Lysosomal basification and decreased autophagic flux in oxidatively stressed trabecular meshwork cells

Porter, Kristine; Jeyabalan, Nallathambi; Lin, Yizhi; Liton, Paloma B.

doi:10.4161/auto.23568

Cited by 104 publications

(110 citation statements)

References 48 publications

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“…34 Indeed, our laboratory has recently demonstrated activation of autophagy in response to chronic oxidative stress in TM cells. 35 Interestingly, the results presented here clearly indicate the induction of autophagy with AA supplementation. Furthermore, higher LC3-II levels, and therefore a greater level of induction, were observed with those concentrations in which AA behaves as an antioxidant.…”

Section: Discussionsupporting

confidence: 54%

“…Interestingly, our laboratory has reported diminished autophagic degradation capacity and impaired proteolytic activation of cathepsins, in particular CTSB, in TM cells subjected to chronic oxidative stress. 35 Ongoing experiments are being conducted to determine whether supplementation of the TM cell cultures with AA could prevent or revert such an impaired autophagic capacity.…”

Section: Discussionmentioning

confidence: 99%

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Ascorbic Acid Modulation of Iron Homeostasis and Lysosomal Function in Trabecular Meshwork Cells

Lin

Porter

et al. 2014

Journal of Ocular Pharmacology and Therapeutics

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Purpose: To investigate the antioxidant properties and biological functions of ascorbic acid (AA) on trabecular meshwork (TM) cells. Methods: Primary cultures of porcine TM cells were supplemented for 10 days with increasing concentrations of AA. Antioxidant properties against cytotoxic effect of H 2 O 2 were evaluated by monitoring cell viability. Redox-active iron was quantified using calcein-AM. Intracellular reactive oxygen species (iROS) production was quantified using H 2 DCFDA. Ferritin and cathepsin protein levels were analyzed by Western blot. Autophagy was evaluated by monitoring lipidation of LC3-I to LC3-II. Lysosomal proteolysis and cathepsins activities were quantified using specific fluorogenic substrates. Results: AA exerts a dual effect against oxidative stress in TM cells, acting as an anti-oxidant or a pro-oxidant, depending on the concentration used. The pro-oxidant effect of AA was mediated by free intracellular iron and correlated with increased protein levels of ferritin and elevated iROS. In contrast, antioxidant properties correlated with lower ferritin and basal iROS content. Ascorbic acid supplementation also caused induction of autophagy, as well as increased lysosomal proteolysis, with the latter resulting from higher proteolytic activation of lysosomal cathepsins in treated cultures. Conclusions: Our results suggest that the reported decrease of AA levels in plasma and aqueous humor can compromise lysosomal degradation in the outflow pathway cells with aging and contribute to the pathogenesis of glaucoma. Restoration of physiological levels of vitamin C inside the cells might improve their ability to degrade proteins within the lysosomal compartment and recover tissue function.

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Section: Discussionsupporting

confidence: 54%

Section: Discussionmentioning

confidence: 99%

Ascorbic Acid Modulation of Iron Homeostasis and Lysosomal Function in Trabecular Meshwork Cells

Lin

Porter

et al. 2014

Journal of Ocular Pharmacology and Therapeutics

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“…Images were acquired using a HCX PL APO 63x/1.4 NA oil immersion objective at a pixel resolution of 240 £ 240 or 80 £ 80 nm and further processed with Photoshop 7.0. In order to make counting of autophagosomes and autolysosomes (yellow and red fluorescent dots, respectively) reliable and basically independent of the operator, 62 the Green and Red Puncta Colocalization ImageJ Plugin (created by Daniel J. Shiwarski, University of Pittsburgh; enhanced and modified by Ruben K. Dagda, University of Nevada School of Medicine and coapproved as an image tool for autophagic flux by Charleen T. Chu, University of Pittsburgh) was used without any setting change or adjustment on a number (indicated in each relevant histogram) of single-cell images extracted from different microscopic fields (3 to 6 for each experimental condition). 63 Autophagy and lysosomal delivery of MT For detection of MT autophagy, a construct (pcDNA3-hMT2A-mCherry) encoding a human (Homo sapiens, Hs) MT2A-mCherry was generated by ligase-independent cloning of the Mt2a cDNA (PCR-amplified from pcDNA3-GFPHsMT2A; Addgene plasmid 11613, deposited by S. Johnson) into the pcDNA3-mCherry LIC cloning vector (6B) (Addgene plasmid 30125, deposited by S. Gradia).…”

Section: Determination Of Autophagic Fluxmentioning

confidence: 99%

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

et al. 2015

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, tandem fluorescent microtubule-associated protein 1 light chain 3 A/B (green/red fluorescent proteins chimera); TNFRSF1A/TNFR1, tumor necrosis factor receptor superfamily member 1A; TNFRSF1B/TNFR2, tumor necrosis factor receptor superfamily member 1B.Lysosomal membrane permeabilization (LMP) induced by oxidative stress has recently emerged as a prominent mechanism behind TNF cytotoxicity. This pathway relies on diffusion of hydrogen peroxide into lysosomes containing redox-active iron, accumulated by breakdown of iron-containing proteins and subcellular organelles. Upon oxidative lysosomal damage, LMP allows relocation to the cytoplasm of low mass iron and acidic hydrolases that contribute to DNA and mitochondrial damage, resulting in death by apoptosis or necrosis. Here we investigate the role of lysosomes and free iron in death of HTC cells, a rat hepatoma line, exposed to TNF following metallothionein (MT) upregulation. Iron-binding MT does not normally occur in HTC cells in significant amounts. Intracellular iron chelation attenuates TNF and cycloheximide (CHX)-induced LMP and cell death, demonstrating the critical role of this transition metal in mediating cytokine lethality. MT upregulation, combined with starvation-activated MT autophagy almost completely suppresses TNF and CHX toxicity, while impairment of both autophagy and MT upregulation by silencing of Atg7, and Mt1a and/or Mt2a, respectively, abrogates protection. Interestingly, MT upregulation by itself has little effect, while stimulated autophagy alone depresses cytokine toxicity to some degree. These results provide evidence that intralysosomal iron-catalyzed redox reactions play a key role in TNF and CHX-induced LMP and toxicity. The finding that chelation of intralysosomal iron achieved by autophagic delivery of MT, and to some degree probably of other ironbinding proteins as well, into the lysosomal compartment is highly protective provides a putative mechanism to explain autophagy-related suppression of death by TNF and CHX.

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“…20 A recent study described lysosomal basification and decreased autophagic flux in travecular meshwork cells in response to chronic oxidative stress, with important implications for the pathogenesis of glaucoma. 21 Furthermore, specific Atg5 deletion in pigment epithelium leads to reduced levels of visual pigments and vision alterations, 22 indicating that autophagy has an important role in sustaining retinal pigment epithelium function.…”

mentioning

confidence: 99%

Lysosomal membrane permeabilization and autophagy blockade contribute to photoreceptor cell death in a mouse model of retinitis pigmentosa

et al. 2014

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Retinitis pigmentosa is a group of hereditary retinal dystrophies that normally result in photoreceptor cell death and vision loss both in animal models and in affected patients. The rd10 mouse, which carries a missense mutation in the Pde6b gene, has been used to characterize the underlying pathophysiology and develop therapies for this devastating and incurable disease. Here we show that increased photoreceptor cell death in the rd10 mouse retina is associated with calcium overload and calpain activation, both of which are observed before the appearance of signs of cell degeneration. These changes are accompanied by an increase in the activity of the lysosomal protease cathepsin B in the cytoplasm of photoreceptor cells, and a reduced colocalization of cathepsin B with lysosomal markers, suggesting that lysosomal membrane permeabilization occurs before the peak of cell death. Moreover, expression of the autophagosomal marker LC3-II (lipidated form of LC3) is reduced and autophagy flux is blocked in rd10 retinas before the onset of photoreceptor cell death. Interestingly, we found that cell death is increased by the induction of autophagy with rapamycin and inhibited by calpain and cathepsin inhibitors, both ex vivo and in vivo. Taken together, these data suggest that calpain-mediated lysosomal membrane permeabilization underlies the lysosomal dysfunction and downregulation of autophagy associated with photoreceptor cell death.

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Lysosomal basification and decreased autophagic flux in oxidatively stressed trabecular meshwork cells

Cited by 104 publications

References 48 publications

Ascorbic Acid Modulation of Iron Homeostasis and Lysosomal Function in Trabecular Meshwork Cells

Ascorbic Acid Modulation of Iron Homeostasis and Lysosomal Function in Trabecular Meshwork Cells

Autophagy of metallothioneins prevents TNF-induced oxidative stress and toxicity in hepatoma cells

Lysosomal membrane permeabilization and autophagy blockade contribute to photoreceptor cell death in a mouse model of retinitis pigmentosa

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