Human Molecular Genetics

Human Molecular Genetics Advance Access originally published online on April 6, 2004

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Human Molecular Genetics, 2004, Vol. 13, No. 11 1193-1204
DOI: 10.1093/hmg/ddh128
Human Molecular Genetics, Vol. 13, No. 11 © Oxford University Press 2004; all rights reserved

Reversal of mutant myocilin non-secretion and cell killing: implications for glaucoma

Yuhui Liu and Douglas Vollrath^*

Department of Genetics, Stanford University School of Medicine, Stanford, CA 94305-5120, USA

Received February 27, 2004; Accepted March 31, 2004

	ABSTRACT

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Glaucoma is a progressive blinding disease characterized by gradual loss of vision due to optic neuropathy and retinal ganglion cell death. Increased intraocular pressure is a common feature of glaucoma that is thought to arise from an increased resistance to outflow of aqueous humor through the trabecular meshwork. Mutations of the myocilin gene are one cause of autosomal dominant juvenile- and adult-onset primary open angle glaucoma, but the mechanism by which mutant myocilins cause disease is poorly understood. We have found that disease-causing myocilin mutants are misfolded, are highly aggregation-prone and accumulate in large aggregates in the endoplasmic reticulum (ER) of human embryonic kidney cells and differentiated primary human trabecular meshwork (HTM) cells. In HTM cells, Pro370Leu mutant myocilin is not secreted under normal culture conditions and prolonged expression results in abnormal cell morphology and cell killing. Culturing HTM cells at 30°C, a condition known to facilitate protein folding, promotes secretion of mutant myocilin, normalizes cell morphology and reverses cell lethality. Our results indicate that myocilin-associated glaucoma is an ER storage disease and suggest a progression of events in which chronic expression of misfolded, non-secreted myocilin leads to HTM cell death, trabecular meshwork dysfunction and, ultimately, a dominant glaucoma phenotype. The beneficial effects of facilitating folding and secretion of mutant myocilin suggest a new type of treatment for this form of glaucoma.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Glaucoma is a heterogeneous eye disease in which affected individuals experience gradual loss of vision and is the second largest cause of bilateral blindness in the world (1). Open angle glaucoma (OAG) is the most common form of the disease, affecting nearly half of the estimated 67 million people with glaucoma worldwide (1). In most cases of OAG, increased resistance to the outflow of aqueous humor results in a rise in intraocular pressure, which eventually leads to loss of retinal ganglion cells. Mutations of the trabecular meshwork inducible glucocorticoid response gene (TIGR) (2), now widely referred to as the myocilin gene (MYOC) (3), have been found in a subset of families with autosomal dominant juvenile-onset OAG and in 3–4% of adult-onset OAG (4).

The function of myocilin protein remains unknown despite intensive study. Myocilin is a secreted, glycosylated protein (5,6) of 504 amino acids with two major domains: a coiled coil domain near the amino terminus, and an olfactomedin-like domain near the carboxy terminus (3). In the human eye, the highest levels of myocilin expression are found in cells of the outflow pathway, including the human trabecular meshwork (HTM) (7), suggesting that one site of action of mutant myocilin is HTM cells. A number of groups have studied the cellular localization of myocilin in an effort to gain clues into its function. These studies have yielded conflicting results with reports of co-localizations as diverse as microtubules (8), mitochondria (HTM cells) (9), Golgi apparatus (Schlemm's canal cells) (10) and ER (HTM cells) (11). ER and Golgi localizations are consistent with the known secretion of myocilin and, as has been suggested, it would be surprising if this extracellular protein also functioned in conjunction with mitochondria or microtubules (12). Recently, a sea urchin protein similar to mammalian myocilin has been shown to form disulfide-bonded networks that mediate intercellular adhesion (13). The relevance of this finding to the role of myocilin in the eye is yet to be demonstrated.

The mechanism by which mutant myocilins cause a dominant glaucoma phenotype is not known. The normal function of myocilin may not be central to understanding this issue because a growing body of evidence points toward a gain-of-function disease model. Of the more than 70 myocilin mutations described, the vast majority are missense substitutions in the olfactomedin domain (14). The clustering of mutations within one protein domain and the notable paucity of glaucoma-associated nonsense mutations argue against a haploinsufficiency model, as does the absence of OAG in an individual hemizygous for MYOC (15). Absence of OAG in an elderly woman homozygous for an early premature termination codon (Arg46Stop) is evidence against a disease mechanism centered on loss of functional myocilin (16). These conclusions are supported by an apparent lack of ocular phenotypes in both Myoc +/– and –/– mice (17).

Biochemical and cell biological studies have provided evidence consistent with a gain-of-function mechanism. Mutant forms of myocilin are not secreted from cells and can diminish secretion of wild-type protein when the two forms are co-expressed (18). Disease-causing mutants of myocilin protein expressed in human embryonic kidney (HEK) cells are relatively insoluble in the non-ionic detergent, Triton X-100, when compared with wild-type protein and controls (19). Detergent insolubility and inefficient secretion of myocilin mutants suggested that deleterious effects on cells of mutant protein retention, distinct from the effect of mutant myocilin on the function of co-expressed wild-type protein, might contribute to myocilin-mediated glaucoma pathogenesis (6,11,18–20).

Many disease-causing mutations do not seriously compromise the synthesis of a polypeptide, but rather produce a protein that cannot fold normally (21). The ER has a quality control system to monitor the folding of secretory and membrane proteins through the association of ER chaperones with unfolded or misfolded polypeptide chains (22). In most cases, secretory or membrane proteins that fail to assume their native structure do not transit to the Golgi. Such misfolded proteins often are subject to ER-associated degradation (ERAD) via retrotransport to the cytosol followed by ubiquitination and proteasomal degradation. In some cases, folding mutants are not efficiently degraded and form ER or cytoplasmic aggregates (23). Cytoplasmic aggregates can compromise proteasomal function (24), while ER retention of mutant proteins has been implicated in the pathogenesis of a variety of diseases (25). Indeed, misfolded protein accumulation and aggregation is thought to be a central event in the initiation of cell death in a number of inherited neurodegenerative diseases (26–29), which, like MYOC-associated OAG, are dominant, delayed-onset disorders.

The discoveries of Triton insolubility and non-secretion of mutant myocilins suggested a disease mechanism in which mutant proteins overwhelm the ERAD pathway (12), leading to aggregate formation and cellular dysfunction or death, while other evidence indicates that mutant myocilins trigger an ER stress response (20). In the present study, we have investigated the cellular fate of disease-causing mutant myocilins with the aim of understanding pathogenic mechanisms of myocilin-associated glaucoma. We have concentrated our efforts on understanding the properties of disease-causing missense mutations, which account for nearly half of all suspected myocilin-associated glaucoma cases (4) and which are by far the predominant form of myocilin mutation found in severe, early-onset cases of OAG (30–32). We asked whether glaucoma-causing myocilin missense mutants are misfolded and find that they are, leading us to investigate their pathway of degradation, their aggregation status and their potential for cell killing. An understanding of these issues has demonstrated that myocilin-associated glaucoma is likely an ER storage disease and has led to the discovery of a promising new avenue for development of myocilin-associated glaucoma therapy.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Mutant myocilin is misfolded
Biochemical analyses of detergent-soluble and detergent-insoluble cell extracts have been used to explore the aggregation state of misfolded disease-causing mutant proteins (28,33,34). We assessed the misfolding and aggregation of wild-type and glaucoma-causing mutant myocilins by analysis of detergent-soluble and -insoluble fractions from HEK cells transiently expressing these proteins (Fig. 1A). In both the soluble and insoluble fractions, myocilin protein with two distinct types of mobility is apparent: a doublet of bands between 56 and 58 kDa that corresponds to unglycosylated and glycosylated (6) FLAG-tagged myocilin monomer, and a low mobility fraction that does not exit the stacking gel and that is indicative of the formation of protein aggregates (33,35). Wild-type myocilin and a control myocilin with a single human to murine substitution (Ala386Ser) (19) show little or no low mobility protein, even at the highest expression levels. In contrast, five mutant myocilins (Asp380Ala, Glu323Lys, Tyr437His, Gly364Val and Lys423Glu) exhibit abundant low-mobility protein that is frequently more than half of the total mutant myocilin produced. Low-mobility mutant protein is present even at lower expression levels. It persists after samples are treated with 100 mM dithiothreitol (DTT) (Fig. 1B), indicating that it does not result from incomplete reduction of disulfide bonds.

View larger version (48K): [in this window] [in a new window]   Figure 1. Mutant myocilin forms low mobility complexes. (A) HEK cells were transiently transfected with myocilin plasmid DNA and cultured for 48 h. Triton-soluble (upper panel; 10 s exposure) or -insoluble fractions (lower panel; 20 s exposure) of cell lysate were prepared and subjected to immunoblot analysis with an anti-FLAG antibody. (B) HEK cells were transiently transfected with 1 µg of plasmid DNA and cultured for 48 h. The soluble (S) and insoluble (I) fractions were treated with 100 mM DTT in Laemmli sample buffer (20 s exposure). (C) HTM cells were transduced with Ad-MYOC-WT or Ad-MYOC-P370L at an MOI of 5 or 2.5 for 48 h. The resulting Triton-soluble fractions were analyzed by immunoblot with an anti-FLAG antibody (30 s exposure). The horizontal lines to the right of the blot images indicate the extent of the stacking (upper line) and separating (lower line) gels.

 
In order to investigate the behavior of myocilin in differentiated primary HTM cells, we constructed replication-deficient, recombinant adenoviruses expressing FLAG-tagged wild-type myocilin (Ad-MYOC-WT) or FLAG-tagged pathogenic Pro370Leu mutant myocilin (Ad-MYOC-P370L) and used them to transduce HTM cells. Most of the myocilin protein partitioned into the soluble fraction (data not shown). Low-mobility mutant, but not wild-type, protein is present in the soluble fraction at a multiplicity of infection (MOI) of 5 (Fig. 1C). At higher MOIs, low-mobility protein is present in both mutant and wild-type samples (data not shown). The HEK and HTM expression data indicate that disease-causing missense mutations of myocilin render the protein prone to misfolding and aggregation, probably by favoring non-native conformations.

Misfolded myocilin is bound by calnexin and calreticulin
Chaperones are expected to interact with misfolded proteins in mammalian cells. We evaluated the roles of calnexin and calreticulin, two ER-resident chaperones that bind glycosylated proteins (22), in the folding of myocilin by determining whether these chaperones interact directly with myocilin expressed in cells. Very little wild-type myocilin co-immunoprecipitates with calnexin or calreticulin in the soluble fraction from HEK cells (Fig. 2A and B). In contrast, significant amounts of Asp380Ala, Gly364Val, Lys423Glu and Pro370Leu mutant myocilins co-immunoprecipitate with calnexin (Fig. 2A) or calreticulin (Fig. 2B) in the soluble fraction. Similar to HEK cells, soluble Pro370Leu mutant myocilin expressed in HTM cells co-immunoprecipitates with calreticulin, while much less wild-type protein binds to this chaperone (Fig. 2C). Binding of calnexin to mutant or wild-type myocilin could not be detected in HTM cells. These results confirm that mutant myocilins are misfolded and suggest that a significant amount of the misfolded protein resides in the ER bound to chaperones.

View larger version (21K): [in this window] [in a new window]   Figure 2. Misfolded myocilin associates with calnexin and calreticulin. (A) HEK cells were transfected with 1 µg of myocilin plasmid DNA and cultured for 48 h. Triton-soluble fractions of cell lysates were immunoprecipitated with an anti-calnexin polyclonal antibody and subjected to immunoblot analysis either with an anti-FLAG (upper panel; 30 s exposure) or with anti-calnexin antibody (lower panel; 10 s exposure). The control lane is soluble protein from HEK cells transfected with 1 µg Pro370Leu myocilin plasmid DNA without immunoprecipitation. (B) HEK cells were treated the same as in (A). Cell lysates were immunoprecipitated with an anti-calreticulin antibody and subjected to immunoblot analysis either with an anti-FLAG (upper panel; 5 s exposure) or with an anti-calreticulin antibody (lower panel; 1 s exposure). The control is the same as in (A). (C) Lysates from recombinant adenovirus-transduced (duration 48 h) HTM cells were immunoprecipitated with an anti-calreticulin antibody and subjected to immunoblot analysis either with an anti-FLAG (upper panel; 2 s exposure) or with an anti-calreticulin antibody (lower panel; 2 s exposure).

 
Misfolded myocilin is degraded by proteasomes
Many misfolded ER proteins are subject to ERAD. We investigated whether misfolded myocilins are degraded by proteasomes. The low mobility and unglycosylated monomer forms of mutant myocilin increase substantially in the insoluble fraction when HTM cells are treated with the proteasome inhibitors ALLN or MG132, while the wild-type protein is only modestly affected (Fig. 3). The amount of myocilin protein in the HTM cell soluble fraction is largely unaffected by proteasome inhibitors (data not shown). Experiments in HEK cells demonstrated that proteasome inhibition causes a substantial increase in mutant myocilin in both the soluble and insoluble fractions for a number of different missense mutants, including Pro370Leu (Supplementary Material, Fig. S1), and that mutant myocilins are conjugated with ubiquitin (Supplementary Material, Fig. S2) and accumulate in the presence of a dominant negative form of ubiquitin (Supplementary Material, Fig. S3). Together, these results demonstrate that a portion of misfolded mutant myocilin is retrotransported out of the ER and degraded by proteasomes.

View larger version (52K): [in this window] [in a new window]   Figure 3. Some misfolded myocilin is degraded by proteasomes. Differentiated HTM cells were transduced with recombinant adenovirus for 48 h and treated overnight with the proteasome inhibitors ALLN or MG132, or with the DMSO vehicle. Insoluble fractions from cell lysates were subjected to immunoblot analysis with an anti-FLAG (upper panel; 40 s exposure) or an anti-beta-actin (lower panel; 40 s exposure) antibody. The horizontal lines to the right of the blot image indicate the extent of the stacking (upper line) and separating (lower line) gels. The upper-most arrow indicates a background protein band.

 
Mutant myocilins form large juxtanuclear aggregates in cells
In some disease states, failure of cells to efficiently degrade misfolded proteins results in formation of large protein aggregates within cells (23). We examined the cellular distribution of mutant myocilins and controls expressed in HEK cells. At lower expression levels (0.2 µg of DNA), both wild-type and mutant myocilins are distributed diffusely around the nucleus (data not shown), consistent with studies of endogenous myocilin expression in HTM tissue (36) and Schlemm's canal cells (37). At higher expression levels (2 µg of DNA), cells expressing the Gly364Val and Pro370Leu mutant myocilin contain striking juxtanuclear aggregates (Fig. 4A, and data not shown). Large juxtanuclear aggregates are evident in 15% of cells expressing the Lys423Glu, Asp380Ala, Glu323Lys or Gly364Val mutants, and only rarely (<1%) in cells expressing wild-type myocilin and the Ala386Ser control (Fig. 4B).

View larger version (64K): [in this window] [in a new window]   Figure 4. Mutant myocilin accumulates in juxtanuclear aggregates. (A) HEK cells were transfected with 2 µg of myocilin plasmid DNA and processed after 48 h for immunofluorescence staining using an anti-FLAG antibody. Arrows indicate myocilin aggregates. Bar: 10 µm. (B) HEK cells were processed as in (A). The number of aggregate-containing transfected cells was counted in 15 randomly selected microscopic fields, each with 100–150 transfected cells. The data represent mean±SE of all the areas and are representative of at least three independent experiments.

 
We next examined the distribution of myocilin in HTM cells. After transduction at an MOI of 25 for 2 days, a marked difference is seen in the distribution of wild-type and Pro370Leu mutant myocilin (Fig. 5A). Wild-type myocilin is distributed diffusely throughout the cell, with the exception of the nucleus, while Pro370Leu mutant myocilin is distributed discontinuously in a granular pattern. At a higher magnification, this pattern is seen to result from multiple large aggregates (Fig. 5A), consistent with other studies (11). Seven days post-transduction, single large juxtanuclear aggregates are seen in some HTM cells transduced with Ad-MYOC-P370L at an MOI of 25, and similar but less numerous aggregates are seen at an MOI of 5 (Fig. 5B). Some aggregates are even seen at an MOI of 1 (data not shown). No large juxtanuclear aggregates were observed in cells transduced with Ad-MYOC-WT, even at an MOI of 25. Thus, in HTM cells, as in HEK cells, mutant myocilin is prone to form large juxtanuclear aggregates, much more so than its wild-type counterpart.

View larger version (37K): [in this window] [in a new window]   Figure 5. Mutant myocilin aggregates in primary HTM cells. (A) HTM cells were transduced with Ad-MYOC-WT (left panel) or Ad-MYOC-P370L (middle panel) at an MOI of 25 for 48 h. The right panel shows a higher magnification of cells transduced with Ad-MYOC-P370L. An anti-FLAG antibody was used to label myocilin. Bar: 10 µm. (B) HTM cells were transduced with Ad-MYOC-P370L at an MOI of 25 (upper panel) or 5 (lower panel) for 7 days. Red corresponds to myocilin and blue to nuclei in both (A) and (B). Bar: 10 µm.

 
Myocilin aggregates are associated with ER chaperones
Two types of protein aggregates within mammalian cells have been characterized: Russell bodies and aggresomes (23). Russell bodies result from aggregation of misfolded protein within the ER and sometimes co-localize with ER chaperones. Aggresomes form in the cytoplasm and can result from retrotranslocation of ER proteins. Our biochemical data suggest that misfolded myocilin is present in the ER, and also that the cytoplasmic ubiquitin–proteasome system (UPS) plays a role in myocilin degradation (Supplementary Material, Figs S1–S3). We investigated the characteristics of mutant myocilin aggregates to determine if they are more similar to Russell bodies or aggresomes.

A key characteristic of aggresomes is that they co-localize with the centrosome due to minus-end directed transport of aggregates to the microtubule organizing center (38). Confocal microscopy of HEK cells expressing myocilin mutants Lys423Glu or Pro370Leu did not show evidence of co-localization of aggregates with pericentrin, a centrosomal marker, suggesting that the aggregates are not aggresomes (Supplementary Material, Fig. S4; and data not shown). However, the aggregates do co-localize with the ER chaperones calreticulin and protein disulphide isomerase (PDI) in both HEK and HTM cells (Fig. 6A and B). PDI is more uniformly distributed in HTM aggregates than in HEK aggregates (Fig. 6B), and calnexin co-localizes with HEK aggregates, but not with HTM aggregates (Fig. 6C), demonstrating differences in the composition of myocilin aggregates in the two cell types.

View larger version (54K): [in this window] [in a new window]   Figure 6. ER-resident chaperones are recruited to myocilin aggregates. (A) HEK cells were transfected with 1 µg of Lys423Glu myocilin plasmid DNA and cultured for 48 h. HTM cells were transduced with Ad-MYOC-P370L at an MOI of 5 and cultured for 7 days. Red corresponds to myocilin and green to calreticulin. (B) Cells were treated the same as in (A). Red corresponds to myocilin and green to PDI. (C) Cells were treated the same as in (A). Red corresponds to myocilin and green to calnexin. Hoechst was used to highlight nuclei (blue) in (A), (B) and (C). Bar: 10 µm.

 
Prolonged expression of mutant myocilin kills differentiated primary HTM cells
Misfolding and aggregation of mutant myocilin could be toxic to differentiated primary HTM cells and may help to explain the dominant glaucoma phenotype resulting from myocilin mutations. We tested the effects of prolonged expression of mutant myocilin on HTM cells. After transduction at low MOIs (1 or 2.5) for 12 days, the morphology of cells expressing mutant myocilin is obviously abnormal (data not shown, and Fig. 7A). Untransduced and Ad-EGFP-transduced HTM cells are evenly distributed on the dish surface and adopt a generally oval shape, while cells transduced with mutant myocilin are stretched into spindle shapes and rounded up (Fig. 7A). The morphology of cells expressing wild-type myocilin is intermediate between that of the Ad-EGFP-transduced and Ad-MYOC-P370L-transduced cells.

View larger version (52K): [in this window] [in a new window]   Figure 7. Prolonged expression of mutant myocilin kills differentiated primary HTM cells. (A) HTM cells were transduced with recombinant adenovirus at an MOI of 2.5 and cultured for 12 days. The images were taken by phase-contrast microscopy. Bar: 20 µm. (B) HTM cells were transduced with recombinant adenovirus at an MOI of 25, cultured for 20 days and then fixed and stained with Hoechst. Bar: 20 µm. (C) HTM cells were treated the same as in (B) with different MOIs of recombinant adenovirus. Cells were fixed and stained with Hoechst. Nuclei were counted in 10 randomly selected microscopic fields. Each microscopic field had 100–400 nuclei. The data represent mean±SE of all the areas. The viability of the cells transduced by Ad-MYOC-P370L is significantly lower than that of cells transduced by Ad-MYOC-WT at the same MOI. (*P<0.001 by Student's unpaired t-test).

 
The morphological changes are dose dependent. At higher MOIs (e.g. 5–25), the abnormal morphology appears more rapidly, is more severe and can be observed in cells transduced with all three viruses (data not shown). Importantly, the relative severity remains the same (Ad-MYOC-P370L>Ad-MYOC-WT>Ad-EGFP). The morphological changes are also time dependent; they were not apparent 3 days after transduction, even at an MOI as high as 25, but are evident as early as 6 days post-transduction, even at MOIs as low as 2.5 and 1 (data not shown).

We assessed the effect of prolonged myocilin expression on HTM cell viability by counting the number of Hoechst-stained nuclei present 20 days post-transduction at MOIs ranging from 1 to 25 (Fig. 7B and C). Although the viability of transduced cells is generally lower than that of untransduced cells, clearly there is a significant reduction (P<0.001 by Student's unpaired t-test) in the viability of Ad-MYOC-P370L-transduced cells, compared with Ad-MYOC-WT- or Ad-EGFP-transduced cells, at higher expression levels (Fig. 7C: MOIs of 5, 10 or 25).

These results demonstrate that prolonged expression of mutant myocilin, even at an MOI as low as 5, is lethal to differentiated primary HTM cells. The toxic effect results initially in abnormal cell morphology and, later, in cell death.

Lower temperature allows secretion of mutant myocilin and promotes cell survival
Protein processing defects arising from mutations in genes such as HERG, TYR, RET and CTFR have been rescued by protein expression at a lower temperature, typically 30°C (39–42). We investigated whether a similar treatment could affect the processing and cell lethality of mutant myocilin. HTM cells were transduced with Ad-MYOC-WT or Ad-MYOC-P370L for 2 days at 37°C and then cultured at 30 or 37°C for an additional 4 days. Analysis of secreted proteins shows that, as expected, wild-type myocilin is secreted at 37°C, while Pro370Leu mutant myocilin is not (Fig. 9A), as previously shown for several other myocilin missense mutants in an immortalized HTM cell line (18). Interestingly, the shift to 30°C partially reverses the block to secretion of Pro370Leu mutant myocilin (Fig. 9A).

View larger version (56K): [in this window] [in a new window]   Figure 9. Lower temperature allows secretion of Pro370Leu mutant myocilin and normalizes cytoplasmic distribution. (A) HTM cells were transduced with recombinant adenovirus for 48 h at 37°C, and then were cultured for an additional 4 days at either 37 or 30°C. The culture medium was collected and mixed with an equal volume of 2x Laemmli sample buffer and subjected to immunoblot analysis with an anti-FLAG antibody. (B) HTM cells were treated the same as in (A) except that total cell lysate was collected and subjected to immunoblot analysis with an anti-FLAG antibody. The upper-most arrow indicates a background protein band. (C) HTM cells were transduced with recombinant adenovirus at an MOI of 2.5 for 48 h at 37°C, and then cultured for an additional 18 days at either 37 or 30°C before immunostaining with an anti-FLAG antibody. Bar: 20 µm.

 
The improved secretion of Pro370Leu protein at 30°C correlates with a striking normalization of the morphology of HTM cells expressing the mutant protein; cell morphology 12 days post-transduction is very similar to that of untransduced cells, even at an MOI of 10 or 25 (Fig. 8A). A similar effect is seen for wild-type myocilin at these higher MOIs.

View larger version (57K): [in this window] [in a new window]   Figure 8. Lower temperature reverses myocilin-induced HTM cell killing. (A) HTM cells were transduced with Ad-MYOC-WT or Ad-MYOC-P370L at an MOI of 10 or 25 for 48 h at 37°C, then cultured for an additional 10 days at either 37 or 30°C. The images were taken by phase-contrast microscopy. Bar: 20 µm. (B) HTM cells were transduced with Ad-EGFP, Ad-MYOC-WT or Ad-MYOC-P370L at different MOIs for 48 h at 37°C, then cultured for an additional 18 days at 30°C. Cells were fixed and stained with Hoechst. Nuclei were counted in 10 randomly selected microscopic fields. Each microscopic field had 100–400 nuclei. The data represent mean±SE of all the areas.

 
Cell viability 20 days post-transduction is also strikingly improved by culturing at 30°C (Fig. 8B). The viability of cells expressing Pro370Leu mutant or wild-type myocilin is almost the same as that of untransduced cells, even at MOIs of 10 and 25. [The viability of untransduced cells is not affected by the temperature shift (P>0.05 by Student's unpaired t-test).]

The improved cell morphology and viability occur despite the fact that expression of myocilin is not diminished in cells cultured at 30°C (Fig. 9B). After 20 days of transduction, myocilin protein is detected in many more cells cultured at 30°C than at 37°C, and the cellular distribution of both wild-type and mutant proteins is very different at the two temperatures at MOI 2.5 (Fig. 9C) and at all other tested MOIs (1, 5, 10 and 25; data not shown); in many cells at 30°C, myocilin is diffusely distributed, while at 37°C myocilin expression is much less uniform.

In summary, culturing differentiated primary HTM cells at 30°C allows partial secretion of an otherwise non-secreted mutant protein, normalizes cell morphology and cellular distribution of myocilin and restores cell viability. These three effects also are seen, to some extent, in cells expressing wild-type protein. These beneficial effects likely arise from improved folding and processing of myocilin at the lower temperature.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
We have demonstrated that mutant myocilins are misfolded, are highly aggregation prone and accumulate in the ER. The result of prolonged expression and retention of mutant myocilin in differentiated HTM cells is abnormal cell morphology and cell death. To our knowledge, this is the first description of cell killing induced by a mutant myocilin. Our findings suggest a gain-of-function disease model in which prolonged expression of mutant protein leads to trabecular meshwork cell dysfunction and death, a consequent increase in resistance to aqueous humor outflow and, ultimately, glaucoma. This model is consistent with the dominant inheritance of myocilin-associated glaucoma and with the observation that patients with primary OAG experience trabecular meshwork cell loss beyond that seen in normal aging (43). Our findings further suggest that the root cause of cell death induced by mutant myocilin is failure of the mutant protein to properly fold. Culturing cells at 30°C, a condition known to facilitate protein folding, allowed secretion of an otherwise non-secreted mutant myocilin and reversed the cell killing observed at the normal physiological temperature. Thus, our findings not only provide a plausible disease mechanism, but also suggest a promising new approach for treating myocilin-associated glaucoma.

Several aspects of our study suggest that it may be a good model for processes that occur within the trabecular meshwork of glaucoma patients. First, we used differentiated primary HTM cells which exhibit many of the key characteristics of trabecular meshwork cells in vivo (44). Second, we observed abnormal cell morphology and killing at relatively low levels of myocilin expression, levels that might well be achieved in vivo because of the marked induction of myocilin by environmental stresses (45). Third, consistent with the chronic progressive nature of myocilin-associated glaucoma, the pace of cell killing was slow, with cell loss becoming evident only several weeks after initial transduction. Our approach contrasts with that of earlier studies (11,20) in which the acute effects of very high levels (MOIs of 500 or 1000) of mutant myocilin–GFP fusion proteins on rapidly proliferating, undifferentiated HTM cells were assessed. These earlier studies described myocilin-induced cytotoxicity, but not the cell death that we observed, perhaps because our approach models more closely the pathogenic mechanisms that occur in glaucomatous eyes.

Differences between cell types
There may be cell-type specific features to mutant myocilin-induced cell lethality. Myocilin is expressed in tissues throughout the body yet, to date, mutations in this gene have been linked exclusively to glaucoma. One factor may be the degree to which myocilin expression can be induced by environmental stimuli in various cell types. Glucocorticoids induce a high level of myocilin expression in HTM cells, but their effect on myocilin expression in Schlemm's canal cells is much more modest (46).

There may also be cell-type specific differences in the way mutant myocilin is processed. We observed significant differences in the characteristics of mutant myocilin between HTM and HEK cells. The ER chaperone calnexin associated with myocilin aggregates in HEK cells but not in HTM cells (Fig. 6C), despite comparable calnexin levels in the two cell types (data not shown). Moreover, a relatively small amount of mutant myocilin was detergent-insoluble in HTM cells, whereas a much larger percentage partitioned into this fraction in HEK cells. Consistent with this, large myocilin aggregates formed more rapidly in HEK cells (2 days) than in HTM cells (7 days) and a smaller percentage of HTM cells contained large aggregates as compared to HEK cells (<1 versus 15%, respectively). These differences were apparent even though recombinant myocilin was expressed at comparable levels in the two cell types under the conditions we employed (data not shown).

Several reports have suggested that small soluble oligomeric intermediates, rather than fully formed insoluble aggregates, are the offending cytotoxic molecules in other protein misfolding diseases (47). The fact that proportionately more misfolded mutant myocilin is detergent soluble in HTM cells and that fewer HTM cells have solitary large aggregates may indicate that HTM cells are not as effective at sequestering misfolded proteins. This may render HTM cells more vulnerable to expression of mutant myocilin than other cell types.

Mechanism of cell killing
The molecular mechanism of cell killing by mutant myocilin is unknown. Cytoplasmic protein aggregates can compromise the ubiquitin–proteasome machinery (24), thus potentially leading to a cycle of dysfunctional protein clearance, increased aggregate formation and a further reduction in proteasome activity. However, we found that only a small portion of myocilin is degraded by proteasomes in HTM cells and we did not observe inhibition of the UPS by over-expression of mutant myocilin in the HEK-derived GFP^U-1 reporter cell line (24) (Y. Liu and D. Vollrath, unpublished data).

Accumulation of misfolded myocilin in the ER may stress HTM cells. Two ER stress responses have been described: the ER overload response (EOR), which activates the nuclear transcription factor NFkappaB (25), and the unfolded protein response (UPR), which leads to up-regulation of UPR target genes including ER resident chaperones such as GRP78 and PDI. Activation of the NFkappaB pathway may be involved in the pathogenesis of primary OAG (48). Recent studies have shown activation of the UPR by over-expression of mutant myocilin–GFP fusion proteins (20), and UPR activation can trigger apoptosis (49). Activation of the EOR and/or UPR by misfolded myocilin may contribute to the HTM cell death that we observe.

We found that higher levels of expression of wild-type myocilin are lethal to HTM cells, although significantly less so than equivalent levels of mutant myocilin (Fig. 7A). This may reflect the cellular stress imposed by the inherent inefficiency of protein synthesis; >30% of newly synthesized polypeptides never attain native structure and are degraded by proteasomes (50). As suggested above, HTM cells may be ill-equipped to handle misfolded protein. Indeed, we detected substantial low-mobility wild-type protein in the detergent-soluble fraction from HTM cells transduced at higher MOIs, while much less misfolded wild-type myocilin was present in HEK cells at comparable expression levels. Thus, similar mechanisms may underlie cell killing induced by mutant and wild-type myocilin. In light of the substantial induction of myocilin synthesis by dexamethasone treatment of cultured HTM cells (45) or organ cultures (51), trabecular meshwork cell killing by high levels of wild-type myocilin may play a role in steroid-induced glaucoma.

Lower temperature reversal of myocilin non-secretion and cell killing
Protein non-secretion appears to be a characteristic of most glaucoma-causing mutant myocilins (18). The partial rescue of non-secretion of Pro370Leu mutant protein probably results from an increase in the thermal stability of the mutant protein at the lower temperature, with the result that more molecules fold properly (52). This improved secretion efficiency is all the more intriguing because patients with Pro370Leu mutations exhibit an aggressive glaucoma phenotype (19), indicating that Pro370Leu is a ‘strong’ disease allele. Because the function of myocilin is unknown, we cannot address the question of whether mutant protein secreted at 30°C is functional. The fact that both glycosylated and unglycosylated forms of the mutant protein are secreted (Fig. 9A), which is also the case for wild-type myocilin at 37°C (6), indicates that the rescued protein probably travels the normal secretion pathway. Lower temperature rescue of misfolded mutant proteins associated with a number of other diseases results in normal trafficking of mutant proteins that are stable and functional (41,42). Rescued mutant myocilin also might be functional. Regardless, improved secretion of mutant myocilin would be expected to increase secretion of co-expressed wild-type protein (18). Because the disease mechanism of myocilin-associated glaucoma may include loss of a functional myocilin component, along with the gain-of-function cell lethality, improved secretion of mutant myocilin may have multiple beneficial effects.

Lower temperature culturing reversed the abnormal morphology and cell killing induced by both mutant and wild-type myocilin across a wide range of expression levels. It is noteworthy that the HTM cell killing induced by enhanced green fluorescence protein (EGFP) at 37°C (Fig. 7C: MOIs of 10 and 25), which has been observed in other cell types (53,54), was not as readily reversed by lower temperature (Fig. 8B). EGFP is a cytoplasmic protein, misfolding of which should not affect the ER. Thus, lower temperature reversal of HTM cell killing by mutant and wild-type myocilin, but not by EGFP, supports a myocilin cell killing mechanism that depends upon the presence of misfolded protein in the ER.

New therapeutic approaches
Our findings suggest a new approach to development of pharmacological and other therapies for the progressive, neurodegenerative disease myocilin-associated glaucoma. The trabecular meshwork is located near the front of the eyeball. Periodic cooling treatments applied to the eye might lower the temperature of the trabecular meshwork enough to be beneficial. Topical medical treatments also might be feasible. Folding of temperature-sensitive mutant proteins can be rescued by exposure of cells to small organic molecules termed chemical chaperones because they are believed to stabilize folding intermediates (55). Several of these molecules occur naturally in the body and have been used as therapeutics for other applications (56). The fact that only partial rescue of protein secretion was necessary to reverse abnormal morphology and cell killing, at least over the time period we monitored (20 days), suggests that treatments which reduce misfolding, while not necessarily eliminating it, may prove effective in slowing progression of myocilin-associated glaucoma. Such treatments may have broader significance if other forms of OAG are found to arise from the sensitivity of HTM cells to misfolded proteins.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Antibodies, plasmids and chemicals
The following antibodies were used in this study: monoclonal anti-FLAG and monoclonal anti-beta-actin (Sigma Chemical), polyclonal anti-calnexin, polyclonal anti-calreticulin and polyclonal anti-PDI (Stressgen Biotechnologies). The construction of the expression plasmids encoding wild-type or mutant myocilin cDNAs tagged with a FLAG epitope was described previously (19). Proteasome inhibitors ALLN (10 µg/ml; Calbiochem) or MG132 (20 µg/ml; Sigma Chemical) were added to culture medium and cells were incubated overnight before harvesting.

Cell culture
HEK 293 cells were cultured and transfected as previously described (19). All transfections were carried out in 6-well plates. Primary HTM cells established from a 30-year-old non-glaucomatous individual and characterized as previously described by Polansky et al. (57) were cultured in 6-well plates as described (58). Fifth passage HTM cells were cultured in maintenance medium (Dulbecco's modified Eagle's medium, 10% fetal bovine serum, 2 mM L-glutamine plus antibiotics) for at least 2 weeks after reaching confluence, which allows HTM cells to differentiate from a fibroblast-like morphology to a stable endothelial-like morphology (58).

Recombinant adenovirus
Replication deficient, recombinant adenoviruses encoding FLAG-epitope-tagged wild-type or Pro370Leu mutant myocilin were constructed by cutting the corresponding pCDNA3 constructs (19) with EcoRI and BamHI and subcloning the myocilin cDNAs into pENTR11 vector (Invitrogen Life Technologies). Viral genomes were generated by recombination of a pENTR11 construct and pAd/CMV/V5-DEST using Gateway^TM technology, thereby placing a myocilin cDNA downstream of a cytomegalovirus (CMV) promoter. Recombinant adenoviruses Ad-MYOC-WT (wild-type) and Ad-MYOC-P370L (Pro370Leu mutant) were amplified by standard methods (59) and purified as previously described (60). The titers of both purified viral stocks were 2x10¹¹ plague-forming units/ml (p.f.u./ml). A recombinant adenovirus encoding EGFP driven by a CMV promoter (Ad-EGFP) with a titer of 2x10¹⁰ p.f.u./ml was purchased from the Gene Transfer Vector Core Facility at University of Iowa. Confluent, differentiated primary HTM cells were transduced with Ad-MYOC-WT, Ad-MYOC-P370L or Ad-EGFP at various MOIs for 48 h at 37°C, and were either harvested or fed with fresh complete medium and cultured at either 37 or 30°C for various lengths of time.

Protein extraction, SDS–PAGE and immunoblot analysis
Cell pellets from transfected HEK cells or transduced HTM cells were collected and Triton X-100-soluble and -insoluble materials were extracted as previously described (34). This extraction protocol, which differs from that used previously (19), was chosen to allow comparison of myocilin to other studies of protein misfolding and aggregation (34). Total protein for each sample was quantified with a BCA kit (Pierce Biotechnology) according to the manufacturer's instructions before resolving by SDS–PAGE. Protein samples were boiled in 1xLaemmli sample buffer (Bio-Rad Laboratories) containing 2.5% beta-mercaptoethanol (Sigma Chemical) or 100 mM DTT (Sigma Chemical) for 10 min before loading. For immunoblot analysis, equivalent amounts of total protein for each sample were separated by 10 or 12% SDS–PAGE and electroblotted to 0.22 µm NitroBind nitrocellulose membrane (Fisher Scientific). Chemiluminescence detection was carried out with an ECL detection kit (Amersham Biosciences).

Immunofluorescence microscopy
Cells were seeded on collagen I-coated coverslips (BD Bioscience) for culturing. Cells were fixed in 4% paraformaldehyde for 15 min at room temperature or in methanol for 5 min at –20°C. After three rinses with 1xDulbecco's phosphate-buffered saline (PBS) buffer (Invitrogen), cells were blocked with 10% normal goat serum (Sigma Chemical) and 0.4% Triton X-100 (Sigma Chemical) in antibody buffer (NaCl 150 mM, Tris base 50 mM, BSA 1%, L-lysine 100 mM, sodium azide 0.04%, pH 7.4) for 1 h. Cells were incubated overnight at 4°C with primary antibody diluted in antibody buffer and washed twice for 10 min each in PBS. Cells were incubated with Alexa 488- or Alexa 633-conjugated anti-mouse IgG or anti-rabbit IgG (Molecular Probes) and Hoechst 33342 (2 µg/ml) (Sigma Chemical) diluted in antibody buffer for 1 h at room temperature and washed four times for 10 min each. Cells were mounted on glass slides and images were taken with a Zeiss LSM 510 confocal laser scanning microscopy system.

Immunoprecipitation
Cell pellets from transfected HEK cells or transduced HTM cells were lysed in 500 µl lysis buffer [50 mM Tris, 150 mM NaCl, 1% Nonidet P-40 (Sigma Chemical)] containing protease inhibitor cocktail (Boehringer Mannheim) for 30 min on ice. Cell lysates were incubated overnight at 4°C with 5 µg of primary antibody. Antigen–antibody complexes were precipitated with protein A–Sepharose beads (Amersham Biosciences), and proteins were dissociated from the beads by boiling for 5 min in 1xLaemmli sample buffer.

	SUPPLEMENTARY MATERIAL

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS SUPPLEMENTARY MATERIAL REFERENCES

 
Supplementary Material is available at HMG Online.

	ACKNOWLEDGEMENTS

 
We thank Dr J.R. Polansky for primary HTM cells and culturing protocol, and Dr N.F. Bence for helpful discussions. This work was supported by a grant from the National Eye Institute (EY11405).

	FOOTNOTES

 
^* To whom correspondence should be addressed. Tel: +1 6507233290; Fax: +1 6507237016; Email: vollrath{at}genome.stanford.edu

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