Aggregated Myocilin Induces Russell Bodies and Causes Apoptosis

Yam, Gary Hin‐Fai; Gaplovská-Kyselá, Katarína; Zuber, Christian; Roth, Jürgen

doi:10.2353/ajpath.2007.060806

Cited by 123 publications

(55 citation statements)

References 74 publications

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“…Initially, to assess for potential differences in how eGLuc2-tagged MYOC is secreted and handled within the cell, we compared the secretion and intracellular profile of untagged WT MYOC and WT MYOC FLAG (FT, used extensively in past experiments 2,37,38 ) to WT MYOC eGLuc2. We found that indeed, all three variants are readily secreted from transfected cells and detectable in the media 48 hours after initial transfection (Fig.…”

Section: Resultsmentioning

confidence: 99%

A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

Zadoo

Nguyen

Zode

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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PurposePrimary open angle glaucoma–associated mutations in myocilin (MYOC) cause protein “nonsecretion,” rendering secreted MYOC difficult to detect using conventional techniques. This study focused on developing and using an assay that can quickly and easily detect mutant MYOC secretion.MethodsWe fused Gaussia luciferase (eGLuc2) to MYOC variants and expressed the constructs in HEK-293T and NTM-5 cells. Secreted and intracellular levels of MYOC eGLuc2 variants were evaluated by Western blotting and compared to untagged and FLAG-tagged MYOC constructs. Secreted and soluble intracellular MYOC eGLuc2 were measured by a GLuc assay. The secretion of nine additional MYOC mutants was assayed in conditioned media from transfected cells to test the applicability of the assay for monitoring other MYOC variants.ResultsMyocilin eGLuc2 behaved similarly to untagged and FLAG-tagged MYOC with respect to secretion, soluble intracellular levels, and in response to drug treatment. The GLuc assay could sensitively detect Y437H MYOC secretion 30 minutes after media change. Gaussia luciferase fused variants followed anticipated trends; nonpathogenic variants (D208E, G244V) were secreted at wild-type (WT) levels, whereas predicted disease-causing variants (C245Y, G246R, E300K, Y437H, I477N) demonstrated substantial secretion defects. Secretion defects caused by the C245Y, G246R, and Y437H mutations were partially rescued by permissive growth temperature. Interestingly, however, this increase in secretion was independent of newly synthesized protein.ConclusionsFusion of eGLuc2 to MYOC does not significantly change the behavior of MYOC. This newly developed MYOC reporter system can be used to study engineered MYOC variants and potentially to identify modulators of MYOC secretion and function.

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Section: Resultsmentioning

confidence: 99%

A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

Zadoo

Nguyen

Zode

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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“…Impaired degradation 74 of myocilin aggregates presumably leads to aberrant accumulation in other relevant cellular compartments 9; 13; 14; 75; 76; 77; 78 , affecting protein folding and other cellular processes more generally. Under these conditions, even degradation by autophagy 79 , which, in the case of mutant myocilin may be assisted by peroxisomes 8 , likely cannot compensate sufficiently for ERAD inhibition, leading to apoptosis 9; 13; 14 . Once TM cells die, it is straightforward to foresee that cellular debris would be detrimental to TM function and proper outflow, leading to IOP elevation.…”

Section: Discussionmentioning

confidence: 99%

“…Several studies note intracellular sequestration of mutant myocilin 8; 9; 10; 11 . Some studies further report an endoplasmic reticulum (ER) stress response 12; 13 , including explicit colocalization of large juxtanuclear myocilin aggregates with known ER chaperones 9; 14 leading to apoptosis and cell death 9; 13; 14 . The majority of myocilin variants examined, located in the ~30 kDa myocilin C-terminal olfactomedin domain (myoc-OLF) 5 , thermally destabilize the domain but in vitro retain secondary structure similar to wild-type (WT); age of diagnosis follows the extent of destabilization of myoc-OLF 15 .…”

Section: Introductionmentioning

confidence: 99%

Amyloid Fibril Formation by the Glaucoma-Associated Olfactomedin Domain of Myocilin

Orwig

Perry

Kim

et al. 2012

Journal of Molecular Biology

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Myocilin is a protein found in the extracellular matrix of the trabecular meshwork (TM) tissue, the anatomical region of the eye involved in regulating intraocular pressure. Wild-type (WT) myocilin has been associated with steroid-induced glaucoma, and variants of myocilin have been linked to early-onset, inherited glaucoma. Elevated levels and aggregation of myocilin hasten increased intraocular pressure and glaucoma-characteristic vision loss due to irreversible damage to the optic nerve. In spite of the reports of intracellular accumulation of mutant and WT myocilin in vitro, cell culture and model organisms, these aggregates have not been structurally characterized. In this work, we provide biophysical evidence for the hallmarks of amyloid fibrils in aggregated forms of WT and mutant myocilin, localized to the C-terminal olfactomedin (OLF) domain. These fibrils are grown under a variety of conditions in a nucleation dependent, self-propagating manner. Protofibrillar oligomers and mature amyloid fibrils are observed in vitro. Full-length mutant myocilin expressed in mammalian cells forms intracellular amyloid-containing aggregates as well. Taken together, this work provides new insights into and raises new questions about the molecular properties of the highly conserved OLF domain, and suggests a novel protein-based hypothesis for glaucoma pathogenesis for further testing in a clinical setting.

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“…Several in vitro studies demonstrated that disease-causing MYOC mutants are secretion incompetent and accumulate in the endoplasmic reticulum (ER), inducing ER stress. 24–32 Using Tg-MYOC Y437H mice expressing mutant human myocilin, we have shown that ER stress is associated with IOP elevation in MYOC-associated glaucoma. 26 We have recently demonstrated that ER stress is also associated with glucocorticoid-induced ocular hypertension.…”

mentioning

confidence: 99%

Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork

Kasetti

Phan

Millar

et al. 2016

Invest. Ophthalmol. Vis. Sci.

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PurposeAbnormal accumulation of extracellular matrix (ECM) in the trabecular meshwork (TM) is associated with decreased aqueous humor outflow facility and IOP elevation in POAG. Previously, we have developed a transgenic mouse model of POAG (Tg-MYOCY437H) by expressing human mutant myocilin (MYOC), a known genetic cause of POAG. The purpose of this study is to examine whether expression of mutant myocilin leads to reduced outflow facility and abnormal ECM accumulation in Tg-MYOCY437H mice and in cultured human TM cells.MethodsConscious IOP was measured at various ages of Tg-MYOCY437H mice using a rebound tonometer. Outflow facility was measured in 10-month-old Tg-MYOCY437H mice. Selected ECM proteins were examined in human TM-3 cells stably expressing mutant myocilin and primary human TM cells (n = 4) as well as in the TM of Tg-MYOCY437H mice by real-time PCR, Western blotting, and immunostaining. Furthermore, TM cells expressing WT or mutant myocilin were treated with 5 mM sodium 4-phenylbutyrate (PBA), and ECM proteins were examined by Western blot and immunostaining.ResultsStarting from 3 months of age, Tg-MYOCY437H mice exhibited significant IOP elevation compared with wild-type (WT) littermates. Outflow facility was significantly reduced in Tg-MYOCY437H mice (0.0195 μl/min/mm Hg in Tg-MYOCY437H vs. 0.0332 μl/min/mm Hg in WT littermates). Increased accumulation of fibronectin, elastin, and collagen type IV and I was observed in the TM of Tg-MYOCY437H mice compared with WT littermates. Furthermore, increased ECM proteins were also associated with induction of endoplasmic reticulum (ER) stress markers, GRP78 and CHOP in the TM of Tg-MYOCY437H mice. Human TM-3 cells stably expressing DsRed-tagged Y437H mutant MYOC exhibited inhibition of myocilin secretion and its intracellular accumulation compared with TM cells expressing WT MYOC. Expression of mutant MYOC in TM-3 cells or human primary TM cells induced ER stress and also increased intracellular protein levels of fibronectin, elastin, laminin, and collagen IV and I. In addition, TM-3 cells expressing mutant myocilin exhibited reduced active forms of matrix metalloproteinase (MMP)-2 and MMP-9 in conditioned medium compared with TM-3 cells expressing WT myocilin. Interestingly, both intracellularly accumulated fibronectin and collagen I colocalized with mutant myocilin and also with ER marker KDEL further suggesting intracellular accumulation of these proteins in the ER of TM cells. Furthermore, reduction of ER stress via PBA decreased selected ECM proteins in primary TM cells.ConclusionsThese studies demonstrate that mutant myocilin induces abnormal ECM accumulation in the ER of TM cells, which may be responsible for reduced outflow facility and IOP elevation in myocilin-associated glaucoma.

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Aggregated Myocilin Induces Russell Bodies and Causes Apoptosis

Cited by 123 publications

References 74 publications

A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

A Novel Luciferase Assay For Sensitively Monitoring Myocilin Variants in Cell Culture

Amyloid Fibril Formation by the Glaucoma-Associated Olfactomedin Domain of Myocilin

Expression of Mutant Myocilin Induces Abnormal Intracellular Accumulation of Selected Extracellular Matrix Proteins in the Trabecular Meshwork

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