166Q, and G83V) on homomeric (Kir4.1) and heteromeric (Kir4.1-Kir5.1) channel function. We compared these with previously characterized EAST/SeSAME mutants (G77R and A167V) in kidney-derived tsA201 cells and in glial cell-derived C6 glioma cells. The membrane potentials of tsA201 cells expressing G77R and G83V were significantly depolarized as compared with WTKir4.1, whereas cells expressing Q212R, L166Q, and A167V were less affected. Furthermore, macroscopic currents from cells expressing WTKir4.1 and Q212R channels did not differ, whereas currents from cells expressing L166Q, G83V, G77R, and A167V were reduced. Unexpectedly, L166Q current responses were rescued when co-expressed with Kir5.1. In addition, we observed notable differences in channel activity between C6 glioma cells and tsA201 cells expressing L166Q and A167V, suggesting that there are underlying differences between cell lines in terms of Kir4.1 protein synthesis, stability, or expression at the surface. Finally, we determined spermine (SPM) sensitivity of these uncharacterized SNPs and found that Q212R-containing channels displayed reduced block by 1 µmSPM. At 100 µmSPM, the block was equal to or greater than WT, suggesting that the greater driving force of SPM allowed achievement of steady state. In contrast, L166Q-Kir5.1 channels achieved a higher block than WT, suggesting a more stable interaction of SPM in the deep pore cavity. Overall, our data suggest that G83V, L166Q, and Q212R residues play a pivotal role in controlling Kir4.1 channel function.


Subject(s)
Mutation, Missense , Polymorphism, Single Nucleotide , Potassium Channels, Inwardly Rectifying/metabolism , Amino Acid Substitution , Animals , Cell Line, Tumor , Potassium Channels, Inwardly Rectifying/genetics , Rats , Kir5.1 Channel
17.
PLoS One ; 10(4): e0125195, 2015.
Article in English | MEDLINE | ID: mdl-25886567

ABSTRACT

Excitotoxicity due to glutamate receptor over-activation is one of the key mediators of neuronal death after an ischemic insult. Therefore, a major function of astrocytes is to maintain low extracellular levels of glutamate. The ability of astrocytic glutamate transporters to regulate the extracellular glutamate concentration depends upon the hyperpolarized membrane potential of astrocytes conferred by the presence of K+ channels in their membranes. We have previously shown that TREK-2 potassium channels in cultured astrocytes are up-regulated by ischemia and may support glutamate clearance by astrocytes during ischemia. Thus, herein we determine the mechanism leading to this up-regulation and assess the localization of TREK-2 channels in astrocytes after transient middle cerebral artery occlusion. By using a cell surface biotinylation assay we confirmed that functional TREK-2 protein is up-regulated in the astrocytic membrane after ischemic conditions. Using real time RT-PCR, we determined that the levels of TREK-2 mRNA were not increased in response to ischemic conditions. By using Western blot and a variety of protein synthesis inhibitors, we demonstrated that the increase of TREK-2 protein expression requires De novo protein synthesis, while protein degradation pathways do not contribute to TREK-2 up-regulation after ischemic conditions. Immunohistochemical studies revealed TREK-2 localization in astrocytes together with increased expression of the selective glial marker, glial fibrillary acidic protein, in brain 24 hours after transient middle cerebral occlusion. Our data indicate that functional TREK-2 channels are up-regulated in the astrocytic membrane during ischemia through a mechanism requiring De novo protein synthesis. This study provides important information about the mechanisms underlying TREK-2 regulation, which has profound implications in neurological diseases such as ischemia where astrocytes play an important role.


Subject(s)
Astrocytes/metabolism , Ischemic Attack, Transient/metabolism , Potassium Channels, Tandem Pore Domain/metabolism , Protein Biosynthesis , Animals , Astrocytes/pathology , Cell Membrane/metabolism , Cells, Cultured , Glial Fibrillary Acidic Protein/metabolism , Humans , Infarction, Middle Cerebral Artery/metabolism , Infarction, Middle Cerebral Artery/pathology , Ischemic Attack, Transient/pathology , Potassium Channels, Tandem Pore Domain/genetics , Rats, Sprague-Dawley , Up-Regulation
18.
PLoS One ; 9(5): e97155, 2014.
Article in English | MEDLINE | ID: mdl-24831221

ABSTRACT

BACKGROUND: Müller cells, the principal glial cells of the vertebrate retina, are fundamental for the maintenance and function of neuronal cells. In most vertebrates, including humans, Müller cells abundantly express Kir4.1 inwardly rectifying potassium channels responsible for hyperpolarized membrane potential and for various vital functions such as potassium buffering and glutamate clearance; inter-species differences in Kir4.1 expression were, however, observed. Localization and function of potassium channels in Müller cells from the retina of crocodiles remain, hitherto, unknown. METHODS: We studied retinae of the Spectacled caiman (Caiman crocodilus fuscus), endowed with both diurnal and nocturnal vision, by (i) immunohistochemistry, (ii) whole-cell voltage-clamp, and (iii) fluorescent dye tracing to investigate K+ channel distribution and glia-to-neuron communications. RESULTS: Immunohistochemistry revealed that caiman Müller cells, similarly to other vertebrates, express vimentin, GFAP, S100ß, and glutamine synthetase. In contrast, Kir4.1 channel protein was not found in Müller cells but was localized in photoreceptor cells. Instead, 2P-domain TASK-1 channels were expressed in Müller cells. Electrophysiological properties of enzymatically dissociated Müller cells without photoreceptors and isolated Müller cells with adhering photoreceptors were significantly different. This suggests ion coupling between Müller cells and photoreceptors in the caiman retina. Sulforhodamine-B injected into cones permeated to adhering Müller cells thus revealing a uni-directional dye coupling. CONCLUSION: Our data indicate that caiman Müller glial cells are unique among vertebrates studied so far by predominantly expressing TASK-1 rather than Kir4.1 K+ channels and by bi-directional ion and uni-directional dye coupling to photoreceptor cells. This coupling may play an important role in specific glia-neuron signaling pathways and in a new type of K+ buffering.


Subject(s)
Ependymoglial Cells/cytology , Photoreceptor Cells, Vertebrate/cytology , Potassium Channels, Inwardly Rectifying/metabolism , Retina/physiology , Alligators and Crocodiles/metabolism , Animals , Fluorescent Dyes/chemistry , Glutamates/metabolism , Ion Channel Gating , Membrane Potentials , Nerve Tissue Proteins/metabolism , Neurons/metabolism , Potassium/chemistry , Potassium Channels, Tandem Pore Domain/metabolism , Protein Structure, Tertiary , Retina/metabolism , Signal Transduction
19.
J Neurosci Neuroeng ; 3(2): 85-91, 2014 12 01.
Article in English | MEDLINE | ID: mdl-25866702

ABSTRACT

Birds which possess high visual acuity, such as eagles and falcons, are known to have retinas with a deep conically curved central foveal pit. There have been different attempts to explain the importance of this particular shape of the fovea in visual resolution. Recently, the function of Müller cells as "light fibers" was discovered, showing how the endfeet of Müller cells trap the light and then transfer it to a single cone photoreceptor. Here we describe how the endfeet of Müller cells line the walls of the foveal pit in the Pied Flycatcher, and how the Müller cell body extends its processes towards individual cones, forming machinery that could allow for light transfer from the pit wall to the photoreceptor layer alongside the pit. We describe how this construction may send an image from the fovea to the cones, and also, how the angular positioning of Müller cells, being optical extensions of the cones, has the advantage of being much denser than on a flat or slightly curved fovea. We, therefore, suggest that this type of optic fiber alignment can be used as a novel type of "amplifying array" that simply increases the amount of megapixels at the photoreceptor cell layer.

20.
Invest Ophthalmol Vis Sci ; 52(7): 4907-13, 2011 Jul 01.
Article in English | MEDLINE | ID: mdl-21642629

ABSTRACT

PURPOSE: To determine the effect of Stokes radius (R(S)) on the diffusion of molecules through Bruch's membrane (BM), and to establish a system suitable for the analysis of diffusion through small (<2 mm(2)) samples of BM. METHODS: Porcine BM/choroid (BM/Ch) was mounted in a modified Ussing chamber. A concentration gradient was simultaneously established for four tracers with R(S) values ranging from <1.0 to 6.15 nm. Samples were collected from both chambers at various time points up to 36 hours and the amount of each tracer was determined using quantitative gel exclusion chromatography. The integrity of samples was determined using scanning electron microscopy. RESULTS: BM/Ch mounted in the chamber exhibited no obvious damage even after 36 hours in the chamber. Flux was significantly (P < 0.05) greater in the BM to Ch direction than that in the Ch to BM direction for only two of the tracers: cytosine and RNase A. Flux also was dependent on R(S); cytosine, the smallest tracer (R(S) < 1 nm), exhibited the greatest flux and ferritin (R(S) = 6.15 nm) the least. Permeability coefficients for each tracer were determined and exhibited a power relationship with R(S). CONCLUSIONS: Flux was dependent on the direction of the concentration gradient and the R(S) of the individual tracers. We have successfully demonstrated that quantitative gel exclusion chromatography can be used to follow diffusion of a mixture of tracers across BM/Ch, and that we can measure flux across BM/Ch preparations with an exposed surface area as small as 1.8 mm(2).


Subject(s)
Bruch Membrane/metabolism , Cell Membrane Permeability/physiology , Macromolecular Substances/pharmacokinetics , Serum Albumin/pharmacokinetics , Animals , Biological Transport , Bruch Membrane/ultrastructure , Chromatography, Gel , Disease Models, Animal , Macular Degeneration/metabolism , Macular Degeneration/pathology , Microscopy, Electron, Scanning , Swine
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