Autophagy deficiency protects against ocular hypertension and neurodegeneration in experimental and spontanous glaucoma mouse models.

Glaucoma is a group of diseases that leads to chronic degeneration of retinal ganglion cell (RGC) axons and progressive loss of RGCs, resulting in vision loss. While aging and elevated intraocular pressure (IOP) have been identified as the main contributing factors to glaucoma, the molecular mechanisms and signaling pathways triggering RGC death and axonal degeneration are not fully understood. Previous studies in our laboratory found that overactivation of autophagy in DBA/2J::GFP-LC3 mice led to RGC death and optic nerve degeneration with glaucomatous IOP elevation. We found similar findings in aging GFP-LC3 mice subjected to chronic IOP elevation. Here, we further investigated the impact of autophagy deficiency on autophagy-deficient DBA/2J-Atg4bko and DBA/2J-Atg4b+/- mice, generated in our laboratory via CRISPR/Cas9 technology; as well as in Atg4bko mice subjected to the experimental TGFß2 chronic ocular hypertensive model. Our data shows that, in contrast to DBA/2J and DBA/2J-Atg4b+/- littermates, DBA/2J-Atg4bko mice do not develop glaucomatous IOP elevation. Atg4b deficiency also protected against glaucomatous IOP elevation in the experimental TGFß2 chronic ocular hypertensive model. Atg4 deletion did not compromise RGC or optic nerve survival in Atg4bko mice. Moreover, our results indicate a protective role of autophagy deficiency against RGC death and ON atrophy in the hypertensive DBA/2J-Atg4b+/- mice. Together, our data suggests a pathogenic role of autophagy activation in ocular hypertension and glaucoma.

Shear stress induces autophagy in Schlemm's canal cells via primary cilia-mediated SMAD2/3 signaling pathway.

The Schlemm's canal (SC) is a circular, lymphatic-like vessel located at the limbus of the eye that participates in the regulation of aqueous humor drainage to control intraocular pressure (IOP). Circumferential flow of aqueous humor within the SC lumen generates shear stress, which regulates SC cell behaviour. Using biochemical analysis and real-time live cell imaging techniques, we have investigated the activation of autophagy in SC cells by shear stress. We report, for the first time, the primary cilium (PC)-dependent activation of autophagy in SC cells in response to shear stress. Moreover, we identified PC-dependent shear stress-induced autophagy to be positively regulated by phosphorylation of SMAD2 in its linker and C-terminal regions. Additionally, SMAD2/3 signaling was found to transcriptionally activate LC3B, ATG5 and ATG7 in SC cells. Intriguingly, concomitant to SMAD2-dependent activation of autophagy, we also report here the activation of mTOR pathway, a classical autophagy inhibitor, in SC cells by shear stress. mTOR activation was found to also be dependent on the PC. Moreover, pharmacological inhibition of class I PI3K increased phosphorylation of SMAD2 at the linker and activated autophagy. Together, our data indicates an interplay between PI3K and SMAD2/3 signaling pathways in the regulation of PC-dependent shear stress-induced autophagy in SC cells.

SEPHS1: Its evolution, function and roles in development and diseases.

Selenophosphate synthetase (SEPHS) was originally discovered in prokaryotes as an enzyme that catalyzes selenophosphate synthesis using inorganic selenium and ATP as substrates. However, in contrast to prokaryotes, two paralogs, SEPHS1 and SEPHS2, occur in many eukaryotes. Prokaryotic SEPHS, also known as SelD, contains either cysteine (Cys) or selenocysteine (Sec) in the catalytic domain. In eukaryotes, only SEPHS2 carries out selenophosphate synthesis and contains Sec at the active site. However, SEPHS1 contains amino acids other than Sec or Cys at the catalytic position. Phylogenetic analysis of SEPHSs reveals that the ancestral SEPHS contains both selenophosphate synthesis and another unknown activity, and that SEPHS1 lost the selenophosphate synthesis activity. The three-dimensional structure of SEPHS1 suggests that its homodimer is unable to form selenophosphate, but retains ATPase activity to produce ADP and inorganic phosphate. The most prominent function of SEPHS1 is that it is implicated in the regulation of cellular redox homeostasis. Deficiency of SEPHS1 leads to the disturbance in the expression of genes involved in redox homeostasis. Different types of reactive oxygen species (ROS) are accumulated in response to SEPHS deficiency depending on cell or tissue types. The accumulation of ROS causes pleiotropic effects such as growth retardation, apoptosis, DNA damage, and embryonic lethality. SEPHS1 deficiency in mouse embryos affects retinoic signaling and other related signaling pathways depending on the embryonal stage until the embryo dies at E11.5. Dysregulated SEPHS1 is associated with the pathogenesis of various diseases including cancer, Crohn's disease, and osteoarthritis.

The physiological and pathophysiological roles of the autophagy lysosomal system in the conventional aqueous humor outflow pathway: More than cellular clean up.

During the last few years, the autophagy lysosomal system is emerging as a central cellular pathway with roles in survival, acting as a housekeeper and stress response mechanism. Studies by our and other labs suggest that autophagy might play an essential role in maintaining aqueous humor outflow homeostasis, and that malfunction of autophagy in outflow pathway cells might predispose to ocular hypertension and glaucoma pathogenesis. In this review, we will collect the current knowledge and discuss the molecular mechanisms by which autophagy does or might regulate normal outflow pathway tissue function, and its response to different types of stressors (oxidative stress and mechanical stress). We will also discuss novel roles of autophagy and lysosomal enzymes in modulation of TGFß signaling and ECM remodeling, and the link between dysregulated autophagy and cellular senescence. We will examine what we have learnt, using pre-clinical animal models about how dysregulated autophagy can contribute to disease and apply that to the current status of autophagy in human glaucoma. Finally, we will consider and discuss the challenges and the potential of autophagy as a therapeutic target for the treatment of ocular hypertension and glaucoma.

Primary cilia and the reciprocal activation of AKT and SMAD2/3 regulate stretch-induced autophagy in trabecular meshwork cells.

Activation of autophagy is one of the responses elicited by high intraocular pressure (IOP) and mechanical stretch in trabecular meshwork (TM) cells. However, the mechanosensor and the molecular mechanisms by which autophagy is induced by mechanical stretch in these or other cell types is largely unknown. Here, we have investigated the mechanosensor and downstream signaling pathway that regulate cyclic mechanical stretch (CMS)-induced autophagy in TM cells. We report that primary cilia act as a mechanosensor for CMS-induced autophagy and identified a cross-regulatory talk between AKT1 and noncanonical SMAD2/3 signaling as critical components of primary cilia-mediated activation of autophagy by mechanical stretch. Furthermore, we demonstrated the physiological significance of our findings in ex vivo perfused eyes. Removal of primary cilia disrupted the homeostatic IOP compensatory response and prevented the increase in LC3-II protein levels in response to elevated pressure challenge, strongly supporting a role of primary cilia-mediated autophagy in regulating IOP homeostasis.

Cathepsin B Localizes in the Caveolae and Participates in the Proteolytic Cascade in Trabecular Meshwork Cells. Potential New Drug Target for the Treatment of Glaucoma.

Extracellular matrix (ECM) deposition in the trabecular meshwork (TM) is one of the hallmarks of glaucoma, a group of human diseases and leading cause of permanent blindness. The molecular mechanisms underlying ECM deposition in the glaucomatous TM are not known, but it is presumed to be a consequence of excessive synthesis of ECM components, decreased proteolytic degradation, or both. Targeting ECM deposition might represent a therapeutic approach to restore outflow facility in glaucoma. Previous work conducted in our laboratory identified the lysosomal enzyme cathepsin B (CTSB) to be expressed on the cellular surface and to be secreted into the culture media in trabecular meshwork (TM) cells. Here, we further investigated the role of CTSB on ECM remodeling and outflow physiology in vitro and in CSTBko mice. Our results indicate that CTSB localizes in the caveolae and participates in the pericellular degradation of ECM in TM cells. We also report here a novel role of CTSB in regulating the expression of PAI-1 and TGFß/Smad signaling in TM cells vitro and in vivo in CTSBko mice. We propose enhancing CTSB activity as a novel therapeutic target to attenuate fibrosis and ECM deposition in the glaucomatous outflow pathway.

Corrigendum to "Inhibition of cAMP/PKA Pathway Protects Optic Nerve Head Astrocytes against Oxidative Stress by Akt/Bax Phosphorylation-Mediated Mfn1/2 Oligomerization".

[This corrects the article DOI: 10.1155/2019/8060962.].

Autophagy in the Aging and Experimental Ocular Hypertensive Mouse Model.

Purpose: To investigate autophagy in the outflow pathway and ganglion cell layer in the aging and ocular hypertensive mouse. Methods: Both 4-month-old and 18-month-old C57BL/6J and GFP-LC3 mice were subjected to unilateral injection of hypertonic saline into a limbal vein, causing sclerosis of the outflow pathway and subsequent elevation of intraocular pressure (IOP). IOP was measured on a weekly basis using a rebound tonometer. Protein expression levels of LC3B, Lamp1, and p62 were evaluated by western blot and/or immunofluorescence. Retinal ganglion cell (RGC) count was performed in whole retinal flat mounts using an anti-Brn3a antibody. Optic nerves were fixed with 4% paraformaldehyde and resin-embedded for axon counts and electron microscopy. Results: In contrast to 18-month-old mice, which developed sustained elevated IOP with a single injection, 4-month-old mice were refractory to high elevations of IOP. Interestingly, both the percentage of animals that developed elevated IOP and the mean ∆IOP were significantly higher in the transgenic mice compared to C57BL/6J. Immunofluorescence and western blot analysis showed dysregulated autophagy in the iridocorneal and retina tissues from 18-month-old mice compared to 4-month-old ones. Moreover, the LC3-II/LC3-I ratio correlated with IOP. As expected, injected hypertensive eyes displayed axonal degeneration and RGC death. RGC and axon loss were significantly exacerbated with aging, especially when combined with GFP-LC3 expression. Autophagic structures were observed in the degenerating axons. Conclusions: Our results indicate dysregulation of autophagy in the trabecular meshwork and retinal tissues with aging and suggest that such dysregulation of autophagy contributes to neurodegeneration in glaucoma.

The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress.

The trabecular meshwork (TM) is a key regulatory tissue of intraocular pressure (IOP) in the anterior chamber of eye. Dysfunction of the TM causes resistance to outflow of aqueous humor, which in turn leads to elevated IOP, a main risk factor of glaucomatous neurodegeneration. Due to variations in IOP, TM cells are continuously exposed to mechanical deformations. We previously reported activation of macroautophagy/autophagy, as one of the physiological responses elicited in TM cells following mechanical strain application. By using biochemical fractionation analysis and imaging techniques, we demonstrate here for the first time the nuclear accumulation of the autophagic marker MAP1LC3/LC3 (microtubule associated protein1 light chain 3)-II, endogenous and exogenously added (AdGFP-LC3, AdtfLC3), in response to cyclic mechanical stress (CMS). Wheat germ agglutinin (WGA) and leptomycin B treatment suggest LC3 to enter the nucleus by passive diffusion, but to exit in an XPO1/CRM1 (exportin 1)-dependent manner in human TM (hTM) cells. While blockage of nuclear export leads to accumulation of LC3 with promyelocytic leukemia (PML) bodies, nuclear LC3 localizes in the nucleolus in cells under CMS. Moreover, nuclear LC3 co-immunoprecipitated with NUFIP1, a ribosome receptor for starvation-induced ribophagy. More interestingly, we further demonstrate that NUFIP1 translocates from the nucleus to LAMP2 (lysosomal associated membrane protein 2)-positive organelles in the stretched cells without triggering ribophagy, suggesting a more general role of NUFIP1 as a selective autophagy receptor for another yet-to-be-identified target in CMS and a surveillance role of nuclear LC3 against stretch-induced damage. ABBREVIATION: AdGFP: adenovirus encoding GFP; ATG: autophagy-related; BSA: bovine serum albumin; CMS: cyclic mechanical stretch; Co-IP: coimmunoprecipitation; DAPI: 4',6-diamidino-2-phenylindole; DFCs: dense fibrillar components; EM: electron microscopy; FCs: fibrillar centers; GCs: granular components; GFP: green fluorescent protein; hTM: human trabecular meshwork; HBSS: Hanks balanced salt solution; IOP: intraocular pressure; LAMP1/2: lysosomal associated membrane protein 1/2; LepB: leptomycin B; MTOR: mechanistic target of rapamacyin kinase; NES: nuclear export signals; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; NLS: nuclear localization signal; NPCs: nuclear pore complexes; NUFIP1: nuclear FMR1 interacting protein 1; NS: non-stretched; PBS: phosphate-buffered saline; PE: phosphatidylethanolamine; pfu: plaque-forming units; PML: promyelocytic leukemia; RFP: red fluorescent protein; RPS15A: ribosomal protein S15a; RPL26: ribosomal protein L26; rRNA: ribosomal RNA; SIRT1: sirtuin 1; SQSTM1/p62: sequestosome 1; tfLC3: mRFP-GFP tandem fluorescent-tagged LC3; TM: trabecular meshwork; WB: western blot; WDR36: WD repeat domain 36; WGA: wheat germ agglutinin; XPO1/CRM1: exportin 1.

Transcriptome analysis reveals autophagy as regulator of TGFß/Smad-induced fibrogenesis in trabecular meshwork cells.

The trabecular meshwork (TM) is a specialized ocular tissue, which is responsible, together with the Schlemm's canal (SC), for maintaining appropriate levels of intraocular pressure. Dysfunction of these tissues leads to ocular hypertension and increases the risk for developing glaucoma. Previous work by our laboratory revealed dysregulated autophagy in aging and in glaucomatous TM cells. In order to gain more insight in the role of autophagy in the TM pathophysiology, we have conducted transcriptome and functional network analyses of TM primary cells with silenced expression of the autophagy genes Atg5 and Atg7. Atg5/7-deficient TM cells showed changes in transcript levels of several fibrotic genes, including TGFß2, BAMBI, and SMA. Furthermore, genetic and pharmacological inhibition of autophagy was associated with a parallel reduction in TGFß-induced fibrosis, caused by a BAMBI-mediated reduced activation of Smad2/3 signaling in autophagy-deficient cells. At the same time, TGFß treatment led to Smad2/3-dependent dysregulation of autophagy in TM cells, characterized by increased LC3-II levels and autophagic vacuoles content. Together, our results indicate a cross-talk between autophagy and TGFß signaling in TM cells.

Inhibition of cAMP/PKA Pathway Protects Optic Nerve Head Astrocytes against Oxidative Stress by Akt/Bax Phosphorylation-Mediated Mfn1/2 Oligomerization.

Glaucoma is characterized by a progressive optic nerve degeneration and retinal ganglion cell loss, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role in glaucomatous neurodegeneration. Here, we investigate the impact of activation of cyclic adenosine 3',5'-monophosphate (cAMP)/protein kinase A (PKA) pathway on mitochondrial dynamics of ONH astrocytes exposed to oxidative stress. ONH astrocytes showed a significant loss of astrocytic processes in the glial lamina of glaucomatous DBA/2J mice, accompanied by basement membrane thickening and collagen deposition in blood vessels and axonal degeneration. Serial block-face scanning electron microscopy data analysis demonstrated that numbers of total and branched mitochondria were significantly increased in ONH astrocytes, while mitochondrial length and volume density were significantly decreased. We found that hydrogen peroxide- (H2O2-) induced oxidative stress compromised not only mitochondrial bioenergetics by reducing the basal and maximal respiration but also balance of mitochondrial dynamics by decreasing dynamin-related protein 1 (Drp1) protein expression in rat ONH astrocytes. In contrast, elevated cAMP by dibutyryl-cAMP (dbcAMP) or isobutylmethylxanthine treatment significantly increased Drp1 protein expression in ONH astrocytes. Elevated cAMP exacerbated the impairment of mitochondrial dynamics and reduction of cell viability to oxidative stress in ONH astrocytes by decreasing optic atrophy type 1 (OPA1), and mitofusin (Mfn)1/2 protein expression. Following combined treatment with H2O2 and dbcAMP, PKA inhibition restored mitochondrial dynamics by increasing mitochondrial length and decreasing mitochondrial number, and this promoted cell viability in ONH astrocytes. Also, PKA inhibition significantly promoted Akt/Bax phosphorylation and Mfn1/2 oligomerization in ONH astrocytes. These results suggest that modulation of the cAMP/PKA signaling pathway may have therapeutic potential by activating Akt/Bax phosphorylation and promoting Mfn1/2 oligomerization in glaucomatous ONH astrocytes.

Ubiquinol promotes retinal ganglion cell survival and blocks the apoptotic pathway in ischemic retinal degeneration.

Coenzyme Q10 (CoQ10) protects retinal ganglion cells (RGCs) in experimental retinal ischemia and glaucoma by scavenging reactive oxygen species. We tested whether a diet supplemented with ubiquinol, the reduced form of CoQ10, promotes RGC survival and blocks the apoptotic pathway in ischemic mouse retina induced by acute high intraocular pressure (IOP) elevation. Ubiquinol (1%) treatment significantly promoted RGC survival at 2 weeks after ischemia/reperfusion. The ubiquinol treatment significantly blocked activation of astroglial and microglial cells in the ischemic retina at 2 weeks. While the ubiquinol treatment significantly decreased active Bax protein expression in the ischemic retina, phosphorylation of Bad at serine 112 and Bcl-xL protein expression were preserved in the ubiquinol-treated ischemic retina at 12 h. Consistently, the ubiquinol treatment prevented apoptotic cell death by blocking caspase-3 cleavage. These results suggest that the ubiquinol enhances RGC survival by modulating the Bax/Bad/Bcl-xL-mediated apoptotic pathway in the ischemic retina. Ubiquinol has therapeutic potential for ameliorating elevated IOP-induced ischemic retinal degeneration.

Optineurin E50K triggers BDNF deficiency-mediated mitochondrial dysfunction in retinal photoreceptor cell line.

Optineurin (OPTN) mutations are linked to glaucoma pathology and E50K mutation shows massive cell death in photoreceptor cells and retinal ganglion cells. However, little is known about E50K-mediated mitochondrial dysfunction in photoreceptor cell degeneration. We here show that overexpression of E50K expression triggered BDNF deficiency, leading to Bax activation in RGC-5 cells. BDNF deficiency induced mitochondrial dysfunction by decreasing mitochondrial maximal respiration and reducing intracellular ATP level in RGC-5 cells. However, BDNF deficiency did not alter mitochondrial dynamics. Also, BDNF deficiency resulted in LC3-mediated mitophagosome formation in RGC-5 cells. These results strongly suggest that E50K-mediated BDNF deficiency plays a critical role in compromised mitochondrial function in glaucomatous photoreceptor cell degeneration.

Elevated intracellular cAMP exacerbates vulnerability to oxidative stress in optic nerve head astrocytes.

Glaucoma is characterized by a progressive loss of retinal ganglion cells and their axons, but the underlying biological basis for the accompanying neurodegeneration is not known. Accumulating evidence indicates that structural and functional abnormalities of astrocytes within the optic nerve head (ONH) have a role. However, whether the activation of cyclic adenosine 3',5'-monophosphate (cAMP) signaling pathway is associated with astrocyte dysfunction in the ONH remains unknown. We report here that the cAMP/protein kinase A (PKA) pathway is critical to ONH astrocyte dysfunction, leading to caspase-3 activation and cell death via the AKT/Bim/Bax signaling pathway. Furthermore, elevated intracellular cAMP exacerbates vulnerability to oxidative stress in ONH astrocytes, and this may contribute to axonal damage in glaucomatous neurodegeneration. Inhibition of intracellular cAMP/PKA signaling activation protects ONH astrocytes by increasing AKT phosphorylation against oxidative stress. These results strongly indicate that activation of cAMP/PKA pathway has an important role in astrocyte dysfunction, and suggest that modulating cAMP/PKA pathway has therapeutic potential for glaucomatous ONH degeneration.

Role of cyclic AMP in the eye with glaucoma.

Glaucoma is characterized by a slow and progressive degeneration of the optic nerve, including retinal ganglion cell (RGC) axons in the optic nerve head (ONH), leading to visual impairment. Despite its high prevalence, the biological basis of glaucoma pathogenesis still is not yet fully understood, and the factors contributing to its progression are currently not well characterized. Intraocular pressure (IOP) is the only modifiable risk factor, and reduction of IOP is the standard treatment for glaucoma. However, lowering IOP itself is not always effective for preserving visual function in patients with primary open-angle glaucoma. The second messenger cyclic adenosine 3',5'-monophosphate (cAMP) regulates numerous biological processes in the central nervous system including the retina and the optic nerve. Although recent studies revealed that cAMP generated by adenylyl cyclases (ACs) is important in regulating aqueous humor dynamics in ocular tissues, such as the ciliary body and trabecular meshwork, as well as cell death and growth in the retina and optic nerve, the functional role and significance of cAMP in glaucoma remain to be elucidated. In this review, we will discuss the functional role of cAMP in aqueous humor dynamics and IOP regulation, and review the current medications, which are related to the cAMP signaling pathway, for glaucoma treatment. Also, we will further focus on cAMP signaling in RGC growth and regeneration by soluble AC as well as ONH astrocytes by transmembrane ACs to understand its potential role in the pathogenesis of glaucoma neurodegeneration. [BMB Reports 2017; 50(2): 60-70].

Mitochondrial pathogenic mechanism and degradation in optineurin E50K mutation-mediated retinal ganglion cell degeneration.

Mutations in optineurin (OPTN) are linked to the pathology of primary open angle glaucoma (POAG) and amyotrophic lateral sclerosis. Emerging evidence indicates that OPTN mutation is involved in accumulation of damaged mitochondria and defective mitophagy. Nevertheless, the role played by an OPTN E50K mutation in the pathogenic mitochondrial mechanism that underlies retinal ganglion cell (RGC) degeneration in POAG remains unknown. We show here that E50K expression induces mitochondrial fission-mediated mitochondrial degradation and mitophagy in the axons of the glial lamina of aged E50K-tg mice in vivo. While E50K activates the Bax pathway and oxidative stress, and triggers dynamics alteration-mediated mitochondrial degradation and mitophagy in RGC somas in vitro, it does not affect transport dynamics and fission of mitochondria in RGC axons in vitro. These results strongly suggest that E50K is associated with mitochondrial dysfunction in RGC degeneration in synergy with environmental factors such as aging and/or oxidative stress.

Coenzyme Q10 inhibits glutamate excitotoxicity and oxidative stress-mediated mitochondrial alteration in a mouse model of glaucoma.

PURPOSE: To test whether a diet supplemented with coenzyme Q10 (CoQ10) ameliorates glutamate excitotoxicity and oxidative stress-mediated retinal ganglion cell (RGC) degeneration by preventing mitochondrial alterations in the retina of glaucomatous DBA/2J mice. METHODS: Preglaucomatous DBA/2J and age-matched control DBA/2J-Gpnmb(+) mice were fed with CoQ10 (1%) or a control diet daily for 6 months. The RGC survival and axon preservation were measured by Brn3a and neurofilament immunohistochemistry and by conventional transmission electron microscopy. Glial fibrillary acidic protein (GFAP), superoxide dismutase-2 (SOD2), heme oxygenase-1 (HO1), N-methyl-d-aspartate receptor (NR) 1 and 2A, and Bax and phosphorylated Bad (pBad) protein expression was measured by Western blot analysis. Apoptotic cell death was assessed by TUNEL staining. Mitochondrial DNA (mtDNA) content and mitochondrial transcription factor A (Tfam)/oxidative phosphorylation (OXPHOS) complex IV protein expression were measured by real-time PCR and Western blot analysis. RESULTS: Coenzyme Q10 promoted RGC survival by approximately 29% and preserved the axons in the optic nerve head (ONH), as well as inhibited astroglial activation by decreasing GFAP expression in the retina and ONH of glaucomatous DBA/2J mice. Intriguingly, CoQ10 significantly blocked the upregulation of NR1 and NR2A, as well as of SOD2 and HO1 protein expression in the retina of glaucomatous DBA/2J mice. In addition, CoQ10 significantly prevented apoptotic cell death by decreasing Bax protein expression or by increasing pBad protein expression. More importantly, CoQ10 preserved mtDNA content and Tfam/OXPHOS complex IV protein expression in the retina of glaucomatous DBA/2J mice. CONCLUSIONS: Our findings suggest that CoQ10 may be a promising therapeutic strategy for ameliorating glutamate excitotoxicity and oxidative stress in glaucomatous neurodegeneration.

Coenzyme Q10 ameliorates oxidative stress and prevents mitochondrial alteration in ischemic retinal injury.

Coenzyme Q10 (CoQ10) acts by scavenging reactive oxygen species for protecting neuronal cells against oxidative stress in neurodegenerative diseases. We tested whether a diet supplemented with CoQ10 ameliorates oxidative stress and mitochondrial alteration, as well as promotes retinal ganglion cell (RGC) survival in ischemic retina induced by intraocular pressure elevation. A CoQ10 significantly promoted RGC survival at 2 weeks after ischemia. Superoxide dismutase 2 (SOD2) and heme oxygenase-1 (HO-1) expression were significantly increased at 12 h after ischemic injury. In contrast, the CoQ10 significantly prevented the upregulation of SOD2 and HO-1 protein expression in ischemic retina. In addition, the CoQ10 significantly blocked activation of astroglial and microglial cells in ischemic retina. Interestingly, the CoQ10 blocked apoptosis by decreasing caspase-3 protein expression in ischemic retina. Bax and phosphorylated Bad (pBad) protein expression were significantly increased in ischemic retina at 12 h. Interestingly, while CoQ10 significantly decreased Bax protein expression in ischemic retina, CoQ10 showed greater increase of pBad protein expression. Of interest, ischemic injury significantly increased mitochondrial transcription factor A (Tfam) protein expression in the retina at 12 h, however, CoQ10 significantly preserved Tfam protein expression in ischemic retina. Interestingly, there were no differences in mitochondrial DNA content among control- or CoQ10-treated groups. Our findings demonstrate that CoQ10 protects RGCs against oxidative stress by modulating the Bax/Bad-mediated mitochondrial apoptotic pathway as well as prevents mitochondrial alteration by preserving Tfam protein expression in ischemic retina. Our results suggest that CoQ10 may provide neuroprotection against oxidative stress-mediated mitochondrial alterations in ischemic retinal injury.

SUMO modification of NZFP mediates transcriptional repression through TBP binding.

The negatively regulating zinc finger protein (NZFP) is an essential transcription repressor required for early development during gastrulation in Xenopus laevis. In this study, we found that NZFP interacts with the small ubiquitin-like modifier (SUMO) conjugation E2 enzyme, Ubc9, and contains three putative SUMO conjugation sites. Studies with NZFP mutants containing mutations at the putative SUMO conjugation sites showed that these sites were able to be modified independently with SUMO. NZFP was found to be localized in the same nuclear bodies with SUMO-1. However, sumoylation of NZFP did not play a role either in the translocation of NZFP into the nucleus or on nuclear body formation. While wild type NZFP showed significant transcriptional repression, SUMO-conjugation site mutants manifested a decrease in transcriptional repression activity which is reversely proportional to the amount of sumoylation. The sumoylation defective mutant lost its TBP binding activity, while wild type NZFP interacted with TBP and inhibited transcription complex formation. These results strongly suggest that the sumoylation of NZFP facilitates NZFP to bind to TBP and the NZFP/TBP complex then represses the transcription of the target gene by inhibiting basal transcription complex formation.