A Common Outer Retinal Change in Retinal Degeneration by Optical Coherence Tomography Can Be Used to Assess Outcomes of Gene Therapy.

Identifying early disease hallmarks in animal models with slow disease progression may expedite disease detection and assessment of treatment outcomes. Using optical coherence tomography, a widely applied noninvasive method for monitoring retinal structure changes, we analyzed retinal optical sections from six mouse lines with retinal degeneration caused by mutations in different disease-causing genes. While images from wild-type mice revealed four well-separated hyper-reflective bands in the outer retina (designated as outer retina reflective bands, ORRBs) at all ages, the second band (ORRB2) and the third band (ORRB3) were merged in retinas of five mutant mouse lines at early ages, suggesting the pathological nature of this alteration. This ORRB change appeared to be degenerating photoreceptor related, and occurred before obvious morphological changes that can be identified on both hematoxylin and eosin-stained sections and electron microscopic sections. Importantly, the merging of ORRB2 and ORRB3 was reversed by treatment with adeno-associated viral vector-mediated gene replacement therapies, and this restoration occurred much earlier than measurable functional or structural improvement. Our data suggest that the ORRB change could be a common hallmark of early retinal degeneration and its restoration could be used for rapid and noninvasive assessment of therapeutic effects following gene therapy or other treatment interventions.

Myocilin Regulates Metalloprotease 2 Activity Through Interaction With TIMP3.

Purpose: To elucidate functions of wild-type myocilin, a secreted glycoprotein associated with glaucoma. Methods: Lysates of mouse eyes were used for immunoprecipitation with affinity-purified antibodies against mouse myocilin. Shotgun proteomic analysis was used for the identification of proteins interacting with myocilin. Colocalization of myocilin and tissue inhibitor of metalloproteinases 3 (TIMP3) in different eye structures was investigated by a multiplex fluorescent in situ hybridization and immunofluorescent labeling with subsequent confocal microscopy. Matrix metalloproteinase 2 (MMP2) activity assay was used to test effects of myocilin on TIMP3 inhibitory action. Results: TIMP3 was identified by a shotgun proteomic analysis as a protein that was coimmunoprecipitated with myocilin from eye lysates of wild-type and transgenic mice expressing elevated levels of mouse myocilin but not from lysates of transgenic mice expressing mutated mouse myocilin. Interaction of myocilin and TIMP3 was confirmed by coimmunoprecipitation of myocilin and TIMP3 from HEK293 cells transiently transfected with cDNAs encoding these proteins. The olfactomedin domain of myocilin is essential for interaction with TIMP3. In the eye, the main sites of myocilin and TIMP3 colocalization are the trabecular meshwork, sclera, and choroid. Using purified proteins, it has been shown that myocilin markedly enhanced the inhibitory activity of TIMP3 toward MMP2. Conclusions: Myocilin may serve as a modulator of TIMP3 activity via interactions with the myocilin olfactomedin domain. Our data imply that in the case of MYOCILIN null or some glaucoma-causing mutations, inhibitory activity of TIMP3 toward MMP2 might be reduced, mimicking deleterious mutations in the TIMP3 gene.

Mutated myocilin and heterozygous Sod2 deficiency act synergistically in a mouse model of open-angle glaucoma.

Glaucoma is a multifactorial optic neuropathy characterized by retinal ganglion cell (RGC) death and axonal degeneration leading to irreversible blindness. Mutations in the myocilin (MYOC) gene are the most common genetic factors of primary open-angle glaucoma. To develop a genetic mouse model induced by the synergistic interaction of mutated myocilin and another significant risk factor, oxidative stress, we produced double-mutant mice (Tg-MYOC(Y437H/+)/Sod2(+/-)) bearing human MYOC with a Y437H point mutation and a heterozygous deletion of the gene for the primary antioxidant enzyme, superoxide dismutase 2 (SOD2). Sod2 is broadly expressed in most tissues including the trabecular meshwork (TM) and heterozygous Sod2 knockout mice exhibit the reduced SOD2 activity and oxidative stress in all studied tissues. Accumulation of Y437H myocilin in the TM induced endoplasmic reticulum stress and led to a 45% loss of smooth muscle alpha-actin positive cells in the eye drainage structure of 10- to 12-month-old Tg-MYOC(Y437H/+)/Sod2(+/-) mice as compared with wild-type littermates. Tg-MYOC(Y437H/+)/Sod2(+/-) mice had higher intraocular pressure, lost about 37% of RGCs in the peripheral retina, and exhibited axonal degeneration in the retina and optic nerve as compared with their wild-type littermates. Single-mutant littermates containing MYOC(Y437H/+) or Sod2(+/-) exhibited no significant pathological changes until 12 months of age. Additionally, we observed elevated expression of endothelial leukocyte adhesion molecule-1, a human glaucoma marker, in the TM of Tg-MYOC(Y437H/+)/Sod2(+/-) mice. This is the first reported animal glaucoma model that combines expression of a glaucoma-causing mutant gene and an additional mutation mimicking a deleterious environment factor that acts synergistically.

Tobacco class I cytosolic small heat shock proteins are under transcriptional and translational regulations in expression and heterocomplex prevails under the high-temperature stress condition in vitro.

Seven genomic clones of tobacco (Nicotiana tabacum W38) cytosolic class I small heat shock proteins (sHSPs), probably representing all members in the class, were isolated and found to have 66 to 92% homology between their nucleotide sequences. Even though all seven sHSP genes showed heat shock-responsive accumulation of their transcripts and proteins, each member showed discrepancies in abundance and timing of expression upon high-temperature stress. This was mainly the result of transcriptional regulation during mild stress conditions and transcriptional and translational regulation during strong stress conditions. Open reading frames (ORFs) of these genomic clones were expressed in Escherichia coli and the sHSPs were purified from E. coli. The purified tobacco sHSPs rendered citrate synthase and luciferase soluble under high temperatures. At room temperature, non-denaturing pore exclusion polyacrylamide gel electrophoresis on three sHSPs demonstrated that the sHSPs spontaneously formed homo-oligomeric complexes of 200 ∼ 240 kDa. However, under elevated temperatures, hetero-oligomeric complexes between the sHSPs gradually prevailed. Atomic force microscopy showed that the hetero-oligomer of NtHSP18.2/NtHSP18.3 formed a stable oligomeric particle similar to that of the NtHSP18.2 homo-oligomer. These hetero-oligomers positively influenced the revival of thermally inactivated luciferase. Amino acid residues mainly in the N-terminus are suggested for the exchange of the component sHSPs and the formation of dominant hetero-oligomers under high temperatures.

Myocilin regulates cell proliferation and survival.

Myocilin, a causative gene for open angle glaucoma, encodes a secreted glycoprotein with poorly understood functions. To gain insight into its functions, we produced a stably transfected HEK293 cell line expressing myocilin under an inducible promoter and compared gene expression profiles between myocilin-expressing and vector control cell lines by a microarray analysis. A significant fraction of differentially expressed genes in myocilin-expressing cells was associated with cell growth and cell death, suggesting that myocilin may have a role in the regulation of cell growth and survival. Increased proliferation of myocilin-expressing cells was demonstrated by the WST-1 proliferation assay, direct cell counting, and immunostaining with antibodies against Ki-67, a cellular proliferation marker. Myocilin-containing conditioned medium also increased proliferation of unmodified HEK293 cells. Myocilin-expressing cells were more resistant to serum starvation-induced apoptosis than control cells. TUNEL-positive apoptotic cells were dramatically decreased, and two apoptotic marker proteins, cleaved caspase 7 and cleaved poly(ADP-ribose) polymerase, were significantly reduced in myocilin-expressing cells as compared with control cells under apoptotic conditions. In addition, myocilin-deficient mesenchymal stem cells exhibited reduced proliferation and enhanced susceptibility to serum starvation-induced apoptosis as compared with wild-type mesenchymal stem cells. Phosphorylation of ERK1/2 and its upstream kinases, c-Raf and MEK, was increased in myocilin-expressing cells compared with control cells. Elevated phosphorylation of ERK1/2 was also observed in the trabecular meshwork of transgenic mice expressing 6-fold higher levels of myocilin when compared with their wild-type littermates. These results suggest that myocilin promotes cell proliferation and resistance to apoptosis via the ERK1/2 MAPK signaling pathway.

Myocilin mediates myelination in the peripheral nervous system through ErbB2/3 signaling.

The glaucoma-associated gene, myocilin, is expressed in ocular and non-ocular tissues including the peripheral nervous system, but its functions in these tissues remain poorly understood. We demonstrate that in sciatic nerve, myocilin is expressed in Schwann cells with high concentrations at the nodes of Ranvier. There, myocilin interacts with gliomedin, neurofascin, and NrCAM, which are essential for node formation and function. Treatment of isolated dorsal root ganglion cultures with myocilin stimulates clustering of the nodal proteins neurofascin and sodium channel Nav1.2. Sciatic nerves of myocilin null mice express reduced levels of several myelin-associated and basal membrane proteins compared with those of wild-type littermates. They also demonstrate reduced myelin sheath thickness and partial disorganization of the nodes. Myocilin signaling through ErbB2/3 receptors may contribute to these observed effects. Myocilin binds to ErbB2/ErbB3, activates these receptors, and affects the downstream PI3K-AKT signaling pathway. These data implicate a role for myocilin in the development and/or maintenance of myelination and nodes of Ranvier in sciatic nerve.

Glucose-regulated protein 94 triage of mutant myocilin through endoplasmic reticulum-associated degradation subverts a more efficient autophagic clearance mechanism.

BACKGROUND: Mutant myocilin accumulates in the endoplasmic reticulum for unknown reasons. RESULTS: Glucose-regulated protein (Grp) 94 depletion reduces mutant myocilin by engaging autophagy. CONCLUSION: Grp94 triages mutant myocilin through ER-associated degradation, subverting autophagy. SIGNIFICANCE: Treating glaucoma could be possible by inhibiting Grp94 and reducing its novel client, mutant myocilin. Clearance of misfolded proteins in the endoplasmic reticulum (ER) is traditionally handled by ER-associated degradation (ERAD), a process that requires retro-translocation and ubiquitination mediated by a luminal chaperone network. Here we investigated whether the secreted, glaucoma-associated protein myocilin was processed by this pathway. Myocilin is typically transported through the ER/Golgi network, but inherited mutations in myocilin lead to its misfolding and aggregation within trabecular meshwork cells, and ultimately, ER stress-induced cell death. Using targeted knockdown strategies, we determined that glucose-regulated protein 94 (Grp94), the ER equivalent of heat shock protein 90 (Hsp90), specifically recognizes mutant myocilin, triaging it through ERAD. The addition of mutant myocilin to the short list of Grp94 clients strengthens the hypothesis that ß-strand secondary structure drives client association with Grp94. Interestingly, the ERAD pathway is incapable of efficiently handling the removal of mutant myocilin, but when Grp94 is depleted, degradation of mutant myocilin is shunted away from ERAD toward a more robust clearance pathway for aggregation-prone proteins, the autophagy system. Thus ERAD inefficiency for distinct aggregation-prone proteins can be subverted by manipulating ER chaperones, leading to more effective clearance by the autophagic/lysosomal pathway. General Hsp90 inhibitors and a selective Grp94 inhibitor also facilitate clearance of mutant myocilin, suggesting that therapeutic approaches aimed at inhibiting Grp94 could be beneficial for patients suffering from some cases of myocilin glaucoma.

Myocilin interacts with syntrophins and is member of dystrophin-associated protein complex.

Genetic studies have linked myocilin to open angle glaucoma, but the functions of the protein in the eye and other tissues have remained elusive. The purpose of this investigation was to elucidate myocilin function(s). We identified α1-syntrophin, a component of the dystrophin-associated protein complex (DAPC), as a myocilin-binding candidate. Myocilin interacted with α1-syntrophin via its N-terminal domain and co-immunoprecipitated with α1-syntrophin from C2C12 myotubes and mouse skeletal muscle. Expression of 15-fold higher levels of myocilin in the muscles of transgenic mice led to the elevated association of α1-syntrophin, neuronal nitric-oxide synthase, and α-dystroglycan with DAPC, which increased the binding of laminin to α-dystroglycan and Akt signaling. Phosphorylation of Akt and Forkhead box O-class 3, key regulators of muscle size, was increased more than 3-fold, whereas the expression of muscle-specific RING finger protein-1 and atrogin-1, muscle atrophy markers, was decreased by 79 and 88%, respectively, in the muscles of transgenic mice. Consequently, the average size of muscle fibers of the transgenic mice was increased by 36% relative to controls. We suggest that intracellular myocilin plays a role as a regulator of muscle hypertrophy pathways, acting through the components of DAPC.

Analysis of glucocorticoid-induced MYOC expression in human trabecular meshwork cells.

To understand the regulatory mechanisms governing glucocorticoid-mediated MYOC induction in human trabecular meshwork (HTM) cells, the expression and degradation of MYOC mRNA were quantified in HTM cells by Northern blot analysis, and the transcriptional activity of constructs containing variable lengths of putative MYOC promoters was assessed by luciferase reporter assay. Here, we confirmed that MYOC is a delayed secondary glucocorticoid-responsive gene by demonstrating that its transcription was not initiated immediately by the addition of dexamethasone (DEX) and was completely inhibited by treatment with cycloheximide. In addition, we demonstrated that MYOC mRNA is degraded very slowly, with approximately half persisting for at least 4 days, suggesting that its mRNA is intrinsically quite stable. Promoter analysis of up to 5271 base pairs upstream of MYOC revealed that luciferase induction by DEX was increased by 280 ± 34% in HTM cells. Moreover, DEX induction required the region between base pairs -2548 and -1541. However, the putative regulatory element exhibited little activity in other cell lines, including TM-5, 293A, SH-SY5Y, and human retinal pigment epithelium (RPE) cells. To our knowledge, this study provides the first evidence for the presence of a cis-acting region for secondary glucocorticoid responsiveness in the 5'-flanking sequences of MYOC. It will be a major step towards understanding the expression pattern of MYOC in HTM cells and TM tissue.

Expression of myocilin mutants sensitizes cells to oxidative stress-induced apoptosis: implication for glaucoma pathogenesis.

Mutations in the myocilin gene are associated with juvenile and adult-onset primary open-angle glaucoma. However, the pathogenic mechanisms of myocilin-induced glaucoma are still largely unknown. To investigate these mechanisms, we developed stably transfected HEK293 cell lines expressing wild-type or mutant (Y437H and I477N) myocilins under an inducible promoter. Expression of two mutant myocilins led to different levels of endoplasmic reticulum stress and increased apoptosis after treatment of cells with hydrogen peroxide. The Y437H mutant myocilin cell line showed the highest sensitivity to the oxidant treatment. Several antioxidant genes were down-regulated in the Y437H mutant myocilin cell line, but not in other cell lines. The Y437H mutant myocilin cell line also produced more reactive oxygen species than other cell lines examined. Consistent with the data obtained in cultured cells, the endoplasmic reticulum stress marker, 78 kDa glucose-regulated protein, was up-regulated, whereas antioxidant proteins, paraoxonase 2 and glutathione peroxidase 3, were down-regulated in the eye angle tissue of 18-month-old transgenic mice expressing Y437H myocilin mutant. In addition, a pro-apoptotic factor, CCAAT/enhancer-binding protein-homologous protein, was up-regulated in the aged transgenic mouse angle tissue. Our results suggest that expression of mutated myocilins may have a sensitization effect, which can lead to a severe phenotype in combination with oxidative stress. Mutant myocilins may confer different sensitivity to oxidative stress depending on the mutation.

KLF4 positively regulates human ghrelin expression.

Ghrelin, an endogenous ligand of the GH (growth hormone) secretagogue receptor, influences many metabolic processes including GH secretion, food intake, energy balance, insulin secretion and adipogenesis. Although ghrelin exhibits a variety of biological functions, the mechanism by which ghrelin expression is regulated is unknown. Ghrelin is expressed in the gastrointestinal tract, predominantly in the stomach, as is KLF4 (Krüppel-like factor 4). Therefore we investigated whether ghrelin expression is associated with KLF4, and found that the tissue distribution of ghrelin corresponded with that of KLF4. Furthermore, treatment with butyrate, an inducer of KLF4 expression, stimulated ghrelin expression, and fasting, which induces ghrelin expression, also increased KLF4 expression, suggesting that ghrelin expression is associated with KLF4. Then, we investigated the effects of KLF4 on the human ghrelin-promoter activity and identified a KLF4-responsive region in the promoter. KLF4 expression specifically stimulated human ghrelin-promoter activity in a dose-dependent manner in human gastric-cancer AGS cells. However, this effect was not seen in response to a mutant KLF4 construct. Transfection studies using mutant constructs containing 5'-deletions in the human ghrelin promoter revealed that the KLF4-responsive element is located between -1228 and -1105. Electrophoretic mobility shift assays using oligonucleotides containing -1165/-1146 revealed the binding of KLF4 to the human ghrelin promoter. Finally, deletion of the -1165/-1146 region abrogated KLF4-induced transactivation of the ghrelin promoter. Collectively, these results indicate that KLF4 positively regulates human ghrelin expression via binding to a KLF-responsive region in the promoter.

Dual localization of wild-type myocilin in the endoplasmic reticulum and extracellular compartment likely occurs due to its incomplete secretion.

PURPOSE: Wild-type myocilin is known to be secreted extracellularly, but a significant amount of the protein is also present in the endoplasmic reticulum (ER). The present study was undertaken to address whether intracellular myocilin is a true ER resident protein. METHODS: Human wild-type myocilin was adenovirally expressed in human trabecular meshwork cells, and general characteristics of both intracellular and extracellular myocilins including molecular weight, pI, glycosylation state, and cleavage site of the signal peptide were examined by biochemical analyses. Topology and decay kinetics of myocilin were also examined by protease protection assay and pulse chase analysis, respectively. The expression pattern and cytopathic effect of myocilin were analyzed in individual cells by immunocytochemistry. RESULTS: Intracellular myocilin were very similar to secreted myocilin in characteristics such as molecular weight, pI, glycosylation state, and cleavage site of the signal peptide. The intracellular protein was found to be present in the lumen of the ER where it appeared to be retained without further export to the Golgi apparatus. The kinetics of myocilin turnover clearly showed that it was intrinsically a very stable but incompletely secreted protein. The expression of myocilin was confined to a subset of cells and accompanied by the upregulation of a 78 kDa glucose-regulated protein, suggesting that it was not properly folded or processed in the ER. CONCLUSIONS: Based on these findings and the fact that myocilin has no known ER retention signals, the ER localization of wild-type myocilin is likely a consequence of its incomplete secretion due to its misfolding.

Crucial roles of neuronatin in insulin secretion and high glucose-induced apoptosis in pancreatic beta-cells.

Neuronatin (Nnat) was initially identified as a selectively-expressed gene in neonatal brains, but its expression has been also identified in pancreatic beta-cells. Therefore, to investigate the possible functions that Nnat may serve in pancreatic beta-cells, two Nnat isotypes (alpha and beta) were expressed using adenoviruses in murine MIN6N8 pancreatic beta-cells, and the cellular fates and the effects of Nnat on insulin secretion, high glucose-induced apoptosis, and functional impairment were examined. Nnatalpha and Nnatbeta were primarily localized in the endoplasmic reticulum (ER), and their expressions increased insulin secretion by increasing intracellular calcium levels. However, under chronic high glucose conditions, the Nnatbeta to Nnatalpha ratio gradually increased in proportion to the length of exposure to high glucose levels. Moreover, adenovirally-expressed Nnatbeta was inclined to form aggresome-like structures, and we found that Nnatbeta aggregation inhibited the function of the proteasome. Therefore, when glucose is elevated, the expression of Nnatbeta sensitizes MIN6N8 cells to high glucose stress, which in turn, causes ER stress. As a result, expression of Nnatbeta increased hyperglycemia-induced apoptosis. In addition, the expression of Nnatbeta under high glucose conditions decreased the expression of genes important for beta-cell function, such as glucokinase (GCK), pancreas duodenum homeobox-1 (PDX-1), and insulin. Collectively, Nnat may play a critical factor in normal beta-cell function, as well as in the pathogenesis of type 2 diabetes.

Therapeutic potential of peroxisome proliferators--activated receptor-alpha/gamma dual agonist with alleviation of endoplasmic reticulum stress for the treatment of diabetes.

OBJECTIVE: Peroxisome proliferator-activated receptor (PPAR) alpha/gamma dual agonists have the potential to be used as therapeutic agents for the treatment of type 2 diabetes. This study evaluated the function of macelignan, a natural compound isolated from Myristica fragrans, as a dual agonist for PPARalpha/gamma and investigated its antidiabetes effects in animal models. RESEARCH DESIGN AND METHODS: GAL4/PPAR chimera transactivation was performed and the expression of PPARalpha/gamma target genes was monitored to examine the ability of macelignan to activate PPARalpha/gamma. Additionally, macelignan was administrated to obese diabetic (db/db) mice to investigate antidiabetes effects and elucidate its molecular mechanisms. RESULTS: Macelignan reduced serum glucose, insulin, triglycerides, free fatty acid levels, and triglycerides levels in the skeletal muscle and liver of db/db mice. Furthermore, macelignan significantly improved glucose and insulin tolerance in these mice, and without altering food intake, their body weights were slightly reduced while weights of troglitazone-treated mice increased. Macelignan increased adiponectin expression in adipose tissue and serum, whereas the expression and serum levels of tumor necrosis factor-alpha and interleukin-6 decreased. Macelignan downregulated inflammatory gene expression in the liver and increased AMP-activated protein kinase activation in the skeletal muscle of db/db mice. Strikingly, macelignan reduced endoplasmic reticulum (ER) stress and c-Jun NH(2)-terminal kinase activation in the liver and adipose tissue of db/db mice and subsequently increased insulin signaling. CONCLUSIONS: Macelignan enhanced insulin sensitivity and improved lipid metabolic disorders by activating PPARalpha/gamma and attenuating ER stress, suggesting that it has potential as an antidiabetes agent for the treatment of type 2 diabetes.

Identification of flotillin-1 as a protein interacting with myocilin: implications for the pathogenesis of primary open-angle glaucoma.

Mutations in MYOC gene encoding myocilin are responsible for primary open-angle glaucoma (POAG). In order to search for protein(s) that can interact with myocilin, we screened a human skeletal muscle cDNA library using yeast two-hybrid system and identified flotillin-1, a structural protein of lipid raft that is detergent-resistant and a liquid ordered microdomain, as a protein interacting with myocilin. The interaction was confirmed by in vitro glutathione S-transferase pulldown and in vivo co-immunoprecipitation studies. In yeast two-hybrid assay, the C-terminus of myocilin, an olfactomedin-like domain in which most mutations related to POAG are scattered, was found to be necessary and sufficient for the interaction. However, myocilins with mutations such as G364V, K423E, and Y437H on the domain failed to interact with flotillin-1. Although the physiological significance of the interaction has yet to be elucidated, our results showed that the alteration of the interaction by mutations in MYOC might be a key factor of the pathogenesis of POAG.

Accumulation of mutant myocilins in ER leads to ER stress and potential cytotoxicity in human trabecular meshwork cells.

MYOC encoding a 55kDa secretory glycoprotein named myocilin is closely linked to primary open-angle glaucoma (POAG). To understand a role played by MYOC in glaucoma, we examined the cellular fate of various mutant myocilins that were adenovirally expressed in human trabecular meshwork cells. Most myocilins with mutations such as G364V, Q368X, K423E, Y437H, and I477N were intrinsically stable, and appeared to have interactions with wild-type myocilin but not with stromelysin and thereby selectively inhibited the secretion of the former protein. The myocilins expressed were identified to be concentrated into fine punctate aggregates in endoplasmic reticulum, but never developed into the formation of aggresomes. In endoplasmic reticulum, the accumulation of the myocilins resulted in the upregulation of 78kDa glucose-regulated protein and protein disulfide isomerase. In addition, the expression of the myocilins led to deformed cellular morphology and diminished cell proliferation, an effect postulated to result in the dysfunction of trabecular cells that could be a cause of glaucoma. Therefore, our results support the statement that gain of function rather than haploinsufficiency is a critical mechanism for POAG in individuals with mutations on MYOC.

Expression of wild-type and truncated myocilins in trabecular meshwork cells: their subcellular localizations and cytotoxicities.

PURPOSE: To investigate the subcellular localizations and potential cytotoxicities of wild-type and truncated (Q368X) myocilin in cultured human trabecular meshwork (TM) cells. METHODS: Full-length wild-type myocilin, truncated myocilin, and stromelysin were expressed as green fluorescence (GFP) or DsRed fusion proteins in TM cells by using adenoviral vectors, and their secretory properties and cytopathic effects were evaluated by Western blot analysis and cell proliferation assay, respectively. To determine the subcellular localizations of myocilins, the cellular organelles of the infected TM cells were stained with organelle-specific antibodies or fluorescent indicators and examined under a confocal microscope. RESULTS: Wild-type myocilin was expressed mainly in the perinuclear region of TM cells and was localized preferentially in endoplasmic reticulum (ER), but not in actin, microtubules, or mitochondria. Truncated myocilin was also localized in ER, and its expression was found to be potentially toxic to TM cells, leading to deformed cellular morphology and diminished cell proliferation, but it had no effect on the secretion of stromelysin. The truncated myocilin was also found to be colocalized with and appeared to aggregate with wild-type myocilin when the proteins were coexpressed. CONCLUSIONS: TM cells participating in the turnover of trabecular extracellular matrix (ECM) components are important in regulating aqueous outflow. The truncated myocilin, colocalized and coaggregated with wild-type myocilin, is believed to cause a dysfunction of the cells, resulting in alterations in structural compartmentalization of trabecular ECM and obstruction of aqueous outflow.