Lipofuscin accumulation and autophagy in glaucomatous human lamina cribrosa cells.
McElnea Elizabeth M,Hughes Emily,McGoldrick Aloysius,McCann Amanda,Quill Barry,Docherty Neil,Irnaten Mustapha,Farrell Michael,Clark Abbot F,O'Brien Colm J,Wallace Deborah M
BACKGROUND:Disease associated alterations in the phenotype of lamina cribrosa (LC) cells are implicated in changes occurring at the optic nerve head (ONH) in glaucoma. Lipofuscin, the formation of which is driven by reactive oxygen species (ROS), is an intralysosomal, non-degradable, auto-fluorescent macromolecule which accumulates with age and can affect autophagy - the lysosomal degradation of a cell's constituents. We aimed to compare the content of lipofuscin-like material and markers of autophagy in LC cells from normal and glaucoma donor eyes. METHODS:The number and size of peri-nuclear lysosomes were examined by transmission electron microscopy (TEM). Cellular auto-fluorescence was quantified by flow cytometry. Cathepsin K mRNA levels were assessed by PCR. Autophagy protein 5 (Atg5) mRNA and protein levels were analysed by PCR and Western blot. Protein levels of subunits of the microtubule associated proteins (MAP) 1A and 1B, light chain 3 (LC3) I and II were analysed by Western blot. Immunohistochemical staining of LC3-II in ONH sections from normal and glaucomatous donor eyes was performed. RESULTS:A significant increase in the number of peri-nuclear lysosomes [4.1 × 10,000 per high power field (h.p.f.) ± 1.9 vs. 2.0 × 10,000 per h.p.f. ± 1.3, p = 0.002, n = 3] and whole cell auto-fluorescence (83.62 ± 45.1 v 41.01 ± 3.9, p = 0.02, n = 3) was found in glaucomatous LC cells relative to normal LC cells. Glaucomatous LC cells possessed significantly higher levels of Cathepsin K mRNA and Atg5 mRNA and protein. Enhanced levels of LC3-II were found in both LC cells and optic nerve head sections from glaucoma donors. CONCLUSIONS:Increased lipofuscin formation is characteristic of LC cells from donors with glaucoma. This finding confirms the importance of oxidative stress in glaucoma pathogenesis. Intracellular lipofuscin accumulation may have important effects on autophagy the modification of which could form the basis for future novel glaucoma treatments.
Autophagic dysregulation in glaucomatous trabecular meshwork cells.
Porter Kristine,Hirt Joshua,Stamer W Daniel,Liton Paloma B
Biochimica et biophysica acta
Primary open angle glaucoma (POAG) is a degenerative disease commonly associated with aging and elevated intraocular pressure (IOP). Higher resistance to aqueous humor (AH) outflow through the trabecular meshwork (TM) generates the elevated IOP in POAG; unfortunately the underlying molecular mechanisms responsible for elevated resistance are unknown. It is widely accepted, however, that differences between normal and POAG TM tissues are presumably a consequence of cellular dysfunction. Here, we investigated the autophagic function and response to chronic oxidative stress in TM cells isolated from glaucomatous and age-matched donor eyes. Glaucomatous TM cells showed elevated senescence-associated-beta-galactosidase (SA-β-Gal) and cellular lipofuscin, together with decreased steady-state levels of LC3B-II, decreased levels of pRPS6K-T389 and reduced proteolysis of long-live proteins. Moreover, the glaucomatous cultures failed to activate autophagy when exposed to hyperoxic conditions. These results strongly suggest mTOR-dependent dysregulation of the autophagic pathway in cells isolated from the glaucomatous TM. Such dysregulated autophagic capacity can have a detrimental impact in outflow pathway tissue, i.e. mechanotransduction, and thus represent an important factor contributing to the progression of the disease.
Evidence of Hypoxic Glial Cells in a Model of Ocular Hypertension.
Jassim Assraa H,Inman Denise M
Investigative ophthalmology & visual science
Purpose:Reoxygenation after hypoxia can increase reactive oxygen species and upregulate autophagy. We determined, for the first time, the impact of elevated IOP on hypoxia induction, superoxide accumulation, and autophagy in a bead model of glaucoma. Method:Ocular hypertension was achieved with magnetic bead injection into the anterior chamber. Before mice were killed, they were injected with pimonidazole for hypoxia detection and dihydroethidium (DHE) for superoxide detection. Total retinal ganglion cells (RGCs) and optic nerve (ON) axons were quantified, total glutathione (GSH) was measured, and retinal and ON protein and mRNA were analyzed for hypoxia (Hif-1α and Hif-2α), autophagy (LC3 and p62), and SOD2. Results:With IOP elevation (P < 0.0001), the retina showed significantly (P < 0.001) decreased GSH compared with control, and a significant decrease (P < 0.01) in RGC density compared with control. Pimonidazole-positive Müller glia, microglia, astrocytes, and RGCs were present in the retinas after 4 weeks of ocular hypertension but absent in both the control and after only 2 weeks of ocular hypertension. The ON showed significant axon degeneration (P < 0.0001). The mean intensity of DHE in the ganglion cell layer and ON significantly increased (P < 0.0001). The ratio of LC3-II to LC3-I revealed a significant increase (P < 0.05) in autophagic activity in hypertensive retinas compared with control. Conclusions:We report a novel observation of hypoxia and a significant decrease in GSH, likely contributing to superoxide accumulation, in the retinas of ocular hypertensive mice. The significant increase in the ratio of LC3-II to LC3-I suggests autophagy induction.
Differential effects of N-acetylcysteine on retinal degeneration in two mouse models of normal tension glaucoma.
Sano Hiroki,Namekata Kazuhiko,Kimura Atsuko,Shitara Hiroshi,Guo Xiaoli,Harada Chikako,Mitamura Yoshinori,Harada Takayuki
Cell death & disease
N-acetylcysteine (NAC) is widely used as a mucolytic agent and as an antidote to paracetamol overdose. NAC serves as a precursor of cysteine and stimulates the synthesis of glutathione in neural cells. Suppressing oxidative stress in the retina may be an effective therapeutic strategy for glaucoma, a chronic neurodegenerative disease of the retinal ganglion cells (RGCs) and optic nerves. Here we examined the therapeutic potential of NAC in two mouse models of normal tension glaucoma, in which excitatory amino-acid carrier 1 (EAAC1) or glutamate/aspartate transporter (GLAST) gene was deleted. EAAC1 is expressed in retinal neurons including RGCs, whereas GLAST is mainly expressed in Müller glial cells. Intraperitoneal administration of NAC prevented RGC degeneration and visual impairment in EAAC1-deficient (knockout; KO) mice, but not in GLAST KO mice. In EAAC1 KO mice, oxidative stress and autophagy were suppressed with increased glutathione levels by NAC treatment. Our findings suggest a possibility that systemic administration of NAC may be available for some types of glaucoma patients.
LOXL1 folding in exfoliation glaucoma.
Bernstein Audrey M,Ritch Robert,Wolosin J Mario
Advances in protein chemistry and structural biology
Exfoliation syndrome (XFS) is an age-related disease defined by the deposition of aggregated fibrous material (XFM) in the peri-cellular space. Principal morbidity occurs in the eye, where XFM accumulates on the anterior ocular tissues. GWAS have found that certain genetic variants of lysyl oxidase-like 1 (LOXL1), a matrix cross-linking enzyme that is required for elastic fiber formation confer risk for the development of XFS, but are not a single causative factor as many genetically affected individuals do not develop XFS or subsequent glaucoma (XFG). We have found that XFG cells display defects in lysosomes, microtubules, autophagy, and mitochondria resembling defects found in cells from age-related syndromes, such as the main neurodegenerative diseases. In the majority of these diseases, the determining cellular factor is a protein containing intrinsically disordered regions (IDRs) and displaying a high propensity for aggregation. We have found that in XFG patient-derived cells, LOXL1 protein is actively subjected to autophagic clearance, suggesting that LOXL1 is undergoing aggregation. In silico analysis demonstrates that LOXL1's first 369 aa constitute an IDR with the highest disorder probability peak centering around the known risk positions. Experimentally, we have found over-expression of either unmodified LOXL1 or fluorescent chimeras preserving the well-structured N-terminus cause copious intracellular aggregation and that aggregation wanes when the high IDR peaks are deleted. Overall, our work suggests that XFS/G results from the aggregation of the LOXL1 protein coupled with a reduction of cellular proteostasis capabilities in aging, resulting in a chronic build-up of LOXL1-containing protein aggregates.
Temporal dynamics of PARK2/parkin and OPTN/optineurin recruitment during the mitophagy of damaged mitochondria.
Wong Yvette C,Holzbaur Erika L F
Damaged mitochondria are selectively degraded via autophagy in a regulated pathway known as mitophagy. Parkinson disease-linked proteins PINK1 (PTEN induced putative kinase 1) and PARK2 (parkin RBR E3 ubiquitin protein ligase) are recruited to the outer mitochondrial membrane upon mitochondrial damage, leading to the PARK2-mediated ubiquitination of mitochondrial proteins. Here, we discuss our recent work demonstrating that OPTN (optineurin) is recruited to damaged mitochondria, serving as an autophagy receptor for autophagosome formation around mitochondria. Using high-resolution live-cell imaging, we find that OPTN is recruited to ubiquitinated mitochondria downstream of PARK2, and induces autophagosome assembly around mitochondria via its LC3-interacting region. Mutations in OPTN are linked to both glaucoma and ALS (amyotrophic lateral sclerosis), and an ALS-associated E478G mutation in OPTN's ubiquitin binding domain leads to defective mitophagy and accumulation of damaged mitochondria. Importantly, our results highlight a role for mitophagy defects in ALS pathogenesis, and demonstrate that defects in the same pathway for mitochondrial homeostasis are causal for both familial Parkinson disease and ALS.
Autophagy in axonal degeneration in glaucomatous optic neuropathy.
Munemasa Yasunari,Kitaoka Yasushi
Progress in retinal and eye research
The role of autophagy in retinal ganglion cell (RGC) death is still controversial. Several studies focused on RGC body death, although the axonal degeneration pathway in the optic nerve has not been well documented in spite of evidence that the mechanisms of degeneration of neuronal cell bodies and their axons differ. Axonal degeneration of RGCs is a hallmark of glaucoma, and a pattern of localized retinal nerve fiber layer defects in glaucoma patients indicates that axonal degeneration may precede RGC body death in this condition. As models of preceding axonal degeneration, both the tumor necrosis factor (TNF) injection model and hypertensive glaucoma model may be useful in understanding the mechanism of axonal degeneration of RGCs, and the concept of axonal protection can be an attractive approach to the prevention of neurodegenerative optic nerve disease. Since mitochondria play crucial roles in glaucomatous optic neuropathy and can themselves serve as a part of the autophagosome, it seems that mitochondrial function may alter autophagy machinery. Like other neurodegenerative diseases, optic nerve degeneration may exhibit autophagic flux impairment resulting from elevated intraocular pressure, TNF, traumatic injury, ischemia, oxidative stress, and aging. As a model of aging, we used senescence-accelerated mice to provide new insights. In this review, we attempt to describe the relationship between autophagy and recently reported noteworthy factors including Nmnat, ROCK, and SIRT1 in the degeneration of RGCs and their axons and propose possible mechanisms of axonal protection via modulation of autophagy machinery.
Functional analysis of optineurin and some of its disease-associated mutants.
Bansal Megha,Swarup Ghanshyam,Balasubramanian Dorairajan
Optineurin is a multifunctional protein involved in a variety of cellular functions such as protein trafficking by vesicles, autophagy, and signal transduction. Certain mutations in optineurin (gene OPTN) are associated with neurodegenerative diseases like glaucoma and amyotrophic lateral sclerosis (ALS). Optineurin is also seen in pathological structures present in several other neurodegenerative diseases. In glaucoma, loss of vision occurs due to progressive degeneration of retinal ganglion cells, and perhaps loss of photoreceptor cone cells as well. Most of the glaucoma-associated mutations of optineurin are heterozygous missense mutations, whereas the ALS-associated mutations include deletion, truncation, and missense mutations. Optineurin mediates its functions by interacting with various proteins, often acting as an adaptor to provide a link between two or more proteins. Disease-causing mutations alter these interactions leading to functional defects in membrane vesicle trafficking, autophagy, signaling, aggregate formation, and other processes. Some of these functional defects, caused by glaucoma-associated mutants of optineurin, led to retinal cell death mediated by apoptosis and therefore may contribute to pathogenesis directly. Other mutations are likely to cause glaucoma by indirect mechanisms involving other cell types. Mechanisms of ALS pathogenesis by optineurin mutations are yet to be investigated in detail; however, some ALS-associated mutants cause defects in signaling, autophagy, and ubiquitin binding, which might contribute to pathogenesis.
Induction of autophagy in rats upon overexpression of wild-type and mutant optineurin gene.
Ying Hongyu,Turturro Sanja,Nguyen Tara,Shen Xiang,Zelkha Ruth,Johnson Elaine C,Morrison John C,Yue Beatrice Y J T
BMC cell biology
BACKGROUND:Optineurin is a gene associated with normal tension glaucoma and amyotrophic lateral sclerosis. It has been reported previously that in cultured RGC5 cells, the turnover of endogenous optineurin involves mainly the ubiquitin-proteasome pathway (UPP). When optineurin is upregulated or mutated, the UPP function is compromised as evidenced by a decreased proteasome β5 subunit (PSMB5) level and autophagy is induced for clearance of the optineurin protein. RESULTS:Adeno-associated type 2 viral (AAV2) vectors for green fluorescence protein (GFP) only, GFP-tagged wild-type and Glu50Lys (E50K) mutated optineurin were intravitreally injected into rats for expression in retinal ganglion cells (RGCs). Following intravitreal injections, eyes that received optineurin vectors exhibited retinal thinning, as well as RGC and axonal loss compared to GFP controls. By immunostaining and Western blotting, the level of PSMB5 and autophagic substrate degradation marker p62 was reduced, and the level of autophagic marker microtubule associated protein 1 light chain 3 (LC3) was enhanced. The UPP impairment and autophagy induction evidently occurred in vivo as in vitro. The optineurin level, RGC and axonal counts, and apoptosis in AAV2-E50K-GFP-injected rat eyes were averted to closer to normal limits after treatment with rapamycin, an autophagic enhancer. CONCLUSIONS:The UPP function was reduced and autophagy was induced when wild-type and E50K optineurin was overexpressed in rat eyes. This study validates the in vitro findings, confirming that UPP impairment and autophagy induction also occur in vivo. In addition, rapamycin is demonstrated to clear the accumulated mutant optineurin. This agent may potentially be useful for rescuing of the adverse optineurin phenotypes in vivo.
Autophagy dysregulation and the fate of retinal ganglion cells in glaucomatous optic neuropathy.
Russo Rossella,Nucci Carlo,Corasaniti Maria Tiziana,Bagetta Giacinto,Morrone Luigi Antonio
Progress in brain research
Glaucoma is a neurodegenerative disease caused by the progressive apoptotic death of retinal ganglion cells (RGCs). The mechanisms leading to the RGC loss are still unknown but it is now clear that, besides elevated intraocular pressure (IOP), which is considered the main risk factor, other IOP-independent determinants are responsible for the development of the optic neuropathy. Autophagy is a highly conserved catabolic pathway by which cellular components are degraded through the lysosomes. Dysfunctional autophagic pathway has been associated with several neuropathological conditions and a considerable number of studies have proved autophagy as a potential target for pharmacological modulation to achieve neuroprotection. Here, we review the current literature bridging the degeneration of RGCs to alterations of the autophagic pathway; we also discuss the possible role of autophagy in the pathogenesis and progression of glaucoma in view of the future application of autophagy modulators for glaucoma therapy.
Glaucoma Genes and Mechanisms.
Wiggs Janey L
Progress in molecular biology and translational science
Genetic studies have yielded important genes contributing to both early-onset and adult-onset forms of glaucoma. The proteins encoded by the current collection of glaucoma genes participate in a broad range of cellular processes and biological systems. Approximately half the glaucoma-related genes function in the extracellular matrix, however proteins involved in cytokine signaling, lipid metabolism, membrane biology, regulation of cell division, autophagy, and ocular development also contribute to the disease pathogenesis. While the function of these proteins in health and disease are not completely understood, recent studies are providing insight into underlying disease mechanisms, a critical step toward the development of gene-based therapies. In this review, genes known to cause early-onset glaucoma or contribute to adult-onset glaucoma are organized according to the cell processes or biological systems that are impacted by the function of the disease-related protein product.
Rapamycin Removes Damaged Mitochondria and Protects Human Trabecular Meshwork (TM-1) Cells from Chronic Oxidative Stress.
He Jing Na,Zhang Shao Dan,Qu Yang,Wang Hai Lin,Tham Clement C,Pang Chi Pui,Chu Wai Kit
Glaucoma is a chronic optic neuropathy that could lead to permanent vision loss. Primary open-angle glaucoma (POAG) is the most common type of glaucoma, with elevated intraocular pressure (IOP) as a major risk factor. IOP is mainly regulated by trabecular meshwork (TM), an important component of the conventional aqueous humor (AH) outflow pathway. TM cells are constantly subjected to oxidative stress. Long-term exposure to oxidative stress has been shown to cause elevation of AH outflow resistance, leading to higher IOP. In this study, we induced chronic oxidative stress in human trabecular meshwork (TM-1) cells with 1 μM rotenone and investigated the levels of reactive oxygen species (ROS), autophagy, and mitochondrial functions. Protective effects of rapamycin, an inducer of autophagy, were also investigated. Our data indicated that rotenone significantly increased oxidative stress, but not autophagy, in TM-1 cells. Rapamycin at 10 nM effectively suppressed the rotenone-induced cell apoptosis, as well as the ROS elevation. The protective effects of rapamycin could be associated to the induction of autophagy and removal of damaged mitochondria in TM-1 cells. Our results suggest autophagy has important roles in protecting TM-1 cells from oxidative stress, which could be further developed into a novel treatment to POAG.
Caveolin-1 regulates human trabecular meshwork cell adhesion, endocytosis, and autophagy.
Wu Zhenggen,Huang Chukai,Xu Ciyan,Xie Lijing,Liang Jia-Jian,Liu Lifang,Pang Chi Pui,Ng Tsz Kin,Zhang Mingzhi
Journal of cellular biochemistry
Impaired trabecular meshwork (TM) outflow is implicated in the pathogenesis of primary open-angle glaucoma (POAG). We previously identified the association of a caveolin-1 (CAV1) variant with POAG by genome-wide association study. Here we report a study of CAV1 knockout (KO) effect on human TM cell properties. We generated human CAV1-KO TM cells by CRISPR/Cas9 technology, and we found that the CAV1-KO TM cells less adhered to the surface coating than the wildtype TM cells by 69.34% ( P < 0.05), but showed no difference in apoptosis. Higher endocytosis ability of dextran and transferrin was also observed in the CAV1-KO TM cells (4.37 and 1.89-fold respectively, P < 0.001), compared to the wildtype TM cells. Moreover, the CAV1-KO TM cells had higher expression of extracellular matrix-degrading enzyme genes ( ADMTS13 and MMP14) as well as autophagy-related genes ( ATG7 and BECN1) and protein (LC3B-II) than the wildtype TM cells. In summary, results from this study showed that the CAV1-KO TM cells have reduced adhesion with higher extracellular matrix-degrading enzyme expression, but increased endocytosis and autophagy activities, indicating that CAV1 could be involved in the regulation of adhesion, endocytosis, and autophagy in human TM cells.
Acteoside inhibits autophagic apoptosis of retinal ganglion cells to rescue glaucoma-induced optic atrophy.
Chen Qianbo,Xi Xiaoting,Zeng Yong,He Zhendan,Zhao Jianfeng,Li Yan
Journal of cellular biochemistry
BACKGROUND:Glaucoma is the world's second biggest cause of blindness, and patients progressively lose their eyesight. The current clinical treatment for glaucoma involves controlling intraocular pressure with drugs or surgery; however, some patients still progressively lose their eyesight. This treatment is also similar to the treatment of traumatic optic neuropathy. Thus, saving retinal ganglion cells (RGCs) from apoptosis is essential. METHODS:The role of Acteoside on autophagy modulation in the 661 W cell line. RESULTS:In this study, we first find that Acteoside inhibits autophagy, Rapamycin alleviates this inhibition and the PI3K inhibitor, 3-MA or LY294002, synergistically promotes it. In a mechanistic study, we find that Optineurin (OPTN) mediates Acteoside regulation of autophagy. OPTN overexpression or knockdown activates or inhibits autophagy, respectively. OPTN is inhibited by autophagy inhibitors, such as Acteoside and 3-MA and is promoted by the autophagy activator, Rapamycin. Meanwhile, PI3K and AKT are elevated by Acteoside and 3-MA and inhibited by Rapamycin. Finally, we find that Acteoside inhibits apoptosis in parallel to autophagy and that this inhibition is also mediated by OPTN. CONCLUSION:In summary, we conclude that Acteoside inhibits autophagy-induced apoptosis in RGCs through the OPTN and PI3K/AKT/mTOR pathway, and glaucoma patients may benefit from Acteoside treatment alone or in combination with other autophagy inhibitors.
Mendelian genes in primary open angle glaucoma.
Sears Nathan C,Boese Erin A,Miller Mathew A,Fingert John H
Experimental eye research
Mutations in each of three genes, myocilin (MYOC), optineurin (OPTN), and TANK binding kinase 1 (TBK1), may cause primary open-angle glaucoma (POAG) that is inherited as a Mendelian trait. MYOC mutations cause 3-4% of POAG cases with IOP >21 mmHg, while mutations in OPTN, TBK1, and MYOC each cause ∼1% of POAG with IOP ≤21 mmHg, i.e. normal tension glaucoma. Identification of these disease-causing genes has provided insights into glaucoma pathogenesis. Mutations in MYOC cause a cascade of abnormalities in the trabecular meshwork including intracellular retention of MYOC protein, decreased aqueous outflow, higher intraocular pressure, and glaucoma. Investigation of MYOC mutations demonstrated that abnormal retention of intracellular MYOC and stimulation of endoplasmic reticular (ER) stress may be important steps in the development of MYOC-associated glaucoma. Mutations in OPTN and TBK1 cause a dysregulation of autophagy which may directly cause retinal ganglion cell damage and normal tension glaucoma. Discovery of these Mendelian causes of glaucoma has also provided a new set of potential therapeutic targets that may ultimately lead to novel, gene-directed glaucoma treatments.
Glaucoma-Associated Mutations in the Optineurin Gene Have Limited Impact on Parkin-Dependent Mitophagy.
Chernyshova Kseniia,Inoue Keiichi,Yamashita Shun-Ichi,Fukuchi Takeo,Kanki Tomotake
Investigative ophthalmology & visual science
Purpose:Glaucoma results in progressive degeneration of the optic nerve and irreversible vision loss. Several mutations in the gene encoding optineurin (OPTN), the receptor for Parkin-dependent mitochondrial autophagy (mitophagy), are associated with glaucoma and amyotrophic lateral sclerosis (ALS). ALS mutations in the ubiquitin-binding domain of OPTN impair Parkin-dependent mitophagy. However, the effects of glaucoma mutations in this region remain unknown. We examined the impact of glaucoma-associated OPTN mutations on Parkin-dependent mitophagy. Methods:The mitochondria-localized, pH-sensitive fluorescent protein mito-Keima was used to monitor mitophagy. HeLa cells expressing Parkin were treated with carbonyl cyanide 3-chlorophenylhydrazone (CCCP) or oligomycin/antimycin A (O/A) to induce Parkin-dependent mitophagy. Two complementary mitophagy receptors, OPTN and NDP52, were deleted in HeLa cells expressing mito-Keima and Parkin (DKO_HeLa). The mutant OPTN genes were re-introduced into DKO_HeLa cells using retroviruses or through transfection. Mitophagy activity and OPTN localization were evaluated via microscopic analyses. OPTN binding to ubiquitin was examined using an immunoprecipitation assay. Results:Parkin-dependent mitophagy was inhibited in DKO_HeLa cells. Introduction of two glaucoma mutations in the ubiquitin-interacting region of OPTN restored mitophagy in CCCP-treated DKO_HeLa cells, whereas the two ALS mutations failed to replicate this effect. Under treatment with CCCP, the two glaucoma-mutant OPTN proteins normally translocated to mitochondria and bound to ubiquitinated proteins. Furthermore, five additional glaucoma-mutant OPTN proteins restored CCCP-induced mitophagy. Moreover, treatment with O/A exhibited similar results. Conclusions:Glaucoma-mutant OPTN proteins retain their normal properties as mitophagy receptors, suggesting that mutations in the OPTN gene cause glaucoma through a mechanism independent of mitophagy defects.
LncRNA NR_003923 promotes cell proliferation, migration, fibrosis, and autophagy via the miR-760/miR-215-3p/IL22RA1 axis in human Tenon's capsule fibroblasts.
Zhao Yang,Zhang Feng,Pan Zheng,Luo Haomin,Liu Ke,Duan Xuanchu
Cell death & disease
Noncoding RNAs (ncRNAs), including long ncRNAs (lncRNA) have manifested an important role in the pathophysiology of many diseases. Glaucoma is a primary cause of irreversible blindness worldwide. However, the involvement of lncRNAs in glaucoma remains largely unknown. Here, we performed the lncRNA expression assay based on clinical tissues and identified a specific functional lncRNA, NR_003923, and investigated its potential role in glaucoma. Knockdown of NR_003923 in human Tenon's capsule fibroblast cells (HTFs) inhibited TGF-β-induced cell migration, proliferation, fibrosis, and autophagy. The dual luciferase reporter assay confirmed that miR-760 and miR-215-3p interacted with NR_003923. miR-760 and miR-215-3p inhibitor reversed the effects of NR_003923 and TGF-β-induced cell apoptosis. Moreover, the expression of miR-760 and miR-215-3p was decreased in glaucoma comparing with control. Furthermore, through microarray we found IL22RA1 was increased in glaucoma and both of miR-760 and miR-215-3p bound to the 3' UTR of IL22RA1. Overexpression of IL22RA1 enhanced HTFs migration and proliferation, while miR-760 and miR-215-3p mimics reversed these promotive biological roles induced by IL22RA1. In conclusion, NR_003923 and IL22RA1 might contribute to glaucoma progression and be a novel and potential biomarkers and therapeutic targets for glaucoma.
Mitochondria and Autophagy Dysfunction in Glucocorticoid-Induced Ocular Hypertension/Glaucoma Mice Model.
Zeng Wen,Wang Wenhuan,Wu Shengyu,Zhu Xiaolu,Zheng Tian,Chen Xiaomin,Ren Jiangbo,Gong Yan,Ke Min
Current eye research
: This study is aimed to investigate the effects of periocular steroids induction on intraocular pressure (IOP), retinal ganglion cells (RGCs) and trabecular meshwork (TM) ultrastructure in glucocorticoid-induced ocular hypertension mice model.: Dexamethasone-21-acetate (Dex-Ace) was administered through periocular conjunctival fornix injection every 3 days in C57BL/6J mice. Intraocular pressure was measured weekly by rebound tonometry. RGCs were examined with immunofluorescent staining of BRN3a at week 1, 4, and 8. TM morphology was visualized with electron microscopy. Autophagy was evaluated with immunoblotting in TM tissues.: Dex-Ace rapidly and significantly induced IOP, which peaked at week 4. The absolute increase in IOP in the Dex-Ace-treated mice was 8.1 ± 1.4 mmHg, a 60% induction ( < .0001) compared with that in the vehicle-treated mice. The IOP sustained a higher level in the Dex-Ace group from week 4 to week 8. Dex-Ace treatment decreased the number of RGCs in a time-dependent manner, suggesting that high IOP resulted in optic neuropathy. In addition, Dex-Ace thickened trabecular beams and decreased intertrabecular spaces, with marked accumulation of fibrillar and amorphous granular extracellular material. Moreover, Dex-Ace induced swollen and elongated mitochondria in TM cells. The average mitochondria area was 0.090 ± 0.044 µm in the vehicle-treated mice, and increased to 0.161 ± 0.094 µm ( < .0001), 0.121 ± 0.029 µm ( = .0223) and 0.171 ± 0.076 µm ( < .0001) in the Dex-Ace-treated mice at weeks 1, 4 and 8, respectively. Autophagy was also increased by Dex-Ace treatment, indicating by the upregulation of LC3-I, LC3-II and beclin-1, and downregulation of p62.: Dex-Ace administration decreased RGCs and changed TM ultrastructure, mimicking hallmarks of human glucocorticoid-induced glaucoma (GIG). In addition, mitochondria and autophagy dysfunction suggested abnormal energy metabolism in TM cells, which warranted further study to fully elucidate the pathogenesis of GIG.
The autophagic protein LC3 translocates to the nucleus and localizes in the nucleolus associated to NUFIP1 in response to cyclic mechanical stress.
Shim Myoung Sup,Nettesheim April,Hirt Joshua,Liton Paloma B
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 TGFβ/Smad-induced fibrogenesis in trabecular meshwork cells.
Nettesheim April,Shim Myoung Sup,Hirt Josh,Liton Paloma B
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 TGFβ2, BAMBI, and SMA. Furthermore, genetic and pharmacological inhibition of autophagy was associated with a parallel reduction in TGFβ-induced fibrosis, caused by a BAMBI-mediated reduced activation of Smad2/3 signaling in autophagy-deficient cells. At the same time, TGFβ 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 TGFβ signaling in TM cells.
Recent advances in risk factors associated with ocular exfoliation syndrome.
Ghaffari Sharaf Mehdi,Damji Karim F,Unsworth Larry D
Exfoliation syndrome is generally considered a progressive age-related systemic disorder of the extracellular matrix, which is clinically characterized through the observation of flaky white aggregates on ocular tissues. Exfoliation syndrome is directly linked to exfoliative glaucoma in elderly patients, where it is known as the most common identifiable cause of open-angle glaucoma. Despite the identification of various risk factors associated with exfoliation syndrome, the exact pathogenesis of this syndrome has not been fully elucidated. There is a growing number of genome-wide association studies in different populations around the world to identify genetic factors underlying exfoliation syndrome. Besides variants in LOXL1 and CACNA1A genes, new loci have been recently identified which are believed to be associated with exfoliation syndrome. Among different genetic factors, functional variants might help to better understand mechanisms underlying this systemic disorder. Besides genetic factors, epigenetic regulation of different gene expression patterns has been thought to play a role in its pathogenesis. Other factors have been also considered to be involved in the development of exfoliation syndrome at cellular organelles level where mitochondrial impairment and autophagy dysfunction have been suggested in relation to exfoliation syndrome. This review addresses the most recent findings on genetic factors as well as cellular and molecular mechanisms involved in both the development and progression of exfoliation syndrome.
Autophagy in glaucoma: Crosstalk with apoptosis and its implications.
Wang Yao,Huang Changquan,Zhang Hongbing,Wu Renyi
Brain research bulletin
Glaucoma is characterized by elevated intraocular pressure that causes progressive loss of retinal ganglion cells (RGCs). Autophagy is a lysosomal degradative process that updates the cellular components and plays an important role in cellular homeostasis. Recent studies have shown that autophagy is involved in the pathophysiological process of glaucoma. The role played by autophagy in glaucoma is complex, and conflicting evidence shows that autophagy promotes both RGC survival and death. The understanding of the major pattern of RGC loss and the crosstalk between autophagy and apoptosis remains limited in glaucoma. This review focuses on the relationship between autophagy and glaucoma, particularly on the influence of autophagy on apoptosis in glaucoma. Further research on autophagy in glaucoma may provide a novel understanding of the glaucoma pathology and novel treatment targets for glaucoma in the future.
A Glaucoma-Associated Variant of Optineurin, M98K, Activates Tbk1 to Enhance Autophagosome Formation and Retinal Cell Death Dependent on Ser177 Phosphorylation of Optineurin.
Sirohi Kapil,Kumari Asha,Radha Vegesna,Swarup Ghanshyam
Certain missense mutations in optineurin/OPTN and amplification of TBK1 are associated with normal tension glaucoma. A glaucoma-associated variant of OPTN, M98K, induces autophagic degradation of transferrin receptor (TFRC) and death in retinal cells. Here, we have explored the role of Tbk1 in M98K-OPTN-induced autophagy and cell death, and the effect of Tbk1 overexpression in retinal cells. Cell death induced by M98K-OPTN was dependent on Tbk1 as seen by the effect of Tbk1 knockdown and blocking of Tbk1 activity by a chemical inhibitor. Inhibition of Tbk1 also restores M98K-OPTN-induced transferrin receptor degradation. M98K-OPTN-induced autophagosome formation, autophagy and cell death were dependent on its phosphorylation at S177 by Tbk1. Knockdown of OPTN reduced starvation-induced autophagosome formation. M98K-OPTN expressing cells showed higher levels of Tbk1 activation and enhanced phosphorylation at Ser177 compared to WT-OPTN expressing cells. M98K-OPTN-induced activation of Tbk1 and its ability to be phosphorylated better by Tbk1 was dependent on ubiquitin binding. Phosphorylated M98K-OPTN localized specifically to autophagosomes and endogenous Tbk1 showed increased localization to autophagosomes in M98K-OPTN expressing cells. Overexpression of Tbk1 induced cell death and caspase-3 activation that were dependent on its catalytic activity. Tbk1-induced cell death possibly involves autophagy, as shown by the effect of Atg5 knockdown, and requirement of autophagic function of OPTN. Our results show that phosphorylation of Ser177 plays a crucial role in M98K-OPTN-induced autophagosome formation, autophagy flux and retinal cell death. In addition, we provide evidence for cross talk between two glaucoma associated proteins and their inter-dependence to mediate autophagy-dependent cell death.
Regulation of TBK1 activity by Optineurin contributes to cell cycle-dependent expression of the interferon pathway.
Weil Robert,Laplantine Emmanuel,Génin Pierre
Cytokine & growth factor reviews
The innate immune system has evolved to detect and neutralize viral invasions. Triggering of this defense mechanism relies on the production and secretion of soluble factors that stimulate intracellular antiviral defense mechanisms. The Tank Binding Kinase 1 (TBK1) is a serine/threonine kinase in the innate immune signaling pathways including the antiviral response and the host defense against cytosolic infection by bacteries. Given the critical roles of TBK1, important regulatory mechanisms are required to regulate its activity. Among these, Optineurin (Optn) was shown to negatively regulate the interferon response, in addition to its important role in membrane trafficking, protein secretion, autophagy and cell division. As Optn does not carry any enzymatic activity, its functions depend on its precise subcellular localization and its interaction with other proteins, especially with components of the innate immune pathway. This review highlights advances in our understanding of Optn mechanisms of action with focus on the relationships between Optn and TBK1 and their implication in host defense against pathogens. Specifically, how the antiviral immune system is controlled during the cell cycle by the Optn/TBK1 axis and the physiological consequences of this regulatory mechanism are described. This review may serve to a better understanding of the relationships between the different functions of Optn, including those related to immune responses and its associated pathologies such as primary open-angle glaucoma, amyotrophic lateral sclerosis and Paget's disease of bone.
Neurodegeneration and Neuroprotection in Glaucoma.
Gauthier Angela C,Liu Ji
The Yale journal of biology and medicine
Glaucoma is the principal cause of irreversible blindness in the world. The disease leads to progressive optic nerve degeneration with a gradual loss of retinal ganglion cells. Neurodegeneration in glaucoma extends beyond the eye into the lateral geniculate nucleus and visual cortex, and the disease even shares some characteristics with other central nervous system degenerative disorders. Glaucoma destroys neurons through oxidative stress, impairment in axonal transport, neuroinflammation, and excitotoxicity. Autophagy may promote or inhibit disease progression. Currently, lowering intraocular pressure is the only way proven to delay glaucoma advancement. However, many new therapies are being developed, including antioxidants, adenosine receptor antagonists, Rho-pathway inhibitors, stem cell therapy, and neurotrophic factors. These therapies focus on neuroprotection, and they may eventually halt glaucoma progression or reverse the process of the disease itself.
Cigarette Smoke Extract Causes Injury in Primary Retinal Ganglion Cells via Apoptosis and Autophagy.
Lee Kwanghyun,Hong Samin,Seong Gong Je,Kim Chan Yun
Current eye research
AIMS:To investigate whether tobacco smoke directly injures retinal ganglion cells (RGCs) and to evaluate the mechanisms of cell death. METHODS:Primary rat RGCs were harvested from 3- or 4-day-old newborn rats and exposed to cigarette smoke extract (CSE). Cell viability was determined by adenosine 5'-triphosphate (ATP) assay. Apoptosis was evaluated by terminal deoxynucleotidyl transferase (TdT)-mediated deoxyuridine triphosphate (dUTP) nick-end labeling (TUNEL) and real-time reverse transcription polymerase chain reaction (RT-PCR) for Bcl-2 family. Autophagy was also assessed by Western immunoblots for light chain (LC) 3B. RESULTS:When the primary RGCs were exposed to CSE for 2 h, cell viability decreased in a dose-dependent manner, as measured by ATP assay. In the presence of 0.05% CSE, the RGC viability was 77.68% ± 7.60% compared to the control cells; in the presence of 1.0% CSE, viability was 47.48% ± 2.56% of the control cells. As determined by TUNEL, CSE increased the apoptotic RGCs in a dose-dependent manner. In the presence of 0.05% CSE, the apoptosis was 26.55% ± 1.97% of the control cells; in the presence of 2.5% CSE, it was 41.07% ± 3.75% of the control cells. When apoptosis was evaluated using real-time RT-PCR, exposure to 0.05% CSE resulted in significantly increased expression of Bad, Bax, Bcl-2, and Bcl-XL mRNA. When autophagy was assessed by Western immunoblots, exposure to 0.05% CSE significantly increased the expression of LC3B II. CONCLUSIONS:Our data suggest that CSE directly injures primary RGCs, and both cell death mechanisms of apoptosis and autophagy seem to be related to this CSE-induced RGC damage.
New strategies for neuroprotection in glaucoma, a disease that affects the central nervous system.
Nucci Carlo,Russo Rossella,Martucci Alessio,Giannini Clarissa,Garaci Francesco,Floris Roberto,Bagetta Giacinto,Morrone Luigi Antonio
European journal of pharmacology
Glaucoma is a disease where retinal ganglion cells (RGC) are specifically affected though a number of evidences endorse the hypothesis that glaucoma is a neuro-degenerative disorder of the central nervous system and suggest a possible connection between glaucomatous damage and cerebrovascular alterations. The mechanisms underlying RGC loss are not yet fully known but alterations of the autophagy machinery have been recently proposed as a potential contributing factor as for Alzheimer's disease. Here we review the current literature on new strategies for neuroprotection in glaucoma, focusing on pharmacologic strategies to minimize RGC damage.
Human TBK1: A Gatekeeper of Neuroinflammation.
Ahmad Liyana,Zhang Shen-Ying,Casanova Jean-Laurent,Sancho-Shimizu Vanessa
Trends in molecular medicine
The importance of TANK binding kinase-1 (TBK1), a multimeric kinase that modulates inflammation and autophagy, in human health has been highlighted for the first time by the recent discoveries of mutations in TBK1 that underlie amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), normal tension glaucoma (NTG) or childhood herpes simplex encephalitis (HSE). Gain-of-function of TBK1 are associated with NTG, whereas loss-of-function mutations result in ALS/FTD or in HSE. In light of these new findings, we review the role of TBK1 in these seemingly unrelated, yet allelic diseases, and discuss the role of TBK1 in neuroinflammatory diseases. This discovery has the potential to significantly increase our understanding of the molecular basis of these poorly understood diseases.
SQSTM1 Mutations and Glaucoma.
Scheetz Todd E,Roos Ben R,Solivan-Timpe Frances,Miller Kathy,DeLuca Adam P,Stone Edwin M,Kwon Young H,Alward Wallace L M,Wang Kai,Fingert John H
Glaucoma is the most common cause of irreversible blindness worldwide. One subset of glaucoma, normal tension glaucoma (NTG) occurs in the absence of high intraocular pressure. Mutations in two genes, optineurin (OPTN) and TANK binding kinase 1 (TBK1), cause familial NTG and have known roles in the catabolic cellular process autophagy. TKB1 encodes a kinase that phosphorylates OPTN, an autophagy receptor, which ultimately activates autophagy. The sequestosome (SQSTM1) gene also encodes an autophagy receptor and also is a target of TBK1 phosphorylation. Consequently, we hypothesized that mutations in SQSTM1 may also cause NTG. We tested this hypothesis by searching for glaucoma-causing mutations in a cohort of NTG patients (n = 308) and matched controls (n = 157) using Sanger sequencing. An additional 1098 population control samples were also analyzed using whole exome sequencing. A total of 17 non-synonymous mutations were detected which were not significantly skewed between cases and controls when analyzed separately, or as a group (p > 0.05). These data suggest that SQSTM1 mutations are not a common cause of NTG.
Autophagy and Mitochondrial Dysfunction in Tenon Fibroblasts from Exfoliation Glaucoma Patients.
Want Andrew,Gillespie Stephanie R,Wang Zheng,Gordon Ronald,Iomini Carlo,Ritch Robert,Wolosin J Mario,Bernstein Audrey M
PURPOSE:To test the hypothesis that autophagy dysfunction is involved in exfoliation syndrome (XFS), a systemic disorder of extracellular elastic matrices that causes a distinct form of human glaucoma. METHODS:Fibroblasts derived from tenon tissue discards (TFs) from filtration surgery to relieve intraocular pressure in XFS patients were compared against age-matched TFs derived from surgery in primary open-angle glaucoma (POAG) patients or from strabismus surgery. Differential interference contrast light, and electron microscopy were used to examine structural cell features. Immunocytochemistry was used to visualize LOXL1 and Fibulin-5, lysosomes, endosomes, Golgi, and microtubules. Light scatter, Cyto-IDTM and JC1 flow cytometry were used to measure relative cell size, autophagic flux rate and mitochondrial membrane potential (MMPT), respectively. Enhanced autophagy was induced by serum withdrawal. RESULTS:In culture, XFS-TFs were 1.38-fold larger (by light scatter ratio, p = 0.05), proliferated 42% slower (p = 0.026), and were morphologically distinct in 2D and 3D culture compared to their POAG counterparts. In extended 3D cultures, XFS-TFs accumulated 8-10 times more Fibulin-5 than the POAG-TFs, and upon serum withdrawal, there were marked deficiencies in relocation of endosomes and lysosomes to the perinuclear area. Correspondingly, the XFS-TFs displayed significant accumulation of the autophagasome marker LC3 II (3.9 fold increase compared to POAG levels, p = 0.0001) and autophagic flux rate as measured by Cyto-ID dye was 53% lower in XFS-TFs than in POAG-TFs (p = 0.01), indicating reduced clearance of autophagasomes. Finally the percent of cells with diminished MMPT was 3-8 times larger in the XFS-TFs than in POAG-TFs (p = 0.02). CONCLUSIONS:Our results provide for the first time a link between XFS pathology to autophagy dysfunction, a major contributor to multiple age related diseases systemically throughout the body, in the brain and in the retina. A diminished capacity for degradation of denatured protein and aging cellular organelles may underpin the development of extracellular protein aggregates in XFS.
From the Cover: Alterations in Optineurin Expression and Localization in Pre-clinical Parkinson's Disease Models.
Wise John Pierce,Cannon Jason
Toxicological sciences : an official journal of the Society of Toxicology
Parkinson's disease (PD) is a progressive neurodegenerative disease that affects ∼5 million people around the world. PD etiopathogenesis is poorly understood and curative or disease modifying treatments are not available. Mechanistic studies have identified numerous pathogenic pathways that overlap with many other neurodegenerative diseases. Mutations in the protein optineurin (OPTN) have recently been identified as causative factors for glaucoma and amyotrophic lateral sclerosis. OPTN has multiple recognized roles in neurons, notably in mediating autophagic flux, which has been found to be disrupted in most neurodegenerative diseases. OPTN(+ )aggregates have preliminarily been identified in cytoplasmic inclusions in numerous neurodegenerative diseases, however, whether OPTN has a role in PD pathogenesis has yet to be tested. Thus, we chose to test the hypothesis that OPTN expression and localization would be modulated in pre-clinical PD models. To test our hypothesis, we characterized midbrain OPTN expression in normal rats and in a rat rotenone PD model. For the first time, we show that OPTN is enriched in dopamine neurons in the midbrain, and its expression is modulated by rotenone treatment in vivo Here, animals were sampled at time-points both prior to overt neurodegeneration and after severe behavioral deficits, where a lesion to the nigrostriatal dopamine system is present. The effect and magnitude of OPTN expression changes are dependent on duration of treatment. Furthermore, OPTN colocalizes with LC3 (autophagic vesicle marker) and alpha-synuclein positive puncta in rotenone-treated animals, potentially indicating an important role in autophagy and PD pathogenesis.
Cellular Functions of Optineurin in Health and Disease.
Slowicka Karolina,Vereecke Lars,van Loo Geert
Trends in immunology
Optineurin (OPTN) was initially identified as a regulator of NF-κB and interferon signaling, but attracted most attention because of its association with various human disorders such as glaucoma, Paget disease of bone, and amyotrophic lateral sclerosis. Importantly, OPTN has recently been identified as an autophagy receptor important for the autophagic removal of pathogens, damaged mitochondria, and protein aggregates. This activity is most likely compromised in patients carrying OPTN mutations, and contributes to the observed phenotypes. In this review we summarize recent studies describing the molecular mechanisms by which OPTN controls immunity and autophagy, and discuss these findings in the context of several diseases that have been associated with OPTN (mal)function.
Autophagy in the eye: Development, degeneration, and aging.
Boya Patricia,Esteban-Martínez Lorena,Serrano-Puebla Ana,Gómez-Sintes Raquel,Villarejo-Zori Beatriz
Progress in retinal and eye research
Autophagy is a catabolic pathway that promotes the degradation and recycling of cellular components. Proteins, lipids, and even whole organelles are engulfed in autophagosomes and delivered to the lysosome for elimination. In response to stress, autophagy mediates the degradation of cell components, which are recycled to generate the nutrients and building blocks required to sustain cellular homeostasis. Moreover, it plays an important role in cellular quality control, particularly in neurons, in which the total burden of altered proteins and damaged organelles cannot be reduced by redistribution to daughter cells through cell division. Research has only begun to examine the role of autophagy in the visual system. The retina, a light-sensitive tissue, detects and transmits electrical impulses through the optic nerve to the visual cortex in the brain. Both the retina and the eye are exposed to a variety of environmental insults and stressors, including genetic mutations and age-associated alterations that impair their function. Here, we review the main studies that have sought to explain autophagy's importance in visual function. We describe the role of autophagy in retinal development and cell differentiation, and discuss the implications of autophagy dysregulation both in physiological aging and in important diseases such as age-associated macular degeneration and glaucoma. We also address the putative role of autophagy in promoting photoreceptor survival and discuss how selective autophagy could provide alternative means of protecting retinal cells. The findings reviewed here underscore the important role of autophagy in maintaining proper retinal function and highlight novel therapeutic approaches for blindness and other diseases of the eye.
Structural insights into the interaction and disease mechanism of neurodegenerative disease-associated optineurin and TBK1 proteins.
Li Faxiang,Xie Xingqiao,Wang Yingli,Liu Jianping,Cheng Xiaofang,Guo Yujiao,Gong Yukang,Hu Shichen,Pan Lifeng
Optineurin is an important autophagy receptor involved in several selective autophagy processes, during which its function is regulated by TBK1. Mutations of optineurin and TBK1 are both associated with neurodegenerative diseases. However, the mechanistic basis underlying the specific interaction between optineurin and TBK1 is still elusive. Here we determine the crystal structures of optineurin/TBK1 complex and the related NAP1/TBK1 complex, uncovering the detailed molecular mechanism governing the optineurin and TBK1 interaction, and revealing a general binding mode between TBK1 and its associated adaptor proteins. In addition, we demonstrate that the glaucoma-associated optineurin E50K mutation not only enhances the interaction between optineurin and TBK1 but also alters the oligomeric state of optineurin, and the ALS-related TBK1 E696K mutation specifically disrupts the optineurin/TBK1 complex formation but has little effect on the NAP1/TBK1 complex. Thus, our study provides mechanistic insights into those currently known disease-causing optineurin and TBK1 mutations found in patients.
Significance of optineurin mutations in glaucoma and other diseases.
Minegishi Yuriko,Nakayama Mao,Iejima Daisuke,Kawase Kazuhide,Iwata Takeshi
Progress in retinal and eye research
Glaucoma is one of the leading causes of bilateral blindness, affecting nearly 57 million people worldwide. Glaucoma is characterized by a progressive loss of retinal ganglion cells and is often associated with intraocular pressure (IOP). Normal tension glaucoma (NTG), marked by normal IOP but progressive glaucoma, is incompletely understood. In 2002, Sarfarazi et al. identified FIP-2 gene mutations responsible for hereditary NTG, renaming this gene "optineurin" (OPTN). Further investigations by multiple groups worldwide showed that OPTN is involved in several critical cellular functions, such as NF-κB regulation, autophagy, and vesicle transport. Recently, OPTN mutations were found to cause amyotrophic lateral sclerosis (ALS). Surprisingly, a mutation in the OPTN interacting protein, i.e., the duplication of TANK binding protein 1 (TBK1) gene, also can cause both NTG and ALS. These phenotypically distinct neuronal diseases are now merging into one common pathological mechanism by these two genes. TBK1 inhibition has emerged as a potential therapy for NTG. In this manuscript, we focus on the OPTN E50K mutation, the most common mutation for NTG, to describe the molecular mechanism of NTG by expressing a mutant Optn gene in cells and genetically modified mice. Patient iPS cells were developed and differentiated into neural cells to observe abnormal behavior and the impact of the E50K mutation. These in vitro studies were further extended to identify the inhibitors BX795 and amlexanox, which have the potential to reverse the disease-causing phenomenon in patient's neural cells. Here we show for the first time that amlexanox protects RGCs in Optn E50K knock-in mice.
The Evolving Functions of Autophagy in Ocular Health: A Double-edged Sword.
Chai Peiwei,Ni Hongyan,Zhang He,Fan Xianqun
International journal of biological sciences
Autophagy plays an adaptive role in cell survival, development, differentiation and intracellular homeostasis. Autophagy is recognized as a 'self-cannibalizing' process that is active during stresses such as starvation, chemotherapy, infection, ageing, and oxygen shortage to protect organisms from various irritants and to regenerate materials and energy. However, autophagy can also lead to a form of programmed cell death distinct from apoptosis. Components of the autophagic pathway are constitutively expressed at a high level in the eye, including in the cornea, lens, retina, and orbit. In addition, the activation of autophagy is directly linked to the development of eye diseases such as age-related macular degeneration (ARMD), cataracts, diabetic retinopathy (DR), glaucoma, photoreceptor degeneration, ocular tumours, ocular infections and thyroid-associated ophthalmopathy (TAO). A high level of autophagy defends against external stress; however, excessive autophagy can result in deterioration, as observed in ocular diseases such as ARMD and DR. This review summarizes recent developments elucidating the relationship between autophagy and ocular diseases and the potential roles of autophagy in the pathogenesis and treatment of these diseases.
A critical role of Hrd1 in the regulation of optineurin degradation and aggresome formation.
Mao Jiahui,Xia Qin,Liu Chunfeng,Ying Zheng,Wang Hongfeng,Wang Guanghui
Human molecular genetics
Mutations in optineurin (OPTN) are associated with several human disorders including amyotrophic lateral sclerosis (ALS) and primary open-angle glaucoma (POAG). OPTN is known to be a multifunctional autophagy receptor that plays important roles in NF-κB signaling, vesicle trafficking, maintenance of the Golgi apparatus and autophagy. Given that a loss of neurons and an abnormal aggregation of disease proteins are two key features of neurodegenerative diseases, protein quality control systems are considered to be tightly associated with neurodegeneration. In this study, we investigated the involvement of the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway, two major intracellular protein quality control systems, in the regulation of wild-type (WT) OPTN, ALS-linked mutant E478G OPTN and POAG-linked mutant E50K OPTN. Our data revealed that the UPS, not the autophagy-lysosome pathway, is the major system for degradation and aggregation of OPTN. Moreover, we found that Hrd1, an E3 ubiquitin ligase, could play an important role in the protein quality control of OPTN. Our results demonstrated that overexpression of Hrd1 increased the proteasomal degradation and microtubule-dependent aggresome formation of OPTN in the microtubular organizing center, whereas knockdown of Hrd1 stabilized OPTN and inhibited aggresome formation of OPTN.
The role and therapeutic potential of melatonin in age-related ocular diseases.
Crooke Almudena,Huete-Toral Fernando,Colligris Basilio,Pintor Jesús
Journal of pineal research
The eye is continuously exposed to solar UV radiation and pollutants, making it prone to oxidative attacks. In fact, oxidative damage is a major cause of age-related ocular diseases including cataract, glaucoma, age-related macular degeneration, and diabetic retinopathy. As the nature of lens cells, trabecular meshwork cells, retinal ganglion cells, retinal pigment epithelial cells, and photoreceptors is postmitotic, autophagy plays a critical role in their cellular homeostasis. In age-related ocular diseases, this process is impaired, and thus, oxidative damage becomes irreversible. Other conditions such as low-grade chronic inflammation and angiogenesis also contribute to the development of retinal diseases (glaucoma, age-related macular degeneration and diabetic retinopathy). As melatonin is known to have remarkable qualities such as antioxidant/antinitridergic, mitochondrial protector, autophagy modulator, anti-inflammatory, and anti-angiogenic, it can represent a powerful tool to counteract all these diseases. The present review analyzes the role and therapeutic potential of melatonin in age-related ocular diseases, focusing on nitro-oxidative stress, autophagy, inflammation, and angiogenesis mechanisms.
[The progress of studies on aqueous humor dynamics abnormality induced by trabecular meshwork and Schlemm canal endothelial cell senescence and its relation with glaucoma].
Song M M,Lei Y,Wu J H,Sun X H
[Zhonghua yan ke za zhi] Chinese journal of ophthalmology
Glaucoma is the second leading cause of blindness in the world next to cataract. Aging is a strong risk factor leading to elevated intraocular pressure (IOP). IOP is associated with aqueous humor circulation. Trabecular meshwork cells and Schlemm canal endothelial cells, which form the conventional outflow pathway, play an important role in maintaining the IOP. Cell senescence induces abnormalities of the aqueous humor dynamics, leading to elevated IOP. Trabecular meshwork cells cause increased intrinsic stiffness, autophagy dysfunction, abnormal expression of microRNA and mitochondrial dysfunction with senescence. The senescence of Schlemm canal endothelial cells decreases cell permeability and mechanotransduction and disrupts the endothelial nitric oxide synthase signaling pathway. The changes will increase aqueous humor outflow resistance and elevate IOP. This review discusses how cell senescence induces aqueous humor dynamics abnormalities and the relation with glaucoma. .
Ghrelin Attenuates Retinal Neuronal Autophagy and Apoptosis in an Experimental Rat Glaucoma Model.
Zhu Ke,Zhang Meng-Lu,Liu Shu-Ting,Li Xue-Yan,Zhong Shu-Min,Li Fang,Xu Ge-Zhi,Wang Zhongfeng,Miao Yanying
Investigative ophthalmology & visual science
Purpose:Ghrelin, a natural ligand for the growth hormone secretagogue receptor type 1a (GHSR-1a), may protect retinal neurons against glaucomatous injury. We therefore characterized the underlying mechanism of the ghrelin/GHSR-1a-mediated neuroprotection with a rat chronic intraocular hypertension (COH) model. Methods:The rat COH model was produced by blocking episcleral veins. A combination of immunohistochemistry, Western blot, TUNEL assay, and retrograde labeling of retinal ganglion cells (RGCs) was used. Results:Elevation of intraocular pressure induced a significant increase in ghrelin and GHSR-1a expression in retinal cells, including RGCs and Müller cells. Western blot confirmed that the protein levels of ghrelin exhibited a transient upregulation at week 2 after surgery (G2w), while the GHSR-1a protein levels were maintained at high levels from G2w to G4w. In COH retinas, the ratio of LC3-II/LC-I and beclin1, two autophagy-related proteins, were increased from G1w to G4w, and the cleavage product of caspase3, an apoptotic executioner, was detected from G2w to G4w. Intraperitoneal injection of ghrelin significantly increased the number of surviving RGCs; inhibited the changes of LC3-II/LC-I, beclin1, and the cleavage products of caspase3; and reduced the number of TUNEL-positive cells in COH retinas. Ghrelin treatment also reversed the decreased levels of p-Akt and p-mTOR, upregulated GHSR-1a protein levels, and attenuated glial fibrillary acidic protein levels in COH retinas. Conclusions:All these results suggest that ghrelin may provide neuroprotective effect in COH retinas through activating ghrelin/GHSR-1a system, which was mediated by inhibiting retinal autophagy, ganglion cell apoptosis, and Müller cell gliosis.
MiR-93-5p targeting PTEN regulates the NMDA-induced autophagy of retinal ganglion cells via AKT/mTOR pathway in glaucoma.
Li Rui,Jin Yiping,Li Qian,Sun Xinghuai,Zhu Haohao,Cui Hongping
Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie
BACKGROUND:Glaucoma is hallmarked with the death of retinal neurons in the ganglion cell layer, which results in irreversible vision loss. The abnormal levels of miRNA have been associated with glaucoma. Our study purposed to explore the underlying molecule mechanism of miR-93-5p in NMDA-induced glaucoma. METHODS:The Sprague-Dawley (SD) rats were used for the establishment of glaucoma model with the injection of NMDA. Vision behavior test were performed on the glaucoma rats. MiR-93-5p expression was determined by real-time PCR. The levels of autophagy-related protein and PTEN were assessed by Western blot assays. TUNEL assay and flow cytometry were performed to analyze cell apoptosis in vivo and in vitro, respectively. And cell viability was examined by CKK-8 assay. The relationship between miR-93-5p and PTEN was confirmed by Dual-Luciferase reporter gene system. RESULTS:NMDA-induced glaucoma rats exhibited less time in the dark box, suggesting the recession of their vision. Moreover, the retinal ganglion cell (RGC) viability was reduced not only in the glaucoma rat models but also in the glaucoma RGC models. The autophagy-related protein was obviously increased in the NMDA-treated rats or RGCs. PTEN regulated the autophagy of RGCs through AKT/mTOR pathway in NMDA-treated RGCs. MiR-93-5p could target regulate PTEN negatively, and exhibit the similar effect of 3-MA on the survival of RGCs. CONCLUSION:Up-regulation of miR-93-5p binding with PTEN suppressed the autophagy of RGCs through AKT/mTOR pathway in NMDA-induced glaucoma.
Is Autophagy Dysfunction a Key to Exfoliation Glaucoma?
Wolosin J Mario,Ritch Robert,Bernstein Audrey M
Journal of glaucoma
In this short report we review previous work toward the identification of the protein and cellular sources of exfoliation glaucoma and described our recent finding on dysfunction of autophagy in Tenon capsule fibroblasts obtained from exfoliation syndrome glaucoma patients at the time of surgery and discuss the potential implications of these findings for understanding the cellular sources of the disease.
MEG3 is involved in the development of glaucoma through promoting the autophagy of retinal ganglion cells.
Sun W,Li Y-N,Ye J-F,Guan Y-Q,Li S-J
European review for medical and pharmacological sciences
OBJECTIVE:In this study, we aimed at investigating whether MEG3 may be involved in the pathogenesis of glaucoma by regulating the autophagy of retinal ganglion cells (RGCs). MATERIALS AND METHODS:We used qRT-PCR to detect the expression of MEG3 in RGC-5s cell line under high hydrostatic pressure. RGC-5s were transfected with a lentiviral vector to achieve MEG3 overexpression or knockdown. The influence of overexpression or inhibition of MEG3 on cell proliferation and apoptosis was observed using CCK-8 test and flow cytometry. After overexpression of MEG3 and/or knockdown of MEG3 or Beclin-1, detection of the expressions of autophagy-related and apoptosis-related proteins was performed using Western blot. RESULTS:MEG3 expression level increased in RGC-5 cells under high hydrostatic pressure, while exogenously decreased MEG3 expression can reverse the impact of the high pressure on RGC-5 cells. Additionally, overexpression of MEG3 can improve Atg3 expression, promote cell apoptosis, inhibit cell proliferation, and enhance autophagy levels. Meanwhile, knockdown of Beclin-1 up-regulated Bcl-2 level. CONCLUSIONS:Upregulation of MEG3 is involved in the pathogenesis of glaucoma through promoting apoptosis of retinal ganglion cells, the mechanism of which may be related to the enhanced autophagy levels.
Optineurin: A Coordinator of Membrane-Associated Cargo Trafficking and Autophagy.
Ryan Thomas A,Tumbarello David A
Frontiers in immunology
Optineurin is a multifunctional adaptor protein intimately involved in various vesicular trafficking pathways. Through interactions with an array of proteins, such as myosin VI, huntingtin, Rab8, and Tank-binding kinase 1, as well as via its oligomerisation, optineurin has the ability to act as an adaptor, scaffold, or signal regulator to coordinate many cellular processes associated with the trafficking of membrane-delivered cargo. Due to its diverse interactions and its distinct functions, optineurin is an essential component in a number of homeostatic pathways, such as protein trafficking and organelle maintenance. Through the binding of polyubiquitinated cargoes via its ubiquitin-binding domain, optineurin also serves as a selective autophagic receptor for the removal of a wide range of substrates. Alternatively, it can act in an ubiquitin-independent manner to mediate the clearance of protein aggregates. Regarding its disease associations, mutations in the optineurin gene are associated with glaucoma and have more recently been found to correlate with Paget's disease of bone and amyotrophic lateral sclerosis (ALS). Indeed, ALS-associated mutations in optineurin result in defects in neuronal vesicular localisation, autophagosome-lysosome fusion, and secretory pathway function. More recent molecular and functional analysis has shown that it also plays a role in mitophagy, thus linking it to a number of other neurodegenerative conditions, such as Parkinson's. Here, we review the role of optineurin in intracellular membrane trafficking, with a focus on autophagy, and describe how upstream signalling cascades are critical to its regulation. Current data and contradicting reports would suggest that optineurin is an important and selective autophagy receptor under specific conditions, whereby interplay, synergy, and functional redundancy with other receptors occurs. We will also discuss how dysfunction in optineurin-mediated pathways may lead to perturbation of critical cellular processes, which can drive the pathologies of number of diseases. Therefore, further understanding of optineurin function, its target specificity, and its mechanism of action will be critical in fully delineating its role in human disease.
Dysfunction of Optineurin in Amyotrophic Lateral Sclerosis and Glaucoma.
Toth Reka P,Atkin Julie D
Frontiers in immunology
Neurodegenerative disorders, including amyotrophic lateral sclerosis (ALS), frontotemporal dementia, and glaucoma, affect millions of people worldwide. ALS is caused by the loss of motor neurons in the spinal cord, brainstem, and brain, and genetic mutations are responsible for 10% of all ALS cases. Glaucoma is characterized by the loss of retinal ganglion cells and is the most common cause of irreversible blindness. Interestingly, mutations in , encoding optineurin, are associated with both ALS and glaucoma. Optineurin is a highly abundant protein involved in a wide range of cellular processes, including the inflammatory response, autophagy, Golgi maintenance, and vesicular transport. In this review, we summarize the role of optineurin in cellular mechanisms implicated in neurodegenerative disorders, including neuroinflammation, autophagy, and vesicular trafficking, focusing in particular on the consequences of expression of mutations associated with ALS and glaucoma. This review, therefore showcases the impact of optineurin dysfunction in ALS and glaucoma.
Altered Functions and Interactions of Glaucoma-Associated Mutants of Optineurin.
Swarup Ghanshyam,Sayyad Zuberwasim
Frontiers in immunology
Optineurin (OPTN) is an adaptor protein that is involved in mediating a variety of cellular processes such as signaling, vesicle trafficking, and autophagy. Certain mutations in OPTN (gene ) are associated with primary open angle glaucoma, a leading cause of irreversible blindness, and amyotrophic lateral sclerosis, a fatal motor neuron disease. Glaucoma-associated mutations of OPTN are mostly missense mutations. OPTN mediates its functions by interacting with various proteins and altered interactions of OPTN mutants with various proteins primarily contribute to functional defects. It interacts with Rab8, myosin VI, Huntigtin, TBC1D17, and transferrin receptor to mediate various membrane vesicle trafficking pathways. It is an autophagy receptor that mediates cargo-selective as well as non-selective autophagy. Glaucoma-associated mutants of OPTN, E50K, and M98K, cause defective vesicle trafficking, autophagy, and signaling that contribute to death of retinal ganglion cells (RGCs). Transgenic mice expressing E50K-OPTN show loss of RGCs and persistent reactive gliosis. TBK1 protein kinase, which mediates E50K-OPTN and M98K-OPTN induced cell death, is emerging as a potential drug target. Autoimmunity has been implicated in glaucoma but involvement of OPTN or its mutants in autoimmnity has not been explored. In this review, we highlight the main functions of OPTN and how glaucoma-associated mutants alter these functions. We also discuss some of the controversies, such as the role of OPTN in signaling to transcription factor NF-κB, interferon signaling, and use of RGC-5 cell line as a cell culture model.
Optineurin: The autophagy connection.
Ying Hongyu,Yue Beatrice Y J T
Experimental eye research
Optineurin is a cytosolic protein encoded by the OPTN gene. Mutations of OPTN are associated with normal tension glaucoma and amyotrophic lateral sclerosis. Autophagy is an intracellular degradation system that delivers cytoplasmic components to the lysosomes. It plays a wide variety of physiological and pathophysiological roles. The optineurin protein is a selective autophagy receptor (or adaptor), containing an ubiquitin binding domain with the ability to bind polyubiquitinated cargoes and bring them to autophagosomes via its microtubule-associated protein 1 light chain 3-interacting domain. It is involved in xenophagy, mitophagy, aggrephagy, and tumor suppression. Optineurin can also mediate the removal of protein aggregates through an ubiquitin-independent mechanism. This protein in addition can induce autophagy upon overexpression or mutation. When overexpressed or mutated, the optineurin protein also serves as a substrate for autophagic degradation. In the present review, the multiple connections of optineurin to autophagy are highlighted.
The autophagic lysosomal system in outflow pathway physiology and pathophysiology.
Liton Paloma B
Experimental eye research
Malfunction of the trabecular meshwork (TM)/schlemm's canal (SC) conventional outflow pathway is associated with elevated intraocular pressure (IOP) and, therefore, increased risk of developing glaucoma, a potentially blinding disease affecting more than 70 million people worldwide. This TM/SC tissue is subjected to different types of stress, including mechanical, oxidative, and phagocytic stress. Long-term exposure to these stresses is believed to lead to a progressive accumulation of damaged cellular and tissue structures causing permanent alterations in the tissue physiology, and contribute to the pathologic increase in aqueous humor (AH) outflow resistance. Autophagy is emerging as an essential cellular survival mechanism against a variety of stressors. In addition to performing basal functions, autophagy acts as a cellular survival pathway and represents an essential mechanism by which organisms can adapt to acute stress conditions and repair stress-induced damage. A decline in autophagy has been observed in most tissues with aging and has been considered responsible, at least in part, for the accumulation of damaged cellular components in almost all tissues of aging organisms. Dysfunction in the autophagy pathway is associated with several human diseases, from infectious diseases to cancer and neurodegeneration. In this review, we will summarize our current knowledge of the emerging roles of autophagy in outflow tissue physiology and pathophysiology, including novel evidence suggesting compromised autophagy in the glaucomatous outflow pathway.
Defects in autophagy caused by glaucoma-associated mutations in optineurin.
Sirohi Kapil,Swarup Ghanshyam
Experimental eye research
Certain mutations in optineurin (gene OPTN) are associated with primary open angle glaucoma. Optineurin is ubiquitously expressed but it shows high level of expression in certain cells and tissues including retinal ganglion cells. It interacts with many proteins, often acting as an adaptor to link two or more proteins. These interactions play a crucial role in mediating various functions of optineurin such as membrane vesicle trafficking, autophagy, signal transduction etc. Autophagy is basically a quality control mechanism to remove damaged proteins and organelles through lysosomal degradation. Optineurin was identified as an autophagy receptor that directly interacts with autophagosomal protein, LC3, and ubiquitin. These interactions are important for autophagy receptor function. Autophagy receptors recruit their cargo and take it to autophagosomes which fuse with lysosomes to form autolysosomes where degradation of proteins takes place. Optineurin interacts with a motor protein, myosinVI, and this interaction is involved in mediating fusion of autophagosomes with lysosomes. A glaucoma-associated mutant of optineurin, E50K, impairs autophagy as well as vesicle trafficking, leading to death of retinal cells by apoptosis. E50K-OPTN-induced block in autophagy is dependent on a GTPase activating protein, TBC1D17. The E50K mutant also causes other changes in the cells such as altered interaction with TBK1 protein kinase, aggregate formation, generation of reactive oxygen species and inhibition of proteasome, which may contribute to pathogenesis. A polymorphism of optineurin, M98K, associated with glaucoma, causes enhanced autophagy leading to transferrin receptor degradation and apoptotic death of retinal cells. M98K-OPTN-induced autophagic cell death is dependent on Rab12 GTPase. Thus, an optimum level of optineurin-mediated autophagy is crucial for survival of retinal cells, and impaired autophagy is likely to contribute to glaucoma pathogenesis. How impaired autophagy caused by optineurin mutants leads to apoptosis and cell death, is yet to be explored.
Targeting the ER-autophagy system in the trabecular meshwork to treat glaucoma.
Stothert Andrew R,Fontaine Sarah N,Sabbagh Jonathan J,Dickey Chad A
Experimental eye research
A major drainage network involved in aqueous humor dynamics is the conventional outflow pathway, which is gated by the trabecular meshwork (TM). The TM acts as a molecular sieve, providing resistance to aqueous outflow, which is responsible for regulating intraocular pressure (IOP). If the TM is damaged, aqueous outflow is impaired, IOP increases and glaucoma can manifest. Mutations in the MYOC gene cause hereditary primary open-angle glaucoma (POAG) by promoting the abnormal amyloidosis of the myocilin protein in the endoplasmic reticulum (ER), leading to ER stress-induced TM cell death. Myocilin accumulation is observed in approximately 70-80% of all glaucoma cases suggesting that environmental or other genetic factors may also promote myocilin toxicity. For example, simply preventing myocilin glycosylation is sufficient to promote its abnormal accretion. These myocilin amyloids are unique as there are no other known pathogenic proteins that accumulate within the ER of TM cells and cause toxicity. Moreover, this pathogenic accumulation only kills TM cells, despite expression of this protein in other cell types, suggesting that another modifier exclusive to the TM participates in the proteotoxicity of myocilin. ER autophagy (reticulophagy) is one of the pathways essential for myocilin clearance that can be impacted dramatically by aging and other environmental factors such as nutrition. This review will discuss the link between myocilin and autophagy, evaluating the role of this degradation pathway in glaucoma as well as its potential as a therapeutic target.
Working your SOCS off: The role of ASB10 and protein degradation pathways in glaucoma.
Keller Kate E,Wirtz Mary K
Experimental eye research
Evidence is accumulating to suggest that mutations in the Ankyrin and SOCS Box-containing protein-10 (ASB10) gene are associated with glaucoma. Since its identification in a large Oregon family with primary open-angle glaucoma (POAG), ASB10 variants have been associated with disease in US, German and Pakistani cohorts. ASB10 is a member of the ASB family of proteins, which have a common structure including a unique N-terminus, a variable number of central ankyrin (ANK) repeat domains and a suppressor of cytokine signaling (SOCS) box at the C-terminus. Mutations in ASB10 are distributed throughout the entire length of the gene including the two alternatively spliced variants of exon 1. A homozygous mutation in a Pakistani individual with POAG, which lies in the center of the SOCS box, is associated with a particularly severe form of the disease. Like other SOCS box-containing proteins, ASB10 functions in ubiquitin-mediated degradation pathways. The ANK repeats bind to proteins destined for degradation. The SOCS box recruits ubiquitin ligase proteins to form a complex to transfer ubiquitin to a substrate bound to the ANK repeats. The ubiquitin-tagged protein then enters either the proteasomal degradation pathway or the autophagic-lysosomal pathway. The choice of pathway appears to be dependent on which lysine residues are used to build polyubiquitin chains. However, these reciprocal pathways work in tandem to degrade proteins because inhibition of one pathway increases degradation via the other pathway. In this publication, we will review the literature that supports identification of ASB10 as a glaucoma-associated gene and the current knowledge of the function of the ASB10 protein. In addition, we present new data that indicates ASB10 expression is up-regulated by the inflammatory cytokines tumor necrosis factor-α and interleukin-1α. Finally, we will describe the emerging role of other SOCS box-containing proteins in protein degradation pathways in ocular cells.
Autophagy and mechanotransduction in outflow pathway cells.
Hirt Joshua,Liton Paloma B
Experimental eye research
Because of elevations in IOP and other forces, cells in the trabecular meshwork (TM) are constantly subjected to mechanical strain. In order to preserve cellular function and regain homeostasis, cells must sense and adapt to these morphological changes. We and others have already shown that mechanical stress can trigger a broad range of responses in TM cells; however, very little is known about the strategies that TM cells use to respond to this stress, so they can adapt and survive. Autophagy, a lysosomal degradation pathway, has emerged as an important cellular homeostatic mechanism promoting cell survival and adaptation to a number of cytotoxic stresses. Our laboratory has reported the activation of autophagy in TM cells in response to static biaxial strain and high pressure. Moreover, our newest data also suggest the activation of chaperon-assisted selective autophagy, a recently identified tension-induced autophagy essential for mechanotransduction, in TM cells under cyclic mechanical stress. In this review manuscript we will discuss autophagy as part of an integrated response triggered in TM cells in response to strain, exerting a dual role in repair and mechanotransduction, and the potential effects of dysregulated in outflow pathway pathophysiology.
Investigation of Association between Autophagy-Related Gene Polymorphisms and Pseudoexfoliation Syndrome and Pseudoexfoliation Glaucoma in a Spanish Population.
de Juan-Marcos Lourdes,Escudero-Domínguez Francisco A,Hernández-Galilea Emiliano,Cruz-González Fernando,Follana-Neira Isora,González-Sarmiento Rogelio
Seminars in ophthalmology
PURPOSE:Cellular stress conditions are important mechanisms implicated in the pathogenesis of pseudoexfoliation syndrome. One of the potential cellular responses to these stress conditions is induction of autophagy. The purpose of this study was to evaluate whether genetic variants in three critical genes of autophagy (ATG16L, ATG2B, ATG5) may be involved in the development of pseudoexfoliation syndrome (XFS) and pseudoexfoliation glaucoma (XFG) in a Spanish population. METHODS:108 patients (64 XFS, 44XFG) and 118 healthy controls were evaluated. The analysis of genetic polymorphisms was performed by standard TaqMan allelic discrimination technique. RESULTS:No significant differences in either genotype distributions or allelic frequencies of the tested polymorphisms were found between patients with XFS/XFG and control subjects. CONCLUSIONS:Our results suggest that these three genes that are critical components of the autophagy pathway (ATG16L, ATG2B, ATG5) are not significant risk factors among Spanish patients with either XFS or XFG.
Why autophagy is good for retinal ganglion cells?
Eye (London, England)
Autophagy is a catabolic pathway that promotes the degradation and recycling of cellular components. Proteins, lipids, and even whole organelles are engulfed in autophagosomes and delivered to the lysosome for elimination. In response to stress, autophagy mediates the degradation of cell components, which are recycled to generate the nutrients and building blocks required to sustain cellular homeostasis. Moreover, it has an important role in cellular quality control, particularly in neurons, in which the total burden of altered proteins and damaged organelles cannot be reduced by redistribution to daughter cells through cell division. Autophagy occurs in all cells and tissues, and it is regulated by the Atg genes. The importance of this pathway has been recently recognized by the Nobel Prize in Physiology and Medicine award to Professor Yoshinori Ohsumi who was the discoverer of the first Atg genes in yeast in the 1990s. Research has only begun to examine the role of autophagy in the visual system. Both the retina and the eye are exposed to a variety of environmental insults and stressors, including genetic mutations and age-associated alterations that impair their function. Here, we review studies that have sought to explain autophagy's importance for retinal ganglion cells, and their implications for diseases like glaucoma and optic neuropathies.
Exfoliation Syndrome: A Disease of Autophagy and LOXL1 Proteopathy.
Bernstein Audrey M,Ritch Robert,Wolosin Jose M
Journal of glaucoma
Exfoliation syndrome (XFS) is an age-related disease involving the deposition of aggregated fibrillar material (exfoliation material) at extracellular matrices in tissues that synthesize elastic fibers. Its main morbidity is in the eye, where exfoliation material accumulations form on the surface of the ciliary body, iris, and lens. Exfoliation glaucoma (XFG) occurs in a high proportion of persons with XFS and can be a rapidly progressing disease. Worldwide, XFG accounts for about 25% of open-angle glaucoma cases. XFS and XFG show a sharp age-dependence, similarly to the many age-related diseases classified as aggregopathies. Progress in understanding the cellular bases for XFS/XFG has been slowed by a lack of experimental models. Working with primary human tenon fibroblasts (TF) derived from trabeculectomies of XFG patients and age-matched primary open-glaucoma controls, we found that TF from XFG cells display many of the functional features observed in cells from other protein aggregate diseases, such as Parkinson, Alzheimer, Huntington, and age-related macular degeneration. We have documented defects in lysosomal positioning, microtubule organization, autophagy processing rate, and mitochondrial health. In regard to failure of lysosomal and autophagosome positioning in XFG cells, we have found that XFG TF are unable to establish the transnuclear microtubule organizing center that is required for efficient centripetal vesicular locomotion along microtubules. In regard to potential sources of the autophagy malfunction, we have directed our attention to a potential role of the lysyl oxidase-like 1 protein (LOXL1), the elastic fiber catalyst that displays variant-dependent association with risk for XFG. Our experiments show that (a) in XFG cells, a substantial fraction of LOXL1 is processed for degradation by the autophagic system; (b) most of the LOXL1 N-terminus domain exists in a highly disordered state, a condition known to greatly increase the frequency of polypeptide misfolding; (c) that maximum misfolding occurs at amino acid position 153, the location of the high risk variant G153D; and (d) that replacement of glycine (G) by aspartate (D) there results in a substantial decrease in disorder within the 20 amino acid surrounding domain. Finally, we show that clusterin, a protein that can be induced by the presence of intracellular, or extracellular aggregates, is uniformly overexpressed in XFG TF. The implications of our results for a theory relating XFG to cellular aggregopathy are discussed.
Contribution of autophagy to ocular hypertension and neurodegeneration in the DBA/2J spontaneous glaucoma mouse model.
Hirt Joshua,Porter Kris,Dixon Angela,McKinnon Stuart,Liton Paloma B
Cell death discovery
Glaucoma is a progressive optic neuropathy characterized by axonal degeneration and retinal ganglion cells loss. Several factors have been postulated to play a role in glaucoma, elevated intraocular pressure (IOP) being the best well-known causative factor. The mechanisms leading to ocular hypertension and glaucoma are still not fully understood. An increasing number of evidence indicates a role of autophagy in the pathophysiological process of ocular hypertension and glaucoma. However, while all of the studies agree that autophagy is induced in RGCs in response to injury, autophagy was found to either protect or promote cell death depending on the experimental model used. In order to gain more insight into both, the role of autophagy in the pathogenesis of glaucoma and the effect of chronic IOP elevation in the autophagy pathway, we have investigated here for the first time autophagy in the iridocorneal angle region, retinal ganglion cell bodies, and ON axons in the spontaneous ocular hypertensive DBA/2J mouse glaucoma model and in the transgenic DBA/2J::GFP-LC3 mice, generated in our laboratory. Our results indicate decreased autophagic flux in the outflow pathway cells in the DBA/2J mice, characterized by increased levels of LC3-II and p62 together with a decrease in the lysosomal marker LAMP1, evaluated by western blot and immunofluorescence. Elevated presence of autophagic vacuoles in the DBA/2J and, in particular, in the DBA/2J::GFP-LC3 mice was also observed. Expression of the GFP-LC3 transgene was associated to higher cumulative IOP in the DBA/2J background. In addition to higher elevation in IOP, DBA/2J::GFP-LC3 were characterized by further RGCs and exacerbated axonal degeneration compared to DBA/2J. This was accompanied by the notable high presence of autophagic figures within degenerating axons. These results strongly suggest overactivation of autophagy as a potential cellular mechanism leading to ON degeneration in the chronic hypertensive DBA/2J mice.
Different contributions of autophagy to retinal ganglion cell death in the diabetic and glaucomatous retinas.
Park Hae-Young Lopilly,Kim Jie Hyun,Park Chan Kee
Diabetes mellitus and glaucoma are the two major causes of selective retinal ganglion cell (RGC) death. To determine the relationship between autophagy and RGC death, we compared autophagy and the related molecular pathways in diabetic and glaucomatous retinas and examined their effect on RGC survival. Biochemical analysis of microtubule-associated protein light chain 3 (LC3)-II and beclin-1 were observed. To determine the pathways involved in autophagy induction, adenosine monophosphate-activated protein kinase (AMPK) and the mechanistic target of rapamycin (mTOR) were also explored. Beclin-1 and the LC3B-II to LC3B-I ratio significantly elevated at 4 and 8 weeks after glaucoma induction; however, only a slight increase was apparent in the diabetic retina. Significant upregulation of phosphorylated AMPK and downregulation of phosphorylated mTOR was evident in the diabetic retina. After autophagy was inhibited with 3-methyladenine (3-MA), apoptosis of RGCs was significantly increased in the diabetic retinas. However, 3-MA inhibition of autophagy decreased the apoptosis of RGCs in glaucomatous retinas. Therefore, our results suggest that RGC death is differentially regulated by autophagy and that the pathways involved differ depending on the triggering injury.
Rapamycin and fasting sustain autophagy response activated by ischemia/reperfusion injury and promote retinal ganglion cell survival.
Russo Rossella,Varano Giuseppe Pasquale,Adornetto Annagrazia,Nazio Francesca,Tettamanti Gianluca,Girardello Rossana,Cianfanelli Valentina,Cavaliere Federica,Morrone Luigi Antonio,Corasaniti Maria Tiziana,Cecconi Francesco,Bagetta Giacinto,Nucci Carlo
Cell death & disease
Autophagy, the cellular process responsible for degradation and recycling of cytoplasmic components through the autophagosomal-lysosomal pathway, is fundamental for neuronal homeostasis and its deregulation has been identified as a hallmark of neurodegeneration. Retinal hypoxic-ischemic events occur in several sight-treating disorders, such as central retinal artery occlusion, diabetic retinopathy, and glaucoma, leading to degeneration and loss of retinal ganglion cells. Here we analyzed the autophagic response in the retinas of mice subjected to ischemia induced by transient elevation of intraocular pressure, reporting a biphasic and reperfusion time-dependent modulation of the process. Ischemic insult triggered in the retina an acute induction of autophagy that lasted during the first hours of reperfusion. This early upregulation of the autophagic flux limited RGC death, as demonstrated by the increased neuronal loss observed in mice with genetic impairment of basal autophagy owing to heterozygous ablation of the autophagy-positive modulator Ambra1 (Ambra1). Upregulation of autophagy was exhausted 24 h after the ischemic event and reduced autophagosomal turnover was associated with build up of the autophagic substrate SQSTM-1/p62, decreased ATG12-ATG5 conjugate, ATG4 and BECN1/Beclin1 expression. Animal fasting or subchronic systemic treatment with rapamycin sustained and prolonged autophagy activation and improved RGC survival, providing proof of principle for autophagy induction as a potential therapeutic strategy in retinal neurodegenerative conditions associated with hypoxic/ischemic stresses.
Involvement of HDAC6 in ischaemia and reperfusion-induced rat retinal injury.
Yuan Haihong,Li Hui,Yu Ping,Fan Qichen,Zhang Xuan,Huang Wei,Shen Junyi,Cui Yongyao,Zhou Wei
BACKGROUND:The role of histone deacetylases 6 (HDAC6) has been elucidated in various neurodegenerative diseases. However, the effect of HDAC6 on retinal degenerative processes remains unknown. The aim of this study was to elucidate the potential role of HDAC6 in the retinal ischaemia and reperfusion (I/R) injury model. METHODS:The retinal pathological lesion was evaluated by haematoxylin and eosin (H&E) staining. HDAC expression or activity was detected by immunohistochemistry, Western blotting assays or colorimetric assays. The expression of apoptotic- and autophagic- related proteins were quantified by Western blotting and RT-PCR. The expression of peroxiredoxin 2 (Prx2) was determined by RT-PCR and ELISA. The levels of acetylated α-tubulin and acetylated histone 3 in the retina were assayed by Western blotting. RESULTS:We found that I/R-induced reduction of the retinal thickness was ameliorated, and the survival of RGCs was increased by the histone deacetylase (HDAC) inhibitor Trichostatin A (TSA) as well as by tubacin (an HDAC6 selective inhibitor). The decreased expression of THY (thymus cell antigen) in the I/R-induced retinas was also reversed by TSA and tubacin. Elevated HDAC6 expression and activity in the retina from I/R injury were significantly inhibited by tubacin, which also attenuated I/R-mediated apoptosis by decreasing TUNEL-positive RGCs and Bax expression and increasing Bcl-2 expression. Additionally, tubacin increased the expression of autophagy-related gene Beclin 1 and microtubule-associated protein 1 light chain 3B (LC3B) and the levels of Prx2. Furthermore, the protective effect of tubacin was associated with acetylated α-tubulin and was independent of acetylated histone 3. CONCLUSIONS:Our findings suggest that tubacin exhibits neuroprotective effects after I/R retinal injury, and HDAC6 may be a potential therapeutic target for the retinal neurodegenerative disease of glaucoma.